the assignment of absolute configuration by nmr · · 2012-03-12optical methods (e.g., circular...

The Assignment of Absolute Configuration by NMR†

Jose Manuel Seco, Emilio Quinoa, and Ricardo Riguera*

Departamento de Quımica Organica and Unidad de RMN de Biomoleculas Asociada al CSIC, Universidad de Santiago de Compostela,E-15782, Santiago de Compostela, Spain

Received November 18, 2002

Contents1. Introduction and Scope 172. Methods Based on Chiral Derivatizing Reagents

(CDAs): General Characteristics19

2.1. Description of the Method 192.2. Characteristics of the Auxiliary Reagents 202.3. The NMR Spectra of the Derivatives 202.4. Importance of the Conformational Equilibrium 212.5. Substrates and Derivatives 22

3. Methods Based on Double Derivatization 223.1. Application to R-Chiral Secondary Alcohols 22

3.1.1. Methoxytrifluoromethylphenylacetic Acid(MTPA): Mosher’s Reagent

22

3.1.2. Methoxyphenylacetic Acid (MPA) 333.1.3. 9-Anthrylmethoxyacetic Acid (9-AMA) and

Related AMAAs40

3.1.4. Other Reagents 453.2. Application to â-Chiral Primary Alcohols 57

3.2.1. MTPA 583.2.2. 9-AMA 61

3.3. Application to R-Chiral Tertiary Alcohols 633.3.1. MTPA 633.3.2. Methoxy-(2-naphthyl)acetic Acid (2-NMA) 633.3.3. The Fucofuranoside Method 64

3.4. Application to R-Chiral Primary Amines 653.4.1. MTPA 653.4.2. MPA 683.4.3. AMAAs 703.4.4. Boc-phenylglycine (BPG) 703.4.5. Other Reagents 74

3.5. Application to Secondary Amines: MTPA 773.6. Application to R-Chiral Carboxylic Acids 79

3.6.1. Ethyl 2-(9-anthryl)-2-hydroxyacetate(9-AHA) and Related Aryl Alcohols

80

3.6.2. Arylcyclohexanols 823.6.3. (S)-Methyl Mandelate. 843.6.4. Phenylglycine Dimethyl Amide (PGDA)

and Phenylglycine Methyl Ester (PGME)85

3.6.5. 1,1′-Binaphthalene-8,8′-diol (BNDO) 883.7. Application to â-Chiral Carboxylic Acids 89

3.7.1. Arylethylamines 893.7.2. PGME 90

3.8. Application to Chiral Sulfoxides: MPA 92

3.9. Application to Chiral PolyhydroxylatedCompounds

92

3.9.1. MPA and 9-AMA 943.9.2. MTPA 96

4. Methods Based on a Single Derivatization 994.1. Application to R-Chiral Secondary Alcohols 101

4.1.1. 9-AMA Esters: Esterification Shifts 1014.1.2. Glycosidation Shifts 1034.1.3. Low-Temperature NMR of MPA Esters 1054.1.4. Complexation of MPA Esters with

Barium(II)107

4.2. Application to R-Chiral Primary Amines:Complexation of MPA Amides with Barium(II)

108

5. New Trends 1095.1. Hyphenated High-Pressure Liquid

Chromatography (HPLC) NMR−MSTechniques for Automatization andMicroscale Analysis

109

5.2. Resin-Bound Auxiliaries: The “Mix andShake” Method

111

6. Concluding Remarks: A Critical Assessment 1137. Acknowledgment 1148. References 114

1. Introduction and ScopeThe interest in determining the absolute stereo-

chemistry of a chiral organic compound stems fromthe widely recognized fact that the stereochemistryoften determines important properties in the chemi-cal, physical, biological, and pharmaceutical aspectsof the compounds. The need to obtain enantiomeri-cally pure pharmaceuticals and chemicals generallyhas produced a fantastic growth of fields such asasymmetric synthesis, asymmetric catalysis, andother processes where the availability of simple andreliable methods for the determination of enantio-meric purity and absolute configuration is a must.

Several instrumental methods exist for the deter-mination of absolute configuration. The most widelyknown are X-ray crystallography, followed by chiro-optical methods (e.g., circular dichroism1a (CD), opti-cal rotatory dispersion (ORD), or specific opticalrotation); however, their use is not devoid of someinconveniences and limitations related to the equip-ment, which is very specific to the method andrequires special training for operation, and to thesample, which, in the case of X-ray diffraction (XRD),requires monocrystals of good quality. Other methodsinclude specific rotation,1b infrared (IR) vibrationalCD,1c and vibrational Raman optical activity.1d

* Author to whom correspondence should be addressed. E-mail:[email protected].† R.R. dedicates this review to the memory of his beloved wife M.Carmen.

17Chem. Rev. 2004, 104, 17−117

10.1021/cr000665j CCC: $48.50 © 2004 American Chemical SocietyPublished on Web 01/14/2004

Recently, different approaches to the problem ofdetermining absolute configuration have emerged,based on NMR spectroscopy. These techniques arevery appealing, because of the undoubted advan-tages, which include the following: (a) the instru-ment is available in most laboratories; (b) an in-depthunderstanding of the fundamentals of the method isnot necessary to apply this method; (c) a smallamount of sample is needed, and this can be recov-ered; and (d) because the analysis is conducted insolution, it is applicable to both solid and liquidsamples.

Two general approaches are known. The firstapproach involves those procedures where derivati-

zation of the substrate whose absolute configurationis studied is not necessary. The sample (i.e., a pureenantiomer) is analyzed by NMR in a chiral environ-ment that is provided by a chiral solvent or by theaddition of a chiral solvating agent2 (CSA) to anonchiral standard NMR solvent. In this approach,there is no covalent linkage between the substrateand the chiral “reagent”, and this advantage is theorigin of its main limitation: The chiral environmentproduces very small differences in chemical shifts forthe two enantiomers whose NMR are very similar;many times, the two enantiomers must be availablefor comparison and no clear-cut correlations betweenthe absolute configuration and the NMR spectra canbe established. For those reasons, the usefulness ofthis method is practically restricted to the determi-nation of enantiomeric purity.3

A clear distintinction between those two objectivesshould be established at this point. For the enantio-meric purity measurement, we usually work with amixture of both enantiomers in unknown ratio andjust need to obtain separate signals in NMR (or peaksin high-pressure liquid chromatography (HPLC), gas-liquid chromatography (GLC), or similar techniques)that are amenable to quantification. Determinationof the absolute configuration is a much more difficulttask, because it is performed on a single enantiomerand, usually, we have no other enatiomer for com-parison; in addition, even if we had both and couldproduce different spectra, we must have criteria toassign the absolute configuration separately for eachenantiomer, not just its ratio.

The second approach involves derivatization of thesubstrate (i.e., a pure enantiomer) with the two

Jose Manuel Seco was born in Dodro (La Coruna, Spain) in 1968. Hereceived his B.Sc. (1991), M. Sc. (1992), and Ph.D. (1997) degrees inChemistry from the University of Santiago de Compostela, the latter underthe supervision of Profs. Ricardo Riguera and Emilio Quinoa. He wasawarded with the Ph.D. Extraordinary Prize in 1998. In 2001, he becameAssistant Professor in Organic Chemistry. His current research interestis focused in the development of new reagents and methods for thedetermination of absolute stereochemistry of organic compounds by NMR.

Emilio Quinoa, a native of Lugo (Spain), obtained his Ph.D. degree (1985)in Chemistry from the University of Santiago de Compostela, for work onmarine natural products from the Galician Coast, under the supervisionof Prof. Ricardo Riguera. From 1985 to 1988, he worked as a postdoctoralfellow with Prof. Phillip Crews at the University of California, Santa Cruz,where he participated in the study of bioactive metabolites from marineorganisms of tropical waters. He returned to Santa Cruz in 1989 and1990 as Visiting Associate Professor. After his return to the University ofSantiago, he became Associate Professor in Organic Chemistry in 1989and Full Professor in 1998. His current research interests include marinenatural products, solid-phase synthesis, and the stereochemistry of organiccompounds. Among other recognition, he has received the Real AcademiaGallega de Ciencias Award.

Professor Ricardo Riguera was born in Lugo (Spain) in 1948. He receivedits M. S. degree in Chemistry from the University of Santiago deCompostela in 1971 and the Ph.D. degree in 1973 under the supervisionof the late Prof. I. Ribas. From 1974 to 1976, he stayed at the UniversityCollege London as a postdoctoral fellow under the supervision of Prof.Peter J. Garratt. After returning to the University of Santiago deCompostela, he was appointed Lecturer of Organic Chemistry in 1978and became Full Professor in 1990. As academic, he has served theuniversity as Chairman of the Department, Dean of the Faculty ofChemistry, and Vice-Chancellor. He also has authored textbooks forstudents. His research interests have involved the fields of bioactive naturalproducts from terrestrial and marine organisms, in regard to new syntheticmethods and some aspects of medicinal chemistry. In the past few years,he has focused on the development of methods for determination of theabsolute configuration by NMR.

18 Chemical Reviews, 2004, Vol. 104, No. 1 Seco et al.

enantiomers of a chiral derivatizing agent4 (CDA),producing two diasteromeric derivatives. In this case,the chiral environment is provided by the auxiliaryreagent; the association with the substrate is covalentand leads to much-greater differences in chemicalshifts than those obtained when CSAs are used.Although, in certain cases, a combination of the twoapproaches has been used5 (i.e., the addition oflanthanide shift reagents to the CDA derivatives), thederivatization with chiral auxiliary reagents is, byfar, the method of choice for the assignment ofabsolute configuration by NMR and constitutes themain object of this review.

Many efforts to develop CDAs that are useful toassign the absolute configuration of different sub-strates have been described;6 however, since itsimplementation in 1973, the so-known “Mosher’smethod”, which uses MTPA (methoxytrifluorphenyl-acetic acid) as the reagent in its original description,has been the most successful, giving way in itsevolution to many new and more-efficient reagentsthat are useful for different substrates and are basedon the same principle.

The importance of this method can be evaluatedfrom the ever-increasing number of papers where itis mentioned; however, in contrast to the determi-nation of enantiomeric purity by NMR, which hasbeen widely covered in the literature,3 exhaustivereviews of this topic are absent. This absence of acomprehensive coverage means that the researcheroften uses inadequate reagents for the problem beingaddressed or performs a nonrigorous evaluation ofthe experimental data, making the assignment ofabsolute configuration a risky exercise.

In this review, we will cover the principles andfoundations of the use of CDAs and the practicalaspects that are most relevant for its application tothe different substrates. It is intended that thisinformation will be of particular use to researcherswho are not necessarily specialists in this field, andthe purpose of this study is to provide a critical viewof the methods available.

2. Methods Based on Chiral DerivatizingReagents (CDAs): General Characteristics

2.1. Description of the MethodAll the NMR-based methods for the determination

of absolute configuration require the transformationof the chiral substrate to two different species thatcan be differentiated by NMR spectroscopy (i.e.,diastereoisomers or conformers). This is achieved byderivatization of the substrate with the CDA, fol-lowed by comparison of the 1H NMR spectra of thosetwo species. Two main procedures exist, dependingon the number of derivates that should be prepared:(a) those in which the substrate (a pure enantiomer)is separately derivatized with the two enantiomersof the chiral auxiliary (Figure 1) and (b) those thatrequire the preparation of a single derivative, usingone enantiomer of a chiral auxiliary only (Figure 2).

In the first case, the chiral substrate [(?)-A] iscoupled separately with the two enantiomers of thechiral auxiliary ((R)- and (S)-CDA), and the NMRspectra of the resulting diastereoisomers ((?)-A-(R)-CDA and (?)-A-(S)-CDA) are compared.

In the second possibility, the chiral substrate ((?)-A) is combined with only one enantiomer of the chiralauxiliary (either (R)- or (S)-CDA). In this way, onediastereoisomer is obtained and the spectrum of thiscompound is recorded. A certain conformationalchange is then induced (e.g., by a change in temper-ature of the probe or by formation of a complex) anda second spectrum is taken (Figure 2). Analysis ofthe differences and comparison with the conforma-tional changes allows the configuration of the sub-strate to be correlated with that of the reagent.Another option that does not require the aforemen-tioned conformational change relies simply on com-parison of the spectrum of the derivative with thatof the substrate prior to derivatization.

Regardless of the chosen procedure, two differentspectra always must be obtained for comparison. Theassignment of configuration is based on the use ofthe NMR to correlate the absolute stereochemistry

Figure 1.

Assignment of Absolute Configuration by NMR Chemical Reviews, 2004, Vol. 104, No. 1 19

of the chiral center of the auxiliary (CR; configurationknown) with that of the substrate (C(1′), configura-tion unknown). To this end, the changes in thechemical shifts of the substituents of the asymmetriccarbon of the substrate (L1 and L2) in the twoderivatives (or conformational species) are consid-ered.

These differences in the chemical shifts are repre-sented by ∆δ, and it is the sign of this parameter(+ or -) that provides information about the config-uration. For a particular substituent (e.g., L1), ∆δ isdefined as the difference of chemical shifts of a givensignal of the substituent (δL1) in the two spectraunder consideration (diastereoisomers or conformers,depending on the routine chosen). As will be de-scribed in this paper, there is a variety of ways toexpress the differences in chemical shifts, theirsignificance, and their characteristics. For example,in Mosher’s method, a chiral secondary alcohol (i.e.,2-pentanol; see Figure 3) is derivatized with the twoenantiomers of an arylmethoxyacetic acid (AMAA)i.e., (R)- and (S)-R-methoxy-R-phenylacetic acid (MPA),the 1H NMR spectra of the two derivatives arerecorded, and the ∆δRS value is calculated for thesubstituents L1 (∆δRSL1) and L2 (∆δRSL2). In eachcase, ∆δRS is the difference between the chemicalshift in the (R)-MPA ester derivative (δ(R)) and inthe (S)-MPA derivative [δ(S)] (see Figure 3).

2.2. Characteristics of the Auxiliary ReagentsIn general, the structure of the chiral auxiliary

reagent (B) must incorporate groups with specificfunctions:

(a) A polar or bulky group (R1) to fix a particularconformation.

(b) A functional group (Z) (e.g., carboxylic acid) thatprovides a site for covalent attachment of the sub-strate.

(c) A group (Y) that is able to produce an efficientand space-oriented anisotropic effect (e.g., aromatic,carbonyl) that selectively affects substituents L1 andL2 of the substrate. This shielding/deshielding will

ensure that the chemical shifts of L1 and L2 aredifferent in the two species (diastereoisomers orconformers) that are used for the determination ofthe conformation.

For example, in the case of MPA, R1 is a methoxygroup, Z is a carboxylic acid, and Y is a phenyl group(Figure 4).

2.3. The NMR Spectra of the Derivatives

As we have said, comparison of the NMR spectraof the two derivatives and evaluation of the differ-ences in the chemical shifts of the signals for thesubstituents L1 and L2 (i.e., ∆δRS) must be performed.The signs of those parameters contain the informa-tion necessary for the configurational assignment,because they indicate the relative position of L1/L2,with respect to the anisotropic group Y; therefore, theobserved differences must not be uncertain and mustbe clearly predictable.

In the methods that are based on the formation oftwo derivatives, it is important that both diastereo-isomers show a certain preference for a particularconformation (NMR-significant conformer) indepen-dent of the particular substrate, and the Y groupmust project its magnetic anisotropy in a selectiveway, i.e., on L1 in one derivative and on L2 in theother.

For example, in the MPA esters (Figure 3), it isassumed that the representative conformer in termsof NMR is the one in which the methoxy, carbonyl,and C(1′)H groups are situated in the same plane.In this way, in the (R)-MPA derivative, the phenylgroup shields L1 (-CH2CH2CH3), whereas, in the (S)-MPA derivative, the shielded group is L2 (-CH3). Thecomparison of both spectra leads to ∆δRSL1 < 0 and∆δRSL2 > 0 (-0.14, -0.23, -0.09, and +0.013 ppm,respectively).

If the absolute configuration of the alcohol were theopposite, the signs of ∆δRS will also be reversed (i.e.,∆δRSL1 > 0, ∆δRSL2 < 0).

Figure 2.


2.4. Importance of the ConformationalEquilibrium

Naturally, the key for the success of these methodslies in the assumption that a certain conformer is themost representative in both derivatives and that itremains so when different substrates are derivatized.

In solution, the CDA-derived diastereoisomers existin a conformational equilibrium (Figure 5). Thisequilibrium is often very complex, because severalbonds are involved and the final balance has asubstantial influence on the ability of the anisotropicgroup Y to shield/deshield the substituents L1/L2selectively.

The balance between the relative populations ofeach conformer and the strength of the effect of Y oneach conformer will be reflected (as an average) in

the resulting NMR chemical shift. Therefore, tocorrelate the information gained from the NMRspectra (the ∆δ values) with the absolute configura-tion, a detailed understanding of the structure andenergy of the main conformers, as well as thestrength and direction of the anisotropic effect of Yon L1 and L2 in each conformation is necessary.Naturally, for the direct use of the method, modelsthat simplify the equilibrium, showing only the mostrepresentative conformer, are preferred (i.e., sp con-former for MPA esters; see Figure 5).

In general, the models described in the literaturehave been formulated as empirical rules. That is, theNMR behavior of the CDA derivatives from severalsubstrates of known absolute configuration are ana-lyzed and a conformational model that could explainthe experimental NMR shifts is devised. In a fewcases, the model proposed is claimed to be supportedby XRD data; however, the differences betweensolution and solid state can be so big that not muchsupport can come from that.

It is only in the past decade that extensive studies,fundamentally theoretical calculations of structureand energy, and experimental dynamic NMR andshielding effect calculations have been performed toknow the contribution of each conformer and that ofthe average to the NMR shift that is observed.

This understanding of the fundamentals of themethods has allowed us to rationally explain not only

Figure 3.

Figure 4.


the way in which the CDAs work but also led to thedevelopment of new and more-efficient CDA reagentsand methodologies giving a plus of reliability to theabsolute configuration assignment process that isnow based on both empirical and theoretical results.

2.5. Substrates and DerivativesThe ranges of substrates whose configuration can

be assigned by these methods have increased signifi-cantly recently and include primary, secondary, andtertiary alcohols, diols, carboxylic acids, primary andsecondary amines, and sulfoxides. Appropriate chiralauxiliary reagents have been developed for each casethat produces derivatives such as esters, amides,hemiacetals, phosphonates, etc. This review coversall those chiral substrates, the reagents and methodsmost commonly used and attempts to offer a criticalassessment of each method.

3. Methods Based on Double Derivatization

3.1. Application to r-Chiral Secondary Alcohols

3.1.1. Methoxytrifluoromethylphenylacetic Acid (MTPA):Mosher’s Reagent

R-Methoxy-R-trifluoromethyl-R-phenylacetic acid(MTPA; see Figure 6) has been the most commonlyused derivatizing reagent for the determination of theabsolute configuration of secondary alcohols by NMR

ever since Mosher first reported7,8 its use in 1973.The procedure starts with the esterification of thealcohol with the two enantiomers of MTPA. After thetwo diastereomeric esters have been prepared, theirNMR spectra are acquired and compared. MTPA isavailable both as the acid and the acid chloride, andusers must remember that, although derivatizationof the alcohol with the (R)-MTPA acid leads to thecorresponding (R)-MTPA ester, the (R)-MTPA chlo-ride leads to the (S)-MTPA ester, which has led toconfusion and some erroneous assignments.9

In his first study, Mosher used 19F NMR spectros-copysparticularly, the chemical shifts of the CF3

groupssfor the assignment. In later studies, how-ever, 1H and 13C NMR were utilized, and the chemicalshifts of the protons and C atoms of the chiral alcoholunder examination were used, respectively. Theabsolute configuration of the substrate is deduced byinterpretation of the signs of ∆δ values, using certainempirical models. A description of those models,classified according to the NMR technique used,follows.

3.1.1.1. The Use of 19F NMR. 19F NMR was usedby Mosher to determine the absolute configurationof secondary alcohols.8a The procedure is based on theanisotropic effect that the carbonyl group of theauxiliary MTPA exerts on the CF3 group. For thispurpose, Mosher assumed that the (R)- and (S)-MTPA esters of secondary alcohols exist in a confor-mation in which C(1′)H, the carbonyl, and the CF3

groups are situated in the same plane (Figure 7) inthe derivative where the phenyl ring is opposite tothe least bulky substituent of the alcohol. In thederivative in which the phenyl ring is situated on thesame side as the most bulky substituent, the copla-narity is lost and, as a consequence, the CF3 movesfrom the deshielding zone to the shielding zone of thecarbonyl group.

When the bulkier substituent (e.g., L1) is on thesame side as the phenyl group, the CF3 resonates ata higher fieldsbecause of greater shielding (Figure7b)sthan if L1 were on the side of the methoxy group(Figure 7a). These differences in chemical shifts are

Figure 5.

Figure 6.


expressed in terms of the parameter ∆δSR(19F)CF3,which is defined as the difference in the chemicalshift of the trifluoromethyl signal in the (S)-MTPAester (δCF3(S)) and the chemical shift of the samesignal in the (R)-MTPA ester (∆δSR(19F)CF3 ) δCF3-(S) - δCF3(R)).

For example, an alcohol with the configurationrepresented in Figure 7, in which L1 is more bulkythan L2, gives a negative value of ∆δSR(19F)CF3 (<0).If the configuration of the alcohol were the opposite,or the size of the substituent L2 was much greaterthan that of L1, the sign of ∆δSR would be positive.

An advantage of this procedure lies in the clean-ness of the 19F NMR spectra, which generally onlycontain signals that are due to the CF3 groups andeliminates the possibility of overlapping signals thatoften complicate proton spectra. Its application forthe configurational assignment will be discussed laterin this review.

3.1.1.2. The Use of 1H NMR. The use of 1H NMRspectroscopy is undoubtedly more common for theassignment of the configuration of secondary alco-hols10 than the use of 19F NMR that has beendescribed previously. This approach is based on theanisotropic effect that the phenyl group of the chiralauxiliary MTPA exerts on the substituents (L1/L2) ofthe alcohol. This effect allows a correlation to be

made, regarding the spatial position of L1 and L2,with respect to the phenyl group of the MTPA moietyon the basis of the signs of ∆δSR of the substituents.In this case, Mosher assumed that the most repre-sentative conformation is that in which C(1′)H, thecarbonyl group, and the CF3 group are situated inthe same plane (Figure 8a).

Accordingly, the protons of substituent L2 areshielded by the phenyl ring in the (R)-MTPA ester,whereas those on L1 remain unaffected (Figure 8a).In the (S)-MTPA derivative, on the other hand, it isL1 and its protons that are shielded while L2 isunaffected (Figure 8b). Therefore, the substituent L1will be more shielded in the (S)-MTPA ester than inthe (R)-MTPA ester and the substituent L2 will bemore shielded in the (R)-MTPA than in the (S)-MTPAester derivative.

These selective shieldings are expressed using theparameter ∆δSR, which is defined as the differencebetween the chemical shift of a certain proton in the(S)-MTPA ester and the chemical shift of the sameproton in the (R)-MTPA derivative. All the protonsshielded in the (R)-MTPA will present a positive ∆δSR

value, whereas those shielded in the (S)-MTPAderivative will present a negative ∆δSR value.

In the case of the alcohol shown in Figure 8c, forexample, the signs for each proton in L1 and L2 are

Figure 7. Model for the correlation of configuration/NMR for 19F and NMR spectra of MTPA esters.

Figure 8. Models proposed by the Mosher model for (a, b) the assignment of configuration by 1H NMR and (c, d) theexpected sign of ∆δSR.


as follows:

If the configuration of the alcohol shown in Figure8c were opposite, then the signs of ∆δSRL1 and ∆δSRL2would also be opposite (see Figure 8d). An analysisof the results obtained with this technique, its scope,and its limitations are included in the correspondingsection of this review.

3.1.1.3. The Use of 13C NMR. This approach forthe assignment of the absolute configuration of thealcohol is similar to that previously described for 1HNMR spectroscopy but using the 13C NMR chemicalshifts11 of the substituents L1 and L2 ((C)L1, (C′)L2)in the (S)- and (R)-MTPA esters. In the exampleshown in Figure 8c, the substituent that results innegative signs of ∆δSR occupies the place of L1,whereas the substituent whose C atoms result in apositive ∆δSR value is L2.

3.1.1.4. Scope and Limitations in the Use of19F, 1H, and 13C NMR of MTPA Esters. The NMRmethod for assignment of the absolute configurationof alcohols using MTPA, as described by Mosher,8d

is entirely empirical, and the models represent nomore than a convenient way to correlate the experi-mental NMR with the absolute configuration of thecompounds. This means that the quality of theassignment is indicated by the number and struc-tural variety of the substrates of known absolutestereochemistry used as test compounds. In addition,the NMR data used for assignment should adhere toseveral prerequisites:

(a) The ∆δSR values must be sufficiently large andbe above the level of experimental error,

(b) The distribution of the signs of the parameter∆δSR must be uniform for a given substituent (i.e.,all the protons on the same side of the plane of theMTPA ester should have the same sign), and, finally,

(c) If the sign of ∆δSR is negative for one substituent(e.g., L1), then the sign of ∆δSR for the other sub-stituent (i.e., L2) must be positive.

