university of groningen chemo-enzymatic routes to enantiopure … · epoxidation reactions using...

University of Groningen

Chemo-enzymatic routes to enantiopure haloalcohols and epoxidesHaak, Robert M.

IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.

Document VersionPublisher's PDF, also known as Version of record

Publication date:2008

Link to publication in University of Groningen/UMCG research database

Citation for published version (APA):Haak, R. M. (2008). Chemo-enzymatic routes to enantiopure haloalcohols and epoxides. s.n.

CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).

Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.

Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.

Download date: 10-10-2020

https://www.rug.nl/research/portal/en/publications/chemoenzymatic-routes-to-enantiopure-haloalcohols-and-epoxides(c24b44f3-2b41-4fa7-9787-5ab756154e52).html

https://www.rug.nl/research/portal/en/publications/chemoenzymatic-routes-to-enantiopure-haloalcohols-and-epoxides(c24b44f3-2b41-4fa7-9787-5ab756154e52).html

Chemo-Enzymatic Routes to Enantiopure Haloalcohols and Epoxides

Robert M. Haak

Franse pagina.doc

The work described in this thesis has been carried out at the Stratingh Institute for Chemistry, University of Groningen, the Netherlands.

The research described in this thesis was part of the program Integration of Biosynthesis and Organic Synthesis (IBOS), with financial support from the Netherlands Organization for Scientific Research (NWO), the Ministry of Ecomic Affairs, Royal DSM N.V. and N.V. Organon.

Cover design by Robert M. Haak and Thomas C. Pijper, photograph taken by Alex Nikada.

Printed by PrintPartners Ipskamp BV, Enschede, the Netherlands

ISBN: 978-90-367-3525-4 (printed version)

978-90-367-3526-1 (digital version)

RIJKSUNIVERSITEIT GRONINGEN


Proefschrift

ter verkrijging van het doctoraat in de Wiskunde en Natuurwetenschappen aan de Rijksuniversiteit Groningen

op gezag van de Rector Magnificus, dr. F. Zwarts, in het openbaar te verdedigen op

maandag 15 september 2008 om 13.15 uur

door

Robert Maurits Haak

geboren op 26 juli 1979 te Delfzijl

Titelblad.doc

Promotores : Prof. dr. B.L. Feringa

Prof. dr. J.G. de Vries

Prof. dr. ir. A.J. Minnaard

Beoordelingscommissie : Prof. dr. J.B.F.N. Engberts

Prof. dr. D.B. Janssen

Prof. dr. F.P.J.T. Rutjes

ISBN: 978-90-367-3525-4 (printed version)

978-90-367-3526-1 (digital version)

5

Table of contents.doc

Table of contents

Table of contents

Preface................................................................................................................................ 9 Chapter 1: General introduction ..................................................................................... 13 1.1 Process intensification ..................................................................................................14 1.2 Stereochemistry and enantiomers ................................................................................14 1.3 Catalysis .........................................................................................................................17 1.3.1 Enzyme catalysis .................................................................................................19 1.3.2 Cascade catalysis..................................................................................................20 1.4 Kinetic resolution..........................................................................................................21 1.4.1 Dynamic kinetic resolution ................................................................................23 1.5 Enantioselective synthesis of epoxides.........................................................................26 1.6 Combination of bio- and chemocatalysis.....................................................................28 1.7 Notes and references .....................................................................................................31 Chapter 2: Epoxidation of olefins in a centrifugal contact separator .............................. 39 2.1 Importance of process intensification – the CCS.........................................................40 2.1.1 Existing biphasic epoxidation reactions .............................................................42 2.2 Exploring epoxidation reactions for use in a CCS .......................................................44 2.2.1 Manganese-catalyzed epoxidation reactions .....................................................44 2.2.2 MTO-catalyzed epoxidation of pinenes .............................................................46 2.3 Biphasic iron-catalyzed epoxidation of olefins............................................................46 2.4 Epoxidation of olefins catalyzed by Fe/phen and Fe/bipy complexes ........................50 2.5 Tungsten-catalyzed epoxidation of cyclooctene in the CCS.......................................57 2.6 Conclusions and outlook...............................................................................................60 2.7 Experimental section.....................................................................................................62 2.7.1 General remarks ..................................................................................................62 2.7.2 Biphasic manganese-catalyzed epoxidation of styrene .....................................62 2.7.3 Biphasic MTO-catalyzed epoxidation of pinene................................................63 2.7.4 Iron-catalyzed epoxidation of olefins in a two-phase system...........................63 2.7.5 Epoxidation of olefins catalyzed by Fe/phen and Fe/bipy complexes ..............64 2.7.6 NaZnPOM-catalyzed epoxidation of cyclooctene.............................................65 2.8 Notes and references .....................................................................................................67

6


Table of contents

Chapter 3: Enantiopure chloroalcohols via enzymatic kinetic resolution...................... 71 3.1 Introduction to haloalcohol dehalogenases .................................................................72 3.2 Diene monoepoxides, chloroalcohols, and their synthesis .........................................73 3.3 Enzymatic nucleophilic ring opening of vinyloxiranes ..............................................75 3.4 Enzymatic ring closure of chloroalcohols to epoxides ................................................77 3.4.1 Kinetic resolution of 3.1 – 3.7 on analytical scale .............................................77 3.4.2 Kinetic resolution on preparative scale..............................................................81 3.5 Hydrolysis of epoxides during enzymatic kinetic resolution......................................83 3.5.1 The role of hydrolysis in HheC-catalyzed enzymatic kinetic resolution.........84 3.5.2 Trapping of epoxide to prevent hydrolysis ........................................................86 3.6 Limitations of this methodology ..................................................................................87 3.7 Conclusions ...................................................................................................................89 3.8 Experimental section.....................................................................................................90 3.8.1 General remarks ..................................................................................................90 3.8.2 Synthesis of substrates 3.1 – 3.7..........................................................................90 3.8.3 Production and purification of the enzyme.......................................................96 3.8.4 General procedure for enzymatic kinetic resolution on analytical scale .........97 3.8.5 General procedure for enzymatic kinetic resolution on preparative scale.......97 3.8.6 Determination of absolute configuration...........................................................98 3.9 Notes and references .....................................................................................................99 Chapter 4: A new approach to the dynamic kinetic resolution of haloalcohols........... 103 4.1 Introduction to dynamic kinetic resolution ..............................................................104 4.2 Results and discussion.................................................................................................109 4.2.1 Substrates...........................................................................................................109 4.2.2 Enzymes.............................................................................................................110 4.2.3 Racemization catalysts ......................................................................................114 4.2.4 Dynamic kinetic resolution experiments.........................................................117 4.3 Conclusions and outlook.............................................................................................129 4.4 Experimental section...................................................................................................130 4.4.1 General remarks ................................................................................................130 4.4.2 Synthesis of haloalcohols 4.3 ............................................................................130 4.4.3 Synthesis of racemic products 4.4 ....................................................................137 4.4.4 Production and overexpression of enzymes E1 – E5.......................................140 4.4.5 Kinetic resolution of 4.3l using E1 – E5...........................................................141 4.4.6 Synthesis of catalysts 4.8a – 4.8c ......................................................................141 4.4.7 General procedure for the dynamic kinetic resolution of compounds 4.3.....142 4.4.8 Dynamic kinetic resolution of 4.3 on preparative scale ..................................143 4.5 Notes and references ...................................................................................................143

7


Table of contents

Chapter 5: Synthetic applications of enantiopure chloroalcohols ................................ 147 5.1 Introduction ................................................................................................................148 5.2 Achmatowicz rearrangement of 2-chloro-1-(furan-2-yl)ethanol ............................148 5.3 Ireland-Claisen rearrangement of (E)-1-chloro-4-phenylbut-3-en-2-yl propionate ..................................................................................................................150 5.4 Johnson orthoester rearrangement of (E)-1-chloro-4-phenylbut-3-en-2-ol ...........154 5.5 Suggestions for further research.................................................................................157 5.6 Conclusions .................................................................................................................158 5.7 Experimental section...................................................................................................158 5.7.1 General remarks ................................................................................................158 5.7.2 Achmatowicz rearrangement ...........................................................................159 5.7.3 Ireland-Claisen rearrangement ........................................................................160 5.7.4 Johnson orthoester rearrangement...................................................................162 5.7.5 Further research................................................................................................163 5.8 Notes and references ...................................................................................................164 Chapter 6 Enantiopure alcohols as amplifiers of 2D chirality....................................... 167 6.1 Introduction ................................................................................................................168 6.2 Synthesis of chiral solvents.........................................................................................171 6.3 Control of enantioselective 2D self-assembly using chiral solvents .........................172 6.4 Conclusions and outlook.............................................................................................176 6.5 Experimental part........................................................................................................177 6.5.1 General remarks ................................................................................................177 6.5.2 Synthesis of chiral solvents...............................................................................178 6.5.3 Scanning tunneling microscopy (STM)............................................................179 6.6 Notes and references ...................................................................................................179 Samenvatting ................................................................................................................. 183 Dankwoord .................................................................................................................... 193

8


Table of contents

9

Preface.doc

Preface

Preface

In the research described in this thesis, methods originating from biocatalysis, organic synthesis, transition metal catalysis, and chemical engineering are combined in order to develop novel multistep routes to enantiopure epoxides and epoxide derivatives. We chose epoxides as the focus of our collaborative efforts, because they are often-used, highly versatile intermediates in chemical reactions. They may be converted by a number of methods leading to a wide variety of substituted alcohols and other products (Scheme 1).

Scheme 1 Synthesis and transformation of epoxides.

Our initial aim was to make the catalytic cascade shown in Scheme 2, starting off with the transition-metal catalyzed epoxidation of a terminal olefin, followed by a stereoselective enzymatic ring opening leading to enantiopure epoxides and haloalcohols. Coupled with transition metal catalyzed racemization of the obtained haloalcohol, this would eventually lead to enantiomerically pure epoxides in quantitative yield.

Furthermore, our aim was to perform these reactions in centrifugal contact separators (CCS), devices for continuous, biphasic processes, that combine intensive mixing with efficient centrifugal separation. Conceivably, the reaction cycle shown in Scheme 2 could be brought about by putting multiple CCSs in series, with the advantage that each individual step is done in a separate vessel, allowing for optimal reaction conditions (Figure 1).

10

Preface.doc

Preface

Scheme 2 Synthesis of enantiopure epoxides by a combination of olefin epoxidation, enzymatic epoxide ring opening, and racemization of haloalcohols. TM catal: transition metal catalyzed.

Figure 1 Envisioned catalytic cascade performed in a series of CCSs.

A number of important methods are combined in the cycle shown in Scheme 2, such as transition-metal catalyzed epoxidation, racemization, and enzyme-catalyzed ring opening of epoxides. In view of the objective of doing these reactions in a CCS, efficient biphasic procedures had to be found or developed for all these different reactions. In due course, the focus of the research described in this thesis shifted from epoxide ring opening to the reverse reaction, enzymatic ring closure of haloalcohols, as will be explained later. Nonetheless, the combined use of enzyme and transition-metal catalysis and the use of CCS reactors will emerge as important themes throughout this thesis.

To give a brief outline, Chapter 1 gives a general introduction on concepts, objectives, and approaches that are central to the entire thesis, such as stereochemistry, enantiomers, enzyme and cascade catalysis, (dynamic) kinetic resolution, and process

11

Preface.doc

Preface

intensification. Furthermore, existing approaches to the synthesis of enantiopure epoxides will be briefly discussed. Finally, some examples will be given of research at the interface between traditional subdisciplines in chemistry. Besides dynamic kinetic resolution, these examples include hybrid catalysts based on (modified) proteins or DNA.

In Chapter 2, the concept of process intensification and the use of a CCS for performing transition-metal catalyzed reactions, in particular epoxidation of olefins, will be introduced. An overview is given of the specific requirements for reactions in CCSs. Based on the literature, two epoxidation methods were selected for further development in the CCS. Besides the study of these methods and the discussion of the experimental results, a number of new Fe-catalysts are presented that can be used for epoxidation reactions using peracetic acid as the oxidant.

In Chapter 3, the use of enzymatic kinetic resolution to obtain enantiopure chloroalcohols is described. In this study, we employed the haloalcohol dehalogenase HheC, a highly active and selective enzyme both in the enantioselective ring closure of halohydrins and the nucleophilic ring opening of epoxides. In this chapter, our investigations of the scope of HheC are described using a variety of substrates, mainly allylic and (hetero)aromatic chloroalcohols. Furthermore, the feasibility of using a haloalcohol dehalogenase on multigram scale is demonstrated.

In Chapter 4, the extension of HheC-catalyzed kinetic resolution to a dynamic kinetic resolution (DKR) is discussed, leading to aromatic epoxides of high enantiopurity. The combination of various HheC mutants and a newly discovered iridium-based racemization catalyst is described. Furthermore, the optimization process for the DKR is discussed in detail, with emphasis on how to avoid the key problems, like deactivation of the racemization catalyst and of the enzyme. The resulting method constitutes a complementary procedure to the well-known lipase-based DKR procedures, in that it provides a direct route from haloalcohols to enantiomerically enriched epoxides.

Chapter 5 gives an overview of a number of synthetic applications of the chloroalcohols obtained by HheC-catalyzed kinetic resolution. Rearrangement reactions were chosen as the focus of these studies, because of their potential to bring about substantial and often stereoselective structural modification in a single step. Three transformations are discussed, namely Achmatowicz rearrangement of 2-chloro-1-furyl-ethanol, Ireland-Claisen rearrangement of 1-chloro-4-phenylbut-3-en-2-yl propionate, and Johnson orthoester rearrangement of 1-chloro-4-phenylbut-3-en-2-ol.

Finally, in Chapter 6, the synthesis and subsequent lipase-catalyzed kinetic resolution of 1-phenyl-1-octanol is discussed. The products have been subsequently used as chiral solvents to control the enantiopreference of the chiral self-assembly of achiral

12

Preface.doc

Preface

molecules on an achiral surface. This result demonstrates for the first time that enantiopure solvent may be used to control chiral molecular self-assembly on a surface.

In conclusion, the results described in this thesis comprise a number of innovative chemo-enzymatic procedures to obtain enantiomerically pure compounds of synthetic interest, in particular haloalcohols and epoxides. Furthermore, the potential of some of these compounds in synthesis and chiral 2D self-assembly is shown. In addition, a novel approach to process intensification is discussed.

Chapter 1 General introduction

In this introductory chapter, several important concepts will be introduced, such as

stereochemistry, enantiomers, enzyme and cascade catalysis, (dynamic) kinetic

resolution, and process intensification. Furthermore, existing methods for the synthesis

of enantiopure epoxides, one of the focus compounds of this thesis, will be briefly

discussed. Finally, the importance of combining approaches from various areas in

chemistry will be discussed and some relevant examples will be mentioned, including

chemoenzymatic dynamic kinetic resolution and hybrid catalysts based on (modified)

proteins or DNA.

14

Chapter 1 - General Introduction.doc

Chapter 1

1.1 Process intensification

One of the major incentives for the research described in this thesis, is the search for chemical processes that are cleaner, more atom- and energy-efficient, and operating under milder conditions and in smaller equipment than many large-scale methods used so far. In bulk chemistry, continuous flow processes are the methods of choice, allowing for the manufacturing of large quantities of chemicals in relatively small devices. In the production of fine and specialty chemicals, batch methods are normally employed, but the use of flow processes has become more common in recent years.1 The technical endeavor towards greater sustainability and safety, especially by using continuous systems, is known as ‘process intensification’.2,3 In this context, especially microreactor technology has become popular.4 Other examples of process intensification include the use of spinning-disk technology, monolithic columns, or catalyst immobilization strategies.3 The concept of process intensification is especially prominent in Chapter 2 of this thesis on epoxidation reactions in a centrifugal contact separator.5

Another important concept is atom economy, introduced by Trost.6 An atom economical reaction is a transformation in which the maximum number of atoms of the reactants appear in the products. In this respect, the use of bio- or transition metal catalyzed reactions are obvious examples of atom economy, since it allows faster transformations under milder conditions and lower temperatures and generates less waste compared to stoichiometric procedures.2 In the context of waste minimization, Sheldon introduced the “E factor”,a which is the amount of waste produced per kilogram of product.7 Examples of efficiently catalyzed reactions can be found throughout this thesis.

1.2 Stereochemistry and enantiomers

Stereochemistry is the branch of chemistry studying the three-dimensional arrangement of atoms within molecules.8 Compounds with the same molecular formula but a different spatial arrangement of their atoms are called isomers. They can be broadly classified as constitutional isomers and stereoisomers. In constitutional isomers, the bonds between their atoms are connected in different ways. For instance, acetic acid and glycolaldehyde (2-hydroxyethanal, the simplest sugar) both share the molecular formula C2H4O2. The other class of isomers consists of stereoisomers, which have the

a Not to be confused with the E value for enzyme enantioselectivity, see for instance Chapters 3 and 4 of this thesis.

15


General introduction

same bond structure, but a different spatial arrangement of their atoms. The type of stereoisomers playing an especially prominent role in this thesis are enantiomers.

Enantiomers are said to be chiral (from the Greek word χειρ for hand), meaning that they are non-superimposable mirror images of each other, like a left and right hand. They have identical physical properties such as boiling and melting point and share the same chemical reactivity patterns in an achiral environment. One of the few physical differences between enantiomers is the fact that they rotate the plane of plane-polarized light in opposite directions, which was discovered by Pasteur in 1848, who later argued that dissymmetry on the molecular level should be at the basis of this phenomenon.9 In 1874, Van 't Hoff and Le Bel independently proposed the tetrahedral arrangement of the four substituents around a central carbon atom, so that two non-superimposable mirror images results if, and only if, the four substituents are different (Figure 1.1).10

Figure 1.1 A stereogenic carbon atom has four different substituents, pointing toward the corners of a regular tetrahedron.

In a chiral environment, enantiomers behave differently from each other. For instance, in the presence of chiral reagents or a chiral catalyst, enantiomers may undergo the same reaction at different rates. Furthermore, since the interactions of molecules with biological receptors are three-dimensional and thus often stereoselective, the physiological properties of enantiomers may differ markedly.11 For example, the (S)-enantiomer of carvone smells like caraway, whereas (R)-carvone has the odor of spearmint. The different aromas of (S)- and (R)-carvone can be explained by the fact that olfactory receptors are chiral, so that they have different interactions with the two enantiomers.

Likewise, stereoselectivity plays an important role in drug action. In the beginning of the twentieth century, Cushny demonstrated that levorotatory hyoscyamine is twice as potent an anticholergenicb agent as racemic but otherwise identical atropine. Furthermore, endogenous levorotatory (R)-adrenaline was shown to be twice as potent b Reducing the effect of acetylcholine on the central and peripheral nervous system.

16


Chapter 1

as synthetic, racemic adrenaline.11,12 These and other observations led Easson and Stedman to propose their theory on the three-point steric interaction of drug molecules and receptors.13 Despite this knowledge on the different physiological effects of enantiomers, chiral medicines have been commonly produced for a long time as equimolar mixtures of enantiomers, or racemates. The arguments for this practice were mainly technical and economical, since the development of efficient routes to enantiopure compounds was perceived as cumbersome and expensive. However, following the understanding that different enantiomers may have qualitatively distinct physiological effects,14 and instigated by stricter regulations from health authorities, a growing number of new drugs are now marketed as single enantiomers.15

Figure 1.2 The enantiomers of carvone, (−)-hyoscyamine, and (R)-(−)-L-adrenaline.

A 50:50 mixture of enantiomers is called racemic, whereas a sample consisting of strictly one of the enantiomers is said to be enantiomerically pure. To indicate the level of enantiopurity for mixtures with a composition in between these two extremes, the term enantiomeric excess (ee) is used (Equation 1.1).

%100×+−

=SRSRee

Equation 1.1 Formula for calculating the ee of a sample containing an excess of the (R)-enantiomer.

A racemic mixture is the typical result if a reaction leading to a chiral compound is conducted in the absence of any chiral elements (such as catalyst, auxiliaries, solvents, or chirality in the starting materials). Racemization is the partial conversion of one enantiomer into another. Usually, this is an unwanted process, but a notable exception will be treated in paragraph 1.4.1, namely dynamic kinetic resolution.

In order to obtain enantiomerically pure compounds, three main strategies are known.16 The first one consists of taking an enantiomerically pure compound from the “chiral pool” − the vast collection of enantiopure compounds available form natural sources − and transform it in such a way that existing stereogenic centers remain intact. Another

17



important strategy is resolution, meaning the separation of a racemate into its enantiomers.

Finally, one can perform asymmetric synthesis on prochiral compounds. A compound is called prochiral if it is not chiral, but can be transformed into a chiral molecule in a single reaction step. Asymmetric synthesis requires a source of chirality, which can for example be a chiral solvent, reagent, auxiliary, or catalyst. Although in the chemical and pharmaceutical industry, resolution techniques are often still the method of choice for obtaining enantiopure target compounds, asymmetric synthesis of active pharmaceutical ingredients has been increasingly employed over the past decade.17 The efforts to develop efficient routes to enantiopure compounds can be summarized using the term “chirotechnology”. As indicated by Sheldon, economic considerations are an important aspect of efficiency in this context.18

1.3 Catalysis

A catalyst is a substance that increases the rate of a reaction without modifying the overall standard Gibbs energy change of the reaction.19 Importantly, the catalyst itself is not consumed in the reaction.

Catalysis can be broadly divided in two groups. Heterogeneous catalysis is based on a solid catalyst which is in a different phase from the reactants. The reactants must adsorb on the surface of the catalyst for reaction to take place. The surface of metals, or metals on a support such as activated carbon, is often used, but a huge variety of minerals can be used, as well as microporous aluminosilicate materials (zeolites). The German physicist Gerhard Ertl has studied reactions on metal surfaces in detail and has been awarded the 2007 Nobel Prize in Chemistry for his efforts to understand the mechanisms of heterogeneous catalytic reactions.20

In homogeneous catalysis, the catalyst is usually dissolved in the reaction medium. Again, various types are known. Soluble complexes based on transition metals can catalyze a wide variety of reactions and have been used for a long time.21,22 Several relevant examples will be treated in this thesis, for instance transition-metal catalyzed epoxidation of olefins (Chapter 2) and the catalysts for racemization of secondary (halo)alcohols, discussed in Chapter 4.

Apart from transition metal species, catalysts from biological sources (typically enzymes) are often used. In paragraph 1.3.1, this approach will be discussed in more detail. A research field of growing importance is organocatalysis, in which relatively small organic compounds are used to catalyze reactions.23 A recent example is the biologically inspired asymmetric aldol reaction catalyzed by proline and other α-amino

18


Chapter 1

acids or derivatives thereof.24 From a mechanistic point of view, proline can be regarded a mimic of Class I aldolases, since the two starting materials are unmodified carbonyl compounds (i.e. no prior enolization is required) and the aldol reaction proceeds via an enamide-based mechanism (Scheme 1.1).25 Other noteworthy contributions to organocatalysis come from MacMillan and coworkers on the asymmetric organocatalytic α-addition of relatively nonpolar hydrocarbon groups to carbonyl compounds,26a enantioselective cross-aldol reaction of aldehydes,26b or enantioselective organocatalytic Diels-Alder reactions,26c to give just a few examples.

Scheme 1.1 Mechanism of the L-proline-catalyzed asymmetric aldol reaction.

Many catalysts do not only speed up reactions, but also make them more selective.21 Selectivity comes in various forms, but the most relevant to the research described in this thesis is stereoselectivity.c Stereoselectivity refers to the preferential formation of one possible stereoisomer of a product over the formation of other one(s). Enantioselectivity is a particular case of stereoselectivity, referring to the preferential formation of one enantiomer of a product over the other. The corresponding branch of catalysis, asymmetric or enantioselective catalysis, has gained tremendously in importance over the years.27

Apart from heterogeneous and homogeneous catalysis, a third group is often distinguished, namely biocatalysis, i.e. catalysis with enzymes, RNA or other complex biomolecules, which is the topic of the next paragraph.

c For definitions of selectivity, chemoselectivity, regioselectivity, and related terminology, see refs. 8 and 19.

19



1.3.1 Enzyme catalysis For many thousands of years, people have used biological techniques, in particular fermentation, to produce and preserve food and drinks, although for a long time without being aware of the molecular details of these processes. Alcoholic beverages such as wine and beer were necessities of life in places where water was not of drinkable quality, for example in large cities before the construction of proper sewer systems. Fermentation is also essential in the production of additives which may help to enhance the taste and to preserve food, such as vinegar, or in the production of sauerkraut from cabbage.

The use of biocatalysis in chemical synthesis, using non-natural compounds as substrates, started more than a century ago. Enzymes are very versatile, active, selective, and environmentally friendly catalysts that generally work under mild conditions, which explains their increasing popularity for synthetic purposes.28

Various sources give a systematic overview of the different organic transformations for which enzyme catalysts have been used.28,29 Especially lipases are popular, since they are relatively inexpensive and stable towards strenuous conditions such as elevated temperatures and organic solvents, besides sharing the high activity and selectivity typical for many enzymes.30 In their natural environment, lipases catalyze the hydrolysis of ester bonds in lipid substrates, but in chemical synthesis they are often used for esterification or transesterification. An example is given in Scheme 1.2, which shows the reduction of a chloroketone followed by highly enantioselective lipase-catalyzed acetylation in a one-pot procedure.31

Scheme 1.2 One-pot reduction of 2-chloro-1-phenylethanone followed by lipase-catalyzed kinetic resolution.

The performance of enzymatic catalysts for synthetic purposes may be improved by a number of means, for instance modification of the reaction medium or substrate.32 However, the most powerful approaches to improve enzyme selectivity or specificity comprise protein engineering techniques, such as random or site-directed mutagenesis or evolutionary methods such as gene shuffling.33 Particularly interesting in this respect are contributions from Reetz and Jaeger, who used random mutagenesis by error-prone

20


Chapter 1

PCRd to increase sequentially the enantioselectivity of an initially unselective lipase-catalyzed ester hydrolysis.34a-c Reetz also reviewed the use of combinatorial and evolution-based methods for enantioselective catalysis.34d

1.3.2 Cascade catalysis Domino or cascade reactions are defined as processes

“involving two or more bond-forming transformations (usually C–C bonds) which take place under the same reaction conditions without adding additional reagents and catalysts, and in which the subsequent reactions result as a consequence of the functionality formed in the previous step.”35,36

A famous example of a cascade reaction is the biosynthesis of lanosterol and other steroids from squalene epoxide.37 Scheme 1.3 illustrates how squalene epoxide, a linear molecule containing only a single stereogenic center, is selectively transformed into the polycyclic steroid ring system containing four new C−C bonds and six new stereogenic centers.37b In 1971, Johnson and coworkers reported the biomimetic synthesis of the related steroid (±)-progesterone.38 Further applications of cascade reactions in total synthesis were recently reviewed by Nicolaou et al.39

Scheme 1.3 Biocatalytic synthesis of lanosterol from squalene epoxide.

For cascade catalysis, Fogg and Dos Santos add the restriction that all transformations should be catalyzed by a single catalytic mechanism. If multiple transformations occur via two or more mechanistically distinct processes, the term “tandem catalysis” should be used.36 Recently, a review on cascade catalysis vs. stepwise synthesis in the generation of molecular complexity was published by Walji and MacMillan, who illustrate their point using the synthesis of taxol as an example.40

d Polymerase chain reaction.

21



Tandem catalysis is an attractive and powerful concept, leading to highly functionalized molecules in a one-pot operation. However, in practice there are some drawbacks, which include potential negative interactions between the catalysts used to bring about the individual transformations, and the probability that a single set of reaction conditions is not optimal for multiple catalytic processes. Furthermore, if the rates of the involved catalytic steps differ to a large extent, the resulting concentration build-up of intermediates may lead to undesired side-reactions. The catalysts or reaction conditions for each step of the transformation may even be mutually incompatible. On a larger scale, difficulties in recovering individual precious metal components can make some tandem catalytic transformations prohibitively expensive. In Chapter 2, a strategy will be presented to generalize the tandem concept by performing the single transformations in separate two-phase reaction vessels, with the catalyst in one of the phases and substrate and product in the other.

In cell metabolism, intricated catalytic cascades are found in which many different enzymes operate together under similar conditions. These highly complex assemblies of interconnected reactions are regulated by a variety of mechanisms, such as product inhibition, feedback inhibition by reversible allosteric control, control proteins, hormones, reversible covalent modification, and proteolytic activation (irreversible conversion of an inactive enzyme into an active one). Furthermore, opposing reaction pathways (biosynthetic and catabolic) are separated from each other by compartmentation.41

It is possible to adopt existing metabolic pathways to the production of complex molecules, as demonstrated for instance by Keasling and coworkers in the synthesis of the antimalarial drug precursor artemisinic acid and other terpenoids.42

1.4 Kinetic resolution

Separating a racemic mixture into its constituent enantiomers is called resolution,19,43 which can be broadly divided into two categories. In the first one, classical resolution, a racemic mixture is allowed to react with a single enantiomer of a resolving agent, thus forming a two diastereomeric compounds which are physically different and can be separated, for instance by crystallization. The other resolution method is kinetic resolution, the achievement of resolution by virtue of unequal rates of reaction of the enantiomers in a racemate with a chiral agent.19 This agent may be a chiral reagent, as in the first published case of non-biochemical kinetic resolution by Marckwald and McKenzie in 1899, the reaction of racemic mandelic acid with optically active (−)-menthol (Scheme 1.4).44

22


Chapter 1

Scheme 1.4 Kinetic resolution as discovered by Marckwald and McKenzie.

Chiral catalysts can also be used for kinetic resolution.45 An example is the use of an enantiopure DMAP-derivative to catalyze the non-enzymatic acylation of arylalkylcarbinols, as described by Fu and coworkers (cf. the results in the lipase-catalyzed acylation of alcohols described in Paragraph 1.3.1).46

Scheme 1.5 Kinetic resolution of alcohols by chiral DMAP-catalyzed acylation.

Another well-known example of chemocatalytic kinetic resolution is the metal-salen catalyzed asymmetric ring opening of terminal epoxides developed by Jacobsen and coworkers (Scheme 1.6).47 This reaction is used on industrial scale for the manufacturing of enantiopure epichlorohydrin.48

Scheme 1.6 Hydrolytic kinetic resolution of terminal epoxides, developed by Jacobsen and coworkers.

23



Also the Sharpless asymmetric epoxidation reagent (vide infra) has been used in kinetic resolutions of allylic alcohols.49 As a final example, the kinetic resolution of chiral alkyl-substituted 2-cyclohexenones and allylic epoxides has been described using enantioselective copper-phosphoramidite catalyzed addition of dialkylzinc reagents.50

However, by far the most frequently used catalysts for kinetic resolution are enzymes, which will be treated extensively in Chapter 3, 4, and 6 of this thesis.

1.4.1 Dynamic kinetic resolution Given the importance of enantiomerically pure compounds, it follows that generally one would like a chiral compound to be configurationally stable, just as it should be chemically stable. However, there is a notable exception. Consider an ideal kinetic resolution, in which one enantiomer of the starting material, for instance SR, is selectively converted to a single product enantiomer PR, whereas SS remains untouched (Scheme 1.7a). No matter how selective the process is, the maximum yield of product PR is 50%. Moreover, PR and SS have to be separated from each other, which may be difficult, expensive and time-consuming. However, if SR and SS are in fast equilibrium, so that there is always some SR to react to PR, a very selective reaction could theoretically yield >99% of PR in >99% ee. This approach is called dynamic kinetic resolution (DKR, illustrated in Scheme 1.7b).51

Scheme 1.7 Schematic illustration of the concepts of a) kinetic resolution and b) dynamic kinetic resolution.

The following obvious question is how such racemization of the substrate enantiomers could take place. As it turns out, there are a number of strategies.8 These will be briefly discussed here, whereas a comprehensive historical overview of DKR will be given in Chapter 4.

First of all, some compounds exhibit thermal racemization. This was reported in 1853 by Pasteur, who succeeded in preparing racemic tartaric acid by heating the cinchonine salt of (+)-tartaric acid to 170 °C, an experiment during which, en passant, meso-tartaric acid was discovered.52 Under milder conditions, racemization is possible via carbocation, carbanion, or other intermediates. For instance, α-methylbenzyl chloride was observed to racemize during column chromatography on silica gel or acidic

24


Chapter 1

alumina, most likely through the PhCH(Me)+ cation.53 α-Amino acids may racemize via carbanion intermediates.54 Ketones with a stereogenic center at the α-position can be quite prone to epimerization, as was observed in the enantioselective synthesis of molecular motors using enantiopure ketones as building blocks.55 A systematic overview of various types of racemization was given in 1997 by Ebbers et al.56

In DKR, racemization through neutral intermediates has been more commonly employed, for instance in the diastereo- and enantioselective preparation of α-substituted β-hydroxy esters by asymmetric hydrogenation, reported by Noyori and coworkers (Scheme 1.8).57 Racemization proceeds via the enol form of the substrate. The spontaneous racemization of 5-hydroxy-5H-furan-2-one and analogous pyyolinones was used by Van der Deen et al. in lipase-catalyzed DKR.58 An extensive overview was given in 1995 by Noyori et al. on DKR using configurationally labile substrates.59

Scheme 1.8 Dynamic kinetic resolution of α-substituted β-ketoesters.

In order to perform DKR on configurationally more robust compounds, very efficient transition-metal based racemization catalysts have been developed. Williams and coworkers developed the dynamic kinetic resolution of 1-phenylethanol, combining various transition metal catalysts for racemization and lipase-catalyzed acetylation. Using a catalyst based on Rh2(OAc)4 and Pseudomonas fluorescens lipase, (R)-1-phenylethyl acetate was obtained with 60% conversion and 98% ee.60 This was followed by an important contribution from the group of Bäckvall, who reported the Ru/lipase-catalyzed conversion of rac-1-phenyl-1-ethanol to (R)-1-phenylethyl acetate in quantitative yield and excellent enantioselectivity.61 Since then, DKR of alcohols using the combination of transition metal catalysts and enzymes has established itself as a leading method for the preparation of enantiopure alcohols and, to a lesser extent, amines.62 The practical applicability of chemo-enzymatic DKR is underscored by its use in the ton-scale industrial production of fine chemicals.63

25



An extensive overview of the applications of racemization in organic synthesis, especially in DKR procedures, was given in 2001 by Bäckvall and coworkers.64 More recently, a review has appeared focusing specifically on the various racemization catalysts that have been developed for DKR of alcohols and amines.65 The common feature shared by these catalysts is that they function by hydrogen abstraction from the alcohol or amine, thus forming a carbonyl compound as intermediate (Scheme 1.9).66 This is also the case for a new class of iridium-based catalysts for alcohol racemization that will be presented in Chapter 4 of this thesis.

Occasionally, mechanisms different from hydrogen transfer are encountered in transition-metal catalyzed racemization. For instance, in the palladium-catalyzed DKR of allylic acetates,67a-b γ-acyloxybutenolides,67c-d or vinyloxiranes,67e and in the DKR of allylic alcohols using the oxovanadium(V) species VO(OSiPh3)3,68 racemization occurs through an allyl metal species which is in equilibrium with both enantiomers of the starting material.

Scheme 1.9 Racemization of alcohols and amines by dehydrogenation / hydrogenation.

The most common approach to DKR nowadays is the combination of transition-metal catalyzed racemization with enzyme-catalyzed acylation of the alcohol or amine.51 By far the most often used enzymes are commercially available lipases such as Candida antarctica B lipase (CALB) or an immobilized form of CALB, Novozym 435.69 Occasionally, Pseudomonas sp. lipase (PSL) or Pseudomonas cepacia lipase (PCL) are used. Some advantages of using commercially available lipases is that they are inexpensive, easy to come by, robust towards non-natural conditions and have a broad substrate range. Moreover, for most alcohols a lipase can be found that shows good selectivity. A disadvantage is the fact that lipases only give access to one of the product enantiomers, usually having the (R)-configuration in case of relatively simple secondary alcohols.70 Kim et al. have developed an (S)-selective DKR by combining Ru-catalyzed racemization with subtilisin as acylation catalyst.71 The relatively small scope in enzymes that have been used in enzyme/metal catalyzed DKR may be explained partly by mutual incompatibility between enzymes and transition metal catalysts, which is often observed.51 More than any other class of enzymes, lipases have shown to be highly compatible with non-natural conditions such as organic solvents, high temperatures, or metal species.28,30 The use of enzymes for the functionalization of alcohols, in particular lipases and oxido-reductases, was recently reviewed.72

26


Chapter 1

1.5 Enantioselective synthesis of epoxides

Epoxides are important intermediates in chemical reactions, both in the laboratory and on industrial scale.73 They are readily accessible, also in enantiomerically pure form, and can be functionalized by a variety of methods leading to different classes of compounds (Scheme 1.10).

Methods for the synthesis of epoxides can broadly be divided into two categories. First of all, there is the epoxidation of olefins using oxidizing agents such as peracids, peroxides, or molecular oxygen. With peracids, epoxidation may proceed uncatalyzed, but in general a catalyst is required. Transition metal salts, aggregates, or complexes,74,75 enzymes,76 and organic compounds77 are all known to catalyze epoxidation of olefins. Many of the epoxidation systems mentioned in ref. 74 − 77 are enantioselective. Three celebrated examples will be discussed here.

Scheme 1.10 Synthesis and further functionalization of epoxides.

Arguably the best-known example of an asymmetric epoxidation reaction is the Katsuki-Sharpless asymmetric epoxidation, using Ti(O-i-Pr)4, (+)- or (−)-tartrate, and tert-butyl hydroperoxide, which was reported in 1980 (Scheme 1.11a).78 It is very effective and selective for allylic and to a lesser degree homoallylic alcohols,78d but differently substituted or unfunctionalized olefins are not oxidized under these conditions.

In the same year Sharpless and Katsuki published their famous method, another asymmetric epoxidation was reported by Juliá et al., using hydrogen peroxide under basic conditions and oligoamino acids as the source of chirality (Scheme 1.11b). The best results were obtained using poly-Ala, poly-Leu, and poly-Ile. However, this Juliá-Colonna epoxidation, as it has become known, is only effective using electron-deficient

27



substrates able to stabilize a negative charge after addition of HOO− to the olefin, specifically α,β-unsaturated ketones such as chalcone derivatives.79

Finally, Jacobsen developed Mn-salen catalysts for the asymmetric epoxidation of unfunctionalized olefins using sodium hypochlorite as the oxidant (Scheme 1.11c).80 His procedure is most effective for cis-substituted internal olefins, whereas yields and enantioselectivities are inferior using terminal olefins (Scheme 1.11c). More recently, Katsuki reported the asymmetric epoxidation of unfunctionalized olefins using a titanium/salalen catalyst and hydrogen peroxide as the oxidant.81 A concise overview of enantioselective epoxidation catalysts was recently given by De Boer.74

Scheme 1.11 Examples of asymmetric epoxidation reactions: a) Katsuki-Sharpless epoxidation, b) Juliá-Colonna epoxidation, c) Jacobsen epoxidation; d) Katsuki epoxidation.

Other catalysts for enantioselective epoxidation include the fructose-derived ketone developed by Shi and coworkers, which is an effective and enantioselective organocatalyst for the epoxidation of unfunctionalized trans-olefins, using potassium peroxomonosulfate as the oxidant.77b,82 Furthermore, metalloporphyrins have been reported as catalysts for enantioselective epoxidation.83 Heterogeneous epoxidation

28


Chapter 1

catalysts are also available in both enantioselective75d,84 and racemic85 form, often based on homogeneous catalysts immobilized by a variety of techniques.86 A general, broad overview of heterogeneous and homogeneous catalytic asymmetric epoxidation methodologies has been given by Xia et al.87 As was shown, the existing methods for asymmetric epoxidation often have the disadvantage that they are limited to specific types of substrates.

The other class of reactions leading to epoxides comprises intramolecular ring closure of alcohols or alcoholates having a suitable vicinal leaving group. This includes sulfur ylide addition to carbonyl compounds, followed by ring closure and expulsion of dimethyl sulfide. This approach was first reported by Johnson in 1958,88a followed by a contribution by Corey and Chaykovsky.88b The alcoholate may be generated by other means, such as in the reaction of aldehydes with diiodomethane and methyllithium, described by Concellón et al.89 Novel, asymmetric approaches using this way of generating epoxides were described recently by Aggarwal and coworkers.90

Also vicinal haloalcohols are precursors of epoxides. In Chapter 3, an efficient and highly enantioselective method will be presented to obtain various enantiopure functionalized chloroalcohols using the haloalcohol dehalogenase HheC. In Chapter 4, the dynamic kinetic resolution of haloalcohols using haloalcohols dehalogenases is described, leading directly and efficiently from racemic halohydrins to enantiopure epoxides.

Epoxides can undergo ring opening reactions with a wide variety of nucleophiles. Analogous to the formation of epoxides, their ring opening may be uncatalyzed, transition-metal catalyzed, biocatalyzed, or organocatalyzed. The reaction of racemic chiral epoxides with a chiral reagent or catalyst may lead to kinetic resolution. Under similar conditions, meso-epoxides can be desymmetrized by enantioface discrimination. Epoxide ring opening has been extensively reviewed.47,91 Also biocatalytic reactions of epoxides have been reviewed.92

Furthermore, epoxides are known to isomerize to carbonyl compounds under enzymatic93a or (Lewis) acid catalyzed93b conditions. This − synthetically usually less interesting − reaction is known as the Meinwald rearrangement.93c

1.6 Combination of bio- and chemocatalysis

In his famous 1959 lecture “There's plenty of room at the bottom”,94 Richard Feynman made strong arguments for a closer collaboration between different scientific disciplines. He foresaw the rapid elucidation of central and fundamental problems biologists were facing at that time, if physicists would succeed in making analytical

29



instruments that allowed for observation at molecular scale. Furthermore, he contended that improved physical detection methods could make chemical analysis a lot easier.e

The need for such an integrative approach comprising several scientific disciplines is widely felt, as is illustrated for instance in a review by Corma for attempts to efficiently combine enzymatic, homogeneous, and heterogeneous catalysis.95 Although science would benefit from increased and improved collaboration between individual researchers, the combination of approaches and techniques from various scientific areas is not a new endeavor. In fact, Nature has been giving the example for four billions of years, by using transition metal complexes as part of biocatalysts. Proteins containing metal centers, or metalloproteins, are omnipresent in all domains of life. Some of them, for example iron-sulfur proteins, can be found in all living beings and play an essential role in diverse processes, such as electron transfer, reduction catalysis and, more specifically, in photosynthesis, the respiratory chain and nitrogen fixation.96 The ubiquitous nature of some of the metalloproteins suggests an ancient evolutionary history.97 In one of the current hypotheses on the origin of life, the so-called iron-sulfur world theory, transition metals − notably iron and nickel − even play a crucial role in explaining the emergence of life itself through a primitive chemoautotrophic metabolic cycle.98

Metal centers in a protein can perform structural, storage, electron transfer, or dioxygen binding functions, but most relevant to the work in this thesis, they may show catalytic activity.99 A detailed overview of metalloproteins and their properties is outside the scope of this introduction and the reader is directed to a multitude of reviews and textbooks in the field.100,101

Transition metal complexes are often used as models for enzymatic behavior. Some of these models have grown out to be successful catalysts in their own right. To give one example, metalloporphyrins were originally synthesized as model systems to study the behavior of cytochrome P450 enzymes,102 a large superfamily of heme-containing proteins,103 but since then they have developed into a class of highly efficient catalysts for various oxidation reactions.75g

Artificial enzymes − synthetic molecules mimicking the catalytic functions of enzymes − have been designed and prepared in order to overcome boundaries imposed by natural limitations in terms of reaction scope and substrate acceptance.104 Especially poly-α-amino acids are relevant in this respect, since they have been used as chiral catalysts in the synthesis of epoxides (Juliá-Colonna epoxidation, see paragraph 1.5).79

e Interestingly, Feynman also expressed the vision that it should be possible to synthesize chemicals by manipulating individual atoms.

30


Chapter 1

Furthermore, inspired by biological systems, the field of biomimetic catalysis has developed.105 The well-known proline-catalyzed aldol reaction, for instance, was developed as a mimic for Class I aldolases.25 Another recent example is a system for aerobic alcohol oxidation reminiscent of biological oxidation, published by Bäckvall and coworkers, which combines ruthenium and Co-salen catalysis.106 However, this introduction will focus on approaches that combine the advantages of transition metal complexes and biologically-derived catalysts or scaffolds.

Chemical/biological hybrid catalysts have been developed by other groups as well. Wilson and Whitesides described the hydrogenation of α-acetamidoacrylic acid to N-acetylalanine with full conversion and about 40% ee (S), using a catalyst based on the protein avidin and the biotin-derived biphosphine ligand shown in Scheme 1.12.107

In the reactions described by Wilson and Whitesides, the active catalyst is prepared in situ and held together by strongf but non-covalent interactions. A related biotin−avidin system was later chemogenetically improved for enantioselective hydrogenation by Ward and coworkers, using a combination of saturation mutagenesis and chemical optimization.108

Scheme 1.12 Asymmetric hydrogenation using a hybrid catalyst based on avidin and a biotin-derived biphosphine (1 = N,N-bis(2-diphenylphosphinoethyl)biotinamide).

Modifying a protein is also possible by selective covalent functionalization of specific residues. For example, Panella and coworkers reported a hybrid catalyst based on papain, modified at Cys-25 with a monodentate phosphite ligand and complexed with Rh(COD)2BF4, which was active in the hydrogenation of methyl 2-acetamidoacrylate (Scheme 1.13).109 Unfortunately, no asymmetric induction was obtained.

A different example of the efficient combination of natural and man-made entities in enantioselective catalysis is the enantioselective catalysis based on DNA, first reported by Roelfes and Feringa.110 In this case, double-stranded DNA is used as the asymmetric scaffold for transition-metal catalyzed Diels-Alder reactions110b-e,111 and Michael

f The association of avidin and biotin or biotin-derivatives like 1 (Scheme 1.12), is effectively irreversible (Kd = 10−12 − 10−15 M), see ref. 107 and references cited therein.

31



additions.110a The catalyst consists of a copper complex of an achiral ligand that allows for simultaneous binding to DNA, such as 4,4'-dimethyl-2,2'-bipyridine (Scheme 1.14).110d

Scheme 1.13 Papain-based hybrid catalyst for olefin hydrogenation by Panella et al.

Scheme 1.14 Example of copper / DNA hybrid catalysis in diastereo- and enantioselective Diels-Alder reactions (st-DNA = salmon testes DNA, MOPS = 3-morpholinopropanesulfonic acid).

Related to the previous examples is the development of metallodeoxyribozymes based on phosphine-containing oligonucleotides for enantioselective palladium-catalyzed allylic nucleophilic substitution.112 A different example of the merging between concepts and practices from biocatalysis and asymmetric transition metal catalysis is the work of Reetz and coworkers on the directed evolution of enantioselective hybrid catalysts.113 The vast field of artificial metalloproteins and other bioinorganic hybrid catalysts has been extensively reviewed.114

A somewhat different crossbreeding is constituted by the field of nanobiotechnology. Key to this approach is the combination of inorganic nanoparticles and proteins or DNA. In this way, biological systems may be tuned by inorganic nanometer-size components, while conversely, the nanoparticles may acquire new functionalities based on biomolecular mechanisms.115

1.7 Notes and references 1 G. Jas and A. Kirschning, Chem. Eur. J. 2003, 9, 5708-5723.

32


Chapter 1

2 J. F. Jenck, F. Agterberg, and M. J. Droescher, Green Chem. 2004, 6, 544-556. 3 A. E. Rubin, S. Tummala, D. A. Both, C. Wang, and E. J. Delaney, Chem. Rev. 2006, 106, 2794-2810. 4 J.-C. Charpentier, Chem. Eng. Technol. 2005, 28, 255-258. 5 J. G. De Vries, G. J. Kwant, and H. J. Heeres, WO 2007/031332, 2007, to DSM IP Assets B.V. 6 a) B. M. Trost, Angew. Chem. Int. Ed. Engl. 1995, 34, 259-281; b) B. M. Trost, Science 1991, 254, 1471-1477. 7 R. A. Sheldon, J. Chem. Technol. Biotechnol. 1997, 68, 381-388. 8 E. L. Eliel, S. H. Wilen, and L. N. Mander, Stereochemistry of Organic Compounds, Wiley, New York, 1994. 9 a) L. Pasteur, Ann. Chim. Phys. 1848, 24, 442-459; b) see also: L. Pasteur, Oeuvres de Pasteur, Vol. 1, J. L. Pasteur Vallery-Radot, Ed., Masson, Paris, 1922. 10 a) J. H. Van 't Hoff, Arch. Neerl. Sci. Exactes Nat. 1874, 9, 445-454; b) J.-A. Le Bel, Bull. Soc. Chim. Fr. 1874, 22, 337-347, reprinted 1995, 132, 6-16. 11 B. Waldeck, Pharmacol. Toxicol. 2003, 93, 203-210. 12 A. R. Cushny, Biological Relations of Optically Isomeric Substances, Baillière, Tinball & Cox, London, 1926. 13 L. H. Easson and E. Stedman, Biochem. J. 1933, 27, 1257-1266. 14 a) G. T. Tucker and M. S. Lennard, Pharmacol. Ther. 1990, 45, 309-329; b) F. Jamali, R. Mehvar, F. M. Pasutto, J. Pharm. Sci. 1989, 78, 695-715; c) D. E. Drayer, Clin. Pharmacol. Ther. 1986, 40, 125-133; d) K. Williams and E. Lee, Drugs 1985, 30, 333-354; e) E. J. Ariëns, Eur. J. Clin. Pharmacol. 1984, 26, 663-668. 15 a) L. Bielory and A. Leonov, Ann. Allergy Asthma Immunol. 2008, 100, 1-9; b) H.-J. Federsel, Nat. Rev. Drug Discov. 2005, 4, 685-697; c) W. H. De Camp, Chirality 1989, 1, 2-6. 16 D. Seebach, Angew. Chem. Int. Ed. Engl. 1990, 29, 1320-1367. 17 V. Farina, J. T. Reeves, C. H. Senanayake, and J. J. Song, Chem. Rev. 2006, 106, 2734-2793. 18 R. A. Sheldon, J. Chem. Technol. Biotechnol. 1996, 67, 1-14. 19 IUPAC Compendium of Chemical Terminology, http://goldbook.iupac.org/index.html 20 G. Ertl, Surf. Sci. 1994, 299/300, 742-754. 21 P. W. N. M. Van Leeuwen, Homogeneous Catalysis: Understanding the Art, Kluwer Academic Publishers, Dordrecht, 2004. 22 a) J. G. De Vries, in Encyclopedia of Catalysis, Vol. 3, pp. 295-347, I. T. Horváth, Ed., Wiley, Hoboken, New Jersey, 2003; b) J. K. Smith, ibid., pp. 447-479.

