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In Memory of my Mother, dedicatedto my Father.

List of Papers

This thesis is based on the following papers, which are referred to in the text

by their Roman numerals.

(I) Srivastava, P.; Engman, L.*

A radical cyclization route to cyclic imines

Tetrahedron Lett. 2010, 51, 1149-1151.

(II) Srivastava, P.; Barman, J.; Pathmasiri, W.; Plashkevych, O.; Wenska,

M.; Chattopadhyaya, J*

Five- and six-membered conformationally locked 2', 4'-carbocyclic ribo-

thymidines: synthesis, structure, and biochemical studies

J. Am. Chem. Soc. 2007, 129, 8362-8379.

(III) Srivastava, P.; Engman, L.*

Palladium catalyzed Sonogashira cross-coupling of organic tellurides with

alkynes

Manuscript

(IV) Appendix. Experimental procedures

Reprints were made with permission from the respective publishers.

Contents

1. Introduction ............................................................................................... 11 1.1 Radicals ............................................................................................... 11 1.2 Radical Reactions ............................................................................... 13 1.3 The chain concept ............................................................................... 14

1.3.1 Initiators ...................................................................................... 15 1.3.2 The tin hydride method ............................................................... 18 1.3.3 Silanes ......................................................................................... 20

1.4 Radical precursors ............................................................................... 21 1.4.1 Organochalcogens as radical precursors ..................................... 21

1.5 Radical cyclization .............................................................................. 22 1.5.1 5-Hexenyl cyclizations ................................................................ 23 1.5.2 6-Heptenyl cyclizations ............................................................... 24

1.6 Fundamentals of nucleic acids ............................................................ 26 1.7 Nucleic acid based therapeutics and significance of locked nucleosides ................................................................................................ 28

2. A radical cyclization route to �1 - pyrrolines (publication I) .................... 30 2.1 Design ................................................................................................. 30

2.1.1 Synthesis ..................................................................................... 31

3. A radical cyclization route to conformationally constrained nucleosides: Synthesis, structure and biochemical studies (publication II) ....................... 34

3.1 Radical reactions in nucleic acids ....................................................... 34 3.1.1 Synthesis of 2',4'-locked carbocyclic nucleoside (carbocyclic LNA-T; 37a/37b) ................................................................................. 35 3.1.2 NMR characterization of carbocyclic LNA-T (37a/37b) ............ 37 3.1.3 Synthesis of 2',4'-locked carbocyclic nucleoside (carbocyclic ENA-T; 48) .......................................................................................... 38 3.1.4 NMR characterization of the carbocyclic-ENA-T (48) ............... 39

3.2 Thermal denaturation studies of antisense oligonucleotides (AONs) . 40 3.3 Evaluation of target affinity and antisense properties of modified AONs ......................................................................................... 43 3.4 Probable mechanism for the nuclease stability of the modified AONs ......................................................................................... 46 3.5 RNase H digestion studies .................................................................. 47

4. Microwave-induced chalcogen extrusion reactions .................................. 49

5. Palladium (0)-catalyzed Sonogashira cross-coupling of organic tellurides with alkynes (Publication III) ........................................................ 51

5.1 Synthesis ............................................................................................. 53

6. Oxime ethers in 5-hexenyl radical cyclizations ......................................... 55 6.1 Attempted synthesis of a pyrrolidinone .............................................. 55

7. Piperidines from amino acids via radical cyclization ................................ 57 7.1 Attempted synthesis of piperidines ..................................................... 58

8. Summary in Swedish ................................................................................. 61

Acknowledgements ....................................................................................... 64

References ..................................................................................................... 67

Abbreviations

A Adenin-9-yl or adenosine Ac Acetyl AIBN 2,2' -Azobisisobutyronitrile AON Antisense oligonucleotide Aza-ENA 2'-N,4'-C-Ethylene bridged nucleic acid 9-BBN 9- Borabicyclo(3.3.1)nonane BNA Bridged nucleic acid Bn Benzyl BtH 1-H-Benzotriazole C Cytosin-1-yl or cytidine CD Circular dichroism COSY Correlation spectroscopy DCC Dicyclohexylcarbodiimide DCM Dichloromethane DEAD Diethyl azodicarboxylate DIPEA Diisopropylethylamine DMAP 4-Dimethylaminopyridine DMSO Dimethylsulfoxide DMTr 4,4'-Dimethoxytriphenylmethyl DNA Deoxyribonucleic acid ENA 2'-O,4'-C-Ethylene bridged nucleic acid FDA Food and Drug Administration G Guanin-9-yl or guanosine HMBC Heteronuclear multiple bond correlation LNA Locked nucleic acid mRNA Messenger RNA NOE Nuclear Overhauser effect PAGE Polyacrylamide gel electrophoresis Ph Phenyl RNA Ribonucleic acid RNaseH Ribonuclease H RNAi RNA interference SVPDE Snake venom phosphodiesterase T Thymin-1-yl or thymidine TBAF Tetrabutylammonium fluoride TBDMSCl tert-Butyldimethylsilyl chloride

THF Tetrahydrofuran TOCSY Total correlation spectroscopy TTMSS Tris(trimethylsilyl)silane

11

1. Introduction

1.1 Radicals Radicals are atoms or molecules which contain unpaired electrons.

Simple examples are hydrogen atoms and bromine atoms which have one

and seven electrons, respectively, in their valence shell. Almost all

radicals can be described as ‘‘free’’ as they exist independently, without

any need of support from the surroundings. Radicals are inherently

unstable and reactive since their major reactions i.e. dimerization,

hydrogen abstraction and disproportionation, are often favoured

thermodynamically and they occur with little or no activation barrier.

Notable exceptions are the naturally occuring oxygen (O2) and nitrogen

monoxide (NO). In 1849 Kolbe described the product obtained from

electrolysis of potassium acetate as ‘methyl radical’ with a formula C2H3.

It was later proven that the product was in fact ethane formed by

dimerization of two methyl radicals in the electrolysis reaction. The key

break-through, however, came in 1900 when Gomberg1 investigated the

reaction of triphenylmethyl bromide (1) with silver (Figure 1). In the

absence of oxygen the reaction produced a white solid which on

dissolution turned into a yellow solution. Gomberg proposed that the

product was hexaphenyl ethane (3) which could exist in equilibrium with

the coloured triphenylmethyl radical (2). It is another story that Gomberg

in fact had isolated another stable dimerization product 4. This was a

major discovery since it established that radicals are capable of

independent existence. Almost 30 years later, Paneth2,3 showed the

existance of the less stable, more reactive, methyl radical. It was not until

12

1937 that radicals were postulated to be intermediates in a variety of

organic reactions.4 Later works proved the involvement of radicals in

biological systems.5

Br + Ag + AgBr

Ph

PhPh

PhPhPh

Gomberg product

H

PhPhPh

Ph

Ph

Actual product

1 2

3 4

Figure 1. The classic Gomberg experiment

In the mid 1970s important contributions by physical chemists increased the

understanding of kinetics of various radical processes.6,7 With the knowledge

of rate constants for most of the common reactions, it was possible to design

experiments, i.e. select temperature and concentrations of radical precursors

and additives, that produced the desired product in synthetically useful yields.

This was the basis for the use of radical reactions in modern synthetic organic

chemistry.7-9

13

1.2 Radical Reactions Most reactions involving carbon or heteroatom centered radicals involve one

or several of the following steps:10 (Figure 2; A, B, C and D represent atoms

or groups, not necessarily representing carbon).

A + B A B

A B A + B

A + e- A-

A+A e-+

+A B C A B + C

A + B D A B D

A B D A + B D

(i)

(ii)

(iii)

(iv)

(v)

(vi) Figure 2. Common radical reactions.

(i) Homolysis: Thermal or photochemical cleavage of a covalent bond

between atoms or molecules results in formation of radicals.

(ii) Recombination: Radicals may combine by sharing their valence electrons

in a covalent bond in a process termed as recombination.

(iii) Electron transfer: Radicals may accept or donate an electron to form

anions and cations, respectively.

(iv) Atom or group transfer: Radicals may add to an atom accompnied by

expulsion of another radical from that atom (the SH2 type of reaction).

14

(v) Addition: A radical may add across a double bond to form a covalent

bond, e.g. Kharasch reaction.11

(vi) �-fission: Expulsion of a radical �� to a radical center accompanied by

double-bond formation. Such reactions are favoured, e.g. in the ring-opening

of cyclopropylcarbinyl or cyclobutylcarbinyl radicals where the ring strain is

relieved.12

In-spite of the diversity of radical reactions, the actual number of practical

protocols available for executing radical reactions are rather few in

number.13,14

1.3 The chain concept

Unlike cations or anions, radicals react with themselves by combination or

disproportionation at rates approching the diffusion controlled limit. The

maintainence of low concentrations of radicals over the course of reaction is

therefore imperative for a successful synthetic outcome. Chain reactions

involving reactions between radicals and non-radicals are ideally suited to

meet this requirement. Radicals generated via some initiation process

undergo propagation steps to generate new radicals leading ultimately to

termination of the chain via coupling or disproportionation reactions. The

chain reaction thus maintains a steady concentration of the reactive species

while converting the starting material to product. Most of the chain reactions

used in synthesis involve a metal hydride as a mediator. The processes are

illustrated in Figure 3.

15

Initiation

Initiator In

In +M H In H +M Propagation

M + R1

R2

X R1 M X+

R1

R2 +M H R2 H + M Termination

In , R1 , R2 , M non-radicals Figure 3. Processes involved in radical chain reactions.

The initiator undergoes homolytic fission thermally or photochemically to

generate the initiator radical In which abstracts hydrogen from the metal

hydride to give a metal radical M . Reaction of the radical precursor R1�X

with M results in transfer of X to generate an alkyl radical R1 . The alkyl

radical may undergo rearrangement (atom or group transfer, addition or �-

fission) to generate another radical R2 which could react with M-H with

hydrogen transfer thereby regenerating the metal radical M and thus

propagating the reaction. All radicals generated in the sequence may also

produce non-radicals via recombination thus terminating the chain.

1.3.1 Initiators For radical initiation one requires a molecule with weak covalent bonds that

can be broken homolytically under mild conditions. The energy required to

cleave the bond can be provided by heating (thermolysis), by ultraviolet light

(photolysis) or by X-rays (radiolysis). A majority of radical reactions useful

16

in synthetic organic chemistry use either photolysis or thermolysis as triggers

for radical initiation. The following compounds are frequently used:

Azo-compounds: Thermolysis of azo-compounds (R-N=N-R) leads to

cleavage of the two carbon–nitrogen bonds and formation of stable dinitrogen

(N2). The rate of decomposition is determined by the stability of the resulting

radical (R ). One of the favorite initiators with organic chemists is 2,2'-

azobisisobutyronitrile15 (AIBN; 5) which can be cleaved either photolytically

or thermally (Figure 4).