Unfortunately, MTPA derivatives frequently do notseem to comply with those criteria, leading to a lossof confidence in the assignment that clearly overcomethe alleged advantages of MTPA as an auxiliaryreagent. In this section, we will present a criticalaccount of the models described, the scope of eachprocedure, and its anomalies and limitations.

In regard to using 19F NMR spectroscopy, theresearcher must remember that the assignment ismade on the basis of a single piece of data: ∆δSR(19F)-CF3 is the difference in the chemical shifts of the CF3group in the two derivatives. In contrast, the use ofthe proton or C chemical shifts from the two substit-uents of the alcohol involves the recording of manysignals and allows any anomaly to be easily detected,because it will produce a nonhomogeneous distribu-tion of ∆δSR signs. This check is not possible using19F NMR, because only one data point is obtained;the data point can be either positive or negative and,as such, will always give a certain configuration.

Indeed, many configurations that had been as-signed on the basis of 19F NMR have since beencorrected.12 A selection of these cases is given inFigure 9.

Apart from that phenomenon, 19F NMR of theMTPA esters is frequently performed with completesuccess for the enantiomeric excess calculation ofalcohols.

∆δSRL1 ) δL1(S) - δL1(R) < 0

∆δSRL2 ) δL2(S) - δL2(R) > 0

Figure 9. Selection of compounds that do not conform tothe 19F NMR Mosher model for assignment.12

Figure 10. ∆δSR values from the 13C (and 1H) NMRspectra of different MTPA esters. Anomalous values areunderlined.


Application of 13C NMR for the assignment ofconfiguration of alcohols presents some advantagesbut also some important limitations.

Although the use of 13C NMR requires a greateramount of sample than does 1H NMR, this techniquecould be especially interesting in cases where 1HNMR is not useful because one of the substituentsL1/L2 has no protons. Nevertheless, those wishing touse it as a general method for assignment should

remember the following points that greatly limit itsapplication on a general basis:

(a) The number of alcohols of known configuration(Figure 10) that have been used to test the validityof the correlations is rather small; therefore, theaccuracy of the configuration assigned to a certainsubstrate has some uncertainty.

(b) The ∆δSR values obtained are very small, incomparison to the 13C chemical shift scale.

Figure 11.


(c) Nonhomogeneous distributions of the signs of∆δSR have been obtained for some secondary alcohols(data underlined in compounds 10.2 and 10.4 inFigure 10).

The most numerous examples of application ofMTPA for the assignment of configuration of alcoholshave been described using 1H NMR. The method hasbeen tested with a fair number of alcohols of knownabsolute configuration; nevertheless, numerous caseshave been described in which the ∆δSR values ob-tained from the MTPA esters do not allow a safeassignment, in accordance with the criteria discussedpreviously,13 either because (a) the magnitude of theshifts is very similar to the experimental error or (b)the distribution of signs is not consistent.

Both the small shifts and the irregular distributionof signs can lead to erroneous assignment of config-uration. This has been demonstrated in certain casesby XRD12 or other methods; however, in other cases,the configuration that is assigned has remainedunchecked.

For example, the MTPA derivatives of siphlenolA14a (Figure 11) result in ∆δSR values with the samesign for both substituents (L1 and L2), which is a factthat makes the assignment impossible, whereas itsepimer, epi-sipholenol A14b (Figure 11), gives oppositesigns for each substituent. Similar situations havebeen described for other cyclic alcohols12b that giveirregular distribution of the signs of ∆δSR, including3-R-cholesterol and friedelan-3â-ol, which possessaxial hydroxyl groups. In contrast, the epimers ofthese two compounds, cholesterol and friedelan-3R-ol, with equatorial hydroxyl groups give a perfectlyhomogeneous distribution of signs for ∆δSR (Figure11).

The anomalous behavior of siphlenol A, 3-R-cholesterol, and friedelan-3â-ol has been explainedin terms of the steric hindrance of the axial hydroxyl

group modifying the conformation of the MTPAesters. However, similar cases are known in whichthis explanation is not applicable, such as sinulari-olide and its epimer 11-episinulariolide,15 which bothproduce anomalous ∆δSR sign distributions and, thus,cannot be assigned a configuration with any degreeof certainty by this method.

Given the frequency with which MTPA esters leadto irregular sign distributions, Kakisawa and co-workers10,12b,16 proposed a new way to evaluate the∆δSR data; this methodology is called the “ModifiedMosher Method.” This procedure involves the analy-sis of all (or the vast majority) of the protons in themolecule, so that a representative sign for ∆δSR ofsubstituents L1 and L2 can then be adopted based onthe majority of the protons for each substituent. Inour opinion, however, the elimination of those reso-nances that produce anomalous signs can only bejustified if (a) those proton(s) are located at a longdistance from the chiral center and (b) there is asufficiently large number of protons close to theasymmetric C atom producing the expected homoge-neous sign distribution.

Another class of compounds in which irregular ∆δSR

signs have been observed is alcohols with aromaticrings in the R-position.17 This situation is illustratedin Figure 12 by the MTPA esters of the alcoholsderived from tautomycin,17a in which irregular ∆δSR

values are found exclusively for the â-protons (un-

Figure 12. R-Aryl-substituted secondary alcohols thatpresent anomalies in the ∆δSR signs of the â-protons (dataunderlined).

Figure 13. (a) Structure of tanabalin (13.1) and ∆δSR

values for the MTPA esters; (b) ∆δSR values for the MTPAesters of N-acetyladrenalin.

Figure 14. Structure of quadrangularic acid L and ∆δSR

values of the MTPA esters.


derlined in Figure 12) whereas the rest of the protonsgive regular sign distributions. The authors suggest17a

that the cause of these irregular sign distributionscould be the fact that these protons are affected notonly by the phenyl ring of the MTPA but also by thearomatic ring at the R-position in the alcohol, whichis a view supported by Molecular Mechanics calcula-tions.

Other examples include tanabalin17b (13.1, Figure13), which is an insect antifeedant isolated fromTanacetum balsamita, and N-acetyl L-adrenalin (13.3,Figure 13).

Irregular distributions in the signs of ∆δSR havealso been found in substrates that do not containaromatic groups competing with MTPA. Quadran-gularic acid L18 (Figure 14) is one such example thatproduces negative signs for ∆δSR of both substituentsL1 and L2. The fact that the dehydro-derivatives ofthese compounds give the correct sign distributionhas led to the belief that the presence of a neighbor-ing polar group should be responsible for that anoma-lous sign distribution.

Unfortunately, the explanation for that incoherenceis not so simple, and there are many other compoundsthat produce nonhomogeneous sign distribution but

have no polar groups or neighboring aromatic ringsin their structures. Some examples are shown inFigure 15.19

Many compounds have been reported whose MTPAesters produce sign distributions in good agreementwith Mosher’s model, despite the presence of hin-dered hydroxyl groups, polar groups, or other aro-matic rings. Their structures are shown in Figures16,20 17,21 and 18.22

One specific class of compounds in which irregularsign distributions are obtained from the MTPA esters

Figure 15. ∆δSR values obtained for the MTPA estersshown.

Figure 16.


warrants special mention. Those are the polyalcoholsthat have been completely derivatized with MTPA.Examples of such systems include penaresidin A andB,23 pyripyropene A,24 and other compounds25 shownin Figure 19. The anomalies observed in thesecompounds have been identified as being due to thepresence of several MTPA units whose effects inter-fere with each other. Such crossed effects cause signdistributions that bear no relation to Mosher’s pre-dictions for monoalcohols where only the effect of asingle aromatic ring must be considered. The assign-ment of the configuration of polyalcohols by NMRspectroscopy must be analyzed in a different way and

will be discussed in the section covering polyfunc-tional compounds.

3.1.1.5. Special Models for Special Cases. It isobvious that no prediction of configuration is possibleby the method described if one of the substituents ofthe substrate has no resonances to examine or ifthose peaks cannot be clearly distinguished becauseof overlapping.

Alternatives have been proposed26 to overcomethese limitations in two particular classes of com-pounds.

Thus, for fluoro-substituted secondary alcoholssuch as the 1,2-difluoroallyl alcohols shown in Fig-ures 20 and 21, where one substituent containsprotons and the other contains only F atoms, thecombined use of 1H and 19F NMR spectroscopy has

Figure 17.

Figure 18.


been described.26a The 1H and 19F NMR spectra ofthe two MTPA esters are recorded and the ∆δSR

values calculated for the protons on one substituentand for the fluoro-substituents of the other substitu-ent. The model proposed assumes that if the config-uration of the alcohol is that shown in Figure 20b,then the protons in L1 must produce a negative ∆δSR

value, whereas the fluoro-substituents in L2 musthave a positive ∆δSR value. When the configurationis the opposite, the signs of ∆δSR then will be reversed(see Figure 20c).

Figure 21 shows the alcohols of known absoluteconfiguration that have been used as support of themixed proton-fluorine NMR approach. Although

most of the ∆δSR signs are in good agreement withthe configuration, some compounds (21.3 and 21.6)exhibit signals (underlined in the figure) that areinconsistent with their configuration and put a limiton the validity of the method.

The purpose of the second alternative described26b

is to solve the difficulties presented by overlappingsignals for L1/L2 in aromatic and heteroaromaticalcohols. It is based on the combined use of theanisotropic effect caused by the aromatic ring of thealcohol on the methoxy group of the auxiliary MTPAand that of the phenyl ring of the auxiliary on thesubstituents L1/L2 of the alcohol.

According to the author, in the (R)-MTPA ester ofthe alcohol shown in Figure 22a, both aryl rings are

Figure 19.

Figure 20.

Figure 21.


on the same side of the plane of the MTPA, and,consequently, neither the signals of the MeO northose of substituent (R) of the alcohol are affectedby the anisotropy of the rings. For its part, in the(S)-MTPA ester (Figure 22b), the aromatic groups areon opposite sides and both the MeO and substituent(R) are under their shielding influence. Applicationof this method requires the derivatization of thealcohol with the two enantiomers of MTPA andcomparison of their NMR spectra.

Negative ∆δSR values for the MeO group resultwhen the configuration is that indicated in Figure22, and positive ∆δSR values are to be obtained if theconfiguration of the alcohol were the opposite.

This model has been tested with 50 alcohols ofknown absolute configuration. The prediction of theconfiguration was correct in 46 cases and incorrectin just 1. In 3 examples, the ∆δSR values werenegligible and did not allow the assignment of con-figuration.

In summary, the data presented indicates thatMTPA frequently presents severe limitations in itsuse for the assignment of configuration of secondaryalcohols: small ∆δSR values, which are very muchaffected by the presence of anisotropic groups in thesubstrate, and irregular sign distributions.

In addition, the explanations that have been putforth for these inconsistenciessusually centered onthe particular characteristics of the substratessarenot satisfactory. They are neither general nor do theyallow the researcher to predict whether this methodcould be used to assign, with complete certainty, theabsolute configuration of a new substrate. We willshow in the next section that the problem is not withthe substrate but rather with the characteristics andlimitations of the MTPA itself.

3.1.1.6. The Mode of Action of MTPA: A Revi-sion of the Model. As we have seen, the assignmentof absolute configuration by 1H NMR of MTPA estersrests with the detection by NMR of the relativeposition of substituents L1 and L2, with respect to thephenyl ring of the auxiliary MTPA. It is this spatialinformation that is contained in the ∆δSR signs of thesubstituents.

The anomalies observed in the use of MTPA sug-gested that perhaps the empirical model describedby Mosher in 1973 is not fully representative of the

real position of the phenyl ring, with respect to thesubstituents, and stressed the need to study theconformational composition and the exact directionof the anisotropic effect in MTPA esters.

In fact, theoretical calculations on the conforma-tions (molecular mechanics, semiempirical and abinitio) and shielding/deshielding contributions (aro-matic ring current increments) were performed27 inconjunction with variable-temperature NMR anddynamic NMR experiments that allowed the fullunderstanding of the fundamentals of the MTPAaction and of the correlation between NMR spectraand absolute configuration.27

The results indicate that the conformational com-position of MTPA esters is very complex and that thesituation cannot be fully represented by the empiricalmodel proposed by Mosher (Figure 8). According tothose studies,27 the main conformational processesinvolve rotation about the CR-CO and CR-Ph bonds(Figure 23). The first generates two conformers: thesp conformer, in which the CF3 has a syn-periplanardisposition, with respect to the carbonyl group, andthe ap conformer, in which these groups have an anti-periplanar disposition. Rotation about the CR-Phbond generates three conformers that differ in theorientation of the phenyl ring (coplanar with the CR-CO, CR-CF3, and CR-OMe bonds, respectively).Consideration of their relative energies indicates thatonly three of the six possible forms (Figure 24) arereally representative: ap1, sp1, and sp2. Their orderof stability is ap1 > sp1 > sp2, although the energydifferences are so small (0.40 and 0.63 kcal/mol,respectively) that they are present in similar popula-tions, and, therefore, significant contribution of eachone to the average spectrum mus be expected.

Figure 22.

Figure 23. Generation of conformers in MTPA esters andthe most-significant characteristics of each.


Conformer ap1 is the most stable conformer andhas the CF3 group anti-periplanar, with respect tothe carbonyl group; its phenyl ring produces adeshielding effect on the substituents of the alcohol.The next conformer, in terms of energy, is sp1; it hasthe CF3 and carbonyl groups in a syn-periplanardisposition, as in the empirical Mosher’s model, andits phenyl ring produces a shielding effect on thealcohol part. The third conformer is sp2, and thisconformer also has a syn-periplanar disposition thatresults in deshielding on the alcohol substituents.The conformers are shown in Figure 24.

In accordance with that composition, the finalchemical shift of substituents L1 and L2 in theaverage spectrum will be the result of the combinedaction of those three similarly populated forms. Eachconformer contributes a different shielding/deshield-ing effect; therefore, some cancellations may result,causing small ∆δSR values.

For example, in the ester that is derived from (R)-MTPA (Figure 25a), substituent L1 is deshielded inconformer ap1, whereas substituent L2 is shielded inconformer sp1 and deshielded in conformer sp2.Therefore, the net effect on substituent L2 will rangefrom slight shielding/deshielding to a mutual cancel-lation of effects.

For its part, in an ester that is derived from (S)-MTPA (Figure 25b), substituent L2 is deshielded inconformer ap1, whereas substituent L1 is shielded inconformer sp1 but deshielded in conformer sp2. Asin the previous example, the net result is a total or

partial cancellation of effects and this results insubstituent L1 being slightly shielded/deshielded orremaining unaffected by the phenyl ring of thereagent.

The final results for substituent L1 will be deshield-ing in the ester-derived form (R)-MTPA, and eitherslightly shielding or just a null effect in the (S)-MTPAderivative. Substituent L2, on the other hand, isdeshielded in the (S)-MTPA derivative but onlyslightly shielded or unaffected in the (R)-MTPAderivative. Negative signs for ∆δSR are then expectedfor all the protons on substituent L1 and positive

Figure 24.

Figure 25. Shielding/deshielding effects in the three most-representative conformers of the MTPA esters.


signs are expected for those on substituent L2:

As indicated, experimental support for those con-clusions has been obtained from the evolution of theNMR spectra, with temperature, of the MTPA estersthat showed the shift of the equilibrium towardconformer ap1 at lower temperatures. Similarly, goodagreement was observed between the experimentalchemical shifts and those expected from the calcu-lated aromatic shielding effect of the phenyl ring onL1 and L2 in each individual conformer and in theaverage mixture.27 Interestingly, both calculationsand experiments demonstrate that the effect onsubstituents L1 and L2 of the phenyl ring of MTPAis net deshielding rather than shielding, as suggestedby Mosher in its empirical rule.8

On that basis, a simplified model representativeof the NMR behavior of the MTPA esters and usefulfor the assignment has been formulated.27 It has theCF3 group, the carbonyl group, and the H atom onthe chiral C atom of the alcohol in the same planeand the CF3 anti-periplanar to the carbonyl group.In this way, the phenyl ring deshields the substituenton the same side of the plane, i.e., L1 in the (R)-MTPAester and L2 in the (S)-MTPA analogue (Figure 26).

3.1.1.7. The Conformations of MTPA Esters.The existence of three main conformers that havesimilar energy and contribute to the chemical shiftswith both shielding and deshielding effects serves toexplain the origin of the two most important limita-tions of this reagent: the small ∆δSR values and thefrequent surge of irregular distributions of the signsof ∆δSR.

The origin of the reduced ∆δSR values has beenalready advanced and resides in the eventual cancel-lation in the average spectrum of the shielding/deshielding that is associated with each individualconformer.

Another consequence of this same fact is reflectedin the case of the R aryl alcohols in Figures 12 and13, where anisotropic groups in the alcohol substit-uents effectively overcome the reduced shielding/deshielding produced by the MTPA, leading to ∆δSR

signs that do not necessarily reflect the configurationof the substrate.

Figure 27a illustrates how the magnetic field of thephenyl ring affects the space occupied by the alcoholsubstituents. When we consider the three mainconformers of a MTPA ester, the space distributionof the magnetic field is different for each conformerand leads to the signs of the ∆δSR value that areindicated in Figure 27b.

As can be observed, it is perfectly possible for asubstituent such as L1 to possess protons located inthe space determined by quadrant 2 and otherprotons in quadrant 3. If this were the case, different∆δSR signs will result in the average spectra and,therefore, an irregular distribution of signs will resultfor the protons in L1.

Furthermore, if one considers the role of theconformational equilibrium in the average spectra,it is easy to understand that small changes in thebalance of conformers may produce certain protonsto pass from a net shielding area to a net deshieldingarea. In fact, even if we assume that conformer ap1

Figure 26. Mosher’s model8 and the revised model27 for the assignment of absolute configuration of MTPA esters.

∆δSRL1 ) δL1(S) - δL1(R) < 0

∆δSRL2 ) δL2(S) - δL2(R) > 0

Figure 27. Spatial distributions of (a) the shielding effecton a MTPA ester and (b) the resulting signs of ∆δSR in theconformers of a MTPA ester.


remains the most significant conformer in all cir-cumstances, small variations in the ratio of the othertwo forms could eventually lead to a different shield-ing/deshielding balance, because each conformer hasa different shielding/deshielding effect.

3.1.1.8. Summary. In summary, the use of MTPAfor the assignment of the absolute configuration ofalcohols is restricted by the conformational limitsimposed by the reagent. Three conformations ofsimilar population are present, and these simulta-neously cause shielding/deshielding effects on thesame substituent. This situation explains many of theobservations regarding this method,13 including (a)the frequency with which small values of ∆δSR arefound that are of little use for the assignment ofconfiguration and (b) irregular sign distributions.

The conformational flexibility of MTPA esters andthe magnetic field distribution facilitates small struc-tural or experimental changes (e.g., NMR solvent ortemperature) to change the balance between theconformers, causing the sign distributions of ∆δSR tofollow patterns that are different from those pre-dicted by the original Mosher model.

Overall, Mosher’s reagent, although still useful forthe determination of enantiomeric purity by NMRspectroscopy, is not recommended for the determi-nation of the absolute configuration of secondaryalcohols by NMR. Such assignments should be madeusing other, more-reliable reagents.

3.1.2. Methoxyphenylacetic Acid (MPA)3.1.2.1. Classical Models for MPA Esters.

R-Methoxy-R-phenylacetic acid (MPA; see Figures 4and 28), along with MTPA, has been one of the mostfrequently used auxiliary reagents for the assignmentof the absolute configuration of secondary alcoholsby NMR. The application of this reagent, togetherwith the use of mandelic acid (MA) and atrolactic acid(Figure 28), was described by Mosher in 1973.8c

However, the utility of this reagent was not fullyaccepted at the time, mainly because of problemsassociated with racemization found during the de-rivatization of the alcohol. It was later found that thisproblem could be easily circumvented if differentderivatization conditions are used.28

The procedure for the determination of absoluteconfiguration with this reagent is the same as thatalready described for MTPA and can be summarizedas follows (Figure 29): (a) derivatization of thealcohol in question with the two enantiomers of thereagent, (b) examination of the NMR spectra of the(R)- and (S)-MPA ester derivatives and evaluationof the corresponding differences in the chemical shiftsof the substituents of the alcohol (∆δRSL1 ) δL1(R)- δL1(S) and ∆δRSL2 ) δL2(R) - δL2(S)). As notedpreviously, the configuration is deduced from thesigns of these differences and their comparison withan empirical model (see Figure 29b).

In this model, the most representative conformerhas the methoxy group of MPA (the hydroxyl groupin MA and atrolactic acid) and the carbonyl andC(1′)H proton of the alcohol in the same plane (seeFigure 29a). These groups are arranged in such a waythat, in the (R)-MPA ester, substituent L1 is shieldedby the phenyl ring while L2 is unaffected, whereas,in the (S)-MPA ester, substituent L2 is the shieldedgroup and L1 remains unaffected (Figure 29a).

In this way, the alcohols in the figure show ∆δRSL1< 0, because substituent L1 is more shielded in the

Figure 28. Isopropyl esters of MPA, MA, and atrolactic acid.

Figure 29. Model for configurational correlation of MPAesters.


(R)-MPA ester than in the (S)-MPA ester, and ∆δRSL2> 0, because substituent L2 is more shielded in the(S)-MPA ester than in the (R)-MPA ester (see Figure29b).

The relative position of the phenyl group of theauxiliary, with respect to L1 and L2 of the samealcohol, is opposite in the MPA and MTPA esters,thus producing opposite signs for the same chirality(see Figure 8). To obtain the same signs with the tworeagents, the differences in chemical shifts are cal-culated as ∆δRS in MPA esters, whereas ∆δSR is usedwith MTPA esters.

3.1.2.2. Conformational Analysis. Historically,the first model used to correlate the absolute config-uration of alcohols with the NMR shifts and signs ofthe ∆δRS values of the MPA esters was proposed byMosher8c and is, as in the case of MTPA derivatives,empirically generated from the study of severalalcohols of known stereochemistry. For this reason,the validity of the method is related to the represen-tativity and scope of the series of alcohols initiallystudied.8c

To evaluate the limitations of this model, studieshave been conducted29 that are comprised of (a)

theoretical structure calculations (molecular mechan-ics, semiempirical (AM1) and ab initio), (b) shielding-effect calculations on the contribution of the phenylgroup to the chemical shift, and (c) dynamic NMR toelucidate the conformational characteristics of thecompounds.

As a result, the main conformers of the MPA esterswere identified. Their populations and the role of thephenyl ring were determined, and this determinationallowed a complete understanding of the fundamen-tals of the method and provided the basis for thedesign of more-efficient reagents.30

It was found29 that the MPA esters of secondaryalcohols are mainly composed of two conformers thatare formed by hindered rotation around the CR-CObond (see Figures 30 and 31). The most stable one(by 0.6-1.0 kcal/mol) is the sp conformer, in whichthe methoxy group, the CR carbon, and the carbonylgroup of the MPA portion and the C(1′)H hydrogenof the alcohol portion are in the same plane, with themethoxy and the carbonyl groups in a syn (syn-periplanar) disposition. In this conformation, thephenyl ring is coplanar with the CR-H bond, whichis the best arrangement to effectively transmit aro-matic shielding to the substituent (L1 or L2) locatedin the same side of the plane (see Figure 31).

The next conformer, in terms of energy (the apconformer), has the same coplanar arrangement ofthe aforementioned groups, but, in this case, themethoxy and carbonyl groups are in an anti-peri-planar relationship and the phenyl group is rotatedso it cannot transmit the shielding effect to substit-uents L1/L2 as efficiently as in the example discussedpreviously (see Figure 31). Thus, in the (R)-MPAester derivative, substituent L1 is shielded by thephenyl ring in the sp conformation while substituentL2 is unaffected (see Figure 32a). In contrast, sub-stituent L2 should be slightly shielded in the apconformation while substituent L1 remains un-affected (see Figure 32a). The opposite situationoccurs in the (S)-MPA ester: substituent L2 isshielded and substituent L1 remains unaffected in the

Figure 30. Main conformers of the MPA esters.

Figure 31. Conformers sp and ap of the isopropyl (R)-MPA ester.


sp conformer, whereas substituent L1 is slightlyshielded and substituent L2 is unaffected in the apconformer (Figure 32b).

As a result of the characteristics discussed above,the aromatic group of the reagent will modify thechemical shifts of substituents L1 and L2 in a veryselective way: In the (R)-MPA ester, it is expectedthat strong shielding will be experienced by substitu-ent L1 but only on the proportion of the molecules inwhich substituent L1 is beside the phenyl group(conformer sp). On the other hand, in the (S)-MPAester, substituent L1 will be slightly shielded, but onlyfor those molecules in the ap conformation. Giventhat the sp conformer is more abundant than the apconformer, substituent L1 is more shielded in the (R)-MPA ester than in the (S)-MPA ester. Similarreasoning for substituent L2 leads to the conclusionthat it will be more shielded in the (S)-MPA esterthan in the (R)-MPA ester. Thus, the differences inchemical shifts (∆δRS) will be positive in one case andnegative in the other (see Figure 32c). If the oppositeconfiguration is considered for the alcohol (i.e., sub-stituent L1 in place of substituent L2), then theopposite distribution of signs is obtained (see Figure32d), thus demonstrating that the sign of ∆δRS is areliable indicator of the spatial arrangement of thesubstituents.