33



23 a) H. Pellissier, Tetrahedron 2007, 63, 9267-9331; b) S. Jaroch, H. Weinmann, and K. Zeitler, ChemMedChem 2007, 2, 1261-1264; c) M. J. Gaunt, C. C. C. Johansson, A. McNally, and N. T. Vo, Drug Discov. Today 2007, 12, 8-27. 24 a) C. F. Barbas III, Angew. Chem. Int. Ed. 2008, 47, 42-47; b) S. Mukherjee, J. W. Yang, S. Hoffmann, and B. List, Chem. Rev. 2007, 107, 5471-5569; c) G. Guillena and D. J. Ramón, Tetrahedron: Asymmetry 2006, 17, 1465-1492. 25 a) W. Notz, F. Tanaka, and C. F. Barbas III, Acc. Chem. Res. 2004, 37, 580-591; b) K. Sakthivel, W. Notz, T. Bui, and C. F. Barbas III, J. Am. Chem. Soc. 2001, 123, 5260-5267; c) B. List, R. A. Lerner, and C. F. Barbas III, J. Am. Chem. Soc. 2000, 122, 2395-2396. 26 a) T. D. Beeson, A. Mastracchio, J.-B. Hong, K. Ashton, and D. W. C. MacMillan, Science 2007, 316, 582-585; b) A. B. Northrup and D. W. C. MacMillan, J. Am. Chem. Soc. 2002, 124, 6798-6799; c) K. A. Ahrendt, C. J. Borths, and D. W. C. MacMillan, J. Am. Chem. Soc. 2000, 122, 4243-4244. 27 Comprehensive Asymmetric Catalysis, E. N. Jacobsen, A. Pfaltz, H. Yamamoto, Eds., Springer, Berlin, 1999. 28 K. Faber, Biotransformations in Organic Chemistry: A Textbook, 4th ed., Springer, Berlin, 2000. 29 B. G. Davis and V. Boyer, Nat. Prod. Rep. 2001, 18, 618-640. 30 a) A. Ghanem, Tetrahedron 2007, 63, 1721-1754; b) A. Ghanem and H. Y. Aboul-Enein, Chirality 2005, 17, 1-15. 31 A. Kamal, M. Sandbhor, and K. V. Ramana, Tetrahedron: Asymmetry 2002, 13, 815-820. 32 M. Adamczak and S. Hari Krishna, Food Technol. Biotechnol. 2004, 42, 251-264. 33 a) N. M. Antikainen and S. F. Martin, Bioorg. Med. Chem. 2005, 13, 2701-2716; b) F. Valetti and G. Gilardi, Nat. Prod. Rep. 2004, 21, 490-511. 34 a) M. T. Reetz and K.-E. Jaeger, Chem. Eur. J. 2000, 6, 407-412; b) K. Liebeton, A. Zonta, K. Schimossek, M. Nardini, D. Lang, B. W. Dijkstra, M. T. Reetz, and K.-E. Jaeger, Chem. Biol. 2000, 7, 709-718; c) M. T. Reetz, A. Zonta, K. Schimossek, K.-E. Jaeger, and K. Liebeton, Angew. Chem. Int. Ed. Engl. 1997, 36, 2830-2832; d) M. T. Reetz, Angew. Chem. Int. Ed. 2001, 40, 284-310. 35 L. F. Tietze, Chem. Rev. 1996, 96, 115-136. 36 D. E. Fogg and E. N. Dos Santos, Coord. Chem. Rev. 2004, 248, 2365-2379. 37 a) I. Abe, M. Rohmer, and G. D. Prestwich, Chem. Rev. 1993, 93, 2189-2206; b) E. J. Corey and S. C. Virgil, J. Am. Chem. Soc. 1991, 113, 4025-4026. 38 a) M. B. Gravestock, W. S. Johnson, B. E. McCarry, R. J. Parry, and B. E. Ratcliffe, J. Am. Chem. Soc. 1978, 100, 4274-4282; b) W. S. Johnson, M. B. Gravestock, and B. E. McCarry, J. Am. Chem. Soc. 1971, 93, 4332-4334. 39 K. C. Nicolaou, D. J. Edmonds, and P. G. Bulger, Angew. Chem. Int. Ed. 2006, 45, 7134-7186. 40 A. M. Walji and D. W. C. MacMillan, Synlett 2007, 1477-1489. 41 L. Stryer, Biochemistry, 4th ed., W. H. Freeman and Company, New York, 1995.

34


Chapter 1

42 a) D.-K. Ro, E. M. Paradise, M. Ouellet, K. J. Fisher, K. L. Newman, J. M. Ndungu, K. A. Ho, R. A. Eachus, T. S. Ham, J. Kirby, M. C. Y. Chang, S. T. Withers, Y. Shiba, R. Sarpong, and J. D. Keasling, Nature 2006, 440, 940-943; b) V. J. J. Martin, D. J. Pitera, S. T. Withers. J. D. Newman, and J. D. Keasling, Nature Biotech. 2003, 21, 796-802; c) J. D. Keasling and S.-W. Bang, Curr. Opin. Biotechnol. 1998, 9, 135-140. 43 For a recent review on resolution techniques, see: E. Fogassy, M. Nógrádi, D. Kozma, G. Egri, E. Pálovics, and V. Kiss, Org. Biomol. Chem. 2006, 4, 3011-3030. 44 W. Marckwald and A. McKenzie, Ber. Deut. Chem. Ges. 1899, 32, 2130-2136. 45 For reviews on non-enzymatic kinetic resolution, see: a) E. Vedejs and M. Jure, Angew. Chem. Int. Ed. 2005, 44, 3974-4001; b) D. E. J. E. Robinson and S. D. Bull, Tetrahedron: Asymmetry 2003, 14, 1407-1446. 46 J. C. Ruble, J. Tweddell, and G. C. Fu, J. Org. Chem. 1998, 63, 2794-2795. 47 E. N. Jacobsen, Acc. Chem. Res. 2000, 33, 421-431. 48 By Daiso Co., Ltd., http://www.daiso-co.com/eng/prd/prd04_02.html 49 a) Y. Gao, R. M. Hanson, J. M. Klunder, S. Y. Ko, H. Masamune, and K. B. Sharpless, J. Am. Chem. Soc. 1987, 109, 5165-5180; b) V. S. Martin, S. S. Woodard, T. Katsuki, Y. Yamada, M. Ikeda, and K. B. Sharpless, J. Am. Chem. Soc. 1981, 103, 6237-6240. 50 a) M. Pineschi, New J. Chem. 2004, 28, 657-665; b) R. Naasz, L. A. Arnold, A. J. Minnaard, and B. L. Feringa, Angew. Chem. Int. Ed. 2001, 40, 927-930. 51 For recent reviews, see: a) H. Pellissier, Tetrahedron 2008, 64, 1563-1601; b) B. Martín-Matute and J.-E. Bäckvall, Curr. Opin. Chem. Biol. 2007, 11, 226-232; c) O. Pàmies and J.-E. Bäckvall, Chem. Rev. 2003, 103, 3247-3261. 52 L. Pasteur, C. R. Acad. Sci. 1853, 37, 162-166. 53 D. B. Denney and R. DiLeone, J. Org. Chem. 1961, 26, 984. 54 A. Neuberger, Adv. Prot. Chem. 1948, 4, 297-383. 55 R. Hoen, New Approaches in Asymmetric Rhodium-catalyzed Hydrogenations with Monodentate Phosphoramidites, Ph.D. thesis, University of Groningen, 2006. 56 E. J. Ebbers, G. J. A. Ariaans, J. P. M. Houbiers, A. Bruggink, and B. Zwanenburg, Tetrahedron 1997, 53, 9417-9476. 57 R. Noyori, T. Ikeda, T. Ohkuma, M. Widhalm, M. Kitamura, H. Takaya, S. Akutagawa, N. Sayo, T. Saito, T. Taketomi, and H. Kumobayashi, J. Am. Chem. Soc. 1989, 111, 9134-9135. 58 H. Van Der Deen, A. D. Cuiper, R. P. Hof, A. Van Oeveren, B. L. Feringa, and R. M. Kellogg, J. Am. Chem. Soc. 1996, 118, 3801-3803. 59 R. Noyori, M. Tokunaga, and M. Kitamura, Bull. Chem. Soc. Jpn. 1995, 68, 36-56.

35



60 P. M. Dinh, J. A. Howarth, A. R. Hudnott, J. M. J. Williams, and W. Harris, Tetrahedron Lett. 1996, 37, 7623-7626. 61 a) B. A. Persson, A. L. E. Larsson, M. Le Ray, and J.-E. Bäckvall, J. Am. Chem. Soc. 1999, 121, 1645-1650; b) A. L. E. Larsson, B. A. Persson, and J.-E. Bäckvall, Angew. Chem. Int. Ed. Engl. 1997, 36, 1211-1212. 62 a) M. A. J. Veld, K. Hult, A. R. A. Palmans, and E. W. Meijer, Eur. J. Org. Chem. 2007, 5416-5421; b) C. Roengpithya, D. A. Patterson, A. G. Livingston, P. C. Taylor, J. L. Irwin, and M. R. Parrett, Chem. Commun. 2007, 3462-3463; c) A. J. Blacker, M. J. Stirling, and M. I. Page, Org. Proc. Res. Devel. 2007, 11, 642-648; d) M.-J. Kim, W.-H. Kim, K. Han, Y. K. Choi, and J. Park, Org. Lett. 2007, 9, 1157-1159; e) S. Gastaldi, S. Escoubet, N. Vanthuyne, G. Gil, and M. P. Bertrand, Org. Lett. 2007, 9, 837-839; f) M. Stirling, J. Blacker, and M. I. Page, Tetrahedron Lett. 2007, 48, 1247-1250; g) J. Paetzold and J.-E. Bäckvall, J. Am. Chem. Soc. 2005, 127, 17620-17621; h) A. Parvulescu, D. De Vos, and P. Jacobs, Chem. Commun. 2005, 5307-5309; i) M. T. Reetz and K. Schimossek, Chimia 1996, 50, 668-669. 63 For instance by DSM, see: a) A. M. Rouhi, Chem. Eng. News 2002, 80 (23), 43-50; b) G. K. M. Verzijl, J. G. De Vries, and Q. B. Broxterman, Tetrahedron: Asymmetry 2005, 16, 1603-1610. 64 F. F. Huerta, A. B. E. Minidis, and J.-E. Bäckvall, Chem. Soc. Rev. 2001, 30, 321-331. 65 Y. Ahn, S.-B. Ko, M.-J. Kim, and J. Park, Coord. Chem. Rev. 2008, 252, 647-658. 66 For reviews with a mechanistic focus, see: M. H. S. A. Hamid, P. A. Slatford, and J. M. J. Williams, Adv. Synth. Catal. 2007, 349, 1555-1575; b) J. S. M. Samec, J.-E. Bäckvall, P. G. Andersson, and P. Brandt, Chem. Soc. Rev. 2006, 35, 237-248. 67 a) Y. K. Choi, J. H. Suh, D. Lee, I. T. Lim, J. Y. Jung, and M.-J. Kim, J. Org. Chem. 1999, 64, 8423-8424; b) J. V. Allen and J. M. J. Williams, Tetrahedron Lett. 1996, 37, 1859-1862; c) B. M. Trost and F. D. Toste, J. Am. Chem. Soc. 2003, 125, 3090-3100; d) B. M. Trost and F. D. Toste, J. Am. Chem. Soc. 1999, 121, 3543-3544; e) B. M. Trost, B. S. Brown, E. J. McEachern, and O. Kuhn, Chem. Eur. J. 2003, 9, 4442-4451. 68 S. Akai, K. Tanimoto, Y. Kanao, M. Egi, T. Yamamoto, and Y. Kita, Angew. Chem. Int. Ed. 2006, 45, 2592-2595. 69 Novozym® 435 is a commercially available preparation of Candida antarctica B lipase, immobilized on acrylic resin, available from Novozymes. 70 R. J. Kazlauskas, A. N. E. Weissfloch, A. T. Rappaport, and L. A. Cuccia, J. Org. Chem. 1991, 56, 2656-2665. 71 M.-J. Kim, Y. I. Chung, Y. K. Choi, H. K. Lee, D. Kim, and J. Park, J. Am. Chem. Soc. 2003, 125, 11494-11495. 72 O. Jurček, M. Wimmerová, and Z. Wimmer, Coord. Chem. Rev. 2008, 252, 767-781. 73 B. E. Rossiter, T. Katsuki, and K. B. Sharpless, J. Am. Chem. Soc. 1981, 103, 464-465. 74 J. W. De Boer, cis-Dihydroxylation and Epoxidation of Alkenes by Manganese Catalysts, Ph.D. thesis, University of Groningen, 2008.

36


Chapter 1

75 For recent reviews, see: a) D. Chatterjee, Coord. Chem. Rev. 2008, 252, 176-198; b) J. Muzart, J. Mol. Catal. A: Chem. 2007, 276, 62-72; c) G. Sello, T. Fumagalli, and F. Orsini, Curr. Org. Synth. 2006, 3, 457-476; d) C. Baleizão and H. Garcia, Chem. Rev. 2006, 106, 3987-4043; e) T. Punniyamurthy, S. Velusamy, and J. Iqbal, Chem. Rev. 2005, 105, 2329-2363; f) E. M. McGarrigle and D. G. Gilheany, Chem. Rev. 2005, 105, 1563-1602; g) E. Rose, B. Andrioletti, S. Zrig, and M. Quelquejeu-Ethève, Chem. Soc. Rev. 2005, 34, 573-583; h) J.-M. Brégeault, Dalton Trans. 2003, 3289-3302; i) G. Grigoropoulou, J. H. Clark, and J. A. Elings, Green Chem. 2003, 5, 1-7; j) R. Noyori, M. Aoki, and K. Sato, Chem. Commun. 2003, 1977-1986. 76 For recent reviews, see: a) V. B. Urlacher and R. D. Schmid, Curr. Opin. Chem. Biol. 2006, 10, 156-161; b) D. Chang, J. Zhang, B. Witholt, and Z. Li, Biocatal. Biotrans. 2004, 22, 113-130. 77 See for instance: a) M. M. K. Boysen, Chem. Eur. J. 2007, 13, 8648-8659; b) Y. Shi, Acc. Chem. Res. 2004, 37, 488-496; c) E. R. Jarvo and S. J. Miller, Tetrahedron 2002, 58, 2481-2495; d) P. I. Dalko and L. Moisan, Angew. Chem. Int. Ed. 2001, 40, 3726-3748. 78 a) E. Höft, Top. Curr. Chem. 1993, 164, 63-77 b) A. Pfenninger, Synthesis 1986, 89-116; c) T. Katsuki and K. B. Sharpless, J. Am. Chem. Soc. 1980, 102, 5974-5976; d) B. E. Rossiter and K. B. Sharpless, J. Org. Chem. 1984, 49, 3707-3711. 79 a) E. A. Colby Davie, S. M. Mennen, Y. Xu, and S. J. Miller, Chem. Rev. 2007, 107, 5759-5812; b) T. Geller, A. Gerlach, C. M. Krüger, and H.-C. Militzer, J. Mol. Catal. A: Chem. 2006, 251, 71-77; c) G. Carrea, S. Colonna, D. R. Kelly, A. Lazcano, G. Ottolina, and S. M. Roberts, Trends Biotechnol. 2005, 23, 507-513; d) G. Carrea, S. Colonna, A. D. Meek, G. Ottolina, and S. M. Roberts, Tetrahedron: Asymmetry 2004, 15, 2945-2949; e) A. Gerlach and T. Geller, Adv. Synth. Catal. 2004, 346, 1247-1249; f) M. W. Cappi, W.-P. Chen, R. W. Flood, Y.-W. Liao, S. M. Roberts, J. Skidmore, J. A. Smith, and N. M. Williamson, Chem. Commun. 1998, 1159-1160; g) S. Banfi, S. Colonna, H. Molinari, S. Juliá, and J. Guixer, Tetrahedron 1984, 40, 5207-5211; h) S. Juliá, J. Masana, and J. C. Vega, Angew. Chem. Int. Ed. Engl. 1980, 19, 929-931. 80 a) T. Katsuki, Coord. Chem. Rev. 1995, 140, 189-214; b) W. Zhang and E. N. Jacobsen, J. Org. Chem. 1991, 56, 2296-2298; b) E. N. Jacobsen, W. Zhang, A. R. Muci, J. R. Ecker, and L. Deng, J. Am. Chem. Soc. 1991, 113, 7063-7064; c) R. Irie, K. Noda, Y. Ito, N. Matsumoto, and T. Katsuki, Tetrahedron Lett. 1990, 31, 7345-7348; d) W. Zhang, J. L. Loebach, S. R. Wilson, and E. N. Jacobsen, J. Am. Chem. Soc. 1990, 112, 2801-2803. 81 a) Y. Sawada, K. Matsumoto, and T. Katsuki, Angew. Chem. Int. Ed. 2007, 46, 4559-4561; b) Y. Sawada, K. Matsumoto, S. Kondo, H. Watanabe, T. Ozawa, K. Suzuki, B. Saito, and T. Katsuki, Angew. Chem. Int. Ed. 2006, 45, 3478-3480; c) K. Matsumoto, Y. Sawada, B. Saito, K. Sakai, and T. Katsuki, Angew. Chem. Int. Ed. 2005, 44, 4935-4939. 82 a) Z.-X. Wang, Y. Tu, M. Frohn, J.-R. Zhang, and Y. Shi, J. Am. Chem. Soc. 1997, 119, 11224-11235; b) Y. Tu, Z.-X. Wang, and Y. Shi, J. Am. Chem. Soc. 1996, 118, 9806-9807. 83 J. P. Collman, X. Zhang, V. J. Lee, E. S. Uffelman, and J. I. Brauman, Science 1993, 261, 1404-1411.

37



84 a) M. Heitbaum, F. Glorius, and I. Escher, Angew. Chem. Int. Ed. 2006, 45, 4732-4762; b) K. Ding, Z. Wang, X. Wang, Y. Liang, and X. Wang, Chem. Eur. J. 2006, 12, 5188-5197; c) M. Holbach and M. Weck, J. Org. Chem. 2006, 71, 1825-1836. 85 a) M. Shokouhimehr, Y. Piao, J. Kim, Y. Jang, and T. Hyeon, Angew. Chem. Int. Ed. 2007, 46, 7039-7043; b) N. R. Candeias and C. A. M. Afonso, J. Mol. Catal. A: Chem. 2005, 242, 195-217; c) D. E. De Vos, B. F. Sels, P. A. Jacobs, Adv. Synth. Catal. 2003, 345, 457-473. 86 a) K. R. Jain and F. E. Kühn, J. Organomet. Chem. 2007, 692, 5532-5540; b) C. Freund, M. Abrantes, and F. E. Kühn, J. Organomet. Chem. 2006, 691, 3718-3729; c) P. McMorn and G. J. Hutchings, Chem. Soc. Rev. 2004, 33, 108-122; Q.-H. Fan, Y.-M. Li, and A. S. C. Chan, Chem. Rev. 2002, 102, 3385-3466. 87 Q.-H. Xia, H.-Q. Ge, C.-P. Ye, Z.-M. Liu, and K.-X. Su, Chem. Rev. 2005, 105, 1603-1662. 88 a) A. W. Johnson and R. B. LaCount, Chem. Ind. 1958, 1440-1441; b) E. J. Corey and M. Chaykovsky, J. Am. Chem. Soc. 1965, 87, 1353-1364. 89 J. M. Concellón, H. Cuervo, and R. Fernández-Fano, Tetrahedron 2001, 57, 8983-8987. 90 a) E. M. McGarrigle, E. L. Myers, O. Illa, M. A. Shaw, S. L. Riches, and V. K. Aggarwal, Chem. Rev. 2007, 107, 5841-5883; b) V. K. Aggarwal and C. L. Winn, Acc. Chem. Res. 2004, 37, 611-620. 91 a) C. Schneider, Synthesis 2006, 3919-3944; b) M. Pineschi, Eur. J. Org. Chem. 2006, 4979-4988; c) I. M. Pastor and M. Yus, Curr. Org. Chem. 2005, 9, 1-29. 92 E. J. De Vries and D. B. Janssen, Curr. Opin. Biotechnol. 2003, 14, 414-420. 93 a) K. Miyamoto, K. Okuro, and H. Ohta, Tetrahedron Lett. 2007, 48, 3255-3257, and references cited therein; b) see for instance: B. M. Smith, E. J. Skellam, S. J. Oxley, and A. E. Graham, Org. Biomol. Chem. 2007, 5, 1979-1982; c) J. Meinwald, S. S. Labana, and M. S. Chadha, J. Am. Chem. Soc. 1963, 85, 582-585. 94 R. P. Feynman, There's Plenty of Room at the Bottom: An Invitation to Enter a New Field of Physics, lecture held on 29 December 1959; at http://www.zyvex.com/nanotech/feynman.html, a transcript can be found. 95 A. Corma, Catal. Rev. 2004, 46, 369-417. 96 B. Lamotte and J.-M. Mouesca, C. R. Acad. Sci. II B 1997, 324, 117-132. 97 H. Sticht and P. Rösch, Prog. Biophys. Mol. Biol. 1998, 70, 95-136. 98 a) G. Wächtershäuser, Chem. Biodiv. 2007, 4, 584-602; b) W. Martin and M. J. Russell, Phil. Trans. R. Soc. B 2003, 358, 59-83; c) G. Wächtershäuser, Proc. Natl. Acad. Sci. USA 1990, 87, 200-204. 99 R. H. Holm, P. Kennepohl, and E. I. Solomon, Chem. Rev. 1996, 96, 2239-2314. 100 S. J. Lippard and J. M. Berg, Principles of Bioinorganic Chemistry, University Science Books, Mill Valley, 1994. 101 a) E. I. Solomon, T. C. Brunold, M. I. Davis, J. N. Kemsley, S.-K. Lee, N. Lehnert, F. Neese, A. J. Skulan, Y.-S. Yang, and J. Zhou, Chem. Rev. 2000, 100, 235-349; b) D. W. Christianson and J. D. Cox, Annu. Rev. Biochem. 1999, 68, 33-57; c) J. Imsande, Plant Physiol. Biochem. 1999, 37, 87-

38


Chapter 1

97; d) R. H. Holm and E. I. Solomon, Eds., Chem. Rev. 1996, 96, 2237, thematic issue on bioinorganic enzymology. 102 H.-C. Liang, M. Dahan, and K. D. Karlin, Curr. Opin. Chem. Biol. 1999, 3, 168-175. 103 C. J. Reedy and B. R. Gibney, Chem. Rev. 2004, 104, 617-649. 104 a) Y. Murakami, J. Kikuchi, Y. Hisaeda, and O. Hayashida, Chem. Rev. 1996, 96, 721-758; b) A. J. Kirby, Angew. Chem. Int. Ed. Engl. 1996, 35, 706-724. 105 a) G. Wulff, Chem. Rev. 2002, 102, 1-27; b) R. Breslow and S. D. Dong, Chem. Rev. 1998, 98, 1997-2011; c) R. Breslow, Acc. Chem. Res. 1995, 28, 146-153. 106 G. Csjernyik, A. H. Éll, L. Fadini, B. Pugin, and J.-E. Bäckvall, J. Org. Chem. 2002, 67, 1657-1662. 107 M. E. Wilson and G. M. Whitesides, J. Am. Chem. Soc. 1978, 100, 306-307. 108 a) G. Klein, N. Humbert, J. Gradinaru, A. Ivanova, F. Gilardoni, U. E. Rusbandi, and T. R. Ward, Angew. Chem. Int. Ed. 2005, 44, 7764-7767; b) C. Letondor, A. Pordea, N. Humbert, A. Ivanova, S. Mazurek, M. Novic, and T. R. Ward, J. Am. Chem. Soc. 2006, 128, 8320-8328. 109 a) L. Panella, J. Broos, J. Jin, M. W. Fraaije, D. B. Janssen, M. Jeronimus-Stratingh, B. L. Feringa, A. J. Minnaard, and J. G. De Vries, Chem. Commun. 2005, 5656-5658; b) J. G. De Vries and L. Lefort, Chem. Eur. J. 2006, 12, 4722-4834. 110 a) D. Coquière, B. L. Feringa, and G. Roelfes, Angew. Chem. Int. Ed. 2007, 46, 9308-9311; b) A. J. Boersma, B. L. Feringa, and G. Roelfes, Org. Lett. 2007, 9, 3647-3650; c) G. Roelfes, Mol. Biosyst. 2007, 3, 126-135; d) G. Roelfes, A. J. Boersma, and B. L. Feringa, Chem. Commun. 2006, 635-637; e) G. Roelfes and B. L. Feringa, Angew. Chem. Int. Ed. 2005, 44, 3230-3232. 111 For pioneering contributions on Lewis-acid catalyzed asymmetric Diels-Alder reactions in water, see: S. Otto and J. B. F. N. Engberts, J. Am. Chem. Soc. 1999, 121, 6798-6806, and references cited therein. 112 L. Ropartz, N. J. Meeuwenoord, G. A. Van Der Marel, P. W. N. M. Van Leeuwen, A. M. Z. Slawin, and P. C. J. Kamer, Chem. Commun. 2007, 1556-1558. 113 a) M. T. Reetz, M. Rentzsch, A. Pletsch, M. Maywald, P. Maiwald, J. J.-P. Peyralans, A. Maichele, Y. Fu, N. Jiao, F. Hollmann, R. Mondière, and A. Taglieber, Tetrahedron 2007, 63, 6404-6414; b) M. T. Reetz, Chimia 2007, 61, 100-103; c) M. T. Reetz, J. J.-P. Peyralans, A. Maichele, Y. Fu, and M. Maywald, Chem. Commun. 2006, 4318-4320; d) M. T. Reetz, M. Rentzsch, A. Pletsch, and M. Maywald, Chimia 2002, 56, 721-723. 114 a) J. Steinreiber and T. R. Ward, Coord. Chem. Rev. 2008, 252, 751-766; b) M. D. Mihovilovic, J. Chem. Technol. Biotechnol. 2007, 82, 1067-1071; c) R. Krämer, Angew. Chem. Int. Ed. 2006, 45, 858-860; d) Y. Lu, Curr. Opin. Chem. Biol. 2005, 9, 118-126; e) D. Qi, C.-M. Tann, D. Haring, and M. D. Distefano, Chem. Rev. 2001, 101, 3081-3111. 115 a) Y. Lu and J. Liu, Curr. Opin. Biotechnol. 2006, 17, 580-588; b) C. M. Niemeyer, Angew. Chem. Int. Ed. 2003, 42, 5796-5800.

Chapter 2 Epoxidation of olefins in a centrifugal contact separator

In this chapter, our investigations towards a continuous transition-metal catalyzed

epoxidation reaction in a centrifugal contact separator (CCS) are described. The results

of two epoxidation methods selected for further study, based on iron and tungsten,

respectively, will be discussed. Furthermore, a number of new iron-based catalysts are

presented that can be used for epoxidations using peracetic acid as the terminal

oxidant.a

a Part of this chapter will be submitted for publication: R. M. Haak, A. J. Minnaard, J. G. de Vries, and B. L. Feringa, Iron-catalyzed epoxidation of olefins, manuscript in preparation.

Part of this chapter will be reported in: G. N. Kraai, B. Schuur, F. van Zwol, R. M. Haak, A. J. Minnaard, B. L. Feringa, H. J. Heeres, and J. G. de Vries, Process Intensification. Continuous Two-Phase Catalytic Reactions in a Table-Top Centrifugal Contact Separator, book chapter on the occasion of the 22nd Biennial ORCS Conference on the Catalysis of Organic Reactions, Richmond, Virginia, 2008.

40

Chapter 2 - Epoxidation of Olefins in a CCS.doc

Chapter 2

2.1 Importance of process intensification − the CCS

Fast and selective epoxidation of olefins remains a challenging goal in organic chemistry.1 Epoxides are especially valuable because of their versatility as intermediates in organic synthesis. The goal of the project described in this thesis is the development of new methods for the production of epoxides and epoxide-derived compounds in high yields and with high stereoselectivities by integration of synthetic, biochemical and chemical engineering methods.

Central to this approach is the shift towards more sustainable and safer processes, especially by using continuous systems, a concept known as process intensification.2 The use of such energetically and environmentally more benign methods is increasingly important based on economic and environmental considerations, but also for reasons of public perception.3

The most often used strategy towards process intensification is the application of continuous flow processes, since they allow the production of large quantities of compound, using equipment that is small compared to batch reactors.4 Within the area of continuous flow reactors, microreactor technology has received a lot of attention.2,5 Compared to conventional reactors, higher heat and mass transfer rates are possible in microreactors. This allows for more selective and higher-yielding reactions, even under more extreme conditions.2 However, there are some disadvantages to microreactors, for instance congestion of the microchannels through fouling. Furthermore, scale-up is only possible by parallel use of a large number of microreactors, which can be costly.6 We set out to investigate another approach towards intensification of processes, namely the use of centrifugal contact separators (CCSs), which are table-top sized flow reactors.

Figure 2.1 Schematic representation of the CCS. In this continuous centrifugal separator, the light and heavy phase are intensively mixed after entering the reactor (indicated by the cross-hatched area) after which they are separated by centrifuging (light gray for the light phase, dark gray for the heavy phase).

41


Epoxidation of olefins in a centrifugal contact separator

CCSs are devices that allow continuous fast mixing and consecutive separation of two phases. They combine very intensive mixing in an annular zone with rapid separation by centrifugation, as illustrated in Figure 2.1. We have investigated the use of a particular CCS, the CINC V-02 separator.7

Since the phases are mixed intensively in the annular zone and the average residence time of reagents in the CCS is of the order of minutes, it should be possible to perform biphasic reactions in the CCS, provided they are fast enough. Following this approach, the lighter, organic stream would contain starting material and eventually product, whereas the heavier phase would consist of an aqueous solution of reagent(s) and catalyst. As shown in Scheme 2.1, a number of CCSs in series would allow a continuous cascade of transformations.

Scheme 2.1 Envisioned catalytic cascade performed in a series of CCSs; TM catal. = transition metal catalyzed.

There are a number of important requirements that reactions should fulfill if they are to be performed in a CCS. First of all, they should be liquid-liquid phase reactions. A common approach is the use of organic-aqueous two-phase systems, but there are organic-organic biphasic systems that can also be used (vide infra). Secondly, reactions conducted in a CCS should be fast, since the residence time of reagents in this type of

42


Chapter 2

reactor is relatively short, of the order of minutes.b Another requirement is that reactions in the CCS should be highly (chemo)selective, since the aim of cascade catalysis requires that the organic outlet stream of one CCS can be used directly as the inlet stream of another reactor.

There are some additional restrictions: gases cannot be used as reactants or produced as by-products, since this would interfere with the separation in the centrifuge. Reactions should run at atmospheric pressure and at a temperature well below the solvent boiling point, since heating is possible in the CCS, but not at reflux conditions.

Based on these considerations − fast, selective, mild conditions, compatible with two-phase systems − reactions catalyzed by enzymes or transition metal catalysts seem most promising.

2.1.1 Existing biphasic epoxidation reactions Initially, we set out to develop the cascade shown in Scheme 2.1, which could, conceivably, be realized by linking multiple CCSs in series. This cascade starts with a transition-metal catalyzed epoxidation, followed by enzymatic enantioselective epoxide ring opening using a halide or other good leaving group as the nucleophile. This biocatalytic reaction is coupled to racemization of the haloalcohol produced by ring opening, so that enantiopure epoxides are eventually obtained in high yield. Hence, for the first step we looked for an epoxidation procedure that fulfilled the requirements for use in the CCS.

Existing procedures for the epoxidation of olefins1 suffer from several drawbacks that complicate their potential use in the CCS. For instance, in phase transfer catalysis a water-soluble reagent is transferred to the organic layer by means of a phase transfer catalyst to perform the reaction.8 In the cascade envisioned (Scheme 2.1), the organic layer is directed to another CCS to engage in a subsequent reaction, and this is complicated if remnants of reagent or catalyst from a previous step are present in the organic stream. Furthermore, existing epoxidation procedures often require reaction times which are too long to be of use in the CCS, are substrate-specific, employ expensive catalysts, or operate at harsh conditions.

A few examples will be evaluated to clarify the specific demands that the use of the CCS places on reactions.

b After technical modifications making it possible to recycle the stream containing the product, this condition became less strict.

43



An elegant system that at first glance seems to be a good candidate for scale-up in general has been reported by Hamilton and coworkers.9 They employ an aqueous solution of sodium hypochlorite for the epoxidation of substrates such as phenanthrene (Scheme 2.2). The required phase-transfer catalyst is available commercially and the reaction conditions are relatively mild (pH 8.5). However, it is effective only for polyaromatic hydrocarbons. Furthermore, reagents such as hypochlorite could damage the CCS.

O

CHCl3, rt

NaOCl aq. pH 8 - 9

[Bu4N]+HSO4− (0.2 - 1 eq.)

90% Scheme 2.2 Epoxidation of phenanthrene using NaOCl.

Another system that employs sodium hypochlorite has been reported by Montanari et al.10 It is based on the manganese-porphyrin catalyst C2.1 (Scheme 2.3) and has the advantage that non-activated olefins such as cyclooctene (S2.1) are epoxidized. However, the catalyst requires a lengthy synthesis and, as in the previous example, the use of hypochlorite in the CCS might not be feasible.

Scheme 2.3 Epoxidation of cyclooctene using a Mn-porphyrin catalyst.

As an oxidant, hydrogen peroxide is clearly preferred over hypochlorite, since its only by-product is water and it does not cause corrosion.11 Various tungstate catalysts have been reported for epoxidation,1 such as that by Venturello et al. in the early eighties, illustrated in Scheme 2.4.12 A range of olefins, including less reactive terminal alkenes such as 1-octene (S2.2), are epoxidized using H2O2 as the terminal oxidant and a catalyst that self-assembles from sodium tungstate and phosphoric acid. As phase transfer catalyst, methyltrioctylammonium chloride is used. Drawbacks of this system include the harsh conditions that are employed (e.g. pH 1.6), as well as the use of environmentally unfriendly dichloroethane. Furthermore, the time-scale of the reaction is too long for application in the CCS.

44


Chapter 2

Scheme 2.4 Tungstate-catalyzed epoxidation of 1-octene (S2.2) under acidic conditions.

An analogous procedure, developed by Noyori and coworkers,11b,13 suffers from similar drawbacks: it employs an expensive ligand and the reaction conditions are relatively harsh. Furthermore, it has an induction time before the epoxidation starts, something more frequently seen in transition-metal catalyzed epoxidations (vide infra). In a batch setup, this is not a problem since the reaction may be allowed to run as long as necessary, but in a CCS there is much less room for variation of the residence time of the reagents. An induction period therefore leads to low conversions.

2.2 Exploring epoxidation reactions for use in a CCS

2.2.1 Manganese-catalyzed epoxidation reactions Considering the drawbacks of several known biphasic procedures for olefin epoxidation (see paragraph 2.1.1), we decided to develop a two-phase epoxidation protocol based on a known fast, clean, and selective method.

Our first attempts were based on the work published by Hage14 and Burgess15 on manganese catalysis. First of all, the manganese-catalyzed epoxidation method developed by Hage and coworkers in collaboration with the group of Feringa was investigated.14 This procedure uses hydrogen peroxide as the oxidant, and in general works under mild conditions. The dinuclear Mn2(μ-O)3 complex C2.2 used as catalyst is based on the 1,4,7-trimethyl-1,4,7-triazacyclononane (tmtacn) ligand. Mechanistic studies performed in our group have revealed, among other things, that the catalyst retains its dinuclear structure throughout the catalytic cycle.16,17

Scheme 2.5 Epoxidation of 1-decene (S2.3) catalyzed by manganese catalyst C2.2.

45



The main disadvantage of this method is the need to add hydrogen peroxide over an extended period of time (using a syringe pump), because of the propensity of C2.2 to catalyse the disproportionation of hydrogen peroxide. In preliminary investigations, it was found that 1-decene (S2.3) was efficiently epoxidized using 0.2 mol% of catalyst and 1.5 equivalent of hydrogen peroxide in the presence of an additive such as glyoxylic acid (Scheme 2.5). Unfortunately, efforts to develop a two-phase system were unsuccessful. Conversion was obtained only in acetonitrile or acetone, whereas in biphasic systems of water and apolar organic solvents, Mn-tmtacn catalyzed epoxidation was ineffective.c

Table 2.1 MnSO4-catalyzed epoxidation of styrene (S2.4).a

Entry Cosolvent t

(h) Conversion

(%) 1 DMF (25 mL) 24 >99 2 DMF (5 mL) / pentane (5 mL) 96 49

a) 15 mL of aqueous NaHCO3 buffer (0.2 M, pH 8) was used.

The manganese-catalyzed epoxidation described by Burgess and coworkers15 provided an alternative method, with the advantage that it uses low-cost, readily available manganese sulfate as catalyst. As expected, it was possible to epoxidize styrene (S2.4) at room temperature using manganese sulfate as catalyst and DMF as cosolvent, but the reaction suffered from reaction times that were prohibitively long for application in the CCS (Table 2.1). Another disadvantage is the fact that 10 equivalents of hydrogen peroxide have to be used, because of the propensity of the manganese salt to catalyze disproportionation of hydrogen peroxide. The subsequent biphasic reaction in a n-pentane/aqueous solvent mixture reached only 46% conversion after 5 days, despite the fact that the concentrations of reagents were much higher than in the single-phase system (Table 2.1, entry 2).

c Subsequent research in our group has shown that certain combinations of water and organic solvents may be used in Mn-tmtacn catalyzed epoxidation and cis-dihydroxylation, see Refs. 16 and 17.

46


Chapter 2

2.2.2 MTO-catalyzed epoxidation of pinenes A number of reactions using methyltrioxorhenium (MTO,18 commercially available) and 30% H2O2 in the presence of pyridine ligands were conducted in dichloromethane, as described by Sharpless and coworkers.19

In preliminary experiments, the substrates 1-octene, (1S)-α-pinene, and (1S)-(−)-β-pinene (S2.2, S2.5, and S2.6, respectively, Figure 2.2) were tested under the conditions of Sharpless et al.19a with the difference that 40 mol% of pyridine was used instead of 12 mol%, to improve reaction rates (Scheme 2.6).

Scheme 2.6 MTO-catalyzed epoxidation of β-pinene (S2.6) using H2O2.

Under these conditions, S2.6 was fully converted in 1.5 h, whereas with S2.5 82% conversion was reached in 1 h. The terminal olefin S2.2 needed 19 h to reach full conversion to P2.2, using 3-cyanopyridine (0.1 eq) instead of pyridine as ligand.19b

The observed reaction rates were too low to be of use in the CCS at that point. Moreover, it was observed that the MTO was predominantly present in the organic phase instead of the aqueous one. This interfered with our intentions to use the CCS for a catalytic cascade as depicted in Scheme 2.1. In the envisioned cascade, the catalyst and reagents constitute one of the streams, typically the aqueous one, whereas the starting material and product are dissolved in the other, usually organic, stream. In conclusion, another epoxidation reaction had to be found for use in the CCS.

2.3 Biphasic iron-catalyzed epoxidation of olefins

Recent years have seen the publication of some notable examples of methods for iron-catalyzed epoxidation. An example is a procedure that uses peracetic acid in combination with an iron(III) catalyst, published by Dubois et al. in 2003.20 Analogous iron systems are known that catalyze the oxidation of alkanes using peracids21 or alkyl hydroperoxides.22 The catalytic mechanism is not known for most of these systems, but it has been observed that “mono and dinuclear iron(III) peroxo complexes were identified in several of them.”22

47



The complexes used as catalyst (C2.3a and C2.3b, Figure 2.4) have been known for several years.23 They form readily from low-cost commercially available starting materials and are active catalysts for the epoxidation of olefins by peracetic acid. Besides alkene epoxidation, they were also reported as catalysts in alkane oxidation.24 First, the original system was investigated (Scheme 2.7).

Scheme 2.7 Fe(III)-catalyzed epoxidation of olefins.

The substrate scope of this epoxidation method is broad. Cyclic olefins such as cyclooctene and cyclohexene are epoxidized efficiently, but the procedure is also effective for terminal alkenes such as 1-octene. Using olefins such as styrene, epoxidation is observed, but in this case the product is unstable under the reaction conditions due to solvolytic ring opening.

In this and the next paragraph, two novel procedures based on this method are described. First of all, a two-phase epoxidation method for application in a centrifugal contact separator, described in this paragraph and secondly, as a spin-off, an epoxidation method that uses a range of iron complexes to catalyze the epoxidation of olefins (Paragraph 2.4). The substrates that were used in these studies are depicted in Figure 2.2.

Figure 2.2 The substrates used in this study, cyclooctene (S2.1), 1-octene (S2.2), 1-decene (S2.3), styrene (S2.4), (1S)-α-pinene (S2.5), and (1S)-(−)-β-pinene (S2.6).

We became interested in the possibility of adapting the iron(III)-based system of Stack and coworkers20 for use in the CCS. First attempts to use catalyst C2.3b in an n-heptane/water biphasic system with water as the (co)solvent were unsuccessful (see Section 2.4). It was thought that the homogeneous system on which it was based, could be converted into a two-phase system by using an alkane as a second solvent in

48


Chapter 2

combination with acetonitrile. In this way, the reactant olefin and the product epoxide would reside preferentially in the hydrocarbon phase, whereas the catalyst and oxidant would stay in the acetonitrile layer. The results of our preliminary studies on batch scale are depicted in Table 2.2. Reactions were run on 10 mmol scale, using 20 mL of both pentane and acetonitrile and 1.0 mol% of catalyst.

Table 2.2 Epoxidation of S2.1 in alkanes / acetonitrile catalyzed by C2.3b.a

Entry t

(h)/cycle Conv. (%)

Solventb Misc.

1a 0.17 73 MeCN/n-pentane slow add. of C2.3bc b 47 93 2a 0.17 63 MeCN/n-pentane slow add. of C2.3b and AcOOHd b 21.5 94 3a 1st 72 MeCN/n-heptane 7 cycles, 15 min each b 2nd 100 c 3rd 91 d 4th 71 e 5th 79 f 6th 72

a) For details see the experimental section; b) Reaction procedure was similar whether n-pentane or n-heptane was used; c) A solution of catalyst C2.3b was added slowly to the reaction; d) Both catalyst C2.3b and peracetic acid were added slowly to the reaction mixture.

As expected, the non-catalyzed reaction takes longer in the two-phase system than in acetonitrile (not shown in the table). Two days are needed to reach >90% conversion. The order of addition of reagents was crucial in obtaining high yields of epoxide. Although the literature mentions slow addition of peracetic acid to a solution already containing the catalyst, we found that adding a stock solution of catalyst to the rest of the reaction mixture gave better results. Epoxidation was fast (73% of P2.1 after 10 min), although the reaction slowed down considerably after this period, suggesting catalyst deactivation (entry 1). Slow addition of both catalyst C2.3b and peracetic acid gave similar results (entry 2). The catalyst loading in this biphasic system was higher than in acetonitrile (1.0 vs. 0.25 mol%). By increasing the catalyst loading even more, we demonstrated that multiple cycles are possible. The experiment shown in entry 3 was an attempt to use a single batch of catalyst (1 equiv. with respect to a single batch of substrate) to catalyze the epoxidation of multiple batches of S2.1. A total of 6 batches of

49



S2.1 (10 mmol each) were epoxidized using 10 mmol of C2.3b, with fair to good conversions after 15 min of reaction for each batch.

In all experiments, gas development was observed in the course of the reaction, suggesting that catalyst C2.3b also catalyzes the disproportionation of peracetic acid. The decline in reaction rate after the first 10 min of reaction further supports this hypothesis. Otherwise, the reactions appear clean, cyclooctene oxide (P2.1) being the only product observed.

Terminal epoxides such as 1,2-epoxyoctane (P2.2) are a valuable class of compounds from an industrial perspective.25 The epoxidation of 1-octene (S2.2) was initially more problematic than that of cyclooctene under these biphasic conditions. However, by increasing the substrate concentration from 0.5 to 1.0 M, S2.2 was converted to the epoxide (Table 2.3). Thus, for a reaction on 10 mmol scale, 10 mL of n-pentane and 20 mL of acetonitrile were used, leading to a maximum of 72% conversion after 24 hours (entry 3). Although the reaction is substantially slower than the corresponding epoxidation of S2.1, (Table 2.2), these results show that biphasic iron-catalyzed epoxidation of less reactive terminal olefins is feasible. Note that under these biphasic conditions, non-catalyzed epoxidation of 1-octene is not observed.

Table 2.3 Epoxidation of S2.2 in alkanes / acetonitrile catalyzed by C2.3b.a

Entryt

(h) Conv. (%)

1 0.17 12 2 3.75 35 3 24 72

a) For details see the experimental section.

There are some drawbacks associated with this alkane/acetonitrile biphasic approach, which make it less convenient for use in the CCS. For instance, the by-product acetic acid has to be separated from the acetonitrile layer in which it is dissolved before this layer can be used as, for instance, the ingoing stream of another CCS. Furthermore, there is considerable decomposition of peracetic acid with concurrent catalyst deactivation during the reaction. These phenomena were most pronounced when both the catalyst and the oxidant are simultaneously added to the acetonitrile layer with no olefin substrate being present. In the CCS setup, the phase containing the substrate is separated from that containing the catalyst and oxidant, compelling us to look for ways

50


Chapter 2

to prevent this highly exothermic and potentially dangerous decomposition of peracetic acid.

It became apparent that another setup of the CCS would be necessary for this reaction. This structural modification would consist of an extra inlet for the catalyst stream, depicted schematically in Figure 2.3. Using this setup, the catalyst could be added to the system slowly. This design was based on the premise that the low-cost catalyst would not need to be recovered.

a) b)

Figure 2.3 a) Setup of the CCS prior to modification: Heptin and MeCNin indicate the ingoing n-heptane and acetonitrile streams, respectively, whereas Heptout and MeCNout indicate the corresponding outgoing streams; b) Setup of the CCS after modification: Catin signifies the ingoing stream of catalyst (dissolved in acetonitrile).

Although it was now thought that the reaction could in principle be conducted safely in the CCS,26 there were some remaining questions about the safety of the iron-catalyzed epoxidation in the CCS reactor, primarily because the reaction is exothermic. Based on calorimetric experiments, performed in order to obtain the adiabatic temperature increase of the epoxidation, the reaction was deemed not to be safe enough for the CCS.27

Successful implementation of this biphasic iron-catalyzed epoxidation method in the CCS will require a more stable catalyst, which should also be less prone to catalysis of peracetic acid decomposition. Given that technical developments make it possible to recycle one or both of the outgoing streams of the CCS (vide infra), slower but less exothermic epoxidation reactions may be feasible, such as Mn-tmtacn catalyzed epoxidation using hydrogen peroxide (see also Paragraph 2.2.1).14,16,17 Unfortunately, there was insufficient time to develop this manganese-catalyzed process in the CCS.

2.4 Epoxidation of olefins catalyzed by Fe/phen and Fe/bipy complexes

Over the course of our investigations, it was discovered that, in addition to C2.3a and C2.3b, other iron complexes catalyze the epoxidation of olefins by peracetic acid. Moreover, some of these complexes, especially complexes of the form FeIIL3, are more

51



stable than μ-oxo complexes C2.3. In the hope of finding a catalyst that combined high activity and stability, we tested a number of iron complexes for their catalytic activity in the epoxidation of olefins.

One of variations investigated was, for instance, the use of 2,2’-bipyridine (bipy) instead of 1,10-phenanthroline (phen) as a ligand. Also iron(II) compounds with the general formulae [FeII(phen)3]X2 and [FeII(bipy)3]X2 were examined. A complete overview of all catalysts employed in these studies is given in Figure 2.4. Complexes C2.4 − C2.6 were prepared as described in the literature.20,23 The blue complex [FeIII(phen)3](ClO4)3 (C2.7) was prepared by oxidation of the corresponding FeII(phen)3SO4 complex with Cl2, followed by precipitation using NaClO4.30,28

Scheme 2.8 Synthesis of C2.8.

Catalyst C2.8, although described in the literature,29,30,31 was synthesized by an alternative, more straightforward method, outlined in Scheme 2.8. When a mixture of iron trichloride hexahydrate and phenanthroline (molar ratio 2:3) is heated at reflux in absolute ethanol for 30 min, the complex precipitates from solution. An overview of the properties of a number of these and similar iron and other transition metal complexes has been given by Figgis and Lewis.32

The iron(II) and iron(III) complexes listed in Figure 2.4 were examined as catalysts in alkene epoxidation using peracetic acid as the oxidant. A number of aspects merit attention. In all reactions, epoxidation takes place, although the yield varies depending on both substrate and catalyst, as does the ratio of epoxidation vs. the major side reaction, decomposition of peracetic acid forming acetic acid and dioxygen. This section will focus on the epoxidation results using a homogeneous solvent system (typically acetonitrile). Iron-catalyzed epoxidation in a two-phase system is described in Section 2.3.

The rate of uncatalyzed epoxidation using peracetic acid is considerable, but dependent on the substrate. For instance, cyclooctene (S2.1) is converted to cyclooctene oxide (P2.1) completely after an overnight reaction (17.5 h). In the case of the terminal olefin 1-octene (S2.2), the reaction is slower, reaching 79% conversion after a reaction period of 24 h. Oxidation of styrene is very fast in the absence of catalyst (64% conversion after 3 h), however, a number of byproducts are formed, notably 2-phenylacetaldehyde.

52


Chapter 2

As already mentioned in the literature, simple iron salts such as Fe(ClO4)3 or Fe(ClO4)2 do not catalyze this epoxidation reaction.20 Furthermore, preliminary experiments with alternative oxidants such as t-BuOOH, cumene hydrogen peroxide, or H2O2, were unsuccessful. It was reported recently that iron-catalyzed epoxidation is possible using peracetic acid formed in situ from hydrogen peroxide and acetic acid.33,34 A preliminary experiment was carried out using this approach. However, epoxidation was not observed.

Figure 2.4 The iron(II) and iron(III) catalysts used in this study.

The results of the screening of substrate S2.1 are described in Table 2.4. In general, reactions were performed using 10 mmol of substrate, 0.5 mol% of catalyst, 2 equivalents of peracetic acid, and 20 mL of acetonitrile.

Complex C2.3a is one of the most extensively used catalysts in this study, together with C2.3b. Since the difference between C2.3a and C2.3b was found to be negligible, they were used interchangeably.20 Epoxidation using C2.3a in acetonitrile, under the

53



conditions mentioned in literature,20 led to full conversion in 5 min (entry 1). In order to investigate to what extent the reaction was compatible with the presence of water, a series of experiments were performed in which the water content of the reaction was increased stepwise from 0 to 40% (entries 1 − 5). The reaction slowed down considerably, showing only 51% conversion after 5 min when 30 or 40% water with respect to acetonitrile was used. A possible explanation is that the presence of water in the reaction mixture favors decomposition of peracetic acid instead of epoxidation. Furthermore, [Fe(phen)3]2+ species are readily formed in the presence of water, which are themselves catalytically inactive, although they may be converted to an active catalyst after an induction period (vide infra).

Table 2.4 Iron-catalyzed epoxidation of cyclooctene (S2.1) by peracetic acid.a

Entry Cat

(mol%) T

(°C) Solvent

t (min)

Induction (min)

Conv. (%)

1 C2.3a 0 → rt MeCN 5 −b >99 2 C2.3a 0 → rt MeCN + 10% H2O 5 −b 64 3 C2.3a 0 → rt MeCN + 20% H2O 5 −b 72 4 C2.3b 0 → rt MeCN + 30% H2O 5 −b 51 5 C2.3b 0 → rt MeCN + 40% H2O 5 −b 51 6 C2.4 0 → rt MeCN 60 −b 96 7 C2.5a rt MeCN 210 n.d.c 98 8 C2.5b rt MeCN 60 n.d.c >99 9 C2.8 rt → 0 MeCN 12 6 100

a) For details, see the experimental section; b) Reaction started immediately; c) Not determined.

Employing C2.4, a μ-oxo FeIII complex with bipyridine ligands (Figure 2.4), led to full conversion after a reaction time of 1 h (entry 6). Use of the [Fe(bipy)3]2+ complexes C2.5a and C2.5b gave good conversion in acetonitrile at room temperature (entries 7 and 8). The bright orange complex C2.8 also turned out to be an efficient catalyst for the epoxidation of S2.1 using peracetic acid, giving full conversion in 6 min after an induction period of 6 min (entry 9).

Epoxidations using catalysts C2.3c and C2.5 − C2.7, both for S2.1 and other substrates, were characterized by an induction period, during which the initial iron(II)-species is presumably oxidized to catalytically active iron(III). Possibly, the active species is the same as in the reactions catalyzed by μ-oxo iron complexes C2.3 and C2.4. During this

54


Chapter 2

activation phase, the complex appears to be going through different oxidation states. This is suggested by striking color changes, going from red to orange to blue to yellow. Catalytic activity is only apparent in the last stage, when the reaction becomes highly exothermic and gas evolution (from decomposition of peracetic acid) is also observed. The induction period ranges from 5 min to 1.5 h (vide infra) and can be shortened by heating the reaction or by including a one-electron oxidant such as CAN. In the reaction using Fe(phen)3(ClO4)2 (C2.6b),35 this series of color changes was: red → brown → blueish grey → light blue → orange → yellow. The different colors likely correspond to iron species of various oxidation states and nuclearity.

Since the color of the final active species is the same in all reactions we performed and the rate of the catalyzed epoxidation itself does not vary significantly with the catalyst, it is presumed that the catalytically active species is the same for all catalysts C2.4 − C2.7. Further evidence to back up this hypothesis still has to be obtained, for example on the basis of ES-MS, UV, or electrochemical measurements. Such measurements may shed light on the molecular origin of the observed lag time, which likely involves ligand dissociation and association processes and possibly dimerization, besides oxidation of the iron center. Crucial for obtaining a molecular understanding of the catalysis in this process will be the identification of the active species. Recently, the epoxidizing agent was identified in other systems, such as Mn-tmtacn catalyzed epoxidation using H2O216,17 and iron-catalyzed epoxidation using H2O2 in the presence of acetic acid,36 using a combination of techniques such as 18O labeling, UV-vis, ESI-MS, GC(-MS), X-band EPR, NMR, and electrochemical measurements. In Mn-tmtacn catalyzed epoxidation and cis-dihydroxylation, it was found that the lag time was caused by the formation of the active catalyst from the resting state of the manganese complex.16,17

Besides S2.1, the linear alkenes S2.2 (1-octene) and S2.3 (1-decene) were investigated in this study. Our interest in the epoxidation of 1-octene stems from the fact that terminal epoxides are very useful synthetic intermediates from an industrial perspective.25 The results are summarized in Table 2.5.