N CNNNC�

CNNC + + NC2N260%

CNNC

40%

t1/2(60oC)= 17 ht1/2(80oC)=1.3 h

5

Figure 4. Thermal decomposition of the initiator AIBN. Only 60 % of the initiator radical is available for initiation in a typical radical chain reaction.

The easy decomposition of AIBN is due to the stability of the 2-cyano-2-

propyl radical. It is stabilized both by resonance (the unpaired electron can be

delocalized to nitrogen) and through bond by electron donating methyl

groups.

NNNC

CN

MeO

OMe

NNNH

HN NH2

H2N

CNNN

NC

6 7 8

O

O

HO

OH

Figure 5. Azo-type radical initiators: (6) 2,2'-Azobis(4-methoxy-2,4-dimethylvaleronitrile); (7) 2,2'-Azobis(2-methylpropionamidine); (8) 4,4'-Azobis(4-cyanopentanoic acid).

17

Recently, azo-type radical initiators that decompose at room temperature

have been used for stereoselective carbon-carbon bond formation, e.g. 2,2'-

azobis(4-methoxy-2,4-dimethylvaleronitrile) (V-70).16(Figure 5; 6)

Hydrophilic radical initiators such as 2,2'-azobis(2-methylpropionamidine)

(Figure 5; 7) as dihydrochloride (V-50)17 and 4,4'-azobis(4-cyanopentanoic

acid) (V-501)18 (Figure 5; 8) have been used at moderate temperature for

initiating radical reactions in aqueous solution.

Peroxides and diacylperoxides: Thermolysis of peroxides has been used in

the study of radical reactions for a long time.11,19,20 On heating or irradiation,

alkoxy and acyloxy radicals, respectively, are formed by cleavage of the O�O

bond. Some commonly used peroxide initiators are shown in Figure 6.

Alkoxyl radicals obtained from di-tert-butyl peroxide (12) abstract hydrogen

atoms at the �- position in amines, ethers and esters.

O

OO

O

O

OO

OO

OO

OO C11H23 O

OO C11H23

O

9 10 11

12 13

Figure 6. Some commonly used peroxide radical initiators. Benzoyl peroxide (9), acetyl peroxide (10), tert-butyl perbenzoate (11), di-tert-butyl peroxide (12) and lauroyl peroxide (13).

Boranes: Trialkylborane21 together with molecular oxygen has been used

in several stereoselective reactions22 since they effectively initiate reactions at

-78 °C.

18

R3B + O2 R

R + O2

+ R3B R2BO2R + R

+ R2BOO

ROO

ROO Figure 7. Alkyl boranes as initiators.

As shown in Figure 7, alkyl radicals (R ) are thought to be the active

initiating species formed as a result of atom transfer. Other boranes such as 9-

BBN have also been used as radical initiators.23

1.3.2 The tin hydride method

To be useful in synthesis, a given chain reaction must generate radicals

site-selectively. A reaction extensively applied in synthesis which meets the

above requirement is the trialkyltin hydride mediated reduction of various

functional groups such as alcohols, halides and various chalcogen containing

R X + Bu3SnH R H + Bu3SnX

X= Xanthate, I, Br, Cl, SPh, SePh, TePh, NO2.

Figure 8. Reduction of halides and pseudohalides with trialkyltin hydride.

compounds (e.g. Barton-McCombie-deoxygenation,24 Ono-reduction25,26)

(Figure 8). Since its introduction by Kuivila27-29 the methodology has also

been extensively used for carbon-carbon bond formation.30-34Since this thesis

is concerned with radical cyclization chemistry, the various steps involved

are outlined in Scheme 1.

Radicals In produced by decomposition of the initiator readily abstract

hydrogen atoms from tributyltin hydride (14) to form the chain carrying

radical Bu3Sn (15). The propagation sequence involves an atom or group

abstraction (step 1) from 16 to provide a 5-hexenyl radical 17.

19

X

19

16 17

Bu3Sn��

X-SnBu3 Step 1

Bu3SnHIn�

In-H

14 15

+

17

Step 3

18

�kc

�

18

Step 4H-SnBu3kH Bu3Sn�

20

�

+

H

17

�

Step 2

Bu3Sn�H-SnBu3kH+

+ +

+

+

Bu3Sn�

Scheme 1. Steps involved in tin-mediated radical cyclization. Depending on concentrations and rates of hydrogen abstraction mixtures of cyclized (20) and reduced (19) products are formed.

Radical 17 may close via a first order 5-exo cyclization to provide

cyclopentylmethyl radical (18) (step 2). In competition with this process,

hydrogen abstraction could produce reduced compound 19 in a bimolecular

reaction (step 3). Finally, radical 18 abstracts a hydrogen from tin hydride to

produce cyclized compound 20 (step 4).

At 25ºC, kc, the rate of cyclization, is approximately 2 x 105 s-1 and kH, the

rate of hydrogen abstraction, is approximately 2 x 106 M-1s-1.35,36 The fate of

radical 17 is determined by the relative rate of cyclization (kc) versus

hydrogen atom abstraction (kH[Bu3SnH]). Thus, the concentration of tin

hydride is a variable by which product distribution can be controlled. At very

high concentrations, reduction product 19 will predominate (kc<kH[Bu3SnH].

At intermediate concentrations mixtures are expected. At low concentrations

20

(<0.05 M), most of radicals 17 will cyclize to 18 prior to hydrogen atom

abstraction (kc>kH[Bu3SnH] to give 20. Although the tin hydride method is

still a favourite among radical organic chemists,37 there is a need for

replacement,38 primarily because of toxicity and environmental concerns.

One such compound that has been in use for more than 20 years is

tris(trimethylsilyl)silane (TTMSS, (Me3Si)3SiH).39

1.3.3 Silanes A comparative study of the reaction of primary alkyl radicals with a

variety of group 14 hydrides revealed that rate constants for

tris(trimethylsilyl)silane and tributyltin hydride are comparable.40 It also

showed that for all kinds of radicals the reactivity of the former is always

lower. In Figure 9, the actual rate constants for hydrogen atom abstraction

from some group 14 hydrides are shown.

105 106 107 108

n-Bu3GeH

(Me3Si)2Si(H)Me (Me3Si)3SiH

Bu3SnH

(Me3Si)3SiH/RSH

(Me3Si)3SiH/ArSH

kH/M-1s-1 Figure 9. Rate constants for hydrogen atom abstraction from a variety of hydrogen donors by primary radicals at 80 ºC.41

Tris(trimethylsilyl)silane was introduced as an alternative to tin hydride in

the late 1980s.42 Toxicity of tin based reducing agents and the problems

associated with removal of tin residues has led to an increasing number of

reports on the use of TTMSS as a reducing agent in radical reactions. Thus,

iodides, bromides, chlorides, selenides, isocyanides, acid chlorides, xanthates

21

and sulphides have all been used as radical precursors in TTMSS mediated

chain reactions.41

1.4 Radical precursors Depending on the ease of preparation, halides, aryl sulphides, aryl selenides,

aryl tellurides, xanthates and other thiocarbonyls, nitro and thiohydroxamate

compounds were the precursors of choice for radical chain reactions.13 Alkyl

chlorides, however, cannot be used as substrates in reactions involving

addition of a radical to a double bond since chlorine atom abstraction by the

organostannyl radical is a slower process than is addition to the electron

deficient olefin. Hydrostannylation of the olefin and recovery of chloride

would result.43

1.4.1 Organochalcogens as radical precursors

Due to their stability and relative ease of preparation, organoselenium

compounds have proven to be very versatile radical precursors. They have

been used in Sn- and Si- mediated radical reactions as well as in group

transfer reactions for the formation of carbon-carbon bonds and carbon-

heteroatom bonds.44 Alkyl aryl selenides have been prepared for generation

of a wide range of carbon-centered radicals. Due to unfavourable expulsion

of phenyl radicals, alkyl phenyl selenides are known to cleanly produce alkyl

radicals. However, a word of caution should be mentioned here. Small

amounts of diphenyl diselenide, present as a result of photochemical

decomposition of the radical precursor or as a carry over from the radical

precursor synthesis, can be detrimental. The explanation to this is the well

established phenomenon of polarity reversal catalysis45,46 wherein the

reduction of the alkyl radical is brought about by benzeneselenol (PhSeH

22

reacts with alkyl radicals faster than Bu3SnH by more than three orders of

magnitude). In fact, the selenol acts as a catalyst for the reduction processes.

The rapid reduction has been taken advantage of in dehalogenation,

deoxygenation and desulphurization reactions47,48 where undesired radical

rearrangements which are sufficiently fast to proceed in the presence of tin

hydride can be avoided.

Phenyl selenides are easily obtained from nucleophilic49 or electrophilic50

selenium reagents.51 Thus, halides, alcohols, lactones, epoxides and aziridines

have been transformed into phenyl selenides and carried like a protecting

group for a radical through several synthetic steps. In fact, in one of the first

tin-mediated addition reactions, phenylselenides were found to give better

yields than the corrosponding iodides.52

The use of organotellurium compounds as precursors for carbon-centered

radicals was first reported by Clive in 1980.53 Ten years later, Barton and

Ramesh54 first applied organotellurium compounds for radical mediated

carbon-carbon bond formation using a thiohydroxamate ester as initiator and

promotor. This work was followed by the first report on organotellanyl group

transfer reactions with alkynes55 and intramolecular group transfer reactions

of acyl tellurides.56 The scope was further extended by intramolecular group

transfer reactions in the synthesis of cyclopentanes and tetrahydrofurans.57-60

The synthetic utility61 reflects the fact that the organochalcogen group

transfer reactions occur as rapidly as those for iodides.62-64

1.5 Radical cyclization It has been known since long that free radicals add to double bonds.11 The

alkyl radical so formed may undergo further reactions such as hydrogen or

halogen abstraction. The peroxide-catalyzed addition of HBr to olefins is an

early example of this type of reaction. Intramolecular addition of radicals to

alkenes and alkynes always proceed more rapidly than the corresponding

23

intermolecular reactions. Radical cyclization reactions have therefore been

extensively used for preparation of both carbocycles and heterocycles.