The model proposed by Mosher8c,28a for the assign-ment of the configuration of MPA esters is, in fact, arepresentation of the most stable conformer of the

equilibrium, which is also the most important, fromthe standpoint of NMR, and is therefore suitable topredict the correct configuration. Figure 33 shows aselection of compounds studied using that model.31

Note that this is not the case for MTPA esters,where the model used by Mosher is not truly repre-sentative of the conformational composition.27

3.1.2.3. MPA versus MTPA. In general, research-ers have not been very critical when choosing be-tween MPA and MTPA for the assignment of sec-ondary alcohols. Quite frequently, they have over-looked the anomalies detected with MTPA13 thatwere mentioned in the previous chapter.

In the case of MTPA esters of secondary alcohols,two factors contribute to these limitations:27,32 (a) thepresence of three main conformers with similarpopulations and (b) the fact that some of theseconformers produce shielding effects and othersproduce deshielding effects on substituents L1/L2 (seeFigure 25), the combination of which produces verysmall ∆δSR values and sometimes unexpected signs.

In contrast, the MPA esters present a simplerconformational composition (see Figure 32) with onlytwo conformers (sp/ap) and a clearer preference forone of those (sp), which, in turn, transmits a shieldingeffect to the substituents to give higher ∆δRS valuesthat are more reliable and homogeneous in terms ofdistribution of the signs (Figure 34).

On the basis of the aforementioned discussion, thechoice between MTPA and MPA as a derivatizingagent for secondary alcohols should be clearly in favorof MPA.

3.1.2.4. New MPA Analogues: The Effect ofStructural Modifications. In the search for moreefficient and reliable reagents, a variety of modifica-

Figure 32. Conformational equilibrium in MPA esters.

∆δRSL1 ) δL1(R) - δL1(S) < 0

∆δRSL2 ) δL2(R) - δL2(S) > 0


tions of the structure of MA have been performed(Figure 35), leading to a series of arylmethoxyaceticacids30,33 (AMAAs). Some of these compounds areinteresting from the practical standpoint and othersare interesting because of the information theyprovide regarding our understanding of the method.The most revealing modifications have been (a) theintroduction of different O-substituents on MA,34 (b)the introduction of different substituents on thephenyl ring30,33 of MPA, and (c) the replacement ofthat phenyl ring by other aryl ring systems.29,30,33

3.1.2.5. O-Substituted Mandelic Acids. Table 1shows the structures of several O-substituted MAsand the ability of their enantiomers to differentiatethe NMR signals34a of (-)-menthol expressed as ∆δRS.As can be seen, the values are very similar in allcases, being somewhat greater in MA and its acetylderivative (see Figure 36). In addition to similar ∆δRS

values, the same distribution of the signs as thatobserved with MPA itself is obtained, indicating thattheir mode of action and conformational compositionis quite probably the same, suggesting that no realimprovement can be expected by changing the O-substituent in MA.

Figure 33.

Figure 34. ∆δRS and ∆δSR values (in ppm) for a selectionof MPA and MTPA esters of secondary alcohols obtainedin (a) Cl3CD and (b) CS2:CD2Cl2 (4:1). MTPA data are notedby an underline.

Figure 35.


A different case is constituted by the THP ether ofMA, which has been investigated34b as a reagent forthe assignment of the absolute configuration of cyclicalcohols (see Figure 37). Unfortunately, an irregulardistribution of ∆δRS signs were obtained in the twocompounds tested, which makes the reagent uselessfor the intended purpose. Only two open-chain sec-ondary alcohols (Figure 37) were studied with thisreagent and showed the same distribution of signsas that for MPA.

3.1.2.6. The Role of the Substituents in theAromatic Ring. Similar studies have been con-ducted30,33 to ascertain whether the introduction ofsubstituents into the phenyl ring of MPA will producean increase in the ∆δRS values.

The results obtained for some of these new re-agents with (-)-menthol as the substrate are shownin Table 2. These data indicate that, in general, thepresence of fluoro or methoxy groups in severaldifferent positions does not produce any improvementin the capability of MPA to separate the signals ofenantiomers. In the case of the pentafluorphenyl

derivative, the ∆δRS values are, in fact, smaller thanthose obtained with MPA and similar to thoseobtained with MTPA, whereas the methoxy-substi-tuted reagents give ∆δRS values that are greater thanthose for MTPA and equivalent to those of MPA.

Conformational studies were performed and re-vealed that the introduction of those substituentsshifts the equilibrium between the sp and ap con-formers and changes the orientation of the phenylring to a less favorable arrangement for shielding.It is the combination of these two facts that result insmaller ∆δRS values.

3.1.2.7. Arylmethoxyacetic Acids (AMAAs). Thestructural modifications discussed in the precedinglines have shown that no improvement of ∆δ can beexpected by introduction of substituents on MPA. Aswill be fully discussed in the corresponding section(Section 3.1.3), the replacement of the phenyl ring ofMPA by a naphthyl or anthryl ring has beenshown to produce the most efficient reagents todate.29,30,33,35-37 More specifically, R-(2-naphthyl)-R-methoxyacetic acid (2-NMA), R-(1-naphthyl)-R-meth-oxyacetic acid (1-NMA), R-(9-anthryl)-R-methoxy-acetic acid (9-AMA), and R-(2-anthryl)-R-methoxy-acetic acid (2-ATMA) are effective reagents (seeFigure 38).

Table 1. ∆δRS Values of O-Substituted MandelicEsters of (-)-Menthol

Table 2. ∆δRS Values Obtained for Phenyl-SubstitutedMPA Esters of (-)-Menthol

Figure 36. ∆δRS values obtained for O-substituted man-delic esters with a selection of secondary alcohols. Data forR ) AcMA is given in italic type, and data given for R )MA is given in boldface type.

Figure 37. ∆δRS values of O-substituted mandelic esters.Data for R ) MPA is given in boldface type; underlineddata present anomalous signs.


3.1.2.8. The Effect of the Temperature on theNMR of MPA Esters. A different and successfulapproach to obtain the best ∆δRS values withoutmodification of the structure of the auxiliary reagentconsists of the controlled modification of the relativepopulations (conformer sp/ap) of the MPA esters bydirectly acting on the thermodynamic parametersgoverning the equilibrium,38 namely the polarity ofthe NMR solvent and the temperature of the NMRprobe.

Although changes in the NMR solvent30 did notlead to any significant improvement of the ∆δRS

values obtained for MPA esters of several secondary

alcohols, when their NMR spectra were recorded atdifferent temperatures, the changes in chemicalshifts consistently produced higher ∆δRS values38 ofpractical interest.

Thus, in the (R)-MPA ester of (-)-menthol, theMe(8′) and Me(9′) protons are strongly shielded bythe phenyl ring in the sp conformer (see Figure 39a,resonances at 0.651 and 0.418 ppm, respectively),whereas Me(10′) is slightly shielded in the ap con-former (0.888 ppm). A decrease of the probe temper-ature should produce an increase in the number ofmolecules in the sp conformation (the most stableone) and a corresponding decrease in the ap popula-

Figure 38. Molecular structure of the arylmethoxyacetic acids (AMAAs).

Figure 39. Conformational composition of the MPA esters of (-)-menthol.


tion (the least stable one). For this reason, as shownin Table 3, at lower temperatures, more molecules

have their Me(8′) and Me(9′) groups shielded (in-creased shielding in the average spectrum; reso-nances at 0.561 and 0.165 ppm) and fewer moleculeshave their Me(10′) shielded by the phenyl group(increased shielding in the average spectrum; reso-nance at 0.898 ppm).

Similarly, in the (S)-MPA ester, the Me(8′) andMe(9′) groups become less shielded at lower temper-atures and the signals are shifted from 0.846 and0.642 ppm to 0.895 and 0.681 ppm, respectively,whereas Me(10′) is more intensely shielded at lowertemperatures, with a change in chemical shift from0.822 to 0.785 ppm for the corresponding signal (seeFigure 39b).

Table 3. Chemical Shifts of MPA Esters of(-)-Menthol at Different Temperaturesa

chemical shift

ester Me(8′) Me(9′) Me(10′)T ) 296 K

(R)-MPA 0.651 0.418 0.888(S)-MPA 0.846 0.642 0.822∆δRS -0.195 -0.224 0.066

T ) 153 K(R)-MPA 0.561 0.165 0.898(S)-MPA 0.895 0.681 0.785∆δRS -0.334 -0.516 0.113

a CS2:CD2Cl2 ratio ) 4:1.

Figure 40. Evolution with temperature of the ∆δRS values of MPA esters of secondary alcohols.

Table 4. ∆δRS Values for a Selection of MPA Esters at Different Temperaturesa

∆δRS (ppm)temp,T (K) H(1′) H(3′) H(4′) H(5′) H(6′a) H(6′e) H(6′ax) H(7′) H(8′) Me(8′) Me(9′) Me(10′)

(-)-Isopulegol298 0.050 0.085 -0.210 -0.250 -0.216 0.039213 0.100 0.175 -0.319 -0.373 -0.348 0.049183 0.100 0.175 -0.346 -0.403 -0.40 0.049

(-)-Menthol298 -0.45 -0.195 -0.224 0.066153 -1.170 -0.334 -0.516 0.103

(-)-Borneol298 0.050 0.255 0.080 -0.200213 0.104 0.499 0.138 -0.312183 0.125 0.596 0.162 -0.331

(R)-2-Butanol298 0.101 -0.095 -0.161263 0.120 -0.127 -0.207230 0.138 -0.137 -0.259203 0.156 -0.166 -0.316

a CS2:CD2Cl2 ratio ) 4:1.


In accordance with those results, the ∆δRS valuesof MPA esters of secondary alcohols become higheras the probe temperature is reduced (see Table 4 andFigure 40).

This trend reflects a general behavior that is wellillustrated by the variety of structures shown inFigure 40. This technique is particularly useful whenthe ∆δRS values obtained at room temperature areso small that reliable e.e. or absolute configurationcannot be derived. In these cases, simply repeatingthe spectrum at a lower temperature can be sufficientto obtain well-separated signals and large ∆δRS

values.3.1.2.9. Other Models for MPA Esters. A new

application of MPA in the determination of theabsolute configuration of secondary alcohols has beendescribed recently,39 based in the exclusive use of thechemical shifts of the CRH protons of the substratein the corresponding (R)- and (S)-MPA esters. Toassign the configuration, interactions between thereagent and substrate moieties of the diasteromericesters must be estimated in the two main forms inthe equilibrium (sp and ap) and compared with thechemical shifts of the CRH protons. The diastereomerwith the greater relative population of the moststable conformer in the equilibrium (sp form) shouldresonate at lower field. This behavior was experi-mentally proven for six alcohols and two amines (seeFigure 41).

This method presents some limitations. One ofthem lies in the fact that it is based on the analysisof data from a single group (∆δRS of the CRH),analogous to Mosher’s Method for 19F NMR (∆δSR ofthe CF3). The sign will always be correlated to acertain configuration, so if an anomaly arises, it willnot be detected. In some cases, another restrictioncomes from the need of a combined use of NMR dataand theoretical studies (i.e., semiempirical calcula-tions) to evaluate the difference of energy betweenboth conformers to compare them with the experi-

mental ∆δRS value. Nevertheless, and despite theaforementioned limitations, the method may be use-ful to analyze structures that lack protons in one ofthe substituents.3.1.3. 9-Anthrylmethoxyacetic Acid (9-AMA) and RelatedAMAAs

The results described to date for MPA, along withthe discovery of the role played by the phenyl ringin influencing the NMR shifts, have led to a searchfor more-efficient reagents by replacement of thephenyl ring by other aryl systems that are able toproduce a more intense aromatic shielding effect onthe substrate.29,30,33,35-37 The most widely studiedsystems in this respect are the arylmethoxyaceticacids (AMAAs; see Figures 38 and 42), which containnaphthyl or anthryl systems. In general, all thesecompounds produce more-intense shielding thanMPA and, consequently, better separation of thesignals is observed for the enantiomers of the sub-strate. This effect is particularly important when9-AMA is used as the reagent with secondary alco-hols: the ∆δRS values obtained are 3-4 times higherthan those with MPA.29,30 In fact, this more pro-nounced separation of the signals for the substrateresults from the combination of two factors: theintense aromatic magnetic field of the anthracenesystem and the greater conformational rigidity of9-AMA, which is a factor that leaves the anthracenering particularly well-oriented, with respect to thesubstrate.29

Figure 41. Alcohols tested with this method.

Figure 42. Structure of the arylmethoxyacetic acids(AMAAs).

Figure 43. Main conformational processes in AMAAesters.

Figure 44. Main conformers of the 9-AMA esters.


3.1.3.1. Conformational Analysis. Conforma-tional studies29 (theoretical and experimental) basedon structure minimizations (molecular mechanics,semiempirical and ab initio, and shielding-effectcalculations) and NMR studies (variable tempera-ture) on the auxiliaries 9-AMA, 1-NMA, and 2-NMA

(no such information is available on 2-ATMA37) haverevealed that, in a way similar to that for the MPAesters, the main conformational process is rotationaround the CR-CO bond, and this process generatesthe same types of conformers as already describedfor MPA esters:29 conformer sp, with the MeO groupsyn to the carbonyl group, is the most stable con-former and conformer ap, where the MeO group isanti, with respect to the carbonyl group, is the leaststable (see Figures 43 and 44).

In regard to rotation around the CR-Ar bond,calculations indicate that the most stable conformeris that in which the CR-H bond is coplanar with thearyl plane, as shown in Figure 44. In those reagentswhere the aromatic substitution is asymmetric (e.g.,1-NMA, 2-NMA, and probably 2-ATMA), the mainportion of the aryl ring is anti, with respect to theCR-H bond (see Figure 45).

Therefore, in the more stable conformer (sp), theCR-Ar bond is almost perpendicular to the CdObond and the plane of the aromatic ring is parallelto the CdO bond. This arrangement makes theorientation of the aryl ring particularly effective, interms of transmission of the shielding effect to thealcohol substituents L1/L2.

From the conformational standpoint, the equilib-rium between the sp and ap conformations is, in allcases, shifted toward the sp conformer, which is themost stable conformer and the most representative

Figure 45. Main conformers generated by rotation aroundthe CR-Ar bond (a) for 1-NMA and (b) for 2-NMA.

Figure 46. Conformational equilibrium in the AMAA esters.


in the NMR process. However, the difference inenergy29 varies with the nature of the aryl ring andfollows the order 9-AMA > 1-NMA > 2-NMA > MPA.

Thus, changing the aromatic ring plays a doublerole, because it affects both the intensity of thearomatic magnetic current and the conformationalorientation of the ring, with respect to the substrate.In fact, the larger the ring, the more intense thearomatic shielding effect and the greater the result-ing difference of energy between the sp and apconformers. The combined effect of these two factorsmakes 9-AMA the most useful reagent of all, produc-ing ∆δRS values that are much greater than thosecaused by other reagents.

The substitution pattern of the ring is also impor-tant because the rotational barriers for the CR-Arand CR-CO bonds will be different. For example, thebarrier in 1-NMA is greater than that for 2-NMA andthis, in turn, affects the NMR results.

3.1.3.2. NMR Spectra of AMAA Esters. TheNMR spectra of the AMAA esters are similar to thoseof the MPA esters, and the contribution of eachconformer to the average NMR spectra also followsthe same trend. Thus, in the (R)-AMAA ester, sub-

stituent L1 is shielded in conformer sp and substitu-ent L2 remains unaffected, whereas in the ap con-former, substituent L2 is shielded and substituent L1is not affected (see Figure 46a). In the ester derivedfrom the (S)-AMAA auxiliary, the opposite situationoccurs: L2 is shielded in the sp conformer and L1 isshielded in the ap conformer (see Figure 46b).

Comparison of the spectra of the (R)- and the (S)-AMAA esters, and considering the greater populationof the sp conformer in comparison to that of the apconformer, leads to the following signs for ∆δRS (theseare the same as those obtained in the MPA esterswith the same configuration):

Naturally, if the absolute configuration of the alcoholis opposite to that shown above, then the signs shouldbe reversed (see Figure 46c and d).

Figure 47 illustrates the power of 9-AMA as areagent for the assignment of the absolute configu-ration. The NMR spectra of the (R)- and (S)-9-AMA

Figure 47. (a, b) NMR spectra of the (R)- and (S)-9-AMA esters of a marine metabolite and (c) comparison of the ∆δRS

values of the 9-AMA esters with those of the MPA esters.

∆δRSL1 ) δL1(R) - δL1(S) < 0

∆δRSL2 ) δL2(R) - δL2(S) > 0


esters of a marine metabolite40 show that the signalsfor protons H(1′), H(2′), H(4′), H(5′), H(6′), andMe(14′) result in negative ∆δRS values and, therefore,take the place of substituent L1 in the model shownin Figure 47. On the other hand, protons H(9′),H(10′), Me(12′), and Me(13′) give positive ∆δRS valuesand are therefore represented by substituent L2. Ascan be seen in Figure 47c, the ∆δRS values obtainedwith 9-AMA are far superior to those obtained usingMPA as the auxiliary (in some cases, by a factor of>4), although the signs are the same.

An experimental demonstration of the reliabilityof the configurational assignment obtained with theseAMAAs, and, consequently, of the model describedin Figure 46, was achieved by examination of a seriesof alcohols of known absolute configuration. In all thecases described41 in Figures 48 and 49, the ∆δRS signsare in accordance with the stereochemistry of thealcohol examined. The magnitude of the ∆δRS valuesfollows the order 9-AMA > 1-NMA > 2-NMA > MPA> MTPA.

The number of alcohols of known absolute config-uration used to validate 2-ATMA37 as the reagent ismuch lower than for the other reagents. In addition,neither structure calculations nor conformationalanalyses have been presented,37 although it does notseem unreasonable to believe that the model shouldbe the same as that presented for the other AMAAs.

On the basis of the nature and orientation of thearomatic ring in the AMAAs and its associatedmagnetic field, it was proposed that some of thesereagents should be particularly suited for studylinear alcohols and others should be suited for cyclicsystems.37b In this respect, the reagents 2-NMA and2-ATMA were proposed36,37 as being the most suitablefor the study of long-chain alcohols, on the basis ofthe expectation that, in the esters, the aromatic ringsof the reagent would lie over the chain of thesubstrate and thus cause significant shift changes onthe protons of a large portion of the chain (see Figure50). Reagent 9-AMA, on the other hand, was expectedto be more efficient when used in conjunction withcyclic alcohols.30

Nevertheless, comparison of the ∆δRS values ob-tained30,36,37 for the esters of 9-AMA, 2-NMA, and2-ATMA with the linear long-chain alcohols shownin Figure 51 demonstrates that the highest ∆δRS

values are always obtained when 9-AMA is used asthe reagent, whereas 2-NMA is the least-efficientreagent of the three.

A graphical representation of the ∆δRS valuesobtained for protons at different distances from theasymmetric C atom for the 9-AMA esters42 derivedfrom 51.1, 51.2, and 51.3, and of the 2-NMA and2-ATMA esters derived from 51.4 and 51.5, is shownin Figure 51. The spatial zone of maximum shieldingis much more concentrated in the 9-AMA esters thanin the other. In the 9-AMA esters, it is experiencedby protons 2-3 carbon bonds away from the asym-metric C atom, decreases rapidly with distance, andat approximately the fifth bond away shows ∆δRS

values equivalent to those produced by 2-NMA and2-ATMA.

In the esters formed with 2-NMA and 2-ATMA, thevariation of shielding intensity with distance is muchmore uniform and the maximum shielding zonecovers the region from approximately two carbonbonds away from the asymmetric C atom up toapproximately six bonds away.

In practice, when the assignment of the configu-ration of an alcohol is made difficult by substantialoverlap of the NMR signals, 9-AMA is better thanboth 2-NMA and 2-ATMA, because the intense, yetrapidly decaying, shielding zone ensures a wide

Figure 48. ∆δRS values for selected 9-AMA and MPAesters.


signal distribution across the spectrum for the rel-evant protons and eliminates signal overlap. Thereagents 2-NMA and 2-ATMA37 are less effective inthis respect, because a large number of protons inthe chain will be shielded to approximately the sameextent and, therefore, considerable signal overlap islikely.

This ability of 9-AMA to separate the signals of thealcohol portion is essential for the correct assignmentof the protons and calculation of the ∆δRS values andis not limited to linear substrates but can also be ofuse for cyclic alcohols.30 An illustrative example ofsuch a case is represented by cis-androsterone.

The 500 MHz NMR spectrum of cis-androsterone30

is shown in Figure 52b and those of its (R)- and (S)-9-AMA ester derivatives in Figure 52a and c, respec-tively. It is clear that an unambiguous assignmentof the signals in Figure 52b is impossible, becausemost of the signals are overlapped in a narrow rangeof ∼1 ppm. In contrast, the 9-AMA esters present anextraordinary dispersion of these signals, and thisphenomenon allows the complete assignment of allthe protons in the molecule.

As predicted, the maximum shielding is located inthe region near to the asymmetric center (rings A andB of the steroid) and is so intense that the chemical

Figure 49. ∆δRS values for selected 9-AMA, 1-NMA, 2-NMA, 2-ATMA, MPA, and MTPA esters of secondary alcohols.


shifts of protons H(1′), H(9′), H(6′), H(5′), and H(7′),which are indistinguishable in cis-androsterone itself,are moved to the right of TMS and is enough to moveprotons in the C ring.

In conclusion, replacement of the phenyl ring ofMPA by naphthyl or anthryl rings is shown to be auseful way to modify these reagents. Of all theAMAAs investigated, 9-AMA is the one that producesthe most marked changes in shifts and ∆δRS values,because (a) its aromatic system produces the mostintense magnetic field and (b) the population of the

sp conformer in its esters is greater than that in theother AMAAs.

3.1.4. Other ReagentsIn addition to the reagents described thus far,

many other compounds have been proposed as CDAsfor the assignment of configuration of secondaryalcohols by NMR. Most of the methods reported inthis section use 1H and 13C NMR, with only a fewsmostly those devoted to e.e. calculations rather thanabsolute configuration assignmentsinvolving 19F

Figure 50.

Figure 51. Variation of ∆δRS with the distance to the asymmetric C atom in 9-AMA, 2-NMA, and 2-ATMA esters oflineal alcohols.


NMR. Generally, these reagents contain a magneti-cally anisotropic group (e.g., phenyl, naphthyl), whichis responsible for the shifts experienced by substit-uents L1/L2, a polar group (e.g., OMe, F) that favorsa certain conformation, and an additional functionalgroup for bonding to the substrate. This linkage is,in most cases, an ester bond, although there areexamples where an ether, a glycoside, or a phospha-mide bond have been used.

In contrast to the cases described for MPA, MTPA,and 9-AMAA, only a few of the procedures describedin this section involve conformational analysis and/or theoretical calculations to support the results. Forthis reason, the reliability of the configuration as-signed by these procedures relies essentially on thenumber and structural variety of the alcohols ofknown absolute configuration used to validate themethod/reagent.

3.1.4.1. 2-Phenylbutyric Acid: Helmchen’sMethod. The commercially available (R)- and (S)-2-phenylbutyric acids ((R)- and (S)-PBA, respectively;see Figure 53a), which are well-known in the kineticmethod described by Horeau,43 have been proposedby Helmchen et al.44 as CDAs for secondary alcohols,and this method has since been extended to assignthe stereochemistry of amines and thiols.

Helmchen’s procedure is similar to that describedby Mosher and consists of the preparation of theesters of the alcohols with the two enantiomers of thereagent (PBA) and subsequent comparison of the 1H

NMR spectra of the diastereomeric esters. The signsof the resulting ∆δRS values are then used to locate,in space, the substituents L1 and L2, in accordance

Figure 52. 1H NMR spectra of cis-androsterone (b) and its (R)-9-AMA (a) and (S)-9-AMA esters (c).

Figure 53. (a) Structure of (R)-PBA and (b) model for theassignment of the configuration of secondary alcohols fromthe NMR spectra of the PBA esters.


with an empirical model (see Figure 53b). Theempirical model was designed to correlate the NMRresults of many secondary alcohols of known absoluteconfiguration with the stereochemistry. Table 5shows a selection of secondary alcohols of knownabsolute configuration, along with the resulting ∆δRS

values, that have been used to validate this proce-dure. This model has also been applied, withoutmodifications, to the assignment of the configurationsof amines45a and thiols.45b

Despite the very small number of substrates ofknown absolute configuration originally used for thevalidation of PBA as a CDA,45 as well as the absenceof other studies that could lend support to the model,PBA has been frequently used to deduce the config-uration of natural products.46 Examples (Figure 54)include the following: the fungus metabolites chryso-gine, which is isolated from Fusarium sambucinum;46a

ripostatin A, B, and C, which is isolated from Sor-angium cellulosum;46b massarinolin B methyl ester46c

(from Massarina tunicata); and streptoketol A46d

(from an Streptomyces sp).The applicability of this reagent for the assignment

of the configuration of secondary alcohols using 13CNMR instead of 1H NMR has also been investigated.47

However, in this case, the number of alcohols ofknown absolute configuration studied as test com-pounds is very limited and more examples should beanalyzed before the procedure is recommended.