Use of C2.3a (entry 1) led to a conversion of 67% in 6 min, whereas the literature mentions full conversion for this reaction.20 Epoxidation using the similar catalyst C2.3b led to fast initial conversion (79% after 15 min), after which the reaction slowed down considerably, possibly due to catalyst deactivation (entry 2). The use of catalyst C2.3c, the chloride equivalent of C2.3a and C2.3b, led to good conversion (86%) after a lag time of 5 min followed by a reaction time of 10 min (entry 3). Interestingly, in the literature it is indicated that C2.3c is inactive as an epoxidation catalyst under these circumstances.20

55



The use of various iron(II) catalysts in principle led to fast reactions. Similar to the epoxidation of substrate S2.1, epoxidation of S2.2 started only after a certain lag time using catalysts C2.5 and C2.6. When C2.5a was used, this period amounted to 24 min, whereas using C2.5b it was about 1 h and 15 min. The subsequent reaction was typically fast, in the order of 10 min. The highest conversion using bipyridine catalysts C2.5 was a moderate 68% (entries 4 − 6).

Results were better using [Fe(phen)3]2+ complexes C2.6 that have 1,10-phenanthroline instead of 2,2'-bipyridine as ligand. For instance, C2.6a gave 91% conversion after a total of 45 min (entry 7), whereas C2.6b gave conversions to P2.2 of 94% after 165 min, including a lag time of 80 min (entry 8). This period could be considerably shortened by heating the mixture before the reaction started (entry 9) or, surprisingly, by including the reducing agent SmI2 (entry 12). Addition of the one-electron oxidizing agent cerium ammonium nitrate ((NH4)2CeIV(NO3)6, CAN) had little effect (entry 10).

Table 2.5 Iron-catalyzed epoxidation of S2.2 and S2.3 by peracetic acid.a

Entry Substrate Cat

(mol%) T

(°C) t

(min) Conv.(%)

Induction (min)

Additive

1 S2.2 C2.3a rt 12 67 − 2a ,, C2.3b 0 15 79 − b ,, ,, ,, 1000 98 − 3 ,, C2.3c 0 10 86 5 4 ,, C2.5a 0 50 50 24 5 ,, C2.5b rt 200 62 80 6 ,, C2.5b Δ→0 5 68 n.d.b 7 ,, C2.6a rt →0 45 91 36 8 ,, C2.6b rt 165 94 90 9 ,, C2.6b Δ→0 66 88 n.d.b 10 ,, C2.6b 0 69 94 50 (NH4)2CeIV(NO3)6c 11 ,, C2.6c rt→0 29 85 19 12 ,, C2.6d 0 12 58 7 SmI2 0.1M in THFc 13 ,, C2.7 rt→0 35 77 30 14 ,, C2.8 rt→0 10 93 4 15 S2.3 C2.3a 0 11 30 n.d.b 16 ,, C2.8 rt→0 12 86 6

a) For experimental details see the experimental section; b) Not determined; c) 1 equivalent w.r.t. the catalyst.

56


Chapter 2

Fe(phen)3Cl2 (C2.6c) gave a good conversion of 85% after 45 min, in contrast with the literature in which it is stated that C2.6c is inactive.20 However, the corresponding PF6−-complex (C2.6d) led to a disappointing conversion (entries 11 and 12). The FeIII-equivalent of C2.6b, C2.7,30,28 showed a disappointing conversion of only 30% (entry 13). Finally, the orange binuclear catalyst C2.8 was used to catalyze the epoxidation of S2.2 efficiently in a short time of 10 min with an lag time of only 4 min (entry 14). It was also observed that FeII-catalysts C2.5, C2.6, and C2.8 catalyzed the decomposition of peracetic acid, hence an excess (2 equivalents) of peracetic acid was used.

Substrate S2.3 (1-decene) was used in reactions using catalysts C2.3 and C2.8 (Table 2.5, entries 15 and 16). The use of catalyst C2.3a led to disappointing conversion (30% after 10 min, entry 1) for unknown reasons. However, using C2.8 led to 86% conversion in 12 min, including an induction period of only 6 min (entry 2).

Substrate S2.4 (styrene) was epoxidized using catalysts C2.3 and C2.8, with mixed results (Table 2.6). Reasonable to good conversions were generally observed. However, the conversion was not selective since considerable amounts of phenylacetaldehyde were also formed. The ratio of P2.4 vs. byproducts is comparable whether the reaction is catalyzed (by C2.3a and C2.8, entries 2 − 4) or not (entry 1). Full conversion was reached only using catalyst C2.8 (entry 4).

Table 2.6 Iron-catalyzed epoxidation of S2.4 by peracetic acid.a

Entry Cat

(mol%) T

t (h)

Induction (min)

Conv. (%)b Misc.

1 − 0°C 2.82 − 63 (38) AcOOH 2.5 eq 2 C2.3a 0°C→rt 0.25 n.d.c 82 (46) 3 C2.3a 0°C→rt 0.08 n.d.c 46 (31) 4 C2.8 rt, 0°C 0.22 8 97 (46)

a) For experimental details see the experimental section; b) In between brackets is the conversion to the epoxide; c) Not determined.

The pinene substrate S2.6 (1S-(−)-β-pinene) was more extensively tested in another reaction, namely MTO-catalyzed epoxidation (see Paragraph 2.2.2). The reaction of S2.6 with peracetic acid catalyzed by C2.3b led to a disappointing yield of 33% after 7 min (Scheme 2.9).

57



Scheme 2.9 Iron-catalyzed epoxidation of (1S)-(−)-β-pinene (S2.6).

In conclusion, we have demonstrated that various iron(II) and iron(III) complexes with phenanthroline or bipyridine ligands are catalytically active in the epoxidation of unfunctionalized olefins using peracetic acid as the oxidant. In general, the use of complexes with phenanthroline ligands (e.g. C2.3 and C2.6) led to faster epoxidation reactions than their bipyridine analogues (C2.4 and C2.5). As expected, dimeric μ-oxo iron(III) / phenanthroline complexes C2.3, some of which have been described before in oxidation catalysis,20 are efficient epoxidation catalysts. When the cationic complexes C2.3a and C2.3b are used, the reaction starts immediately, whereas the neutral complex C2.3c had a lag time of 5 min. Much longer induction times (up to 90 min) are observed when iron(II) complexes C2.5 and C2.6 and iron(III) complex C2.7 are used as catalyst, although the epoxidation itself is as fast as when using catalysts C2.3. The induction period can be shortened most efficiently by heating the reaction mixture until epoxidation starts. Complex C2.8 has a short lag time of 5 min.

It was observed that the presence of water in the reaction mixture is detrimental to the conversion of the epoxidation reaction. In terms of substrate scope, unfunctionalized olefins such as cyclooctene (S2.1) or 1-octene (S2.2) are converted cleanly to the corresponding epoxides. However, styrene (S2.4), which yields an acid-sensitive epoxide, is converted to a number of products, most notably styrene oxide and phenylacetaldehyde.

2.5 Tungsten-catalyzed epoxidation of cyclooctene in the CCS37

In later stages of the project, developments in the setup of the CCS allowed the recycling of one or both phases. Thus, it became feasible to try other, slower but less violent, epoxidation reactions, such as the one reported by Alsters et al.,38 that uses hydrogen peroxide as the oxidant in the presence of a polyoxometalate catalyst based on tungsten. We chose this reaction because i) it employs hydrogen peroxide, one of the more atom-efficient and environmentally friendly of oxidants, ii) this catalyst is known to have a low extent of aproductive catalase activity, a commonly encountered side-reaction in transition-metal catalyzed oxidations, iii) it lacks an induction period prior to the reaction starting, otherwise common in tungsten-catalyzed epoxidations, iv) the reaction conditions are mild: only slightly elevated temperature and near-neutral pH, v)

58


Chapter 2

the catalyst loading is low, vi) this reaction is performed in a two-phase system (see also Section 2.1 for a list of requirements for reactions that are to be performed in a CCS), and vii) perhaps most importantly, the polyoxometalate has no organic ligands, which are notorious weak spots in oxidation catalysis. The characteristics mentioned under iv and vi were important reasons to prefer POM-based epoxidation over tungsten-catalyzed epoxidation as reported by Noyori and coworkers,11b which employs (aminomethyl)-phosphonic acid as an additive.

Polyoxometalates (POMs) are molecular clusters with a wide variety of structures and physical properties.39 They are applied in various areas such as nanotechnology, biology, medicine, surfaces, supramolecular and molecular materials, and sensors.40 Especially the area of catalysis has been investigated in depth.40,41 One of the developments is the epoxidation on large scale of a number of olefins using the polyoxometalate Na12[WZn3(ZnW9O34)2] (hereafter abbreviated NaZnPOM).38 This POM is conveniently obtained by self-assembly of low-cost, commercially available starting materials, according to the equation:42

19 Na2WO4 + 5 Zn(NO3)2 + 16 HNO3 → Na12[WZn3(ZnW9O34)2] + 26 NaNO3 + 8 H2O

The significance of the work described in this paragraph is that it constitutes the first example of a transition-metal catalyzed reaction in a centrifugal contact separator, and demonstrates that such devices may be used to perform highly sophisticated and valuable chemical transformations at a large scale.

The reaction, that was first optimized in common laboratory glassware, is depicted in Table 2.7. The reactions were performed at 25 mmol scale, using 5 mL of toluene, 5 mL 0.1 M aqueous solution of NaZnPOM (so 0.5 mmol W), 0.25 mmol of phase transfer salt (0.5 equivalent w.r.t. W), and 2.5 equivalents of hydrogen peroxide, at a temperature of 60 °C. To simulate the conditions in the CCS as good as possible, stirring was performed mechanically at 600 rpm.

It was found that the replacement of [(n-Oct)3(Me)N]Cl by [(n-Oct)3(Me)N]HSO4 improved reaction rates considerably (compare entries 1 and 2 in Table 2.7). Other modifications from the literature procedure38 include raising of the catalyst loading from 0.1% to 2% and doubling the amount of hydrogen peroxide used (1.5 eq to 3 eq). The quality of hydrogen peroxide was found to be essential in obtaining good

59



conversions, "medicinal extra pure" being the quality of choice.d Under these new conditions, S2.1 was fully converted to P2.1 after 160 min.

Table 2.7 Optimization of NaZnPOM-catalyzed epoxidation of cyclooctene.a

Entry QX t

(min) Conv. (%)b

1 a [(n-Oct)3(Me)N]Cl 120 46 b 1,300 95 2 a [(n-Oct)3(Me)N]HSO4 90 87 b 160 99

a) For details, see the experimental section; b) Conversion was periodically monitored using GC.

The system was now considered ready for the CCS (Figure 2.5). Nevertheless, extensive optimization was required. The first CCS experiments were conducted with an aqueous stream consisting of 200 g of aqueous NaZnPOM stock solution (0.1 mmol W / g of solution), 320 mL of 30% medicinal extra pure H2O2, and 10 mmol of a phase transfer salt. The organic stream consisted of a solution of 1.5 mol of cyclooctene and 40 mmol of n-dodecane as the internal standard in 2.5 L of toluene. The two vessels containing the aqueous and organic feed streams were heated to 65 °C and two peristaltic tube pumps were used to pump the two solutions through the CCS at a flow rate of 10 mL/min for both streams. After an hour, disproportionation of hydrogen peroxide was observed in the the storage vessel for the aqueous feed stream. The vessel was removed from the heat source and the reaction was stopped. No conversion to the epoxide was observed.

Also in subsequent experiments, disproportionation of H2O2 was observed when the aqueous storage vessel was heated for 1 − 1.5 h at 65 °C. Although NaZnPOM was chosen as a catalyst because it was reported that it only catalyzed the disproportionation of hydrogen peroxide to a minor extent,38 it was concluded that in the absence of olefin, the catalyst behaves differently. This was confirmed by an experiment in which the NaZnPOM solution, [(n-Oct)3(Me)N]HSO4, and H2O2 were combined and heated to 80 d Hydrogen peroxide solutions may contain sequestrants for metal ions that interfere with transition-metal catalyzed epoxidation (personal communication by Dr. J. W. de Boer). Medicinal grade hydrogen peroxide is stabilized using small amounts (0.015%) of phosphate buffer, which is compatible with the reaction.

60


Chapter 2

°C in the absence of olefin. After 5 min, evolution of O2 was observed, indicating H2O2 decomposition.

Figure 2.5 Schematic representation of the setup of the CCS. The aqueous phase consisted of 500 g of NaZnPOM solution (0.1 mmol W/g) and [(n-Oct)3(Me)N]HSO4 (0.5 g, 0.1 mmol) at 70 °C. The organic phase consisted of cyclooctene (135.1 g, 1.23 mol), dodecane (12.34 g, 72.4 mmol), [(n-Oct)3(Me)N]HSO4 (9.68 g, 20.7 mmol) and toluene (total volume 2 L) at 70 °C.

The CCS was then modified to allow separate addition of H2O2 from the rest of the aqueous stream. Further modifications included a doubling of the amount of [(n-Oct)3(Me)N]HSO4 and dissolving it in the organic stream instead of the aqueous one, since its solubility in toluene is much higher than in water. Intially, no conversion was observed under these conditions, but when the aqueous stream was recycled, 20% conversion to P2.1 was observed (Figure 2.5). Details regarding the optimization process are given in the experimental section, Paragraph 2.7.6.

The observation that conversion is only seen after recycling the aqueous stream, might be explained by a possible induction period of the catalyst. This is a common phenomenon in tungsten-catalyzed epoxidation.43 NaZnPOM was chosen specifically because of its reported absence of induction time compared to several other systems.38 However, it is conceivable that NaZnPOM has a very short induction time, e.g. of the order of a few minutes. In a measurement on the time-scale of hours, such a short induction would not be noticed, but in CCS experiments it could have a large effect owing to the short contact time of reagents in the appparatus.

2.6 Conclusions and outlook

As part of the development of a catalytic cascade from olefins to enantiopure epoxides on industrial scale, we aimed to set up an efficient biphasic epoxidation in a centrifugal contact separator (CCS). A number of epoxidation methods were examined, taking into account the specific requirements of the CCS, after which two of them were chosen for

61



further development. These were the iron/phenanthroline-catalyzed epoxidation of various olefins using peracetic acid, and the tungsten-catalyzed epoxidation of cyclooctene using hydrogen peroxide.

Based on an existing homogeneous reaction,20 a fast and selective method was developed for iron-catalyzed epoxidation of unfunctionalized olefins in a two-phase system consisting of acetonitrile and a hydrocarbon such as n-heptane, using peracetic acid as the oxidant. Although this method requires the use of higher loadings of catalysts C2.3a and C2.3b than the original procedure, as well as longer reaction times, it has the advantage that it could be used in a biphasic continuous flow process such as in the CCS. However, some of its features limit its applicability. For instance, the procedure may be too exothermic and too prone to peracetic acid decomposition to be safely performed in the CCS. Furthermore, the need for a separate inlet for the solution containing the catalyst, in order to prevent premature catalyst deactivation and disproportionation of peracetic acid, is inconvenient and makes recycling of the catalyst loop impossible.

As a spin-off of the former project, a method was developed for epoxidation of olefins using a variety of iron(II) and iron(III) catalysts. This method also employs peracetic acid as oxidant. The catalysts, which have a longer shelf-life than C2.3a and C2.3b, especially in stock solution, are conveniently available from various low-cost iron(II) and iron(III) salts using bipyridine or phenanthroline as ligands, the latter being the most efficient. Catalysts C2.3c and C2.5 − C2.7 go through an induction period (ranging from 5 to 90 min, depending on the catalyst) before the catalytically active species is formed. Further experimental data on the formation of the catalytically active species and the mechanism of epoxidation still have to be obtained.

In a later stage of the project, technical improvements on the CCS allowed for the use of elevated temperatures, separate addition of catalyst and other reagents, and facile recycling of the two streams. This made it possible to perform reactions that initially were considered too slow for the CCS. Thus, tungsten-catalyzed epoxidation of cyclooctene in a centrifugal contact separator was investigated, using H2O2 as the oxidant. After a single pass of both streams, no conversion to cyclooctene oxide was observed, however, recycling of the aqueous stream (containing the catalyst and reagents) led to a conversion of 20%. Compared to a batch setup, giving conversions of 87% after 90 min and 99% after 160 min in our hands, the result in the CCS is somewhat disappointing.

If the CCS approach is to be successful, it would be highly beneficial if it were possible to have better control over the average residence time of the reagents in the apparatus, so that high conversions can be obtained with a single pass of both product and catalyst stream.

62


Chapter 2

2.7 Experimental section

2.7.1 General remarks Starting materials were purchased from Aldrich or Acros and used as received unless stated otherwise. All solvents were reagent grade and, if necessary, dried and distilled prior to use. Demineralized water was used in the preparation of all aqueous solutions. In epoxidation reactions, hydrogen peroxide (30%, medicinal extra pure grade)e was used.

CCS experiments were performed in a CINC V-02 separator, also known as the CIT V-02 separator, of either stainless steel or Hastelloy®,44 as indicated for each experiment. Two Verder VL 500 control peristaltic tube pumps equipped with a double pump head (3,2 x 1,6 x 8R) were used to feed the CCS. To operate the reactor at elevated temperature, it was equipped with a jacket which was connected to a temperature controlled water bath that has a temperature accuracy of ± 0.01 °C. 1H NMR spectra were recorded on a Varian VXR300 (299.97 MHz) or a Varian AMX400 (399.93 MHz) spectrometer in CDCl3 unless stated otherwise. Chemical shifts are reported in δ values (ppm) relative to the residual solvent peak (CHCl3, 1H = 7.24). Splitting patterns are indicated as follows: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad).

Mass spectra (HRMS) were performed on a Jeol JMS-600H. GC-MS spectra were recorded on a Hewlett Packard HP6890 equipped with an HP1 column and an HP 5973 Mass Selective Detector.

GC analysis was performed on a Shimadzu GC-17A or a Hewlett Packard HP6890 spectrometer equipped with an HP1 column. n-Dodecane was used as internal standard. To monitor reactions, 0.1 mL aliquots were periodically taken from the reaction mixture, filtered over a short plug of silica, diluted with ether, and analyzed using GC or GC-MS.

2.7.2 Biphasic manganese-catalyzed epoxidation of styrene The procedure given by Burgess et al.15 was modified. Thus, to a stirred mixture of DMF (5 mL), n-pentane (5 mL), MnSO4•H2O (16.92 mg, 0.1 mmol, 1 mol%), and styrene (S2.4, 1.15 mL, 1.04 g, 10.0 mmol), a mixture of H2O2 30% (10 mL, 11 g, 97 mmol) and

e See Footnote d on page 59.

63



NaHCO3 bufferf (0.2 M, pH 8, 15 mL) was added dropwise over 0.5 h. After 96 h, maximum conversion was reached (49% by GC).

2.7.3 Biphasic MTO-catalyzed epoxidation of pinene Slightly modified conditions compared to those reported by Sharpless et al.19 were used. Thus, a mixture of MTO (13.3 mg, 0.05 mmol), (1S)-(−)-β-pinene (S2.6, 1.57 mL, 1.35 g, 10 mmol), pyridine 0.33 mL (0.32 g, 4.0 mmol), 30 % H2O2 (1.6 mL, 1.78 g, 15.6 mmol), and 10 mL of DCM, was stirred at room temperature. When the reaction had finished, the mixture was washed successively with water, NaHSO3 aq 10% to remove traces of peroxide, NaHCO3 sat. and brine, dried over MgSO4, filtered and evaporated. Spectral data were in accordance with the literature.45

2.7.4 Iron-catalyzed epoxidation of olefins in a two-phase system Stock solutions of catalysts C2.3a and C2.3b were prepared as described by Dubois et al.20 from Fe(ClO4)3 or Fe(NO3)3 and 1,10-phenanthroline. A representative general procedure for the biphasic epoxidation is as follows:

To a 250 mL three-necked flask equipped with a mechanical stirrer, thermometer, and dropping funnel were added n-heptane (20 mL), acetonitrile (5 mL), cyclooctene (S2.1; 1.37 mL, 1.15 g, 10.4 mmol), and peracetic acid 35% (3 mL, 3.39 g, 15.6 mmol). While this mixture was stirred (600 rpm), a solution of C2.3b in 15 mL acetonitrile was added dropwise to the solution.

When the reaction had finished (as judged from GC measurements), the layers were separated, the acetonitrile layer was extracted with n-heptane, the hydrocarbon layers combined, dried over MgSO4, filtered and evaporated. The spectroscopic data of product P2.1 were in accordance with the literature.20

The corresponding reaction of 1-octene (S2.2) was performed analogously, with the exception that only 10 mL of hydrocarbon solvent was used. The spectral data of product P2.2 were in accordance with the literature.20

f A mixture of NaHCO3 (16.80 g, 0.2 mol) and Na2CO3 (100 mg, 0.9 mmol) in 1 L of H2O, see Supporting Information of Ref. 15.

64


Chapter 2

2.7.5 Epoxidation of olefins catalyzed by Fe/phen and Fe/bipy complexes

Catalyst preparation Stock solutions of catalysts C2.3 and C2.4 were prepared as described by Dubois et al.20 from Fe(ClO4)3 or Fe(NO3)3 and 1,10-phenanthroline (phen) or 2,2'-bipyridine (bipy) in acetonitrile.

Stock solutions (100 mM) of catalysts [FeII(bipy)3]X2 (C2.5) and [FeII(phen)3]X2 (C2.6) were prepared by dissolving the appropriate FeII-salt (1.0 mmol) and 2,2'-bipyridine or 1,10-phenanthroline (3.0 mmol) in H2O or acetonitrile (10 mL). The resulting solutions were pinkish red (complexes with bipyridine) or dark red (complexes with phenanthroline).

To isolate the resulting complexes, FeII-salts (10 mmol) and bipyridine or phenanthroline (30 mmol) were dissolved in demineralized water, after which the solution was concentrated until the pinkish red (bipyridine as ligand) or dark-red (phenanthroline as ligand) complexes precipitated out of solution. They were collected on a glass filter and washed with a small amount of ice-cold water and ether, respectively. Complex C2.6 could also be precipitated from the reaction mixture by the addition of 2 equivalents of NH4PF6, giving C2.6d.

[FeIII(phen)3](ClO4)3 (C2.7) was prepared by oxidizing C2.6b according to the procedure described by Plowman et al.23a

[FeIII(phen)2Cl2]+[FeIII(phen)Cl4]− (C2.8)29,30,31 was prepared by refluxing a solution of FeCl3·6H2O (5.41 g; 20.0 mmol) and 1,10-phenanthroline (5.41 g; 30.0 mmol) in EtOH until an orange solid precipitated, which was filtered and dried in a vacuum desiccator. Yield: 8.70 g (10 mmol, quant); mp 270 − 273 °C; Anal. calc. for C36H24Fe2N6Cl6•H2O: C 49.0, H 2.97, N 9.52, found: C 49.1, H 2.96, N 9.20. When dissolved in water, the solution turned from orange to light brown and the resulting complex was found to be paramagnetic. 1H NMR (D2O) δ 23.74 (br), 18.45 (br), 17.33 (br), 16.26 (s), 15.95 (s), 15.82 (br), 15.21 (s), 14.20 (br), 12.02 (br), 10.79 (s), 8.32 (s).

General procedure for iron-catalyzed olefin epoxidation Epoxidation reactions were conducted according to the following general procedure: The catalyst (C2.4 − C2.7, 0.05 mmol) was suspended in MeCN (20 mL), then substrate (S2.1 − S2.6, 10 mmol) and AcOOH (32% in AcOH, 4.5 mL, 5.1 g, 21 mmol) were added. After an induction period, during which the catalytically active species formed, the reaction started. The reaction was highly exothermic and evolution of dioxygen was

65



observed. The progress of the reaction was monitored by periodically checking samples from the mixture by GC.

2.7.6 NaZnPOM-catalyzed epoxidation of cyclooctene

Preparation of NaZnPOM The procedure of Alsters et al.38 was used. Thus, for the preparation of NaZnPOM, Na2WO4·2H2O (3.3 g, 10.0 mol) was dissolved in 9 mL of water and this mixture was heated to 85 °C. Then, HNO3 conc. (0.67 mL, 0.94 g, 15.0 mmol) was added to the mixture, resulting in a yellow precipitate that gradually dissolved. The mixture was subsequently heated to 95 °C and a solution of [Zn(NO3)2·4H2O] (0.68 g, 2.6 mmol) in 5 mL of water was added dropwise to the mixture, during which care was taken that the resulting white precipitate had completely disappeared before another drop of solution was added to the reaction mixture. When all [Zn(NO3)2·4H2O] had been added, the mixture was decanted in 40 mL of water at room temperature, after which water was added to this solution to give a total weight of 100 g, resulting in a NaZnPOM solution containing 0.1 mmol W / g of solution.

Epoxidation of cyclooctene in batch setup To a 250 mL three-necked flask equipped with a mechanical stirrer, a dropping funnel, and a thermometer, were successively added: dodecane (internal standard, 227 μL, 1 mmol), cyclooctene (3.27 mL, 25 mmol), toluene (distilled over Na, 5 mL), [(n-Oct)3(Me)N]HSO4 (116 mg, 0.25 mmol), NaZnPOM (5 mL 0.1 M solution, 0.5 mmol W), and hydrogen peroxide (medical grade, 30%, 8 mL, 70 mmol). This mixture was stirred at 600 rpm at 60 °C. The conversion was monitored periodically by GC.

Optimization of tungsten-catalyzed epoxidation in the CCS The experiments were performed in collaboration with G. N. Kraai. First, the CCS was fed with pure toluene and pure water at the indicated flow rates. Subsequently, the centrifuge was started (40 Hz, which corresponds to 2400 rpm) and the setup was allowed to equilibrate for a period of 1 h. At this point, the toluene feed stream was replaced by the organic feed stream (composition given below for each experiment). After equilibration for 15 min, the reaction in the CCS was started by replacing the water stream with the aqueous feed stream (composition given below for each experiment). Samples were taken at regular intervals and analysed by GC.

First CCS experiment: A stainless steel CCS was used. The low mix bottom plate was applied. The aqueous stream consisted of aqueous NaZnPOM stock solution (0.1 mmol

66


Chapter 2

W/g, 200 g),38,42 H2O2 30% medicinal extra pure (320 mL), and [(n-Oct)3(Me)N]HSO4 (4.69 g, 10.0 mmol). The organic stream consisted of 2.5 L of a solution of cyclooctene (S2.1, 173.88 g, 1.58 mol) and n-dodecane (internal standard, 9.08 mL, 6.84 g, 40 mmol) in toluene. The two vessels containing the aqueous and organic feed streams were heated to 65 °C. The flow rate for both streams was 10 mL/min. Otherwise, the general procedure was followed. After 60 min, evolution of gas and an increasing temperature were observed in the storage vessel for the aqueous feed stream, indicating the decomposition H2O2. The vessel was removed from the heat source and the reaction was stopped. No conversion to the epoxide was observed.

Second CCS experiment: A Hastelloy CCS was used. The composition of the aqueous and organic feed streams was equal to the first experiment. The feed streams (flow rate 10 mL/min for both) were heated to 65 °C and the mantle of the apparatus to 75 °C. Otherwise, the experiment was performed as the first one. After 90 min, evolution of gas and an increasing temperature were observed in the storage vessel for the aqueous feed stream, indicating the decomposition H2O2. The vessel was removed from the heat source and the reaction was stopped. No conversion to the epoxide was observed.

To probe the propensity of NaZnPOM to catalyze disproportionation of hydrogen peroxide, 360 mg (0.77 mmol) of [(n-Oct)3(Me)N]HSO4 was dissolved in a mixture of 15.29 g aqueous NaZnPOM solution (0.1 mmol W/g, 1.53 mmol) and 24.4 mL of H2O2 (30%, medicinal extra pure grade). This mixture was heated to 80 °C. After 5 min, evolution of gas and an increasing temperature were observed in the storage vessel for the aqueous feed stream, indicating the decomposition of H2O2.

Third CCS experiment: A stainless steel CCS was used. In this experiment, hydrogen peroxide was fed to the CCS separate from the rest of the aqueous stream via an extra inlet and was not heated prior to addition. Both other streams were heated to 65 °C and the mantle to 75 °C, as in the previous reaction. Instead of 200 g, 500 g of NaZnPOM solution was used. The phase transfer salt was dissolved in the organic stream instead of the aqueous one. Flow rates: 5 mL/min for both aqueous streams and 10 mL/min for the organic stream. No reaction was observed, neither in the CCS nor in any of the storage vessels.

In the fourth CCS experiment, the conditions were analogous to those in the third experiment, with the difference that twice the amount of [(n-Oct)3(Me)N]HSO4 was used (10 g instead of 5 g) and that it was dissolved in the toluene stream, in which it is more soluble than in water. For the rest, the conditions were analogous to those in the third CCS experiment. No conversion to the epoxide was observed.

Fifth CCS experiment: A stainless steel CCS was used. The high mix bottom plate was applied. Also in this experiment, hydrogen peroxide was fed to the CCS separate from

67



the rest of the aqueous stream via an extra inlet and was not heated prior to addition. Both other streams were heated to 70 °C and the mantle to 80 °C. Flow rates: 3 mL/min for both aqueous streams and 6 mL/min for the organic stream. Otherwise, the reaction was performed analogously to the previous one. When the aqueous supply had depleted, it was recycled, which resulted in 20% conversion to the epoxide in the organic outgoing stream (quantified by GC and GC-MS).

2.8 Notes and references 1 For recent reviews, see: a) G. Sello, T. Fumagalli, and F. Orsini, Curr. Org. Synth. 2006, 3, 457-476; b) Q.-H. Xia, H.-Q. Ge, C.-P. Ye, Z.-M. Liu, and K.-X. Su, Chem. Rev. 2005, 105, 1603-1662; c) E. M. McGarrigle and D. G. Gilheany, Chem. Rev. 2005, 105, 1563-1602; d) E. Rose, B. Andrioletti, S. Zrig, and M. Quelquejeu-Ethève, Chem. Soc. Rev. 2005, 34, 573-583; e) B. S. Lane and K. Burgess, Chem. Rev. 2003, 103, 2457-2473. 2 J.-C. Charpentier, Chem. Eng. Technol. 2005, 28, 255-258. 3 J. F. Jenck, F. Agterberg, and M. J. Droescher, Green Chem. 2004, 6, 544-556. 4 G. Jas and A. Kirschning, Chem. Eur. J. 2003, 9, 5708-5723. 5 V. Hessel, P. Löb, and H. Löwe, Curr. Org. Chem. 2005, 9, 765-787. 6 J. G. De Vries, G. J. Kwant, and H. J. Heeres, WO 2007/031332, 2007, to DSM IP Assets B.V. 7 D. H. Meikrantz, L. L. Macaluso, H. W. Sams III, C. H. Schardin Jr., and A. G. Federici, US5762800, 1998, to Costner Industries Nevada, Inc. 8 T. Ooi and K. Maruoka, Angew. Chem. Int. Ed. 2007, 46, 4222-4266. 9 a) H. E. Fonouni, S. Krishnan, D. G. Kuhn, and G. A. Hamilton, J. Am. Chem. Soc. 1983, 105, 7672-7676; b) S. Krishnan, D. G. Kuhn, and G. A. Hamilton, J. Am. Chem. Soc. 1977, 99, 8121-8123. 10 F. Montanari, M. Penso, S. Quici, and P. Viganò, J. Org. Chem. 1985, 50, 4888-4893. 11 a) J. Brinksma, J. W. De Boer, R. Hage, and B. L. Feringa, Manganese-based Oxidation with Hydrogen Peroxide, in Modern Oxidation Methods, J.-E. Bäckvall, Ed., Wiley-VCH, Weinheim, 2004, pp. 295-326; b) R. Noyori, M. Aoki, and K. Sato, Chem. Commun. 2003, 1977-1986, and references cited therein. 12 C. Venturello, E. Alneri, and M. Ricci, J. Org. Chem. 1983, 48, 3831-3833. 13 a) K. Sato, M. Aoki, M. Ogawa, T. Hashimoto, D. Panyella, and R. Noyori, Bull. Chem. Soc. Jpn. 1997, 70, 905-915; b) K. Sato, M. Aoki, M. Ogawa, T. Hashimoto, and R. Noyori, J. Org. Chem. 1996, 61, 8310-8311. 14 a) J. W. De Boer, J. Brinksma, W. R. Browne, A. Meetsma, P. L. Alsters, R. Hage, and B. L. Feringa, J. Am. Chem. Soc. 2005, 127, 7990-7991; b) J. Brinksma, L. Schmieder, G. Van Vliet, R. Boaron, R. Hage, D. E. De Vos, P. L. Alsters, and B. L. Feringa, Tetrahedron Lett. 2002, 43, 2619-

68


Chapter 2

2622; c) J. Brinksma, R. Hage, J. Kerschner, and B. L. Feringa, Chem. Commun. 2000, 537-538; d) R. Hage, J. E. Iburg, J. Kerschner, J. H. Koek, E. L. M. Lempers, R. J. Martens, U. S. Racherla, S. W. Russell, T. Swarthoff, M. R. P. Van Vliet, J. B. Warnaar, L. Van Der Wolf, and B. Krijnen, Nature 1994, 369, 637-639. 15 B. S. Lane, M. Vogt, V. J. DeRose, and K. Burgess, J. Am. Chem. Soc. 2002, 124, 11946-11954. 16 a) J. W. De Boer, W. R. Browne, J. Brinksma, P. L. Alsters, R. Hage, and B. L. Feringa, Inorg. Chem. 2007, 46, 6353-6372. 17 J. W. De Boer, cis-Dihydroxylation and Epoxidation of Alkenes by Manganese Catalysts, Ph.D. thesis, University of Groningen, 2008. 18 a) First prepared: I. R. Beattie and P. J. Jones, Inorg. Chem. 1979, 18, 2318-2319; b) First used as a catalyst for epoxidation: W. A. Herrmann, R. W. Fischer, and D. W. Marz, Angew. Chem. Int. Ed. Engl. 1991, 30, 1638-1641. 19 a) J. Rudolph, K. L. Reddy, J. P. Chiang, and K. B. Sharpless, J. Am. Chem. Soc. 1997, 119, 6189-6190; b) C. Copéret, H. Adolfsson, and K. B. Sharpless, Chem. Commun. 1997, 1565-1566. 20 G. Dubois, A. Murphy, and T. D. P. Stack, Org. Lett. 2003, 5, 2469-2472. 21 T. A. Van Den Berg, J. W. De Boer, W. R. Browne, G. Roelfes, and B. L. Feringa, Chem. Commun. 2004, 2550-2551. 22 S. V. Kryatov, E. V. Rybak-Akimova, and S. Schindler, Chem. Rev. 2005, 105, 2175-2226, and references therein. 23 a) J. E. Plowman, T. M. Loehr, C. K. Schauer, and O. P. Anderson, Inorg. Chem. 1984, 23, 3553-3559; b) P. C. Healy, B. W. Skelton, and A. H. White, Aust. J. Chem. 1983, 36, 2057-2064. 24 S. Ménage, J. M. Vincent, C. Lambeaux, G. Chottard, A. Grand, and M. Fontecave, Inorg. Chem. 1993, 32, 4766-4773. 25 O. Pàmies and J.-E. Bäckvall, J. Org. Chem. 2002, 67, 9006-9010. 26 Based in part on a personal communication by safety advisors at the Akzo safety lab, Deventer, the Netherlands, in a meeting on 18 February 2005. 27 The calorimetric experiments were conducted by O. Post at the Department of Process Chemistry of NV Organon, Oss, the Netherlands. 28 C. M. Harris and T. N. Lockyer, Chem. Ind. 1958, 1231. 29 a) B. N. Figgis, J. M. Patrick, P. A. Reynolds, B. W. Skelton, A. H. White, and P. C. Healy, Aust. J. Chem. 1983, 36, 2043-2055; b) R. B. Berrett, B. W. Fitzsimmons, and A. A. Owusu, J. Chem. Soc. A Inorg. Phys. Theor. 1968, 1575-1579. 30 A. E. Harvey Jr. and D. L. Manning, J. Am. Chem. Soc. 1952, 74, 4744-4746. 31 A. S. Abushamleh and H. A. Goodwin, Aust. J. Chem. 1982, 35, 1053-1056. 32 B. N. Figgis and J. Lewis, Prog. Inorg. Chem. 1964, 6, 37-239. 33 M. Fujita and L. Que Jr., Adv. Synth. Catal. 2004, 346, 190-194.

69



34 M. C. White, A. G. Doyle, and E. N. Jacobsen, J. Am. Chem. Soc. 2001, 123, 7194-7195. 35 J. Baker, L. M. Engelhardt, B. N. Figgis, and A. H. White, J. Chem. Soc. Dalton Trans. 1975, 530-534. 36 R. Mas-Ballesté and L. Que Jr., J. Am. Chem. Soc. 2007, 129, 15964-15972. 37 The experiments described in this paragraph were performed in collaboration with G. N. Kraai. 38 P. T. Witte, P. L. Alsters, W. Jary, R. Müllner, P. Pöchlauer, D. Sloboda-Rozner, and R. Neumann, Org. Proc. Res. Devel. 2004, 8, 524-531. 39 Thematic issue on POMs: C. L. Hill, Chem. Rev. 1998, 98, 1-2. 40 D.-L. Long, E. Burkholder, and L. Cronin, Chem. Soc. Rev. 2007, 36, 105-121, and references contained therein. 41 The following review systematically covers the fundamental aspects of POM chemistry: M. T. Pope and A. Müller, Angew. Chem. Int. Ed. Engl. 1991, 30, 34-48. 42 C. M. Tourné, G. F. Tourné, and F. Zonnevijlle, J. Chem. Soc. Dalton Trans. 1991, 143-155. 43 R. Neumann and M. Dahan, J. Am. Chem. Soc. 1998, 120, 11969-11976. 44 Hastelloy® is the registered trademark name of Haynes International, Inc. The trademark is applied as the prefix name of a range of over twenty different highly corrosion resistant metal alloys, with nickel as the typical predominant ingredient. 45 A. Bordoloi, F. Lefebvre, S.B. Halligudi, J. Mol. Catal. A: Chem. 2007, 270, 177-184.

70


Chapter 2

Chapter 3 Enantiopure chloroalcohols via enzymatic kinetic resolution

Alkenyl and heteroaryl chloroalcohols have been obtained in excellent enantiomeric

excess (> 99%) by enzymatic kinetic resolution using the haloalcohol dehalogenase

HheC. Yields were close to the theoretical maximum for the majority of substrates.

Furthermore, the applicability of this methodology on multigram scale has been

established. In addition, our efforts to extend the scope of this methodology to other

substrates and transformations are described in this chapter.a

a Part of this chapter has been published: R. M. Haak, C. Tarabiono, D. B. Janssen, A. J. Minnaard, J. G. de Vries, and B. L. Feringa, Org. Biomol. Chem. 2007, 5, 318-323.

72

Chapter 3 - Enzymatic Kinetic Resolution of Chloroalcohols.doc

Chapter 3

3.1 Introduction to haloalcohol dehalogenases

Haloalcohol dehalogenases are enzymes that catalyze the interconversion of haloalcohols and epoxides (Scheme 3.1).1

Scheme 3.1 HheC-catalyzed ring closure of haloalcohols.

Haloalcohol dehalogenases can be divided in three groups, called the A, B, and C type.2 Of these, especially the C type has proven to be a useful biotechnological tool in enantioselective transformations. HheC is a haloalcohol dehalogenase produced by Agrobacterium radiobacter AD1, a soil-dwelling bacterium that is able to use halogen-containing organic compounds as its sole carbon source. The enzyme was discovered some years ago, when its role in the degradation pathway of halogenated xenobiotic compounds such as 1,3-dichloro-2-propanol was disclosed.3

Recently, the structure of HheC has been solved by X-ray crystallography.4 The active form is a homotetramer, or rather a dimer of dimers, of four identical monomers. Each of the 28 kD monomers has its own active site. However, only the tetrameric state is catalytically active since the monomer is in a catalytically inactive conformation.

Scheme 3.2 Mechanism of HheC-catalyzed ring closure of chloroalcohols, adapted from Refs. 1a and 2.

73


Enantiopure chloroalcohols via enzymatic kinetic resolution

The kinetics and catalytic mechanism of HheC have been elucidated.2,4,5 The substrate binds near a catalytic triad (Ser132-Tyr145-Arg149), of which the tyrosine residue activates the hydroxy group of the haloalcohol. Concurrent SN2-type attack on the vicinal carbon atom by the hydroxy group leads to ring closure and expulsion of the halide anion (Scheme 3.2).

Notably, the biocatalytic potential of HheC has been the subject of investigation6 and its substrate scope was found to be remarkably wide.7 HheC catalyzes the reversible ring-closure of various haloalcohols to form epoxides, as well as the irreversible ring-opening of epoxides with a number of non-halide nucleophiles (Scheme 3.3), such as cyanide,7a nitrite,7b and azide.7c

Scheme 3.3 HheC-catalyzed ring opening of epoxides.

3.2 Diene monoepoxides, chloroalcohols, and their synthesis

Enantiomerically pure epoxides8 and their immediate precursors, such as chloroalcohols, are valuable synthetic building blocks.9 Especially functionalized vicinal chloroalcohols like 3.1 - 3.7 (Scheme 3.4) are highly valuable building blocks in synthesis.10 A number of strategies have been used to prepare these compounds in enantiomerically pure form. Chloroalcohols 3.5 − 3.7 have been prepared by asymmetric transfer hydrogenation of the corresponding chloroketones.11 There is a recent, detailed study on the preparation of chiral chloroalcohols by ruthenium-catalyzed reduction of aromatic chloroketones.10 Furthermore, enantiomerically pure 3.6 has been obtained by lipase-catalyzed kinetic resolution of the racemic chloroalcohol,12 and a route to enantiomerically pure 3.5 by Red-Al reduction of the enantiomerically pure alkyne is known.13 Though not reported for compounds 3.1 − 3.7, reduction using alcohol dehydrogenases is a potential method of obtaining these chloroalcohols.14

74


Chapter 3

Scheme 3.4 Preparation of functionalized chloroalcohols 3.1 – 3.7.

Enantiomerically pure 3.1 − 3.4 have not been reported before and a general, convenient, and highly enantioselective method for preparing enantiopure 3.1 − 3.7 is lacking. Kinetic resolution using a haloalcohol dehydrogenase (Scheme 3.5) is an attractive method to obtain these compounds from racemic chloroalcohols.

Scheme 3.5 Enzymatic kinetic resolution of chloroalcohols using HheC.

We chose to concentrate on chloroalcohols functionalized with unsaturated and heteraromatic moieties, since these are especially versatile synthetic scaffolds. The compounds studied in this project were prepared using a method published by Lautens and coworkers15 and are summarized in Scheme 3.4.

The products of ring closure of 3.1 − 3.5 are vinyloxiranes. Like all epoxides, vinyloxiranes (vinylepoxides) are valuable intermediates in synthesis. They are even more versatile than normal epoxides, since in principle three positions are available for nucleophilic attack, depending on the conditions that are chosen.16,17

75



In the preparation of terminal vinylepoxides, one encounters the problem that they are not available by oxidation of terminal dienes. Such oxidation would lead to the epoxidation of the more electron-rich internal double bond.18

Protocols for the preparation of terminal vinyloxiranes are therefore usually based on the ring closure of an alcohol or alcoholate with a suitable vicinal leaving group. For instance, a common method for their preparation is the addition of dimethylsulfonium methylide to an α,β-unsaturated aldehyde, followed by ring closure to the epoxide and expulsion of dimethylsulfide (Scheme 3.6).19

Scheme 3.6 Synthesis of vinyloxiranes from α,β-unsaturated carbonyl compounds.

Unsaturated chloroalcohols can undergo ring closure to yield vinylepoxides.15 We envisioned that, using a haloalcohol dehalogenase (vide supra), this could be done with preference for one of the enantiomers (usually R), leading to kinetic resolution.

Interestingly, methodology similar to the one presented in Scheme 3.4 can be used to directly produce epoxides. Thus, if an aldehyde is subjected to diiodomethane in the presence of methyl lithium, epoxides are obtained in good yields, as reported by Concellón et al. (Scheme 3.7).20 Using this method, cyclohexyl oxirane (3.9) was prepared in 85% yield (lit. 72%) as a substrate for HheC-catalyzed nucleophilic ring opening with azide in a centrifugal contact separator.21

Scheme 3.7 Synthesis of cyclohexyl oxirane.

3.3 Enzymatic nucleophilic ring opening of vinyloxiranes

The initial goal of our investigations was the HheC-catalyzed azidolysis of vinyloxiranes, depicted in Scheme 3.8. Using bulky nucleophiles and under basic conditions, vinyloxiranes are often attacked at the terminal position.22 However, attack at the 2- or 4-position is frequently observed, especially under conditions that favor formation of the allylic cation.16 By using halohydrin dehalogenase HheC (from

76


Chapter 3

Agrobacterium radiobacter AD1) as catalyst, regioselective azidolysis on the 1-position was expected to be possible. It was anticipated that this would be an enantioselective reaction as well, leading to kinetic resolution. Since 1,2-epoxy-3-octene (3.10) is not commercially available, it was synthesized by reaction of hept-2-enal and trimethylsulfonium methylsulfate23 or trimethylsulfonium iodide.19

Scheme 3.8 Reaction pathways in the HheC-catalyzed azidolysis of 1,2-epoxy-3-octene.

In preliminary experiments the enzyme showed some activity, but the blank azidolysis was faster than the enzyme-catalyzed reaction (Table 3.1). It was considered that, by keeping the azide concentration low, it would be possible to improve this ratio, but this turned out not to be the case. Given the disappointing results in terms of reactivity, the enantioselectivity was not further investigated.

Table 3.1 HheC-catalyzed azidolysis of 1,2-epoxy-3-octene (3.10).a

Entry Addition

time azide

Init. rate enzymatic azidolysis

(mmol•min−1)b

Init. rate uncatal. azidolysis

(mmol•min−1)

Final ratio enzymatic / uncatalyzed

1 at once 1.6 (0.41) 5.9 16/84 2 45 minc 1.0 (0.25) 4.0 15/85 3 100 minc 0.5 (0.13) 1.6 15/85

a) Conditions: 200 μL of a solution of 3.10 (0.5 M in DMSO) and 500 μl of an aqueous solution of purified HheC (8 mg•ml−1) were dissolved in 20 ml of Tris-SO4 buffer (200 mM, pH 8.5), after which sodium azide (1 equiv. as a 750 mM aqueous solution) was added. Samples (1.0 mL) were periodically taken from the mixture, extracted with Et2O, and the resulting samples analyzed by GC; b) In parentheses the initial enzyme activity (in U•mg−1) is given; c) Using a syringe pump.

We investigated if it was possible to start from 1-chloro-3-octene-2-ol (3.3) as illustrated in Scheme 3.9. The hydrolysis and blank azidolysis are first order in 3.10.

77



The enzyme-catalyzed reaction is governed by Michaelis-Menten kinetics (vide infra), so that the rate of reaction is independent of the concentration of 3.10 if the concentration is sufficiently higher than the KM (see also Equation 3.1). Hence, provided that the KM is sufficiently low, it could be possible to suppress hydrolysis and uncatalyzed azidolysis of 3.10 while the HheC-catalyzed azidolysis still proceeds at maximum rate.

Scheme 3.9 Enzyme-catalyzed tandem reaction from 3.3 to 3.13.

It turned out that spontaneous hydrolysis of the epoxide in this case is faster than ring-opening by azide. The observed azidolysis is even more in favour of the uncatalyzed reaction than when the epoxide is directly employed as substrate (enzymatic / uncatalyzed = 96 : 4). However, importantly, in a sample taken after 20 min, only (S)-3.3 could be observed, whereas (R)-3.3 had completely reacted away. Based on this observation, we turned our attention to the enzymatic kinetic resolution of this compound and similar chloroalcohols.

3.4 Enzymatic ring closure of chloroalcohols to epoxides

3.4.1 Kinetic resolution of 3.1 − 3.7 on analytical scale We studied the synthesized chloroalcohols 3.1 − 3.7 as substrates for HheC, initially on analytical scale. The results of this screening are summarized in Table 3.2. Enzymatic activity towards each of the substrates is expressed both as initial enzyme activity (μmol•min-1•mg-1 of enzyme) and as turnover frequency (s-1). In the last column, the selectivity factor E is given for each of the substrates.

A clear trend in reactivity can be observed with the linear substrates: the shorter the chain, the faster the enzymatic conversion. This is expected on the basis of previous observations.7 We attribute the observed reactivity pattern to increasing difficulty of the substrate to fit into the active site of the enzyme in a reactive conformation as it gets bulkier. Based on such steric arguments, the reactivity pattern of substrates 3.1 − 3.5 can

78


Chapter 3

be rationalized. Variations in substrate binding between 3.1 − 3.5, possibly influenced by steric factors or mutual differences in binding affinities, may also partly explain the marked difference in reactivity between the otherwise comparable substrates 3.6 and 3.7.

Table 3.2 HheC-catalyzed kinetic resolutions of substrates 3.1 − 3.7 on analytical scale.a

Entry Substrate R = init. enz. activ.b

(μmol·min−1·mg−1) TOF(s−1)c Ed

1 3.1 48 22.4 >200 2 3.2 29 13.5 177 3 3.3 8 3.7 >200 4 3.4 12 5.6 102

5 3.5

10 4.7 >200

6 3.6

47 21.9 >200

7 3.7

11 5.1 65

a) General conditions: 0.2 mmol scale, 10 mM in Tris-sulfate pH 8.1; b) Initial enzyme activity (μmol of product per min per mg of HheC); c) Per enzyme subunit; d) Obtained by fitting measured data points (concentration v. time) against the mathemetical curves for competitive Michaelis-Menten kinetics using MicroMath® Scientist®.

The enantioselectivity of these transformations is high in all cases, and even the lowest E observed (65, Table 3.2, entry 7) is excellent for a kinetic resolution.

Equation 3.1 Competitive Michaelis-Menten kinetics.

a) Rk

KKSR

RVdtdR

cRmS

m

R

⋅−

⋅⎟⎟⎠

⎞⎜⎜⎝

⎛++

⋅−=

1

max

b) Sk

KKRS

SVdtdS

cSmR

m

S

⋅−

⋅⎟⎟⎠

⎞⎜⎜⎝

⎛++

⋅−=

1

max

79



a) b)

0 50 100 150 2000

1

2

3

4

5

0 50 100 150 2000

1

2

3

4

5

0 50 100 150 2000 50 100 150 2000

1

2

3

4

5

0

1

2

3

4

51-chloro-pent-3-en-2-ol

conc

.(m

M)

t (min)

0 50 100 150 200 2500

1

2

3

4

5

6

0 50 100 150 200 2500

1

2

3

4

5

61-chloro-hex-3-en-2-ol

0 50 100 150 200 2500 50 100 150 200 250

t (min)

conc

.(m

M)

0

1

2

3

4

5

6

0

1

2

3

4

5

6

c) d)

0 50 100 150 200 250 3000

1

2

3

42-chloro-1-furan-2-yl-ethanol

0 50 100 150 200 250 3000

1

2

3

4

0 50 100 150 200 250 3000 50 100 150 200 250 3000

1

2

3

4

0

1

2

3

4

t (min)

conc

.(m

M)

0 50 100 150 200 250 300 3500

1

2

3

4

5

0 50 100 150 200 250 300 3500

1

2

3

4

5

0 50 100 150 200 250 300 3500 50 100 150 200 250 300 3500

1

2

3

4

5

0

1

2

3

4

52-chloro-1-thiophen-2-yl-ethanol

conc

.(m

M)

t (min) Figure 3.1 Progress curves for enzymatic conversion of 3.1 (a), 3.2 (b), 3.6 (c), and 3.7 (d).

80


Chapter 3

Equation 3.2 Calculating E from kinetic parameters.

Sm

S

Rm

R

KV

KV

Emax

max

=

In Equation 3.1a and b, R and S represent the concentrations of both enantiomers, VmaxR, VmaxS, KMR and KMS are the relevant Michaelis-Menten parameters, and kc is the first-order rate constant of chemical hydrolysis. After fitting these equations by numerical integration to the obtained data points,24 the E-value was calculated from Equation 3.2.25

Other methods for calculating the E-value of an enzymatic conversion are given in Equation 3.3. They are not extensively used in this chapter, but are relevant for the studies described in other chapters, as well as for the discussion in general. In Equation 3.3a25, the conversion and the ee of the product are used to calculate E, whereas Equation 3.3b26 is independent of conversion and is based on the ee's of both substrate and product. Since these methods are time-independent, they do not take into account possible chemical background reactions.