1.5.1 5-Hexenyl cyclizations

The kinetically controlled rearrangement of variously substituted 5-hexenyl

radicals to cyclopentylmethyl radicals has been extensively

investigated,10,19,20,65,66 both from a mechanistic and synthetic point of view

by Beckwith,10,67-69 Giese70, Curran,32,33 Stork,71 and Rajanbabu.72-75 It

constitutes an excellent method for preparation of 5-membered carbocycles

and heterocycles.31

�

��

5-exo product 6-endo product

k6-endok5-exo

Figure 10. Rearrangement of a 5-hexenyl radical to a cyclopentylmethyl radical (5-exo) or a cyclohexyl radical (6-endo).

In light of Baldwin’s rules76,77 for ring closure, the preference for 5-

exo- over 6-endo- cyclization (Figure 10) is well understood in terms of a

relatively strain-free chair-like transition state78 which fulfills the

stereoelectronic requirements for radical addition to a double bond. For the

unsubstituted 5-hexenyl radical k5-exo is approximately 2 x 105 s-1 and k6-endo is

4 x 103 s-1 thus resulting in formation of cyclopentylmethyl radical as the

major product. The Beckwith-Schiesser, Houk79 model also provides

satisfactory explanations for the observed stereoselectivities in the cyclization

of 2-, 3- and 4-monosubstituted 5-hexenyl radicals43 (Figure 11). This can be

explained assuming the substituent adopts a pseudo-equatorial position in the

24

transition state,74 i.e. , the 2- and 4-methyl-5-hexenyl radicals cyclize with

preferential formation of trans-disubstituted cyclopentanes whereas 1- and 3-

methyl-5-hexenyl radicals predominantly afford cis-disubstituted products.

The efficiency and regiochemistry of intramolecular cyclization addition

reactions12 have been shown to be controlled by (i) the initial radical

structure, (ii) the steric effects resulting from the olefin substitution pattern,10

(iii) the geometric constraints on the chain linking the radical centre and the

tethered double bond and (iv) the presence of a heteroatom in the chain.80-83

major minor

major

major

major

minor

minor

minor

2-substituted 5-hexenyl radical




Figure 11. Diastereoselectivities in the cyclization of 1-, 2-, 3- and 4-substituted 5-hexenyl radicals.43

1.5.2 6-Heptenyl cyclizations

Whereas 5-hexenyl radical cyclization comprises the ultimate tool for making

five membered rings, the use of 6-heptenyl cyclization for making 6-

membered rings is less important. The formation of 6-membered rings by

radical cyclization presents several problems (Figure 12). First, the rate of

cyclization of the parent 6-heptenyl radical is almost two orders of magnitude

slower than that of the 5-hexenyl radical. Second, ring-closure is

25

considerably less regioselective (kexo/kendo~ 7, for the 6-heptenyl radical10,78 vs

kexo/kendo ~ 50, for the 5-hexenyl radical).12,63 Third, the 6-heptenyl radical is

prone to rearrangement via [1,5]-hydrogen atom transfer to give an allylic

radical.12

�

�

�

H � H�

k6-exo

k7-endo

[1,5]-shif t

6-exo product

7-endo product

rearranged product

Figure 12. Rearrangement of a 6-heptenyl radical to a cyclohexylmethyl radical (6-exo) or a cycloheptyl radical (7-endo). Competing [1,5]-hydrogen atom transfer.

Cyclization of methyl substituted 6-heptenyl radicals was recently

studied84 and the poor selectivity was attributed to the conformational

flexibility inherent in the radical. Difficulties associated with cyclization of

6-heptenyl radicals have been overcome by several methods: (i) Restriction

of the degrees of freedom in the heptenyl chain either by incorporation of

rings10 or substituents (Thorpe-Ingold effect85) (ii) Activation of the double

bond. The presence of an electron-withdrawing group at the terminal vinyl

position of the 6-heptenyl radical significantly improved both the rate of

cyclization and the regioselectivity in the ring-closure. Highly regioselective

6-exo cyclizations with moderate to good stereoselectivity proceeding via a

chair-like transition state have been reported.86-88 Our attempts to use 6-

heptenyl cyclizations in nucleoside synthesis and for the preparation of

substituted piperidines will be discussed in chapters 3 and 7.

26

1.6 Fundamentals of nucleic acids The nucleic acids,89,90 i.e. deoxyribonucleic acid (DNA) and ribonucleic acid

(RNA) are the carriers of genetic information in all living organisms. DNA

acts as the storehouse of genetic information in the nucleus while the RNA

acts as the carrier of the genetic information to the ribosome, where it is

processed and translated into proteins (notable exceptions are the retro

viruses where the RNA acts as the carrier of the genetic information). The

nucleic acids are made-up of nucleotide building blocks. Each nucleotide

consists of a nucleobase, a sugar and a phosphate linker. The nucleobase

generally consist of one of the following: 9-adeninyl (A), 9-guaninyl (G), 1-

thyminyl (T) or 1-cytosinyl (C) in DNA while in RNA, 1-thyminyl is

replaced by 1-uracilyl (U). Adenine and guanine are purines, while thymine,

uracil and cytosine are pyrimidines. The major difference between DNA and

RNA is the absence of the 2'-hydroxyl in DNA (2'-�-D-deoxyribofuranosyl)

which is present in RNA (2'-�-D-ribofuranosyl). This subtle change in the

sugar gives rise to a distinctly different structure as well as function of the

two molecules. Each sugar residue is linked to the neighbouring sugar

residues by a 3',5'-phosphodiester bond (3'-carbon atom of a sugar with the

5'-carbon of the neighbouring sugar). While the sugar moiety and the sugar-

phosphate backbone of DNA/RNA primarily play a structural role, the

genetic information is determined by the sequence of the nucleobases. In the

cell, the RNA usually exists in single strands, while the DNA adopts a double

helical91 structure in which the two DNA strands are held together by

noncovalent interactions i.e. intra-strand stacking of nucleobase moieties and

weak hydrogen bonds between intra-strand pairs of nucleobases. The

hydrogen bonding occurs between laterally opposite bases, ‘The base pair’,

of the two strands of the DNA duplex according to Watson-Crick base-

pairing rules91 (A specifically hydrogen bonds to T and G specifically to C).

27

Normally, G�C-pairs (linked by three hydrogen bonds) are stronger than

A=T-pairs (linked by two hydrogen bonds). The double stranded structures of

DNA and RNA form different types of helices with the two strands oriented

in opposite directions (3'�5' of one strand and 5'�3' of the other). The

DNA:DNA duplexes normally form a B-type helix which is characterized by

an extended structure with a distance of ~3.4 Å between adjacent bases and

approximately 10 base-pairs per helical turn, which are nearly perpendicular

to the helical axis. Also, the B-type helix has a narrow and deep minor

groove (~6 Å) and a wide major groove (~12 Å). In contrast, the RNA:RNA

duplexes normally adopt an A-type helix which has a more compressed

structure (the distance between adjacent bases is ~2.6 Å). Thus, A-type

duplexes exhibit approximately 11 base-pairs per helical turn, which are

tilted approximately 20° with respect to the helical axis. Also, unlike the B-

type helix, they have a very wide (~11 Å) and shallow minor groove and a

very narrow (~3 Å) and deep major groove. The DNA:RNA duplexes

typically adopt an intermediate A/B-type structure, with an overall structure

resembling the A-type. It is also noteworthy that A-type duplexes normally

have higher duplex stability as compared to the B-type ones. With identical

nearest neighbours the RNA:RNA is most stable while the order of stability

between DNA:DNA and RNA:DNA depends on the sequence context.92,93 In

general, the order of stability is RNA:RNA> DNA:RNA> DNA:DNA. The

helical geometry described above can also be explained by the furanose

conformation94-96 adopted in the nucleosides and the �/�-angle of the

phosphate backbone.97 The furanose ring of a nucleoside can adopt numerous

conformations in solution due to puckering98 of the sugar ring. In solution,

the major population of 2'-deoxy pentofuranose adopts a 2'-endo (DNA-type)

conformation while in RNA the sugar adopts a 3'-endo conformation (RNA-

type) also known as South- and North- type respectively.

28

1.7 Nucleic acid based therapeutics and significance of locked nucleosides Targeting proteins with small molecules, which is the basis of the traditional

drug discovery approach is difficult. Moreover, it has been estimated that

only 10-14 % of the proteins have appropriate binding sites (druggable) for

small molecules.99 Therefore, instead of targeting the protein itself, if one can

target DNA or messenger RNA (mRNA) by an oligonucleotide

complementary to the target sequence, it might be possible to stop the protein

production.100 Thus, nucleic acid based therapy offers a seemingly simple and

straight-forward mechanism to down-regulate a gene of interest. The

technique used to treat a disease at the genomic level is called anti-gene

approach.101,102 In this method an artificial, short, modified DNA or RNA

sequence is synthesized which forms a stable triple helix (triplex)103,104 with

the double stranded genomic DNA through the major groove via Hoogsteen

hydrogen bonding.105,106 The other popular oligonucleotide based therapy is

called antisense technology.107 The traditional antisense technology exploits

the ability of a single stranded oligodeoxynucleotide (ODN) of 15-25

nucleotides to bind to the accessible regions of the target mRNA via Watson-

Crick base pairing in a sequence specific manner, thereby inhibiting protein

synthesis either by sterically blocking the ribosomal assembly or by RNase H

mediated degradation of the mRNA.108

The antisense technology has found increased acceptance from the

pharmacological perspective which is evident from the recent approval by

FDA of the first oligonucleotide based antisense drug called Vitravene®.