More recently, the possibility of using the isomer3-phenylbutyric acid (3-PBA) as a CDA has beenevaluated48 (Figure 55a). However, the presence ofthe phenyl group and asymmetric center at positionâ, rather than at position R (as in PBA, MPA, MTPA,or 9-AMA), and the higher conformational freedominherent in this system leads to a reduction of thearomatic shielding/deshielding on substituents L1/L2,which is a situation that gives smaller ∆δRS values.In fact, comparison of the ∆δRS values obtained forthe ester derivatives of diacetone-D-glucose and 2-bu-tanol with 3-PBA, MPA, and MTPA as reagentsshowed that the smallest shifts were obtained with3-PBA.

The model presented in Figure 55b is empirical andwas designed to correlate the NMR spectra of the3-PBA esters of just five alcohols of known absoluteconfiguration with their stereochemistry.

The authors assume that the most representativeconformation is the one in which the H atoms at theâ-carbon of the acid, the carbinol, and the carbonylgroup are syn-periplanar. In accordance with thismodel, substituent L1 should be shielded in the (R)-ester, while substituent L2 is shielded in the (S)-ester;therefore, ∆δRS must be negative for substituent L1and positive for substituent L2.

In conclusion, the applicability of both PBA and3-PBA as CDAs is limited by several considerations:(a) the small number of substrates of known absoluteconfiguration that have been used to validate theassignment, (b) the absence of other experimental ortheoretical studies that might support the correlationmodels, and (c) (in the case of 3-PBA) the small ∆δRS

values.3.1.4.2. Naphthylpropionic Acids. In the past

few years, several naphthylpropionic acids have beeninvestigated as CDAs.49 One of the most commonlyused is 2-methoxy-2-(1-naphthyl)propionic acid(MRNP; see Figure 56), which is already known as aderivatizing agent for HPLC resolution of alcohols.50

In the (R)-MRNP ester of the alcohol shown inFigure 57, substituent L1 experiences a shieldinginfluence from the naphthyl group, whereas, in the(S)-MRNP ester, the shielded group is substituent L2.Therefore, substituent L1 results in a negative ∆δRS

value and substituent L2 yields a positive ∆δRS value.

Table 5. ∆δRS Values Obtained for the (R)- and(S)-PBA Esters of a Selection of Secondary Alcoholsof Known Configuration

L1 L2 PBA

C6H13a δ 1.11 CH3 δ 1.16 R

δ 1.28 δ 1.05 S∆δ -0.17 ∆δ 0.11

C6H5 CH3 δ 1.45 Rδ 1.35 S∆δ 0.10

COOEt δ 1.09/4.04 CH3 δ 1.41 Rδ 1.23/4.15 δ 1.36 S∆δ -0.14/-0.11 ∆δ 0.05

bornylb δ 0.63 Rδ 0.81 S∆δ -0.18

a Shift of the CH2 group. b Signal corresponding to Me(10).

Figure 54. Natural products of microbial origin studiedby Helmchen’s method.


This model was derived from the study of just fivealcohols of known absolute configuration, and thestructures and ∆δRS values for these are shown inFigure 58.49a

The authors proposed a variant using the racemicmixture of the auxiliary (()-MRNP. In this case, themixture of MRNP esters first should be separated anda small sample of one diastereoisomer transformedto the corresponding methyl ester. Inspection of theCD spectra allows the identification of the (R)- and(S)-MRNP esters (the (S)-MRNP has a negative CDband and the (R)-MRNP has a positive band at 280nm). After the (R)- and the (S)-MRNP esters havebeen identified, their NMR spectra are recorded and

the model shown in Figure 57 allows the configura-tion to be assigned.49a

In addition to MRNP, the potential of some othernaphthylpropionic acids to act as CDAs has beenevaluated using (-)-menthol as a test compound, onthe assumption that the correlation model of MRNPis still valid.

Table 6 shows the ∆δRS values obtained for the (-)-menthol esters of 2-hydroxy-2-(1-naphthyl)propionicacid49c (HRNP), 2-hydroxy-2-(2-naphthyl)propionicacid (HâNP), and 2-methoxy-2-(2-naphthyl)propionicacid49d (MâNP), as well as, for comparative purposes,those obtained with several AMAA reagents de-scribed previously in this reviewsincluding MPA andMTPA.

Examination of the data shows that the ∆δRS

values of the naphthylpropionic acids mentioned inthis section follow the order HRNP > MRNP > HâNP> MâNP, and, in fact, the values for MâNP are verysmall and the distribution of the signs is irregular(for example, see H(10′)).

Figure 55. (a) Structure of (R)-3-PBA and (b) model for the assignment of the configuration of secondary alcohols fromthe NMR spectra of the 3-PBA esters.

Figure 56. Structure of the 2-methoxy-2-(1-naphthyl)-propionic acids.

Figure 57. Model for the assignment of configuration byNMR of MRNP esters.

Figure 58. Alcohols of known configuration studied byNMR of their MRNP esters and the resulting ∆δRS values.


Comparison of these reagents with the AMAAsystems discussed previously shows that the anthrylreagent 9-AMA produces the highest ∆δRS values ofall, with the second-highest values resulting from thenaphthyl acid, 1-NMA. In addition, comparison be-tween the 1-naphthyl-substituted reagents MRNPand 1-NMA indicates that 1-NMA is the more effec-tive reagent. Similarly, among the 2-naphthyl-substituted systems, it is evident that 2-NMA is moreeffective than MâNP and this clearly suggests thatthe replacement of the H atom in the R-position by amethyl group (as in MRNP and MâNP) leads to asignificant loss of efficacy of the reagents sprobablya consequence of a reduction in the energy differencebetween the conformers.

In conclusion, the use of naphthylpropionic acidsas CDAs is limited by (a) the small number ofsubstrates of known absolute configuration used tovalidate the method and (b) the absence of informa-tion about their conformational characteristics. Theshifts obtained are smaller than those observed forother, better-known naphthyl-substituted reagentsand, therefore, do not provide any advantage.

3.1.4.3. Axially Chiral CDAs (MBNC andMNCB). Several compounds of the binaphthyl andbiphenyl classes with axial chirality have beenproposed as CDA reagents.51 The most important

examples are 2′-methoxy-1,1′-binaphthyl-2-carboxylicacid (MBNC) and 2-(2′-methoxy-1′-naphthyl-)-3,5-dichlorobenzoic acid (MNCB). These compounds havebeen evaluated for the assignment of the configura-tion of secondary alcohols by means of 1H and 13CNMR spectroscopy51a,b (see Figure 59).

This protocol is not unusual in any respect andconsists of the derivatization of the substrate withthe two enantiomers of the reagent and subsequentcomparison of their 1H or 13C NMR spectra.

The interpretation of the NMR data follows themodel shown in Figure 60. Thus, in the case of theesters of MNCB (Figure 60a), the most representativeconformer is assumed to be that which the phenylgroup of the acid (Ar1), the carbonyl group, and thehydrogen bond to the chiral center of the alcohol arein the same plane (plane “CB”). In addition, the Ar1-Ar2 bond and the CdO group should be anti-peri-planar, with the aromatic rings (Ar1, Ar2) perpen-dicular to each other.

This geometry allows the naphthyl ring to transmitits shielding to the substituents of the alcohol but,unfortunately, not in a very selective manner, be-cause both substituents in each conformer areshielded, although one is slightly more affected thanthe other.

Table 6. Structure of the Naphthylpropionic AcidsEvaluated as CDA Reagents and the ∆δRS ValuesObtained for Their Esters with (-)-Menthol andThose of Other AMAAs

Figure 59. Structure of axially chiral acids MBNC andMNCB.

Figure 60. Model for the assignment of the configurationof alcohols using the NMR of their MBNC and MNCBesters.


The signal for Ha appears in the (aS)-MNCB esterat higher field than in the (aR)-MNCB ester; thereverse situation is observed for Hb. In this way, theresulting differences (∆δ ) δaS - δaR) are ∆δHbSR

> 0 and ∆δHaSR < 0.Figures 61 and 62 show the structures of the

alcohols of known configuration used to validate themodel and the corresponding ∆δSR values (1H and 13CNMR data). The proton NMR data of the linealalcohols are in good agreement with the model;however, the use of cyclic alcohols results in an

irregular distribution of signs for ∆δSR (see 61.8, 61.9,and 61.12).

In regard to the 13C NMR data (Figure 62), theresulting ∆δSR values are too small to ensure a safeassignment. A similar situation had been observedpreviously with other reagents (MTPA; see Section3.1.1.4) and this is not surprising, given the lowsensitivity of 13C chemical shifts to the anisotropiceffect.

The ∆δ values shown in Figure 61 are clearlysmaller than those obtained with other naphthyl-containing reagents described in this review, such asthe AMAAs (1-NMA and 2-NMA). The explanationfor this less-marked effect is evident after performingcalculations51c on the structure and conformation ofthe compounds in question. For example, molecularmechanics (MM), semiempirical (PM3 and AM1), andab initio calculations have revealed that these com-pounds exist in solution as two main conformers inequilibrium: the sp conformer (with the Ar1-Ar2bond and the CdO group syn-periplanar; see Figure63) and the ap conformer (where the aforementionedgroups are anti-periplanar). In both cases, the Ar1ring, the CdO group, and the C(1′)-H bond remaincoplanar, whereas Ar2 is perpendicular to Ar1.

Although the ap conformer has a better geometrythan the sp conformer, in terms of transmission ofthe effect of Ar2 to the substituents of the alcohol(R1/R2), the calculations reveal that the most stableform is the sp conformer, in which the aromatic ringis poorly oriented for transmission purposes.

Despite the fact that the ap conformer is lessabundant, it is considered to be the most importantconformer, in terms of NMR shifts, and its use in thecorrelation model shown in Figure 60 is justified bythe results of NMR studies at low temperature andby aromatic shielding calculations. A second aspectthat must be considered concerns the fact that inneither of the two conformers can the aromatic ringproduce selective shielding on just one of the sub-stituents (R1/R2), which is a situation that is contraryto that observed for the AMAA derivatives. In thecase in question here, both substituents are moder-ately shielded in the ap conformer, whereas in thesp conformer, they are only slightly shielded.

These two points explain the small shifts obtainedwith these reagents and the need for more efforts toimprove their performance. With this intent in mind,the reagent 1-(10-bromo-1-anthryl)-2-naphthoic acid(BANA, Figure 64) was prepared and its ability toproduce separate signals for diastereomeric (-)-menthol esters was evaluated. Although the ∆δSR

values obtained are slightly better than those ob-tained with MNCB, they are still too small to justify

Figure 61. Secondary alcohols and ∆δ (1H NMR) valuesobtained from their MNCB (plain, ∆δSR) and MBNC (inparentheses, ∆δRS) esters.

Figure 62. Secondary alcohols and ∆δ (13C NMR) valuesobtained from their MNCB (∆δSR) and MBNC (∆δRS) esters.MBNC data given in parentheses.

Figure 63. Conformational equilibrium in the esters ofMNCB.


the extensive use of these reagents for the assign-ment of secondary alcohols.

3.1.4.4. Difluorodinitrobenzene. An empiricalmethod based on the use of 1,5-difluoro-2,4-di-nitrobenzene (FFDNB) as a CDA for the assignmentof secondary alcohols and primary amines has beendescribed.52 In the case of alcohols52 (the applicationto amines is reviewed in Section 3.4.5.1), the sub-strate and the difluorodinitrobenzene reagent arelinked through an ether bond and a second auxiliaryreagent is then introduced. Therefore, the procedurefor derivatization entails the reaction of the chiralalcohol with FFDNB and the resulting fluoro-dini-trobenzene (FDNB) ether is subsequently made toreact separately with the two enantiomers of 1-phe-nylethylamine (second auxiliary). The resulting di-astereomeric alcohol-DPEA derivatives are thenexamined by NMR (Figure 65).

Interpretation of the NMR spectra of the deriva-tives and the assignment of the configurations of thealcohols are based on a model proposed by theauthors (based on fast atom bombardment (FAB), UVand 2D NMR spectra, and NOE experiments) inwhich the proton on the asymmetric C atom of thealcohol and H(6) on the benzene ring are coplanar(see Figure 66). According to this conformation, theDPEA derivative containing (R)-1-phenylethylaminehas substituent L2 shielded by the phenyl group ofthe amine, whereas, in the derivative containing the(S)-1-phenylethylamine fragment, it is substituent L1that is shielded. Consequently, the difference in thechemical shifts (∆δ ) δR - δS) for an alcohol withthe stereochemistry shown in Figure 66 is negativefor substituent L2 and positive for substituent L1.

This model has been experimentally assessed withfive alcohols of known absolute configuration (Figure67). The signs of ∆δ are in good agreement with thestereochemistry and the shifts are large. However,note that the number and structural variety ofcompounds used to validate the method are too smallto guarantee its general applicability to other sub-strates. This fact, together with the two-step deriva-tization procedure, represents the main limitation ofthis reagent.

3.1.4.5. 5(R)-Methyl-1-(chloromethyl)-2-pyrro-lidinone. 5(R)-Methyl-1-(chloromethyl)-2-pyrrolidi-none (68.1) has been proposed53 as an auxiliaryreagent for absolute configuration (and % ee) deter-minations of chiral secondary alcohols by the forma-tion of the corresponding N-(alkoxymethyl)-2-pyrro-lidinone derivative 68.2 (see Figure 68).

Theoretical calculations have shown that thosederivatives exist in equilibrium between two confor-mational forms obtained by rotation of the CH2ORunit around the C-N bond. The more stable one hasan NCOC angle of ∼90°, whereas the conformationwith a linear arrangement (NCOC angle of ∼180°)has greater energy. In both, the lactam ring is

Figure 64.

Figure 65. Derivatization procedure for secondary alco-hols.

Figure 66. Conformational model and predicted ∆δ signsfor the assignment of the configuration of secondaryalcohols from the NMR of their alcohol-DPEA derivatives.

Figure 67. Secondary alcohols of known absolute config-uration and ∆δ values obtained from their alcohol-DPEAderivatives.

Figure 68. Preparation of N-(alkoxymethyl)-2-pyrrolidi-none derivatives.


essentially planar and the methyl group has apseudo-axial orientation.

In accordance with that prediction, experimentaldata indicated that the NMR shifts of the NCH2Oprotons are determined by the anisotropic effect ofthe CdO bond and the polar effect of the O atom ineach conformer. Under those conditions, a relation-ship between the chemical shifts and the relativeposition of those protons, with respect to the carbonylgroup, and the absolute stereochemistry of the de-rivatives can be established. In fact, application ofthe proposed model to six chiral alcohols (Figure 69)predicted the correct configuration.

The authors warn about the convenience of work-ing with both diastereomeric derivatives; that is tosay, both enantiomers of the chiral alcohol arenecessary. This is a clear limitation of the method,and the report explicitly says that if only one enan-tiomer of the alcohol is available for derivatization,assignment of the correct absolute configuration isrisky. Nevertheless, the procedure works well whensubstituents L1 and L2 are clearly different in size.

3.1.4.6. Diazacyclophosphamides. The use ofdiazacyclophosphamide chlorides as reagents forsecondary alcohols and primary amines has beendescribed54 (see Figure 70a). In this procedure, thelinkage between the substrate and the reagent is aphosphate or phosphamide bond and the configura-tion of the substrate is determined by 1H NMR onthe basis of the models indicated in Figure 70b.

This method is totally empirical and conforma-tional studies have not been performed to support themodel shown. In addition, the number of substratesused to validate this model is extremely small and

the NMR data are very limited: Only two alcoholssone primary and the other secondarysand sevenamines have been studied, and, even then, only theshift of one of the substituents of the substrate (L1or L2) is considered.

A model for the correlation of the 31P NMR shiftswith the configuration is not proposed by the authors;therefore, the best application of this method is inthe field of e.e. calculation rather than the assign-ment of configuration.

3.1.4.7. Tetra-O-benzoylglucosyl Bromides: TheGlucosylation Method. There have been severalattempts to use sugars as CDAs for the assignmentof the configuration of alcohols that are derivatizedas glycosides55-57 and examined by 13C and 1H NMR.In a few cases, a single derivative is used55 (compari-son of the spectra of the alcohol with that of thederivative) and these will be discussed in the corre-sponding section (Section 4.1.2). In this part of thereview, however, we are only concerned with casesin which the assignment is performed by comparisonof the L and D glycosides.56

The most commonly used of these proceduresconsists of the preparation of the tetra-O-benzoyl-â-D-glucopyranoside and tetra-O-benzoyl-â-L-gluco-pyranoside of the alcohol and comparison of their 1HNMR spectra.56

The interpretation of the spectra and the configu-rational assignment is based on the observation that,in both derivatives, protons anti to the endocyclicoxygen (substituent L2 in â-D-glucopyranoside; seeFigure 71) are shielded, probably because of the effectof the benzoyl group at C-2, whereas those with thesyn arrangement (substituent L1 in â-D-glucopyra-noside) are deshielded, presumably because they areclose to the endocyclic O atom (O-5).

In this way, the differences in the chemical shifts(∆δ ) δD - δL) for an alcohol with the configurationshown in Figure 71 are negative for substituent L2and positive for substituent L1.

Nine alcohols of known absolute configuration werestudied, and these are shown in Figure 72. Theresults obtained are in agreement with the predic-

Figure 69. Alcohols of known absolute configurationstudied by this method.

Figure 70. Structure of the diazacyclophosphamide chlo-rides and correlation model for configurational assignmentof alcohols.

Figure 71. Structure and correlation models of â-D- andâ-L-glucopyranosides.


tions and, therefore, validate the method. Only in themethylene group of (-)-methyl-3-hydroxybutyrateare the signs unexpected, and this is probably dueto the anisotropic action of the two carbonyl groups.

The authors also mention that a correlation ap-parently exists between the chemical shift of theproton at C(1′)H, the “carbinylic carbon”, and theabsolute configuration: For the derivatives with theR configuration, ∆δ is positive, whereas ∆δ is nega-tive when the configuration at that C atom is S (seeTable 7). Unfortunately, cholesterol and cholestanolare two exceptions to this rule, and the authorsexplain this phenomenon as being due to the absenceof substituents in the â position.

3.1.4.8. Fucofuranose Tetra-O-acetate: TheFucofuranoside Method. A procedure has beendescribed57 that involves comparison of the NMRspectra of the â-D- and â-L-fucofuranosides (Figure73) in chloroform or pyridine as the solvent and theapplication of totally empirical rules (see Figure 74).

These rules are based on the study of seven examplesof known absolute configuration (Figure 75), althoughthe range of structures is not very diverse (six arecyclic alcohols and just one is an open-chain alcohol)and, as such, the use of this reagent for the assign-ment of the configuration of a secondary alcohol isnot yet fully established. In addition, the preparationof the auxiliary reagent and the derivatization of thealcohol are more complex and time-consuming thanthat with glucopyranosides or AMAAs. Finally, theshifts obtained with the fucofuranosides are smallerthan those obtained with the tetra-O-benzoylgluco-sides, and, for this reason, its use does not offer anyadvantages.

If the configurational assignment is to be per-formed using 1H NMR spectroscopy, the spectra ofthe â-L-fucofuranoside and the â-D-fucofuranoside aretaken in pyridine and the differences between thechemical shifts (∆δH

p) is calculated (∆δ ) δL - δD).In the case where the alcohol has the configurationshown in Figure 74, substituent L1 should result ina positive ∆δH

p and substituent L2 should result in anegative value. In this procedure, only the signs forthe protons on the â-carbon of substituents L1/L2 areconsidered.

Figure 72. Secondary alcohols of known absolute config-uration studied as tetra-O-benzoyl-â-glucopyranosyl de-rivatives and the corresponding ∆δ values.

Figure 73.

Table 7. Relationship between the AbsoluteConfiguration of the Carbinylic Carbon and the Signof ∆δ

alcoholcarbinyl carbon

configuration (C1′) δD - δL

(-)-2-octanol R +0.09(+)-2-octanol S -0.09(-)-menthol R +0.16(+)-menthol S -0.16(-)-neomenthol R +0.16(+)-neomenthol S -0.16(-)-borneol R +0.23(+)-borneol S -0.23(3b)-cholesterol S 0.00(3b)-cholestanol S 0.00(17b)-testosterone S -0.03dimethyl-D-malate R -0.14dimethyl-L-malate S +0.14(-)-methyl 3-hydroxy-butyrate R +0.02(+)-methyl 3-hydroxy-butyrate S -0.02

Figure 74.


Figure 75 shows the structures of the compoundsstudied by this method, along with the corresponding∆δH

p data. It can be seen that, with the exception offurospongin 1, the signs of ∆δH

p for the protons onthe â-carbons correlate well with the stereochemistry.However, the other protons do not show any correla-tion and different signs are obtained for protons inthe same substituent.

A study by the same authors on a series of just fourcompounds (Figure 76) led them to suggest that therecould be a correlation between the configuration andthe signs of ∆δH

p for the protons on the asymmetriccarbon of the alcohol (carbinyl protons, C(1′)H): thesign of ∆δH

p is positive for those classified as S-typealcohols and negative for R-type alcohols. The smallnumber of examples investigated, along with the lackof studies on the fundamental aspects of this method,represents a clear limitation for the general ap-plicability of this approach.

A procedure very similar to that previously de-scribed but involving 13C NMR shifts has beendescribed, and the results are represented in Figure77. Once again, only the shifts of the C atoms at the

â-position are considered; because the signs for theother C atoms are unreliable, the absolute values arevery small and, furthermore, the degree of substitu-

Figure 75. ∆δHp values observed for â-D- and â-L-fuco-

furanoside derivatives.

Figure 76. ∆δHp for the carbinyl protons.

Figure 77. Chemical-shift differences (13C NMR, ∆δC) inpyridine-d5 (∆δC

p) and CDCl3 (∆δCc) of â-D- and â-L-

fucofuranosides. CDCl3 data given in parentheses.


tion at that C atom should be taken into consider-ation for the assignment.

3.1.4.9. Noe Lactols. The lactols derived fromcamphor are known as Noe lactols, and these havebeen used as CDAs for secondary alcohols58 in whichone of the substituents (L1 or L2) is a phenyl, nitrile,ethynyl, or formyl group. One example of this typeof reagent is (+)- and (-)endo-octahydro-7,8,8-tri-methyl-4,7-4,7-methanobenzofuran-2-ol ((+)- and(-)-endo-MBF-OH), the structure of which is shownin Figure 78.

The method is totally empirical and consists of thederivatization of the alcohol with the two enanti-omers of the reagent MBF-OH, followed by analysisof the 13C or 1H NMR spectra of the resultingdiastereomeric lactols.

The stereochemistry at carbon 2 (Figure 78) in thestarting hemiketal is not fixed; therefore, when singleenantiomers of the auxiliary and the substrate aremade to react, two epimers at C-2 result (types A andB). The predominant epimer in the mixture is thetype-A lactol (see Figure 79).

The four stereoisomers obtained from a singleenantiomer of the auxiliary and the two enantiomers(named A and B) of the alcohol are represented inFigure 79 and classified according to their origin asAA, AB, BB, and BA.

The assignment of the configuration involves de-termining the differences in the chemical shifts (δAB

- δAA) of protons H(7a), H(2) in the auxiliary portionand H-C* in the alcohol portion (Figure 79). Alter-natively, the corresponding C-atom signals can beconsidered. The chemical-shift differences should becalculated and the signs interpreted, using the so-called “b-pl rules” (where b denotes bulky and pldenotes plane/lineal) indicated in Table 8.

For example, starting from the lactol MBF-OH oftype A and a secondary alcohol of unknown config-uration, we can obtain two derivatives, arbitrarilydesignated as A-A and A-B (see Figure 79a). If thedifferences ∆δ ) δΗAB - δΗAA obtained are as shownin Table 8, derivative AA is formed from an alcoholwith configuration type A and the derivative denotedas AB comes from the enantiomeric alcohol of con-figuration B. If the ∆δ signs obtained were oppositeto those shown in Table 8, the configuration of thealcohol would also be opposite.

The chemical shifts (1H and 13C NMR) and theirdifferences (∆δ) are shown in Table 9 for the threemost-relevant signals of a series of alcohols of knownabsolute configuration. The results obtained validatethe rules for the assignment previously discussed andindicate that the method works well with secondaryalcohols containing aryl, heteroaryl, nitrile, carbonyl,carboxyl, or ethynyl groups directly bonded to theasymmetric center (the pl substituent), as well as incases where the substituents can be easily classifiedaccording to their “b-pl” character.

Figure 78.

Figure 79.

Table 8. The “b-pl” Rules for the Assignment ofConfiguration from Noe Lactols

∆δ ) δHAB - δHAA

pl 2-H 7a-H C*-H

aryl >0 <0 <0CN, CO, CtC <0 >0 <0


These limitations certainly restrict the use of Noe’smethod for other alcohols but, at the same time,makes this procedure advantageous, because it al-lows the assignment of the configuration of alcoholsthat do not have protons in one of the substituentsL1/L2sa case where the classical Mosher methodcannot be used (see Section 3.1.1).

3.1.4.10. Fluoroacetic Acids. As mentioned pre-viously in this review, with the exception of MTPA,very few reagents that contain F atoms have beenexplored59,60 as auxiliary reagents for the assignmentof the absolute configuration of substrates through19F NMR. In general, the structures of these reagentsare derived from that of MTPA (Figure 80) and,although some have been successfully used for thedetermination of the e.e. of alcohols, their applicationas CDAs has been hampered by the absence of a trulygeneral model that can be applied to any othersubstrate.