Equation 3.3 Calculation of E from conversion and ee (a) or ee of both substrate and product (b).

a) ( )( )[ ]( )( )[ ]R

R

eeceec

E+−−−

=11ln11ln

b) ( ) ( )[ ]( ) ( )[ ]PSS

PSS

eeeeeeeeeeee

E+++−

=11ln11ln

As is shown in Figure 3.1, the reaction rate of the slow-reacting S-enantiomer can be appreciable. This is the case especially for the linear substrates 3.1 − 3.4. Naturally, this behavior should be taken into account when these reactions are to be performed on preparative scale (vide infra).

81



3.4.2 Kinetic resolution on preparative scale Having established the remarkable efficiency of this enzymatic kinetic resolution, we set out to transform substrates 3.1 and 3.3 − 3.7 on preparative scale. In view of the excellent selectivities, our initial aim was to isolate both the remaining enantiomerically pure chloroalcohol and the produced epoxide.

Preparative scale reactions were performed on 2.0 mmol scale at a concentration of 10 mM in Tris-sulfate buffer, i.e., analogous to the analytical reactions. In this manner it proved possible to isolate enantiomerically pure chloroalcohols (ee >99%) in fair to high yields (Table 3.3, entries 1 − 4). Our attempts to isolate the produced epoxides as well failed, since it turned out that they rapidly hydrolyse in situ to form the corresponding diols (Scheme 3.10).

Scheme 3.10 Hydrolysis of the product epoxides in HheC-catalyzed ring closure of halohydrins.

Attempts were made to suppress this hydrolysis by performing the reaction in a two-phase system (Table 3.3, entries 5 and 6), but even in those cases hydrolysis proved inevitable. The role of this spontaneous hydrolysis will be examined in more detail further on.

Accordingly, our efforts focused on obtaining the enantiomerically pure halohydrins, which were isolated with excellent enantioselectivities (Table 3.3). In general, the yields were good, allowing for the isolation of 3.1 and 3.5 − 3.7 in 40 − 47% yield. Using substrates 3.3 and 3.4, yields were around 30%. The cause of these lower yields is not clear.

The absolute configuration of the remaining chloroalcohols was S in all cases, in agreement with previous studies showing that HheC is R-selective for the majority of substrates.6,7 A variety of methods were used to elucidate the absolute configuration of the slow-reacting enantiomers of the chloroalcohols. The absolute configuration of (S)-(E)-1-chloro-4-phenyl-but-3-en-2-ol ((S)-3.5) could be deduced from a crystal structure using the Bijvoet method with chloride as heavy atom (CCDC 605888).27 The optical rotations of (S)-3.6 and (S)-3.7 were identical to literature values for these compounds.11,12 Finally, (S)-3.1, (S)-3.3, and (S)-3.4 were hydrogenated to 3.14 and 3.15 using Wilkinson’s catalyst and dihydrogen (Scheme 3.11). For these saturated

82


Chapter 3

analogues, specific rotations are known, allowing the absolute configuration to be established by structural correlation.28

Table 3.3 Enzymatic kinetic resolutions of unsaturated and heteroaromatic vicinal chloroalcohols on preparative scale.

Entry Substrate R = Chloroalcohol (ya, ee, conf.)

Diol (ya)

Ed

1 3.1b 40%, >99%, S n.i.c >200 3 3.3d 31%, >99%, S n.i.c 177 4 3.4e 29%, >99%, S 24%f >200

5 3.5d

47%, >99%, S 19%f 102

6 3.6g

42%, 98.5%, S n.i.c >200

7 3.7h

47%i, >99%, S 49%i >200

a) Isolated yield (based on 50% maximum); b) 1.0 mmol scale, 10 mM in Tris-sulfate buffer (pH 8.1); c) Not isolated; d) 2.0 mmol scale, 10 mM in Tris-sulfate buffer (pH 8.1); e) 1.5 mmol scale, 10 mM in Tris-sulfate buffer (pH 8.1); f) Non-optimized yield of a mixture of diols; g) 16 mmol scale, 1 : 1 toluene / Tris-sulfate (pH 8.1); h) 120 mmol scale, 1 : 10 toluene / Tris-sulfate (pH 8.1); i) crude yield.

Scheme 3.11 Hydrogenation of (S)-3.1, (S)-3.3, and (S)-3.4. to (S)-3.14 and (S)-3.15.

We were especially interested in the possibilities of performing this resolution as a preparative procedure. It was indeed possible to perform this kinetic resolution on multigram scale (Table 3.3, entries 5 and 6). For instance, starting from 20.9 g of racemic 2-chloro-1-thiophen-2-yl-ethanol (3.7), 9.8 g (94% of the theoretical yield) of

83



(S)-3.7 was obtained with an excellent ee of >99%. No modifications to the enzyme (e.g. immobilization) were needed to achieve these results and the resulting compounds are stable for several months at 4 °C. To avoid the use of excessive amounts of buffer solution, the latter two reactions were performed in a two-phase system of toluene and Tris-buffer (pH 8.1).

3.5 Hydrolysis of epoxides during enzymatic kinetic resolution

It was observed that the epoxides that were produced during HheC-catalyzed kinetic resolution, almost immediately hydrolyzed spontaneously to the corresponding diols (vide supra). This was initially perceived as a drawback of the system, since it interfered with our intentions of isolating both the remaining chloroalcohols and the epoxides. However, comparison of our results with those previously reported for the related compound 2-chloro-1-(4-nitro-phenyl)-ethanol (3.16) disclosed a more positive role of hydrolysis in the kinetic resolution of chloroalcohols.

[ ] [ ][ ]holchloroalco

HClepoxideKeq⋅

=

Scheme 3.12 Incomplete conversion of (R)-2-chloro-1-(4-nitro-phenyl)-ethanol ((R)-3.16).

As illustrated in Scheme 3.12, when a kinetic resolution is performed on rac-3.16, the R-selective enzyme HheC converts the R-enantiomer at a much faster rate than the S-enantiomer. However, the Keq of the equilibrium between (R)-3.16 and (R)-3.17 is 40 mM,7c in other words when equilibrium is reached, the ratio between chloroalcohol and epoxide will be about 2.5 / 97.5, indicating that a small percentage of (R)-3.16 will always remain present if the epoxide is in equilibrium with the chloroalcohol. In this case, the maximum ee of the remaining chloroalcohol will be about 95% (in practice: 92%).6e,7c

The results obtained in this study indicate that the spontaneous hydrolysis of the formed epoxide results in a shift of the chloroalcohol-epoxide equilibrium to the

84


Chapter 3

product side by removing the epoxide from the reaction, thus ensuring excellent ee’s of the remaining chloroalcohols.

3.5.1 The role of hydrolysis in HheC-catalyzed enzymatic kinetic resolution Contrary to our expectations, in the case of substrate 3.7 (Table 3.3, entry 6) hydrolysis led to the formation of racemic diol (Scheme 3.13), probably resulting from approximately equal rates for nucleophilic attack on the terminal and internal carbon atom of the (enantiomerically pure) epoxide.29

Scheme 3.13 Formation of racemic diol 3.19 from enantiopure epoxide 3.18.

Under the mildly basic reaction conditions of the enzymatic kinetic resolution, it is unlikely that racemization results through the intermediacy of a (hetero)benzylic carbocation. A better explanation for the formation of racemic diol is a 1 : 1 ratio of attack on the sterically favored terminal carbon atom of the epoxide and the electronically favored benzylic carbon atom.29 Evidence for this hypothesis could be provided by the following experiments: 18O labeling experiments (Scheme 3.14) and pH dependence on product distribution (Scheme 3.15).

In MS (EI+) measurements, the [M − CH2OH]+ peak has a high abundance. The mass of this fragment will be 113 for diol 3.19a, resulting from terminal attack of water, and 115 for diol 3.19b, resulting from attack of labeled water on the benzylic position. By measuring the ratio of these fragments, one can determine the regioselectivity of the epoxide ring opening.

Thiophen-2-yl-oxirane (3.18) was synthesized by reaction of thiophene carboxaldehyde and trimethylsulfonium iodide in the presence of potassium hydroxide30 and was isolated in 60% yield after Kugelrohr distillation. The hydrolysis experiment was performed by adding 5 μL of epoxide to 30 μL of H218O. These conditions do not completely reflect the experimental conditions of enzymatic kinetic resolution, since in this case the pH is around 7, whereas the enzymatic kinetic resolution is conducted at pH 8. Diols 3.19b and 3.19a were obtained in a 4 : 1 ratio. This proves that attack taking place at the terminal position is a significant pathway, even under neutral conditions.

85



Scheme 3.14 Ring opening of thiophenyl oxirane by H218O.

Experiments on the pH-dependence of the hydrolysis of (R)-3.18 are in agreement with the findings in the previous paragraph. Enantiomerically pure (R)-3.18 was obtained by K2CO3-catalyzed ring closure of (S)-3.7 in acetone.b Subsequently, hydrolysis experiments were conducted at different pH values (Scheme 3.15). At pH 8.1, nearly racemic 3.19 was obtained, whereas at pH 10.5 an excess of (R)-3.19, the product with retention of configuration, is obtained. This indicates that the proportion of diol resulting from attack on the terminal carbon atom of the epoxide, increases with increasing pH.31

Scheme 3.15 pH-dependence of the hydrolysis of (R)-3.18.

To summarize, nucleophilic attack at the terminal carbon atom of 3.18 is a significant pathway, even at pH values lower than during enzymatic kinetic resolution. Furthermore, the ratio of terminal and internal attack increases with increasing pH. In conclusion, competition between attack on the terminal and internal carbon atom on

b Various bases (NaH, NaOH, K2CO3), solvents (DMF, THF, Et2O, IPA, H2O) and conditions (presence or absence of additives like NaI or silver salts) were examined. Reproducible results were obtained using K2CO3 in acetone.

86


Chapter 3

the epoxide is the most likely explanation for the formation of racemic 3.19. It is possible that other mechanisms are involved in the hydrolysis of the other substrates.29

3.5.2 Trapping of epoxide to prevent hydrolysis Spontaneous hydrolysis of the epoxide product limits the applicability of this − in principal − highly efficient kinetic resolution. To prevent hydrolysis of the formed epoxide, it might be feasible to convert it in situ to a product which is more stable towards hydrolysis, using a strong nucleophile (Scheme 3.16). Besides finding a nucleophile that is reactive enough to accomplish this under the given reaction conditions, two selectivity issues have to be addressed: the nucleophile should react selectively with the epoxide as opposed to the chloroalcohol (chemoselectivity) and it should favor attack on the terminal epoxide carbon as opposed to the internal carbon atom (regioselectivity).

Scheme 3.16 Trapping of epoxide to prevent hydrolysis.

If a nucleophile such as azide, nitrite or cyanide is used, the ring opening reaction will also be enzyme-catalyzed.32 An advantage of this approach is the regioselectivity of HheC-catalyzed azidolysis of epoxides. Exclusively the terminal adduct will be produced (Scheme 3.17). However, for chloroalcohol 3.20 it was experimentally verified that the enzymatic ring opening of the epoxide with azide is two orders of magnitude slower than the enzymatic ring closure of the chloroalcohol under the same conditions.

The specific enzyme activity is 14.1 μmol·min−1·mg−1 for the ring closure of the chloroalcohol (3.20 → 3.21) and 0.19 μmol·min−1·mg−1 for the ring opening with azide (3.21 → 3.24), at their respective pH optima. Therefore, the epoxide accumulates to such an extent that spontaneous hydrolysis becomes inevitable.33

In a different approach, thiophenolate was used as the nucleophile (Scheme 3.18). Under the reaction conditions for enzymatic kinetic resolution (pH = 8), thiophenol will be deprotonated (pKa = 6.6) and hence reactive enough to attack the epoxide. Moreover, it is known from the literature that nucleophilic attack of thiophenols on epoxides can be highly regioselective.34

87



Scheme 3.17 Trapping of epoxide 3.21 to prevent hydrolysis.

Scheme 3.18 Attempted trapping of epoxide by thiophenol.

A difficulty we encountered when testing this approach, was the undesirable oxidative dimerization shown in Scheme 3.19. It was tried to suppress this side-reaction by working under oxygen-free conditions, but dimer 3.26 remained the main product.

Scheme 3.19 Formation of 3.26 during attempted thiophenol addition to 3.21.

A more thorough screening of sulfur nucleophiles might lead to the desired outcome.

3.6 Limitations of this methodology

To extend the scope of the kinetic resolution procedure developed so far, a number of other substrates were tested. Furthermore, a variation of this approach was tested, employing formate esters instead of haloalcohols.

88


Chapter 3

After the successful results obtained in the kinetic resolution of unsaturated and aromatic chloralcohols (vide supra), we became interested in the possibility of using this methodology for the preparation of the ferrocenyl-derived chloroalcohol 3.28. Such a chiral chloroalcohol could serve as, for instance, a useful starting material in the preparation of enantiomerically pure ferrocenyl-based ligands for asymmetric catalysis.35 It would also be the first organometallic compound to act as a substrate in this transformation.

Racemic 3.28 was prepared by Friedel-Crafts acylation of ferrocene with chloroacetyl chloride yielding 3.27 in 20% yield (not optimized).36 Reduction of the resulting chloroketone 3.27 using sodium borohydride37 led to chloroalcohol 3.28 in quantitative yield (Scheme 3.20).

Scheme 3.20 Synthesis and enzymatic kinetic resolution of 3.28.

In the kinetic resolution, the conversion of compound 3.28 turned out to be very slow. A maximum ee of 14% in the starting material was reached. Probable causes for this include the steric bulk of the compound and its very low solubility in the aqueous reaction medium.

Another substrate that did not show efficient kinetic resolution using HheC, was 1-chloro-4-trimethylsilanyl-but-3-yn-2-ol (3.30) depicted in Scheme 3.21.

Compound 3.30 was prepared in the usual way from the corresponding aldehyde 3.29, which in turn was obtained by formylation of trimethylsilyl acetylene using DMF (Scheme 3.5).38 No reactivity was observed when 3.30 was subjected to the conditions of kinetic resolution that had been effective for substrates 3.1 − 3.7. As in the case of ferrocenyl-substituted chloroalcohol 3.28, the lack of reactivity was attributed to steric bulk, in this case due to the presence of the trimethylsilyl moiety. Attempts to remove the TMS-moiety using TBAF were unsuccessful.

89



Scheme 3.21 Synthesis and attempted enzymatic kinetic resolution of 3.30.

Another approach that was studied to extend the scope of the developed methodology, included the use of leaving groups other than halides. This idea was prompted by the observation that in the reverse reaction, the ring opening of epoxides, other nucleophiles than halides can be used effectively.

We chose to investigate the ring closure of 2-hydroxyalkyl formates, since formate had been observed to act as a nucleophile, albeit not a very active one,39 in the reverse reaction. The formates used in this reaction have the advantage that they are easily available from the corresponding diols (Scheme 3.22).40,41

Scheme 3.22 Attempted enzymatic ring closure of 2-hydroxyalkyl formates.

However, when tested under the conditions that were successfully applied in the enzymatic kinetic resolution of chloroalcohols, no enzyme activity was observed for the ring closure of formates 3.31 and 3.32 (Scheme 3.22).

3.7 Conclusions

In conclusion, a highly efficient kinetic resolution protocol was developed for functionalized vicinal chloroalcohols. The majority of these compounds have not been reported before in their enantiomerically pure form. Various unsaturated and heteroaromatic chlorohydrins were resolved in high yields and with excellent enantioselectivities. This resolution was shown to be effective on multigram scale, making it highly practical as a preparative method.

Limitations of this methodology include the poor reactivity of bulky substrates and the need to perform this reaction in aqueous solution. The hydrolysis of the epoxide

90


Chapter 3

product that is observed in this reaction, might be instrumental in obtaining high ee's of the chloroalcohols, but limits the applicability of this method.


3.8.1 General remarks Starting materials were purchased from Aldrich or Acros and used as received unless stated otherwise. All solvents were reagent grade and, if necessary, dried and distilled prior to use. Demineralized water was used in the preparation of all aqueous solutions.

Column chromatography was performed on silica gel (Aldrich 60, 230-400 mesh). TLC was performed on silica gel 60/Kieselguhr F254 or neutral aluminum oxide 60 F254 where indicated. 1H and 13C NMR spectra were recorded on a Varian VXR300 (299.97 MHz for 1H, 75.48 MHz for 13C) or a Varian AMX400 (399.93 MHz for 1H, 100.59 MHz for 13C) spectrometer in CDCl3 unless stated otherwise. Chemical shifts are reported in δ values (ppm) relative to the residual solvent peak (CHCl3, 1H = 7.24, 13C = 77.0). Carbon assignments are based on APT 13C experiments. Splitting patterns are indicated as follows: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad).

Mass spectra (HRMS) were obtained on a Jeol JMS-600H. GCMS spectra were recorded on a Hewlett Packard HP6890 equipped with a HP1 column and an HP 5973 Mass Selective Detector.

GC analysis was performed on a Shimadzu GC-17A or a Hewlett Packard HP6890 spectrometer equipped with the columns indicated for each compound separately.

HPLC analysis was performed on a Shimadzu HPLC system equipped with two LC-10AD vp solvent delivery systems, a DGU-14A degasser, a SIL-10AD vp auto injector, an SPD-M10A vp diode array detector, a CTO-10A vp column oven, and an SCL-10A vp system controller using the columns indicated for each compound separately.

Optical rotations were measured on a Schmidt and Haensch Polartronic MH8 using a 10 cm cell.

3.8.2 Synthesis of substrates 3.1 - 3.7 Substrates 3.1 − 3.7 were synthesized according to a literature procedure.15 Yields, spectroscopic data and chromatographic separation conditions will follow for each of the substrates.

91



1-Chloro-pent-3-en-2-ol (3.1).42

Obtained as a colorless oil (2.21 g; 18.3 mmol; 73%) after flash chromatography (n-pentane − Et2O 4 : 1); 1H NMR (CDCl3) δ 5.76-5.80 (m, 1H), 5.48 (dd, J = 15.4, 6.6 Hz, 1H), 4.26 (m, 1H), 3.58 (ddABX, J = 11.0, 3.7 Hz, ΔνAB = 45.0 Hz, 1H), 3.47 (ddABX, J = 11.0, 7.3 Hz, ΔνAB = 45.0 Hz, 1H), 2.17 (d, J = 4.0 Hz, 1H), 1.71 (d, J = 6.6 Hz, 3H); 13C NMR (CDCl3) d 129.7 (d), 129.2 (d), 72.3 (d), 49.6 (t), 17.7 (q); MS (EI+) m/z = 122 (M+), 120 (M+), 107, 105, 71, 53, 41; chiral GC: Chiraldex B-PM, 30m × 0.25 mm × 0.25 μm, He-flow: 1.1 mL/min, 80 °C isothermic, Tr = 10.2 min (S), Tr = 10.9 min (R).

1-Chloro-hex-3-en-2-ol (3.2).

Obtained as a colorless oil (1.77 g; 13.2 mmol; 66%) after flash chromatography (n-pentane − Et2O 6 : 1, gradient to 4 : 1); 1H NMR (CDCl3) δ 5.84 (dtd, J = 15.8, 6.2, 1.1 Hz, 1H), 5.44 (ddt, J = 15.4, 6.6, 1.5 Hz, 1H), 4.28 (br, 1H), 3.59 (ddABX, J = 11.0, 3.7 Hz, ΔνAB = 46.1 Hz, 1H), 3.47 (ddABX, J = 11.0, 7.3 Hz, ΔνAB = 46.1 Hz, 1H), 2.22 (d, J = 3.7 Hz, 1H), 2.06 (qdd, J = 7.3, 6.6, 1.5 Hz, 2H), 0.98 (t, J = 7.3 Hz, 3H); 13C NMR (CDCl3) δ 136.4 (d), 127.0 (d), 72.3 (d), 49.7 (t), 25.2 (t), 13.1 (q); MS (EI+) m/z = 134 (M+), 105, 85, 67, 55; HRMS (EI+) calc.: 134.0498, measured: 134.0491; chiral GC: Chiraldex B-TA, 30m × 0.25 mm × 0.25 μm, He-flow: 1.0 mL/min, 85 °C isothermic, Tr = 16.7 min (S), Tr = 18.0 min (R).

1-Chloro-oct-3-en-2-ol (3.3).15,43

Obtained as a colorless oil (903 mg; 5.55 mmol; 56%) after flash chromatography (n-pentane − Et2O 6 : 1, Rf = 0.32); 1H NMR (CDCl3) δ 5.79 (dtd, J = 15.4, 7.0, 1.1 Hz, 1H), 5.44 (ddt, J = 15.4, 6.6, 1.5 Hz, 1H), 4.2 − 4.35 (m, 1H), 3.58 (ddABX, J = 11.0, 3.7 Hz, ΔνAB = 45.0 Hz, 1H), 3.47 (dd, J = 11.0, 7.7 Hz, ΔνAB = 45.0 Hz, 1H), 2.19 (d, J = 4.2 Hz, 1H), 1.99 − 2.09 (br, 2H), 1.2 − 1.4 (br, 4H), 0.87 (t, J = 7.3 Hz, 3H); 13C NMR (CDCl3) δ 135.2 (d), 127.9 (d), 72.4 (d), 49.9 (t), 31.9 (t), 31.0 (t), 22.1 (t), 13.9 (q); MS (EI+) m/z = 162 (M+), 113, 95, 57; chiral GC: CP Chiralsil Dex CB, 25m × 0.25 mm × 0.25 μm, He-flow: 1.0 mL/min, 120 °C isothermic, Tr = 12.6 min (S), Tr = 13.0 min (R).

92


Chapter 3

1-Chloro-octa-3,5-dien-2-ol (3.4).

Obtained as a colorless oil (1.29 g; 8.0 mmol; 79%) after flash chromatography (n-pentane − Et2O 7 : 1, Rf = 0.26); 1H NMR (CDCl3) δ 6.30 (dd, J = 15.0, 10.3 Hz, 1H), 6.02 (dd, J = 15.0, 10.3 Hz, 1H), 5.79 (dt, J = 15.0, 6.6 Hz, 1H), 5.54 (dd, J = 15.4, 5.9 Hz, 1H), 4.35 (br, 1H), 3.60 (ddABX, J = 11.0, 3.7 Hz, ΔνAB = 47.5 Hz, 1H), 3.48 (ddABX, J = 11.0, 7.3 Hz, ΔνAB = 47.5 Hz, 1H), 2.23 (d, J = 3.7 Hz, 1H), 2.09 (dt, J = 13.9, 7.3 Hz, 2H), 0.99 (t, J = 7.3 Hz, 3H); 13C NMR (CDCl3) δ 138.4 (d), 133.4 (d), 128.1 (d), 128.0 (d), 72.1 (d), 49.6 (t), 25.6 (t), 13.2 (q); MS (EI+) m/z = 162 (M+), 160 (M+), 111, 93, 55; HRMS (EI+) calc. 160.0655, measured: 160.0662; chiral GC: CP Chiralsil Dex CB, 25m × 0.25 mm × 0.25 μm, He-flow: 1.0 mL/min, 125 °C isothermic, Tr = 14.8 min (S), Tr = 15.6 min (R).

(E)-1-Chloro-4-phenyl-but-3-en-2-ol (3.5).11,13,15,43,44

Obtained after flash chromatography (n-pentane − Et2O 5 : 1, Rf = 0.24) as a colorless oil (2.34 g; 12.8 mmol; 64%), which crystallized upon standing; 1H NMR (CDCl3) δ 7.20 − 7.40 (m, 5H), 6.71 (dd, J = 16.1, 1.1 Hz, 1H), 6.19 (dd, J = 16.1, 6.0 Hz, 1H), 4.52 (br m, 1H), 3.71 (ddABX, J = 11.0, 3.7 Hz, ΔνAB = 48.9 Hz, 1H), 3.58 (ddABX, J = 11.0, 7.3 Hz, ΔνAB = 48.9 Hz, 1H), 2.38 (d, J = 4.4 Hz, 1H); 13C NMR (CDCl3) δ 136.0 (s), 132.7 (d), 128.6 (d), 128.1 (d), 127.2 (d), 126.6 (d), 72.3 (d), 49.6 (t); MS (EI+) m/z = 184 (M+), 182 (M+), 133, 115, 105; HRMS (EI+) calc. 182.0498, measured: 182.0507; chiral HPLC: Chiralcel OD, 40 °C, n-heptane / IPA 92 : 8, 1.0 mL/min, Tr = 11.5 min (S), Tr = 16.0 min (R).

2-Chloro-1-fur-2-yl-ethanol (3.6).11,12

Obtained as a light yellow oil (1.87 g; 12.7 mmol; 64%) after flash chromatography (n-pentane − Et2O 4 : 1, gradient to 3 : 1, Rf,3:1 = 0.40); for the resolution on 2.3 g scale, different preparations were combined; 1H NMR (CDCl3) δ 7.39 (s, 1H), 6.36 (s, 2H), 4.93 (m, 1H), 3.83 (m, 2H), 2.53 (d, J = 5.5 Hz, 1H); 13C NMR (CDCl3) δ 152.6 (s), 142.6 (d), 110.4 (d), 107.6 (d), 68.0 (d), 47.7 (t); MS (EI+) m/z = 148 (M+), 146 (M+), 97; chiral GC: Chiraldex G-TA, 30 m × 0.25 mm × 0.25 μm, He-flow: 0.5 mL/min, 120 °C isothermic, Tr = 5.1 min (R), Tr = 5.4 min (S).

93



2-Chloro-1-thiophen-2-yl-ethanol (3.7).12,45

Obtained as a colorless oil (2.86 g; 17.6 mmol; 88%) after flash chromatography (n-pentane − Et2O 4 : 1, Rf = 0.33); for the 20 g scale resolution, this compound was prepared analogously (64%); 1H NMR (CDCl3) δ 7.29 (dd, J = 5.1, 1.1 Hz, 1H), 7.03 (ddd, J = 3.7, 1.1, 0.7 Hz, 1H), 6.99 (dd, J = 5.1, 3.7 Hz, 1H), 5.15 (ddd, J = 8.1, 4.0, 0.7 Hz, 1H), 3.80 (ddABX, J = 11.4, 4.0 Hz, ΔνAB = 27.4 Hz, 1H), 3.72 (ddABX, J = 11.4, 8.1, ΔνAB = 27.4 Hz, 1H), 2.81 (br, 1H); 13C NMR (CDCl3) δ 143.2 (s), 126.9 (d), 125.4 (d), 124.7 (d), 70.2 (d), 50.4 (t); MS (EI+) m/z = 164 (M+), 162 (M+), 113; HRMS (EI+, for C6H37ClOS) calc. 163.9877, measured: 163.9881; chiral GC: Chiraldex B-PM, 30 m × 0.25 mm × 0.25 μm, He-flow: 1.1 mL/min, 135 °C isothermic, Tr = 14.2 min (S), Tr = 14.8 min (R).

2-Cyclohexyloxirane (3.9) 7a,20

Prepared from cyclohexanecarbaldehyde (3.8) as described in Ref. 20. Spectroscopic data were in accordance with the literature.7a

1,2-Epoxy-3-octene (3.10) 15,19

Prepared from n-hept-2-enal as described in Ref. 19. Spectroscopic data were in accordance with the literature.15

Thiophenyloxirane (3.18)

Prepared as described in Ref. 20. (R)-3.18 was prepared by ring closure of (S)-3.7 with K2CO3 in acetone overnight at 0 °C → rt. 1H NMR (CDCl3) δ 7.23 (d, J = 5.1 Hz, 1H), 7.11 (d, J = 3.7 Hz, 1H), 6.96 (dd, J = 5.1, 3.7 Hz, 1H), 4.09 (dd, J = 4.4, 2.6 Hz, 1H), 3.20 (ddABX, J = 5.1, 4.4 Hz, ΔνAB = 58.9 Hz, 1H), 2.98 (ddABX, J = 5.1, 2.6 Hz, ΔνAB = 58.9 Hz, 1H); 13C NMR (CDCl3) δ 141.3 (s), 127.0 (d), 126.3 (d), 125.2 (d), 51.6 (t), 49.3 (d); chiral HPLC: Chiralcel AS, 40 °C, n-heptane / IPA 96 : 4, 1.0 mL/min, Tr = 5.7 min (S), Tr = 6.9 min (R).

94


Chapter 3

1-Thiophen-2-yl-ethane-1,2-diol (3.19).46

1H NMR (CDCl3) δ 7.26-7.24 (m, 1H), 7.00 − 6.96 (m, 2H), 5.03 (dd, J = 7.3, 3.7 Hz, 1H), 3.83 − 3.72 (m, 2H), 3.05 (br, 1H), 2.50 (br, 1H); chiral HPLC: Chiralcel OD, 40 °C, n-heptane / IPA 95 : 5, 1.0 mL/min, Tr = 20.4 min (R), Tr = 23.3 min (S).

Ferrocenyl chloromethyl ketone (3.27).36

To a flamedried flask under an atmosphere of nitrogen were subsequently added: AlCl3 (400 mg, 3.0 mmol), freshly distilled dichloromethane (40 mL), and chloroacetyl chloride (240 μL, 340.8 mg, 3.0 mmol). This mixture was stirred for 30 min at 0 °C and, subsequently, ferrocene (465 mg, 2.5 mmol) was added slowly to the reaction mixture as a solution in dichloromethane (20 mL). Stirring at 0 °C was continued for 2 h, after which the reaction was quenched by addition of water. After separation of the layers, the organic layer was washed with a saturated solution of NaHCO3, dried over MgSO4, filtered and concentrated in vacuo. Purification by chromatography over silica gel (eluent: n-pentane / diethyl ether 4 : 1) yielded three fractions: ferrocene (not isolated), 3.27 (124 mg (472 μmol) of orange crystals), and the corresponding diacylated product (45 mg (133 μmol) of a red oily substance). 1H NMR (CDCl3) δ 4.82 (d, J = 1.5 Hz, 2H), 4.58 (d, J = 1.8 Hz, 2H), 4.40 (d, J = 2.2 Hz, 2H), 4.23 (d, J = 2.2 Hz, 5H); 13C NMR (CDCl3) δ 195.3 (s), 73.1 (d), 70.2 (d), 69.5 (d), 46.0 (t); MS (EI+) m/z = 264 (M+), 263 (M+), 262 (M+), 212, 185, 169, 158, 129, 121, 91, 78, 56.

2-Chloro-1-ferrocenyl-1-ethanol (3.28)

Obtained by reduction of ferrocenyl chloromethyl ketone (3.27) using sodium borohydride (0.5 equiv.) in methanol37 as a red crystalline material in 94% yield. Subsequent purification was achieved by column chromatography over silica gel (eluent: n-pentane / diethyl ether 4 : 1). 1H NMR (CDCl3) δ 4.56 (ddd, J = 7.7, 4.0, 3.3 Hz, 1H), 4.28 (s, 1H), 4.22 − 4.19 (m, 8H), 3.69 (ddABX, J = 11.0, 4.0 Hz, ΔνAB = 39.7 Hz, 1H), 3.59 (ddABX, J = 11.0, 7.7, ΔνAB = 39.7 Hz, 1H), 2.42 (d, J = 3.3 Hz, 1H); 13C NMR (CDCl3) δ 89.1 (s), 70.0 (d), 68.7 (d), 68.3 (d), 67.2 (d), 65.8 (d), 49.6 (t); MS (EI+) m/z = 266 (M+), 265 (M+), 264 (M+), 215, 187, 186, 163, 156, 138, 121, 108, 91, 65, 56; HRMS (EI+, for C12H1356Fe35ClO) calc. 264.0004, measured: 263.9999; chiral HPLC: Chiralcel AD, 40 °C, n-heptane / IPA 80 : 20, 1.0 mL/min, Tr = 7.6 min (R), Tr = 11.5 min (S).

95



Trimethylsilanyl-propynal (3.29).38

A flamedried 250 mL three-necked flask under an atmosphere of nitrogen was charged with freshly distilled diethyl ether (50 mL) and trimethylsilyl acetylene (3.56 mL, 2.46 g, 25 mmol), after which the temperature of the setup was lowered to −40 °C. Then, n-BuLi (2.5 M in hexane, 10 mL, 25 mmol) and DMF (2.33 mL, 2.20 g, 30 mmol) were added, respectively, and the mixture was stirred for 45 min while the temperature was allowed to increase to rt. The reaction mixture was then added to a vigorously stirring mixture of 10% aqueous NaH2PO4 (125 mL) and diethyl ether (125 mL). After separation, the aqueous layer was extracted once more with diethyl ether, the combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo. The resulting product (90% yield) was not purified further. The spectroscopic data of the obtained compound were in accordance with those given for 3.29 in the literature.38

1-Chloro-4-trimethylsilanyl-but-3-yn-2-ol (3.30)

Obtained by a literature procedure15 as a colorless oil (1.54 g, 8.73 mmol, 87%) after column chromatography over silica gel (n-pentane − Et2O 19 : 1). 1H NMR (CDCl3) δ 4.56 (ddd, J = 7.0, 5.9, 4.0 Hz, 1H), 3.70 (ddABX, J = 11.4, 4.0 Hz, ΔνAB = 35.3 Hz, 1H), 3.61 (ddABX, J = 11.4, 7.0, ΔνAB = 35.3 Hz, 1H), 2.37 (d, J = 5.9 Hz, 1H), 0.16 (s, 9H); 13C NMR (CDCl3) δ 102.1 (s), 91.6 (s), 62.8 (d), 48.8 (t), −0.4 (q).

2-Hydroxy-butyl formate (3.31).40,41

A 100 mL two-necked flask was fitted with a distillation setup and charged with 1,2-butanediol (4.5 mL, 4.5 g, 50 mmol) and triethyl orthoformate (8.15 mL, 7.25 g, 48.9 mmol). This mixture was heated until no more ethanol evolved, then water (15 mL) was added and the mixture was stirred for 1.5 h at rt. Subsequently, the mixture was concentrated in vacuo, yielding 5.46 g (92%) of a 1.5 : 1 mixture of 3.31 and its regioisomer 1-hydroxy-butyl formate, respectively. After purification by column chromatography, this mixture was employed in kinetic resolution experiments. 1H NMR (CDCl3) δ 8.08 (s, 1H), 4.19 (ddABX, J = 11.4, 3.3 Hz, ΔνAB = 51.4 Hz, 1H), 4.02

96


Chapter 3

(ddABX, J = 11.4, 7.0, ΔνAB = 51.4 Hz, 1H), 3.76 (ddd, J = 7.0, 5.9, 3.3 Hz, 1H), 2.40 (br s, 1H), 1.53 − 1.40 (m, 2H), 0.95 (t, J = 7.0 Hz, 3H).

2-Hydroxy-2-thiophen-2-yl-ethyl formate (3.32)

Prepared analogous to 3.31, yielding a 5 : 1 mixture of 3.32 and its regioisomer 1-hydroxy-2-thiophen-2-yl-ethyl formate, respectively. This mixture was employed in kinetic resolution experiments after purification using column chromatography (n-pentane − Et2O 2 : 1, 30%). 1H NMR (CDCl3) δ 8.11 (s, 1H), 7.30 (dd, J = 5.1, 1.1 Hz, 1H), 7.04 (ddd, J = 3.3, 1.1, 0.7 Hz, 1H), 6.99 (dd, J = 5.1, 3.3 Hz, 1H), 5.23 (dd, J = 7.7, 3.7 Hz, 1H), 4.42 (ddABX, J = 11.4, 3.7, ΔνAB = 30.9 Hz, 1H), 4.35 (ddABX, J = 11.4, 7.7, ΔνAB = 30.9 Hz, 1H), 2.58 (br s, 1H).

3.8.3 Production and purification of the enzyme5a,47 Solutions of purified HheC were prepared by C. Tarabiono. Halohydrin dehalogenase was expressed in E. coli MC1061. The hheC gene was amplified by PCR from pGEFHheC and cloned into pBAD/Myc-HisA between NcoI and PstI sites. Plasmid DNA was transformed by electroporation to E. coli cells, which were then plated on LB plates containing ampicillin and incubated overnight at 30 °C. A preculture was started by inoculating 100 mL of TB containing 50 μg/mL ampicillin with the transformants from a plate to a starting OD600 of 0.1. After overnight incubation at 30 °C, the preculture was diluted in 1 L of TB, containing 50 μg/mL ampicillin, 2.5 mM betaine, 0.5 M sorbitol and 0.02 % arabinose, and the culture was incubated for two days at 37 °C. The cells were centrifuged, washed, and resuspended in 50 mL of TEMG buffer (10 mM Tris-SO4, 1 mM EDTA, 1 mM β-mercaptoethanol, and 10% glycerol, pH 7.5) containing a protease inhibitor cocktail (Complete Protease Inhibitor Cocktail Tablets, Roche). Cells were broken by sonication and the extract was centrifuged (50,000 rpm, 45 min, 4 °C). The supernatant was applied on a 50-mL Q-Sepharose anion exchange column and elution was carried out with a gradient of 0 to 0.45 M ammonium sulfate in TEMG. The collected fractions that displayed enzymatic activity were pooled and concentrated. The enzyme was stored at 4 °C for short-term storage or −20 °C for long-term storage.

97



3.8.4 General procedure for enzymatic kinetic resolution on analytical scale To 20 mL of Tris-SO4 buffer (100 mM, pH 8.1) at room temperature, 200 μL of a 1 M stock solution of substrate in DMF was added. Then, 20 μL of a solution of HheC in TEMGc of known activityd was added. Periodically, 1.0 mL aliquots were taken from the reaction mixture, which were extracted with 1.0 mL of toluene containing 5.0 mM of n-dodecane as an internal standard. The resulting organic solutions were then analyzed by chiral GC.

In case of substrate 3.5, another internal standard was used (cinnamyl alcohol, present in the reaction mixture instead of the extraction solvent) as well as another extraction solvent, n-heptane. Reactions with this substrate were analyzed by chiral HPLC.

3.8.5 General procedure for enzymatic kinetic resolution on preparative scale Typically, reactions were performed analogous to the procedure described for the kinetic resolutions on analytical scale, but on a scale of 1.0 − 2.0 mmol. This general procedure is for a reaction on 2.0 mmol scale. To 200 mL of Tris-sulfate buffer (100 mM, pH 8.1) at room temperature, 2.0 mL of a 1 M stock solution of substrate in DMF was added. Then, 50 μL of a solution of HheC in TEMG was added.d When the reaction had finished as determined by HPLC (3.5) or GC (the other substrates), the mixture was extracted with diethyl ether (or ethyl acetate if the goal was to isolate the formed diol as well), the combined organic layers dried on Na2SO4, filtered, and the solvents evaporated. The crude product(s) obtained were purified by column chromatography, using the conditions described for the racemic substrates. Details are given in Table 3.4.

Substrates 3.6 and 3.7 were resolved on a larger scale, in a two-phase system consisting of toluene in addition to aqueous Tris-sulfate buffer. Although some enzyme deactivation was observed under these conditions, it remained possible to perform these transformations using very low catalyst loadings: 1.5•10−4 mol% and 3.0•10−4 mol% for 3.6 and 3.7, respectively.

c The buffer solution consisted of 0.15 M (NH4)2SO4 in TEMG (10 mM Tris-sulfate, pH 7.5, 3 mM EDTA, 0.1% 2-mercaptoethanol, 10% glycerol). d The solution contained 3.0 – 6.0 mg/ml of active enzyme. Thus, the amount of HheC used was 60 – 120 μg for analytical scale resolutions and 150 – 300 μg for preparative scale resolutions. The actual amount of active HheC in each individual resolution was checked prior to the reaction by a spectrophotometric assay, according to a method described in: J. H. Lutje Spelberg, L. Tang, M. Van Gelder, R. M. Kellogg and D. B. Janssen, Tetrahedron: Asymmetry 2002, 13, 1083.

98


Chapter 3

Table 3.4 Isolated yields and ee’s for the kinetic resolution of 3.1 and 3.3 − 3.5.

Substrate Scale

(mmol) Enzyme

(μg) Reaction time

(h) Isol. y. chloroalcohol

(mg, mmol, %) (S)-3.1 1.0 200 5 47, 0.40, 40 (S)-3.3 2.0 250 16 100, 0.62, 31 (S)-3.4 1.5 300 66 70, 0.44, 29 (S)-3.5 2.0 740b 3 173, 0.95, 47

a) Ee’s were >99% in each case; b) A large amount of enzyme was used to shorten the reaction time.

Resolution of substrate 3.6 To a mixture of 50 mL Tris-sulfate (2 M, pH 8.1) and 50 mL toluene was added a 1 : 1 w/w solution of racemic 3.6 (2.32 g, 16.1 mmol) in DMF. Next, 225 μg HheC was added. Since, after 8 h, the conversion as based on GC analysis turned out to proceed slower than expected, another 255 μg HheC was added, followed by another 176 μg after 32 h (total amount of enzyme: 656 μg). The reaction was stopped after 48 h. Flash chromatography (SiO2, eluent n-pentane − Et2O 4 : 1, Rf,chloroalc = 0.33) yielded 989 mg (6.75 mmol, 42%) of (S)-3.6 with an ee of 98.5%.

Resolution of substrate 3.7 A 1 : 1 v/v solution of racemic 3.7 (20.9 g; 129 mmol) in DMF was added to a mixture of 1 L Tris-sulfate (1 M, pH 8.1) and 100 mL toluene. Subsequently, a solution containing 2.68 mg of active HheC was added, followed by another 1.99 mg after 7 h, 0.88 mg after 24.5 h, 1.395 mg after 33.5 h, 1.395 mg after 54 h, 1.53 mg after 76 h, and 0.396 mg after 79.5 h (total amount of enzyme: 10.266 mg). After 4 d, the reaction mixture was extracted with toluene (3×). Subsequently, the combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo, yielding 9.8 g (60.3 mmol, 47%) of crude (S)-3.7 with an ee of >99%. To extract the formed diol from the reaction mixture, the residual aqueous layer was evaporated and the resulting salt slurry extracted with dimethoxypropane and ethyl acetate, yielding 9.1 g (63.1 mmol, 49%) of nearly racemic 1-thiophen-2-yl-ethane-1,2-diol (3.19).

3.8.6 Determination of absolute configuration The absolute configurations of the remaining enantiomers of the chloroalcohols were determined using several methods.

For (S)-(E)-1-chloro-4-phenyl-but-3-en-2-ol ((S)-3.5) a crystal structure (Cl used as heavy atom) was obtained (CCDC 605888).27 Suitable crystals were obtained by slow diffusion of n-pentane into a concentrated solution of (S)-3.5 in diethyl ether.

99



(S)-3.6 and (S)-3.7 could be correlated to known compounds by the sign of their optical rotation.11,12

3.6: [α]20D = +29.2 (c 0.452, CHCl3), hence the configuration is S (lit. (S)-3.6 [α]20D = +23.0 (c 0.52, CHCl3)).11

3.7: Identified as (S)-3.7 using the sign of rotation (+). (lit. (S)-3.7 [α]20D = +28.5 (c 0.53, CHCl3)).11

Compounds 3.1, 3.3, or 3.4 had not been described before in enantiomerically pure form. Therefore, they were hydrogenated to their saturated analogues 3.14 and 3.15, for which optical rotations are known.28 Since, in preliminary experiments, hydrogenation using Pd/C as a catalyst proved unsatisfactory, Wilkinson’s catalyst (Rh(PPh3)3Cl) was employed.

The unsaturated chloroalcohol (1 mmol) was dissolved in 4 mL methanol together with 5 mol% of Wilkinson’s catalyst, and this mixture was stirred until the catalyst had dissolved. After various vacuum / N2 cycles the reaction mixture was put under an atmosphere of H2 (25 bar) and was allowed to react overnight. Then it was filtered over a plug of silica, the solvent evaporated and the residue analyzed. After the product had been positively identified as the saturated chloroalcohol (NMR), the crude material was purified by flash chromatography and the optical rotation measured.

3.14 (from 3.1): [α]20D = +1.3 (c 4.6, CHCl3), hence the configuration is S (lit. (S)-3.14 [α]20D = +1.1 (c 2.9, CHCl3)).28

3.15 (from 3.3): [α]20D = +1.1 (c 6.5, CHCl3), hence the configuration is S (lit. (S)-3.15 [α]20D = +1.4 (c 3.1, CHCl3)). 28

3.15 (from 3.4): Identified as (S)-3.15 using the sign of rotation (+).28

3.9 Notes and references 1 a) E. J. de Vries and D. B. Janssen, Curr. Opin. Biotechnol. 2003, 14, 414-420; b) N. Kasai, T. Suzuki, and Y. Furukawa, J. Mol. Catal. B: Enzym. 1998, 4, 237-252. 2 J. E. T. van Hylckama Vlieg, L. Tang, J. H. Lutje Spelberg, T. Smilda, G. J. Poelarends, T. Bosma, A. E. J. van Merode, M. W. Fraaije, and D. B. Janssen, J. Bacteriol. 2001, 183, 5058-5066. 3 A. J. van den Wijngaard, D. B. Janssen, and B. Witholt, J. Gen. Microbiol. 1989, 135, 2199-2208.

100


Chapter 3

4 a) R. M. de Jong, J. J. W. Tiesinga, A. Villa, L. Tang, D. B. Janssen, and B. W. Dijkstra, J. Am. Chem. Soc. 2005, 127, 13338-13343; b) R. M. de Jong, H. J. Rozeboom, K. H. Kalk, L. Tang, D. B. Janssen, and B. W. Dijkstra, Acta Crystallogr. Sect. D: Biol. Crystallogr. 2002, 58, 176-178. 5 a) L. Tang, A. E. J. van Merode, J. H. Lutje Spelberg, M. W. Fraaije, and D. B. Janssen, Biochemistry 2003, 42, 14057-14065; b) L. Tang, J. H. Lutje Spelberg, M. W. Fraaije, and D. B. Janssen, Biochemistry 2003, 42, 5378-5386. 6 a) L. Tang, D. E. Torres Pazmiño, M. W. Fraaije, R. M. de Jong, B. W. Dijkstra, and D. B. Janssen, Biochemistry 2005, 44, 6609-6618; b) J. H. Lutje Spelberg, L. Tang, R. M. Kellogg, and D. B. Janssen, Tetrahedron: Asymmetry 2004, 15, 1095-1102; c) J. H. Lutje Spelberg, L. Tang, M. van Gelder, R. M. Kellogg, and D. B. Janssen, Tetrahedron: Asymmetry 2002, 13, 1083-1089; d) L. Tang, J. E. T. van Hylckama Vlieg, J. H. Lutje Spelberg, M. W. Fraaije, and D. B. Janssen, Enzyme Microb. Technol. 2002, 30, 251-258; e) J. H. Lutje Spelberg, J. E. T. van Hylckama Vlieg, T. Bosma, R. M. Kellogg, and D. B. Janssen, Tetrahedron: Asymmetry 1999, 10, 2863-2870. 7 a) M. Majerić Elenkov, B. Hauer, and D. B. Janssen, Adv. Synth. Catal. 2006, 348, 579-585; b) G. Hasnaoui, J. H. Lutje Spelberg, E. de Vries, L. Tang, B. Hauer, and D. B. Janssen, Tetrahedron: Asymmetry 2005, 16, 1685-1692; c) J. H. Lutje Spelberg, J. E. T. van Hylckama Vlieg, L. Tang, D. B. Janssen, and R. M. Kellogg, Org. Lett. 2001, 3, 41-43. 8 M. Pineschi, Eur. J. Org. Chem. 2006, 4979-4988. 9 O. Pàmies and J.-E. Bäckvall, J. Org. Chem. 2002, 67, 9006-9010. 10 S. P. Tanis, B. R. Evans, J. A. Nieman, T. T. Parker, W. D. Taylor, S. E. Heasley, P. M. Herrinton, W. R. Perrault, R. A. Hohler, L. A. Dolak, M. R. Hesterf, and E. P. Seest, Tetrahedron: Asymmetry 2006, 17, 2154-2182. 11 T. Hamada, T. Torii, K. Izawa, and T. Ikariya, Tetrahedron 2004, 60, 7411-7417. 12 Z. Gercek, D. Karakaya, and A. S. Demir, Tetrahedron: Asymmetry 2005, 16, 1743-1746. 13 T. Schubert, W. Hummel, and M. Müller, Angew. Chem. Int. Ed. 2002, 41, 634-637. 14 a) K. Nakamura and T. Matsuda, Curr. Org. Chem. 2006, 10, 1217-1246; b) W. Kroutil, H. Mang, K. Edegger, and K. Faber, Curr. Opin. Chem. Biol. 2004, 8, 120-126; c) W. Kroutil, H. Mang, K. Edegger, and K. Faber, Adv. Synth. Catal. 2004, 346, 125-142. 15 M. Lautens, M. L. Maddess, E. L. O. Sauer, and S. G. Ouellet, Org. Lett. 2002, 4, 83-86. 16 J. A. Marshall, Chem. Rev. 1989, 89, 1503-1511. 17 a) M. Pineschi, F. Del Moro, P. Crotti, V. Di Bussolo, and F. Macchia, Synthesis 2005, 334-337; b) V. Di Bussolo, M. Caselli, M. R. Romano, M. Pineschi, and P. Crotti, J. Org. Chem, 2004, 69, 8702-8708; c) M. Pineschi, F. Del Moro, P. Crotti, V. Di Bussolo, and F. Macchia, J. Org. Chem. 2004, 69, 2099-2105; d) F. Bertozzi, P. Crotti, F. Del Moro, B. L. Feringa, F. Macchia, and M. Pineschi, Chem. Commun. 2001, 2606-2607; e) F. Bertozzi, P. Crotti, F. Macchia, M. Pineschi, and B. L. Feringa, Angew. Chem. Int. Ed. 2001, 40, 930-932; f) F. Bertozzi, P. Crotti, F. Macchia, M. Pineschi, A. Arnold, and B. L. Feringa, Org. Lett. 2000, 2, 933-936.

101



18 M. L. Merlau, C. C. Borg-Breen, and S. B. T. Nguyen, in Encyclopedia of Catalysis, I. T. Horváth, Ed.-in-Chief, Wiley, Hoboken, New Jersey, 2003, Vol. 3, pp. 155-246, and references therein. 19 a) E. J. Corey and M. Chaykovsky, J. Am. Chem. Soc. 1965, 87, 1353-1364; b) For an asymmetric epoxidation of aldehydes, see: D. M. Badine, C. Hebach, and V. K. Aggarwal, Chem. Asian J. 2006, 1, 438-444. 20 J. M. Concellón, H. Cuervo, and R. Fernández-Fano, Tetrahedron 2001, 57, 8983-8987. 21 HheC-catalyzed reactions in a centrifugal contact separator: unpublished results by C. Tarabiono and G. Kraai. 22 J. Gorzynski Smith, Synthesis 1984, 629-656. 23 P. Mosset and R. Grée, Synth. Commun. 1985, 15, 749-757. 24 The computer program MicroMath® Scientist® was used. 25 C.-S. Chen, Y. Fujimoto, G. Girdaukas, and C. J. Sih, J. Am. Chem. Soc. 1982, 104, 7294-7299. 26 A. J. J. Straathof and J. A. Jongejan, Enzyme Microb. Technol. 1997, 21, 559-571. 27 X-ray crystallography performed by A. Meetsma. C10H11ClO, Mr = 182.65, orthorhombic, P212121, a = 4.9735(7), b = 10.531(2), c = 17.521(3) Å, V = 917.7(3) Å3, Z = 4, Dx = 1.322 g cm–3, F(000) = 384, μ = 3.63 cm–1, λ(MoKα) = 0.71073 Å, T = 100(1) K, 7911 reflections measured, GooF = 1.078, wR(F2) = 0.0698 for 2109 unique reflections and 153 parameters and R(F) = 0.0311 for 1994 reflections obeying Fo 4.0 σ(Fo) criterion of observability. The asymmetric unit consists of one molecule of the title compound; the molecules are linked into an infinite one-dimensional chain by O–H•••O intermolecular bonds. The stereogenic center of C9 showed the S-configuration. 28 T. Sakai, K. Wada, T. Murakami, K. Kohra, N. Imajo, Y. Ooga, S. Tsuboi, A. Takeda, and M. Utaka, Bull. Chem. Soc. Jpn. 1992, 65, 631-638. 29 For background literature on this phenomenon, see for instance: a) N. W. Boaz, Tetrahedron: Asymmetry 1995, 6, 15-16; b) H. E. Audier, J. F. Dupin, and J. Jullien, Bull. Soc. Chim. Fr. 1968, 3844-3850. 30 a) E. Borredon, M. Delmas, and A. Gaset, Tetrahedron Lett. 1982, 23, 5283-5286; b) C. Lemini, M. Ordoñez, J. Pérez-Flores, and R. Cruz-Almanza, Synth. Commun. 1995, 25, 2695-2702. 31 For similar pH dependence in the ring opening of epoxides by ammonium halides, see: M. Chini, P. Crotti, C. Gardelli, and F. Macchia, Tetrahedron 1992, 48, 3805-3812. 32 M. Majerić Elenkov, L. Tang, B. Hauer, and D. B. Janssen, Org. Lett. 2006, 8, 4227-4229. 33 J. H. Lutje Spelberg, Enantioselective Biocatalytic Conversions of Epoxides, Ph.D. thesis, University of Groningen, Groningen, the Netherlands, 2003. 34 a) C. H. Behrens, S. Y. Ko, K. B. Sharpless, and F. J. Walker, J. Org. Chem. 1985, 50, 5687-5696; b) C. H. Behrens and K. B. Sharpless, J. Org. Chem. 1985, 50, 5696-5704.