Other oligonucleotide-based therapeutics are in the pipeline.109

Although the natural phosphodiester oligonucleotides are easy to synthesize,

their use is limited as they are degraded by intracellular endo-and

exonucleases. This has warranted chemical modification of the

oligonucleotides in order to utilize them for therapeutic applications. Recent

29

years have seen development of conformationally constrained bicyclic110,111

and tricyclic112,113 nucleotides, in which the sugar is locked in a definite

puckered conformation. Such oligonucleotides show promising properties

with respect to target RNA binding and nuclease resistance. Among several

molecules reported, short nucleotides containing locked nucleic acid

(LNA)114 or bicyclo-nucleic acid (BNA111) modifications have shown

unprecedented thermal stability (+3 to +8 °C per modification depending

upon the sequence context). The enhanced target binding property of the

conformationally constrained bicyclic sugar units in these nucleotides has

been attributed to the improved stacking between the nearest neighbours and

quenching of concerted local backbone motions by LNA nucleotides in

ssLNA so as to reduce the entropic penalty in the free energy of stabilization

for the duplex formation with RNA.115 The unique features of LNA/BNA

have led to the synthesis of a number of closely related analogues, in which

the 2',4'-bridge has been altered.116-119 Studies120 with several sugar modified

nucleotides show that substituents in the modified sugar play an important

role in conformational steering, hydration, hydrophobic/hydrophilic

interactions, electrostatic interactions121 and thus influencing the interaction

of modified oligonucleotides with other nucleotides and/or enzymes present

in the system.122

30

2. A radical cyclization route to �1 - pyrrolines (publication I)

Pyrrolines are important structural motifs in many pharmacologically

important alkaloids and several synthetic methodologies were used for their

preparation.123-129 As mentioned in the introduction (section 1.5), free radical

methodology has been used for synthesis of 5- and 6-membered nitrogen

containing heterocycles.59,130 However radical methodologies for the

synthesis of pyrrolines are few in number and based primarily on generation

of iminyl radicals.131-134

2.1 Design

As presented in Scheme 2, readily available �-phenylselenenyl ketones

could, after imine formation with an appropriate allylic amine, serve as

precursors of carbon-centered radicals which would hopefully cyclize to �1-

pyrrolines under reductive conditions. A variety of methods are available for

introduction of PhSe-groups into the �-position of ketones and aldehydes.

We found the two-step procedure involving reaction of methyl ketones with

PhSeCl3, followed by reduction of the resulting Se,Se-dichloride, convenient

for large scale preparation.135-137 Imine formation was tried using allyl amine

together with TiCl4 as a dehydrating agent. The reactions went smoothly to

completion and we were able to isolate crude phenylselenenyl imines in good

yields.

31

R

OSePh

R

NSePh

R

N

R

N

R

N

+R

NSePh

N

R

Scheme 2. Radical cyclization route to cyclic imines.

Syn/anti-isomerization in imines has been well studied.138,139 For N-alkyl

imines the energy barrier to interconversion is low. We envisaged (Scheme 2)

that under radical cyclization conditions, resonance in the radical

intermediate would allow for conversion of the unreactive (in cyclization) Z-

isomer into the reactive E-isomer. Our attempts to bring about cyclization by

slow addition of tin hydride led mostly to reduced product. Better results

were obtained using tris(trimethylsilyl)silane as a hydrogen donor.140

2.1.1 Synthesis In the representative reaction sequence shown in Scheme 3, acetophenone

(21) was reacted with phenylseleninyl trichloride in dry ether overnight.

O OSePh

NSePh

O

+

N N

SePh+

(i), (ii) (iii)

(iv)

95% 80%

15% 46%

21 22 23

21 24 25

Scheme 3. Reagents and conditions: (i) PhSeCl3, dry ether, r.t., overnight; (ii) Na2S2O5, water, ether; (iii) Allylamine, TiCl4, dry ether, -78°C to r.t. overnight; (iv) AIBN, TTMSS, C6H6, reflux, 8 h.

32

The white precipitate of crystalline Se(IV)-dichloride formed was filtered off

and isolated in quantitative yield.

Reduction with aqueous Na2S2O5 provided selenide 22 in 95 % yield after

two steps. Reaction with allylamine and TiCl4 afforded the crude imine 23 in

80 % yield as a mixture of syn/anti-isomers. Reductive radical cyclization

using TTMSS/AIBN in refluxing benzene afforded cyclic imine 24 in 46 %

yield along with 15 % of reduced product 21, and a trace of group transfer

product 25. Application of this methodology to other methyl ketones afforded

substituted imines in moderate yields (Table 1).

Table 1. Cyclic imines prepared.

Product

Yielda

Crudeb (%)/isolated (%) N 52/47

N

MeO

50 /46

N

S

33/-

N 49/38 cis/trans=82/18

N 18/-

aYield over two steps (imine formation and cyclization). bAs determined by 1H NMR using DMAP as an internal standard.

Low molecular weight pyrrolines such as those prepared from pinacolone and

2-acetylthiophene were quite difficult to isolate. Unfortunately, our efforts to

33

extend the methodology to cyclic �-phenylselenenyl ketones and �-

phenylselenenyl aldehydes failed at the imine-forming step.

34

3. A radical cyclization route to conformationally constrained nucleosides: Synthesis, structure and biochemical studies (publication II)

3.1 Radical reactions in nucleic acids

As early as 1964, Johnson141 reported formation of C-5' to C-7 linked

adenosine upon anaerobic photolysis of vitamin B12 coenzyme. From that

time and on nucleic acids142 and sugars72-75,143-153 have been frequently used

as substrates in radical mediated transformations. Apart from its synthetic

utility, the radical chemistry of sugars and nucleosides have also contributed

to the understanding of several biological phenomenon such as charge

transfer154 and radical induced damage in DNA and RNA.155,156 The

conversion of ribonucleotides to deoxyribonucleotides catalyzed by

ribonucleotide reductase is one of the most fascinating radical reactions in

nature.157,158

Recent developments in the synthesis of conformationally constrained

nucleoside analogues led us to try free radical carbon-carbon bond formation

to constrain sugar pucker in a definite conformation. We were successful in

synthesizing two conformationally constrained carbocyclic analogues of

thymidine by free radical intramolecular carbon-carbon bond formation. Our

results are presented below.

35

3.1.1 Synthesis of 2',4'-locked carbocyclic nucleoside (carbocyclic LNA-T; 37a/37b)

The synthesis starts from a known sugar precursor 26114 which was

selectively benzylated to give 27 using a known procedure.117 The remaining

primary alcohol in sugar 27 was oxidized to the corresponding aldehyde 28

by Swern oxidation.159 A vinyl group at C-4' was then introduced by Wittig

methodology160 using crude aldehyde to give the sugar 29 (87 % in two steps

from 27). Anti-Markovnikov addition of water to olefin 29 was effected by

successive hydroboration – oxidation using 9-BBN/NaOH-H2O2 to give

O

OO

HO

OBnHO

O

OO

BnO

OBnHO

O

OO

BnO

OBnO

(i) (ii) (iii)O

OO

BnO

OBn

O

OAcOAc

BnO

OBn

O

OAc

BnO

OBn

NH

N

O

OO

OH

BnO

OBn

NH

N

O

OO

OCSOPh

BnO

OBn

NH

N

O

O

(vi)

26 27 28 29

O

OO

BnO

OBnHO

O

OO

BnO

OBn

OHO

OH

NH

N

O

O

CH3

ODMTrO

OH

NH

N

O

O

CH3

ODMTrO

O

NH

N

O

O

CH3PNO CN

(iv) (v)

(vii) (viii) (ix)

(x) (xi) (xii) (xiii)

30

31 32 34 35

36a: major (70 %) 7'R36b: minor (30 %) 7'S 37a/37b 38a/38b

66% 87% 95% 70%

80% 72%

73% 76% 74% 80%

39a/39b

3'

5'

7'6'

4' 2' 1'OBnO

OBn

NH

N

O

O

CH3

3'

5'

7'6'

4' 2' 1'

33

Scheme 4. Reagents and conditions: (i) NaH, BnBr, DMF, �5 °C to r.t. overnight; (ii) oxalyl chloride, DMSO,�78 °C, DIPEA, DCM; (iii) PPh3

+CH3Br�,1.6 M butyllithium in hexane, THF, �78 °C to r.t. overnight,;(iv) 9-BBN, THF, 3 N NaOH, 33 % H2O2; (v) oxalyl chloride, DMSO, �78 °C, DIPEA, DCM, PPh3

+CH3Br�, 1.6 M butyllithium in hexane, THF, �78 °C to r.t. overnight; (vi) acetic acid, acetic anhydride, triflic acid; (vii) persilylated thymine, TMSOTf, CH3CN, 0°C to r.t. overnight; (viii) 27 % methanolic NH3, overnight; (ix) DMAP, O-phenylchlorothiocarbonate, pyridine; (x) Bu3SnH, toluene, AIBN, 4 h; (xi) 20 % Pd(OH)2/C, ammonium formate, methanol, reflux, 8 h; (xii) DMTr-Cl, pyridine, overnight; (xiii) 2-cyanoethyl N,N-diisopropylphosphoramidochloridite, DIPEA, dry THF, overnight.

36

alcohol 30 in 95 % yield, which was again subjected to Swern oxidation /

Wittig 160 olefination to give the required sugar 31 (70 % in two steps from

30) with a strategically placed propenyl side chain at C-4' as an accepter for

radical cyclization. Compound 31 was subjected to acetolysis using a mixture

of acetic anhydride, acetic acid and triflic acid to give the corresponding

diacetate 32, quantitatively as �/� anomeric mixture (single spot on TLC and

proven by 1H-NMR). The crude diacetate 32, after bicarbonate workup, was

subjected to a modified Vorbruggen reaction161 involving in situ silylation of

thymine and subsequent trimethylsilyl triflate mediated coupling to give

thymine nucleoside 33 in 80 % yield in two steps from 31. The �-

configuration of product 33 was confirmed by a 1D differential NOE

experiment, which showed 3 % enhancement of H-2', and 1 % enhancement

of H-3' upon irradiation of H-6 (dH6-2' ~ 2.3 Å for the �� anomer).

Deacetylation of compound 33 using 27 % methanolic ammonia overnight,

and subsequent esterification using O-phenylchlorothiocarbonate yielded the

desired precursor 35 (72 % in two steps from 33) for radical cyclization. The

key free radical cyclization reaction was carried out using Bu3SnH with

AIBN initiation at 115 °C in degassed (N2) toluene. To ensure that the radical

generated has adequate lifetime to capture the double bond before quenching,

the concentrations of Bu3SnH and AIBN were kept low by high dilution and

slow, drop-wise addition. 5-Exo cyclization occurred exclusively to give the

expected,10 5-membered, 2',4'-cis-fused carbocyclic product with a bicyclo

[2.2.1] heptane skeleton as an inseparable diastereomeric mixture of

compound 36a (major compound 70 %, 7'R) and 36b (minor compound 30

%, 7'S). The formation of the bicyclic nucleosides 36a and 36b, with a North

fused conformationally constrained pentofuranosyl moiety was confirmed by

long range 1H-13C NMR correlation (HMBC)162 and 1H-1H (TOCSY)163 for

both isomers. A 1D differential NOE experiment established that the

exocyclic methyl at C-7' is in close proximity to H-1' in the major isomer (d7'-

37

Me/H-1' ~2.8 Å for R and 4.4 Å for S configured C-7') of the bicyclic structure.