In a study describing the application of AFPA (2-fluoro-2-phenylacetic acid) to secondary alcohols,60a-d

the authors indicate that the corresponding estersexist in two conformations (I and II). However,

although conformer I is the most representativespecies from the standpoint of 1H NMR, only con-former II is used as a model (see Figure 81) for thecorrelation of the 19F shifts with the configuration ofthe alcohol. This situation arises because it is thisconformer in which the F atoms are subjected to theelectronic effects of substituents L1/L2 of the alcohol(the γ-gauche-type effect). Figure 82 shows a selectionof the substrates of known absolute configurationthat produce 19F shifts coherent with the modelindicated.

Another fluorine-containing compound used forthis purpose is R-cyano-R-fluoro-p-tolylacetic acid60e

(CFTA). The structure of this compound is shown inFigure 80, and it is used for secondary alcohols (1HNMR) and amines (19F NMR; see Section 3.4.5.1). Theprocedure requires the preparation of the esters fromthe two enantiomers of CFTA and measurement ofthe ∆δSR value (δ(S) - δ(R)) for the substituents (L1/L2) of the alcohol. The assignment is based on thefact that, in the ester derived from (S)-CFTA, sub-stituent L1 is shielded by the fluorophenyl groupwhereas in the (R)-CFTA ester, the shielded sub-stituent is L2 (see Figure 83).

Figure 84 shows the structures of the compoundsof known absolute configuration used to validate thisprocedure, along with the ∆δSR values that areobtained. The data demonstrate that, in all cases, thesigns of ∆δSR are coherent with the configuration andthe model.

Table 9. Selected 1H and 13C NMR Data for the NoeDerivatives of Secondary Alcohols of Known AbsoluteConfigurationa

hydrogen carbon

2H 7a-H C*-H C-2 C-7a C*

b ) CH3, pl ) CNδ 5.38 4.42 4.30 108.9 90.1 60.5δ 5.51 4.19 4.54 106.5 90.3 59.0∆δ -0.13 0.23 -0.24 2.4 -0.2 1.5

b ) C6H5b, pl ) CN

δ 5.35 4.51 5.33 108.1 90.6 66.2δ 5.71 4.23 5.51 107.0 90.8 65.6∆δ -0.36 0.28 -0.18 1.1 -0.2 0.6

b ) CH3, pl ) CtCHδ 5.45 4.38 4.40 106.6 89.3 61.4δ 5.61 4.21 4.46 105.5 89.3 60.1∆δ -0.16 0.17 -0.06 1.1 0.0 1.3

b ) C6H5b, pl ) CtCH

δ 5.23 4.40 5.37 105.9 89.7 66.9δ 5.82 4.22 5.45 105.7 89.7 66.2∆δ -0.59 0.18 -0.08 0.2 0.0 0.7

b ) CH3, pl ) CHdOδ 5.39 4.31 3.94 108.7 90.2 78.0δ 5.44 4.27 4.17 107.9 89.9 76.3∆δ -0.05 0.04 -0.23 0.8 0.3 1.7

b ) C6H5b, pl ) CHdO

δ 5.38 4.37 5.00 107.7 90.4 83.2δ 5.58 4.07 5.14 107.6 90.1 81.6∆δ -0.20 0.30 -0.14 0.1 0.3 1.6

b ) CH3, pl ) C6H5b

δ 5.49 3.95 4.73 106.7 89.5 73.0δ 5.12 4.32 4.78 105.6 89.0 72.9∆δ 0.37 -0.37 -0.05 1.1 0.5 0.1

b ) i-Pr, pl ) C6H5b

δ 5.40 3.61 4.07 109.1 89.1 84.7δ 5.08 4.30 4.26 105.3 89.3 82.1∆δ 0.32 -0.69 -0.19 3.8 -0.2 2.6

a In the table, “b” denotes bulky and “pl” denotes plane/lineal. The first-line δ value indicates Type AB, whereas thesecond-line d value denotes Type AA; the third line is ∆δ. b Inthis case, because both substituents are plane/lineal, thephenyl group at the â position is considered to be the bulkygroup.

Figure 80. Structures of R-fluoroacetic acids explored asCDAs.

Figure 81. Main conformations of the esters of AFPA.

Figure 82. Secondary alcohols studied by NMR of theirAFPA esters.


Theoretical calculations on the geometry and con-formational composition of these CFTA esters, usingab initio (GAUSSIAN 98, RHF/6-31+G(d)) methods,revealed that there is an equilibrium between twomain rotamers around the CR-CdO bond. One

rotamer, denoted sp, has the C-F and CO bonds ina syn-periplanar arrangement and the other, desig-nated ap, contains the aforementioned bonds in ananti-periplanar relation. According to the calcula-tions, the most stable conformer is sp, and this isrepresented in Figure 83.

The 19F NMR spectra of these esters have also beenreported; however, a convincing model that allows thecorrelation of the absolute configuration of the alcoholwith the signs of ∆δSR has yet to be proposed.

3.1.4.11. Chiral Silylating Reagents. Attemptsto use chiral silylating reagents to determine theabsolute stereochemistry of chiral allylic alcohols ledto the proposal of (+)- and (-)-chloromenthoxydiphen-ylsilanes and (+)- and (-)-2-bromo-R-methylbenzyl-oxychlorodiphenylsilanes as CDAs.61 Although theyshowed promising results, the small number ofalcohols tested (two and one examples, respectively)prevents their use as a general method.

3.2. Application to â-Chiral Primary AlcoholsThe extension of the NMR methods for the assign-

ment of the configuration of secondary alcoholsdescribed previously to other substrates is particu-larly appealing, because systems such as primaryalcohols with the chiral center in the position â tothe OH group, are frequently found in nature inchiral forms or are obtained by a reduction of thecarboxylic acids and aldehydes.

Figure 83.

Figure 84.

Figure 85. Empirical models for the determination of the absolute configuration of primary alcohols by NMR of theirMTPA esters in the presence of Eu(fod)3. Rm represents the medium-sized group and Rl represents the large-sized group.


Two main difficulties are apparent when dealingwith primary alcohols:

(1) The distance between groups L1/L2 of thesubstrate and the aryl ring of the auxiliary reagentis greater than that in secondary alcohols, becausethe asymmetric center is at the â-position, and

(2) The presence of an extra C-C bond betweenthe chiral center and the auxiliary reagent reducesthe conformational preference by increasing therotational freedom. As a result, the ∆δRS values forprimary alcohols would be expected to be smaller andless reliable than those usually obtained for second-ary alcohols.

Very few papers have been published on thisparticular subject. MTPA and recently 9-AMA are thereagents most amply used in the assignment ofabsolute configuration of such substrates.

3.2.1. MTPAA quarter of century ago, Yasuhara and Yama-

guchi62a developed a procedure that is based on theuse of a shift reagent with MTPA derivatives for thedetermination of the absolute configuration of pri-mary carbinols with a chiral center at the â-position.This approach was an extension of a previous method

Figure 86. Lanthanide-induced shift (Eu(fod)3; ∆LISOMe) LISA - LISB) obtained for the OMe group of diatestereo-meric (R)-MTPA esters.

Figure 87. Natural compounds studied with MTPA and steroidal side chains obtained by synthesis and used as models.


used for secondary alcohols4a,62b,c and consisted of theconversion of a mixture of the two enantiomers of aprimary alcohol into the corresponding mixture of(R)-(+)-MTPA esters. The magnitude of the lan-thanide-induced shift (LISOMe) produced by Eu(fod)3on the OMe group of the MTPA ester showed acorrelation with the absolute configuration. Thediastereomeric (R)-(+)-MTPA esters with the largerLISOMe value had configuration A and the diastere-omer with the smaller LISOMe value had configurationB (see Figure 85).

The results were rationalized in terms of themodels shown in Figure 85, where the complexationconstant KA is larger than KB, because of the less-marked steric interaction between Eu(fod)3 and theH atom at the chiral center in A′ than with Rm in B′.The fact that the magnitudes of ∆LISOMe for the

esters of 2-phenylethanols with bulky Rm groups arelarger than those with smaller Rm groups is consis-tent with the model. The procedure was tested on the12 substrates shown in Figure 86, where the chiralcenter is not present as part of a ring in any of thecases.

The application of this method to cyclic alcoholswas explored by Takahashi et al.63a with the terpenes87.1 and 87.2 shown in Figure 87. The compoundswere transformed to the corresponding (R)- and (S)-MTPA esters and the resulting ∆LISOMe valuesindicated configurations that were in agreement withthe known configurations deduced by alternativecorrelation procedures.

The same method has also been applied to thedetermination of the stereochemistry of a variety ofsteroidal side chains. Minale et al.63b,c assigned the

Table 10. Selected NMR Data (CD3OD) for Side-Chain Signals in Synthetic 24-Methyl-26-hydroxy Steroids and inNatural Compounds 87.6 and 87.7

compound 26-CHH2 27-CH3 28-CH3 C-24 C-27 C-28(R)-MTPA ester

(26-CH2)

∆22 Series(87.8a) (24R,25S) 3.28 dd, 3.60 dd 0.90 d 0.95 d 39.7 13.8 17.1 4.19 dd, 4.31 dd(87.9a) (24S,25R) 3.29 dd, 3.59 dd 0.90 d 0.97 d 39.5 13.8 16.8 4.13 dd, 4.38 dd(87.9b) (24S,25S) 3.34 dd, 3.53 dd 0.87 d 1.02 d 39.2 13.6 19.0 4.21 br d(87.8b) (24R,25R) 3.34 dd, 3.52 dd 0.88 d 1.02 d 39.3 13.6 19.2 4.16 dd, 4.21 dd

Saturated Series(87.10a) (24R,25S) 3.38 dd, 3.55 dd 0.83 d 0.81 d 35.1 14.8 12.0 4.23 br d(87.11a) (24S,25R) 3.38 dd, 3.49 dd 0.81 d 0.81 d 35.1 15.1 11.6 4.14 dd, 4.34 dd(87.11b) (24S,25S) 3.36 dd, 3.58 dd 0.93 d 0.92 d 36.8 17.5 14.4 4.22 dd, 4.32 dd(87.10b) (24R,25R) 3.37 dd, 3.57 dd 0.91 d 0.91 d 36.1 17.4 14.1 4.16 dd, 4.38 dd

Natural Compounds(87.6) (24R,25S) 3.46 dd, 3.58 dd 0.91 d 0.97 d 40.4 14.5 17.5 (R)-MTPA: 4.17 dd, 4.33 dd

(S)-MTPA: 4.06 dd, 4.46 dd(87.7) (24S,25S) 3.34 dd, 3.62 dd 0.92 d 0.92 d 36.7 17.4 14.3 (R)-MTPA: 4.24 dd, 4.32 dd

(S)-MTPA: 4.16 dd, 4.37 dd

Figure 88. Shape of the partial NMR spectra of the 26-methylene protons in (R)- and (S)-MTPA esters of (a) a 25Ssteroid and (b) its 25R isomer.


stereochemistry at C-25 of a series of polyhydroxy-lated steroids, such as 87.3 or 87.4 (Figure 87), whichwere isolated from different starfish species.

It has been observed empirically that the LISOMesignal in the 1H NMR spectrum of a primary carbinol(+)-MTPA ester with R chirality at the â-carbon (ora (â-S) carbinol (-)-MTPA ester) is greater than thatof its diastereomer. This information was used in theassignment of the C-25 configuration of steroid 87.5(Figure 87), which was obtained after acid metha-nolysis of pavoninin 1, isolated from the defensesecretion of the sole P. pavoninus.63d The addition ofan equimolar amount of Eu(fod)3 to a mixture of the(+)- and (-)-MTPA esters (ca. 10:7) of 87.5 in CDCl3shifted the two OMe signals by 0.68 and 0.66 ppm,respectively, and the addition of 2.8 molar equiva-lents Eu(fod)3 shifted them by 2.05 and 2.00 ppm,respectively. The larger downfield shift observed forthe more intense OMe signal of the (+)-MTPA estershowed the configuration at C-25 to be R.

For those cases where the steroidal side chains hadan extra chiral center at C-24, several compounds ofknown stereochemistry were synthesized and usedas model systems. This was the case for two naturallyoccurring marine steroids from a starfish and anophiuroid: 87.6 and 87.7, respectively64 (see Figure87).

Also, the stereoisomers of ∆22E and the saturated24-methyl-26-hydroxy steroids 87.8-87.11 (Figure87) were prepared by Claisen rearrangement and therelative stereochemistries (threo or erythro) werededuced by comparison of 1H and 13C NMR spectra(see Table 10) and application of the Yasunara-Yamaguchi method that established the configura-tion at C-25.

Similar NMR studies of MTPA esters showed thatthere was a correlation between the shape of thesignals due to the CH2OH (C-26) group in the (R)-and (S)-MTPA esters and the absolute configurationat C-25. This new approach (i.e., comparison of thechemical shifts of the methylene protons at C-26 inthe spectra of the (R)- and (S)-MTPA esters) has theadvantage of avoiding the use of europium salts andhas been widely used by researchers interested in thestereochemistry of steroid side chains.65

It can be seen from Figure 88 that, in the spectraof the MTPA esters of a 25S isomer, the C-26methylene protons resonate much closer to each otherin the spectrum of the (R)-MTPA ester than in the(S)-MTPA derivative, whereas the reverse situationoccurs for the MTPA esters of a 25R isomer.

The assignment of the configuration of the marinepolyhydroxylated steroids 88.1 and 88.2 was con-ducted using this technique. In the case of 88.1, thesignals due to the methylene at C-26 in the (R)-MTPAderivative appeared as two well-separated doublet ofdoublets (dd) at δ 4.14 (J ) 6.6 and 10.8 Hz) and 4.28ppm (J ) 6.4 and 10.8 Hz) (∆δ ca. 0.14 ppm). The(S)-MTPA derivative, on the other hand, exhibitedvery similar dd signals, at 4.19 and 4.23 ppm (∆δ ca.0.04 ppm), thus indicating an R configuration for thecompound in question. The same procedure wasapplied to 88.2; however, unfortunately, the presenceof the bulky sulfate group at C-15 made the differ-

ences between the methylene group in the (R)- and(S)-MTPA esters very small (δ 4.15/4.25 and 4.15/4.23, respectively). Removal of the sulfate groupproduced the expected sets of doublet of doublets atδ 4.14/4.27 and 4.19/4.23, respectively. This caseillustrates the limitations imposed by the presenceof bulky substituents in ring D of steroids.66a

Other examples of the use of this technique include2,6-dimethylheptyl sulfate, 89.1 (see Figure 89),which was isolated from marine ascidians and itsconfiguration at C-2 was inferred as R, from theapplication of the same method to its desulfatedderivative 89.2.66b Kobayashi et al. also used thistechnique on a variety of marine metabolites suchas the ecdysteroids palythoalones A (89.3) and B(89.4) 66c and the amphidinolides T (89.5)66d and E(89.6)66e In these latter cases, the absolute configu-ration was deduced after degradation of the naturalproduct (89.7, 89.8, 89.9, and 89.10), subsequentderivatization with MTPA, and the use of compoundswith known absolute configuration, obtained bysynthesis, as models for comparison of the NMRspectra.

Figure 89. Chiral primary alcohols used in these studies.


In 1996, Seebach and co-workers67 proposed amethod based on 19F NMR analysis of the MTPAesters and, more specifically, directed toward thedetermination of the absolute configuration of hy-droxy-sulfonamides. The study was comprised of sixcyclic hydroxy-sulfonamides, one cyclic hydroxy-ester,and just one acyclic primary alcohol (2-methylbu-tanol) of known configuration. A correlation betweenthe relative configuration of the MTPA esters withthe 19F NMR chemical shift of the CF3 group wasdeduced. Figure 90 shows the structures, chemicalshifts, and the empirical model used to explain thecorrelation.

3.2.2. 9-AMAThe most general approach to the assignment of

the absolute configuration of primary alcohols isbased on the use of 9-AMA,68,69 because this reagent

has been shown29,30 to possess two very importantfeatures: (a) a higher conformational preference (thesyn-periplanar form, sp), than other AMAA reagents,and (b) the anthryl ring in the main conformer (sp)is particularly well-oriented, in terms of projectingits strong aromatic magnetic cone on substituent L1or L2.

No such advantages are expected from MPA orMTPA, because they have lower conformationalpreferences, less-favorable orientation of the phenylring (MTPA), and a weaker magnetic cone (phenylversus anthryl). Indeed, when these three reagentswere tested using 2-methylpropan-1-ol as a modelcompound, completely separate signals for the di-astereotopic methyl groups were obtained only in the9-AMA esters69 (∆δRS ) 0.033 versus ∆δRS ) 0.002ppm for 9-AMA and MPA respectively, and noseparation for MTPA).

Figure 90. 19F NMR chemical shifts of MTPA esters of primary hydroxy-sulfonamides and empirical models.


Complex theoretical studies (including molecularmechanics, semiempirical, ab initio, and aromaticshielding-effect calculations), complemented by DNMR, were performed69 with simple model com-pounds to assess the viability of 9-AMA esters for thispurpose. The results of these studies indicated thatthe main conformational processes involve rotationaround ω1 and ω2 bonds (see Figure 91 a), generatingthree main low-energy conformations (syn-peripla-nar-gauche+/anti (sp-g+/a), syn-periplanar-anti/anti (sp-a/a), and syn-periplanar-gauche-/anti (sp-g-/a); see Figure 91b), with one of these (sp-a/a)becoming the most significant, in terms of NMRspectroscopy in most cases. In all these conforma-tions, the 9-AMA substructure maintains its most-stable sp form.

Because conformer sp-a/a is the more stable andrepresentative, from the standpoint of NMR, for aprimary alcohol with the stereochemistry shown inFigure 92, substituent L1 will be more shielded inthe (R)-9-AMA ester, whereas substituent L2 will bein the (S)-9-AMA ester. Therefore, a negative ∆δRS

sign is expected for substituent L1 and a positive ∆δRS

sign is expected for substituent L2.

Sixteen 9-AMA esters of primary alcohols werestudied,69 and, in most cases (93.1-93.13; see Figure93), the conformational preferences and the modelpreviously described do, in fact, apply. This observa-tion means that the NMR spectra of this set of9-AMA esters could be reliably interpreted on thebasis of the corresponding model, and NMR spec-troscopy can be used to correlate the absolute con-figuration at the asymmetric center of the 9-AMA andthat of the alcohol.

Unfortunately, in three examples involving cyclicstructures or bulky substituents (93.14-93.16), thecalculations showed that the sp-a/a forms were notthe preferred forms and that the sp-g-/a and sp-g+/aforms make a very significant contribution. In thesecases, it is clear that the sp-a/a model is not validfor interpretation of the NMR spectra of the 9-AMAesters.

In conclusion, to assign the absolute configurationof a primary alcohol by this method, a preliminarycalculation must be performed (being the best forcefield, PCff), especially when the chiral center is partof a ring, or when strong steric interactions could bein operation, to confirm that the anti form is the most

Figure 91. Low-energy conformers (perspective and Newman projections) for (R)-9-AMA esters of primary alcohols. Inmost cases, sp-a/a is the most populated and relevant for NMR studies.


stable one and, consequently, that the model shownin Figure 92 is representative and can be applied tothe interpretation of the chemical shifts.

The use of the auxiliary reagents MTPA and MPAdid not generate either the required ∆δRS values orhomogeneity in the sign distribution, and this situ-ation confirmed that these compounds could not beused as auxiliary reagents with chiral primary alco-hols.

A good example of the application of this procedurefor the determination of the configuration of acomplex substance is shown in Figure 94, whichdepicts oxazinin-1, a cytotoxic compound that hasbeen isolated from the digestive glands of an ediblemussel (Mytilus galloprovincialis). That was recentlypublished by Fattorusso et al.70 In that paper, MMcalculations were initially performed to check thereliability of the NMR method with that particularsubstrate, and the stereochemistry was determinedusing the 9-AMA esters only after a positive resulthad been obtained.

3.3. Application to r-Chiral Tertiary AlcoholsAlthough the determination of the configuration of

chiral secondary alcohols by NMR has been satisfac-torily accomplished through the use of the variousalternatives already discussed (see Section 3.1), ageneral methodology for chiral tertiary alcohols hasnot yet been established. This fact is mainly due tointrinsic problems associated with these substrates,such as difficulties in making derivatives (i.e., esters)in high yields and their unsuitable conformationalcharacteristics. A few attempts have been made tofind solutions to these problems but usually the

number of substrates studied is small and theirstructural variety is limited; therefore, the methodsare applicable only to the cases reported.

3.3.1. MTPA

The use of MTPA with tertiary alcohols71 wasexplored for just two examples: pinan-2-ols 95.1 and95.2 (see Figure 95).

A combination of MM2 calculations and NOE andanisotropic effects (measured as ∆δSR of the methylgroups) was used to predict the configuration of thesesubstrates. The authors state that MTPA can be usedin combination with conformational analysis; how-ever, the method was not expanded to include otheralcohols.

3.3.2. Methoxy-(2-naphthyl)acetic Acid (2-NMA)

A method for the prediction of the absolute config-uration of acyclic R-methyl-substituted tertiary al-cohols has been published and involves the use ofmethoxy-(2-naphthyl)acetic acid (2-NMA) as an aux-iliary reagent.72

The method is based on an analysis of the NMRspectra of the 11 compounds of known configurationshown in Figure 96. According to the authors, the∆δRS signs obtained are systematically distributed aspositive and negative for substituents L1 and L2.This arrangement can be represented by the modelshown in Figure 97, and it was used by the authorsfor the assignment of configuration. The â-methyleneand methyl protons directly bonded to the tertiaryhydroxyl group should not be considered for theassignment.

Figure 92. Conformational model for the determination of the absolute configuration of â-chiral primary alcohols byNMR of their 9-AMA esters.


Other drawbacks associated with this method arethe low yields reported for the esterification (i.e.,27%) and the racemization problems that are en-countered. MTPA and MPA were also tested; how-ever, they resulted in smaller ∆δ values than thosefor 2-NMA.

3.3.3. The Fucofuranoside MethodKobayashi73 applied a method that was devised for

the determination of the absolute configuration ofsecondary alcohols to the series of six chiral tertiaryalcohols (substituted with methyl and two methylenegroups) shown in Figure 98. The method involves thederivatization of the alcohol as â-D- and â-L-fucofuran-osides, comparison of the NMR spectra (1H and 13C)of the derivatives taken in pyridine-d5, and evalua-tion of the ∆δ values (δL - δD) and signs for theproximate protons (1H NMR) or â-carbons (13C NMR).

The experimental data show that the ∆δ values aregenerally positive for the protons in the right-handsegment (Rr) and negative for those in the left-handsegment (Rl); however, in several cases, both positiveand negative signs coexist on the same methylenegroup.

The ∆δ values from the 13C NMR study are positivefor the right-hand â-carbon and negative for the left-hand â-carbon, except for the five-membered ringcompound, which did not follow this trend.

These results can be interpreted on the basis of themodel shown in Figure 99. This model does not haveany proven conformational meaning, because NOE

Figure 93. ∆δRS values for the 9-AMA esters of primaryalcohols.

Figure 94. Structure of oxazinin-1.

Figure 95.

Figure 96. R-Methyl-substituted tertiary alcohols used byKobayashi et al.

Figure 97. Empirical model to determine the absoluteconfiguration of R-methyl-substituted tertiary alcohols.


or low-temperature NMR studies or geometry calcu-lations were not performed to establish the confor-mational characteristics of the derivatives.

Indeed, the author states that the method may notbe valid for tertiary alcohols with more-complexsubstitution patterns. In addition, the very low yieldsobtained in the derivatization step (in the range of2%-23%) or, in some cases, even the failure to obtainthe fucofuranosides, represents a major limitation tothe use of this method.

3.4. Application to r-Chiral Primary Amines3.4.1. MTPA

Another of the applications of “Mosher’s method”is the determination by 1H NMR spectroscopy of the

absolute configuration of primary amines8c,74 thathave a chiral center at the R-position. The model usedto correlate the stereochemistry with the chemicalshifts is analogous to that proposed for secondaryalcohols and, although a procedure based on 19F NMRstudies of MTPA esters has been formulated foralcohols (Figure 8), a similar application with amineshas very rarely been used.

The method is standard and involves the prepara-tion of the corresponding MTPA amides from the twoenantiomers of MTPA and the amine of unknownconfiguration. The NMR spectra of the two deriva-tives are then recorded and the differences in thechemical shifts (∆δSR) are calculated. Mosher pro-posed that the most-relevant conformer would be theone in which the CF3, carbonyl, NH, and C(1′)Hgroups are in the same plane, with the CF3 and thecarbonyl units in a syn-periplanar disposition. Inaccordance with this model (see Figure 100), sub-stituent L2 is shielded in the (R)-MTPA amide andsubstituent L1 is shielded in the (S)-MTPA amide.For an amine with the configuration shown in Figure100c, substituent L1 has negative ∆δSR values andsubstituent L2 has positive values. In the case wherethe amine has the opposite configuration, as inFigure 100d, the signs are also opposite to those inthe example in Figure 100c.