102


Chapter 3

35 For more information on ferrocene-based ligands, see: R. Gómez Arrayás, J. Adrio, and J. C. Carretero, Angew. Chem. Int. Ed. 2006, 45, 7674-7715. 36 H. J. Hwang, J. R. Carey, E. T. Brower, A. J. Gengenbach, J. A. Abramite, and Y. Li, J. Am. Chem. Soc. 2005, 127, 15356-15357. 37 J. Hiratake, M. Inagaki, T. Nishioka, and J. Oda, J. Org. Chem. 1988, 53, 6130-6133. 38 N. Krause, A. Hoffmann-Röder, and J. Canisius, Synthesis 2002, 1759-1774. 39 G. Hasnaoui-Dijoux, M. Majerić Elenkov, J. H. Lutje Spelberg, B. Hauer, and D. B. Janssen, ChemBioChem 2008, 9, 1048-1051. 40 G. Crank and F. W. Eastwood, Aust. J. Chem. 1964, 17, 1392-1398. 41 E. C. Tuazon, S. M. Aschmann, and R. Atkinson, Environ. Sci. Technol. 1998, 32, 3336-3345. 42 a) A. N. Pudovik and B. E. Ivanov, J. Gen. Chem USSR 1956, 26, 2129-2132; b) F. G. Ponomarev, O. G. Kharenko, and M. F. Shavkova, J. Gen. Chem. USSR 1957, 27, 1309-1313. 43 M. Lautens and M. L. Maddess, Org. Lett. 2004, 6, 1883-1886. 44 a) O. Grummitt and R. M. Vance, J. Am. Chem. Soc. 1950, 72, 2669-2674; b) I. E. Muskat and L. B. Grimsley, J. Am. Chem. Soc. 1930, 52, 1574-1580. 45 a) A. Kamal, G. B. R. Khanna, R. Ramu, and T. Krishnaji, Tetrahedron Lett. 2003, 44, 4783-4787; b) C. Corral, V. Darias, M. P. Fernández-Tomé, R. Madroñero, and J. del Río, J. Med. Chem. 1973, 16, 882-885; c) H. Hopff and R. Wandeler, Helv. Chim. Acta 1962, 45, 982-986. 46 M. P. Sibi and R. Sharma, Synlett 1992, 497-498. 47 J. Sambrook and D. W. Russell, Molecular Cloning: A Laboratory Manual, 3rd edn., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001.

Chapter 4 A new approach to the dynamic kinetic resolution of haloalcohols

In this chapter, the dynamic kinetic resolution of a variety of racemic haloalcohols to

their corresponding epoxides is described, with excellent enantioselectivity especially

using chloroalcohols. The aim of this research was to enlarge the synthetic potential of

haloalcohol dehalogenases by combining enzyme-catalyzed ring closure of haloalcohols

with transition-metal catalyzed racemization, leading directly from racemic

halohydrins to enantiomerically pure epoxides.a

a Submitted for publication: R. M. Haak, F. Berthiol, T. Jerphagnon, A. J. A. Gayet, C. Tarabiono, C. P. Postema, M. Pfeffer, D. B. Janssen, A. J. Minnaard, B. L. Feringa, and J. G. de Vries, Dynamic Kinetic Resolution of Racemic β-Haloalcohols: Direct Access to Enantioenriched Epoxides.

104

Chapter 4 - Dynamic Kinetic Resolution of Haloalcohols.doc

Chapter 4

4.1 Introduction to dynamic kinetic resolution

Enzymatic kinetic resolution is a powerful strategy to obtain chiral compounds in high enantiomeric purity.1,2 However, being a resolution method, it suffers from the drawback of a maximum yield of 50%. Therefore, a powerful related approach has arisen in recent years: to couple asymmetric conversion with racemization of the remaining starting material. This type of second-order asymmetric transformation3 has become known as dynamic kinetic resolution (DKR).4 Ideally, it results in full conversion to an enantiomerically pure product.

DKR requires that the two enantiomers of a given starting material 1) react to different stereoisomers of the product, 2) undergo this reaction at different rates, and 3) are in continuous, fast equilibrium with each other. A number of strategies has been developed to ensure that the last condition is met.

Scheme 4.1 Dynamic kinetic resolution via ring opening of chiral azlactones by (L)-α-amino acid methyl esters.

In the initial approach to DKR, the intrinsic stereochemical lability of certain stereocenters is used, for instance as a result of facile enolization. In 1966, Weygand and coworkers described the reaction of (L)-α-amino acid esters with racemic azlactones, leading to an excess of one of the possible diastereoisomers.5 Importantly, the authors described and demonstrated the kinetic requirement that one of the enantiomers of the starting material reacts faster with the chiral reagent than the other, and racemization is faster than both of these reaction rates (Scheme 4.1). This understanding makes the

105


A new approach to the dynamic kinetic resolution of haloalcohols

example of Weygand et al. the first unequivocal example of an intentionally performed dynamic kinetic resolution.

Racemization due to the stereochemical lability of stereogenic centers has been used since then in a number of similar studies, including biochemical conversions. Examples are the production of D-phenylglycine-related α-amino acids by immobilized microbial cells6 or the enzymatic second-order asymmetric hydrolysis of ketorolac esters.7

In a seminal publication by Noyori and coworkers,8 the term “dynamic kinetic resolution” was introduced. This group described the asymmetric hydrogenation of racemic 2-substituted 3-oxo carboxylic esters. As a result of the configurational lability at the 2-position, this approach led to an effective dynamic kinetic resolution. β-Hydroxyesters were obtained in remarkable enantiomeric and diastereomeric excess (Scheme 4.2).

Scheme 4.2 Dynamic kinetic resolution of α-substituted β-ketoesters.

The approach of using the configurational lability of the starting material to effect dynamic kinetic resolution has found widespread use and several examples are described in literature.9 These include lipase-catalyzed procedures for DKR of aldehydes to optically active cyanohydrin acetates10 and DKR of furanones.11

Spontaneous or base-assisted interconversion of substrate enantiomers has yielded many examples of efficient DKR. However, to enlarge the applicability, researchers began to look for ways to employ starting materials with more robust stereogenic centers, while maintaining mild reaction conditions. The first example of a transition-metal catalyzed racemization in DKR has been given by Allen and Williams in 1996.12 They combined lipase-catalyzed deacylation of a racemic allyl acetate with palladium-catalyzed racemization, as depicted in Scheme 4.3. The same group later reported lipase-catalyzed

106


Chapter 4

acetylation of rac-1-phenylethanol coupled with racemization using various transition metal catalysts. The best results were obtained using [Rh(cod)Cl]2 (76% conversion, 80% ee) and Rh2(OAc)4 (60% conversion, 98% ee).13

Scheme 4.3 DKR using a combination of enzyme and palladium catalysis.

A further breakthrough was provided by the work of Bäckvall and coworkers. They developed an efficient procedure to convert racemic 1-phenylethanol to (R)-1-phenylethyl acetate in high yield and excellent enantiopurity (Scheme 4.4).14 Their approach features the use of the ruthenium-based Shvo catalyst (Figure 4.1),15 p-chlorophenyl acetate as the acyl donor, and immobilized thermophilic Candida antarctica lipase B (Novozym 435).16

Scheme 4.4 Highly efficient DKR of phenethyl alcohol by combination of lipase and transition metal catalysis.

Since then, numerous publications have appeared on the dynamic kinetic resolution of secondary alcohols using a combination of enzymatic and transition metal catalysis.4 In particular, several catalysts have been developed for efficient racemization.17 Besides the Shvo catalyst, complexes 4.118 and 4.219 (Figure 4.1) have been used, which are able to racemize secondary alcohols at room temperature. This is an important development, since it allows for the use of thermally labile enzymes.

107



Figure 4.1 Ruthenium catalysts for the racemization of secondary alcohols.

Usually employed acyl donors include e.g. p-chlorophenyl acetate, i-propenyl acetate, or vinyl acetate.4 Furthermore, it has been shown that, under the proper conditions, simple esters like ethyl acetate can also be used.20

Following Kazlauskas’ rule,21 lipases are R-selective for secondary alcohols. In order to obtain products with the S-configuration, the serine protease subtilisin has been employed in enzyme/transition-metal catalyzed DKR.22 Another development of interest is the DKR of primary amines, which are generally much harder to racemize than the corresponding secondary alcohols.23

Given the fact that transition-metal based racemization catalysts are active in hydrogen-transfer processes, good racemization catalysts are in general also good catalysts for transfer hydrogenation. This observation has inspired Park et al.24 to extend the scope of DKR by using carbonyl compounds or enol acetates as the starting material (Scheme 4.5).

Scheme 4.5 Ru/lipase mediated conversion of ketones or enol acetates to enantiomerically enriched actetates.

While research into dynamic kinetic resolution of secondary alcohols in general has yielded several practical examples (vide supra), they do not often include the DKR of vicinal haloalcohols. Bäckvall and Pàmies reported the only systematic study so far.25 A possible reason for this lack of attention is the fact that the most common, ruthenium-based racemization catalysts are poorly active for halogenated substrates. This lack of racemization activity might be due to the fact that chloroketones, probably

108


Chapter 4

intermediates in the catalytic cycle,19a are inhibitors of these racemization catalysts.b Nonetheless, β-haloalcohols are highly valuable structural building blocks in asymmetric synthesis, for instance as precursors of chiral epoxides or β- and γ-amino alcohols (Scheme 4.6). Alternatively, they serve as starting materials in the synthesis of side-chains for statins, a class of hypolipidemic drugs used against elevated cholesterol levels.26

Scheme 4.6 Haloalcohols as synthetic intermediates for epoxides and aminoalcohols.

In the study mentioned above,25 Bäckvall and Pàmies have shown that it is possible to obtain enantiomerically pure epoxides from racemic chloroalcohols in a two-step process (Scheme 4.7).

Scheme 4.7 Preparation of optically active epoxides by DKR of chloroalcohols followed by intramolecular ring closure.

Scheme 4.8 Enantiopure epoxides by HheC-catalyzed DKR of halohydrins.

A dynamic kinetic resolution based on the use of haloalcohol dehalogenases would lead to a one-step procedure for the preparation of enantiopure epoxides from vicinal haloalcohols. Given the excellent results we obtained in the enzymatic kinetic b In preliminary experiments by V. Ritleng, it was found that after addition of small quantities of chloroketone in the Ru-catalyzed racemization of enantiopure 2-chloro-1-phenylethanol, no more racemization took place.

109



resolution of β-chloroalcohols (see Chapter 3), we decided to develop such a procedure, as outlined in Scheme 4.8.

Dynamic kinetic resolutions using haloalcohol dehalogenases has been reported,27 but in that case the system was fully enzymatic, based on the irreversible enzyme-catalyzed ring opening of epibromohydrin by azide. The required racemization of the starting material occurred due to the simultaneous and reversible ring opening of epibromohydrin by bromide, leading to the formation of meso-1,3-dibromopropanol as an intermediate in the reaction. A procedure for the use of haloalcohol dehalogenases in combination with transition-metal catalyzed racemization of haloalcohols 4.3 would lead to a more general procedure for the synthesis of enantiopure epoxides 4.4.

4.2 Results and discussion

4.2.1 Substrates In Chapter 3, the highly selective enzymatic kinetic resolution of unsaturated and heteroaromatic haloalcohols is described. The remaining enantiomers of the haloalcohol substrates (3.1 – 3.7) could be isolated in high yields and with high ee’s. Nevertheless, the use of these compounds as substrates for DKR was quickly discarded, since the epoxide products turned out to be unstable in the aqueous reaction medium needed for the enzymatic reaction. It was therefore decided to use a class of related compounds, the substituted aromatic haloalcohols depicted in Figure 4.2.

Of these substrates, 4.3a is commercially available, both in racemic form and as the (R)- or (S)-enantiomers. Compounds 4.3b and 4.3j – 4.3q were prepared by sodium borohydride reduction of the corresponding haloketones.28 Substrates 4.3c, 4.3d, 4.3h, and 4.3i were prepared by reaction of chloroiodomethane with the corresponding aldehydes in the presence of n-butyllithium (See also Chapter 3).29 A notable aspect in the synthesis of aliphatic substrate 4.3i was that α-deprotonation of the aldehyde − which could be followed by a number of undesirable side-reactions − apparently does not occur. Finally, 4.3e – 4.3g were prepared by ring opening of the corresponding epoxide using in situ formed Li2CuCl4 (Scheme 4.9).30,31

Scheme 4.9 Synthesis of 2-chloro-1-(4-nitrophenyl)-ethanol (4.3e).

110


Chapter 4

Although a poor regioselectivity is reported when styrene oxide is used as the starting material, 4.3e was obtained in a regioselectivity of 95:5 in favor of the terminally ring-opened product. We contribute this to the presence of the electron-withdrawing nitro-substituent on the para position, which destabilizes the development of partial positive charge at the benzylic carbon atom and thus suppresses nucleophilic attack at that position. The synthesis of m-nitro substituted 4.3f proceeded with a regioselectivity of 73 : 27, lower than that in the case of 4.3e but still in favor of the product of terminal ring opening. By contrast, in the synthesis of o-nitro substituted 4.3g, the product of chloride attack on the internal carbon was not observed at all. This remarkable regioselectivity may be contributed to a favorable combination of the electron-withdrawing effect of the nitro-group and the steric hindrance it exerts on the internal carbon atom of the oxirane.

Figure 4.2 Haloalcohols used as substrates in this study.

4.2.2 Enzymes In the course of our investigations, several haloalcohol dehalogenases were investigated. First of all wild-type HheC (E1, a haloalcohol dehalogenase from Agrobacterium radiobacter AD1),32 and furthermore a number of mutants of this enzyme (E2 − E4), as well as the chemically modified variant E5:33

111



E1: HheC. This is the wild-type enzyme, isolated from Agrobacterium radiobacter AD1 and overexpressed in E. coli.

E2: HheC Cys153Ser (C153S). The replacement of the cysteine residue at position 153 by serine increased the oxidative stability of the enzyme, presumably since it reduces the chance that disulfur bridges between cysteine residues − a common oxidative inactivation pathway in enzymes − are formed.34

E3: HheC Trp249Phe (W249F). The replacement of a tryptophan residue at position 249 by phenylalanine increased the enantioselectivity of the enzyme for aromatic substrates.35

E4: HheC Cys153Ser Trp249Phe (C153S W249F). This double mutant was expected to have both higher oxidative stability and higher enantioselectivity than wild-type HheC.

E5: Chemically modified HheC W249. Using Lomant’s reagent (4.5),36 lysine residues at the outer surface of the enzyme can be cross-linked through amide bonds. This lysine modification was chosen because it was thought that complexation of lysine to the racemization catalyst initiated enzyme degradation. Hence, protection of the lysine residues was thought to increase the stability of the enzyme towards the racemization catalyst that was used for our dynamic kinetic resolution experiments (vide infra).

Cleavage of the resulting dimers can be effected by treatment with DTT at room temperature, or by boiling a solution of the enzyme in mercaptoethanol. In both cases, the lysine residues remain protected with a 3-mercaptopropylamide moiety. Both the cross-linked and the cleaved, but still protected, enzymes can be used in subsequent experiments.

Kinetic resolution experiments to test E1 − E5 Using 4.3a and 4.3l as model substrates, enzymes E1 − E5 were used in enzymatic kinetic resolution in order to probe the relative enantioselectivity of the enzymes.

Several kinetic resolution experiments were carried out using substrate 4.3a at a 0.2 mmol scale (Table 4.1). It was observed that in a two-phase system, the enzymatic conversion using E1 becomes slower than in a homogeneous system, whereas the enantioselectivity increases slightly (entries 1 and 2). The use of phosphate buffer led to an even slower reaction and a decreased enantioselectivity (entry 3), whereas borate seemed to be well suited for kinetic resolutions using E1 (entry 4). Experiments using double mutant E4 revealed that the high enantioselectivity of this enzyme is more

112


Chapter 4

constant than that of E1, which is very high only when using borate buffer (entries 4 − 7). Hence, subsequent experiments were done using this enzyme. It was discovered later that the buffer system most compatible with the conditions of the biphasic DKR we were developing, was 2-(4-(2-hydroxyethyl)piperazin-1-yl)ethanesulfonic acid (HEPES). As can be expected, the strength of the buffer solution is critical. When 10 mM HEPES buffer was used, the enzyme hardly showed any activity (entry 8). After 16 h, a conversion of 13% was reached. Moreover, it was observed that the pH of the solution had decreased from 8 to 6.5, indicating that a stronger buffer would be necessary for the reaction. Subsequent experiments revealed that a buffer concentration of 100 mM was sufficient.

Table 4.1 Kinetic resolution experiments using 4.3a and the enzymes E1 and E4.a

Entry Enzyme Init. activ.

(U)b Solvent systemc pHd

Volume (mL)

E

1 E1 1.1 Tris-sulfate 8.1 20 74 2 E1 0.4 Tris-sulfate / toluene 8.1 10 / 10 93 3 E1 0.3 Phosphate 7.2 20 56 4 E1 0.8 Borate 8.2 20 >200 5 E4 2.5 Tris-sulfate 8.1 20 154 6 E4 1.0 Tris-sulfate / toluene 8.1 5 / 2 >200 7 E4 1.0 Tris-sulfate / toluene 8.1 2 / 2 142 8 E4 0.1 HEPESe / toluene 8 5 / 4 >200

a) 200 μmol of substrate was added to the indicated solvent mixture, after which the reaction was started by adding 10−50 μL of enzyme solution to the mixture; b) The same amount of active enzyme was used in each reaction: in case of E1 60 mg, in case of E4 50 mL of a 176 U/mL solution (the activity was measured using 4.3l as substrate according to Ref. 37); c) All buffer solutions were 100 mM; d) In case of a biphasic system, this column refers to the pH of the aqueous buffer; e) 10 mM.

A systematic study of enzyme behaviour was conducted using substrate 4.3l, to get an indication of the relative enantioselectivity of enzymes E1 – E5. The results of this screening are summarized in Table 4.2 and give a good indication of the relative differences in enantioselectivity between the enzymes. Since bromoalcohols, including 4.3l, are more sensitive to base-catalyzed, unselective ring closure than chloroalcohols such as 4.3a, reactions were performed at a pH of 7.5 instead of 8.

Based on previous research, it had been assumed that the C153S mutation would increase the stability of the HheC without affecting its enantioselectivity.34 The numbers in Table 4.2 indicate, however, that there is an adverse effect: the C153S

113



mutant shows lower enantioselectivity than wild-type HheC (entries 1 and 2), and the double mutant is less enantioselective than the mutant containing only W249F (entries 3 and 4). In Paragraph 4.2.4 it will be shown that, despite the superior enantioselectivity of E3 in kinetic resolution, the double mutant E4 gives optimal results in DKR.

Table 4.2 Screening of haloalcohol dehalogenases using substrate 4.3l.a

Entry Enzyme Eapp E

1 − kc = 3.17·10-6 s−1 2 E1 HheC wild-type 27 31 3 E2 C153S (more stable towards oxidation) 17 19 4 E3 W249F (more enantioselective) 83 (100)b 94 (120) 5 E4 C153S W249F 37 (40)b 45 (54) 6 E4 C153S W249F, two-phase system 31 44 7 E5c W249F modified with Lomant’s reagent 35 46

a) A solution of 200 μmol of 4.3l in in 0.5 mL of DMSO was added to 10 mL of aqueous HEPES buffer (50 mM, pH 7.5). The reaction was started by adding 4 U of enzyme to the mixture. Samples were taken every 10 min and analyzed by chiral HPLC; b) Numbers in parentheses indicate the outcome of duplo experiments; c) See also Table 4.3, entries 4 − 6.

Chemically modified enzyme E5 was subjected to closer investigation. In Table 4.3, we summarize our observations when E5 was screened using 4.3l as substrate.

At pH 7, the apparent enantioselectivity of the enzyme is highest, presumably since lowering the pH suppresses competing non-selective chemical ring closure of the bromoalcohol to the epoxide. However, at this pH the reaction does not go to completion. When the results at 120, 180, and 240 min are compared (entries 1 − 3), the conversion is roughly the same (around 39%). Also the ee of the remaining substrate remains equal (91%), whereas the ee of the product decreases significantly, from 76% to 65% to 50% (in earlier stages of the reaction the product ee was as high as 95%). This indicates that the incomplete conversion is not due to enzyme deactivation, but instead that the reaction has reached equilibrium. At pH 7.5 equilibrium is reached at a higher level of conversion, 48% (entries 4 − 6). When DTT is added to the reaction mixture, the chemical cross-links (via Lomant’s reagent) between the peptide chains in the enzyme should be disconnected. However, this turns out to have only a small effect on the enzymatic conversion. The apparent E-value (33) and the conversion at equilibrium (50%) are roughly similar to the values observed in the absence of DTT (35

114


Chapter 4

and 48%, respectively, compare entries 7 − 9 and 4 − 6). The dynamic kinetic resolution experiments that have been done using E5 are described in Paragraph 4.2.4.

Table 4.3 Ring closure of 4.3l catalyzed by modified enzyme E5.a

Entry pH Additive Eapp t

(min) Conv. (%)

ee 4.4 (%)

ee 4.3 (%)

1 7 − 51 (73)b 120 36 76 91 2 180 39 65 91 3 240 39 50 90 4 7.5 − 35 (46) 120 45 47 97 5 180 48 28 96 6 240 48 28 96 7 7.5 DTT 33 (39) 120 50 75 95 8 180 52 57 95 9 240 48 39 95

a) A solution of 200 μmol 4.3l in 0.5 mL DMSO was added to 10 mL HEPES 50 mM at the indicated pH. The reaction was started by adding 3 U of enzyme to the mixture; b) Value in parentheses indicates the E value corrected for spontaneous ring closure.

4.2.3 Racemization catalysts Recently, metallocycles based on ruthenium, rhodium, and iridium were reported by the groups of Pfeffer38 and Davies.39 Since some of these metallocycles are active catalysts in transfer hydrogenation of ketones,40 it was expected that they might act as efficient racemization catalysts for sec-alcohols via a dehydrogenation / hydrogenation sequence. In preliminary experiments, iridacycle 4.8a (Scheme 4.10) was identified as the most effective racemization catalyst for β-haloalcohols such as 4.3a.41 Complex 4.8a and similar iridacycles are prepared by cyclo-iridation of the corresponding amines 4.7 using the commercially available iridium precursor [Cp*IrCl2]2 (4.6), as illustrated in Scheme 4.10.

Iridacycle 4.8a itself, a dark yellow complex, is inactive in alcohol racemization and needs to be activated. Treatment with potassium tert-butoxide leads to a deep-red complex with a much higher solubility in toluene, the solvent of choice for the racemization reaction (Scheme 4.11). This activated complex, with the presumable structure 4.9, is a very efficient catalyst for racemization of secondary alcohols.

115



Scheme 4.10 Cyclometallation of N-methylbenzylamine with [Cp*IrCl2]2.

Scheme 4.11 Preparation of alcohol racemization catalyst 4.9.

NMR and HRMS experiments are currently being performed in order to elucidate the exact nature of the racemization catalyst. The most probable structure is 4.9, since an analogous active species was observed in Ru-catalyzed racemization, where the catalyst precursor also needs activation by t-BuOK.19a

Scheme 4.12 Synthesis of ligand 1-(benzylamino)-2-methylpropan-2-ol (4.7b).

For the majority of DKR experiments, 4.9 was used as catalyst. However, also complex 4.8b (Figure 4.3, prepared analogous to 4.8a) and 4.8c were considered as catalyst precursors. Ligand 4.7b (1-(benzylamino)-2-methylpropan-2-ol) was synthesized from benzylamine and 2,2-dimethyl-oxirane,42 as illustrated in Scheme 4.12, in order to mimic t-BuO− as part of the ligand. Since exchange of t-BuO− with water was considered an important catalyst deactivation mechanism, it was envisioned that the stability of the catalyst might improve if a coordinating alcohol moiety was present within the molecule.

116


Chapter 4

The corresponding iridacycle 4.8b could be obtained from 4.7b and 4.6 analogously to 4.8a. Iridacycle 4.8c was obtained from 4.6 and 4.7a (Scheme 4.10) in the presence of air.c Probably, the initially formed complex 4.8a is oxidized under these conditions.

Figure 4.3 Iridacycles 4.8b and 4.8c.

Using R-4.3a as substrate, the catalysts obtained from precursors 4.8a − c after activation with t-BuOK were tested for their racemization activity in toluene at room temperature. The results are summarized in Table 4.4.

Using 5 mol% of 4.8a, 4.3a was completely racemized in 30 min (Table 4.4, entry 1a). Subsequently, a second batch of substrate was added to the reaction mixture, which was partly racemized (entry 1b). In a two-phase system of water and toluene, the catalyst prepared from 4.8a (4.9) shows activity as well (entry 2), although the reaction is slower than in a homogeneous system. We observed that in this two-phase system of water and toluene, mixing should not be too vigorous, since this inactivates the catalyst. When aqueous Tris-sulfate buffer is used instead of plain water, 4.9 completely racemizes R-4.3a, although after 3 days the catalyst does not show reactivity any longer (entry 3).

The catalyst obtained from 4.8b racemized 4.3a in 16h after activation with 1 eq of t-BuOK. However, a second batch of 4.3a was not racemized anymore, indicating a lower stability of 4.8b than 4.8a (entry 4). In another racemization experiment using 4.8b as catalyst precursor, the ee of 4.3a was 68% after 30 min and an overnight reaction was required to completely racemize the compound (entry 5). This confirms the lower activity of the catalyst prepared from 4.8b compared to 4.9. Furthermore, it turns out that after this overnight reaction, the catalyst prepared from 4.8b has lost its activity, since a second batch of R-4.3a is no longer racemized (entry 5c).

Also using a catalyst prepared from 4.8c, an overnight reaction was required to fully racemize 4.3a. Combined with the racemization results previously obtained using a

c Alternatively, iridacycle 4.8c can be obtained using N-benzylidenemethanamine as ligand (T. Jerphagnon, unpublished results).

117



number of similar iridacycles,41 it was decided to use 4.8a as a catalyst precursor in subsequent DKR experiments.

Table 4.4 Racemization of R-4.3a.a

Entry Catalyst t

(h) ee

(%) Remarks

1 a 4.8a 0.5 4 b 0.5+0.66 39 at t=0.5h, 2nd batch R-4.3a added (ee 52%)

2 a 4.8a 16 1 toluene / H2O b 16+2.33 6 at t=16h, 2nd batch R-4.3a added (ee 52%)

3 a 4.8a 64 2 toluene / Tris-sulfate b 64+2.25 53 at t=64h, 2nd batch R-4.3a added (ee 52%)

4 a 4.8b 16 0 b 16+1.75 55 at t=16h, 2nd batch R-4.3a added (ee 55%)

5 a 4.8b 0.5 68 b 16.75 0 c 18+2.5 55 at t=18h, 2nd batch R-4.3a added (ee 55%)

6 a 4.8c 0.58 89 b 2.08 64 c 17.42 0

a) In a thoroughly flame-dried Schlenk flask under an atmosphere of nitrogen, 10 μmol of catalyst and 12 μmol of t-BuOK were dissolved in 3 mL of freshly distilled toluene, after which 200 μmol of R-4.3a were added. The reaction was monitored by periodically taking 0.1 mL aliquots from the mixture, filtering them over silica gel (eluent: Et2O) and analyzing the resulting samples by chiral GC.

4.2.4 Dynamic kinetic resolution experiments Various combinations of substrates, enzymes, racemization catalysts, and other conditions have been investigated. In this paragraph, we will first discuss the experiments done with 2-chloro-1-phenyl-ethanol (4.3a) and 2-bromo-1-(4-nitrophenyl)ethanol (4.3l), respectively. Next, we will discuss the general patterns that can be derived from those experiments, and finally treat the DKR of the remaining substrates.

DKR experiments using 4.3a Initial screening experiments were carried out using 2-chloro-1-phenylethanol (4.3a, 0.2 mmol), 5 mol% of catalyst precursor 4.8a, and enzyme E4 (HheC C153S W249F).

118


Chapter 4

The substrate is typically added to the reaction mixture as a solution in DMF or DMSO. The influence of the cosolvent on the DKR is discussed in more detail below. Enzymatic ring closure takes place at room temperature in aqueous buffer (pH 8), whereas the racemization catalyst is activated with t-BuOK in a separate vessel in freshly distilled toluene under an atmosphere of nitrogen, after which this solution is slowly added to the reaction mixture. The results of this screening are summarized in Table 4.5.

Table 4.5 DKR experiments using substrate 4.3a, racemization catalyst precursor 4.8a, and enzyme E4 in a biphasic system of toluene / HEPES buffer.a

Entry Buffer (mL)

Enzb (μl)

Cosolventc (mL)

N2d Tole Misc. Conv. (%)

ee 4.3a (%)

ee 4.4a (%)

1 2 500 − − − 47 36 98 2 2.5 300 − − − 36 63 95 3 2 1000 − − + 43 73 >98 4 2.5 500 − + − 42 65 >98 5 5 50 − + + 17 4 >98 6 5 25 − + + Trisf 47 80 >98 7 5 50 − + − Trisf 10 2 >98 8 5 50 DMF (0.2) + + Trisf 14 2 >98 9 5 50 DMF (0.2) − + 15 12 >98

10 5 100 DMF (0.4) − − 41 5 >98 11 5 4×25g DMF (0.5) − − 33 29 >98 12 5 100 DMF (1.0) − − 34 4 >98 13 10 50 DMF (0.5) − − 43 14 >98 14 20 50 DMF (0.5) − − addh 64 26 95 15 10 50 DMSO (0.5) − − 53 72 >98 16 10 100 DMSO (0.5) − − BSAi 90 >98 >98

a) A general procedure for dynamic kinetic resolution can be found in the experimental section. Reactions were done on 200 μmol scale. The addition time of the racemization catalyst solution was 0.5 mL/h; b) Prior to reaction, the activity of the enzyme solution was determined to be 370 U/mL for substrate 4.3l using a standard spectrophotometric assay described in ref. 37; c) In between brackets the quantity of cosolvent (in mL) is indicated; d) Indicates if the reaction was performed under a nitrogen atmosphere and using degassed solvents (NB the solution of racemization catalyst is always prepared using flamedried glassware and under nitrogen); e) Indicates if a quantity of toluene (1 mL) was present in the reaction mixture from the outset; f) Tris-SO4 100 mM pH 8.1 was used as buffer; g) every 1.5h, 25 μL was added; h) Addition 4.8a started at 50% substrate conversion; i) BSA (10 eq w.r.t. E4) was added to the reaction.

119



In our initial experiments, we generally observed low conversion to the epoxide, despite the facts that racemization was efficient (see column “ee 4.3”) and the enzyme retained high enantioselectivity (column “ee 4.4”).

At first, it was considered that the problems with enzyme stability were a result of oxidation phenomena. Consequently, we performed a number of reactions in degassed solvent under a nitrogen atmosphere (entries 4 − 8). However, no effect on the course of the DKR was observed. In one experiment the enzyme was added in portions over time, in an attempt to compensate for the loss of activity, but this had an adverse effect (Compare entries 10 and 11).

We noticed that the racemization catalyst slowly lost its activity in a two-phase system. Therefore, in a number of reactions (entries 3, 5, 6, 8, and 9) a small quantity of freshly distilled toluene was added at the outset of the reaction, since it was anticipated that having some organic solvent present from the start could help to reduce the contact between catalyst and water, and thus prolong catalyst lifetime. However, this modification had no effect.

The stirring speed was found to have a major influence on the reaction. When the stirring speed is high, the racemization catalyst is quickly deactivated, and an ordinary kinetic resolution takes place. However, very slow or no stirring leads to a slow enzymatic reaction, presumably due to mass transfer limitations (entry 5). In general, the optimal speed has to be established by repeated experimentation.

The buffer that is used, has some influence on the reaction performance. It had already been established (Table 4.1) that buffers with considerable capacity at pH 8 (the pH optimum for the enzymatic ring closure of 4.3a) were required. Two common buffer salts that satisfy this condition are Tris (2-amino-2-hydroxymethyl-1,3-propanediol) and HEPES, of which HEPES was most extensively used in the studies described in this chapter. Overall, the nature of the buffer does not seem to have a major influence on the course of the reaction. The volume of buffer has some influence, since the concentration of chloride in the solution influences the position of the chloroalcohol − epoxide equilibrium. Therefore, using a larger amount of buffer leads to higher conversions (entries 13 − 16). Release of chloride could also interfere with the racemization catalyst. However, the slow addition of a solution of the activated catalyst using a syringe pump effectively prevents these deleterious interactions, since in most cases the chloroalcohol is at least partly racemized at the end of the reaction (see the column “ee 4.3a”).

During our studies, also the amount of enzyme was varied. Normally, all enzyme was added to the reaction mixture at the beginning of the reaction. An exception is described in entry 11, where the enzyme was added in 4 portions every 1.5h.

120


Chapter 4

Unexpectedly, this approach did not lead to a higher conversion to the epoxide. The addition time of the racemization catalyst was initially varied, but the optimum was found to be 0.5 mL/h. Hence, 6 h are needed for complete addition of the catalyst solution.

In the course of our investigations, it was discovered that the haloalcohol dehalogenases underwent irreversible deactivation and eventually denaturation under the conditions of the reaction. This could be observed visually, first as a turbidity of the reaction mixture that increased as the reaction progressed, and eventually as a definite precipitation in the mixture. Using gel electrophoresis and subsequent MALDI-TOF experiments, it was determined that this precipitation consisted mainly of peptide chains. Some of these chains corresponded to the mass expected for one monomer of the haloalcohol dehalogenase, but other chains showed a lower mass, indicating fragmentation of the peptide chain.

In the absence of racemization catalyst, no such precipitation was observed. A conceivable deactivation pathway for the enzyme could be the initial complexation of the iridium catalyst to certain residues on the enzyme’s surface (notably lysine residues), followed by iridium-assisted fragmentation of the peptide chain.

A practical solution to avoid enzyme deactivation was found in starting the addition of racemization catalyst when the conversion of 4.3a had reached almost 50% (entry 14). This approach led to a significant improvement in product yield. Finally, switching to DMSO as a cosolvent and adding an excess of bovine serum albumine (BSA) to the reaction mixture made it possible to reach full conversion to enantiopure epoxide 4.4a, even when addition of the racemization catalyst was started at the outset of the reaction (entry 16). The reasons for the effect of BSA on the reaction are not fully understood in the present case. Perhaps, BSA helps to diminish interactions between the iridium catalyst and the enzyme at the organic-aqueous interface, by virtue of its abundance in the reaction mixture.d Alternatively, the well-known property of albumin to bind reversibly to a wide variety of ligands could play a role.43

Another possibility to improve enzyme stability, not yet investigated in our system, is the treatment of the enzyme with a nonionic surfactant such as polyoxyethylene(10)-n- cetyl ether (n-C16H33(OCH2CH2)nOH, n ~10; trade name, Brij 56) prior to use in DKR. It is known from the literature that subtilisin shows a much higher activity and stability in the presence of organic solvents after treatment with Brij 56, than when the enzyme is used in its natural state.22

d BSA was added in 10-fold excess (w/w) with respect to the total amount of proteins in the enzyme sample.

121



DKR experiments using 4.3l In addition to our experiments with chloroalcohol 4.3a, bromoalcohol 4.3l was used in the screening of suitable DKR conditions. Conditions were generally analogous to those used for 4.3a. Thus, catalyst precursor 4.8a (5 mol%) and enzyme E4 were used. Substrate 4.3l (0.2 mmol) is typically added to the reaction mixture as a solution in 0.5 mL of DMF or DMSO. Enzymatic ring closure takes place at room temperature in aqueous buffer, however at a pH of 7.5 instead of 8 to suppress non-enzymatic ring closure of the bromoalcohol (vide infra). The racemization catalyst was activated with t-BuOK in a separate vessel in freshly distilled toluene under an atmosphere of nitrogen, after which this solution was slowly added to the reaction mixture. The results of these screening experiments are summarized in Table 4.6.

The use of a bromoalcohol as substrate has a number of consequences for the reaction. For instance, bromoalcohols are more prone to non-selective chemical ring closure than the corresponding chloroalcohols. Therefore, slightly lower ee's are to be expected and the product ee's are expected to diminish over time. The higher reactivity can have the positive effect that the DKR may be finished before the enzyme is completely deactivated, in other words, we were expecting higher conversions than using 4.3a. On the other hand, bromide is a better nucleophile in the ring opening of epoxides than chloride. This should lead to an equilibrium reaction, so 100% conversion cannot be expected when bromoalcohols are employed in this DKR.

The initial conditions we tested using 4.3l were largely based on our previous experiments using 4.3a. DKR experiments of 4.3l at pH 8 led to a low product ee (entries 1 and 2), hence subsequent reactions were conducted at pH 7.5. The conversion generally reaches high values if buffer volumes of ≥10 mL are used (e.g. entries 2,3, and 5). However, when the racemization catalyst is added with 1.0 mL/h instead of 0.5 mL/h, the enzyme is deactivated before high conversions are reached (entry 4).

It is necessary to add the substrate as a solution in a suitable cosolvent, since crystalline 4.3l is not sufficiently soluble in the aqueous reaction medium. As for 4.3a, two different cosolvents were tested in the DKR of 4.3l: DMF (entries 1 − 5) and DMSO (entries 8 − 24). Results with DMSO were superior to those obtained using DMF. Also, DMSO was noticeably better in dissolving 4.3a in the aqueous reaction medium, possibly because of its slightly higher polarity.44 In two experiments, 4.3l was added as a solution in toluene (entries 6 and 7). This led to a constant but low ee if the solution was added at once (entry 6a and b) or to an ee that increased during the course of the reaction but remained low overall if the substrate solution was added dropwise, like the racemization catalyst (entries 7a − c). Using DMSO as cosolvent gave better results, with a conversion of 86% to product 4.4e and an eeP of 90% (entry 8).

122


Chapter 4

Conducting the reaction at pH 7 was expected to show improvements in enantioselectivity, but the results turned out to be comparable with the reaction at pH 7.5 (Compare entries 9 and 10 with entry 8). However, there is an influence on the conversions, which are lower. Presumably, at lower pH the position of the equilibrium shifts to the left. In this particular case, the addition rate of the solution of racemization catalyst (0.5 mL/h vs. 1.0 mL/h) does not have a major influence on enantioselectivity.

Variation of the addition rate of the racemization catalyst was systematically examined at pH 7.5. A gradient in addition rate was tested (gradient from fast addition in the beginning towards slower addition at the end), but this did not have a significant effect (entry 11). The addition rate of the racemization catalyst was varied in two other experiments (entries 12 and 13). These experiments confirm that the epoxide is initially produced with high ee (around 85%), however the ee decreases, since the enzymatic conversion slows down over time, while the rate of chemical ring closure is constant (entry 12b).

Since the rate of enzymatic conversion is proportional to the enzyme concentration, whereas chemical ring closure is constant at a given concentration of substrate, it would be expected that lowering the amount of enzyme leads to a lower ee. Remarkably, however, lowering the amount of enzyme has no influence on the enantioselectivity of ring closure, but only affects the conversion (entry 14).

Table 4.6 DKR experiments using substrate 4.3l, 4.8a, and E4.a

Entry Enz

(μL)b Buffer (mL)

Coc Addn. rate

ml/h t

(h) Conv.(%)

ee 4.3l (%)

ee 4.3l (%)

Miscellaneous

1 50 5 DMF 0.5 16 74 58 54 pH 8 2 50 10 DMF 0.5 17 90 51 79 pH 8 3 50 10 DMF 0.5 16 81 23 87 4 50 10 DMF 1.0 3.5 59 41 78 5 50 20 DMF 0.5 20 89 55 83 6 a 50 10 toluene 0.6 2.5 36 8 77 b 6.5 61 4 77

7 a 50 10 toluene 0.6 3 27 3 54 slow addn. 4.8a, 4.3ld b 4.5 26 4 60 c 22 71 83 68

8 50 10 DMSO 0.5 16 86 65 90 9 50 10 DMSO 0.5 22 75 51 89 pH 7.06

123



Entry Enz

(μL)b Buffer (mL)

Coc Addn. rate

ml/h t

(h) Conv.(%)

ee 4.3l (%)

ee 4.3l (%)

Miscellaneous

10 50 10 DMSO 1.0 6 65 39 90 pH 7.06 11 50 10 DMSO grade 6.5 68 − 86 12 a 100 10 DMSO 1.0 6 79 20 84 b 22 88 75 78

13 a 100 10 DMSO 2.0 1 52 5 85 b 16 84 79 68

14 25 10 DMSO 0.5 30 57 − 87 15 a 50 10+3f DMSO 1.2 3.5 60 10 73 slow add 4.8a, E4f

b 6.5 72 35 70 16 a 50 10+3f DMSO 2.4 1.5 27 6 81 slow add 4.8a, E4f b 23 78 83 67

17 50 10 DMSO 0.6 27.5 74 49 78 IRA-458g 18 a 50 10 DMSO 0.6 4 64 28 82 t-PentOK b 22 84 86 78

19 50 10 DMSO 0.6 23 87 85 71 t-PentOK + IRA-458g 20 50 10 DMSO 1.2 22 90 89 58 HEPES 500 mM 21 a 50 10 DMSO 1.2 2.5 54 12 83 BSAh b 19 85 82 65

22 a 2×50i 10 DMSO 1.2 1.5 52 12 84 BSA + IRA-458g b 22 85 86 62

23 100 10 DMSO 0.6 23 90 84 71 BSA 24 4×25j 10 DMSO 0.6 22 90 87 68 BSA

a) All reactions were allowed to run for a minimum of 16 h. For a general procedure, see experimental section; b) The activity of the enzyme solution was determined to be 370 U/mL prior to reaction by a spectrophotometric assay described in ref. 37; c) In all cases, 0.5 mL of cosolvent was used; d) Both the substrate (dissolved in 3 mL of toluene) and the catalyst solution were slowly added to the reaction mixture; e) A gradient in addition rate was used: 1.5 mL/h for 50 min, then 0.8 mL/h for 75 min, and finally 0.5 mL/h for 90 min; f) Both the racemization catalyst and the enzyme (dissolved in 3 mL of buffer solution) were added slowly; g) Amberlite™ IRA-458 is a strongly basic anion exchange resin; h) Bovine serum albumine; i) Second batch of E4 added after 1.5 h; j) A new batch (25 μL) was added every 30 min.

A number of strategies were tested to improve the stability and activity of the enzyme. Portionwise addition of the enzyme was used (entries 22 and 24) but this did not lead to improvement (compare e.g. entries 23 and 24). Thus, although good conversions are reached, the final ee is on the low side. Also, the slow addition of both the racemization catalyst and the enzyme solution did not increase the ee of the reaction (entries 15 and 16). In these two experiments, also the influence of addition rate was investigated (1.2 mL/h vs. 2.4 mL/h). As in similar experiments (entries 12 an 13), this influence turns out to be minor.

The bromide that is released during ring closure, is not only a nucleophile in the reverse reaction, but it is also an inhibitor of the enzyme.37 Therefore, we investigated the use of an ion exchange resin to capture the released bromide and thus improve conversion

124


Chapter 4

and ee. We used AmberliteTM IRA-458, a commercially available ion exchange resin based on a quaternary ammonium chloride. Since chloride is also a nucleophile in the ring opening of the epoxide, as well as an inhibitor of the enzyme,e the chloride was replaced by sulfate by repeated washing with concentrated sulfuric acid, after which the resin was used as an additive in DKR experiments (Table 4.6, entries 17, 19, and 22). However, no improvement on the reaction was observed.

To diminish interactions of the racemization catalyst and the enzyme, the racemization catalyst was activated using potassium tert-pentoxide, which is more hydrophobic than t-BuOK and should give the catalyst more affinity for the organic phase. However, it can be seen in entries 18 and 19 that this approach was unsuccessful. It is known that HheC-type enzymes generally are more stable in media with a high salt concentration.45 However, in our hands a 10-fold increase in buffer strength led to a disappointing enantioselectivity (entry 20). Notably, the addition of BSA did not lead to improvement in results, contrary to the DKR of 4.3a. In entries 21 − 24 it is shown that, although conversions of 90% are reached, the ee of the product is rather low, despite addition of BSA and addition of the enzyme in portions.

Table 4.7 DKR of 4.3l using various enzymes.a

Entry Enzymeb Cosolventc Add

(mL/h) t (h)

Conv (%)

ee 4.3l ee 4.4e

1 a E1 DMF 0.5 16 91 23 41 2 a E3 DMSO 2.0 4.5 74 25 82 b 21 87 79 73 3 a E3 DMSO 4.0 1 49 11 91 b 4.5 72 53 77 c 21 86 93 63

a) For a general procedure, see the experimental section; b) The activity of enzyme added to the reactions was 18.5 U, determined prior to reaction under standard enzymatic kinetic resolution conditions using the photospectrometric method described in Ref. 37; c) 0.5 mL of the indicated cosolvent was used.

When the reactions are followed over time (the results are shown for a number of experiments in Table 4.6), it can be seen that often the epoxide is initially produced

e According to Ref. 37, the inhibition constant I50 for the ring closure of 250 μM (R)-4.3e (i.e. the nucleophile concentration at which the initial rate of ring closure of 250 μM (R)-4.3e is 50% of the initial rate in the absence of the nucleophile) is 4.3 mM for Br− and 13 mM for Cl−.

125



with high enantioselectivity, while in a later stage the ee drops. This effect seems to be most pronounced when high addition rates of the racemization catalyst are used (entries 13, 16, and 22).

A number of DKR experiments of 4.3l were done with enzymes E1 and E3. The results are listed in Table 4.7. Conditions are analogous to those used in Table 4.6.

A good conversion but low ee are obtained when wild-type HheC (E1) is used in this DKR (entry 1). This finding confirmed what was already predicted based on earlier kinetic resolution experiments (Table 4.2), although the effect is slightly more pronounced than expected. The use of the highly enantioselective Trp249Phe mutant of HheC (E3) did not lead to a breakthrough in our DKR experiments (entries 2 and 3). It was observed that enzyme E3 was more susceptible than E4 to the denaturation phenomena described earlier. This can also be concluded from the fact that the enantioselectivity of the ring closure strongly decreased during the course of the reaction (entries 2 and 3). This observation led to the choice of employing primarily E4 in our DKR experiments.

In conclusion, the best results for the DKR of 4.3l are given in Table 4.6, entry 8, employing enzyme E4 and Ir-catalyst 4.8a (86% conversion, 90% ee).

Dynamic kinetic resolution using other enzymes, racemization catalysts, and substrates. The modified HheC variant E5 was also tested as the enzyme in this DKR. The results are depicted in Table 4.8. Using substrate 4.3a, a very low conversion is reached (entry 1). Also with 4.3l, conversions are disappointing (maximum 80%, entry 3b), although ee's are good (e.g. entries 2 and 4a).

Furthermore, the two Ru-based catalysts 4.118 and 4.219 were tested in the DKR of substrate 4.3l. These experiments showed that the performance of 4.8b, 4.1, and 4.2 was inferior to 4.8a, as illustrated in Table 4.9. It can be seen that under biphasic DKR conditions, these racemization catalysts are quickly deactivated (see column “ee 4.3l”), leading to a low product ee when the conversion exceeds 50% (entries 2, 3c, and 4c).

Finally, all substrates 4.3, listed in Figure 4.2, were tested using the optimal conditions established for 4.3l. The best results for each substrate are given in Table 4.10.

Styrene oxide 4.4a was obtained from 4.3a in 90% conversion and with an excellent ee of >98% (entry 1; see also Table 4.5). Product 4.4b (2-(4-fluorophenyl)oxirane) was formed in low ee (entry 2). Chloroalcohols 4.3c – 4.3g were converted to their corresponding epoxides with excellent ee (91 – 98%, entries 3 – 8). However, the conversions were dependent on the substrate, ranging from 28% for the sterically hindered o-nitro substituted 4.3g to 80% for its p-nitro equivalent 4.3e. Substrate 4.3e

126


Chapter 4

was also reacted at a scale of 1 mmol, with similar results compared to the reaction on analytical scale (entry 6). Using m-methoxy substituted 4.3h, epoxide 4.4h was obtained in 64% conversion and with aan ee of 85% (entry 9). The aliphatic chloroalcohol 4.3i was subjected to DKR conditions, but could not be racemized using 4.8a, resulting in a static enzymatic kinetic resolution (entry 10).

Table 4.8 DKR experiments performed with E5.a

Entry Substrate Addn. rate

(mL/h) t

(h) conv. (%)

ee 4.3

(%) ee 4.4 (%)

Miscellaneous

1 4.3a 0.6 22 25 39 >98 2 a 4.3l 0.6 6 60 9 89 pH 7 b 22 62 12 88 3 a 4.3l 0.6 5.5 70 6 82 b 24 80 12 78 4 a 4.3l 1.2 3 57 8 85 b 22 69 27 80

a) For a general procedure, see the experimental section.

Table 4.9 Attempted DKR of 4.3l using catalysts 4.1, 4.2, and 4.8b.a

Entry Catalyst Add.

(mL/h) t

(h) Conv. (%)

ee 4.3l (%)

ee 4.4e (%)

1 a 4.1 2.0 2.5 56 83 72 b 6.5 62 93 59 c 22.5 73 93 35 2 a 4.2 2.0 1.5 53 76 87 b 4.5 57 92 71 c 22 65 >98 51 3 a 4.8a 2.0 4.5 74 25 82 b 21 87 79 73 4 4.8b 1.0 16 90 >98 13

a) For a general procedure, see the experimental section.

127



Compared to chloroalcohols, bromoalcohols 4.3j – 4.3q in general lead to lower ee's, due to their higher rate of chemical ring closure leading to the racemic product. Furthermore, conversions are often incomplete, due to the reversibility of the reaction. For instance, styrene oxide 4.4a is obtained from 4.3j in 57% conversion and with an ee of 56% (entry 11, cf. entry 1). Better results were obtained using 4-(2-bromo-1-hydroxyethyl)benzonitrile (4.3k), which was converted to the corresponding epoxide 4.4d in 89% conv. and 86% ee (entry 12, cf. entry 4).

The DKR of 4.3l resulted in a conversion of epoxide 86% to 4.4e, with an ee of 90% (Table 4.10, entry 13, cf. Table 4.6, entry 5). Epoxide 4.4f (2-(3-nitrophenyl)oxirane) was obtained from bromoalcohol 4.3m with good conversion (86%), but low ee (43%, entry 14). The ee of substrate 4.3m is 4% at this point, indicating an efficient racemization reaction. Therefore, we conclude that the low ee of product 4.4f is due to a lack of enantioselectivity of the enzymatic conversion using substrate 4.3m.

More detailed data were obtained using 2-bromo-1-(naphthalen-2-yl)ethanol (4.3n). The E-value for 4.3n using E4 is 40. The racemization of 4.3n was tested in toluene, using an equimolar mixture of S-4.3n and R-4.4j (both ee > 98%) and 5 mol% of 4.8a (w.r.t. S-4.3n) activated with one equivalent of t-BuOK. After 1 h and 20 min, the ee of 4.3n had decreased to 24%, however the ratio of 4.4j vs. 4.3n was now found to be 87:13, leading us to conclude that this substrate is rather susceptible to non-selective ring closure. After 18 h, the ratio 4.4j / 4.3n ratio again was found to be 87:13, whereas 4.3n had an ee of 8%. The DKR results for 4.3n are summarized in Table 4.10, entry 15. Despite racemization of the substrate, the conversion to the epoxide does not exceed 41%. When the reaction is allowed to proceed overnight, the conversion stays the same while the product ee drops from 82% (entry 15) to 66% (not shown in the table). The most likely explanation is that the reaction 4.3n → 4.4j has reached equilibrium at a conversion of 41%. A reaction conducted at pH 8 (not shown in the table) led to a conversion to 4.4j of 83%, but with a disappointing ee of 13%.