The benzyl groups in the bicyclic nucleosides 36a/36b were removed using

Pd(OH)2/C and ammonium formate in methanol to give the corresponding

dihydroxy compounds 37a and 37b respectively. Several attempts to separate

this diastereomeric mixture of 37a/37b failed. We therefore subjected it

directly to 5'-dimethoxytritylation (74 %) to give 38a/38b followed by 3'-

phosphitylation (80 %) to give the phosphoramidite 39a/39b using standard

conditions.119

3.1.2 NMR characterization of carbocyclic LNA-T (37a/37b)

The 1H NMR spectrum at 600 MHz of the ring-closure products 36a/36b

revealed a diastereomeric mixture of sugar-fused 5-membered bicyclic 3', 5'-

di-O-benzyl protected nucleosides (Scheme 4). However, because of the

overlap of the H-7' and H-2' peaks, firm NMR evidence could not be

obtained for the carbon-carbon ring closure between C-2' and C-7'. The H-7'

and H-2' peaks in the deprotected compounds 37a/37b were however fully

resolved, and hence could be used for full NMR characterization.

The 1H spectrum showed the presence of two diastereomers, a major 37a

as well as a minor isomer 37b in ca. 7:3 ratio. The upfield H-2' at � 2.43 ppm

along with H-7' at � 2.65 ppm and their proton-proton couplings, proven by

detailed double decoupling and by COSY experiments, showed that the C-2'

substituent is the C-7' methine-carbon. The NOE enhancement (~12 %,

corresponds to ca. 2.6 Å) between H-6 (thymine) and H-3' of 37a/37b, in

addition to 3JH1',H2' = 0 Hz, further confirmed that the sugar is indeed locked

by the fused carbocycle in the North conformation as observed for other

North-locked nucleosides such as ENA164, LNA114, and aza-ENA117. This

further showed that the 1-thyminyl moiety is in the �-configuration with an

anti-conformation across the glycoside bond. The fact that the NOE

38

enhancement of 6.5 % for H-1' upon irradiation on CH3 at C-7' of 37a was

found, showed that the methyl group on C-7' is in close proximity of H-1' (ca.

2.8 Å), thereby confirming the R configuration for C-7'. The NOE

enhancement of 4.5 % for H-7' in 37b upon irradiation on H-1', on the other

hand, confirmed that the H-7' is in close proximity of H-1' (ca. 2.2 Å) and

hence the S configuration was assigned for C-7'. HMBC correlations between

H-7' and C-2' for compounds 37a/37b unequivocally proved that the oxa-

bicyclo[2.2.1]heptane ring system was formed in the ring closure reaction.

3.1.3 Synthesis of 2',4'-locked carbocyclic nucleoside (carbocyclic ENA-T; 48)

For synthesis of a carbocyclic-ENA-T analog, a 6-exo heptenyl type

radical cyclization was desired, and thus the C-4' allylated sugar 31 described

in Scheme 4 was subjected to another sequence of hydroboration-oxidation,

Swern oxidation159 and Wittig160 reactions described previously (yields of 40

and 42 were 90 % and 72 %, respectively). The sugar 42 was subjected to

acetolysis followed by a modified Vorbruggen-type161 coupling to give 44

with a �-configured thymine in 70 % yield over two steps. Deacetylation

followed by esterification using O-phenylchlorothiocarbonate yielded the

ester 46 (60 % yield after two steps). Purified ester was then subjected to free

radical cyclization. Although 7-endo cyclization usually occurs only 6-7

times more slowly than 6-exo cyclization, we were able to isolate exo-product

47 in a decent 76 % yield.

39

O

OO

BnO

OBn

O

OO

BnO

OBnHO

O

OO

BnO

OBnO

O

OO

BnO

OBn

O

OAcOAc

BnO

OBn

O

OAc

BnO

OBn

NH

N

O

OO

OH

BnO

OBn

NH

N

O

OO

OCSOPh

BnO

OBn

NH

N

O

O

OHO

NH

N

O

O

OH CH3

ODMTrO

NH

N

O

O

OH CH3

ODMTrO

NH

N

O

O

O CH3P

N

ONC

47: (8'S)

31 40 41 42 43

44 45 46

48 49 50

(i) (ii) (iii) (iv)

(v) (vi) (vii) (viii) (ix)

(x) (xi)

90% 72%

70% 60% 76% 82%

80% 84%1'2'3'4'

5'

6'7' 8'

OBnO

NH

N

O

O

OBn CH3

1'2'3'4'

5'

6'7' 8'

Scheme 5. Reagents and conditions: (i) 9-BBN, THF, overnight, 3 N NaOH, 33 % H2O2; (ii) oxalyl chloride, DMSO, �78°C, DIPEA, CH2Cl2; (iii) PPh3

+CH3Br�, THF, 1.6 M butyllithium in hexane, �78 °C to r.t. overnight; (iv) acetic acid, acetic anhydride, triflic acid; (v) persilylated thymine, TMSOTf, CH3CN, 0 °C to r.t. overnight; (vi) 27 % methanolic NH3, overnight; (vii) DMAP, O-phenylchlorothiocarbonate, pyridine, r.t. overnight; (viii) Bu3SnH, toluene, AIBN, 4 h, reflux; (ix) 20 % Pd(OH), ammonium formate, methanol, reflux,12 h; (x) DMTr-Cl, pyridine, r.t. overnight; (xi) 2-cynoethyl N,N-diisopropylphosphoramidochloridite, DIPEA, dry THF.

The bicyclic nucleoside 47 was deprotected using Pd(OH)2/C and

ammonium formate in methanol to give the corresponding diol 48 in 82 %

yield. Dimethoxytritylation (80 %) followed by phosphitylation using

standard conditions119 gave the fully protected phosphoramidite 50 in 84 %

yield.

3.1.4 NMR characterization of the carbocyclic-ENA-T (48)

For compound 48, the upfield H-2' shift at � 2.26 ppm along with H-8' at �

2.20 ppm and their vicinal proton-proton couplings, proven by the double

decoupling as well as COSY experiments, shows that the C-2' substituent is

the C-8' methine-carbon. Strong NOE enhancement (8.6 %, corresponding to

40

ca. 2.6 Å) between H-6 (thymine) and H-3' in compound 48, in addition to 3JH1',H2' = 0 Hz, further confirms that the sugar is indeed locked in the North-

conformation and that the 1-thyminyl moiety is in the �-configuration with an

anti conformation across the glycoside bond. The NOE enhancement of 3.0

% for H-1' upon irradiation at CH3(C-8') proved that the CH3(C-8') group is

in close proximity of H-1', hence the C-8' chiral center is R-configured. The

vicinal coupling of H-2' with H-8' as evidenced by double decoupling

experiments and COSY spectra also unequivocally showed that the bicyclo

[3.2.1] octane ring system had indeed been formed in the ring-closure

reaction (Scheme 5). This conclusion was further corroborated by the

observation of a long range 1H-13C connectivity of H-8' with C-2', C-3', and

C-1', that of H-7' with C-2' and that of H-2' with CH3(C-8'), C-7', and C-8' in

an HMBC experiment.

3.2 Thermal denaturation studies of antisense oligonucleotides (AONs) The phosphoramidites 39a/39b and 50 were incorporated by mono-

substitution into a 15-mer DNA sequence through automated synthesis on an

Applied Biosystems 392 RNA/DNA synthesizer for further studies. The

stepwise coupling yields of the modified phosphoramidite were 96 % and 98

%, respectively. Dicyanoimidazole was used as the activating agent for

39a/39b, whereas tetrazole was used to activate 50 with 10 min coupling

time for modified phosphoramidites, followed by deprotection of all base-

labile protecting groups with 33 % aqueous ammonia at 55 °C to give AONs

1-17 (Table 2).

41

Table 2. Thermal denaturation of native and modified AONs in the duplexes with complementary RNA or DNA targets.a

AON Sequence Tm/°C with RNA

b�Tm Tm/°C with DNA

c�Tm

1. 3'-d(CTTCTTTTTTACTTC)-5' 44 45

2. 3'-d(CTT(LNA)CTTTTTTACTTC)-5' 48 +4 47 +2

3 3'-d(CTTCT T(LNA)TTTTACTTC)-5' 49 +5 46.5 +1.5

4 3'-d(CTTCTTTT(LNA)TTACTTC)-5' 49 +5 45.0 0.0

5 3'-d(CTTCTTTTTT(LNA)ACTTC)-5' 49 +5 46 +1

6 3'-d(CTT(5-carbo)CTTTTTTACTTC)-5' 47.5 +3.5 45 0.00

7 3'-d(CTTCTT(5-carbo)TTTTACTTC)-5' 49 +5 44 -1.00

8 3'-d(CTTCTTTT(5-carbo)TTACTTC)-5' 48 +4 44 -1.00

9 3'-d(CTTCTTTTTT(5-carbo)ACTTC)-5' 47.5 +3.5 43.0 -2.00

10 3'-d(CTT(6-carbo)CTTTTTTACTTC)-5' 45.5 +1.5 43.5 -1.5

11 3'-d(CTTCTT(6-carbo)TTTTACTTC)-5' 45.5 +1.5 39.5 -5.5

12 3'-d(CTTCTTTT(6-carbo)TTACTTC)-5' 45.5 +1.5 40.0 -5.0

13 3'-d(CTTCTTTTTT(6-carbo)ACTTC)-5' 45.5 +1.5 39.5 -5.5

14 3'-d(CTT(aza-ENA)CTTTTTTACTTC)-5' 48 +4 44.5 -0.5

15 3'-d(CTTCTT(aza-ENA)TTTTACTTC)-5' 46.5 +2.5 42.5 -2.5

16 3'-d(CTTCTTTT(aza-ENA)TTACTTC)-5' 47.5 +3.5 42 -3

17 3'-d(CTTCTTTTTT(aza-ENA)ACTTC)-5' 48 +4 42 -3

42

a Tm values measured as the maximum of the first derivative of the melting curve (A260 vs. temperature) and are average of at least three runs recorded in medium salt buffer (60 mM Tris-HCl at pH 7.5, 60 mM KCl, 0.8 mM MgCl2 and 2 mM DTT) with temperature range 20 to 70 oC using 1μM concentrations of the two complementary strands; b�Tm = Tm relative to RNA compliment; c�Tm = Tm relative to DNA compliment.