In an effort to establish the scope and limitationsof this method, theoretical calculations (MM, semi-empirical AM1) and NMR studies (aromatic anisot-ropy increments, dynamic NMR) have recently beenconducted.75

This conformational study showed that the mainprocesses are those due to rotations around the CO-NH, CR-CO, and CR-Ph bonds (see Figure 101).Rotation around the CO-NH bond generates tworotamers (Z and E). The Z rotamer is predominant,because of its greater stability, in terms of energy.Rotation around the CR-CO bond produces the twousual sp (CF3 and carbonyl syn-periplanar) and ap(CF3 and carbonyl anti-periplanar) conformers. Rota-tion around the CR-Ph bond results in three mainorientations, with the phenyl group coplanar witheither the CR-OMe, CR-CO, or CR-CF3 bonds.

Figure 98. Tertiary alcohols used in this study.

Figure 99. Models for assignment of tertiary alcohols asâ-D- and â-L-fucofuranosides.

Figure 100. (a, b) Mosher’s model for the correlation of the configuration with the 1H NMR shifts and (c and d) expectedsigns of ∆δSR.


The combination of these three orientations of thephenyl group with the two sp and ap conformersgenerates six possible situations (as in the case ofesters).27 These six possibilities can be reduced to justthree (sp1, ap3, and ap1; see Figure 101)sas inMTPA esters27son the basis of theoretical calcula-tions (MM and AM1). The most stable conformer issp1, with the other two conformers (ap3 and ap1)being energetically less stable (see Figure 102).

In the sp1 conformer, the CF3 group is syn-periplanar to the carbonyl and the phenyl ring iscoplanar with the CR-OMe bond. Overall, this ar-rangement leads to selective shielding on one of thesubstituents (L1/L2) of the amine (see Figure 102a).In the other two conformers (ap1 and ap3), the CF3is anti-periplanar to the carbonyl group. However,in conformer ap1, the phenyl ring is coplanar withthe CR-OMe bond, causing deshielding on one sub-stituent of the amine (see Figure 102b), whereas inconformer ap3, the phenyl ring is coplanar to theCR-CF3 bond, which produces shielding (see Figure102c).

These shielding/deshielding effects were quantita-tively demonstrated by calculating the contributions

of the aromatic ring to the chemical shifts (aromaticanisotropy increments) of each conformer.

In the conformational equilibrium, each conformerplays a different role (shielding or deshielding) andthe overall result is the weighted average among theconformer populations and their shielding/deshield-ing effects. From the NMR standpoint, the L1 sub-stituent of an (S)-MTPA amide is shielded in con-former sp1 (see Figure 103a), while substituent L2is virtually unaffected because it is deshielded inconformer ap1 and shielded in conformer ap3 (Figure103a). The magnitude of the shielding/deshieldingeffects on substituent L2 depends on the differencein the populations of the two conformers. Similarly,in the (R)-MTPA amide, substituent L2 is shieldedin conformer sp1 and substituent L1 is almost un-affected (deshielded in conformer ap1 and shieldedin conformer ap3) (Figure 103b).

The overall result is that the L1 substituent is moreshielded in the (S)-MTPA than in the (R)-MTPAamide. Conversely, substituent L2 is more shieldedin the (R)-MTPA than in the (S)-MTPA amide. Whenthe differences in the chemical shifts are calculatedfor an amine with the configuration shown in Figure

Figure 101. Generation of Z-conformers in MTPA amides and the most significant characteristics of each one.


100c, ∆δSR has a negative value for substituent L1and a positive value for substituent L2. In the casewhere the configuration is opposite, so too are thesigns for ∆δSR (see Figure 100d).

Further experimental evidence to support thisequilibrium and the structures of the conformers wasobtained from the study of the low-temperature 1HNMR spectra of the MTPA amides of (+)-bornyl-amine. The results (Figure 104a) are consistent withthe previously discussed predictions and illustratethe effect of the phenyl ring on the substrate in eachconformer.

Because of the greater significance of conformer sp1in comparison to conformers ap1 and ap3, conformersp1 can effectively be used as a correlation model forthe assignment of the configuration of amines fromthe NMR spectra. This simplified model is shown inFigure 104b and is in full agreement with thatoriginally proposed by Mosher (see Figure 100a). Forinstance, Me(10′) is shielded by the phenyl group inthe (R)-MTPA amide of (+)-bornylamine and H(6′)is shielded in the (S)-MTPA amide, causing the ∆δSR

sign to be positive for Me(10′) and negative for H(6′).The reliability of this model for the prediction of

the configuration was confirmed experimentally bycomparing the NMR spectra of the MTPA amidesderived from a series of R-substituted primary aminesof known absolute configuration.74,75 The signs of∆δSR are consistent with the model in all the ex-amples studied, and a selection of data is shown inFigure 105.

The MTPA derivatives of alcohols27 and amines75

both have a conformational equilibrium that isconsiderably more complex than that of other re-agents such as MPA (MTPA results in three mainconformers with close populations instead of two29).However, there is a difference between MTPA estersand amides that is of significant practical impor-tance: the major conformer in esters causes deshield-ing, whereas in amides, it causes shielding, and,therefore, larger ∆δSR values are obtained for amidesthan esters. For this reason, the use of MTPA as aCDA for the determination of the configuration ofalcohols is frequently hampered by the small changesFigure 102. Main conformers for the MTPA amides.

Figure 103. Low-energy conformations of (a) (S)-MTPA and (b) (R)-MTPA, as obtained by MM and AM1 calculations.


in shift obtained and the uneven distribution of ∆δSR

signs;13,27 such problems for MTPA amides have notbeen reported.

3.4.2. MPA

The application of MPA to the determination of theconfiguration of R-substituted primary amines is avery recent development.76,77 At present, there aretwo methods available for this reagent. The methoddescribed in this chapter involves the use of the twoenantiomers of MPA,76a whereas only one enantiomeris necessary in the procedure described in section 4.2of this review.77

The method consists of the derivatization of theamine with the two enantiomers of MPA, acquisitionof the 1H NMR spectra of the corresponding (R)- and(S)-MPA amides (preferably in a nonpolar solvent76a),and analysis of the ∆δRS parameters.

The ∆δRS signs for substituents L1/L2 are correlatedto the absolute configuration of the amine throughout a nonempirical model, which has been establishedfrom a detailed study of aromatic shielding effectsand structural calculations76a (molecular mechanics(MM), semiempirical (AM1, PM3) and ab initio), aswell as by NMR experiments76a (low-temperature anddynamic NMR).

The conformational analysis shows that the mainconformational processes involve rotations aroundthe CO-NH and CR-CO bonds (see Figure 106). Two

Z and E rotamers result from rotation around theCO-NH bond, with the Z arrangement being thepredominant one. The CR-CO rotation generates thetwo usual conformers for this type of system: sp(OMe and carbonyl in a syn-periplanar disposition)and ap (OMe and carbonyl in an anti-periplanardisposition) (Figure 106). In both the sp and ap forms,the OMe is anti, with respect to the CR-Ar bond,and the phenyl group is coplanar to the CR-H bond.

Theoretical calculations showed that the ap con-former has a lower energy than the sp conformer (thisis opposite to the case for MPA esters, where sp isthe most stable conformer). The evolution of the NMRspectra of the MPA amide of (+)-bornylamine atdifferent temperatures confirmed these conforma-tional characteristics.

The results of all these studies indicate that, insolution, the MPA amides exist in a conformationalequilibrium composed of two main conformers: ap-Z(MeO and carbonyl, anti-periplanar) and sp-Z (MeOand carbonyl, syn-periplanar; carbonyl and C(1′)Hbond, syn-periplanar). In both cases, the methoxygroup, the carbonyl group, the N-H bond, and theCR-H bond are in the same plane and the phenylring is almost coplanar to the CR-H bond (Figure106).

Accordingly, in (R)-MPA amides, the L2 substituentis shielded by the phenyl ring in the ap conformerwhereas substituent L1 is shielded in the sp con-

Figure 104. (a) Low-energy conformations of the (R)-MTPA amide of (+)-bornylamine. Solid arrows show shielding, anddashed arrows show deshielding effects. (b) Simplified models for (R)- and (S)-MTPA amides.


former (Figure 107a). In the (S)-MPA amides, how-ever, substituent L1 is shielded in the ap conformerand substituent L2 is shielded in the sp conformer(see Figure 107b). Given that the ap conformer ismore populated than the sp conformer, the L2 sub-stituent is more shielded in the (R)-MPA than in the(S)-MPA amides and, conversely, substituent L1 ismore shielded in the (S)-MPA than in the (R)-MPAamides. Comparison of the spectra of the two deriva-tives of an amine with the configuration shown inFigure 107c shows that the ∆δRS value is positive forsubstituent L1 and negative for substituent L2 (withopposite signs for the enantiomeric amine) (seeFigure 107d). In practice, this method effectivelyconsiders the ap conformer as a simplified model forthe assignment of configuration of the amine bycorrelation of the ∆δRS signs with the configuration.

The reliability of the assignment obtained from themodel shown in Figure 107c and d has been demon-strated experimentally with a series of amines ofknown absolute configuration (see Figure 108); in allcases, the signs of the ∆δRS values are consistent withthe predictions.

3.4.2.1. MPA versus MTPA. When determiningthe configuration of a substrate, it is important toselect the most suitable CDA available. In the caseof amines, note that there are no significant differ-ences in the magnitude of ∆δ obtained for MPA(∆δRS) and MTPA (∆δSR) amides, indicating thatneither of these reagents has a clear advantage overthe other75 (see Figure 108). This situation may seemsurprising initially, because the population of thepredominant conformer in MTPA amides is smallerthan that in MPA amides. However, in the former

Figure 105. ∆δSR values (ppm, CDCl3) of the (R)- and (S)-MTPA amides of a selection of amines of known configuration.


case, the phenyl ring is better orientated for shieldingthan in MPA amides and the combination of the twofactors leads to similar shift changes.

3.4.2.2. The Effect of the Polarity of the Sol-vent on the NMR of MPA Amides. MM calcula-tions on MPA amides revealed that the dipolarmoment of the predominant ap-Z conformer is smallerthan that of the sp-Z conformer.76a Thus, the popula-tion of ap-Z (and, therefore, the ∆δRS values) shouldincrease if the polarity of the NMR solvent is reduced.

This proposal has been experimentally proven76a

by acquiring NMR spectra in several solvents, whichall showed the expected trend for ∆δRS values: ∆δRS-CS2 > ∆δRSCDCl3 > ∆δRS(CD3)2CO (see Figure 109).Therefore, one way to increase the efficacy of MPAas a CDA for amines (larger ∆δRS values) involvesrecording the spectra of the corresponding amides innonpolar solvents such as CS2.

A similar improvement in the ∆δRS data for MPAesters was obtained by reducing the temperature ofthe NMR probe. This technique was described inSection 3.1.2.8.

3.4.3. AMAAs

Other than MPA and MTPA, other arylmethoxy-acetic acids with different aryl rings (9-AMA and1-NMA) have been explored as CDAs for amines.76a

The predominant conformer in the amides derivedfrom both reagents was ap-Z (as in MPA amides).Despite the presence of an anthryl or naphthyl ringin these reagents, NMR experiments show that the∆δRS values obtained for 9-AMA and 1-NMA amidesare no higher than those obtained with MPA orMTPA. This is probably due to a lower population ofthe shielding conformer or to an unfavorable orienta-tion of the aryl ring. In either case, the practicalresult is small ∆δRS values; therefore, the use of9-AMA and 1-NMA as CDAs for amines does notprovide any advantages over MTPA or MPA.

3.4.4. Boc-phenylglycine (BPG)

The use of Boc-phenylglycine (BPG; see Figure 110)as an auxiliary reagent78 fills a gap that neitherMTPA, MPA, nor the other AMAA reagents were able

Figure 106. Generation and main conformers of MPA amides.


to cover due to the small magnitude of the differencesbetween the chemical shifts (∆δRS or ∆δSR) theygenerate.

The procedure consists of the derivatization of theamine of unknown configuration with the two enan-tiomers of BPG, recording of the 1H NMR spectra of

Figure 107. (a, b) Conformational equilibrium for MPA amides. (c, d) Prediction of ∆δRS signs.

Figure 108. Selected ∆δRS values (ppm) obtained from the 1H NMR spectra of (R)- and (S)-MPA amides (in CDCl3) of theillustrated amines. For the sake of comparison, ∆δSR values obtained with MTPA are shown in parentheses.


the corresponding diastereoisomeric BPG amides andmeasuring the differences in the chemical shifts(∆δRS) of substituents L1/L2. The signs of ∆δRS arethen correlated with the absolute configuration bymeans of a nonempirical model that was establishedby detailed conformational analysis,78 including MMand semiempirical (AM1) calculations, and experi-mentally proven by exhaustive NMR studies.

The main conformational processes involved areshown in Figure 111, and their study led to BPGamides being characterized as follows:

(a) In the amide bond (CO-NH), the Z isomer ismore stable than the E isomer.

(b) Rotation around the ω1 (CR-CO) bond gener-ates the common sp and ap conformations (CR-Hbond and carbonyl group, syn-periplanar and anti-periplanar, respectively).

(c) The phenyl group is coplanar with the CR-Hbond by rotation around ω2 (CR-Ph).

(d) The CR-H, N-H, and CdO bonds have atendency to remain coplanar, the CR-H and N-Hbonds (ω3) have a tendency to remain in an antidisposition, and the N-H bond have a tendency tobe in an anti disposition, with respect to the CdObond (ω4).

Both the theoretical calculations and the NMRstudies revealed that the most important conforma-tional process is the equilibrium between the ap andsp conformers, with the former being the most stable(see Figure 112).

In this way, substituent L1 is shielded by thephenyl group in the ap conformer and substituent L2is shielded in the sp conformer in the case of (R)-BPG amides (see Figure 113a). Conversely, substitu-ent L2 is shielded in the ap conformer and substituentL1 is shielded in the sp conformer in (S)-BPG amides(see Figure 113b). Because the ap population is largerthan the sp population, L1 is the most shieldedsubstituent in (R)-BPG amides and L2 is the mostshielded substituent in (S)-BPG amides.

Thus, when the differences in chemical shifts aremeasured (∆δRS), a negative value is obtained forsubstituent L1 and a positive value is obtained forsubstituent L2 if the amine has the configurationshown in Figure 113c. Opposite signs are clearlyobtained if the configuration of the amine is opposite(see Figure 113d).

This prediction has been experimentally proven ina series of amines of known absolute configurationand with a variety of structures (see Figure 114).

Figure 109. Plots showing the dependence of ∆δRS

(expressed as a percentage of ∆δRS in (CD3)2CO) versus thepolarity of the solvent (as (ε - 1)/(2ε + 1), where ε is thedielectric constant): Line 1, Me(10′) of (+)-bornylamine;Lines 2 and 3, H(3′) and H(4′) of L-valine methyl ester,respectively; and Lines 4 and 5, H(2′) and H(7′) of L-tryptophan methyl ester, respectively.

Figure 110. Structures of (R)- and (S)-BPG.

Figure 111. Generation of conformers of BPG amides.


The ap conformer is the major component in themixture and, therefore, is the most significant, fromthe NMR standpoint. As such, it can be used as asimplified model to correlate the ∆δRS signs with theabsolute configuration of the amines. This simplifiedmodel is shown in Figure 115 and can be used toplace substituents L1 and L2 on the amine by simplyconsidering the relevant ∆δRS signs.

For the sake of comparison, Figure 116 shows the∆δ values observed in a selection of BPG (∆δRS), MPA(∆δRS), and MTPA (∆δSR) amides derived from aminesof known configuration. The data show beyond doubtthat BPG produces the largest values and, hence, themost-accurate assignments.

The reason for the advantage that is inherent inBPG systems, in comparison to the other reagents,

Figure 112. Main conformers of BPG amides: (a) spconformer and (b) ap conformer.

Figure 113. Conformational equilibrium in BPG amides.

Figure 114. Selected ∆δRS values for (R)- and (S)-BPGamides.


lies in the coexistence of two factors that only worktogether in BPG amides: (a) the excellent positionof the phenyl group to transmit its shielding effectto substituents L1/L2 of the amine in the most stableconformer (ap; see Figure 117a) and (b) a conforma-tional equilibrium of just two conformers (sp/ap). Thegood orientation of the phenyl ring is also present inMTPA amides75 (Figure 117c); however, its effect isreduced by a three-conformer composition. In MPAamides, the equilibrium is simple76 but the orienta-tion of the phenyl is not as good as that observed inthe other two cases (see Figure 117b).

In conclusion, BPG has structural and conforma-tional features that make it the reagent of choice forthe assignment of the absolute configuration ofprimary amines. It is commercially available in its

two enantiomerically pure forms, and, in addition,it is cheaper than any of the alternative reagents.

3.4.5. Other ReagentsIn addition to the reagents already described in this

section, several other CDAs have been used withamines, and these compounds are discussed in thissection.

Several methods and reagents are specifically notcovered, because they have already been discussedin other sections (i.e., Helmchen’s reagent44,45 (PBA)and diazacyclophosphamides54 are used for bothalcohols and amines) or have been extensively de-scribed in other review articles4a (e.g., 2-[1-(9-an-thryl)-2,2,2-trifluoroethoxy]acetic acid79 (ATEA) andR-methyl-R-methoxypentafluorophenylacetic acid80

(MMPA)) for which no new relevant contributionshave appeared since their last publication.

3.4.5.1. 1-Fluoro-2,4-dinitrophenyl-5(R,S)-phen-ylethylamine [(R,S)-FDPEA]. This is the samereagent that is used in the difluoronitrobenzenemethod for the determination of the absolute config-uration of secondary alcohols.52 In its application toamines,81 (R)- and (S)-FDPEA (Figure 118) arereacted with the amine of unknown configuration togive the corresponding diastereomers. On the basisof UV and NMR studies, a predominant conformationhas been proposed that has the R-protons of theamine and phenylethylamine moieties, as well as thephenyl H6, located in the same plane (see Figure119a). In accordance with this model, in the (R)-FDPEA derivative, substituent L2 is shielded by thephenyl group of the phenylethylamine while L1remains unshielded. On the other hand, in the (S)-FDPEA derivative, substituent L1 is shielded whereasL2 is not (Figure 119a). When the differences in thechemical shifts (∆δRS) are calculated for an aminewith the configuration shown in Figure 119b, sub-stituent L2 should show a negative ∆δRS value andsubstituent L1 should show a positive ∆δRS value.

Table 11 shows the ∆δRS values for the threeFDPEA amides of known absolute configurationsthat have been used to validate the previouslydescribed predictions. However, despite the fact thatthe signs are as expected and the ∆δSR values arelarger than those obtained with MTPA (included inTable 11 for comparison), the number of test com-

Figure 115. Simplified model for the assignment of the configuration of BPG amides.

Figure 116. Comparison of the ∆δ values of BPG (∆δRS),MTPA (∆δSR), and MPA amides (∆δRS). Data for BPG givenin boldface type, data for MTPA given in normal typeface,and data for MPA given in parentheses.


pounds is too small to recommend the method forapplication to amines in general.

Despite this limitation, the method has been usedto determine the configuration of microginin (Figure120), which is a natural product from cyanobacteria.81

3.4.5.2. (R)-O-Aryllactic Acid (ROAL). As dis-cussed previously, many O-aryllactic acids have beenused as derivatizing agents for the determination ofthe enantiomeric purity3a,60d of alcohols and aminesby 19F NMR, for the assignment of the absoluteconfiguration of amines82 and as solvating agents83

in the determination of the enantiomeric purity of

amines. The O-aryllactic acids (ROAL) shown inFigure 121 have been proposed for the determinationof the absolute configuration of R-substituted primaryamines, on the basis of theoretical and experimentalstudies.82

MM and semiempirical calculations, together withNMR data, suggest that the amides derived from theROAL preferentially exist in the ap-Z conformationin solution. In this conformation, the aryl and car-bonyl groups are anti-periplanar (Figure 122), as inMPA amides, but are different from the main con-formation of their esters with secondary alcohols,where the CR-H and CdO bonds are syn.

According to this conformational model, the ROALamide derived from an amine with the configurationshown in Figure 122a has substituent L1 shieldedwhile substituent L2 is not. Conversely, if the aminehad the configuration shown in Figure 122b, then theshielded group would be substituent L2.

Figure 117. Main orientation of the phenyl ring and direction of maximum shielding in (a) BPG, (b) MPA, and (c) MTPAamides.

Figure 118. Structure of (R)- and (S)-FDPEA.


Therefore, determination of the configuration ofR-substituted primary amines or R-amino acids ispossible by comparison of the chemical shifts of therelevant substituents.

Experimental validation of the predictions givenby this method was provided by the ROAL deriva-tives of the amines shown in Figure 123. The avail-able ∆δRS data is presented in Table 12, and some

limitations are apparent: Only one acid (121.1) wastested with all the amines, whereas the others weretested with just one, two, or three amines.

In most cases, only the ∆δ values for one substitu-ent (either L1 or L2) are reported. All the O-aryllacticacids described have the (R)-configuration (i.e., theyare prepared from (S)-ethyl lactate) and examples ofthe application of both enantiomers of the reagentto one enantiomer of the amine have not been

Figure 119. Model used to correlate the configuration with the 1H NMR shifts and ∆δRS signs.

Table 11. Comparison of ∆δ Values between FDPEA and MTPA Amides

L-isoleucinol L-phenylalaninolposition FDPEA MTPA FDPEA MTPA

D-phenylalaninol,FDPEA

R-CH 0.0045 -0.0420 -0.0725 0.0400 0.0240â-CH 0.3120 0.0000â-CH2 0.2410 -0.0270 -0.2880

0.2855 -0.0010 -0.3410â-CH3 0.2260 -0.0420â′-CH2 -0.43750 0.0500 -0.4825 0.0570 0.4355

-0.5085 0.0700 -0.5360 0.0540 0.4880γ-CH2 0.3910 -0.0840

0.3095 -0.0900γ-CH3 0.2830 -0.0560γ-NH 0.0020 -0.1420 -0.0230 0.1210 -0.0695

Figure 120. ∆δRS values for microginin-FDPEA.

Figure 121. Structures of the O-aryllactic acids.

Figure 122. Model for the assignment of the configurationof (R)-O-aryllactic acid amides.


performed. Thus, in its present form, this methodol-ogy can only be used when the two enantiomers ofthe chiral amine are available.

3.4.5.3. R-Cyano-R-fluoro-p-tolylacetic Acid(CFTA). R-Cyano-R-fluoro-p-tolylacetic acid (CFTA)has been described as a CDA for the determinationof the absolute configuration of secondary alcohols61

(by 1H NMR) and e.e. calculation (by 19F NMR). Itsuse for the determination of the absolute configura-tion of R-amino esters by 19F NMR has also beendescribed84 and is based on the study of the 19F NMRspectra of the CFTA amides of a series of (S)-aminoesters. The spectra of these compounds showed thatthe fluorine signals of the amides derived from (R)-CFTA ((R,S)-amides) resonate at higher field by 2-6ppm, compared to those derived from (S)-CFTA((S,S)-amides). Molecular orbital calculations84 sug-gest a conformational equilibrium between two con-formers: ap (the most stable; CR-F bond anti-periplanar to the carbonyl) and sp (both groups syn-periplanar) (Figure 124). The calculations also helpto explain the shift of the fluorine signal to higherfield in the ap conformer, with respect to the sp. Thelarge difference in ap/sp populations between the(R,S)- and (S,S)-amides also helps to explain the factthat the fluorine signal resonates at higher field inthe (R,S) compound than in the (S,S) compound.

Table 13 shows the NMR data obtained for CFTAand MTPA amides (included for comparative pur-poses). In all cases, the fluorine chemical shifts (δF)and their differences (∆δ) are coherent with thetrends discussed, with larger ∆δ values obtained forthe CFTA amides.

3.5. Application to Secondary Amines: MTPAA procedure for the assignment of the absolute

configuration of cyclic secondary amines with asym-metric carbons in the R- or â-positions has recentlybeen proposed by Hoye et al.,85 using MTPA as theCDA. This method is based on the standard compari-son of the 1H NMR spectra of the corresponding (R)-and (S)-MTPA amides; however, in this case, thefoundations of the correlation between NMR shiftsand configuration and the analysis of the spectrawarrant a more detailed explanation.

In general, the 1H NMR spectra of these MTPAamides show the presence of two rotamers aroundthe amide bond (syn and anti; see Figure 125) thatare in equilibrium. The signals of these rotamers can

Figure 123. Structures of the amines used in this study.

Table 12. Selected ∆δ Values for (R)-O-Aryllactic AcidAmides

∆δa

ROAL amine Ha Hb Hc Hd

121.1 123.1 0.09 -0.15121.3 0.11 -0.21121.4 0.04 -0.25121.1 123.2 0.08121.1 123.3 0.08121.2 0.07121.3 0.10121.4 0.11121.1 123.4 0.10121.1 123.5 -0.05 0.08121.1 123.6 -0.08 0.09 0.18 0.07121.3 -0.11 0.1 0.24 0.22121.1 123.7 -0.06 0.06121.1 123.8 0.13 -0.18/-0.07 -0.25

a ∆δRS) δRR - δRS.