Also using 4.3o, we experienced difficulties with the racemization reaction, as outlined in Table 4.10, entry 16. Product 4.4k was unstable under the conditions of this DKR, it probably hydrolyzed (cf. the epoxides in Chapter 3). However, the kinetic resolution of substrate 4.3q is highly efficient, the remaining enantiomer S-4.3q having an ee of >98% (entry 10).

For 4.3p, a chiral separation method could only be established for product 4.4l, not for the starting materials. Thus, information concerning racemization has to be inferred from the data shown in entry 17. At near-neutral pH, the epoxide 4.4l is obtained with an ee of 82% but the conversion is only 42%, amounting to an ordinary kinetic resolution of substrate 4.3p (entry 17). In a reaction at pH 8, a conversion of 57% was reached, but in this case the ee is only 40%, indicating a large contribution of chemical

128


Chapter 4

ring closure to the formation of the product (not shown in the table). Probably, racemization is slow or even absent for this substrate.

Table 4.10 DKR of all investigated substrates.a

Entry Substrateb t

(h) Conv. (%)

Ee 4.3 (%)

Product Ee 4.4 (%)

Miscellaneous

1 4.3a 16 90 >98 4.4a 98 2 4.3b 22 60 72 4.4b 78 3 4.3c 16 58 n.d.c 4.4c 98 4 4.3d 16 67 n.d.c 4.4d 95 5 4.3e 16 80 78 4.4e 95 6 4.3e 24 76 49 4.4e 97 1 mmol scaled 7 4.3f 16 75 n.d.c 4.4f 97 8 4.3g 16 28 n.d.c 4.4g 91 9 4.3h 16 64 n.d.c 4.4h 85 10 4.3i 16 50 84 4.4i 72 11 4.3j 21 57 >98 4.4a 56 12 4.3k 18 89 78 4.4d 86 13 4.3l 16 86 65 4.4e 90 14 4.3m 16 79 16 4.4f 52 15 4.3n 5.5 41 30 4.4j 82 16 4.3o 6 48 84 4.4k 46 17 4.3p 5 46 n.d.c 4.4l 82 18 4.3q 6 50 >98 No producte No producte

a) Reactions were performed on 200 μmol scale and run overnight. If the indicated time is shorter than that, the optimal results are shown and further reaction did not improve them. The catalyst solution was added at a rate of 0.6 mL/h. 6 U of enzyme were used, enzyme activity was routinely measured on substrate 4.3l using the photospectrometric method described in Ref. 37; c) Not determined; d) Reaction performed at 1 mmol scale. 300 μL of enzyme solution were initially added, after 16h 100 μL extra enzyme was added; e) No product was obtained.

129



Finally, the product of the ring closure of 4.3q is hydrolytically unstable. Under DKR conditions, no product was obtained, but a highly efficient enzymatic kinetic resolution, leading to (S)-4.3q in >98% ee (entry 18, see Chapter 3 for similar examples of enzymatic kinetic resolution).

To summarize, numerous variations on this general scheme have been tested in the course of our studies. These variations include changes in racemization catalyst, catalyst addition rate, substrate structure, buffer composition, buffer strength, pH, and volume. HEPES buffer at 50 mM was found to give best results, the pH optimum is 7.5 for bromoalcohols and 8.0 for chloroalcohols, and, using 0.2 mmol of substrate and 0.5 mL of DMF, a buffer volume of 10 mL was found to be optimal (using more has little effect, using less complicates reactions by allowing unfavorable equilibria to establish). DMSO as a cosolvent was found to be compatible with our reaction conditions. When the use of cosolvent was completely avoided, both rate and enantioselectivity of the reaction were disappointingly low. Using these optimzed conditions, epoxides can be obtained in high conversion and with excellent ee from racemic haloalcohols (Table 4.10, in particular entries 1, 3 – 7, 9, 12, and 13).


A dynamic kinetic resolution has been developed that uses a haloalcohol dehalogenase in combination with a novel iridium catalyst for racemization. This is the first chemoenzymatic DKR using haloalcohol dehalogenases, giving optically active epoxides from racemic haloalcohols in a single step. The optimal results we obtained for all substrates 4.3 are summarized Table 4.10.

A future development might consist of lipase-catalyzed acylation of vicinal haloalcohols, followed by ring closure to the corresponding epoxides, ideally in a one-pot procedure. Although preliminary experiments with 4.3a, 4.8a and Novozym 43516 have not yet led to the identification of a suitable combination of acyl donor, lipase and racemization catalyst, the approach is expected to work based on various examples in the literature.4

Scheme 4.13 Enantiopure epoxides from haloketones.

130


Chapter 4

This DKR might be extended by using catalyst 4.8a or a similar complex as catalyst for transfer hydrogenation of the corresponding haloketones and as racemization catalysts of the resulting haloalcohols. Previous research by Park et al. indicates that this approach could be practical.24 The sequence of reactions, which would constitute a novel and competitive method for the preparation enantiopure epoxides from haloketones, is outlined in Scheme 4.13. It would be complementary to other chemoenzymatic procedures for the preparation of enantiopure epoxides from haloketones.46


4.4.1 General remarks For general information, see Chapters 2 and 3.

4.4.2 Synthesis of haloalcohols 4.3 Racemic 4.3a is available from Alfa Aesar.

The synthesis of 4.3c, 4.3d, and 4.3f – 4.3h was performed by F. Berthiol.

Haloalcohols 4.3b and 4.3j – 4.3q were synthesized by reduction of the corresponding haloketones using sodium borohydride. Purification was achieved by recrystallization from CH2Cl2 / n-pentane in the case of solid compounds. In those cases where the crude products were oils, they were purified by column chromatography over silica gel using mixtures of n-pentane and Et2O as eluent. A typical procedure is given:

2-Bromo-1-(4-bromophenyl)ethanone (5.02 g, 18.06 mmol) was dissolved in 100 mL of MeOH. This solution was brought to 0 °C using an ice bath. Subsequently, NaBH4 (205 mg, 5.42 mmol) was added in portions. After the addition, the ice bath was removed and the mixture was allowed to stir for one hour at rt. The reaction mixture was then concentrated in vacuo and subsequently diluted with water. This aqueous mixture was extracted with Et2O (3x), the combined organic fractions were washed with aqueous NH4Cl (sat.) and brine, respectively, and dried over MgSO4. After filtration, the solvent was removed under reduced pressure. The crude product was purified by recrystallization from CH2Cl2 / n-pentane, yielding 4.775 g (17.1 mmol, 94%) of 4.3p.

Haloalcohols 4.3e – 4.3g were synthesized by ring opening of the corresponding epoxides using copper(II) chloride and lithium chloride.30 Purification was achieved by recrystallization from CH2Cl2 / n-pentane in the case of solid compounds. In case the

131



crude products were oils, they were purified by column chromatography over silica gel using mixtures of n-pentane and AcOEt as eluent. A typical procedure is given for 4.3e:

A flame-dried Schlenk vessel under an atmosphere of nitrogen was charged with LiCl (1.05 g, 25 mmol) and CuCl2 (1.68 g, 12.5 mmol). Subsequently, freshly distilled THF (22 mL) was added. After stirring the mixture for 30 min at rt, a solution of p-nitrostyrene oxide 4.4e (825 mg, 5 mmol) in 5 mL of freshly distilled THF was added dropwise to the reaction. The mixture was allowed to stir at room temperature for 16 h, after which the reaction was quenched by the addition of aqueous HEPES buffer (50 mM, pH 7) and extracted with Et2O (3×). The combined organic layers were washed with brine, dried over MgSO4, filtered and concentrated in vacuo. The crude product (944 mg) was recrystallized from CH2Cl2 / n-pentane, giving 743 mg (3.69 mmol, 74%) of pure 4.3e.

Haloalcohols 4.3c, 4.3d, 4.3h, and 4.3i were synthesized by addition of chloromethyllithium to the corresponding aldehydes, using chloroiodomethane and n-butyllithium.29 Purification was achieved by column chromatography over silica gel using mixtures of n-pentane and AcOEt as eluent. A typical procedure is given for 4.3h:

A flame-dried Schlenk vessel was charged with m-anisaldehyde (1.5 g, 11 mmol) and freshly distilled THF (30 mL) was added. The mixture was then stirred at −78 °C, and chloroiodomethane (2.91 g, 16.5 mmol) was added to the mixture. Then n-butyllithium (10.4 mL of a 1.6M solution in hexane, 1.5 equiv.) was added dropwise by syringe over 30 min. After addition, the mixture was stirred at −78 °C for 1 h, after which the reaction was quenched by the addition of 30 mL of aqueous NH4Cl (sat.) and the mixture was allowed to warm to room temperature. After addition of 20 mL of Et2O to the mixture, the phases were separated, and aqueous layer was extracted with Et2O (2×). The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo. The crude product was purified by column chromatography over silica gel (n-pentane / AcOEt 19:1), yielding 412 mg of m-anisaldehyde (27% recovery) and 1.49 g (8 mmol, 72%) of pure 4.3h.

2-Chloro-1-phenylethanol (4.3a)25

1H NMR (CDCl3) δ 7.38 − 7.29 (m, 5H), 4.88 (dt, J = 8.8, 3.3 Hz, 1H), 3.73 (dd, J = 11.0, 3.3 Hz, 1H), 3.63 (dd, J = 11.0, 8.8 Hz, 1H), 2.68 (d, J = 3.3 Hz, 1H); 13C NMR (CDCl3) δ 139.9 (s), 128.6 (d), 128.4 (d), 126.0 (d), 74.0 (d), 50.8 (t); MS (EI+) m/z = 156 (M+), 152, 141, 139, 107, 105, 91, 79, 77, 51; chiral GC: Chiraldex G-TA, 30 m x 0.25 mm x 0.25 μm, He-flow: 0.5 mL/min, temperature program: start 50°C, 10°C/min to

132


Chapter 4

95°C, hold 10 min, 10°C/min to 125°C, hold 20 min, 10°C/min to 50°C, Tr = 11.7 min (S-4.4a), 14.1 min (R-4.4a), 24.4 min (R-4.3a), 26.1 min (S-4.3a).f

2-Chloro-1-(4-fluoro-phenyl)-ethanol (4.3b)25

1H NMR (CDCl3) δ 7.36 − 7.32 (AA'MM' m, 2H), 7.07 − 7.02 (AA'MM' m, 2H), 4.86 (dd, J = 8.8, 3.3 Hz, 1H), 3.69 (dd, J = 11.0, 3.3 Hz, 1H), 3.59 (dd, J = 11.0, 8.8 Hz, 1H), 2.69 (s, 1H); 13C NMR (CDCl3) δ 162.6 (d, 1JC-F = 246.8 Hz), 135.7 (d, 4JC-F = 3.3 Hz), 127.8 (dd, 3JC-F = 8.1 Hz), 115.5 (dd, 2JC-F = 21.4 Hz), 73.4 (d), 50.7 (t); MS (EI+) m/z = 174 (M+), 126, 125, 123, 97, 95, 77; HRMS (EI+) calcd. for C8H835ClFO: 174.0248, measured: 174.0240; chiral chiral GC: Chiraldex G-TA, 30 m x 0.25 mm x 0.25 μm, He-flow: 0.5 mL/min, temperature program: start 50°C, 10°C/min to 95°C, hold 10 min, 10°C/min to 125°C, hold 20 min, 10°C/min to 50°C, Tr = 12.1 min (R-4.4b), 13.1 (S-4.4b), 23.6 (R-4.3b), 26.1 (S-4.3b), or: Chiraldex B-PM, 30 m x 0.25 mm x 0.25 μm, He-flow: 1.1 mL/min, 50°C to 180°C 5°C/min, hold 5 min, 10°C/min to 50°C, Tr = 21.7 min (4.4b), 22.1 min (4.4b), 23.4 min (4.3b), 23.9 min (4.3b).

2-Chloro-1-(4-trifluoromethyl-phenyl)-ethanol (4.3c)

1H NMR (CDCl3) δ 7.62 (d, J = 8.0 Hz, 2H), 7.48 (d, J = 8.0 Hz, 2H), 4.93 (dd, J = 8.0, 3.6 Hz, 1H), 3.73 (dd, J = 11.6, 3.6 Hz, 1H), 3.52 (dd, J = 11.6, 8.0 Hz, 1H), 3.36 (s, 1H); 13C NMR (CDCl3) δ 144.1, 130.8 (q, 2JC-F = 32.3 Hz), 126.7, 125.8 (q, 3JC-

F = 3.7 Hz), 130.8 (q, 1JC-F = 269.9 Hz), 73.6, 50.6; HRMS (EI+) calcd. for C9H8OF335Cl: 224.0216, found: 224.0225; chiral HPLC: Chiralpak AS, 40 °C, n-heptane/IPA 100:0, 1.0 mL/min, Tr = 8.9 min (R-4.4c), 10.8 min (S-4.4c), 56.0 min (R-4.3c), 60.9 min (S-4.3c).

f Varying amounts of 2-phenylacetaldehyde - a common rearrangement product of styrene oxide - were observed in GC measurements (Tr = 12.4 min). This compound was never observed using other techniques of analysis (e.g. NMR), furthermore a chemically pure sample of styrene oxide also shows a peak of phenylacetaldehyde on GC. Therefore, we conclude that the rearrangement resulting in the formation of 2-phenylacetaldehyde takes place on the GC column. See also J. W. de Boer, cis-Dihydroxylation and Epoxidation of Alkenes by Manganese Catalysts, PhD thesis, University of Groningen, 2008.

133



4-(2-Chloro-1-hydroxy-ethyl)-benzonitrile (4.3d)

1H NMR (CDCl3) δ 7.59 (d, J = 8.0 Hz, 2H), 7.48 (d, J = 8.04 Hz, 2H), 4.94 (dd, J = 7.6, 3.6 Hz, 1H), 3.70 (dd, J = 11.2, 4.0 Hz, 1H), 3.60 (dd, J = 11.2, 8.0 Hz, 1H), 3.53 (s, 1H); 13C NMR (CDCl3) δ 145.9, 132.6, 127.2, 118.9, 111.9, 73.3, 50.2; HRMS (EI+) calcd. for C9H8NOCl: 181.0294, found: 181.0305; chiral HPLC: Chiralpak AD, 40°C, n-heptane/IPA 99.25:0.75 20 min, gradient (1 min) to 95:5, hold 24 min, gradient (1 min) to 99.25:0.75, hold 14 min (total time: 60 min), 1.0 mL/min, Tr = 19.5 min (R-4.4d), 21.3 min (S-4.4d), 47.0 min (R-4.3d), 48.9 min (S-4.3d).

2-Chloro-1-(4-nitro-phenyl)-ethanol (4.3e)30

1H NMR (CDCl3) δ 8.22 (AA'XX' d, Japp = 8.8 Hz, 2H), 7.57 (AA'XX' d, Japp = 8.8 Hz, 2H), 5.02 (dt, J = 8.1, 3.7 Hz, 1H), 3.77 (dd, J = 11.4, 3.7 Hz, 1H), 3.62 (dd, J = 11.4, 8.1 Hz, 1H), 2.83 (d, J = 3.7 Hz, 1H); 13C NMR (CDCl3) δ 146.8 (s), 127.0 (d), 123.8 (d), 73.0 (d), 50.3 (t); MS (EI+) m/z = 201 (M+), 152, 106, 94, 77, 63, 51; HRMS (EI+) calcd. for C8H835ClNO3: 201.0192, found: 201.0204; chiral HPLC: Chiralcel AS, 40 °C, n-heptane/IPA 93:7, 1.0 mL/min, Tr = 10.0 min (R-4.4e), 13.1 min (S-4.4e), 17.9 min (R-4.3e), 21.3 min (S-4.3e).

2-Chloro-1-(3-nitro-phenyl)-ethanol (4.3f)47

1H NMR (CDCl3) δ 8.26 (s, 1H), 8.16 (d, J = 8.1 Hz, 1H), 7.73 (d, J = 7.5 Hz, 1H), 7.55 (t, J = 8.1 Hz, 1H), 5.03 (dd, J = 8.1 Hz, 1H), 3.78 (dd, J = 11.1, 3.9 Hz, 1H), 3.66 (dd, J = 11.1, 8.1 Hz, 1H), 3.27 (s, 1H); 13C NMR (CDCl3) δ 148.2, 142.4, 132.5, 129.9, 123.5, 121.5, 73.1, 50.5; HRMS (EI+) calcd. for C8H835ClNO3: 201.0193, found: 201.0202; Chiralpak AD, 40°C, n-heptane/IPA 99.25:0.75 20 min, gradient (1 min) to 95:5, hold 24 min, gradient (1 min) to 99.25:0.75, hold 14 min (total time: 60 min), 1.0 mL/min, Tr = 15.6 min (S-4.4f), 17.6 min (R-4.4f), 39.0 min (R-4.3f), 41.1 min (S-4.3f).

134


Chapter 4

2-Chloro-1-(2-nitro-phenyl)-ethanol (4.3g)48

1H NMR (CDCl3) δ 7.99 (d, J = 7.8 Hz, 1H), 7.88 (d, J = 7.6 Hz, 1H), 7.69 (t, J = 7.8 Hz, 1H), 7.49 (t, J = 7.8 Hz, 1H), 5.48 (d, J = 6.8 Hz, 1H), 4.01 (dd, J = 11.1, 3.0 Hz, 1H), 3.66 (dd, J = 11.1, 7.8 Hz, 1H), 3.10 (s, 1H); 13C NMR (CDCl3) δ 147.9, 135.7, 134.1, 129.4, 128.9, 125.0, 69.7, 50.2; Elem. Anal.: calcd. C, 47.66; H, 4.00; N, 6.95; found C, 48.13; H, 4.04; N, 6.86; chiral HPLC: Chiralpak AS, 40 °C, n-heptane/IPA 93:7, 1.0 mL/min, Tr = 5.7 min (R-4.4g), 6.5 min (S-4.4g), 10.0 min (R-4.3g), 10.7 min (S-4.3g).

2-Chloro-1-(3-methoxy-phenyl)-ethanol (4.3h)49

1H NMR (CDCl3) δ 7.25 (t, J = 8.0 Hz, 1H), 7.02 - 6.92 (m, 3H), 4.90 - 4.72 (m, 1H), 4.00 - 3.27 (m, 6H); 13C NMR (CDCl3) δ 160.0, 142.2, 129.9, 118.7, 114.1, 112.0, 74.2, 55.5, 50.7; HRMS (EI+) calcd. for C9H1135ClO2: 186.0448, measured: 186.0455; chiral HPLC: Chiralpak AS, 40 °C, n-heptane/IPA 99:1, 1.0 mL/min, Tr = 8.1 min (R-4.4h), 11.5 min (S-4.4h), 24.6 min (R-4.3h), 25.9 min (S-4.3h).

2-Chloro-1-cyclohexyl-ethanol (4.3i)50

1H NMR (CDCl3) δ 3.68 (d, J = 8.8 Hz, 1H), 3.54 (d, J = 8.8 Hz, 1H), 2.09 (d, J = 4.4 Hz, 1H), 1.89 (d, J = 12.8 Hz, 1H), 1.76 − 1.72 (m, 2H), 1.67 − 1.63 (m, 2H), 1.54 − 1.44 (m, 1H), 1.34 − 1.02 (m, 6H); 13C NMR (CDCl3) δ 75.6 (d), 49.1 (t), 41.2 (d), 28.9 (t), 28.3 (t), 26.2 (t), 26.0 (t), 25.9 (t); MS (EI+) m/z = 113, 105, 95, 83, 82, 69, 67, 55; MS (CI+) m/z = 180 (M+NH4+); HRMS (EI+) calcd. for C7H13O+: 113.0966, found: 113.0962; chiral GC: CP Chiralsil Dex CB, 25 m x 0.25 mm x 0.25 μm, He-flow: 1.0 mL/min, temperature program: start 50°C, 5°C/min to 103°C, hold 5 min, 10°C/min to 160°C, hold 5 min, 10°C/min to 50°C, Tr = 13.8 min (R-4.4i), 14.0 min (S-4.4i), 22.1 min (S-4.3i), 23.2 min (R-4.3i).

135



2-Bromo-1-phenylethanol (4.3j)25

1H NMR (CDCl3) δ 7.38 − 7.29 (m, 5H), 4.91 (dt, J = 8.8, 3.3 Hz, 1H), 3.63 (dd, J = 10.6, 3.3 Hz, 1H), 3.53 (dd, J = 10.6, 8.8 Hz, 1H), 2.63 (d, J = 2.9 Hz, 1H); 13C NMR (CDCl3) δ 140.3 (s), 128.6 (d), 128.4 (d), 125.9 (d), 73.7 (d), 40.1 (t); MS (EI+) m/z = 202 (M+), 200 (M+), 107, 103, 91, 79; HRMS (EI+) calcd. for C8H979BrO: 199.9836, found: 199.9827; chiral GC: Chiraldex G-TA, 30 m x 0.25 mm x 0.25 μm, He-flow: 0.5 mL/min, temperature program: start 50°C, 10°C/min to 95°C, hold 10 min, 10°C/min to 125°C, hold 20 min, 10°C/min to 50°C, Tr = 11.7 min (S-4.4a), 14.1 min (R-4.4a), 30.6 min (R-4.3j), 32.0 min (S-4.3j).

4-(2-Bromo-1-hydroxy-ethyl)-benzonitrile (4.3k)51

1H NMR (CDCl3) δ 7.66 (AA'BB' d, Japp = 8.4 Hz, 2H), 7.50 (AA'BB' d, Japp = 8.4 Hz, 2H), 4.97 (dt, J = 8.4, 3.3 Hz, 1H), 3.63 (dd, J = 10.6, 3.7 Hz, 1H), 3.48 (dd, J = 10.6, 8.4 Hz, 1H), 2.75 (d, J = 3.7 Hz, 1H); 13C NMR (CDCl3) δ 145.5 (s), 132.4 (d), 126.7 (d), 118.5 (s), 112.0 (s), 72.7 (d), 39.3 (t); MS (EI+) m/z = 227 (M+), 225 (M+), 132, 104, 89, 77, 51; HRMS (EI+) calcd. for C9H879BrNO: 224.9789, found: 224.9798; chiral HPLC: Chiralpak AD, 40°C, n-heptane/IPA 99.25:0.75 20 min, gradient (1 min) to 95:5, hold 24 min, gradient (1 min) to 99.25:0.75, hold 14 min (total time: 60 min), 1.0 mL/min, Tr = 19.5 min (R-4.4d), 21.3 min (S-4.4d), 47.9 min (R-4.3k), 50.9 min (S-4.3k).

2-Bromo-1-(4-nitro-phenyl)-ethanol (4.3l)28

1H NMR (CDCl3) δ 8.20 (AA'XX' d, Japp = 9.0 Hz, 2H), 7.56 (AA'XX' d, Japp = 8.8 Hz, 2H), 5.03 (dt, J = 8.4, 3.7 Hz, 1H), 3.65 (dd, J = 10.6, 3.7 Hz, 1H), 3.51 (dd, J = 10.6, 8.4 Hz, 1H), 2.84 (d, J = 3.7 Hz, 1H); 13C NMR (CDCl3) δ 147.7 (s), 147.3 (s), 126.9 (d), 123.8 (d), 72.6 (d), 39.3 (t); MS (EI+) m/z = 248 (M+), 247 (M+), 246 (M+), 245 (M+), 153, 152, 122, 106, 105, 94, 91, 78, 77, 51; HRMS (EI+) calcd. for C8H881BrNO3: 246.9667, found: 246.9678; chiral HPLC: Chiralpak AS, 40 °C, n-heptane/IPA 93:7, 1.0 mL/min, Tr = 10.1 min (R-4.4e), 13.3 min (S-4.4e), 18.0 min (R-4.3l), 21.1 min (S-4.3l).

136


Chapter 4

2-Bromo-1-(3-nitro-phenyl)-ethanol (4.3m)52

1H NMR (CDCl3) δ 8.27 (t, J = 1.8 Hz, 1H), 8.17 (m, 1H), 7.72 (d, J = 7.7 Hz, 1H), 7.55 (m, 1H), 5.04 (ddd, J = 8.4, 3.6, 3.3 Hz, 1H), 3.67 (dd, J = 10.6, 3.3 Hz, 1H), 3.53 (dd, J = 10.6, 8.4 Hz, 1H), 2.79 (d, J = 3.7 Hz, 1H); 13C NMR (CDCl3) δ 148.2 (s), 142.4 (s), 132.1 (d), 129.6 (d), 123.2 (d), 121.1 (d), 72.4 (d), 39.3 (t); MS (EI+) m/z = 247 (M+), 245 (M+), 152, 121, 105, 94, 91, 77, 65, 51; HRMS (EI+) calcd. for C8H879BrNO3: 244.9687, found: 244.9688; chiral HPLC: Chiralpak AD, 40°C, n-heptane/IPA 99.25:0.75 20 min, gradient (1 min) to 95:5, hold 24 min, gradient (1 min) to 99.25:0.75, hold 14 min (total time: 60 min), 1.0 mL/min, Tr = 15.6 min (S-4.4f), 17.6 min (R-4.4f), 40.0 min (R-4.3m), 42.3 min (S-4.3m).

2-Bromo-1-naphthalen-2-yl-ethanol (4.3n)51

1H NMR (CDCl3) δ 7.84 – 7.82 (m, 4H), 7.51 – 7.43 (m, 3H), 5.06 (m, 1H), 3.69 (dd, J = 10.6, 3.3 Hz, 1H), 3.61 (dd, J = 10.6, 8.8 Hz, 1H), 2.84 (d, J = 3.3 Hz, 1H); 13C NMR (CDCl3) δ 137.6 (s), 133.2 (s), 133.1 (s), 128.5 (d), 128.0 (d), 127.7 (d), 126.4 (d), 126.2 (d), 125.1 (d), 123.5 (d), 73.8 (d), 40.0 (t); MS (EI+) m/z = 252 (M+), 250 (M+), 158, 157, 153, 141, 129, 128, 127, 77; HRMS (EI+) calcd. for C12H1179BrO: 249.9993, found: 250.0005; chiral HPLC: Chiralpak AS, 40°C, n-heptane/IPA 99:1, 1.0 mL/min, Tr = 7.9 min (R-4.4j), 9.2 min (S-4.4j), 23.9 min (S-4.3n), 27.2 min (R-4.3n).

2-Bromo-1-p-tolyl-ethanol (4.3o)52

1H NMR (CDCl3) δ 7.25 (AA'BB' d, Japp = 8.1 Hz, 2H), 7.17 (AA'BB' d, Japp = 8.1 Hz, 2H), 4.87 (dt, J = 8.8, 3.3 Hz, 1H), 3.60 (dd, J = 10.3, 3.3 Hz, 1H), 3.52 (dd, J = 10.3, 8.8 Hz, 1H), 2.57 (d, J = 3.3 Hz, 1H), 2.33 (s, 3H); 13C NMR (CDCl3) δ 138.1 (s), 137.3 (s), 129.2 (d), 125.8 (d), 73.5 (d), 39.9 (t), 21.1 (q); MS (EI+) m/z = 216 (M+), 214 (M+), 121, 93, 91, 85, 83, 77, 65, 51; HRMS (EI+) calcd. for C9H1181BrO: 215.9973, found: 215.9967; chiral HPLC: Chiralcel OD, 40°C, n-heptane/IPA 99:1, 1.0 mL/min, Tr = 5.5 min (S-4.4k), 5.9 min (R-4.4k), 21.4 (S-4.3o) min, 24.5 min (R-4.3o).

137



2-Bromo-1-(4-bromo-phenyl)-ethanol (4.3p)51

1H NMR (CDCl3) δ 7.47 (AA'MM' d, Japp = 8.4 Hz, 2H), 7.23 (AA'MM' d, Japp = 8.4 Hz, 2H), 4.85 (dt, J = 8.4, 3.3 Hz, 1H), 3.57 (dd, J = 10.3, 3.3 Hz, 1H), 3.46 (dd, J = 9.9, 9.2 Hz, 1H), 2.71 (d, J = 3.3 Hz, 1H); 13C NMR (CDCl3) δ 139.2 (s), 131.7 (d), 127.6 (d), 122.3 (s), 73.0 (d), 39.8 (t); MS (EI+) m/z = 282 (M+), 280 (M+), 278 (M+), 187, 185, 159, 157, 91, 78, 77, 51; HRMS (EI+) calcd. for C8H879Br2O: 277.8941, found: 277.8954; chiral HPLC: Chiralpak AS, 40°C, n-heptane/IPA 98:2, 1.0 mL/min, Tr = 6.2 min (R-4.4l), 7.9 min (S-4.4l), 14.9 min (4.3p).

2-Chloro-1-(4-methoxy-phenyl)-ethanol (4.3q)25

1H NMR (CDCl3) δ 7.28 (AA'XX' d, Japp = 8.8 Hz, 2H), 6.88 (AA'XX' d, Japp = 8.8 Hz, 2H), 4.85 (dd, J = 7.3, 3.7 Hz, 1H), 3.78 (s, 3H), 3.58 (dd, J = 10.3, 3.7 Hz, 1H), 3.51 (dd, J = 10.6, 8.8 Hz, 1H), 2.59 (d, J = 2.9 Hz, 1H); 13C NMR (CDCl3) δ 159.6 (s), 132.4 (s), 127.2 (d), 114.0 (d), 73.4 (d), 55.2 (q), 40.2 (t); MS (EI+) m/z = 232 (M+), 230 (M+), 151, 137, 121, 109, 94, 91, 77, 65, 51; HRMS (EI+) calcd. for C9H1179BrO2: 229.9942, found: 229.9938; chiral HPLC: Chiralcel OD, 40°C, n-heptane/IPA 96:4, 1.0 mL/min, Tr = 14.0 min (S-4.3q), 16.9 min (R-4.3q).

4.4.3 Synthesis of racemic products 4.4 Racemic 4.4a is available from Acros, Alfa Aesar, or Sigma-Aldrich. Racemic 4.4c, 4.4d, and 4.4f – h were prepared by F. Berthiol.

Racemic products 4.4d – 4.4f and 4.4j – 4.4l were synthesized by treating a solution of the corresponding bromoketone in methanol with an excess of NaBH4 (workup analogous to 4.3p).

Racemic product 4.4g was prepared from the corresponding aldehyde following a literature procedure.53

Racemic products 4.4b, 4.4c, 4.4h, and 4.4i were synthesized by ring closure of the corresponding haloalcohols, using potassium carbonate as base and methanol as solvent.54 Subsequently, they were purified by recrystallization from EtOH or by column chromatography over silica gel, using mixtures of n-heptane and ethyl acetate or n-pentane and diethyl ether as eluent.

138


Chapter 4

Styrene oxide (4.4a)25

For a separation method on chiral GC, see 4.3a and 4.3i.g

2-(4-Fluoro-phenyl)-oxirane (4.4b)25

1H NMR (CDCl3) δ 7.23 (AA'MM' dd, Japp = 8.8, 5.5 Hz, 2H), 7.02 (AA'MM' t, Japp = 8.8 Hz, 2H), 3.83 (dd, J = 4.0, 2.6 Hz, 1H), 3.12 (dd, J = 5.5, 4.0 Hz, 1H), 2.75 (dd, J = 5.5, 2.6 Hz, 1H); For a separation method on chiral GC, see 4.3b.

2-(4-(Trifluoromethyl)phenyl)oxirane (4.4c)55

1H NMR (CDCl3) δ 7.60 (d, J = 8.1 Hz, 2H), 7.40 (d, J = 8.1 Hz, 2H), 3.80 - 3.98 (m, 1H), 3.18 (dd, J = 5.7, 4.5 Hz, 1H), 2.76 (dd, J = 5.7, 2.7 Hz, 1H); 13C NMR (CDCl3) δ 142.1, 130.6 (q, 2JC-F = 32.2 Hz), 126.0, 125.7 (q, 3JC-F = 3.7 Hz), 124.3 (q, 1JC-F = 269.6 Hz), 51.9, 51.6; HRMS (EI+) calcd.: 188.0449, found: 188.0437; For a separation method on chiral HPLC, see 4.3c.

4-Oxiranyl-benzonitrile (4.4d)56

1H NMR (CDCl3) δ 7.61 (AA'MM' d, Japp = 8.4 Hz, 2H), 7.36 (AA'MM' d, Japp = 8.1 Hz, 2H), 3.88 (dd, J = 4.0, 2.2 Hz, 1H), 3.17 (dd, J = 5.5, 4.0 Hz, 1H), 2.73 (dd, J = 5.5, 2.6 Hz, 1H); 13C NMR (CDCl3) δ 142.8 (s), 131.7 (d), 125.6 (d), 118.1 (s), 111.0 (s), 51.0 (t), 51.0 (d); MS (EI+) m/z = 145 (M+), 144, 115, 102, 88, 75, 63, 51; HRMS (EI+) calcd.: 145.0528, found: 145.0523; For a separation method on chiral HPLC, see 4.3d and 4.3k.

g Whenever possible, GC or HPLC methods were used which allowed for separation of both the haloalcohol and the epoxide in a single measurement.

139



2-(4-Nitro-phenyl)-oxirane (4.4e)56

1H NMR (CDCl3) δ 8.17 (AA'XX' d, Japp = 8.8 Hz, 2H), 7.42 (AA'XX' d, Japp = 8.8 Hz, 2H), 3.94 (dd, J = 4.0, 2.2 Hz, 1H), 3.20 (dd, J = 5.5, 4.0 Hz, 1H), 2.75 (dd, J = 5.5, 2.6 Hz, 1H); 13C NMR (CDCl3) δ 147.4 (s), 145.2 (s), 126.0 (d), 123.5 (s), 51.5 (t), 51.2 (d); MS (EI+) m/z = 165 (M+), 152, 148, 118, 89, 77, 63, 51; HRMS (EI+) calcd.: 165.0426, found: 165.0425; For a separation method on chiral HPLC, see 4.3e and 4.3l.

2-(3-Nitro-phenyl)-oxirane (4.4f)52

1H NMR (CDCl3) δ 8.15 – 8.12 (m, 2H), 7.60 – 7.48 (m, 2H), 3.94 (dd, J = 4.0, 2.6 Hz, 1H) 3.19 (dd, J = 5.5, 4.0 Hz, 1H), 2.78 (dd, J = 5.5, 2.6 Hz, 1H); 13C NMR (CDCl3) δ 140.0 (s), 131.4 (d), 129.5 (d), 123.1 (d), 120.5 (d), 51.4 (t), 51.4 (d); MS (EI+) m/z = 165 (M+), 148, 135, 118, 105, 89, 77, 63, 51; HRMS (EI+) calcd.: 165.0426, found: 165.0424; For a separation method on chiral HPLC, see 4.3f and 4.3m.

2-(2-Nitro-phenyl)-oxirane (4.4g)48,57

1H NMR (CDCl3) δ 8.15 (d, J = 8.0 Hz, 2H), 7.60 – 7.75 (m, 3H), 4.49 (dd, J = 4.0, 2.6 Hz, 1H), 3.30 (dd, J = 5.6, 4.4 Hz, 1H), 2.67 (dd, J = 5.6, 2.6 Hz, 1H); 13C NMR (CDCl3) δ 148.0, 134.9, 134.5, 128.8, 127.2, 124.9, 50.9, 50.8; Elem. Anal.: calcd. C, 58.18; H, 4.27; N, 8.48; found C, 58.24; H, 4.27; N, 8.45; For a separation method on chiral HPLC, see 4.3g.

2-(3-Methoxy-phenyl)-oxirane (4.4h)58

1H NMR (CDCl3) δ 7.26 (t, J = 7.7 Hz, 1H), 6.78 - 6.93 (m, 3H), 3.85 (dd, J = 4.0, 2.8 Hz, 1H), 3.81 (s, 3H), 3.13 (dd, J = 5.6, 4.2 Hz, 1H), 2.78 (dd, J = 5.6, 2.4 Hz, 1H); 13C NMR (CDCl3) δ 159.9, 139.3, 129.6, 118.0, 113.9, 110.5, 55.3, 52.3, 51.2; HRMS (EI+) calcd.: 150.0681, found: 150.0684; For a separation method on chiral HPLC, see 4.3h.

140


Chapter 4

2-Cyclohexyl-oxirane (4.4i)53

4.3i (425 mg, 2.62 mmol) was dissolved in 10 mL THF, after which potassium carbonate (490 mg, 3.55 mmol) was added. This mixture was stirred for 4 days at room temperature and subsequently filtered and evaporated to dryness. The spectral data for this compound corresponded to those described for compound 3.9 (Chapter 3). For a separation method on chiral GC, see 4.3i.

2-Naphthalen-2-yl-oxirane (4.4j)59

1H NMR (CDCl3) δ 7.86 – 7.79 (m, 4H), 7.50 – 7.44 (m, 2H), 7.32 (d, J = 8.4, 1H), 4.02 (dd, J = 4.0, 2.6 Hz, 1H), 3.21 (dd, J = 4.4, 4.0 Hz, 1H), 2.90 (dd, J = 4.4, 2.6 Hz, 1H); 13C NMR (CDCl3) δ 135.0 (s), 133.3 (s), 133.2 (s), 128.4 (d), 127.7 (d, 2C), 126.3 (d), 126.0 (d), 125.1 (d), 122.6 (d), 52.6 (d), 51.2 (t); For a separation method on chiral HPLC, see 4.3n.

2-p-Tolyl-oxirane (4.4k)56

1H NMR (CDCl3) δ 7.27 (AA'BB' d, Japp = 8.1 Hz, 2H), 7.18 − 7.12 (AA'BB' m, 2H), 3.81 (dd, J = 4.0, 2.6 Hz, 1H), 3.11 (dd, J = 5.5, 4.0 Hz, 1H), 2.78 (dd, J = 5.5, 2.6 Hz, 1H), 2.33 (s, 3H); For a separation method on chiral HPLC, see 4.3o.

2-(4-Bromo-phenyl)-oxirane (4.4l)60

1H NMR (CDCl3) δ 7.45 (AA'MM' d, Japp = 8.4 Hz, 2H), 7.13 (AA'MM' d, Japp = 8.4 Hz, 2H), 3.80 (dd, J = 4.0, 2.6 Hz, 1H), 3.12 (dd, J = 5.5, 4.0 Hz, 1H), 2.73 (dd, J = 5.5, 2.6 Hz, 1H); 13C NMR (CDCl3) δ 136.6 (s), 131.6 (d), 127.1 (d), 122.0 (s), 51.8 (d), 51.2 (t); For a separation method on chiral HPLC, see 4.3p.

4.4.4 Production and overexpression of enzymes E1 − E533 All enzymes were prepared by C. Tarabiono.

141



For E2 − E4, mutations were introduced in the hheC gene by PCR, using the QuikChange® Site-Directed Mutagenesis Kit from Stratagene.61 As a template, pBADHheC wild type was used and the mutagenic primers were supplied by Sigma. The mutations were confirmed by sequencing.

E5 was obtained by modifying an aqueous solution of purified E3 with Lomant’s reagent.45

Expression and purification of the mutant enzymes are analogous to the procedure described for E1 (wild-type HheC) in Chapter 3.

4.4.5 Kinetic resolution of 4.3l using E1 – E5 A solution of 200 μmol of 4.3l in in 0.5 mL of DMSO was added to 10 mL of aqueous HEPES buffer (50 mM, pH 7.5) at room temperature. The reaction was started by adding about 6 U of enzyme to the mixture.37 Samples (0.5 mL) were taken every 10 min, extracted with Et2O, concentrated in vacuo, redissolved in n-heptane/IPA 93:7 and analyzed by chiral HPLC. The apparent E-value for the enzymes was calculated from the ee’s of product and starting material using the formula:62

( ) ( )[ ]( ) ( )[ ]PSS

PSS

eeeeeeeeeeeeE

+++−

=11ln11ln

4.4.6 Synthesis of catalysts 4.8a − 4.8c

The procedure described by Pfeffer et al. for analogous compounds38 was used. Thus, a 50 mL 2-necked flask was thoroughly flame-dried and put under an atmosphere of nitrogen, after which the following compounds were added, respectively: [Cp*IrCl2]2 (120 mg, 0.15 mmol), KPF6 (110 mg, 0.60 mmol), NaOH (12 mg, 0.30 mmol), N-benzylmethylamine (39.0 μL, 36.4 mg, 0.30 mmol), and acetonitrile (4 mL). This mixture was stirred at 45 °C for 2d. The mixture was then cooled down to room temperature, washed with n-heptane (3x) and filtered over neutral aluminum oxide (eluent: MeCN). The resulting solution was concentrated in vacuo. Subsequent stripping with dry Et2O yielded 4.8a (136.2 mg, 215 mmol, 90%) as a dark yellow foam. 1H NMR (CDCl3) δ 7.34 (d, J = 6.2 Hz, 1H), 7.05 (d, J = 7.0 Hz, 1H), 6.98 (t, J = 7.1 Hz, 1H), 6.90 (t, J = 7.2 Hz, 1H), 4.39 (br s, 1H), 4.15 (br s, 1H), 3.66 (br s, 1H), 3.09 (br s, 3H), 2.37 (s, 3H), 1.67 (s, 15H); 13C NMR (CDCl3) δ 150.2 (s), 146.6 (s),

142


Chapter 4

135.2 (d), 127.4 (d), 123.6 (2x d), 89.8 (s), 67.2 (t), 44.8 (q), 8.9 (q); HRMS (EI+) calcd. for C18H25IrN+ (= M+ − CH3CN/PF6−): 448.1616, found: 448.1617.

1-Benzylamino-2-methyl-propan-2-ol (4.7b).42

Benzylamine (2.73 mL, 2.68 g, 25.0 mmol) and 2,2-dimethyloxirane (2.5 mL, 2.03 g, 28.1 mmol) were stirred together under reflux for 24 h. The mixture was then cooled down to room temperature and excess dimethyloxirane was evaporated under reduced pressure. It was observed that crystallization of this compound was exothermic. 1H NMR (CDCl3) δ 7.34 − 7.22 (m, 5H), 3.82 (s, 2H), 2.54 (s, 2H), 2.5 − 2.0 (br s, 2H), 1.16 (s, 6H).

4.8b was prepared analogously to 4.8a, using 4.7b as the amine. 1H NMR (CDCl3) δ 7.56 (d, J = 7.0 Hz, 1H), 7.17 (d, J = 7.3 Hz, 1H), 7.07 (t, J = 7.0 Hz, 1H), 6.96 (t, J = 7.3 Hz, 1H), 6.36 (br s, 1H), 4.15 – 4.00 (m, 2H), 3.61 (br s, 1H), 2.84 (m, 1H), 2.37 (br s, 1H), 1.86 (s, 2H), 1.65 (s, 15H), 1.30 (s, 3H), 1.29 (s, 3H); 13C NMR (CDCl3) δ 157.1 (s), 146.9 (s), 135.8 (d), 127.6 (d), 124.3 (d), 123.2 (d), 87.5 (s), 82.5 (s), 64.0 (t), 61.0 (t), 24.3 (q), 23.7 (q), 9.1 (q).

4.8c was prepared by conducting the preparation of 4.8a in the presence of air.h 1H NMR (CDCl3) δ 8.33 (s, 1H), 7.67 (d, J = 7.0 Hz, 1H), 7.56 (d, J = 7.3 Hz, 1H), 7.20 (t, J = 7.1 Hz, 1H), 7.11 (t, J = 7.1 Hz, 1H), 3.94 (s, 3H), 2.37 (s, 3H), 1.76 (s, 15H); 13C NMR (CDCl3) δ 178.5 (d), 161.8 (s), 146.9 (s), 134.4 (d), 132.1 (d), 128.8 (d), 123.6 (d), 91.1 (s), 50.3 (q), 9.0 (q); HRMS (EI+) calcd. for C18H23IrN+ (= M+ − CH3CN/PF6−): 446.1459, found: 446.1455.

4.4.7 General procedure for the dynamic kinetic resolution of compounds 4.3 The DKR of 4.3c, 4.3d, and 4.3f – 4.3h was performed by F. Berthiol.

h See Footnote c on page 116.

143



A 50 mL 2-necked flask was filled with 10 mL of HEPES buffer (50 mM, pH 7.5 for bromoalcohols, pH 8 for chloroalcohols), a solution of 200 μmol of substrate in 0.5 mL of distilled DMSO, 35 mg of BSA, and a sample of enzyme containing 6 U of haloalcohol dehalogenase (measured on substrate 4.3l under standard conditions, see Ref. 37). Then, a solution of 4.8a (6.3 mg, 10 μmol, 5 mol%) activated with t-BuOK (1.2 mg, 11 μmol) in 3 mL of freshly distilled toluene (under anhydrous conditions and a nitrogen atmosphere) was added to the solution over 6 h using a syringe pump. To analyze the composition of the reaction mixture, 0.1 mL samples were taken from the organic layer, filtered over silica (eluent: Et2O), evaporated, redissolved in n-heptane/IPA and analyzed by chiral HPLC or GC.

4.4.8 Dynamic kinetic resolution of 4.3 on preparative scale DKR on preparative scale was performed analogous to the reaction on analytical scale, but using 1.0 mmol of substrate dissolved in 2.5 mL of DMSO, 50 mL of HEPES buffer (50 mM, pH 8), 150 mg of BSA, and an enzyme sample containing 30 U of HheC Cys153Ser Trp249Phe. The catalyst solution consisted of 31.5 mg (50 μmol, 5 mol%) of 4.8a and 6.0 mg (53 μmol) of t-BuOK, dissolved in 15 mL of freshly distilled toluene. After the reaction had finished (monitored by HPLC), the mixture was extracted using toluene. Separation of the aqueous and organic layers was effected by a high-speed centrifuge (12 000 rpm).

4.5 Notes and references 1 For a review, see: A. Ghanem, Tetrahedron 2007, 63, 1721-1754. 2 K. Faber, Biotransformations in Organic Chemistry: A Textbook, 4th edn., Springer, Berlin, 2000. 3 J. D. Morrison, in Asymmetric Synthesis; Academic Press, New York, 1983, Vol. 1, pp 3-6. 4 For recent reviews, see: a) H. Pellissier, Tetrahedron 2008, 64, 1563-1601; b) B. Martín-Matute and J.-E. Bäckvall, Curr. Opin. Chem. Biol. 2007, 11, 226-232; c) O. Pàmies and J.-E. Bäckvall, Chem. Rev. 2003, 103, 3247-3261. 5 F. Weygand, W. Steglich, and X. Barocio de la Lama, Tetrahedron 1966, 22, Suppl. 8, 9-13. 6 H. Yamada, S. Shimizu, H. Shimada, Y. Tani, S. Takahashi, and T. Ohashi, Biochimie 1980, 62, 395-399. 7 G. Fülling and C. J. Sih, J. Am. Chem. Soc. 1987, 109, 2845-2846.

144


Chapter 4

8 R. Noyori, T. Ikeda, T. Ohkuma, M. Widhalm, M. Kitamura, H. Takaya, S. Akutagawa, N. Sayo, T. Saito, T. Taketomi, and H. Kumobayashi, J. Am. Chem. Soc. 1989, 111, 9134-9135. 9 For reviews on the earlier developments of DKR, see: a) H. Pellissier, Tetrahedron 2003, 59, 8291-8327; b) R. S. Ward, Tetrahedron: Asymmetry 1995, 6, 1475-1490; c) For a comprehensive overview of non-enzymatic (dynamic) kinetic resolution, see: E. Vedejs and M. Jure, Angew. Chem. Int. Ed. 2005, 44, 3974-4001. 10 M. Inagaki, J. Hiratake, T. Nishioka, and J. Oda, J. Am. Chem. Soc. 1991, 113, 9360-9361. 11 H. van der Deen, A. D. Cuiper, R. P. Hof, A. van Oeveren, B. L. Feringa, and R. M. Kellogg, J. Am. Chem. Soc. 1996, 118, 3801-3803. 12 J. V. Allen and J. M. J. Williams, Tetrahedron Lett. 1996, 37, 1859-1862. 13 P. M. Dinh, J. A. Howarth, A. R. Hudnott, J. M. J. Williams, and W. Harris, Tetrahedron Lett. 1996, 37, 7623-7626. 14 A. L. E. Larsson, B. A. Persson, and J.-E. Bäckvall, Angew. Chem. Int. Ed. Engl. 1997, 36, 1211-1212. 15 N. Menashe and Y. Shvo, Organometallics 1991, 10, 3885-3891. 16 Novozym® 435 is a commercially available preparation of Candida antarctica lipase B, immobilized on acrylic resin, available from Novozymes. 17 Y. Ahn, S.-B. Ko, M.-J. Kim, J. Park, Coord. Chem. Rev. 2008, 252, 647-658. 18 a) J. H. Choi, Y. K. Choi, Y. H. Kim, E. S. Park, E. J. Kim, M.-J. Kim, and J. Park, J. Org. Chem. 2004, 69, 1972-1977; b) J. H. Choi, Y. H. Kim, S. H. Nam, S. T. Shin, M.-J. Kim, and J. Park, Angew. Chem. Int. Ed. 2002, 41, 2373-2376. 19 a) B. Martín-Matute, M. Edin, K. Bogár, F. B. Kaynak, and J.-E. Bäckvall, J. Am. Chem. Soc. 2005, 127, 8817-8825; b) B. Martín-Matute, M. Edin, K. Bogár, and J.-E. Bäckvall, Angew. Chem. Int. Ed. 2004, 43, 6535-6539. 20 G. K. M. Verzijl, J. G. de Vries, and Q. B. Broxterman, Tetrahedron: Asymmetry 2005, 16, 1603-1610. 21 R. J. Kazlauskas, A. N. E. Weissfloch, A. T. Rappaport, and L. A. Cuccia, J. Org. Chem. 1991, 56, 2656-2665. 22 a) L. Borén, B. Martín-Matute, Y. Xu, A. Córdova, and J.-E. Bäckvall, Chem. Eur. J. 2006, 12, 225-232; b) M.-J. Kim, Y. I. Chung, Y. K. Choi, H. K. Lee, D. Kim, and J. Park, J. Am. Chem. Soc. 2003, 125, 11494-11495. 23 a) M. A. J. Veld, K. Hult, A. R. A. Palmans, and E. W. Meijer, Eur. J. Org. Chem. 2007, 5416-5421; b) C. Roengpithya, D. A. Patterson, A. G. Livingston, P. C. Taylor, J. L. Irwin, and M. R. Parrett, Chem. Commun. 2007, 3462-3463; c) A. J. Blacker, M. J. Stirling, and M. I. Page, Org. Proc. Res. Devel. 2007, 11, 642-648; d) M.-J. Kim, W.-H. Kim, K. Han, Y. K. Choi, and J. Park, Org. Lett. 2007, 9, 1157-1159; e) S. Gastaldi, S. Escoubet, N. Vanthuyne, G. Gil, and M. P. Bertrand, Org. Lett. 2007, 9, 837-839; f) M. Stirling, J. Blacker, and M. I. Page, Tetrahedron Lett.