The sequence is targeted to the coding region of the SV40 large T-antigen

(TAg)165,166 and has been used in the study of antisense activity of (N)-

Methanocarba-T substituted oligonucleotide167 and as well as in the study of

antisense and nuclease stability assays of oxetane, 118 azetidine,119 and aza-

ENA117 modified oligonucleotides. Thermal denaturation studies of AONs

(Table 2) containing carbocyclic-LNA-T or carbocyclic-ENA-T showed an

increase in Tm of 3.5 to 5.0 °C/modification. (AONs 6-9 in Table 2) and 1.5

°C/modification (AONs 10-13 in Table 2), respectively, for the AONs

duplexes with complementary RNA. However, a net decrease of up to 5.5

°C/modification has been observed for the AON duplexes with

complementary DNA. The DNA/DNA duplex is known to have a shallow

minor groove and the presence of a water backbone all along the

oligonucleotide further contributes to the stability of the duplex90. A decrease

in the thermodynamic stability of a modified AON/DNA duplex may be due

to an increase in steric crowding in the shallow minor groove. Since the

DNA/RNA heteroduplex is known to have a wider but shallow minor groove,

it is evident that the exocyclic methyl is well tolerated in the modified

AON/RNA heteroduplex. The 6-membered carbocyclic-ENA-T counterpart

did not show any sequence dependent change in Tm (+1.5 °C/modification).

A higher decrease in the stability of the 6-membered carbocyclic-ENA-T

modified AONs with DNA further suggests that an additional hydrophobic

methylene linker introduces more steric crowding in the minor groove of the

duplex in addition to disrupting the extensive backbone water network.

However, since 5-membered carbocyclic-LNA-T was used as a

43

diastereomeric mixture of R- and S- isomers oriented differently in the minor

groove, we might be observing averaged steric effects. Further studies with

pure diastereomers are warranted in addition to X-ray crystallographic

evidence of hydration disruption by the exocylic methyls oriented differently

in the minor groove space.

3.3 Evaluation of target affinity and antisense properties of modified AONs

The stability of AONs towards nucleases is necessary in order to develop

any therapeutic oligonucleotides (antisense,108 RNAi,168, microRNA169,170 or

triplexing agents171). In order to study the nuclease stability, if any, of the

carbocyclic-LNA (37a/37b) and carbocyclic-ENA (48) modified AONs a

single modification was introduced at the 3' end and were digested in snake

venom phosphodiesterase (SVPDE) and human blood serum which mainly

comprises 3'-exonucleases. In order to study the effect of a hydrophobic 2'

modification a comparative nuclease stability assay was done with LNA114

and aza-ENA117 modified AONs as shown in the PAGE autoradiograms

(insets A, B, C and D in Figures 13 and 14).

44

Figure 13. Autoradiograms of 20 % denaturing PAGE showing degradation patterns of 5'-32P-labeled AONs in human blood serum (see Table 2 for AON sequences). Inset A: AON 1 and LNA-modified AONs. Inset B: Carbocyclic LNA-modified AONs, Inset C: Carbocyclic–ENA modified AONs, and Inset D: aza-ENA-modified AONs. Time points are 0 h, 0.5 h 1 h, 2 h, 5 h, 7 h, 9 h and 12 h after incubation with blood serum.

Investigation by using circular dichroism (CD) did not reveal any change

in the global structure of the hetero or homoduplex. Since only singly

modified oligonucleotides were used in the CD studies, we assumed that

there is little effect of the structural modification on the structure of the 15-

mer sequence and that there must be minor structural changes adjacent to the

modification which would be reflected in the enzyme digestion studies.

45

Figure 14. Autoradiograms of 20 % denaturing PAGE showing degradation pattern of 5�-32P-labeled AONs in SVPDE (see Table 2 for AON sequence). Inset A: AON 1 and LNA-modified AONs. Inset B: Carbocyclic-LNA modified AONs. Inset C: Carbocyclic-ENA modified AONs. Inset D: aza-ENA modified AONs. Time points are 0 h, 1 h, 2 h, 24 h, 48 h and 72 h after incubation with the enzyme.

The comparison of the modified AON stability in blood serum and

SVPDE, owing to various modifications, becomes very clear as we compare

the digestions of AONs 2, 6, 10 and 14 with T(LNA), T(5-carbo), T(5-carbo) and

T(aza-ENA) each having a specific single modification at the position 3 from the

3'-end: The site of 3'-exonuclease promoted hydrolysis was dictated by the

site of the incorporation of the modification in the AON (Figures 13 and 14).

The isosequential parent LNA modified AONs were found to be rapidly

digested within 30 min similarly to the native heteroduplex. On the other

hand, the 3'-terminal nucleotide is hydrolyzed by 3'-exonuclease (SVPDE

and human serum) in AON substituted by the 5- or 6-membered carbocyclic

residue (AONs 6 and 10) at position 3 to give only the n-1 fragment.

46

3.4 Probable mechanism for the nuclease stability of the modified AONs

The nuclease digestion studies with modified AONs show that a single

modification of AON with carbocyclic-LNA-T or with carbocyclic-ENA-T

nucleotides at position 3 from the 3'-end in AONs 6 and 10 has provided

unprecedented nuclease stability. It has been suggested that replacement of

substituents involved in natural enzyme-substrate complex results in poor

recognition and processing by the nucleolytic enzymes, thereby resulting in

the nuclease stability.172 It was also shown that the native ribonucleoside with

a 2'-O-alkyl substituent either by its bulk173 or by its stereoelectronic

modulation120,122,173,174of hydration can bring about nucleolytic stability. Egli

et al.175 have demonstrated that charge effects and hydration properties are

important factors in influencing the nuclease stability of AONs with a normal

phosphodiester backbone.

AONs containing 5-membered carbocyclic-LNA and 6-membered

carbocyclic-ENA show enhanced, but similar, blood serum stability, thereby

indicating that the steric bulk is relatively unimportant.173 This was in sharp

contrast to the conclusion drawn by comparison of the LNA versus ENA176

modified AONs, in that the latter was found to be 2.5-3 times more stable

than the former,164 wherein the extra methylene linker was suggested to be

the structural cause of observed stability.

Since the carbocyclic AONs were completely devoid of polar effect at C-

2',177 the explanations invoking charge or steric effects is not applicable to

explain the nuclease stability of the carbocyclic AONs in the blood serum.

The enhanced stabilities of the carbocyclic-AONs with respect to their

bicyclic 2'-O-(LNA and ENA) and aza-ENA analogs suggests that either the

accessibility of water or the active site of the enzyme to the 2'-O- in LNA or

to the 2'-N- in aza-ENA substituent in a modified nucleotide is probably more

important in order to cleave the vicinal 3'-phosphodiester bond by the

47

exonucleases of the blood serum. A calculation of the solvation energy for

various 2'-modified nucleosides suggests that the 5-membered and 6-

membered carbocyclic nucleosides, on account of their hydrophobic nature,

are not as well solvated as compared to their LNA, ENA and aza-ENA

counterparts, thereby showing that hydration around a scissile phosphate is

probably important for the nuclease promoted hydrolysis in carbocyclic

modified AONs devoid of charge effects.

3.5 RNase H digestion studies

In the antisense strategy, RNase H recruitment by the modified AON, with

long nuclease stability as exhibited by the carbocyclic modifications, and the

target RNA heteroduplexes is an important step in the design of potential

therapeutics for engineering specific gene silencing effects.108

Hence, RNase H mediated cleavage of the RNA strand complementary to

the modified AON strands (with carbocyclic-LNA, carbocyclic-ENA, aza-

ENA and LNA modifications) was studied, using the native AON/RNA

duplex as a reference, in order to address the issue if the recruitment of

RNase H by the heteroduplexes is in any way compromised as a result of

carbocyclic modifications incorporated in the AON strand. We used E. coli

RNase H in the studies because of its availability and the fact that its

properties are known to be similar to the mammalian enzyme.178 The RNA

complementary to AONs formed duplexes and were found to be good

substrates for RNase H but with varying cleavage sites (Figure 15),

depending on the site of modification in the AONs.

48

Figure 15. The RNase H1 cleavage pattern of AON/RNA heteroduplexes. Vertical arrows show the RNase H cleavage sites, with the relative length of the arrow showing the extent of the cleavage. The square boxes around a specific sequence shows the stretch of the RNA, which is resistant to RNase H cleavage.

49

4. Microwave-induced chalcogen extrusion reactions

The carbon-chalcogen bond is weakened as we traverse the chalcogen

group of the periodic table from top to bottom. Extrusion reactions resulting

in carbon-carbon bond formation are feasible but many of them require harsh

conditions such as flash vacuum pyrolysis used to induce formation of

benzocyclobutenes from dihydrobenzotellurophenes179 or pyrolysis to effect

deselenation180 and desulphurization181 during cyclophane synthesis.

Sometimes extrusion had to be assisted in order for carbon-carbon bonds to

form. For example, degassed Raney nickel was found to produce biaryls from

diaryl tellurides.182 We were curious to see if microwave-heating would

induce such chalcogen extrusion reactions. Indeed, we observed that certain

6- or 7-membered cyclic chalcogenoanhydrides when heated with

microwaves afforded diketones as a result of chalcogen extrusion

accompanied by carbon-carbon bond formation (Table 3). Thus, when

selenodiphenic anhydride (51) was heated in a sealed vial in chlorobenzene at

250 ºC for 2 h, phenanthroquinone (52) was quantitatively formed. Seleno-

and telluroanhydride starting materials were easily prepared from acid

chlorides and Na2Se and Na2Te generated in situ.183-185 Tellurodiphenic

anhydride decomposed to give phenanthroquinone already during attempted

recrystallization. Telluroglutaric anhydride (53) afforded 1,2-

cyclopentanedione (54) in 46 % yield when heated at 210 ºC in the

microwave cavity. However, we were unable to produce

benzocyclobutanedione from seleno- or tellurophthalic anhydride at 250 ºC

under similar reaction conditions. Our results are summarized in Table 3.

50

Table 3. Microwave-assisted chalcogen extrusion reactions.

Chalcogenohydride Reaction conditions Product Yield

Se

O

O 51

Microwaves, chlorobenzene,

250 ºC, 2 h O

O

52

100 %

M

O

O

M=Se, Te


250 ºC, 4 h

No reaction

SeO O


250 ºC, 4 h

No reaction

Te OO 53


210 ºC, 2 h OH

O

54

46 %

Attempts to extend the chalcogen extrusion methodology to the preparation

of biaryls and alkylated aromatics was met with little success. We therefore

turned the attention to metal catalyzed coupling reactions.