Figure 124. Conformational equilibria of CFPA (Ar ) Ph)and CFTA (Ar ) p-tol) amides.

Table 13. ∆δF Values for CFTA and MTPA Amides

Figure 125. Conformational equilibrium in the (R)-MTPAamide of (R)-2-methylpiperidine.


be identified because the populations of each aredifferent, because of steric effects. The most abundantrotamer has the anti configuration when only onesubstituent is present.

The differentiation between the spectra of the tworotamers is essential because, in this procedure, onlythe signals of the rotamer with the asymmetriccarbon of the MTPA syn to the asymmetric carbonof the amine under examination should be consid-ered.

For instance, in the assignment of the configurationof carbon-2 of (R)-2-methylpiperidine (Figure 125),only the signals due to the syn rotamer (Figure126a,b) in the (S)-MTPA and the (R)-MTPA amidesare relevant and only the ∆δSR values correspondingto these rotamers are valid for assignment purposes.In contrast, the anti conformers (Figure 126c, d)should not be considered.

The second requirement of this procedure is thatonly the chemical shifts of axial substituents in theaforementioned syn rotamer should be compared toobtain the ∆δSR value. This is due to the fact thatthe axial substituents are more exposed to theshielding/deshielding effects of the CDA.

Thus, in (R)-2-methylpiperidine, the resonances forthe syn rotamer in the spectra of the (S)- and (R)-MTPA amides should be identified first and thesignals due to the axial methyl group at C-2 and theaxial proton at C-3 (Figure 126a and b) used to obtainthe ∆δSR value.

In this situation, the axial methyl group is moreshielded in the (S)-MTPA amide (Figure 126a) thanin the (R)-MTPA amide (Figure 126b), and the H-3

Figure 126. (a, b) Syn and (c, d) anti rotamers for the(S)- and (R)-MTPA amides of (R)-2-methylpiperidine.

Figure 127. (a, b) Syn and (c, d) anti rotamers for the (S)- and (R)-MTPA amides of (2S,6S)-(2-carbomethoxy-methyl)-6-methylpiperidine.


proton is more shielded in the (R)-MTPA derivative(Figure 126b) than in the (S)-MTPA amide (Figure126a). The resulting ∆δSR values are negative for themethyl at C-2 and positive for the proton at C-3.Conversely, ∆δSR signs will indicate the reverseconfiguration for the amine.

If the cyclic amine had more than one asymmetricC atom near the N atom, e.g., (2S,6S)-(2-carbometh-oxymethyl)-6-methylpiperidine (Figure 127), the sameprinciples would apply; however, the situation ismore complex and the spectra of both the syn andanti rotamers should be considered.

For example, in the assignment of the absoluteconfiguration at C-2, the NMR spectra of the axialgroups in the syn conformers of the (R)- and (S)-MTPA amides should be examined (see Figure 127a,b). For the structure shown, the axial methylene atC-2 results in a negative ∆δSR value and the axial Hatom at C-3 results in a positive ∆δSR value (seeFigure 127c).

The assignment of the configuration at C-6 requiresexamination of the spectra of the axial groups in theanti rotamers (Figure 127 c,d) and, for the structureshown, the axial substituent at C-6 gives a positive∆δSR and the axial hydrogen at C-5 a negative ∆δSR

value (Figure 127f).The generality of this correlation between the ∆δSR

signs and the absolute stereochemistry has beentested with the cyclic secondary amines of knownabsolute configuration shown in Figure 128. In allcases, the signs are as expected from the stereochem-istry and, therefore, the procedure can be used forthe assignment of other amines.

In addition, the absolute values obtained for ∆δSR

are much larger than those observed for the MTPAesters or amides; this condition is probably relatedto the proximity of the groups under examination tothe phenyl group and also to the favorable orientationof the phenyl ring for effective shielding.

3.6. Application to r-Chiral Carboxylic AcidsCarboxylic acids are frequently found in nature as

optically active compounds with the chiral center atthe R-position, and general approaches for the de-termination of the absolute stereochemistry of thesesystems are rare.

An important decision that had to be made in thedevelopment of new reagents for this purpose con-cerned the selection of the type of bond used to linkthe auxiliary reagent to the substrate carboxylic acid.Several studies were undertaken for the purpose ofobtaining suitable reagents, and ester29,30 andamide75-78,86 bonds seemed to be the most appropriatecandidates.

The main issue in relation to the selection of chiralreagents for the assignment of absolute configuration

Figure 128. Cyclic secondary amines of known configu-ration examined by NMR of their MTPA amides.

Figure 129. Conformers ap and sp in carboxylic acids.

Figure 130. Conformational preference in the ester and amide bonds.


of carboxylic acids is to demonstrate the existence ofan NMR significant conformational preference thatremains the same in the R and S derivatives and isindependent of the nature of the carboxylic acid.

In accordance with previous experience, alcoholsand amines can be considered potentially good can-didates to that purpose.

To determine the conformational characteristics ofcarboxylic acids, molecular mechanics and semi-

empirical calculations (AM1, PM3) on more than 20carboxylic acids, and their methyl esters and methyl-amides, were performed87 and showed that the mainconformational process in all those compounds is therotation around the ω1 bond (Ca-CdO; see Figures129 and 130). This generates two main conformers:ap (CR-H and CdO in anti-periplanar) and sp (syn-periplanar) (see Figure 129). In the case of the esters,the ap conformer is the more stable conformer;however, this preference is absent in amides.

In addition, it is known29 that, although, in theesters, the (CdO)-O fragment has a clear preferencefor a Z orientation, the rotation around the (CdO)-NH fragment in amides is more complex75-78 andratios between the two main rotamers from 90:10 to50:50 have been reported86 (see Figure 130).

From that data, one can conclude that the esterbond is more convenient than the amide bond to linkthe auxiliary to the substrate, because the last oneis conformationally less reliable and, as a conse-quence, the best auxiliary reagents for carboxylicacids should be found among secondary alcohols andnot among amines.

3.6.1. Ethyl 2-(9-anthryl)-2-hydroxyacetate (9-AHA) andRelated Aryl Alcohols

With that information on hand and using theAMAAs,29,30 as models, many aryl alcohols have beenevaluated as CDAs for R-chiral carboxylic acids, using

Figure 131.

Figure 132. Conformational equilibrium in the esters of (R)- and (S)-9-AHA with (a, b) an R-chiral carboxylic acid and(c) predicted ∆δRS signs. Energies calculated by AM1 for (S)-2-methylbutiric acid.


(S)-2-methylbutyric acid as the model substrate.Their NMR showed that the higher ∆δRS values wereobtained with ethyl 2-hydroxy-2-(9-anthryl) acetate(9-AHA; see Figure 131) as the auxiliary, and,therefore, 9-AHA was chosen for further studies asa CDA.

Theoretical and experimental studies showed thesuitability of this reagent for the assignment of the

configuration of carboxylic acids: MM and AM1/PM3methods were used to calculate the geometries andrelative stabilities of the conformers of the 9-AHAesters; D NMR data were used to provide experimen-tal evidence of the theoretical findings; and aromaticshielding effect.87

The studies previously described all establishedthat the conformational composition of any ester of

Figure 133. ∆δRS values for the 9-AHA esters of compounds 133.1-133.16.

Figure 134. Conformational model for the determination of the absolute configuration of R-chiral carboxylic acids byNMR of their 9-AHA esters.


9-AHA with an R-chiral carboxylic acid is as repre-sented in Figure 132: two main forms, ap (major)and sp (minor), are in conformational equilibrium.The L1 substituent in the (R)-9-AHA ester is shieldedin the ap conformation, while the L2 substituentremains unaffected. In contrast, in the sp conforma-tion, the L2 substituent is shielded while the L1

substituent remains unaffected (see Figure 132a). Inthe case of the (S)-9-AHA ester, substituent L2 isshielded in the ap conformation and substituent L1

is shielded in the sp conformation (see Figure 132b).Because the population of the ap conformer is higherthan that of the sp conformer, substituent L1 is moreshielded in the (R)-9-AHA ester than in the (S)-derivative (∆δRS < 0) and, conversely, substituent L2

is more shielded in the (S)-9-AHA ester than in the(R)-ester (∆δRS > 0). The signs of the ∆δRS valuesallow the assignment of the spatial position of sub-stituents L1/L2 by comparison of the 1H NMR spectraof both diastereoisomers (see Figure 132c).

Examination by 1H NMR spectroscopy of the (R)-and (S)-9-AHA esters of a large number of carboxylicacids of known absolute configuration and with awide variety of structural features gave full experi-mental support to the aforementioned theory. The∆δRS values shown in Figure 133 serve to illustratetwo important facts. First, the distribution of thesigns of ∆δRS shown in Figure 133 is perfectlyhomogeneous along the series and is consistent withboth the absolute configurations of these compoundsand the conformational composition shown in Figure132.

Figure 134 shows the model for the assignment ofthe configuration of an acid of unknown configurationby comparison of the 1H NMR spectra of its (R)- and(S)-9-AHA esters. In this model, the ap/sp equilibri-um is simplified by considering the dominant ap form(Figure 130) as the only relevant one and using thisto fix the spatial location of substituents L1 and L2.According to this model, the substituent that is moreshielded in the (R)-9-AHA ester than in the (S)-9-AHA ester (∆δRS < 0) should be assigned as L1 inFigure 134c and, conversely, the substituent that ismore shielded in the (S)-9-AHA ester than in the (R)-9-AHA ester (∆δRS > 0) corresponds to L2 (Figure

134c). Identification of substituents L1/L2 and theirplacement around the asymmetric center can thenbe directly obtained from the signs of ∆δRS, as shownin Figure 134.

3.6.2. ArylcyclohexanolsOther aryl alcohols different from the open-chain

1-aryl alcohols shown in Figure 131 also have beenexamined.87b Figure 135 shows their structures.

When the ∆δRS values obtained for the esters ofthe arylcyclohexanols 135.1-135.4 and a series ofcarboxylic acids of known absolute configuration areexamined, it was observed that the commerciallyavailable (1R,2S)- and (1S,2R)-2-phenyl-1-cyclohex-anol (135.1) gave ∆δRS values that were very similarto those obtained with 9-AHA. These data alsoindicate the following: (a) the signs of the shiftsproduced by 135.1 are regularly distributed in all thecompounds examined (∆δRS is positive for protons insubstituent L1 and negative for protons in substituentL2); (b) the signs obtained are opposite to thoseobtained with 9-AHA (negative for substituent L1 andpositive for substituent L2); (c) the absolute ∆δRS

values obtained with 135.1 are, in all cases, similarto those produced by 9-AHA for protons near thechiral center. These facts indicate that both 135.1and 9-AHA essentially show the same ability to

Figure 135. Structure of trans-arylcyclohexanols.

Figure 136. ∆δRS values for the esters of the carboxylicacids shown with trans-2-phenyl-1-cyclohexanol and 9-AHA.Data for 9-AHA given in boldface type.


separate the resonances of the enantiomeric protonsof the substrates (see Figure 136).

Aromatic shielding-effect calculations corroboratedthe shieldings obtained with this reagent. From apractical standpoint, 135.1 has an advantage over9-AHA in that it is commercially available; on theother hand, the signals of 9-AHA in the NMR spectra

of its esters almost never overlap with those of thesubstrate, whereas the presence in 135.1 of numer-ous different protons is a clear limitation in thisrespect.

To analyze the role of the aryl ring in 135.1, thepossibility of increasing the ∆δRS values by changingthe aromatic ring was explored and new reagents

Figure 137. Representation of the direction of the maximum shielding in the esters of (a) 9-AHA and (b) 135.1. Modelsfor the determination of the absolute configuration on R-chiral carboxylic acids by NMR of their trans-2-phenyl-1-cyclohexanolesters (c, d and e).


with 1-naphthyl, 2-naphthyl, and 9-anthryl rings(135.2, 135.3, and 135.4, respectively; see Figure 135)were synthesized and tested. Unfortunately, signifi-cant improvements in the ∆δRS values were not foundwhen compared to 135.1. The fact that compoundswith naphthyl and anthryl rings do not producehigher shifts than 135.1, which has a practicallyidentical conformation, seems to be in conflict withdata obtained for AMAA esters. Nevertheless, in-spection of the structures of the esters of the aryl-cyclohexanols indicates that the direction of maxi-mum shielding is focused on the area immediatelysurrounding the asymmetric C atom (Figure 137a,

b). Thus, when the phenyl group in the arylcyclohex-anols is replaced by a larger aromatic system, theshielding area becomes very much larger and thisshould now affect not only the substituent on thesame side (L2) but also the other substituent (L1), tosome extent; therefore, on average, the ∆δRS valueswill not be as high as expected. In the case ofphenylcyclohexanols, the focus of the shielding coneis so far away from the asymmetric center that evenwhen its effect is greatly increased, it will neverinfluence more than one substituent in each con-former.

Panels c-e in Figure 137 show a graphic summarythat illustrates how the absolute configuration of anR-chiral carboxylic acid can be deduced from the signsof the ∆δ values obtained from its esters with thetwo enantiomers of any trans-2-aryl-1-cyclohexanol.

3.6.3. (S)-Methyl MandelateOther than the auxiliary reagents described by

Riguera’s group in the preceding pages, very fewpapers have been devoted to the determination byNMR of absolute configuration of carboxylic acids. Inone such example, however, (S)-methyl mandelatewas tested as a chiral auxiliary with racemic and

Figure 138. ∆δSR values obtained for enantiomeric mixtures of the acids shown, using (S)-methyl mandelate as the reagent(chemical shift of the (S)-acid-(S)-methyl mandelate minus that of the (R)-acid-(S)-methyl mandelate).

Figure 139. Simplified model for arylalkoxyacetic acidesters of a chiral acid according to (a) Tyrrel88 and (b)Riguera.87

Figure 140. (a) Structure of PGDA and PGME amides and (b) empirical model for the assignment of the absoluteconfiguration of carboxylic acids.


enriched mixtures of eight acids.88 Although thenumber of chemical shifts reported was very limited(only the methyl groups labeled a and b in Figure138), a coherent shift pattern, with a homogeneousdistribution of signs for substituents L1/L2 and useful∆δSR values was obtained.

The results obtained on using (S)-methyl mande-late as the reagent were interpreted assuming aconformational equilibrium and preferred conforma-tion (CR′-H not coplanar with CdO, and L2 anti tothe CdO group; see Figure 139) completely differentfrom those described for the esters of 9-AHA (seeFigures 132 and 134). Although this model caneffectively be used to correlate the NMR shifts withthe structure of the substrates, it is inconsistent withthe well-documented coplanarity of CR′-H withCdO, and therefore does not represent the realconformational composition of the compoundsstudied;29,75-78,87 indeed, specific experimental datawere not advanced to support the model. Therefore,the success of this model in the prediction of correctR/S configuration is purely accidental and can beexplained because the spatial location of substituentsL1/L2, relative to the aryl ring, is approximately thesame as that if the model corresponding to 9-AHA(Figure 132) were considered.

3.6.4. Phenylglycine Dimethyl Amide (PGDA) andPhenylglycine Methyl Ester (PGME)

Phenylglycine dimethyl amide (PGDA) and phen-ylglycine methyl ester (PGME) were proposed byKusumi and co-workers89 as auxiliary reagents forR-chiral carboxylic acids (Figure 140a). In this pro-cedure, the acid of unknown configuration is con-densed with the two enantiomers of the reagent andthe ∆δSR values of substituents L1/L2 are measuredin the corresponding amides.

The empirical model shown in Figure 140b wasproposed for the interpretation of the experimentaldata: substituents Hx, Hy, Hz, which correspond toL2, are more shielded by the phenyl group in the (S)-PGDA (or (S)-PGDE) amides than in the (R)-amides,and the opposite case occurs for substituents Ha, Hb,Hc (L1). Thus, ∆δSR (L2) < 0 and ∆δSR (L1) > 0.

Although PGDA was tested with just one carboxylicacid of known absolute configuration (141.1; seeFigure 141), and, therefore, its use as an auxiliaryreagent for acids cannot be recommended, the reli-ability of the assignments produced using PGME wasdemonstrated with the series of carboxylic acids ofknown configuration shown in Figure 141.

An XRD study and NOE experiments were pre-sented to support the model, although other studieshave shown that, in solution, the CdO, N-H, andC-H adopt a coplanar disposition.75-78 If this is thecase, then the model should be modified to the oneshown in Figure 142, but this change does not affecteither the shielding distribution or the configurationassigned, because the position of substituents L1/L2,relative to the phenyl group, is approximately thesame in both cases.

When PGME was applied to compound 143.1(Figure 143), an irregular distribution of ∆δSR signswas obtained90 for several protons. The authorshypothesized that a phenyl-phenyl interaction couldbe the cause of this phenomenon, but when othersubstrates that contain phenyl groups were studied(Figure 144), the signs were as one would expect fromthe configuration of the substrates and no discrep-ancies were found. Steric hindrance or the effect of a

Figure 141. ∆δSR values for the PGDA and PGME amides of the carboxylic acids shown.

Figure 142. Alternative model for the assignment of theabsolute configuration of carboxylic acids with PGDA orPGME.

Figure 143. ∆δSR values for the PGME amides of com-pound 143.1.


nearby O atom were proposed as possible causes ofthe problem.

PGME has been used on several occasions todeduce the absolute configuration of natural prod-ucts. Some examples in this field include the follow-ing: compound 145.1, which is a degradation productfrom the marine cycloperoxide plakortin;91a the HIV-1integrase inhibitor of microbial origin, integric acid91b

145.2 (Figure 145); and carboxyYTX, which is ananalogue of yessotoxin, isolated from mussels.91c

More recently, PGME was applied to the assign-ment of the configuration of R-oxy tertiary carboxylicacids92 (R-oxy-R,R-disubstituted acetic acids). In these

cases, the empirical model shown in Figure 146 wasproposed and several acyclic and cyclic R-oxy-car-boxylic acids of known absolute configuration, includ-ing some natural products, were used as substratesto prove the reliability of the procedure (Figure 147).

The method was first applied to determine theconfiguration at C-18 of polyether macrolide pect-enotoxin-692a (148.1, Figure 148). The absoluteconfiguration of lactone 148.2, which is a derivativeof tanikolide, isolated from cyanobacteria93 was alsoassigned this way.

Figure 144. ∆δSR values for the amides of the carboxylic acids shown with PGME (obtained from the (S)-PGME amidesof the racemic acids).

Figure 145.

Figure 146. Empirical model for the assignment of theabsolute configuration of R-oxy-R,R-disubstituted aceticacids with PGME.


The applicability of PGME to R-oxy-R-monosubsti-tuted acetic acids, using the empirical model pro-posed for R-oxy-R,R-disubstituted acetic acids inwhich one of the R-substituents is a hydrogen, wasalso investigated.94 Unfortunately, only four ex-amples of known absolute configuration were usedto support the reliability of this model (Figure 149).

In addition, Fusetani and co-workers95 found that,when PGME was used for the study of the furoic acid

derivatives shown in Figure 150, the same ∆δ signswere obtained for all the relevant protons of thePGME amides. The model proposed by Kusumi isconsistent with those results.

Figure 147. R-Oxy-R,R-disubstituted acetic acids studied with PGME.

Figure 148. (a) Structure of Pectenotoxin-6, (b) ∆δSR values in C5D5N, (c) ∆δSR values in CDCl3, and (d) structure ofTanikolide lactone.

Figure 149. R-Oxy-R-monosubstituted acetic acids studiedwith PGME.

Figure 150. ∆δ values for furoic acid derivatives withPGME.


3.6.5. 1,1′-Binaphthalene-8,8′-diol (BNDO)A completely different type of reagent was proposed

in 1996 by Japanese workers96 for the stereochemicalassignment of carboxylic acids; this is 1,1′-binaph-thalene-8,8′-diol (BNDO; see Figure 151a), whichpresents axial chirality and is available in the twoenantiomeric forms.

Several acids of known absolute configuration wereesterified with (aR)-, (aS)-, and/or racemic BNDO,and a method to predict the configuration wasdevised. The idealized conformation of a BNDOmonoester is depicted in Figure 151b: The naphtha-lene rings are orthogonal, the carbonyl and thehydroxyl groups are syn, and the a proton is situatednear the center of the facing naphthalene ring.Because of the effect of the aromatic ring, the signal

due to Ha of the (aR)-BNDO monoester shouldappear downfield, relative to that in the (aS)-BNDOmonoester. The reverse should hold true for Hb

Figure 151. (a) Structure of (aR)-BNDO. (b) Ideal conformation of an (aR)-BNDO monoester. (c) Position of the acidsubstituents in the diasteromeric esters. (d) Model for the assignment of configuration from the ∆δ values.

Figure 152. (a) 1H ∆δaSaR values for the BNDO esters of the carboxylic acids shown. (b) 13C ∆δaSaR values.

Figure 153.


(Figure 151c). The chemical-shift difference wasdefined as ∆δ ) δaS- δaR; thus, in the modeldepicted in Figure 153d, positive and negative valuesare associated with the left- and right-hand sides ofthe substrate, respectively. Isobutyric acid and sev-eral derivatives were used for 1H NMR analyses to

confirm the preponderance of the ideal conformationin solution.

The 1H ∆δ values for a very limited number (four)of chiral acids are shown in Figure 152a. 13C ∆δvalues for three of those compounds were also re-ported (Figure 152b).

3.7. Application to â-Chiral Carboxylic Acids

3.7.1. ArylethylaminesAcyclic carboxylic acids containing remote stereo-

centers constitute a class of substrates for whichmethods to determine their configurations were alsopursued. In fact, â-substituted acids represent aspecific structural unit that is of interest in thecontext of natural products chemistry. Hoye et al.97

chose the amide bond to link the reagent to thesubstrate and explored the use of 1-arylethylamines153.1-153.4 as reagents (see Figure 153a).

These compounds were tested with the homologousseries of acids 153.5-153.8 (see Figure 153b), usingthe signals of the methyl groups of the amides toassess the effectiveness of these systems as reagents.As expected, the anthryl derivative 153.4, followedby the naphthyl derivative 153.2, resulted in thelargest shifts. Amines 153.1 and 153.2 are com-mercially available in optically pure form and weretherefore adopted for further studies with a series of3-substituted acids of known absolute configuration.

The results summarized in Table 14 show how theauthors differentiate two stereochemical possibilities(defined as syn/anti, according to the relative orienta-tion of the benzylic methyl group and the methylsubstituent at C(3)) and calculate the parameter ∆δ(defined as the difference between the chemical shiftsin the syn and anti diastereomers) for the â-methyland other groups.

The data obtained show that the â-methyl groupsare always more deshielded in the syn series than

Table 14. ∆δ Values of the â-Methyl and OtherDiagnostic Resonances in Diastereomeric Syn andAnti 1-Arylethylamides from 3-MethylcarboxylicAcids

Figure 154. Conformational model for the determination of the sign of ∆δ ) δ(syn) - δ(anti) of protons in R1 and R2.

Figure 155. ∆δ ) δ(syn) - δ(anti) values for amides from â-chiral acids with known relative configurations Ar ) 1-Npdata given in boldface type.


in the anti series, whereas protons in the otherâ-substituent are more shielded in the syn isomersswith the exception of the vinylic H(4) proton in thelast entry in Table 14.

A working model (Figure 154) based on the NMRresults and routinely accessible calculations (AMBERforce field, as implemented in the MacroModel soft-ware package) has been proposed. This model isbased on two assumptions: (1) the predominantrotamer around the bond between the benzylic Catom and the N atom is that in which the benzylicproton is eclipsed with the carbonyl group, and (2)one of the two nonhydrogen substituents at C(3)occupies the anti position, relative to the carbonylgroup.

In the case of the anti diastereomer, this situationresults in preferential shielding of R1, whereaspreferential shielding of R2 should be observed in thesyn diastereomer. As a consequence, the ∆δ valuesfor protons in R1 are positive and those in R2 arenegative.

The scope of this method/reagent was extended bythe same authors to include carboxylic acids withsubstituents other than a methyl group at C(3),including the substrates shown in Figure 155 and sixother examples of known absolute configuration

taken from the literature. These studies confirmedthe applicability of this method.98

3.7.2. PGMEPhenylglycine methyl ester (PGME; see Figure

156a), proposed by Kusumi et al.89 for the determi-nation of the configuration of R-chiral carboxylicacids, has also been used with acids that have thechiral center at the â-position. The eight carboxylicacids of known configuration shown in Figure 157were used as test compounds. The signs of the ∆δRS

values of the corresponding PGME amides werecoherent with their stereochemistry and the modeloutlined in Figure 156c.89b

Figure 156. (a) Structure of (S)-PGME. (b) Suggestedmost stable conformation of a PGME amide. (c) Model forthe assignment of the configuration.

Figure 157. ∆δRS values for the amides of the â-chiral carboxylic acids tested with PGME.

Figure 158.

Figure 159. Proposed conformation for the PGME amidesof the gambieric acids based on NMR analysis. ∆δSR

(instead of ∆δRS) values were reported; the inconsistentvalue is highlighted.

Figure 160. Formation of N-(methoxyphenylacetyl)sul-foximines from sulfoxides.

Figure 161. Model for the determination of configurationof N-(methoxyphenylacetyl)sulfoximines from sulfoxides.