145



2007, 48, 1247-1250; g) J. Paetzold and J.-E. Bäckvall, J. Am. Chem. Soc. 2005, 127, 17620-17621; h) A. Parvulescu, D. De Vos, and P. Jacobs, Chem. Commun. 2005, 5307-5309; i) M. T. Reetz and K. Schimossek, Chimia 1996, 50, 668-669. 24 a) M.-J. Kim, Y. Ahn, and J. Park, Curr. Opin. Biotechnol. 2002, 13, 578-587; b) M.-J. Kim, M. Y. Choi, M. Y. Han, Y. K. Choi, J. K. Lee, and J. Park, J. Org. Chem. 2002, 67, 9481-9483; c) H. M. Jung, J. H. Koh, M.-J. Kim, and J. Park, Org. Lett. 2000, 2, 2487-2490; d) H. M. Jung, J. H. Koh, M.-J. Kim, and J. Park, Org. Lett. 2000, 2, 409-411. 25 O. Pàmies and J.-E. Bäckvall, J. Org. Chem. 2002, 67, 9006-9010. 26 S. C. Davis, J. H. Grate, D. R. Gray, J. M. Gruber, G. W. Huisman, S. K. Ma, L. M. Newman, R. Sheldon, and L. A. Wang, US2007161094, 2007, to Codexis, Inc. 27 J. H. Lutje Spelberg, L. Tang, R. M. Kellogg, and D. B. Janssen, Tetrahedron: Asymmetry 2004, 15, 1095-1102. 28 M. Kapoor, N. Anand, K. Ahmad, S. Koul, S. S. Chimni, S. C. Taneja, and G. N. Qazi, Tetrahedron: Asymmetry 2005, 16, 717-725. 29 M. Lautens, M. L. Maddess, E. L. O. Sauer, and S. G. Ouellet, Org. Lett. 2002, 4, 83-86. 30 a) C. Bonini and G. Righi, Synthesis 1994, 225-238; b) J. A. Ciaccio, E. Heller, and A. Talbot, Synlett 1991, 248-250; b) For an alternative to conversion, see: S. Raina, D. Bhuniya, and V. K. Singh, Tetrahedron Lett. 1992, 33, 6021-6022. 31 The synthesis and DKR of substrates 4.3c, 4.3d, and 4.3f – 4.3h were performed by F. Berthiol. 32 A. J. van den Wijngaard, D. B. Janssen, and B. Witholt, J. Gen. Microbiol. 1989, 135, 2199-2208. 33 All enzymes were prepared by C. Tarabiono. 34 L. Tang, J. E. T. van Hylckama Vlieg, J. H. Lutje Spelberg, M. W. Fraaije, and D. B. Janssen, Enzyme Microb. Technol. 2002, 30, 251-258. 35 a) M. Majerić Elenkov, L. Tang, B. Hauer, and D. B. Janssen, Org. Lett. 2006, 8, 4227-4229; b) L. Tang, D. E. Torres Pazmiño, M. W. Fraaije, R. M. de Jong, B. W. Dijkstra, and D. B. Janssen, Biochemistry 2005, 44, 6609-6618; c) L. Tang, A. E. J. van Merode, J. H. Lutje Spelberg, M. W. Fraaije, and D. B. Janssen, Biochemistry 2003, 42, 14057-14065. 36 A. J. Lomant and G. Fairbanks, J. Mol. Biol. 1976, 104, 243-261. 37 J. H. Lutje Spelberg, L. Tang, M. van Gelder, R. M. Kellogg, and D. B. Janssen, Tetrahedron: Asymmetry 2002, 13, 1083-1089. 38 J.-B. Sortais, N. Pannetier, A. Holuigue, L. Barloy, C. Sirlin, M. Pfeffer, and N. Kyritsakas, Organometallics 2007, 26, 1856-1857. 39 D. L. Davies, O. Al-Duaij, J. Fawcett, M. Giardiello, S. T. Hilton, and D. R. Russell, Dalton Trans. 2003, 4132-4138.

146


Chapter 4

40 J.-B. Sortais, V. Ritleng, A. Voelklin, A. Holuigue, H. Smail, L. Barloy, C. Sirlin, G. K. M. Verzijl, J. A. F. Boogers, A. H. M. de Vries, J. G. de Vries, and M. Pfeffer, Org. Lett. 2005, 7, 1247-1250. 41 A. J. A. Gayet, unpublished results. 42 J. F. Kerwin, G. E. Ullyot, R. C. Fuson, and C. L . Zirkle, J. Am. Chem. Soc. 1947, 69, 2961-2965. 43 D. C. Carter and J. X. Ho, Adv. Prot. Chem. 1994, 45, 153-203. 44 Y. Marcus, Chem. Soc. Rev. 1993, 22, 409-416. 45 C. Tarabiono, unpublished results. 46 a) T. M. Poessl, B. Kosjek, U. Ellmer, C. C. Gruber, K. Edegger, K. Faber, P. Hildebrandt, U. T. Bornscheuer, and W. Kroutil, Adv. Synth. Catal. 2005, 347, 1827-1834; b) R. Tanikaga, K. Hosoya, and A. Kaji, J. Chem. Soc., Chem. Commun. 1986, 836-837. 47 Barantsevich, E. N.; Temnikova, T. I.; Kovaleva, E. V. J. Org. Chem. USSR 1967, 3, 106-107, translated from Zh. Org. Khim. 1967, 3, 110-112. 48 F. Arndt, B. Eistert, and W. Partale, Ber. Deut. Chem. Ges. 1928, 61, 1107-1118. 49 T. Hamada, T. Torii, K. Izawa, R. Noyori, and T. Ikariya, Org. Lett. 2002, 4, 4373-4376. 50 S. L. Shapiro, H. Soloway, and L. Freedman, J. Am. Chem. Soc. 1958, 80, 6060-6064. 51 H. C. Brown and G. G. Pai, J. Org. Chem. 1985, 50, 1384-1394. 52 A. C. Knipe, J. Chem. Soc., Perkin Trans. 2 1973, 589-585. 53 J. M. Concellón, H. Cuervo, and R. Fernández-Fano, Tetrahedron 2001, 57, 8983-8987. 54 T. R. Nieduzak and A. L. Margolin, Tetrahedron: Asymmetry 1991, 2, 113-122. 55 M. K. Tse, M. Klawonn, S. Bhor, C. Döbler, G. Anilkumar, H. Hugl, W. Mägerlein, and M. Beller, Org. Lett. 2005, 7, 987-990. 56 J. H. Lutje Spelberg, R. Rink, A. Archelas, R. Furstoss, and D. B. Janssen, Adv. Synth. Catal. 2002, 344, 980-985. 57 N. Hashimoto, T. Aoyama, and T. Shioiri, Heterocycles 1981, 15, 975-979. 58 Fuchs, R. J. Am. Chem. Soc. 1956, 78, 5612-5613. 59 S. Kulasegaram and R. J. Kulawiec, J. Org. Chem. 1997, 62, 6547-6561. 60 D. B. Cordes, T. J. Kwong, K. A. Morgan, and B. Singaram, Tetrahedron Lett. 2006, 47, 349-351. 61 For the manual of this kit, see: http://www.stratagene.com/manuals/200518.pdf 62 A. J. J. Straathof and J. A. Jongejan, Enzyme Microb. Technol. 1997, 21, 559-571.

Chapter 5 Synthetic applications of enantiopure chloroalcohols

This chapter is centered on the use of chloroalcohols in a variety of reactions with the

aim of increasing their functionality and demonstrating their potential as chiral

building blocks in synthesis. We focused on rearrangement reactions such as the

Achmatowicz, Ireland-Claisen, and Johnson orthoester rearrangements. Initial results

are promising, but further research is required to demonstrate the scope and limitations

of these transformations.

148

Chapter 5 - Synthetic applications of enantiopure chloroalcohols.doc

Chapter 5

5.1 Introduction

Chloroalcohols are often employed as intermediates in organic synthesis, for instance as precursors of epoxides or aminoalcohols.1,2 Since we developed a very efficient system to obtain enantiomerically pure chloroalcohols by enzymatic kinetic resolution (see Chapter 3), we became interested in the synthetic applications of these compounds. In particular, we looked into the possible application of enantiopure chloroalcohols in rearrangement reactions such as the Achmatowicz, Ireland-Claisen, and Johnson orthoester rearrangements.

5.2 Achmatowicz rearrangement of 2-chloro-1-(furan-2-yl)ethanol

The Achmatowicz rearrangement (Scheme 5.1), first described in 1971 by Achmatowicz and coworkers, is a useful reaction that converts 2-furanylcarbinols into highly functionalized pyranones.3 Enantiopure pyranones like 5.2 are building blocks in the synthesis of, for example, carbohydrates,4,5,6,7 biologically active compounds such as daumone,8 tirandamycin B,9 and patuline,10 as well as other compounds such as functionalized spirocyclic pyrans.11 Various conditions are described in the literature for this transformation, most importantly via bromination using molecular bromine3,12 or NBS,8,13 or oxidation with m-CPBA,9 dimethyldioxirane,14 or PhI(OAc)2-Mg(ClO4)2.15 Catalytic procedures include the use of t-butyl hydroperoxide / VO(acac)2,16a and H2O2 / titanium silicalite.17 The related aza-Achmatowicz rearrangement has been used in the synthesis of naturally occurring alkaloids.18

Scheme 5.1 Achmatowicz rearrangement of 1-(furan-2-yl)ethanol to 6-hydroxy-2-methyl-2H-pyran-3(6H)-one.

In the Achmatowicz rearrangement, the configuration of the alcohol moiety is preserved. As a result, kinetic resolution of 2-furylcarbinols such as rac-5.1 is possible by using Sharpless asymmetric epoxidation, followed by Achmatowicz rearrangement of the initially formed epoxide.16

149


Synthetic applications of enantiopure chloroalcohols

A recent application is the synthesis of the η3-oxopyranyl- and η3-oxopyridinylmolybdenum complexes TpMo(CO)2(η3-oxopyranyl) and TpMo(CO)2(η3-oxopyridinyl) by oxa- and aza-Achmatowicz reactions, both in racemic and enantiomerically pure form.19,a

Chloroalcohol 5.3 could conceivably be converted into pyranone 5.4 by an Achmatowicz rearrangement (Scheme 5.2).

Scheme 5.2 Envisioned Achmatowicz rearrangement of (S)-5.3 to (S)-5.4.

We looked for suitable reaction conditions using non-chlorinated furanyl carbinol 5.1. The use of bromine3,12 in acetonitrile or methanol led to black tar, but using NBS in a mixture of THF and water8 gave 5.2 in 85% isolated yield as a mixture (65:35) of two diastereomers.

These conditions were then applied to racemic 5.3 (Scheme 5.2) leading, however, to a disappointing yield of 26%. The product was observed to be unstable on silica gel, as well as upon prolonged standing, but could be crystallized from diethyl ether. However, the reaction turned out not to be reproducible, despite multiple attempts. Also, a subsequent reaction with enantiomerically enriched 5.3 (S-enantiomer, 90% ee) failed to give 5.4. An alternative procedure using m-CPBA did not give the product either.9 We concluded that that pyranone 5.4 is relatively stable once isolated, but unstable under the conditions of the reaction or the workup, due to the chlorine substituent present in the compound.

Since the reaction failed to work using 5.3, it was attempted to replace chlorine by a benzyloxy substituent and perform the rearrangement subsequently. The substitution of chlorine for benzylate on vicinal chloroalcohols different from 5.3 was described for example by the groups of Beauchamp20 and Doyle.21 The new approach is outlined in Scheme 5.3.

a Tp = hydridotrispyrazolylborato.

150


Chapter 5

Scheme 5.3 Attempted substitution of chlorine on 5.3, followed by Achmatowicz rearrangement.

Several attempts were made to obtain 5.5 from 5.3, for instance using PhCH2ONa in DMF at reflux,20 PhCH2ONa and NaI in THF at room temperature, and PhCH2OH (neat) and NaI at room temperature. In all cases, the reaction resulted in the formation of black tars. Appropriate, mild conditions for this transformation have to be established. Consequently, it has not been tested on the enantiomerically pure chloroalcohol yet.

5.3 Ireland-Claisen rearrangement of (E)-1-chloro-4-phenylbut-3-en-2-yl propionate

The ester enolate Claisen rearrangement, or Ireland-Claisen rearrangement, is a variety of the Claisen [3,3]sigmatropic rearrangement developed in the 70's by Ireland and coworkers.22 It takes place at milder conditions than the regular Claisen rearrangement, is versatile, and exhibits high levels of stereocontrol.23 By controlling the E/Z stereochemistry of the enolate formed upon deprotonation, the syn/anti stereochemistry of the resulting rearrangement product can be selectively established.22,24

It had been demonstrated that Ireland-Claisen rearrangement reaction was possible on substrates such as (E)-4-phenylbut-3-en-2-yl malonates (Scheme 5.4).25

Scheme 5.4 [3,3]Sigmatropic rearrangement of an allylic malonate followed by decarboxylation.

Furthermore, the Ireland-Claisen rearrangement has been applied in the total synthesis of the antitumor alkaloid, (+)-pancratistatin (Scheme 5.5)26 and it was used in the synthesis of chiral subunits for macrolide synthesis.27 These and other examples show

151



the remarkable stereocontrol of this rearrangement, which makes it possible to predict the stereochemical configuration and double bond geometry in the product by examination of the corresponding properties in the starting material.28

Scheme 5.5 Application of the Ireland-Claisen rearrangement in the total synthesis of (+)-pancratistatin.

Furthermore, Sakaitani and Ohfune described the intramolecular ring closure of carbamate-substituted allylic chlorides using silver fluoride (Scheme 5.6).29

Scheme 5.6 Ring closure of carbamate-substituted allylic chloroalcohols using silver fluoride.

Scheme 5.7 Ireland-Claisen rearrangement of allylic acylates 5.7 followed by intramolecular ring closure to chiral lactones 5.9.

Inspired by these previous results, we envisioned the possibility of using allylic chloroacylates 5.7 en route to chiral lactones, as outlined in Scheme 5.7. An Ireland-Claisen rearrangement leading to silyl-protected carboxylic acids 5.8, followed by intramolecular ring closure, would thus provide an enantioselective entry into chiral lactones 5.9. Following the route outlined in Scheme 5.7, a product with three

152


Chapter 5

consecutive stereocenters would be formed from a starting material containing only one.

The esters used as starting material are available in quantitative yield from the corresponding chloroalcohols and the appropriate anhydride, as shown in Scheme 5.8.30

Scheme 5.8 Synthesis of propyl esters 5.7 from allylic chloroalcohols 5.10.

Table 5.1 Ireland-Claisen rearrangement of (E)-1-chloro-4-phenylbut-3-en-2-yl propionate (5.7b).

Entry Base

(equiv.) Additive T TMSX

Conv. (%)

1 LDA HMPA −80→rt TMSCl 45 (0)a 2 LDA HMPA −80→rt − 35 (0)a 3 LDA − −80→rt TMSCl 20 (0)a 4 LiHMDS − −80→rt TMSCl 5.7b:5.11 ~ 1:1b 5 LiHMDS HMPA −80→rt TMSCl 5.11 (46), 5.12 (11), 5.7b (43) 6 LiHMDS HMPA −80→Δc TMSCl 5.7b and 5.12 only 7 LiHMDS − −80→rt TMSCl primarily 5.7b 8 NaHMDS (1.2 eq) − −60→rt TMSOTf 5.7bd 9 LiHMDS (1.2 eq) − −65→rt TMSOTf 5.7b and 5.12 (trace)

10 NaHMDS (3 eq) − −60→rt TMSOTf 5.7b and 5.12 11 LiHMDS (3 eq) − −80→rt TMSOTf 5.7b and 5.12 12 NaHMDS (3 eq) HMPA −80→rt TMSOTf n.i.e 13 NaHMDS HMPA −80→rt TBDMSCl n.i.e 14 LiHMDS HMPA −80→rt TBDMSCl n.i.e

a) Between brackets is the conversion to product 5.11; b) Considerable amount of unidentified byproducts; c) Heated to reflux; d) Additional unidentified byproducts; e) Unidentified mixture of products.

153



Conditions for the Ireland-Claisen reaction were screened primarily on 5.7b. Various bases (LDA, LiHMDS, NaHMDS) and silylating reagents (TMSCl, TMSOTf, TBDMSCl) were tested, as well as the influence of using HMPA as an additive (Table 5.1). In the end no feasible conditions could be established for this reaction. The best results were obtained using LiHMDS, HMPA, and TMSCl in THF at − 80 °C → rt: 46% conversion to the product, 43% starting material, and 11% dechlorinated starting material (Table 5.1, entry 5).

For the reactions described in Table 5.1, in entries 13 and 14, a different silylating reagent was used (t-butyldimethylsilyl chloride) according to a procedure described by Ko et al.26

Alternative bases, such as NaH, K2CO3, or DIPA, did not give conversion. Some other bases, for instance DBU, n-BuLi, or KOH, give partial or complete conversion to unwanted poducts. Some frequently observed byproducts are (E)-4-phenylbut-3-en-2-one (5.13), (E)-4-phenylbuta-1,3-dien-2-yl propionate (5.14), and ((1E,3E)-4-chlorobuta-1,3-dienyl)benzene (5.15), shown in Figure 5.1.

Figure 5.1 Side-products observed in [3,3]sigmatropic rearrangement of 5.7b.

Despite extensive efforts, we have been unable to establish reaction conditions which give full conversion of 5.7b to products 5.11 or 5.9. A possible reason for the low conversion to the anticipated products might be direct ring closure of the enolate with chloride as the leaving group, as shown in Scheme 5.9.

Scheme 5.9 Possible explanation for low conversion of 5.7b to product 5.11.

What also remains to be done is the development of the ring closure to butyrolactones 5.9. As already mentioned, there are literature precedents for this reaction, since the ring closure of silyl-protected carboxylic acids29 and esters31 has been described.

154


Chapter 5

5.4 Johnson orthoester rearrangement of (E)-1-chloro-4-phenylbut-3-en-2-ol

The Johnson orthoester rearrangement, or Johnson-Claisen rearrangement, was published in 1970 by Johnson and coworkers.32 It involves heating a mixture of an allylic alcohol and an orthoester in the presence of a weak acid, such as propionic acid, after which the resulting ketene acetal undergoes [3,3]sigmatropic rearrangement.23b In 1993, the synthesis was described of bicyclic lactones via orthoester rearrangement followed by selenolactonization / oxidative elimination.33 Because of its ease of performance and, generally, high level of stereocontrol, the Johnson rearrangement has been used extensively in the synthesis of natural products.34

Johnson orthoester rearrangement has been performed with >97% transfer of chirality on the styryl-substituted alcohols 5.16,35 5.17,36 and 5.18,37 shown in Figure 5.2.

Figure 5.2 Literature examples of substrates for Johnson orthoester rearrangement.

We anticipated to perform a Johnson orthoester rearrangement using chloroalcohols 5.10 as substrate, followed by ring closure of the initially formed esters 5.19 to highly functionalized enantiopure butyrolactones 5.9. This sequence of reactions is outlined in Scheme 5.10.

Scheme 5.10 Synthesis of chiral lactones 5.9 by Johnson orthoester rearrangement of 5.10 and consecutive ring closure.

Initial reaction of (E)-1-chloro-4-phenylbut-3-en-2-ol (5.10a) with triethyl orthopropionate in refluxing toluene in the presence of a catalytic amount of propionic acid gave 90% conversion (Table 5.2, entry 1). However, the isolated yield of 5.19a was

155



only 48% and the two diastereomers were formed in equimolar ratio. Stereocontrol may be improved by using starting materials containing trisubstituted double bonds, as shown in an example from the literature.38 We tried to develop a shorter reaction under milder conditions in order to improve stereocontrol. Microwave heating seemed an attractive alternative, however the levels of conversion were similar and the diastereomeric ratio improved only slightly (entries 2 and 3). The use of alternative catalysts such as bentonite clay33 or the Lewis acid scandium triflate led to disappointing results (entries 4 and 5).

Table 5.2 Johnson orthoester rearrangement of 5.10.a

Entry Substrate Orthoester Product T Conv. (%)

Remarks

1 5.10a EtC(OEt)3 5.19a Δ 90 48% isol. y., dr 1:1b 2 5.10a EtC(OEt)3 5.19a Δ, μ 80 dr 1:1.5b 3c 5.10a EtC(OEt)3 5.19a Δ, μ 90 dr 1:1b 4 5.10a EtC(OEt)3 5.19a Δ, μ black tar cat: bentonite clay K-10 5 5.10a EtC(OEt)3 5.19a rt >98, no 5.19ad cat: Sc(OTf)3 6 5.10a MeC(OEt)3 5.19b Δ, μ 85 7 (S)- 5.10a MeC(OEt)3(S)-5.19b Δ >98 ee (5.19a) 41% 8 5.10c EtC(OEt)3 5.19c Δ, μ >98 dr 56:44b

a) For reaction conditions, see the experimental section; b) Diastereomeric ratio; c) Duplo of entry 2; d) Full conversion to unidentified products.

In an attempt to circumvent the formation of diastereomers, some reactions were also performed using triethyl orthoacetate (Table 5.2, entries 6 and 7). The conversion to product 5.19b was similar compared to the reactions using triethyl orthopropionate. However, a disencouraging result was obtained when using enantiopure starting material. Starting from (S)-5.10a (>99% ee), product 5.19b was obtained in only 41% ee (Scheme 5.11), despite complete transfer of chirality (>97%) on structurally similar substrates such as 5.16 – 5.18 (Figure 5.2).35,36,37

It is not clear whether loss of enantiomeric excess stems from racemization of the chloroalcohol prior to rearrangement, or if the rearrangement itself has poor

156


Chapter 5

stereocontrol. Unfortunately, there was no sufficient time to further test any of these hypotheses or establish more favorable reaction conditions.

Scheme 5.11 Partial racemization or incomplete transfer of chirality in the Johnson orthoester rearrangement of (S)-5.10a.

Finally, (E)-1-chloropent-3-en-2-ol (5.10c) was subjected to Johnson rearrangement conditions using triethyl orthopropionate (Table 5.2, entry 8). Although full conversion was reached, diastereoselectivity was practically absent, similar to the results with substrate 5.10a.

In conclusion, the functionalization of chiral allylic chloroalcohols by orthoester Claisen rearrangement turned out to be more challenging than expected, despite the progress that has been made. Conversions are excellent, although the diastereoselectivity is unexpectedly low. Possibilites for improvement include the use of alternative catalysts such as a suitable Lewis acid or possibly a transition metal catalyst. Furthermore, suitable conditions have to be established for the ring closure reaction. Considering prior art in this area,31 this is not expected to pose significant problems.

Scheme 5.12 Synthesis of chiral butyrolactones from allylic chloroalcohol.

As illustrated in Scheme 5.12, it would still be possible to obtain only one diastereomer of the ring-closed product, despite the lack of diastereoselectivity in the rearrangement reaction. Deprotonation of lactones 5.9, e.g. by DBU, followed by reprotonation, would selectively lead to the more stable trans-substituted butyrolactones.

157



5.5 Suggestions for further research

Numerous possible applications for chiral chloroalcohols can be envisioned. Here, a number of ideas will be described which have a lot of potential, but could not be completed due to lack of time.

For instance, the hydroxy moiety in enantiopure (E)-1-chloro-4-phenylbut-3-en-2-ol (5.10a) could be converted into a better leaving group in a stereospecific fashion, creating a molecule with two neighbouring leaving group next to a double bond (5.20). A tandem of two consecutive regio- and stereoselective copper-catalyzed allylic substitution reactions would, via intermediate 5.21, lead to highly functionalized enantiopure products 5.22 (Scheme 5.13). Copper-catalyzed asymmetric alkylations39 usually show the desired γ-selectivity, as opposed to catalysts based on e.g. palladium which often give alkylation at the α-position.40

Scheme 5.13 Tandem copper-catalyzed allylic alkylation. Nu1 and Nu2 are the first and second nucleophile, respectively.

To initiate this chemistry, 5.10a was converted into (E)-(3,4-dichlorobut-1-enyl)benzene (5.20a) using triphenylphosphine in tetrachloromethane.41 However, initial allylic alkylation experiments were unsuccessful and there was insufficient time to optimize the reaction conditions.

Related to the previous idea, it could be possible to perform transition-metal catalyzed allylic substitutions on vinyloxiranes under aqueous conditions. Vinyloxiranes 5.23 are the products of enzymatic kinetic resolution of chloroalcohols described in Chapter 3, and there lability to hydrolysis stood in the way of their isolation. However, transforming them in situ by way of allylic substitution would yield the much more

158


Chapter 5

stable product 5.24, provided it is possible to do such a transformation in an aqueous environment (Scheme 5.14).

Scheme 5.14 Transition metal catalyzed allylic alkylation of vinyloxiranes under aqueous conditions.

Fortunately, there are indications in the literature that this might be possible. Recently, conditions have been described for Pd/C-mediated allylic substitution in water42 and palladium-catalyzed allylic substitution leading to lactones in a water / EtOAc biphasic system.43 Both examples use allyl acetates as substrates. Also, palladium-catalyzed allylic substitution using allyl alcohols as allylating agents in aqueous environment has been described.44

5.6 Conclusions

Chloroalcohols 5.3, 5.10a, and 5.10c have been used in Achmatowicz, Ireland-Claisen, and Johnson orthoester rearrangements. Initial results are promising, nevertheless extensive further research has to be done for these reactions to reach their full potential. In the case of the Achmatowicz rearrangement of 5.3, product 5.4 has been obtained in 26% yield, whereas the yield of the corresponding non-chlorinated compound 5.2 was 85%. Moreover, 5.4 was found to be significantly less stable than 5.2.

Ireland-Claisen rearrangement of unsaturated chloropropionate 5.7b so far gives a maximum conversion of 46% to product 5.11, whereas several side-products are observed. Hence, optimum conditions have yet to be established for this transformation. In the case of the Johnson orthoester rearrangement of chloroalcohols 5.10, the conversion to products 5.19 is high (80 − >98%), but lack of stereocontrol is an issue that still has to be resolved.


5.7.1 General remarks For general remarks, see Chapters 2 and 3.

159



5.7.2 Achmatowicz rearrangement

1-(Furan-2-yl)ethanol (5.1)

Prepared by Grignard addition of in situ prepared MeMgI to furfural. Reactions conditions, work-up procedure, and spectral data were in accordance with the literature.45

6-Hydroxy-2-methyl-2H-pyran-3(6H)-one (5.2) 8

Synthesized according to a literature procedure8 as a 65:35 mixture of diastereoisomers. 1H NMR (CDCl3) δ 6.92 (d, J = 10.3 Hz, 1H, minor isomer), 6.89 − 6.85 (m, 1H, major isomer), 6.12 (d, J = 10.3 Hz, 1H, minor), 6.07 (d, J = 10.3 Hz, 1H, major), 5.65 (br s, 1H, minor), 5.60 (br d, J = 2.2 Hz, 1H, major), 4.68 (qd, J = 7.0, 1.1 Hz, 1H, major), 4.20 (q, J = 6.6 Hz, 1H, minor), 3.94 (br s, 1H, minor), 3.60 (br s, 1H, major), 1.43 (dd, J = 6.6, 1.5 Hz, 1H, minor), 1.37 (dd, J = 7.0, 1.5 Hz, 1H, major); MS (EI+) m/z = 128 (M+), 111, 84, 55, 43.

2-Chloro-1-(furan-2-yl)ethanol (5.3) The synthesis of 2-chloro-1-(furan-2-yl)ethanol (5.3) from furfural and chloroiodomethane is described in Chapter 3 (compound 3.6).

2-(Chloromethyl)-6-hydroxy-2H-pyran-3(6H)-one (5.4)

2-Chloro-1-(furan-2-yl)ethanol (5.3, 150 mg, 1.0 mmol) was dissolved in a THF/H2O 3:1 (4 mL) after which the mixture was cooled down to 0 °C. Subsequently, NaHCO3 (170 mg, 2.0 mmol) and NaOAc•3H2O (138 mg, 1.0 mmol) were added and the mixture was stirred until all components had dissolved. Then, NBS (180 mg, 1.0 mmol) was added and the mixture, which turned yellow, was allowed to stir at 0 °C for 2 h. The reaction was then quenched by addition of aq. NaHCO3 sat. (4 mL, the mixture turned red upon addition), extracted with Et2O (3 ×), dried over MgSO4, filtered and

160


Chapter 5

evaporated, yielding a blue powder (154 mg). Subsequent recrystallization yielded light brown crystals (43 mg, 0.265 mmol, 26%). NMR showed a single diastereomer. 1H NMR (acetone-d6, δ 2.05) δ 7.12 (dd, J = 10.3, 3.7 Hz, 1H), 6.13 (d, J = 6.2 Hz, 1H), 6.07 (d, J = 10.3 Hz, 1H), 5.69 (dd, J = 6.2, 3.3 Hz, 1H), 4.88 (dd, J = 5.1, 3.0 Hz, 1H), 3.98 (d, J = 11.7, 5.1 Hz, 1H), 3.86 (d, J = 11.7, 3.0 Hz, 1H); 13C NMR (acetone-d6, Ccarbonyl δ 206.2) δ 193.9 (s), 148.0 (d), 127.0 (d), 88.4 (d), 74.3 (d), 44.0 (t); MS (EI+) m/z = 162 (M+), 145, 126, 109, 99, 84, 55, 43; chiral GC: Chiraldex G-TA, 25m x 0.25 mm x 0.25 μm, He-flow: 1.0 mL/min, 50 °C to 150 °C, 3 °C/min, 150 °C to 180 °C, 10 °C/min, hold 10 min, 180 °C to 50 °C, 10 °C/min, stop, Tr = 38.5 min (first enantiomer), Tr = 40.7 (second enantiomer).

5.7.3 Ireland-Claisen rearrangement

(E)-1-Chloro-4-phenylbut-3-en-2-yl acetate (5.7a)

(E)-1-Chloro-4-phenylbut-3-en-2-ol (5.10a, 0.91 g, 5.0 mmol) was mixed with pyridine (1.0 mL) and acetic anhydride (1.0 mL, 2 eq) at 0 °C. This mixture was allowed to stir overnight, during which the temperature slowly rose to room temperature. Excess reagents were removed by evaporation and the product was purified by flash chromatography over SiO2 (eluent: n-pentane / Et2O 9:1, Rf 0.29, quant.). 1H NMR (CDCl3) δ 7.40 − 7.24 (m, 5H), 6.70 (d, J = 16.1 Hz, 1H), 6.15 (dd, J = 15.7, 7.3 Hz, 1H), 5.61 (dd, J = 12.4, 5.9 Hz, 1H), 3.68 (d, J = 5.1, 2H), 2.12 (s, 3H); 13C NMR (CDCl3) δ 169.9 (s), 135.6 (s), 134.8 (d), 128.6 (d), 128.4 (d), 126.7 (d), 123.6 (d), 73.6 (d), 45.6 (t), 21.0 (q); MS (EI+) m/z = 224 (M+), 188, 146, 133, 128, 115, 103, 91, 77, 55, 51; HRMS (EI+) calcd. for C12H1335ClO2: 224.0604, found: 224.0608.

(E)-1-Chloro-4-phenylbut-3-en-2-yl propionate (5.7b)

Prepared analogous to 5.7a, in quantitative yield. Purified by flash chromatography over SiO2 (eluent: n-pentane / Et2O 9:1, Rf 0.36). 1H NMR (CDCl3) δ 7.39 − 7.24 (m, 5H), 6.70 (d, J = 15.8 Hz, 1H), 6.15 (dd, J = 16.1, 7.3 Hz, 1H), (dd, J = 12.4, 6.2 Hz, 1H), 3.68 (d, J = 6.2, 2H), 2.40 (qd, J = 7.3, 1.5 Hz, 2H), 1.17 (t, J = 7.3 Hz, 3H); 13C NMR (CDCl3) δ 173.3 (s), 170.2 (s), 134.6 (d), 128.6 (d), 128.3 (d), 126.7 (d),

161



123.7 (d), 73.3 (d), 45.6 (t), 27.6 (t), 9.0 (q); MS (EI+) m/z = 238 (M+), 202, 146, 129, 115, 103, 91, 77, 57, 51; HRMS (EI+) calcd. for C13H1535ClO2: 238.0761, found: 238.0752.

(E)-1-Chlorooct-3-en-2-yl propionate (5.7c)

(E)-1-Chlorooct-3-en-2-ol (5.10b, 0.81 g, 5.0 mmol) was mixed with pyridine (2.5 mL) and acetic anhydride (2.5 mL, 5 eq) at 0 °C. This mixture was stirred overnight, during which the temperature slowly rose to room temperature. The reaction was then quenched with aq. HCl 2M and extracted with Et2O (3×). The combined organic fractions were washed with aq. NaHCO3 and brine, respectively, dried over MgSO4, filtered and evaporated, giving 5.7c (1.03 g, 4.74 mmol, 95%). 1H NMR (CDCl3) δ 5.86 − 5.76 (m, 1H), 5.45 − 5.38 (m, 2H), 3.56 (br d, J = 3.7 Hz, 2H), 2.34 (q, J = 7.7 Hz, 2H), 2.05 − 1.99 (m, 2H), 1.37 − 1.24 (m, 4H), 1.13 (t, J = 7.3 Hz, 3H), 0.86 (t, J = 7.0 Hz, 3H); 13C NMR (CDCl3) δ 173.4 (s), 137.0 (d), 124.6 (d), 73.5 (d), 45.8 (t), 31.9 (t), 30.8 (t), 27.7 (t), 22.1 (t), 13.8 (q), 9.0 (q); MS (EI+) m/z = 218 (M+), 183, 126, 109, 97, 67, 57, 41; HRMS (EI+) calcd. for C11H19O2 (M+ − Cl): 183.1385, found: 183.1379.

General procedure for Ireland-Claisen rearrangement of substrates 5.7 A flame-dried 50 mL flask under an atmosphere of nitrogen was charged with 5.7a, 5.7b, or 5.7c (0.25 mmol), HMPA (1.5 equiv.), and 2 mL of freshly distilled THF. The reaction mixture was then cooled to − 80 °C and a 1.0 M solution of base (typically 0.3 mL) was added, followed after 15 min of stirring by freshly distilled TMSCl (0.1 mL, 0.78 mmol). Subsequently, the reaction mixture was allowed to stir overnight, during which the temperature gradually increased to room temperature. The reaction was then quenched by addition of NH4Claq sat., extracted with Et2O (3×), the combined organic fractions washed with aq. NaHCO3 and brine, respectively, dried over MgSO4, filtered and evaporated. Product composition was determined using GC-MS and 1H NMR.

(E)-4-Phenylbuta-1,3-dien-2-yl propionate (5.14)

(E)-1-Chloro-4-phenylbut-3-en-2-yl propionate (5.7b, 242 mg, 1.0 mmol) was dissolved in THF (5 mL), after which DBU (0.15 mL) was added. The resulting solution was heated at reflux for an hour, after which a white precipitate appeared, which was filtered and washed with freshly distilled THF. The filtrate was

162


Chapter 5

concentrated in vacuo and the crude product thus obtained was purified by flash chromatography on SiO2 (eluent: n-pentane / EtOAc gradient 50:1 to 20:1). The main isolated fraction was identified as (E)-4-phenylbuta-1,3-dien-2-yl propionate (129 mg, 0.64 μmol, 64%). 1H NMR (CDCl3) δ 7.42 − 7.22 (m, 5H), 6.59 (q, J = 15.8 Hz, 2H), 5.11 (s, 1H), 4.96 (s, 1H), 2.57 (qd, J = 7.3, 1.8 Hz, 2H), 1.26 (td, J = 7.3, 1.8 Hz, 3H); 13C NMR (CDCl3) δ 172.2 (s), 151.8 (s), 136.0 (s), 129.8 (d), 128.6 (d), 128.2 (d), 126.8 (d), 122.7 (d), 106.0 (t), 27.6 (t), 9.2 (q). MS (EI+) m/z = 202 (M+), 146, 145, 128, 117, 102, 91, 77, 57, 51.

5.7.4 Johnson orthoester rearrangement

(E)-6-Chloro-2-methyl-3-phenyl-hex-4-enoic acid ethyl ester (5.19a)

(E)-1-Chloro-4-phenylbut-3-en-2-ol (5.10a, 54 mg, 0.3 mmol) was dissolved in 2 mL of freshly distilled toluene, along with triethyl orthopropionate (0.5 mL, 443 mg, 2.5 mmol) and 2 drops of propionic acid. This solution was stirred at reflux for 1 d. Following addition of water, the mixture was extracted with Et2O (2x), the combined organic fractions washed with aq. NaHCO3 and brine, respectively, dried over MgSO4, filtered and the solvent evaporated. The crude product thus obtained was purified by column chromatography over silica, using n-pentane / Et2O 19:1 as eluent. (E)-Ethyl 6-chloro-2-methyl-3-phenylhex-4-enoate (5.19a) was obtained in 48% yield as an oil, consisting of an equimolar mixture of cis/trans isomers that was not further separated. 1H NMR (CDCl3) δ 7.39 − 7.14 (m, 2 × 5H), 5.97 − 5.83 (m, 2 × 1H), 5.75 − 5.59 (m, 2 × 1H), 4.17 − 3.82 (m, 2 × 4H), 3.50 (td, J = 9.5, 2.2 Hz, 1H), 3.44 (m, 1H), 2.85 − 2.74 (m, 2 × 1H), 1.26 (t, J = 7.0 Hz, 3H), 1.25 (t, J = 7.0 Hz, 3H), 0.96 (d, J = 7.0 Hz, 3H), 0.95 (d, J = 7.0 Hz, 3H); 13C NMR (CDCl3) δ 175.4 & 174.9 (s), 141.6 & 140.7 (s), 136.3 & 135.3 (d), 128.7 (d), 128.6 (d), 128.5 (d), 128.03 (d), 128.00 (d), 127.7 (d), 126.9 & 126.7 (d), 60.4 & 60.1 (t), 52.1 & 52.0 (d), 45.3 & 45.0 (d), 44.7 & 44.6 (t), 15.8 & 15.6 (q), 14.2 & 13.9 (q); MS (EI+) m/z = 267 (M+), 231, 165, 157, 129, 115, 102, 91, 77, 65, 51; HRMS (EI+) calcd. for C15H19O2 (M+ − Cl): 231.1385, found: 231.1377.

163



(E)-6-Chloro-3-phenyl-hex-4-enoic acid ethyl ester (5.19b)

Prepared analogous to 5.19a. 1H NMR (CDCl3) δ 7.44 − 7.18 (m, 5H), 5.93 (dd, J = 15.0, 7.3 Hz, 1H), 5.65 (dtd, J = 15.0, 7.0, 1.3 Hz, 1H), 4.08 (q, J = 7.0 Hz, 2H), 4.01 (d, J = 7.0, 2H), 3.88 (q, J = 7.3 Hz, 1H), 2.78 − 2.66 (m, 2H), 1.78 (t, J = 7.0 Hz, 3H); 13C (CDCl3) δ 171.5 (s), 141.8 (s), 136.9 (d), 128.6 (d), 127.5 (d), 126.9 (d), 126.4 (d), 60.5 (t), 44.7 (t), 44.2 (d), 40.4 (t), 14.1 (q); MS (EI+) m/z = 217, 170, 165, 142, 129, 115, 103, 91, 77, 65, 51; HRMS (EI+) calcd. for C14H17O2 (M+ − Cl): 217.1229, found: 217.1220; E.e. determination using chiral HPLC: Chiralcel OD, 40 °C, n-heptane/IPA 99:1, 1.0 mL/min, Tr = 10.2 min (S), 12.9 min (R).

Using (S)-5.10a (>98% ee) as starting material, 5.19b was obtained with 41% ee.

(E)-6-Chloro-2,3-dimethyl-hex-4-enoic acid ethyl ester (5.19c)

Prepared analogous to 5.19a and obtained as a 1:1 mixture of diastereomers. 1H NMR (CDCl3) δ 5.72 − 5.52 (m, 2H), 4.08 (q, J = 7.3 Hz, 2H), 3.99 (t, J = 6.2, 2H), 2.45 (q, J = 7.0 Hz, 1H), 2.33 (qdd, J = 7.3, 7.0, 6.6 Hz, 1H), 1.22 (d, J = 7.0 Hz, 3H), 1.07 (t, J = 6.6 Hz, 3H), 0.99 (d, J = 6.6 Hz, 3H); 13C (CDCl3) δ 138.3 & 137.7 (d), 126.6 & 126.0 (d), 60.6 & 60.3 (t), 45.0 (t), 44.9 & 44.7 (d), 39.4 & 39.1 (d), 18.2 (q), 16.5 (q), 14.5 (q), 14.2 (q), 13.7 (q); MS (EI+) m/z = 169, 159, 153, 141, 131, 123, 113, 103, 958, 85, 74, 67, 55, 41; MS (CI+) m/z = 224 (M + NH4+), 222 (M + NH4+).

5.7.5 Further research

(E)-(3,4-dichlorobut-1-enyl)benzene (5.20a).

Prepared from 5.10a, PPh3 and CCl4, according to a literature procedure.41b 1H NMR (CDCl3) δ 7.47 − 7.24 (m, 5H), 6.71 (d, J = 15.4 Hz, 1H), 6.19 (dd, J = 15.8, 8.8 Hz, 1H), 4.68 (m, 1H), 3.86 (dd, J = 11.0, 5.5 Hz, 1H), 3.76 (dd, J = 11.0, 8.1 Hz, 1H); 13C NMR (CDCl3) δ 135.4 (s), 134.9 (d), 128.7 (d), 128.6 (d), 126.9 (d), 125.8 (d), 61.1 (d), 47.7 (t); Chiral HPLC: Chiralcel OD, 40 °C, n-heptane/IPA 99:1, 1.0 mL/min, Tr = 8.9, 10.5 min.

164


Chapter 5

5.8 Notes and references 1 O. Pàmies and J.-E. Bäckvall, J. Org. Chem. 2002, 67, 9006-9010. 2 S. P. Tanis, B. R. Evans, J. A. Nieman, T. T. Parker, W. D. Taylor, S. E. Heasley, P. M. Herrinton, W. R. Perrault, R. A. Hohler, L. A. Dolak, M. R. Hester, and E. P. Seest, Tetrahedron: Asymmetry 2006, 17, 2154-2182. 3 O. Achmatowicz Jr., P. Bukowski, B. Szechner, Z. Zwierzchowska, and A. Zamojski, Tetrahedron 1971, 27, 1973-1996. 4 A. C. Comely, R. Eelkema, A. J. Minnaard, and B. L. Feringa, J. Am. Chem. Soc. 2003, 125, 8714-8715. 5 L. Zhu, A. Talukdar, G. Zhang, J. P. Kedenburg, and P. G. Wang, Synlett 2005, 1547-1550. 6 M. H. Haukaas and G. A. O'Doherty, Org. Lett. 2002, 4, 1771-1774. 7 R. S. Babu, M. Zhou, and G. A. O'Doherty, J. Am. Chem. Soc. 2004, 126, 3428-3429. 8 H. Guo and G. A. O’Doherty, Org. Lett. 2005, 7, 3921-3924. 9 S. J. Shimshock, R. E. Waltermire, and P. DeShong, J. Am. Chem. Soc. 1991, 113, 8791-8796. 10 M. Bennett, G. B. Gill, G. Pattenden, A. J. Shuker, and A. Stapleton, J. Chem. Soc., Perkin Trans. 1, 1991, 929-937. 11 S. J. Hobson and R. Marquez, Org. Biomol. Chem. 2006, 4, 3808-3814. 12 M. P. Georgiadis, K. F. Albizati, and T. M. Georgiadis, Org. Prep. Proced. Int. 1992, 24, 95-118. 13 M. P. Georgiadis and E. A. Couladouros, J. Org. Chem. 1986, 51, 2725-2727. 14 B. M. Adger, C. Barrett, J. Brennan, M. A. McKervey, and R. W. Murray, J. Chem. Soc., Chem. Commun. 1991, 1553-1554. 15 A. De Mico, R. Margarita, and G. Piancatelli, Tetrahedron Lett. 1995, 36, 3553-3556. 16 a) M. Kusakabe, Y. Kitano, Y. Kobayashi, and F. Sato, J. Org. Chem. 1989, 54, 2085-2091; b) Y. Kobayashi, M. Kusakabe, Y. Kitano, and F. Sato, J. Org. Chem. 1988, 53, 1586-1587. 17 J. Wahlen, B. Moens, D. E. De Vos, P. L. Alsters, and P. A. Jacobs, Adv. Synth. Catal. 2004, 346, 333-338. 18 C. A. Leverett, M. P. Cassidy, and A. Padwa, J. Org. Chem. 2006, 71, 8591-8601. 19 T. C. Coombs, M. D. Lee IV, H. Wong, M. Armstrong, B. Cheng, W. Chen, A. F. Moretto, and L. S. Liebeskind, J. Org. Chem. 2008, 73, 882-888. 20 L. M. Beauchamp, B. L. Serling, J. E. Kelsey, K. K. Biron, P. Collins, J. Selway, J.-C. Lin, and H. J. Schaeffer, J. Med. Chem. 1988, 31, 144-149.

165



21 M. P. Doyle, J. S. Tedrow, A. B. Dyatkin, C. J. Spaans, and D. G. Ene, J. Org. Chem. 1999, 64, 8907-8915. 22 a) R. E. Ireland, R. H. Mueller, and A. K. Willard, J. Am. Chem. Soc. 1976, 98, 2868-2877; b) R. E. Ireland and A. K. Willard, Tetrahedron Lett. 1975, 16, 3975; c) R. E. Ireland and R. H. Mueller, J. Am. Chem. Soc. 1972, 94, 5897-5898. 23 a) L. Kürti and B. Czakó, Strategic Applications of Named Reactions in Organic Synthesis, Elsevier Academic Press, 2005, pp. 90-91; b) ibid., pp. 226-227. 24 T. J. Gould, M. Balestra, M. D. Wittman, J. A. Gary, L. T. Rossano, and J. Kallmerten, J. Org. Chem. 1987, 52, 3889-3901. 25 C. Fehr and J. Galindo, Angew. Chem. Int. Ed. 2000, 39, 569-573. 26 H. Ko, E. Kim, J. E. Park, D. Kim, and S. Kim, J. Org. Chem. 2004, 69, 112-121. 27 R. E. Ireland and J. P. Daub, J. Org. Chem. 1981, 46, 479-485. 28 C. McFarland, J. Hutchison, and M. C. McIntosh, Org. Lett. 2005, 7, 3641-3644. 29 M. Sakaitani and Y. Ohfune, J. Am. Chem. Soc. 1990, 112, 1150-1158. 30 Analogous to the preparation of (E)-4-bromo-4-chloro-5,5,5-trifluoro-1-phenylpent-1-en-3-ol according to: T. Takagi, M. Nakamoto, K. Sato, M. Koyama, A. Ando, and I. Kumadaki, Tetrahedron, 1996, 52, 12667-12676. 31 Y. Ohfune, K. Hori, and M. Sakaitani, Tetrahedron Lett. 1986, 27, 6079-6082. 32 W. S. Johnson, L. Werthemann, W. R. Bartlett, T. J. Brocksom, T.-T. Li, D. J. Faulkner, and M. R. Petersen, J. Am. Chem. Soc. 1970, 92, 741-743. 33 G. B. Jones, R. S. Huber, and S. Chau, Tetrahedron 1993, 49, 369-380. 34 M. Lounasmaa, Curr. Org. Chem. 1998, 2, 63-90. 35 E. Brenna, C. Fuganti, F. G. Gatti, M. Passoni, and S. Serra, Tetrahedron: Asymmetry 2003, 14, 2401-2406. 36 C. Agami, F. Couty, and G. Evano, Tetrahedron Lett. 2000, 41, 8301-8305. 37 T. Hiyama, K. Kobayashi, and M. Fujita, Tetrahedron Lett. 1984, 25, 4959-4962. 38 G. W. Daub, J. P. Edwards, C. R. Okada, J. Westran Allen, C. Tata Maxey, M. S. Wells, A. S. Goldstein, M. J. Dibley, C. J. Wang, D. P. Ostercamp, S. Chung, P. Shanklin Cunningham, and M. A. Berliner, J. Org. Chem. 1997, 62, 1976-1985. 39 For a recent example, see: A. W. van Zijl, F. López, A. J. Minnaard, and B. L. Feringa, J. Org. Chem. 2007, 72, 2558-2563. 40 See for instance the following reviews: a) H. Yorimitsu and K. Oshima, Angew. Chem. Int. Ed. 2005, 44, 4435-4439; b) B. M. Trost and M. L. Crawley, Chem. Rev. 2003, 103, 2921-2943; c) B. M. Trost and C. Lee, In Catalytic Asymmetric Synthesis, 2nd ed., I. Ojima, Ed., Wiley-VCH, New York, 2000, pp. 593-649.

166


Chapter 5

41 a) M. Pineschi, F. Bertolini, R. M. Haak, P. Crotti, and F. Macchia, Chem. Commun. 2005, 1426-1428; b) C. N. Barry and S. A. Evans Jr., J. Org. Chem. 1981, 46, 3361-3364. 42 F.-X. Felpin and Y. Landais, J. Org. Chem. 2005, 70, 6441-6446. 43 H. Kinoshita, H. Shinokubo, and K. Oshima, Angew. Chem. Int. Ed. 2005, 44, 2397-2400. 44 K. Manabe and S. Kobayashi, Org. Lett. 2003, 5, 3241-3244. 45 A. J. M. Janssen, A. J. H. Klunder, and B. Zwanenburg, Tetrahedron 1991, 47, 7645-7662.

Chapter 6 Enantiopure alcohols as amplifiers of 2D chirality

In this chapter, the preparation of enantiomerically pure (R)- and (S)-1-phenyl-1-

octanol by enzymatic kinetic resolution of the corresponding racemic acetate is

described. Using lipase from Pseudomonas cepacia, both enantiomers were obtained in

>98% ee. Subsequently, the products were used as chiral solvents to control the

enantiopreference of the chiral self-assembly of achiral molecules on an achiral surface.

These results demonstrate for the first time that enantiopure solvents may be used to

control chiral self-assembly on a surface, providing an elegant and low-cost method to

form large enantiomerically pure organic surfaces.a,1

a Part of this chapter has been published: N. Katsonis, H. Xu, R. M. Haak, T. Kudernac, Ž. Tomovic, S. George, M. van der Auweraer, A. P. H. J. Schenning, E. W. Meijer, B. L. Feringa, and S. de Feyter, Angew. Chem. Int. Ed. 2008, 47, 4997-5001.

168

Chapter 6 - Enantiopure alcohols as amplifiers of 2D chirality.doc

Chapter 6

6.1 Introduction

Asymmetric transformations using enzymes are very efficient means of obtaining enantiopure compounds, because of their generally high activity and selectivity (see, for instance, Chapters 3 and 4 of this thesis).2 A popular class of enzymes for organic reactions are lipases (EC 3.1.1.3), owing to the mild reaction conditions they allow, their availability at low cost, their stability in various media, in particular organic solvents, their broad substrate range and high stereoselectivity. Also called “the workhorses of biocatalysis”, they have been used for years in the enantioselective hydrolysis or formation of esters. The first reaction is performed in aqueous media, while the latter often proceeds in organic solvents.3

In this chapter, lipase-catalyzed preparation of enantiopure alcohols (R)- and (S)-6.1 (Figure 6.1) is combined with nanoscale investigations on the control of two-dimensional (2D) chirality. The study of chiral surfaces has become an important scientific field in recent years, initial interest coming from the area of heterogeneous catalysis.4 A recent example is the single-molecule imaging of a manganese-porphyrin catalyst at work during an oxidation reaction, reported by Elemans and coworkers.5 Furthermore, asymmetric catalysis on solid supports has great potential as an efficient, sustainable methodology in the synthesis of optically active chemicals.6

The importance of surface chemistry, especially to catalytic processes, is emphasized by the awarding of the 2007 Nobel Prize in Chemistry to Gerhard Ertl.7 However, the study and selective functionalization of chiral surfaces is also relevant for purposes such as chiral recognition, new thin film devices for optical and electronic applications,4 and nanotechnology.8 The critical influence of surface/molecule interactions on expression of chirality at the fluid/solid interface, which is a relevant topic for the development of material sciences, has also been recently reviewed.4,9

Self-assembly, i.e. the spontaneous formation of highly organized structures from molecular components by noncovalent interactions, is an elegant way to achieve surface functionalization.8,10 The supramolecular assemblies formed by self-assembly can be studied using a variety of techniques, but for the observation of surface patterns the most prominent techniques are high-resolution microscopy techniques such as atomic force microscopy (AFM) and scanning tunneling microscopy (STM).11 STM, originating in the early 1980's,12 is a convenient technique to study chirality in 2D supramolecular self-assemblies, since it allows for the resolution of structures on a surface at submolecular or even atomic level.8 Hembury et al. have recently given an overview of various supramolecular systems for chirality-sensing purposes, including some examples of direct chirality observation on surfaces.11 Besides pointing out some advances that

169


Enantiopure alcohols as amplifiers of 2D chirality

have been made in this area, these authors emphasize that the exact nature of the mechanisms of chirality transfer from the molecular to the supramolecular level in these systems is often poorly understood. Moreover, the 2D supramolecular structures described so far are mostly only locally chiral. This means that, even when chiral domains are spontaneously formed on a surface, both enantiomorphic domains are statistically formed in equal amounts, so overall, the samples are racemic. For future applications, it is essential to control the formation of homochiral domains in order to obtain an excess of a single enantiomorph on a surface. An example of such an amplification of chirality was recently reported for heptahelicene monolayers on Cu(111) in ultra-high vacuum, showing a strongly positive non-linear dependence of lattice chirality on the enantiomeric excess of the heptahelicene.13

Another possibility to create macroscopically chiral monolayers, in contrast to locally chiral but globally racemic monolayers, could be the use of chiral solvents. So far, optically active solvents have been used in areas unrelated to the research described here, such as double stereodifferentiation in enantioselective reactions14 or chromatographic separation of α-amino acid enantiomers using a chiral eluent.15 The use of chiral solvents to achieve control over supramolecular stereochemistry on surfaces has not been reported.