51

5. Palladium (0)-catalyzed Sonogashira cross-coupling of organic tellurides with alkynes (Publication III)

The rare element tellurium has a surprisingly rich organic chemistry. A

variety of organotelluriums with well-defined stereochemistry are available

and they are often stable enough to be taken through several synthetic

transformations. In addition to their application as radical precursors (section

1.4.1), organotelluriums have been extensively used in other carbon-carbon

bond forming reactions.186-189 Synthetically useful carbodetelluration was

demonstrated in the carbonylation of aryltellurium chlorides with Ni(CO)4.190

Carbodetelluration was also shown to occur when aryltellurium (IV)-

chlorides were reacted with olefins in the presence of a stoichiometric

amount of palladium.191 In the absence of olefins, biaryls were obtained in

good yields. In the presence of carbon monoxide, carbonylation occured to

yield aromatic acids in good yields using Pd(II) in stoichiometric amount.192

In 1988 Barton193 reported homocoupling of diaryl and alkyl aryl tellurides in

the presence of a stoichiometric amount of Pd(OAc)2. Diaryl ditellurides

which are the precursors to alkyl aryl and diaryl tellurides were also shown to

undergo homocoupling. Until now, most of the palladium assisted

carbodetelluration reactions occured with stoichiometric amounts of rather

expensive palladium reagents. It was not until 1996 that Uemura reported

catalysis of palladium in cross-coupling reactions.194,195 Under Fujiwara-Heck

conditions in the presence of a suitable oxidant, aryl tellurides were coupled

with alkenes to give aryl alkenes in good yields. Whereas diaryl tellurides did

not form any homocoupling products, alkenyl tellurides did react in the

52

presence of a catalytic amount of palladium to provide 1,3-dienes. The cross-

coupling of organic tellurides using catalytic palladium has been extensively

used in Stille,196 Heck,197 Negishi,198 Suzuki,199 and Sonogashira type

reactions of vinylic,197,200,201 and heteroatomic202,203 tellurides. Recently

Pd(0)-catalyzed coupling stimulated by ultrasound was successful for biaryl

synthesis using butyl aryl tellurides as starting materials.204 In our hands this

reaction did not proceed as described. However, from the previous literature

it is obvious that the success of these coupling reactions depends rather

critically on the choice of palladium catalyst, base, solvent and additives. We

therefore decided to find out more about the suitability of diaryl and alkyl

aryl tellurides as coupling partners in transition metal-catalyzed reactions.

Te

(i) +55

56 570%44%

Scheme 6. Reagents and conditions: (i) Pd(OAc)2 (2.0 eq.), Et3N (2.0 eq.), CH3CN, reflux overnight.

When butyl 2-naphthyl telluride (55) was allowed to react (Scheme 6) with a

2-fold molar excess of Pd(OAc)2, 2,2'-binaphthyl (56) was isolated in low (44

%) yield and none of the homocoupling product 57 was formed. This is in

contrast to the early Barton193 paper which claims formation of such products.

When diaryl tellurides were tried under these conditions, biaryls were formed

in similarly low yields (e.g. bis(4-methoxyphenyl) telluride gave 4, 4'-

dimethoxybiphenyl in 28 % yield). Catalytic reactions carried out in the

presence of an oxidant (CuCl2) did not produce any of the desired coupling

products. Rather, the organotellurium compounds were oxidised to the

corresponding tellurium (IV)-species which could be reduced to regenerate

the starting materials. We therefore turned to Sonogashira type cross-

coupling reactions205 which are known to proceed in a catalytic fashion in the

absence of an oxidant.

53

5.1 Synthesis Alkyl aryl and diaryl telluride starting materials were prepared by standard

methods206-208 from the corresponding diaryl ditellurides. Thus, diphenyl

ditelluride (58) 209 was reduced with NaBH4 with careful exclusion of air and

n-butyl bromide was added to produce butyl phenyl telluride (59) in 97 %

yield. In another experiment, diphenyl ditelluride (58) was refluxed with Cu

powder in benzene for 12 h to produce diphenyl telluride (61) in 95 % yield.

TeTe

Tei ii

iii

Te iv

59

61

95 %

68 %

36 %

97 %

58 60

60 Scheme 7. Reagents and conditions: (i) NaBH4, n-butyl bromide, EtOH; (ii) 10 mole % Pd(PPh3)4, 1.5 equivalents of phenylacetylene and CuI, NEt3, reflux, 8 h; (iii) C6H6, Cu powder, reflux, 12 h; (iv) As in (ii) but 2.0 equivalents each of phenylacetylene and CuI were used.

For Sonogashira-type reactions it was found that heating at reflux with 10

mole-% of Pd(PPh3)4, CuI (1.5-2.0 eq) and phenylacetylene (1.5-2.0 eq) with

NEt3 as solvent produced fair to good yields of diarylacetylene coupling

products. Ideally, diaryl tellurides would give rise to two equivalents of

coupling products for every equivalent of telluride used. However, judging

from the low yields of diphenylacetylenes (Table 4) isolated when diphenyl

telluride (61) and other diaryl tellurides were used as coupling partners, only

one of the aryls seems to be transferable. The fate of the remaining aryl group

is as yet unclear. n-Butyl phenyl telluride (59) produced diphenyl acetylene

(60) in 68 % yield as shown in Table 4. Other n-butyl aryl tellurides also

furnished coupling products in fair yields (Table 4). It would therefore seem

more atom economic to use alkyl aryl tellurides as coupling partners rather

than diaryl tellurides. It remains to be seen if the butyl group that we have

54

used so far is optional or if a smaller alkyl such as methyl or ethyl would be

an even better choice. Table 4. Sonogashira cross-coupling of organic tellurides.

Telluride Alkyne Product Yielda

Te

68 %b

Te

36 %c

Te

67 %

Te

35 %c

Te

H3CO H3CO

78 %

Te

OCH3H3CO

H3CO

40 %c

Te

Me2N NMe2

Me2N

19 %c

a Isolated yield; bYield determined by 1H NMR using an internal standard; cYield calculated on the basis that 1.0 mmol of reactant would give 2.0 mmol of product. Two equivalents each of phenylacetylene and CuI were used.

55

6. Oxime ethers in 5-hexenyl radical cyclizations

5-Hexenyl radical cyclization in oxime ethers has been used for

construction of lactones,210 carbocyclic amine derivatives211 and

pyrrolidinones (Figure 16).210 The approach to construct pyrrolidinones from

amino acid precursors has, to the best of our knowledge, not been tried using

radical cyclization chemistry.

N

NOBn

O N

NHOBn

O

EtEt3B (8 eq.)toluene, ref lux

OHOH

47%

Figure 16. Tandem radical addition-cyclization to oxime ethers for pyrrolidinone synthesis.

6.1 Attempted synthesis of a pyrrolidinone

Our approach, exemplified with the commercially available phenylalaninol

(62), is shown in Scheme 8. TBDMS protection of the hydroxyl group in

pyridine afforded 63 in 98 % yield. Coupling with the O-benzyl oxime of

glyoxylic acid212 with dicyclohexylcarbodiimide afforded amide 64 with an

appropriately positioned double bond for 5-exo radical cyclization (70 %

yield). Deprotection with tetrabutylammonium fluoride gave alcohol 65 in 96

% yield. Attempted thiocarbamoylation of the alcohol with

thiocarbonyldiimidazole was unsuccessful in our hands. However, treatment

56

with O-phenylchlorothiocarbonate afforded the desired radical precursor 66

in moderate yield (62 %).

NH2

HO

NH2

OTBDMS

NH

NTBDMSO

O

OBn

NH

NHO

O

OBn

NH

NO

O

OBnSPhO

NH

O

NHOBn

i ii

iii iv v

O

HO NOBn

63 64

65 66

62

98% 70%

96% 62%

Scheme 8. Reagents and conditions: (i) TBDMSCl, DMAP, pyridine, overnight, (ii) DCC, DCM, overnight, (iii) TBAF, THF, 4 h, (iv) O-phenylchlorothiocarbonate, DMAP, pyridine, overnight, (v) TTMSS, AIBN, toluene, reflux. Radical cyclization was tried with tris(trimethylsilyl)silane and AIBN

initiation in refluxing toluene. The reaction product turned out to be a

complex mixture from which it was not possible to identify any cyclized

material.

57

7. Piperidines from amino acids via radical cyclization

The piperidine ring is an important structural motif in natural products and

synthetic pharmaceuticals and hence there is a need for development of

stereocontrolled methodologies for piperidine synthesis.213 The problem of

diastereocontrol in radical reactions has received considerable attention.214

Several excellent reviews215-220 are available on the topic including the radical

cyclization approch.87,221-228

Taking clue from previous work229,230 in our group (Scheme 9) with 2-

substituted 3-aza-5-hexenyl radicals, wherein it was demonstrated that bulky

N-substituents direct cyclization to occur preferentially with formation of cis-

2,4-disubstituted pyrrolidines, we set out to investigate the effect of the N-

substituent in cyclizations of 2-substituted 3-aza-6-heptenyl radicals.

N

PhSe

R

P=H

P= Ph2PO

(i)

(i)

NR

PNR

P

NR

PNR

P

P

trans:cis= 3.3:1

trans:cis= 1:10

Scheme 9. Control of diastereoselectivity by the N-substituent during radical cyclization. Reagents and conditions (i) n-Bu3SnH, AIBN, C6H6, h� (15 °C)

58

7.1 Attempted synthesis of piperidines The initial plan for radical precursor preparation involved homoallylation of

an appropriate amine with 4-bromo-1-butene in analogy with previous

work.229,230 However, we found alkylation difficult, probably due to the low

reactivity of homoallyl bromide and the presence of a bulky �-substituent in

the amino acid derived amines. We were able to circumvent the problem as

shown in Scheme 10.