In this chapter, the enantioselective formation of chiral monolayers of achiral molecules on achiral surfaces is investigated by means of STM at the liquid/solid interface. In the next paragraphs, it will be shown that chiral surface patterns can be formed from achiral molecules on an achiral surface, by using enantiomerically pure 1-phenyl-1-octanol (6.1, Figure 6.1) as chiral solvent.

The choice for 6.1 was motivated by the fact that it is the logical hybrid form between 1-phenyloctane and 1-octanol, typical solvents for scanning tunneling microscopy (STM) imaging at the liquid − solid interface.4,8,9,16 Since we used 1-phenyloctyl acetate (6.2, Figure 6.1) as a precursor for enantiomerically pure 6.1 (see paragraph 6.2), this solvent was also employed as solvent in STM imaging (vide infra).

Figure 6.1 The chiral solvents used in this study, (R)- and (S)-1-phenyl-1-octanol ((R)-6.1 and (S)-6.1) and (R)- and (S)-1-phenyloctyl acetate ((R)-6.2 and (S)-6.2).

170


Chapter 6

The compound used as adsorbate was a hydrogen-bonding achiral diamino triazine oligo-(p-phenylenevinylene) oligomer (A-OPV4T, Figure 6.2). It had already been shown that its chiral analogue, (S)-OPV4T17 (Figure 6.2) assembles exclusively in a counterclockwise rosette motif at the liquid-solid interface, using graphite as substrate and 1-phenyloctane as solvent.18,19 Such transfer of molecular chirality to a surface, creating enantiomorphous patterns, has been observed more often.11 Furthermore, amplification of chirality in dynamic supramolecular aggregates has been observed in solution, as recently reviewed by Meijer et al.20 In most cases, the self-assembly of achiral molecules on an atomically flat surface also involves the breaking of symmetry leading to the formation of locally chiral, enantiomorphic structures.8,9,21 Although it has been demonstrated that the structure of the surface has a dramatic influence on the chiral properties of the monolayers,9 solvents had not yet been demonstrated to be a symmetry breaking agent with respect to surface organisation.

More specifically, it was not known whether an achiral oligo-(p-phenylenevinylene) such as A-OPV4T (Figure 6.2), would adsorb from a chiral solvent onto graphite by forming a chiral pattern similar to (S)-OPV4T, and if so, whether the configuration of the resulting chiral surface structures could be influenced by choosing an appropriate chiral solvent.

Figure 6.2 a) A-OPVT, the compound used in this study; b) (S)-OPV4T, the chiral counterpart of A-OPVT.

171



6.2 Synthesis of chiral solvents

The synthesis and kinetic resolution of 1-aryl-1-alkanols with long alkyl chains was described in 1990 by Mori and Bernotas, with the objective of applying these compounds in liquid crystal technology.22 First, racemic 6.1 was prepared by Grignard addition of n-heptyl magnesium bromide to benzaldehyde, as depicted in Scheme 6.1.

Scheme 6.1 Synthesis of rac-1-phenyl-1-octanol.

Subsequent esterification using acetic anhydride in pyridine yielded 6.2 in quantitative yield (Scheme 6.2).

Scheme 6.2 Synthesis of rac-1-phenyloctyl acetate.

Mori and Bernotas reported that lipase-catalyzed transesterification of 1-phenyloctanol with vinyl and 2-propenyl acetate in organic solvents such as benzene and n-heptane proceeded slowly and with low enantioselectivity.22 However, lipase-catalyzed hydrolysis of the acetate in aqueous phosphate buffer − i.e. the reverse reaction − gave excellent results. We adopted their system and obtained (R)-6.1 and (S)-6.2 in good yield and excellent enantioselectivity (>98% ee for both compounds, determined using chiral HPLC), after an initial screening of a number of Pseudomonas lipases, of which Pseudomonas cepacia gave the best results (Scheme 6.3). As an extension of this approach, we successfully scaled up this protocol from 1 to 20 mmol.

The enantiomers (S)-6.1 and (R)-6.2 were obtained by acetylation of (R)-6.1 and base-assisted deprotection of (S)-6.2, respectively.

172


Chapter 6

Scheme 6.3 Enzymatic kinetic resolution of 1-phenyl-1-octyl acetate.

Thus, four solvents were prepared in high (enantio)purity for use in STM studies, namely (R)- and (S)-1-phenyl-1-octanol and (R)- and (S)-1-phenyloctyl acetate.

6.3 Control of enantioselective 2D self-assembly using chiral solvents

Nanoscale investigation on 2D self-assembly of OPVs has been carried out by STM at the interface between highly oriented pyrolytic graphite (HOPG) and a solution of A-OPV4T in enantiomerically pure 6.1. STM images demonstrate the formation of monolayers of A-OPV4T (Figure 6.3). Within the monolayer, A-OPV4T self-assembles into star-shaped features with six bright arms, hereafter called “rosettes” (Figure 6.3). These bright arms correspond to the conjugated OPV backbone (Figure 6.2) with the molecular axis lying parallel to the surface. The alkyl chains are adsorbed in the low-contrast areas, but in high-resolution STM images they are visible (e.g. Figure 6.3b and c).

Since the OPV units at opposite sides of the rosettes are not in line, but show a clear non-radial orientation (Figure 6.3a, b, and c), they can be classified as clockwise (CW) or counterclockwise (CCW), which are mirror images of each other. In other words, the rosettes show 2D chirality.

Using (S)-6.1 as solvent, a clear bias towards the formation of CW rosettes is observed, whereas using (R)-6.1 primarily CCW rosettes are formed. This bias demonstrates the control which the chiral solvent exerts on supramolecular surface chirality. Furthermore, not only the chirality of the rosettes themselves is dependent on the solvent, but also the − chiral − orientation of the rosettes with respect to each other. This next level of hierarchical self-assembly is illustrated in Figure 6.3b and c, where the dashed marker lines are longer than the solid ones, representing a non-superimposable 2D-chiral arrangement of the rosettes.

In both (R)- and (S)-6.1, many ordered domains of variable size have been observed. Within a given domain, the rosettes are ordered in rows and form a homochiral crystalline lattice characterized by the following unit cell parameters: a = 6.11 ± 0.06

173



nm, b = 6.13 ± 0.04 nm, γ = 60 ± 1° in (S)-6.1 (Figure 6.3c) and a = 6.09 ± 0.06 nm, b = 6.04 ± 0.05 nm, γ = 62 ± 2° in (R)-6.1 (Figure 6.3b). These values are identical − within experimental error − to those of enantiopure (S)-OPV4T at the interface of 1-phenyloctane and HOPG.18 A-OPV4T self-assembles into a chiral pattern in accordance with the plane group p6, one of the five possible chiral space groups on surfaces.23,24

Figure 6.3 Enantioselective formation of rosettes on a surface using chiral solvents as visualized by STM. a) and b) are STM images of an A-OPV4T monolayer at the (R)-6.1 − HOPG interface. A) In addition to several domains of CCW rosettes, one CW domain is observed as marked. Scale bar is 10 nm. B) High-resolution image of the CCW rosette. Individual OPV units are indicated to emphasize the non-radial orientation (top right corner). The rotation direction is highlighted by the white arrow. Scale bar is 3 nm. c) Molecular resolution STM image of an A-OPV4T monolayer at the (S)-6.1 − HOPG interface. The CW rotation direction is highlighted by the white arrow. Scale bar is 3 nm. d) Proposed hydrogen bonding motif of the CCW rotating rosette, involving six A-OPV4T molecules. Arrows indicate the nitrogen atoms which remain free to interact by hydrogen bonding with the solvent molecules.

The observed solvent-induced asymmetry at the liquid-solid interface has been quantitatively confirmed by statistical analysis, wherein a large number of individual rosettes (>1000 per experiment) were indexed as CW or CCW. This analysis has been

174


Chapter 6

carried out using several batches of solvent and substrate in order to ensure reproducibility of the results. As is shown in Table 6.1, monolayer formation in enantiopure 6.1 is characterized by solvent-controlled asymmetric induction. However, complete induction of asymmetry is never observed, possibly because of the slow kinetics of the ordering process. The measured enantiomeric ratios (CCW : CW) range from 17 : 83 in (S)-6.1 (entry 2) to 91: 9 in (R)-6.1 (entry 1), comparable values within the experimental error.

Table 6.1 Asymmetric induction in monolayers of A-OPV4T on HOPG using various solvents.a

Entry Solvent Rosettes analyzed

(#)

Distinctly rotating rosettes

(%)b

CCW:CW (Std. dev.)

1 (R)-6.1 2209 86 91 : 9 (9) 2 (S)-6.1 1948 71 17 : 83 (14) 3 rac-6.1 4190 78 54 : 46 (14) 4 (R)-6.2 1019 44 55 : 45 (15) 5 (S)-6.2 1194 42 48 : 52 (12)

a) All STM images were registered at least one hour after deposition on the surface, to allow the monolayers to organize in view of the dynamics taking place. Typically, the waiting time was longer for rac-6.1 than for the corresponding enantiomerically pure solvent. A significant number of areas per solvent was probed: (R)-6.1: 16, (S)-6.1: 17, rac-6.1: 52, (R)-6.2: 13, (S)-6.2: 15. The standard deviation of the weighted mean of the enantiomeric ratio (that is, corrected for the number of chiral rosettes per area) is given in parentheses. Note that the standard deviation for a constant number of probed rosettes should become smaller by scanning larger areas, which is limited though by the need for high spatial resolution; b) CCW and CW combined.

Three other solvents have been investigated, racemic 1-phenyl-1-octanol (rac-6.1) and both enantiomers of 1-phenyloctyl acetate ((R)- and (S)-6.2). Using rac-6.1, a comparable percentage of distinctly ordered rosettes is observed, however, as expected, without enantiomeric bias (Table 6.1, entry 3). Also when (R)- and (S)-6.2 are employed, there is no preference for any one of the rosette enantiomers. Moreover, the number of distinctly rotating rosettes compared to other surface structures is noticeably lower (entries 4 and 5). These results suggest that hydrogen bonding interactions between enantiomerically pure 6.1 and A-OPV4T are of key importance in inducing the preferred surface chirality, probably through H-bonding of the hydroxyl moiety of the solvent with the unbound nitrogen atoms in triazine hydrogen-bonded rosettes, as illustrated in Figure 6.3d.

Further insight into the mechanism of induction of preferred surface chirality was provided by circular dichroism (CD) measurements of A-OPV4T in either (R)- or (S)-6.1, using a typical concentration for STM (c = 3 x 10-5 M). No CD effects were observed

175



in solution, revealing that neither potential pre-formation of the rosettes nor formation of other chiral assemblies are involved. The fact that rosettes are formed exclusively at the liquid-solid interface is also demonstrated by STM images recorded a few minutes after deposition on the surface, showing disordered monolayers typically observed when achiral solvents are used. Crucially, large areas of the ordered structure formed by the rosettes only develop with time, indicating that the chiral solvent is not directly incorporated in the rosettes, but is in dynamic interaction with the surface.

The emergence of chiral OPV4T monolayers could also be explained by the formation of a solvent monolayer acting as a chiral template for the rosettes to form on. However, this hypothesis is unlikely, since deposition of pure (R)- or (S)-6.1 on HOPG has never resulted in the observation of any ordered layer. In addition, the unit cell parameters of ordered rosette domains are identical in all different solvents used. Therefore, we attribute the emergence of enantiopreference to dynamic interactions of the enantiopure solvent on top of the rosettes. Interestingly, the 2D stereochemistry of enantiopure (S)-OPV4T18 is not affected by the chiral nature of the solvent when experiments are performed in (R)- or (S)-6.1. This means that the effect of molecular chirality overrules the effect of solvent chirality.

Figure 6.4 Time-dependent emergence of preferred chirality. Evolution of the enantiomeric ratio (CCW/(CCW+CW)) and the number of rosettes of a given orientation (CCW, CW, or NO orientation) as a function of time.

176


Chapter 6

STM has also been used to observe in real time how enantiomeric excess emerges, by recording a series of STM images at the (R)-6.1 − HOPG interface over a period of 50 min. The evolution in time of the number of rosettes (CW and CCW) and ill-defined cyclic hexamers without identifiable orientation (NO) is depicted in Figure 6.4. The emergence of order, i.e. the decrease of NO-labeled hexamers, and the increase in enantioselectivity, i.e. the increase of the CCW / CW ratio, are clearly correlated. In this time-dependent sequence, the enantiomeric ratio (CCW : CW) increases from about 50 : 50 in the beginning to 80 : 20 after 50 min.b

In all experiments, this evolution from non-ordered rosettes to CCW or CW rosettes has been observed. Also other changes, such as the evolution of CW into CCW rosettes (or vice versa, depending on the chirality of the solvent) or the transition of e.g. dimers of molecules into rosette-type objects have been identified.

The detailed mechanism for the emergence of 2D enantiomorphic selectivity remains unknown. When an isolated rosette is considered, the energy difference between the two possible orientations is likely to be small. However, in the 2D lattice, the addition of small energy differences at the supramolecular level will lead to the preference of a conglomerate 2D lattice over the racemic lattice.


It has been demonstrated that chirality on a supramolecular level can emerge by self-assembly of achiral molecules on an achiral surface, through the use of an enantiopure solvent. More specifically, p-phenylenevinylene oligomer A-OPV4T (Figure 6.2) self-assembles on a HOPG surface in the form of chiral rosettes, which can be clockwise (CW) or counterclockwise (CCW). Using (R)-1-phenyl-1-octanol ((R)-6.1), there is a preference for CCW rosettes, whereas the use of (S)-6.1 leads to an excess of CW rosettes.

The mechanism of chirality transfer from the solvent to the supramolecular structure likely involves hydrogen bonding, since the corresponding enantiopure acetates 6.2 did not lead to any enantiopreference in the supramolecular surface structure. However, the role of π-π interactions cannot be ruled out. Experiments using an analogous aliphatic chiral solvent, for instance 1-cyclohexyl-1-octanol, could provide additional evidence to understand the mechanism of emergence of enantiopreference.

b When a different area of the same sample was scanned three hours later, the enantiomeric ratio was already at a high level and no longer changed significantly with time.

177



There are reports in the literature about the hydrogenation of chiral 1-phenyl-1-alkanols to the corresponding 1-cyclohexyl-1-alkanols, where the stereochemical integrity remained intact. An example is the PtO2-catalyzed hydrogenation in glacial acetic acid described by Levene et al.,25 later employed by Cram and Tadanier.26 Both groups use this reaction in stereochemical studies. However, in our hands, attempts to hydrogenate 6.1 to 6.3 using PtO2 as the catalyst have repeatedly led to hydrogenolysis of the hydroxy moiety, furnishing 1-phenyloctane 6.4 (Scheme 6.4).

Scheme 6.4 Attempted hydrogenation of 6.1 to 6.3, leading to 6.4.

Given these disappointing results in the PtO2-catalyzed hydrogenation of 6.1, other catalysts should be considered. A possible candidate is rhodium on carbon, which was used by Minnaard et al. to catalyze the hydrogenation of (S)-phenylglycine to (S)-cyclohexylglycine.27 A related reaction described in the literature is the hydrogenation of acetophenone to 1-cyclohexylethanol using a rhodium catalyst28 or nanoparticles of ruthenium or rhodium on carbon nanofibers.29 Furthermore, alternative routes to 6.3 should be taken into consideration, such as enantioselective reduction of ketone 6.5. This could be done in a variety of ways, for example biocatalytically,30 using asymmetric (transfer) hydrogenation,31 or other chiral reduction methods.32

6.5 Experimental part

6.5.1 General remarks For general information, see Chapters 2 and 3.

178


Chapter 6

6.5.2 Synthesis of chiral solvents

rac-1-Phenyl-1-octanol (6.1) Racemic 1-phenyl-1-octanol (6.1) was prepared by Grignard addition of in situ prepared heptyl magnesium bromide to benzaldehyde in Et2O using standard techniques. Purification was achieved by column chromatography over SiO2 (gradient

n-heptane − n-heptane/EtOAc 9:1). To obtain samples for use in STM measurements, the compound was further purified by Kugelrohr distillation. 1H NMR (CDCl3) δ 7.38-7.20 (m, 5H), 4.63 (dd, J = 7.3, 5.9 Hz), 1.90 (s, 1H), 1.83-1.62 (m, 2H), 1.44-1.16 (m, 10H), 0.86 (t, J = 6.6 Hz, 3H); 13C NMR (CDCl3) δ 144.9 (s), 128.4 (d), 127.4 (d), 125.9 (d), 74.7 (d), 39.1 (t), 31.8 (t), 29.5 (t), 29.2 (t), 25.8 (t), 22.6 (t), 14.0 (q); MS (EI+): m/z = 206 (M+), 188, 117, 107, 104, 82, 79, 77; HRMS (EI+): calc. for C14H22O: 206.1671, found: 206.1681; Chiral HPLC: Chiralcel OD, 40°C, n-heptane/IPA 99:1, 1.0 mL/min, Tr = 14.5 min ((R)-6.1), 16.6 min ((S)-6.1).

rac-1-phenyloctyl acetate (6.2) Synthesis of 1-phenyloctyl acetate (6.2) was achieved by subjecting 1-phenyl-1-octanol (6.1) to acetic anhydride in pyridine at 0 °C overnight. The crude acetate thus obtained was purified using column chromatography over silica gel

(eluent: n-pentane/Et2O 50:1). 1H NMR (CDCl3) δ 7.35 − 7.25 (m, 5H), 5.72 (ddd, J = 7.7, 6.2, 1.5 Hz, 1H), 2.05 (d, J = 1.8 Hz, 3H), 1.97 − 1.85 (m, 1H), 1.80 − 1.70 (m, 1H), 1.38 − 1.15 (m, 10H), 0.86 (t, J = 6.6 Hz, 3H); 13C NMR (CDCl3) δ 170.3 (s), 140.9 (s), 128.3 (d), 127.8 (d), 126.5 (d), 76.14 (d), 36.3 (t), 31.7 (t), 29.3 (t), 29.1 (t), 25.5 (t), 22.6 (t), 21.2 (q), 14.0 (q); MS (EI+): m/z = 248 (M+), 206, 188, 149, 117, 107, 105, 104, 91; HRMS (EI+): calculated for C16H24O2: 248.1776, found: 206.1782; Chiral HPLC: Chiralcel OB-H, 40°C, n-heptane/IPA 99:1, 1.0 mL/min, Tr = 10.6 min ((R)-6.2), 16.9 min ((S)-6.2).

(R)-1-phenyl-1-octanol ((R)-6.1) and (S)-1-phenyloctyl acetate ((S)-6.2) Kinetic resolution was performed using the procedure of Mori and Bernotas22 on a larger scale. Thus, 20.5 mmol of rac-1-phenyloctyl acetate (rac-6.2) was dissolved in 100 mL of acetone and added to 900 mL of phosphate buffer (100 mM, pH 6.9). Furthermore, 40 drops of Triton-X100 were added. Finally, 1.94 g of Pseudomonas cepacia lipase was added. After 16 d, the mixture was extracted with Et2O (3×), the combined organic layers were washed with a saturated solution of NaHCO3 sat. and brine, respectively, dried over MgSO4, filtered and the solvent evaporated. Purification

179



was achieved by column chromatography over silica gel using a gradient of n-pentane/Et2O 19:1 – 1:1. (R)-6.1 and (S)-6.2 were both obtained in >98% ee and further purified using kugelrohr distillation.

(S)-1-phenyl-1-octanol ((S)-6.1) and (R)-1-phenyloctyl acetate ((R)-6.2) Enantiomerically pure (S)-1-phenyl-1-octanol ((S)-6.1) and (R)-1-phenyloctyl acetate ((R)-6.2) were obtained by deprotection of (S)-6.2 (K2CO3 in MeOH at rt) and acetylation of (R)-6.1 (Ac2O in pyridine at 0 °C → rt), respectively. Spectral data were in accordance with those obtained for (R)-6.1 and (S)-6.2 and both (S)-6.1 and (R)-6.2 were obtained with >98% ee.

6.5.3 Scanning tunneling microscopy (STM) STM measurements were performed by N. Katsonis and T. Kudernac (University of Groningen) and H. Xu (University KU Leuven). All experiments were performed at room temperature. Pt/Ir STM tips were prepared by mechanical cutting from Pt/Ir wire (80:20, diameter 0.25 mm). Prior to imaging, A-OPV4T or (S)-OPV4T molecules were dissolved in the solvents by sonication (few min) and heating at 40 °C (15 min). The solutions obtained had a concentration ranging between 10-4 to 10-5 M.

Subsequently, a drop of the solution was applied to a freshly cleaved surface of highly oriented pyrolytic graphite (HOPG, from Goodfellow), and then the STM tip was immersed into the drop. The system was then allowed to cool down for at least 30 min before measuring, after which STM imaging was performed at the solution – HOPG interface on two PicoSPM machines (Molecular Imaging, Scientec), using constant current mode. Images shown are subjected to a first-order plane-fitting procedure to compensate for sample tilt.

6.6 Notes and references 1 The STM measurements described in this chapter were performed by N. Katsonis and T. Kudernac (University of Groningen) and H. Xu (University KU Leuven). A-OPV4T was provided by the group of Prof. E. W. Meijer (Eindhoven University of Technology). 2 A. Ghanem, Tetrahedron 2007, 63, 1721-1754. 3 K. Faber, Biotransformations in Organic Chemistry: a Textbook, 4th Ed., Springer, Berlin, 2000. 4 K. H. Ernst, In Supramolecular Surface Chirality, Top. Curr. Chem. 2006, 265, 209-252.

180


Chapter 6

5 B. Hulsken, R. van Hameren, J. W. Gerritsen, T. Khoury, P. Thordarson, M. J. Crossley, A. E. Rowan, R. J. M. Nolte, J. A. A. W. Elemans, and S. Speller, Nature Nanotechnol. 2007, 2, 285-289. 6 A. Corma and H. Garcia, Adv. Synth. Catal. 2006, 348, 1391-1412. 7 a) http://nobelprize.org/nobel_prizes/chemistry/laureates/2007/; b) G. Ertl, Surf. Sci. 1994, 299/300, 742-754. 8 S. De Feyter and F. C. De Schryver, Chem. Soc. Rev. 2003, 32, 139-150. 9 N. Katsonis, E. Lacaze, and B. L. Feringa, J. Mater. Chem. 2008, 18, 2065-2073. 10 Science 2002, 295, 2313-2556, thematic issue on supramolecular chemistry and self-assembly. 11 G. A. Hembury, V. V. Borovkov, and Y. Inoue, Chem. Rev. 2008, 108, 1-73, and references contained therein. 12 G. Binning, H. Rohrer, C. Gerber, and E. Weibel, Phys. Rev. Lett. 1982, 49, 57-61. 13 R. Fasel, M. Parschau, and K.-H. Ernst, Nature 2006, 439, 449-452. 14 E. L. Eliel, S. H. Wilen, and L. N. Mander, Stereochemistry of Organic Compounds, Wiley, New York, 1994, pp. 965-971. 15 R. Wernicke, J. Chromatogr. Sci. 1985, 23, 39-47. 16 J. P. Rabe and S. Buchholtz, Science 1991, 253, 424-427. 17 A. P. H. J. Schenning, P. Jonkheijm, E. Peeters, and E. W. Meijer, J. Am. Chem. Soc. 2001, 123, 409-416. 18 A. Miura, P. Jonkheijm, S. De Feyter, A. P. H. J. Schenning, E. W. Meijer, and F. C. De Schryver, Small 2005, 1, 131-137. 19 P. Jonkheijm, A. Miura, M. Zdanowska, F. J. M. Hoeben, S. De Feyter, A. P. H. J. Schenning, F. C. De Schryver, and E. W. Meijer, Angew. Chem. Int. Ed. 2004, 43, 74-78. 20 A. R. A. Palmans and E. W. Meijer, Angew. Chem. Int. Ed. 2007, 46, 8948-8968. 21 N. Katsonis, A. Minoia, T. Kudernac, T. Mutai, H. Xu, H. Uji-i, R. Lazzaroni, S. De Feyter, and B. L. Feringa, J. Am. Chem. Soc. 2008, 130, 386-387. 22 K. Mori and R. Bernotas, Tetrahedron: Asymmetry 1990, 1, 87-96. 23 S. M. Barlow and R. Raval, Surf. Sci. Rep. 2003, 50, 201-341. 24 K. E. Plass, A. L. Grzesiak, and A. J. Matzger, Acc. Chem. Res. 2007, 40, 287-293. 25 a) P. A. Levene, J. Biol. Chem. 1936, 115, 275-277; b) P. A. Levene and P. G. Stevens, J. Biol. Chem. 1930, 89, 471-477; c) P. A. Levene and P. G. Stevens, J. Biol. Chem. 1930, 87, 375-391. 26 D. J. Cram and J. Tadanier, J. Am. Chem. Soc. 1959, 81, 2737-2748. 27 A. J. Minnaard, W. H. J. Boesten, and H. J. M. Zeegers, Synth. Commun. 1999, 29, 4327-4332.

181



28 I. Bergault, P. Fouilloux, C. Joly-Vuillemin, and H. Delmas, J. Catal. 1998, 175, 328-337. 29 a) Y. Motoyama, M. Takasaki, K. Higashi, S.-H. Yoon, I. Mochida, and H. Nagashima, Chem. Lett. 2006, 35, 876-877; b) I. S. Park, M. S. Kwon, N. Kim, J. S. Lee, K. Y. Kang, and J. Park, Chem. Commun. 2005, 5667-5669. 30 a) J. C. Moore, D. J. Pollard, B. Kosjek, and P. N. Devine, Acc. Chem. Res. 2007, 40, 1412-1419; b) S. M. A. de Wildeman, T. Sonke, H. E. Schoemaker, and O. May, Acc. Chem. Res. 2007, 40, 1260-1266; c) K. Goldberg, K. Schroer, S. Lütz, and A. Liese, Appl. Microbiol. Biotechnol. 2007, 76, 249-255; d) K. Goldberg, K. Schroer, S. Lütz, and A. Liese, Appl. Microbiol. Biotechnol. 2007, 76, 237-248. 31 a) T. Ikariya and A. J. Blacker, Acc. Chem. Res. 2007, 40, 1300-1308; b) X. Wu and J. Xiao, Chem. Commun. 2007, 2449-2466; c) H.-U. Blaser, C. Malan, B. Pugin, F. Spindler, H. Steiner, and M. Studer, Adv. Synth. Catal. 2003, 345, 103-151; d) R. Noyori and T. Ohkuma, Angew. Chem. Int. Ed. 2001, 40, 40-73. 32 a) P. Daverio and M. Zanda, Tetrahedron: Asymmetry 2001, 12, 2225-2259; b) E. J. Corey and C. J. Helal, Angew. Chem. Int. Ed. 1998, 37, 1986-2012.

182


Chapter 6

183

Samenvatting.doc


Samenvatting

In de organische chemie houdt men zich bezig met het maken van moleculen, wat organische synthese genoemd wordt, en het bestuderen van hun eigenschappen. Moleculen zijn de kleinste deeltjes van een stof die nog de chemische eigenschappen van die stof bezitten. Moleculen zijn zelf opgebouwd uit atomen, ook wel elementen genoemd, die met elkaar verbonden zijn door chemische bindingen. Tijdens een chemische reactie worden sommige bindingen verbroken en andere gevormd, en zodoende kan de ene stof omgezet worden in de andere. Inzicht in chemische reactiviteit op moleculair niveau − welke bindingen worden precies verbroken en gevormd onder welke omstandigheden − is van groot belang, om selectief die stof te kunnen maken die gewenst is, bijvoorbeeld nieuwe medicijnen of kunststoffen met verbeterde eigenschappen. Verder is het van belang om niet alleen datgene te maken wat je wilt, maar ook zo snel en goedkoop mogelijk, onder energiezuinige omstandigheden en met zo weinig mogelijk afvalproducten.

Om de synthese van stoffen te versnellen, bestaan verschillende technieken. Zo verlopen chemische reacties doorgaans sneller bij hogere temperatuur. Een nadeel hierbij is dat het vaak ook lastiger te controleren is wat er op moleculair niveau precies gebeurt, waardoor de reactie minder selectief wordt. Ζo kunnen er allerlei ongewenste bijproducten ontstaan, die vervolgens weer van het product gescheiden moeten worden. Een andere manier om reacties sneller te maken, is het gebruik van katalysatoren. Dit zijn stoffen die een reactie versnellen zonder daarbij zelf verbruikt te worden, wat dus betekent dat je in principe aan een zeer kleine hoeveelheid katalysator genoeg hebt. Bijkomend voordeel is dat katalysatoren een reactie vaak niet alleen sneller, maar ook selectiever maken.

In het onderzoek beschreven in dit proefschrift, zijn twee typen katalysatoren gebruikt, waarvan het eerste type is gebaseerd op metalen met daaromheen zogenaamde liganden. Dit zijn organische moleculen die de eigenschappen van het metaal beïnvloeden. De aldus gevormde metaalcomplexen brengen de uitgangsstof en de overige reactanten bij elkaar in de buurt en bevorderen op die manier de reactie. Daarnaast is gebruik gemaakt van enzymen, complexe eiwitverbindingen die door levende cellen worden gebruikt om biologische processen te versnellen, vaak met een factor van een miljoen of nog hoger. Met de juiste technieken kunnen enzymen echter ook buiten de cel gebruikt worden voor de katalyse van chemische reacties. Verder kunnen metaalkatalysatoren en enzymen ook in combinatie worden toegepast, om

184

Samenvatting.doc

Samenvatting

meerdere reactiestappen tegelijkertijd te versnellen en zodoende ingewikkelder reacties mogelijk te maken.

Dit onderzoek concentreerde zich voornamelijk op twee typen verbindingen, epoxiden en haloalcoholen (zie Figuur 1). Dit zijn veelgebruikte en veelzijdige bouwstenen in de synthese. Met name epoxiden − het epoxide is de drieringverbinding gevormd door twee koolstofatomen en een zuurstofatoom − worden veel gebruikt. Haloalcoholen − dit zijn alcoholen met tevens een halogeenatoom in het molecuul, zoals chloor of broom − zijn in één stap om te zetten in epoxides. Zowel epoxiden als haloalcoholen kunnen reageren met een grote verscheidenheid aan andere stoffen.

Figuur 1 a) Een epoxide, b) een chloroalcohol, c) een bromoalcohol. De "R" staat voor een willekeurige zijgroep.

Een belangrijk begrip dat op veel plaatsen in dit proefschrift terugkomt, is chiraliteit. Dit woord is afgeleid van het Griekse χειρ (cheir, hand). Een voorwerp is chiraal als het niet door rotatie of verplaatsing volledig identiek te maken is met zijn spiegelbeeld. In het dagelijks leven zijn veel dingen chiraal, denk maar aan handen of voeten. Ook veel moleculen zijn chiraal, waarbij de twee spiegelbeelden enantiomeren worden genoemd. Enantiomeren worden onderscheiden door de voorvoegsels R en S. Enantiomeren hebben dezelfde fysische en chemische eigenschappen in een niet-chirale omgeving, zoals bijvoorbeeld smeltpunt of kookpunt, maar verschillen van elkaar onder chirale omstandigheden. Zo kan het zijn dat het (R)-enantiomeer van een molecuul sneller reageert met een andere chirale verbinding of katalysator dan het (S)-enantiomeer, of andersom. In dit proefschrift komen een aantal duidelijke voorbeelden hiervan naar voren.

Ook in het lichaam, dat voor een belangrijk deel uit chirale moleculen is opgebouwd, kunnen enantiomeren verschillende effecten hebben. Dit komt doordat enantiomeren een verschillende interactie hebben met de receptoren voor bijvoorbeeld geur- en smaakstoffen. Dit is enigszins te vergelijken met een handschoen, waarvan de linker beter om de linkerhand past, terwijl de rechter juist een “betere interactie heeft” met de rechterhand. Een voorbeeld is carvon, waarvan het (R)-enantiomeer naar munt ruikt en het (S)-enantiomeer naar karwij (Figuur 2). Ook de enantiomeren van medicijnen kunnen verschillende effecten hebben, waarbij het regelmatig voorkomt dat slechts één van de enantiomeren het gewenste medicinale effect heeft, terwijl het andere enantiomeer inactief of zelfs schadelijk is.

185

Samenvatting.doc


Figuur 2 Beide enantiomeren van carvon. De vetgedrukte binding steekt naar voren en de gestippelde binding naar achteren, (R)- en (S)-carvon zijn dus spiegelbeelden van elkaar.

Hoofdstuk 1 geeft een inleiding op het onderzoek beschreven in dit proefschrift, waarbij verscheidene belangrijke concepten worden uitgelegd en in perspectief geplaatst. Hierbij gaat het om onderwerpen als procesintensificatie, chiraliteit, enzymkatalyse, en (dynamische) kinetische resolutie. Verder wordt een aantal baanbrekende resultaten aangestipt van onderzoek waarin methoden uit bio- en chemokatalyse worden gecombineerd. Afgezien van dynamische kinetische resolutie valt hierbij te denken aan kunstmatige enzymen of DNA-katalyse.

In Hoofdstuk 2 wordt het onderwerp procesintensificatie verder uitgediept. In dit onderzoek is een speciaal type reactor gebruikt met als doel reacties efficiënter te laten verlopen. Deze reactor heet "Centrifugal Contact Separator" (CCS), dat wil zeggen dat het een snel ronddraaiend binnenwerk heeft waarin twee niet-mengbare vloeistofen snel en intensief met elkaar in contact gebracht worden, waarna ze gescheiden de reactor weer verlaten (Figuur 3). Een praktisch voorbeeld van deze laatste eigenschap is de toepassing van CCS-technologie om ruwe olie van zeewater te scheiden na olierampen.

Het innovatieve concept was om een apparaat van dit type als reactor te gebruiken voor zogenaamde twee-fasen reacties. In zulke reacties bevinden de uitgangsstof en het uiteindelijk gevormde product zich in de ene fase, bijvoorbeeld een oplossing op basis van tolueen, een vloeistof die niet met water mengt. In de andere fase, wat doorgaans een waterige oplossing is, bevinden zich de reagentia en de katalysator voor de reactie. Het potentiële voordeel van deze aanpak is duidelijk: door het intensieve mengen in het binnenwerk van de CCS kan de reactie efficiënt plaatsvinden, terwijl het product en katalysator / reagentia het apparaat gescheiden verlaten, wat de zuivering van het product een stuk eenvoudiger maakt. Verder kan hierdoor de uitgaande productstroom direct worden gebruikt als ingaande stroom voor een volgende CCS. Met andere woorden, de reactoren kunnen in serie gezet worden, waardoor een cascade van reacties mogelijk wordt. Normaalgesproken kan dit niet, aangezien het product dan

186

Samenvatting.doc

Samenvatting

eerst in een aparte stap gescheiden moet worden van restanten van de katalysator en andere afvalproducten.

Figuur 3 Schematische voorstelling van de CCS, waarbij het lichtgrijze gebied de lichte fase aanduidt en het donkergrijze gebied de zware fase. Het gearceerde gebied is dat deel van de reactor waar menging van de fasen plaatsvindt.

Een ander groot voordeel is dat de CCS een continue reactor is. Nadat hij eenmaal is opgestart, kan hij in theorie voortdurend aan blijven staan, waarbij hij een continue stroom product levert van een continue kwaliteit. Vooral bij het maken van grote hoeveelheden materiaal hebben continue processen veel voordelen, ze zijn namelijk ook gemakkelijker in de hand te houden en daarmee een stuk veiliger dan zogenaamde batch-processen, reacties waarbij telkens een bepaalde hoeveelheid van een product wordt gemaakt in een grote reactor. Dit levert vaak kwaliteitsverschillen tussen verschillende batches op.

Op basis van de literatuur zijn twee epoxidatiereacties doorontwikkeld tot bruikbare twee-fasen processen, waarvan er tenslotte één daadwerkelijk is toegepast in de CCS. Het eerste proces is een ijzergekatalyseerde epoxidatie met perazijnzuur als oxidator. Deze methode was al bekend als homogene reactie, maar kan ook worden toegepast in een twee-fasen systeem. Het gebruik van water was echter niet mogelijk, omdat in dat geval de reactie erg langzaam verliep. Vandaar dat heptaan en acetonitril als oplosmiddelen gekozen werden, twee vloeistoffen die evenmin met elkaar mengen. De katalysator en perazijnzuur zitten in de acetonitril-laag, terwijl de uitgangsstof, een alkeen, en het product, een epoxide, een sterke voorkeur hebben voor de heptaan-fase (Schema 1a). Bij deze reactie komt echter veel warmte vrij, wat een veiligheidsrisico zou kunnen opleveren op grote schaal. Hoewel goede resultaten werden behaald op laboratoriumschaal, is dit procédé niet uitgevoerd in de CCS. Bij de tweede onderzochte epoxidatie, op basis van een wolfraamkatalysator en waterstofperoxide als oxidant, kwam minder warmte vrij. Hierdoor kon deze reactie wel worden toegepast in de CCS, wat een omzetting van 20% van cyclo-octeen naar cyclo-octeenoxide opleverde

187

Samenvatting.doc


(Schema 1b). Hoewel deze opbrengst aan de bescheiden kant is, toont dit resultaat aan dat de CCS inderdaad gebruikt kan worden als continue reactor voor katalytische reacties.

Schema 1 a) IJzergekatalyseerde epoxidatie met perazijnzuur in een tweefasensysteem. Fe-fenantroline katalysator = [((phen)2(H2O)FeIII)2(μ-O)](ClO4)4; b) Epoxidatie met waterstofperoxide gekatalyseerd door wolfraam (NaZnPOM = Na12[WZn3(ZnW9O34)2]).

Verder worden in dit hoofdstuk verscheidene nieuwe epoxidatiekatalysatoren gepresenteerd, alle op basis van ijzer met fenantroline als ligand. Het bijzondere hierbij was dat, uitgaande van verschillende ijzerverbindingen, toch steeds een zeer actieve katalysator verkregen werd voor epoxidatie van olefines met perazijnzuur. Wel was in een aantal gevallen sprake van een inductieperiode waarin nog geen reactie plaatsvond omdat de actieve katalysator zich nog moest vormen. Deze periode varieerde van enkele minuten tot anderhalf uur, afhankelijk van de gebruikte ijzerzouten en andere condities, bijvoorbeeld de reactietemperatuur.

De reacties die in het vervolg van dit proefschrift worden beschreven, zijn op kleinere schaal in conventioneel laboratoriumglaswerk uitgevoerd. Desalniettemin geldt ook voor deze reacties dat ze in principe doorontwikkeld zouden kunnen worden tot geschikte processen voor de CCS.

In Hoofdstuk 3 wordt een enzymatische kinetische resolutie van chloroalcoholen beschreven. Met behulp van een enzym, dat in de natuur door bacteriën gebruikt wordt om giftige chloorhoudende stoffen af te breken, wordt hierbij een van de enantiomeren van een chloroalcohol omgezet in een epoxide, terwijl het andere enantiomeer achterblijft. In het ideale geval levert kinetische resolutie 50% enantiomeerzuivere uitgangsstof op en 50% enantiomeerzuiver product. In dit proces bleek dat het product, het epoxide, niet bestand was tegen de waterige condities waaronder de reactie gedaan moest worden – er vond hydrolyse van het epoxide plaats, in chemische termen. Niettemin was het mogelijk om de enantiomeerzuivere uitgangsstof in goede opbrengsten te isoleren, zoals het chloroalcohol in Schema 2, dat verkregen werd met >99% e.e. en in 47% opbrengst. Hierbij dient bedacht te worden dat in een kinetische

188

Samenvatting.doc

Samenvatting

resolutie de maximale opbrengst 50% is, 47% is dus uitstekend te noemen. Behalve het voorbeeld in Schema 2 werden op deze manier nog verscheidene andere chloroalcoholen in goede opbrengst en met hoge ee verkregen (zie Tabel 3.3).

Schema 2 Voorbeeld van kinetische resolutie van chloroalcoholen met behulp van het enzym HheC. Het (R)-chloroalcohol wordt omgezet in het epoxide, het (S)-enantiomeer blijft achter.

De maximumopbrengst van 50% van elk van beide producten in een kinetische resolutie is een nadeel wanneer men, zoals vaak voorkomt, slechts geïnteresseerd is in één van beide verbindingen. Door de uitgangsstof te racemiseren, blijft er steeds voldoende van het 'favoriete' enantiomeer aanwezig dat door het enzym kan worden omgezet naar enantiomeerzuiver product. Deze aanpak staat bekend als dynamische kinetische resolutie (DKR) en levert optisch actieve verbindingen in hoge opbrengsten (Schema 3).

Schema 3 Illustratie van het concept dynamische kinetische resolutie. Door racemisatie van de uitgangsstof kunnen enantiomeerzuivere producten in hoge opbrengsten worden verkregen.

In Hoofdstuk 4 wordt de DKR beschreven van aromatische chloro- en bromoalcoholen, waarbij een nieuwe combinatie van enzym en racemisatiekatalysator is gebruikt. Het enzym is een variant van HheC die enantioselectiever is voor aromatische moleculen en beter bestand is tegen oxidatie. De racemisatiekatalysator is een metaalcomplex op basis van iridium. Zoals te zien is in Schema 4, bevindt het iridiumatoom zich in het midden omringd door een aantal organische liganden, stoffen die een binding zijn aangegaan met iridium. Met deze procedure, geïllustreerd in Schema 4, kunnen optisch actieve epoxiden in één stap gemaakt worden vanuit racemische haloalcoholen. Een interessant aspect van deze procedure is dat het om een twee-fasenreactie gaat. De enzymatische omzetting vindt plaats in een waterige bufferfase, terwijl de racemisatie plaatsvindt in

189

Samenvatting.doc


tolueen. Dit kenmerk van de reactie biedt mogelijkheden voor eventuele toepassing op grotere schaal in een reactor zoals de CCS.

Schema 4 Dynamische kinetische resolutie van chloro- en bromoalcoholen, gekatalyseerd door een combinatie van een enzym en een iridiumcomplex.

De allylische en heteroaromatische chloroalcoholen beschreven in Hoofdstuk 3, zijn belangrijke bouwstenen in de synthetische chemie, een aspect dat terugkomt in Hoofdstuk 5. In dat hoofdstuk wordt een aantal omleggingsreacties beschreven die leiden tot een geheel andere opbouw van het koolstofskelet van deze chloroalcoholen (Schema 5). Op basis hiervan kunnen nieuwe klassen van verbindingen gemaakt worden. De reacties verliepen met wisselend succes. Met name in het geval van de Johnson ortho-esteromlegging waren de opbrengsten hoog, echter de stereoselectiviteit was niet optimaal.

Schema 5 Omleggingsreacties van chloroalcoholen, van boven naar onder de Achmatowicz, de Ireland-Claisen en de Johnson ortho-ester omlegging.

190

Samenvatting.doc

Samenvatting

Tenslotte wordt in Hoofdstuk 6 de synthese, kinetische resolutie en het gebruik in zelfassemblagestudies van 1-phenyl-1-octanol beschreven. Voor de kinetische resolutie werd een lipase gebruikt, een stabiel en actief enzym dat de hydrolyse van esters katalyseert (Schema 6). In het dagelijks leven zijn lipasen bijvoorbeeld te vinden in wasmiddelen, waar ze de hydrolyse van vetten versnellen en zo helpen olie- en vetvlekken te verwijderen. Door middel van dit enzym kon vanuit racemisch 1-phenyloctylacetaat het (R)-alcohol chemisch en optisch zuiver worden verkregen. Ontscherming van het achterblijvende acetaat leverde (S)-1-phenyloctanol op.

Schema 6 Kinetische resolutie van 1-fenyloctylacetaat met behulp van een lipase.

Vervolgens zijn beide alcoholen gebruikt als oplosmiddel in enantioselectieve zelfassemblage. Het achirale molecuul A-OPV4T (Figuur 4) vormt chirale assemblages op het oppervlak van grafiet, gekenmerkt door links- dan wel rechtsdraaiende rozetten bestaande uit 6 moleculen A-OPV4T (Figuur 5). Het gebruik van (R)- of (S)-1-phenyl-1-octanol als oplosmiddel stuurde de enantioselectieve vorming van de rozetten. Zo leverde (R)-1-phenyl-1-octanol een overmaat op van rozetten die tegen de klok indraaien, terwijl in de aanwezigheid van (S)-1-phenyl-1-octanol de rozetten met de klok meedraaien. Met behulp van Scanning Tunneling Microscopy (STM), een techniek waarmee objecten op atomaire schaal kunnen worden waargenomen, zijn deze rozetten duidelijk te zien (Figuur 5). Dit baanbrekende resultaat toont voor het eerst aan dat enantiomeerzuivere oplosmiddelen gebruikt kunnen worden om chirale zelfassemblage aan een oppervlak te sturen.

Figuur 4 A-OPV4T, de verbinding die gebruikt werd in de studies van enantioselectieve 2D zelfassemblage.

191

Samenvatting.doc


Figuur 5 STM-afbeeldingen van chirale zelfassemblage van A-OPV4T. In (R)-1-fenyloctanol draaien de meeste rozetten tegen de klok in (b en het grootste gedeelte van a), in (S)-1-fenyloctanol worden met voornamelijk met de klok meedraaiende rozetten gevormd (c). Figuur d geeft een idee van de vorming van de rozetten op moleculair niveau.

Concluderend kan gesteld worden dat het onderzoek beschreven in dit proefschrift aantoont hoe technieken uit verschillende subdisciplines van de chemie – biokatalyse, organische synthese, organometaalkatalyse en chemische technologie – met succes geïntegreerd kunnen worden. In het bijzonder de combinatie van enzymkatalyse met traditionele organische synthese en metaalkatalyse is in deze context zeer vruchtbaar gebleken, getuige de uitstekende resultaten op het gebied van enzymatische kinetische resolutie en dynamische kinetische resolutie, beschreven in de Hoofdstukken 3 en 4.

192

Samenvatting.doc

Samenvatting

193

Dankwoord.doc

Dankwoord

Dankwoord

Dit was het dan. Tot mijn opluchting en blijdschap moet ik vaststellen dat het schrijven van mijn proefschrift erop zit. Hieraan is een hoop inspanning van mijn kant vooraf gegaan, maar zonder de inbreng van vele anderen zou het niet gelukt zijn. Al deze mensen wil hierbij van harte bedanken.

Allereerst gaat mijn dank uit naar mijn promotoren, de professoren Ben Feringa, Hans de Vries en Adri Minnaard. Het was een groot voorrecht om onder jullie begeleiding onderzoek te mogen doen. Daarnaast heb ik ook de verscheidenheid van jullie inbreng zeer gewaardeerd de afgelopen tijd. Ben, jouw enthousiasme voor goede wetenschap, je rijkdom aan ideeën en tomeloze energie zijn fenomenaal en voor mij een bron van inspiratie. Hans, jouw altijd optimistische blik op het onderzoek was zeer stimulerend en je iets “industriëlere” kijk op de chemie heeft erg geholpen het geheel in breder perspectief te plaatsen. Adri, ik heb enorm geprofiteerd van je nuchtere en didactische kijk op de wetenschap, tijdelijk zelfs vanuit het verre Duitsland. Ik wil jullie alle drie bedanken voor de vrijheid en bijbehorende verantwoordelijkheid die jullie me hebben gegeven.

De leescommissie, bestaande uit de professoren Jan Engberts, Dick Janssen en Floris Rutjes, ben ik zeer erkentelijk voor de vlotte correctie van het manuscript en de goede suggesties ten aanzien van de inhoud.

Dick, ik wil je ook bedanken voor je bijdrage aan het IBOS-project en je goede ideeën, uiteraard in het bijzonder met betrekking tot het biochemische gedeelte. In dit kader gaat mijn dank tevens uit naar prof. Erik Heeres, de industriële begeleidingscommissie in de personen van Claudia Kronenburg (Organon) en Theo Sonke (DSM) en tevens alle anderen die in de loop van de tijd betrokken zijn geweest bij het IBOS-project, voor hun suggesties en opbouwende kritiek.

I especially want to thank my IBOS colleagues for our fruitful collaboration throughout the project on all the topics mentioned in this thesis and for the excellent company during the many meetings and conferences we attended together. Chiara, Vincent, Arnaud, Florian, and Gerard: grazie mille, merci beaucoup, en dankjewel!

I am grateful to Wesley Browne for his critical and encouraging comments on chapter 2. Ook Hans de Boer wil ik bedanken voor de achtergrondinformatie betreffende dit hoofdstuk. Wat betreft het enzymatische gedeelte heb ik, zeker aan het begin van mijn onderzoek, veel gehad aan de discussies met de leden van de biochemie-groep, in het bijzonder Jeffrey en Ghannia. Bedankt voor jullie verhelderende commentaar. Thomas,

194

Dankwoord.doc

Dankwoord

thanks for your input on the DKR part of this research and for your critical comments on chapter 4. I also want to thank prof. Michel Pfeffer for his contribution to the DKR. Nathalie, Tibor, prof. Steven De Feyter and all others involved in the research on chiral solvents in STM, thank you for giving me the chance to collaborate on this great project. Nathalie, thanks also for your comments on chapter 6. Furthermore, I would like to thank all members of the subgroup asymmetric catalysis for the discussions and good suggestions.

Voor de onmisbare technische ondersteuning ben ik Theodora (GC en HPLC), Albert (massaspectroscopie), Hans (elementenanalyse), Wim, Klaas en Pieter (NMR), Auke (kristallografie), Evert (reparaties) en Ebe (autoclaven) veel dank verschuldigd. Ook Olaf Post (Organon) wil ik bedanken voor het doen van de calorimetrische metingen die in Hoofdstuk 2 worden genoemd. Thom, dankjewel voor je geduldige hulp met computer-gerelateerde zaken.

Dirk en Gerlof, bedankt dat jullie mij terzijde willen staan bij mijn verdediging. Dirk, de afgelopen tijd kon ik altijd terugvallen op jouw persoonlijke ervaring met een heel scala aan zaken die in de afrondende fase van de promotie de kop opsteken. Voor deze “coördinerende” rol kan ik je niet genoeg bedanken! Gerlof, dankjewel voor je kritische blik op de samenvattingen en stellingen en natuurlijk voor je bereidheid om voor de gelegenheid terug te komen naar je oude studiestad.

Then, of course, there are all the members of the organic chemistry group, past and present. Their support, both profesionally and on a personal level, has been fantastic. Special thanks go out to my colleagues of lab room 16.238N, who repeatedly had to endure my versatile taste in music and my singing. The population of our lab has been very dynamic while I did my PhD there and I would probably forget many of you if I tried to mention everyone, but you all deserve a big thank you! I also want to thank my office mates Mike, Koen, and Martin for their pleasant company and for sharing the occasional frustrations of a writing chemist, and the C-wing as well as the rest of the lab for the great working atmosphere.

Over these years I made a lot of good friends both in the lab and outside, who have helped me in many different ways. At this place, I want to thank them all for their support, their great company, and for the fun we had at borrels, parties, barbecues, poker evenings, dinners and on many other occasions. I would like to thank you all personally, but I'm afraid the entire list of names is too long – you know who you are. Be sure that I am deeply grateful to all of you!

Alle leden van studentenkoor Gica, in het bijzonder de bassensectie, wil ik bedanken voor de gezelligheid en de mooie concerten die we gegeven hebben. Ook mijn vrienden van buiten het lab, de oude garde, wil ik van harte bedanken voor de constante

195

Dankwoord.doc

Dankwoord

gezelligheidsfactor de afgelopen jaren. Martin, Renate, Johan, Ronald, Bert, Marc, Foppe, Anton, Annemarie en iedereen die ik nu nog vergeet, bedankt!

Papa, mama, Marjolein, Margriet en Kasper, Niels, opa Haak en de rest van de familie, soms heb ik misschien wat weinig van me laten horen als ik het weer eens druk had – mijn dankbaarheid voor jullie steun is er niet minder om. Dank jullie wel voor jullie oprechte belangstelling, waar ter wereld jullie ook waren. Jullie zijn een voorbeeld voor mij en de afgelopen jaren heb ik ook van jullie een heleboel geleerd. Helaas kunnen opa en oma Meerman en oma Haak de afronding van mijn promotie niet meer meemaken, maar ik bewaar warme herinneringen aan hen en aan hun nieuwsgierigheid naar waar ik mee bezig was – studeerde ik nu nog of was ik al aan het werk?

També vull agraïr a Mariano, Mercedes, Analía, a la abuela i a tots els amics del Palmar per la seua càlida hospitalitat quan estic al seu poble.

But most of all, I want to thank Alicia for always standing beside me despite the distance, for her love and her patience, and for always continuing to believe in me and in our future together.

Robert

university of groningen chemo-enzymatic routes to enantiopure … · epoxidation reactions using...

Documents