HN

R O

OMe

Bt

NH

R

HO

NH

R

O OMe

N

RNH

R

PhSe

NR

PhSe

R

67a, R= Ph

i ii

iv

iii

v

67b, R= Bn68a, R= Ph, (57 %)68b, R= Bn, (62 %)

69a, R= Ph, (85 %)69b, R= Bn, (61 %)

70a, R= Ph, (80 %)70b, R= Bn, (85 %)

71a, R= Ph, (73 %)71b, R= Bn, (70 %)

72a, R= Ph, (97 %)72b, R= Bn, (0 %)

Scheme 10. Reagents and conditions: (i) Allyltrimethylsilane, BF3-etherate, THF, (ii) NaBH4, EtOH, reflux, (iii) PPh3, DEAD, DCM, (iv) PhSeSePh, NaBH4, TFA, EtOH, (v) Diphenylphosphinoyl chloride, DMAP, NEt3. Bt= Benzotriazolyl. Starting materials were previously prepared amino acid esters 67a and

67b,231,232 where the amine had been alkylated with

formaldehyde/benzotriazole (BtH), and an allyl group installed by treatment

with allyltrimethylsilane/BF3-etherate giving 68a and 68b. Reduction with

NaBH4 or LiAlH4 afforded the corresponding alcohols 69a and 69b which

under Mitsunobu conditions (DEAD and PPh3) provided aziridines 70a and

70b in 80 % and 85 % yields, respectively. Ring-opening with

59

benzeneselenol gave the radical precursors 71a and 71b in 73 % and 70 %

yields, respectively.

Radical cyclization of these compounds with TTMSS and AIBN-initiation

gave complex mixtures of reduced and cyclized products.

Introduction of a bulky diphenylphosphinoyl group before cyclization was

also attempted. However, whereas N-phosphinoylation of phenylalanine

derived radical precursor 71b failed under standard conditions

(diphenylphosphinoyl chloride, DMAP, Et3N), the corresponding

phenylglycine derivative 71a was readily phosphinoylated to give 72a (97 %

yield).

TTMSS-mediated cyclization of 72a resulted in a complex mixture of

products. Cyclization was also tried with Bu3SnH/AIBN. TFA treatment to

remove the diphenylphosphinoyl group, followed by basic workup, yielded

3-butenyl 2-phenylethylamine (73), as the only product (92 %). This material

has probably formed via a neophyl rearrangement233 (Scheme 11).

N

PhSei, ii,iii

72a 73R

N N N

NH

Bu3SnH

R R R Scheme 11. Formation of amine 73 via a neophyl rearrangement. Reagents and conditions: (i) Bu3SnH, AIBN, toluene, reflux, (ii) TFA, reflux, (iii) NaOH (aq). Obviously, 6-heptenyl cyclization onto an unactivated double bond does not

60

occur rapidly enough for the reaction to be synthetically useful for piperidine

synthesis. Instead, other processes such as reduction and rearrangement

reactions will be responsible for product formation.

61

8. Summary in Swedish

Som forskare inom området organisk kemi är man inte bara intresserad av att

studera hur atomer och molekyler reagerar utan även hur

reaktionsprodukterna kan användas för att förbättra livskvaliteten för

människor på olika sätt. Traditionellt sett framställs nya kol-kol bindningar

genom reaktioner mellan reagens som har utpräglat positiv (katjonisk)

karaktär och sådana som har en utpräglat negativ (anjonisk) karaktär.

Därutöver finns emellertid även reaktioner där reagensen saknar dessa

utpräglade negativa eller positiva laddningar. Här kan fria radikaler nämnas

som ett exempel. Radikaler är intressanta både ur ett kemiskt och biologiskt

perspektiv. Radikalreaktioner kan genomföras vid mycket låga temperaturer.

De sker på samma sätt oberoende av vilket lösningsmedel man väljer. På

senare tid har man utvecklat radikalreaktioner som uppvisar hög selektivitet

både vad gäller regiokemi och stereokemi. Detta har bidragit till att radikal-

medierade reaktioner numer ingår i den organiske kemistens vertygslåda för

framställning av nya föreningar.

Grundämnena selen och tellur brukar ofta förknippas med giftighet och

illaluktande organiska föreningar. Den organiska kemin för dessa två ämnen

är dock förvånansvärt rik och en mycket stor mängd selen- och

tellurorganiska föreningar finns beskrivna i litteraturen. I denna avhandling

har vi omvandlat lätttillgängliga selenorganiska föreningar på ett sådant sätt

att den radikal som bildas vid homolytisk klyvning av kol-selen bindningen

undergår en cykliseringsreaktion och ger upphov till kväveheterocykliska

föreningar – pyrroliner (Figur 9). Pyrroliner påträffas naturligt som

biosyntetiska mellanprodukter och som en del av strukturen hos feromoner,

62

alkaloider, hem- och klorofyllföreningar samt diverse marina toxiner. Dessa

kväveinnehållande molekyler är p.g.a. sin biologiska aktivitet av stort

intresse.

N

Figur 17. En substituerad pyrrolin.

Vi har även försökt skapa nya kol-kol bindningar från lättillgängliga selen-

och tellurorganiska föreningar genom att hetta upp dem m.h.a. mikrovågor.

Det har lyckats ibland att termiskt extrudera heteroatomen men metodiken

kan inte anses generellt användbar. Vi har därför även undersökt

möjligheterna att katalysera processen med hjälp av övergångsmetaller. Vi

har sålunda beskrivit reaktionsbetingelser där den aromatiska delen av

diaryltellurider och alkyl aryl tellurider kan kopplas till acetylener i närvaro

av katalytisk mängd av Pd(PPh3)4 (Sonogashirareaktionen).

Under det senaste årtiondet har man försökt hitta nya tillvägagångssätt för

behandling sjukdomar. ”Antisensterapi”, en metod baserad på den

biofysikaliska kemin hos DNA och RNA, har rönt stor uppmärksamhet där-

OO N

NH

O

O

CH3O

OO N

NH

O

O

OCH3

OO N

NH

O

O

OO

OO N

NH

O

O

O O

A B C D Figur 18. Nukleosidanaloger med intressanta biofysikaliska egenskaper. (A) Locked nucleic acid (LNA); (B) Etylene bridged nucleic acid (ENA); (C) Karbocyklisk LNA. (D) Karbocyklisk ENA. Strukturerna C och D är nyligen framtagna och diskuteras i denna avhandling.

63

vidlag, även om de terapeutiska tillämpningarna förmodligen ligger en bit

fram i tiden. I denna avhandling beskriver vi syntes av två nya

nukleosidanaloger (C och D; Figur 18) som har visat intressanta egenskaper

både vad gäller stabilitet och aktivitet. Nyckelsteget i syntesen av de båda

nya nukleosidanalogerna är en radikalcyklisering. De nya analogerna C och

D utvärderades med avseende på stabilitet och specificitet i jämförelse med

de redan kända strukturerna A och B. Den förlängda livslängden av dessa

karbocykliska analoger C och D i blodserum är en intressant farmakologisk

egenskap som borde kunna medföra att de kan doseras i mindre mängd.

Förhoppningsvis kan vår forskning bidra till att påskynda utvecklingen av

nya antisens- eller RNAi-terapeutiska läkemedel.

64

Acknowledgements

In the words written below I take the opportunity to thank everybody who

stood by me and supported me in this journey. I am already at loss of

words.‘Words’ mean much more than they say.

I would like to express my sincere gratitude to my supervisor Professor Lars

Engman for agreeing to be my supervisor, for good science and above all for

treating me like a human being.

Professor Adolf Gogoll, you were my pillar of support during these years. I

think I ran to you everytime I felt like giving up and had nowhere to go. A

big reason why I could reach the brighter side of the tunnel is YOU. I am

forever indebted.

Professor Lars Balzer for helping me out and showing me a different world.

Professor Helena Grenneberg for moral support and an ever smiling face.

Professor Joseph Samec for tolerating me and my smelly compounds. I wish

I had an iota of enthusiasm that you have.

I also wish to thank Prof. Henrik Ottoson, Prof. Olle Mattson and Prof. Pher

G. Anderson for being kind to me.

Thanks to Prof. Jyoti Chattopadhyaya and department of biorganic

chemistry for a fascinating chemistry.

Special thanks to Dr. Per Lowdin for excellent support and help. I am

indebted.

I would like to express my gratitute to department secretaries, Eva Pylvänen

and Johanna Johansson for helping me out with everything in the department.

65

Henrik Johansson the lab king. You have been very kind to me and

tolerated all my sayings and all that blah blah blah…, It seems I had a saying

for every occasion. I wish all the best for your future. Be happy.

Bobo for all the help and friendly disposition. Your sincerity, deligence and

calm are your virtues. Keep up the good spirit. Lycka till.

Jonas Rydfjord for being a friendly, honest and a ‘cool coworker’. For all the

help, encouraging discussions and for a memorable movie night with kids.

Your Dalarna souvenir will be cherished forever. I wish you a bright and

happy future ahead. Lycka till.

Zeyed Abdulkarim, the hardworking late night guy. For letting a sur gubbe

accompany the kids to a nice movie. I wish you a bright and happy future

ahead. Lycka till

Supaporn Sawadjoon for giving me nice and peaceful company. You are a

hard and sincere worker. I wish that your hard-work is rewarded. Good luck.

Sawadee

Dr. Khadijeh Bakhtiari the Dokhtar for being a such a good soul, for Hafiz,

Sadi, kalle pacche, kufteh-tabrizi, Shiraz, Isfahan, NMR, tellurium, fresh air,

coffee……the list continues. I wish happiness for you in future.

Anwar Merzae the Peshmerga birader for tolerating me and for being a nice

coworker also for cakes, chips……fresh air…...the list continues. I wish

good luck for your future.

I wish to thank Adam, Andreas, Alexander, Alvi, Christian, Diana, Johan,

Julius, Rikard, Sarah and special thanks to my NMR teacher Claes Henrik. I

thanks all other people from the department whose name I have missed, for

being friendly and kind to me.

I take this opportunity to express my love to my family in Uppsala.

Smita, Nimesh and Adwait; Pratima, Radha Raman and Prashant; Julie,

Varghese and Evleyn; Madhuri, Pradeep and Mayank; Navya and Sandeep,

Fatima, Faizan and Zidan; Kali and Sulena. The ‘bacchas’ in the family

66

Gaurav, Tanmoy and Prasoon. Special thanks to Jitendra the mamu of

‘bacchas’.

I will not be able to pen down my emotions here for each of you. You

have tolerated me over the years, assuaged my pain and insecurities and

above all changed me by providing so much of affection. I wish a happy and

fulfilling life to all the members.

Special Thanks to Jharna, the angry girl for all the discussions, food and

caring attitude.

To Subhrangshu for all the help and caring for me even when you are far

away. To Wimal for a great collaboration and his wife Ayesha for great food

and family atmosphere.

To my world, my mummy (this event came too late for you) and papa and my

sisters Nutan, Ushma and Mili for all the little amount of happiness that is

destined for me. To Madhukarjijaji, Ashwani Jijaji and Rahul for love

affection and care. Love to my kids Kislaya, Ananya, Arnav and Dhruv.

67

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