This situation is rationalized on the assumptionthat steric interactions cause the PGME amide of a

â-chiral carboxylic acid to exist in essentially theconformation shown in Figure 156b. However, theo-

Figure 162. N-MPA-sulfoximides used in this study.

Figure 163. Polyhydroxylated compounds studied as MTPA esters.


retical calculations were not performed to confirmthis hypothesis.

This reagent/model was used to establish thestereochemistry of several natural products such as158.1, which is a degradation product from a diter-penoid isolated from an insect wax99a (see Figure 158)and the gambieric acids, which are polyethers iso-lated from marine dinoflagellates.99b In the lattercase, the authors found it necessary to ensure thatthe proposed PGME model was acceptable for thesecompounds by performing a conformational analysis(NOE, HOHAHA). The resulting modified conforma-tion is shown in Figure 159.

3.8. Application to Chiral Sulfoxides: MPA

A procedure for the determination of the absoluteconfiguration of chiral sulfoxides has been devel-oped100 and requires the introduction of a chiralanisotropic moiety by transformation of the sul-foxides into N-(methoxyphenylacetyl)sulfoximines.The method consists of the amination of the sulfoxidewith O-mesitylsulfonylhydroxylamine, which pro-ceeds with complete retention of chirality at the Satom, followed by introduction of (R)- and (S)-MPAat the N atom (Figure 160).

Subsequent application of the empirical modelshown in Figure 161 allows the configuration to bededuced on the basis of the ∆δSR values (∆δSR ) δ(S)- δ(R)).

The utility of this model and the reliability of theconfigurational assignments are based on the coher-ence of the NMR data obtained for the MPA-derivedsulfoximides shown in Figure 162, which are derivedfrom eleven chiral sulfoxides of known absoluteconfiguration (162.5-162.15). In addition, four ra-cemic sulfoxides were derivatized with racemic MPAand the NMR data of the corresponding MPA-derivedsulfoximides 162.1-162.4 were shown to produceshifts that apparently confirmed the model: the ∆δsigns are systematically arranged on both sides ofthe MPA plane, but the absolute configuration of eachderivative was not available for proper validation.

3.9. Application to Chiral PolyhydroxylatedCompounds

In many fields of chemistrysnatural products,organic synthesissresearchers often must determinethe absolute configuration of substrates that haveseveral chiral centers and functional groups, such aspolyols. Usually, when facing these situations throughthe use of the NMR methods, the direct applicationto polyalcohols of the models originally developed formonoalcohols was the solution that was used, withoutconsidering that such a solution could be highlyinappropriate. This situation was exposed in a work13

where several examples taken from the literatureillustrate the magnitude and characteristics of theproblem.

In those reports, attempts were made to assign theabsolute configuration of all the asymmetric centersof a polyalcohol by application of the NMR methodto the fully esterified derivatives with (R)- and (S)-MTPA; however, the ∆δSR data obtained generallydid not allow a completely unambiguous assignmentof configuration.

Figure 164. Analysis of the diastereomeric nonequivalence in the (R)-MPA esters of the enantiomers of tanshindiol (164.1).The shielding on the methylene protons is different in each case.

Figure 165. Polypropionate metabolites from the marinemollusk Onchidium sp. (165.1 and 165.2) and 1H NMRchemical shifts of chiral bis-MPA esters of (2R,4R)-(-)-pentanediol, indicating the different shielding caused bythe auxiliary reagents.


Figure 163 shows how the hexa-MTPA ester of5-desacetylaltohyrtin A101 (163.1) generates ∆δ val-ues around C-5, C-35, C-38, C-42, and C-47 that donot present the necessary alternation of signs aroundsubstituents L1/L2 and the same occurs with C-16,C-19, and C-24 of squamostatin D25b (19.5; see Figure19). Other examples of this type include annonaceousacetogenins such as bullatacin12a,102a (rolliniastatin-2, 163.2; see Figure 163), sootepensin A25a (19.4; seeFigure 19), 4-deoxyannomontacin102b (163.3); a don-naienin derivative102c (163.4); and sponge metabolitespenaresidins23 (19.1 and 19.2; see Figure 19).

This lack of homogeneity of the ∆δSR signs involvesa clear risk of misassignment and means that, tosome extent, the considerations known to be valid formonoesters are not fulfilled in those bis- or tris-MTPA esters. The aforementioned article13 remindsus that the fundamentals of the NMR method arerelated to aromatic shielding, which is a through-space phenomenon that affects protons several bondsaway from the hydroxylic C atom that has theauxiliary reagent. Therefore, the ∆δSR values ob-served in those polyderivatized compounds are, infact, the result of the combined effects (shielding/deshielding) of all the MTPA phenyl rings and cannotbe interpreted as if they were produced by just onephenyl group as in monofuntional compounds.

In practice, the assignment of the configuration ofcompounds that have several functional groups withthe same reactivity (i.e. 2,3-pentanediol) can beaddressed in two possible ways: either the appropri-ate protection/deprotection protocols are used toachieve selective derivatization of each hydroxylgroup or, more simply, the two alcohol groups arederivatized together in a single reaction.

In the first case, the configuration at each asym-metric C atom is examined separately, using themodels developed for monofunctional compounds;however, the protection/deprotection operations willbe time- and sample-consuming and this is a signifi-cant drawback.

There could be special cases where the reactivityof the functional groups with the CDA is sufficientlydifferent to achieve selective derivatization of one ofthe groups without protecting the other. One par-ticularly noteworthy example of this situation usingMPA as the reagent is represented by tanshindiol(164.1; see Figure 164), which is a plant metabolitethat possesses vicinal primary and tertiary hydroxylgroups.103 When both enantiomers of 164.1 werederivatized with (R)-MPA, single esters on the pri-mary hydroxyl group were obtained and their NMRwere used for the assignment on the basis of themodels shown in Figure 164. Note that MPA is not a

Figure 166. (R)- and (S)-MPA diester of an anti-1,2-diol and expected ∆δRS signs. Arrows show the shielding producedby the phenyl groups.


reliable reagent for assignment of configuration ofprimary alcohols (see section 3.2 of this review) andthat its application in the case of tanshindiol (164.1)is determined by the chelating ability of the tertiaryhydroxyl group.

In the second and more general approach, the twofunctional groups are made to react with the auxil-iary at the same time and the preparation of the bisderivatives is straightforward. However, as men-tioned previously, the shifts observed in the NMRspectra of the fully derivatized compound (i.e., thebis-ester of the diol) cannot be interpreted using themodels for monofunctional compounds. New andspecific models that correlate the absolute stereo-chemistry of diols with the NMR spectra of their bis-AMAA esters have recently been described and willbe presented in the next section of this review.

3.9.1. MPA and 9-AMAThe use of MPA on substrates that have several

chiral hydroxyl groups was first described by Rigueraet al.104 in a study on polypropionate metabolites fromthe marine mollusk Onchidium sp. The configura-tions of the onchitriols 165.1 and 165.2 were estab-lished by comparison of the ∆δRS values of the bis-MPA esters with those of selected diols of knownabsolute configuration, such as (2R,4R)-(-)-pen-tanediol (Figure 165).

Years later, the correspondence between the abso-lute configuration of a series of diols of knownconfiguration and the NMR spectra of their bis-AMAA derivatives was studied by the same group,and a general methodology to determine the config-uration of 1,n-diols (1,2-diols, 1,3-diols etc.) from theNMR spectra of the bis esters was presented.105a Inthis procedure, the diol is fully esterified with the Rand the S auxiliary (MPA or 9-AMA) and the ∆δsigns are interpreted as being the result of thecombined action of the two auxiliary units.

This approach is based on the fact that the aro-matic shielding is a through-space phenomenon; eachreagent unit contributes to the chemical shift of agiven proton in a different way (shielding or deshield-ing) and with a different strength, depending on theactual structure of the diol. As a consequence, thechemical shifts (and ∆δRS values) observed are theresult of the additivity of the shielding/deshieldingeffects produced by all the auxiliary reagents presentin the bis ester. This overall effect can be predictedfor each stereochemical combination if the conforma-tional composition of the AMAA esters is known. Infact, the predicted ∆δRS signs are fully in agreementwith the experimental results for many diols ofknown absolute configuration.

Figure 167. (R)- and (S)-MPA diester of a syn-1,2-diol, and expected ∆δRS signs. Arrows show the shielding produced bythe phenyl groups.


Clearly, if the experimental ∆δ values were inter-preted as if they were caused by just one reagent unit(and neglecting the effect of the other), using themodels for monofunctional compounds, the resultingassignment may be incorrect.

If we consider the conformational characteristicsof the AMAA esters of secondary alcohols previouslydiscussed, and apply them to the case of a vicinal diol,the schematic diagrams that are presented in Figure166 result.

In Figure 166a, the shielding/deshielding zonesoriginated by the aryl group of an anti-1,2-diolderivatized as bis-MPA ester are presented: in thebis-(R)-MPA ester, the R2 group and CR′′H areshielded, whereas in the bis-(S)-MPA ester, only theR1 group and CR′H are shielded. Thus, negative ∆δRS

(δR,R - δS,S) values should be expected for R2, andCR′′H, and positive ∆δRS values are expected for R1,and CR′H. If the configuration of the diol were theopposite, the signs also would be opposite.

When a syn-1,2-diol is considered (Figure 167),caution must be taken into consideration, becauseonly the signals due to the protons in R positions areuseful for assignment. Figure 167 shows that R1 andR2 are both shielded in the bis-(R)- and (S)-MPA

diester, which makes prediction of the sign of their∆δRS values impossible. For its part, the CR′H andCR′′H are shielded only in the bis-(S)-MPA-diesters;thus, a positive ∆δRS should be expected for bothprotons CR′H and CR′′H. Once again, if the config-uration of the diol were the opposite, the signs alsowould be the opposite.

The same reasoning can be used to predict thedistribution of the ∆δRS signs in other diols differentthan 1,2-diols. Figure 168 shows the distinctive ∆δRS

patterns of the four stereoisomers of 1,2-, 1,3-, and1,4-diols and constitutes a guide for the assignmentof the configurations.

Note that, although for monofunctional compounds,the ∆δRS signs provide sufficient information todistinguish between the two enantiomers (R or S),in diols with two asymmetric centers, four stereo-chemical situations are possible (the syn and antipairs: RR, SS, RS, or SR). In fact, four differentcombinations of shielding/deshielding effectssand,consequently, four different sets of ∆δRS signssarepredicted and experimentally obtained for those fourstereochemical arrangements: one for each stereo-isomer. Thus, the absolute configurations of the twochiral centers in any of the four stereochemical

Figure 168. Diagnostic ∆δRS (9-AMA, MPA) and ∆δSR (MTPA) signs for the four stereoisomers of (a) 1,2-diols, (b) 1,3-diols and (c) 1,4-diols. R3 represents the substructure between the chiral centers in diols other than 1,2 systems.


combinations of the diol can be deduced from just theNMR of the corresponding bis-AMAA esters.

After the diol of unknown configuration has beenderivatized with the two enantiomers of the reagent(MPA or 9-AMA), the ∆δRS values and signs of thebis esters are calculated in the usual manner. Thenext stage involves comparison of the signs of ∆δRS

of the diagnostic signals CR′H and CR′′H with thoseshown in Figure 168. If both the CR′H and CR′′Hhave the same ∆δRS sign (either both positive or bothnegative), then the ∆δRS signs for R3 (but not for R1/R2) should also be examined and all three signsshould be compared with those of Figure 168 forassignment.

In case CR′H and CR′′H present opposite ∆δRS signs(one positive and the other negative), the ∆δRS signsfor R1 and R2 (but not for R3) then should beconsidered and all four signs should be comparedwith Figure 168 for assignment.

A selection of the diols of known absolute config-uration that have been used to demonstrate theseconclusions is shown in Figure 169 and includes 1,2-and higher-order diols as substrates and MPA,9-AMA (169.1-169.9) and 2-NMA102a (169.11 and169.12) as reagents.

Extension of this methodology to the assignmentof nonsymmetrical 1,n-diols with two secondary(chiral) hydroxyl groups, or with primary and sec-ondary (chiral) hydroxyl groups, as well as aminoalcohols, has been conducted and shown to besuccessful.105b

3.9.2. MTPAIf the MTPA bis esters of the diols are submitted

to the same analysis as the MPA esters, the resultingshielding/deshielding areas around the diol frame-work and the sign distributions are different fromthose obtained with MPA and AMAA. This is due tothe diverse conformational composition of MTPAesters; however, from a practical standpoint, thesame set of signs shown in Figure 168 for AMAA bisesters can be applied by simply using the experimen-tal ∆δSR values of the MTPA esters as if they were∆δRS. The data reported106 for compound 169.10 serveto illustrate the application of this rule.

There are some examples in the literature of theapplication of MTPA to the assignment of configu-ration of diols where the NMR data are interpretedin a variety of ways. Interestingly, note that applica-tion of the general model of Figure 168 to the NMRdata provided in those papers for the bis-MTPAesters lead, in all cases, to the correct assignment ofabsolute configuration giving further support to thegenerality of the models developed by Riguera’sgroup.

Thus, Ichikawa and co-workers107 studied the as-signment of syn and anti diols by NMR of theirMTPA esters; unfortunately, only a very small num-ber of compounds (one syn (170.1) and two anti (170.2and 170.3); see Figure 170) were examined (their

configurations were not checked by independentmethods: in two cases, the work was done withracemates and the ∆δSR values inferred from the (R)-MTPA derivatives).

According to the authors, the ∆δRS values of theprotons on the asymmetric carbons and their vicinalC atoms are the only ones that can be considered forassignment purposes in the case of syn diols. Theremainder protons should be neglected. This isrelated to the absence of the necessary homogeneity

Figure 169. Structure and ∆δRS values obtained for bis-MPA, 9-AMA (169.1-169.9), 2-NMA (169.11-169.12), andMTPA (169.10). Data given in boldface type correspondsto that of MPA and data given in normal typeface cor-respond to that of 9-AMA.


of ∆δRS signs in those protons in the syn diols (thisis a requirement for reliable assignments frequentlynot fulfilled when MTPA is used as CDA; see Figure171). A different model applies to anti diols (Figure170).

In another paper108 by the same group, the assign-ment by NMR of the bis-MTPA esters of diols thathave aromatic groups was studied, as shown inFigure 172 (the inadequacy of MTPA as a reagentfor alcohols that possess aromatic groups had alreadybeen noted by Ohtani17a). This study showed that, forthe monoaromatic anti-glycols 172.1, 172.2, and172.3, the model showed in Figure 170 was operativebut fail for the monoaromatic syn-glycol 172.4 andthe bis-aromatic syn-glycol 172.5. The fact that themethod did work with the aliphatic syn-glycols 172.6and 172.7 suggested that π-π stacking could be thecause of the disagreement between the prediction andthe experimental data.

Analysis of the NMR data, following the rules ofFigure 168, shows discrepancies in the ∆δSR signsobtained for the protons at CR and C-R′ of monoaro-matic syn-glycol 172.4 and bis-aromatic syn-glycol172.5. This finding reinforces the idea that MTPAshould not be recommended as CDA for the assign-ment of syn-glycols that posses aromatic groups norfor any diol of unknown relative configuration witharomatic groups.

In a more detailed and recent publication,109 aseries of 1,3-diols (see Figure 173) was studied as bis-MTPA esters and their NMR spectra analyzed in the

manner proposed by Riguera et al. for 1,n-diols. Theauthors concluded that the method was only ap-plicable to syn-1,3-diols, where the values of substit-uents L1 and L2 are diagnostic (but not those of the

Figure 170. (a) Diols used in this study. Models for thedetermination of configuration of (b) syn- and (c) anti-1,2-diols with MTPA. Only protons inside the box fit the rulefor syn diols.107

Figure 171. ∆δSR values for a syn diol. The lack ofhomogenity in the signs obtained for a same L substituentare highlighted.

Figure 172. ∆δSR signs obtained from the bis-MTPAesters of glycols. Discrepancies with the models of Figure170 are highlighted in 172.4, 172.5, and 172.7.

Figure 173. Experimental ∆δSR signs obtained for the bis-MTPA esters of the syn- and anti-acyclic 1,3-diols shown.


central part). They also state that it should not beapplied to anti-1,3-diols, where substituents L1/L2

cannot be considered and the central part is, in theiropinion, not surely diagnostic. However, in all thecases, the ∆δSR signs obtained experimentally (Figure173) are in full agreement with those predicted byRiguera’s model for those configurations as shown inFigure 168.

In the same work, six-membered cyclic systemswere examined and four zones with distinct ∆δvalues, depending on the position around the ring,were proposed (see Figure 174). Five cyclohexane-1,3-diols derived from dihydroxyvitamin D3 and withknown absolute configuration were examined as bis-MTPA esters. Figure 174a shows the experimental∆δSR sign distributions and Figure 174b shows theempirical models that have been used.

In the field of natural products, numerous ex-amples can be found of the assignment of configura-tion by NMR of the bis- or tris-MTPA esters. Only ina few cases have the authors based their assignmenton the use of simpler synthetic diols of knownconfiguration as models for comparison. For example,Minale et al. determined the configuration of the sidechains of marine polyhydroxysteroids110a such as175.1 and 175.2, using synthetic 2,3-dimethylpen-tane-1,2-diols and 2-methyl-3-ethylheptane-1,2-diols,respectively, as models.110b

The bis-MTPA derivatives of C27- and C29-alkane-6,8-diols (175.3; see Figure 175) were prepared inanother report;110c however, their NMR data wereused simply to discriminate among stereoisomers (infact, only the small shifts at Me-1 were considered!)and the absolute configuration was not proposed.

Figure 174. (a) Signs of the ∆δSR obtained for the bis-MTPA esters of syn- and anti-cyclohexane-1,3-diols used in thisstudy, and (b) predicted ∆δSR values.


4. Methods Based on a Single DerivatizationAs we have shown, all the procedures in the

preceding pages involve the derivatization of thesubstrate with the two enantiomers of the reagent

and comparison of the NMR spectra of the corre-sponding diasteroisomeric derivatives. Nevertheless,recently, some effort has been devoted to methodsthat require the preparation of only one derivative.The existence of such a procedure would reduce thetime and amount of sample necessary for the analysisby a factor of 2, because only one derivative and onederivatization step would be needed.

Two general approaches can be distinguished. Thefirst applies to secondary alcohols and is based onextensive NMR data, indicating that, when a strongauxiliary reagent such as 9-AMA (but not MPA orMTPA) is used, comparison of the NMR spectra ofthe secondary alcohol with that of one of its 9-AMAesters (either the R or the S) allows the safe assign-ment of the absolute configuration of the alcohol. Theprocedure, which is summarized in Section 4.1.1, isbased on the comparison of the chemical shifts ofmany secondary alcohols of known absolute config-uration and the corresponding (R)- or (S)-9-AMAester. A related method, which makes use of glyco-sydation shifts, is described in Section 4.1.2.

The other approach applies to secondary alcoholsand primary amines. It consists on the controlledmodification of the conformational equilibrium of theMPA esters and MPA amides and is based on ourknowledge of the shielding/deshielding effects associ-ated with each conformer.

Figure 175. Natural products studied as MTPA esters.

Figure 176. Partial 1H NMR spectra (300 MHz) of (a) the (R)-9-anthrylmethoxyacetic acid ester of alcohol 176.1; (b)alcohol 176.1; (c) the (S)-9-anthrylmethoxyacetic acid ester of alcohol 176.1.


If the sp/ap conformer ratio is modified in a certainway (i.e., an increase of the sp conformer), the NMRspectra should translate such a change in ratio intoa predictable modification in the chemical shiftsassociated with the substituents (i.e., greater popula-tion of the sp conformer in the (R)-MPA ester leadsto greater shielding of substituent L1 in the averagespectrum). In this way, the spatial position of sub-

stituents L1/L2, with respect to the phenyl group ofthe auxiliary (absolute configuration known), can bedetermined by the NMR response to the disturbancein the equilibrium.

Two different procedures have been described toinduce the conformational change:

(a) Modification of the equilibrium by reducing thetemperature of the NMR probe. This approach has

Figure 177. (a) Esterification shifts (∆δAR values) measured for the (R)-9-AMA ester of 176.1. (b) Esterification shifts(∆δAS values) measured for the (S)-9-AMA ester. Larger shifts are highlighted.

Figure 178. ∆δAR and ∆δAS values for (R) and (S)-9-AMA esters of alcohols 178.1-178.5. (Larger values are given inboldface type.)


found application in the assignment of the absoluteconfiguration of secondary alcohols using MPA as anauxiliary reagent (see Section 4.1.3).

(b) Modification of the equilibrium by selectivechelation of one of the conformers. This procedure hasbeen described for the assignment of secondaryalcohols and primary amines using MPA as anauxiliary reagent and barium(II) salts as chelators(Sections 4.1.4 and 4.2, respectively).

4.1. Application to r-Chiral Secondary Alcohols

4.1.1. 9-AMA Esters: Esterification ShiftsA procedure for the assignment of the absolute

configuration of secondary alcohols involving the

preparation of only one derivative has been recentlypresented by Riguera et al.35 and consists of acomparison of the NMR spectrum of the alcohol withthat of one of its esters prepared with either the (R)-or the (S)-9-AMA chiral auxiliary.29,30 When the tworesulting spectra are compared, the signals of sub-stituents L1 and L2 can be easily distinguished,because the protons of one of those substituents (theone that is under the shielding cone of the anthrylring in the derivative) move upfield in the 9-AMAester, with respect to the free alcohol, whereas theprotons of the other substituents (unaffected by thearomatic shielding) resonate at practically the samefield in both the 9-AMA ester and in the free alcohol.

Figure 179. Diagram for the assignment of the absolute configuration of a secondary alcohol from ∆δAR or ∆δAS values.

Figure 180. Esterification shifts for the (R)- and (S)-9-AMA esters of alcohols 176.1 and 178.1-178.6, expressed as meanvalues (∆δmL1 and ∆δmL2).


Consideration of the absolute configuration of theauxiliary reagent ((R)- or (S)-9-AMA) and the spatiallocation of its anthryl ring allows the placement ofsubstituents L1/L2 around the asymmetric C atom.

An example illustrating the esterification shiftsobserved in the marine metabolite40 176.1 is shownin Figure 176.

In the (R)-9-AMA ester (Figure 176a), H(6′), H(5′),and H(4′) are intensely shielded, with respect to thesame protons in the free alcohol (Figure 176b), whileH(9′), H(10′), Me(12′), and Me(13′) resonate at practi-cally identical chemical shifts in the ester and in thealcohol (the opposite is observed in the (S)-9-AMAester; see Figure 176c). These esterification shifts arequantitatively expressed for each individual protonas the difference between the chemical shift in thefree alcohol and that in the ester (∆δAR or ∆δAS,depending on which 9-AMA ester has been exam-ined). Figure 177 shows the ∆δAR and ∆δAS valuesobtained for compound 176.1.

Several linear secondary alcohols of known abso-lute stereochemistry (Figure 178) have been exam-ined in this way, and the results corroborate theexistence of the aforementioned correlation betweenthe spatial position of substituents L1/L2 of thealcohol, the configuration of the auxiliary, and theNMR shifts.

Thus, in the (R)-9-AMA esters represented inFigure 178, the most intensely shielded protons areall located in the right-hand substituent (L1), whereasthose that are only slightly shielded are located inthe left-hand substituent (L2) (∆δARL1 . ∆δARL2):The reverse occurs in the (S)-9-AMA esters, where∆δASL1 , ∆δASL2.

In this way, the configuration of a secondaryalcohol such as sec-butanol (178.1) is R (as indicatedin Figure 178) if the more intensely shielded protonsin the (R)-9-AMA ester are in the right-hand side ofthe projection (i.e., the ethyl substituent) and thosethat are only very slightly shielded are on the left-hand side (i.e., the methyl substituent). This ar-rangement results in ∆δARL1 . ∆δARL2. The reverse(∆δASL1 , ∆δASL2) is observed when the enantiomericreagent (S)-9-AMA is used and also when the con-figuration of the sec-butanol is S (opposite to thatshown in Figure 178). A graphical resume for ap-plication of this procedure is shown in the model ofFigure 179.

The plot shown in Figure 180 represents theintensity of the ∆δ parameter (∆δm) produced by9-AMA on the alcohols 176.1 and 178.1-178.5, andmeasured as mean values (∆δm) of all the protons insubstituents L1 and L2 (∆δmL1 and ∆δmL2).

Application of this methodology to cyclic alcoholsis also possible, and compounds 181.1-181.5 (Figure181) presented esterification shifts that were coher-ent with their stereochemistry and the model ofFigure 179. The use of the mean values ∆δmL2 and

Figure 181. Cyclic alcohols studied with this method.

Figure 182. (a) 1H NMR spectrum of a free alcohol. (b)1H NMR spectrum of its (R)-9-AMA ester. (c) Inferredabsolute configuration from the ∆δAR values.

Figure 183. Glucosides studied by the procedure ofglucosidation shifts in 13C NMR.

Figure 184. Tetra-O-acetylglucosides studied by theprocedure of glucosidation shifts in 1H NMR.


∆δmL1 is recommended for assignment of the config-uration of these cyclic structures.

From a theoretical standpoint, the different esteri-fication shifts observed for the shielded and non-shielded substituents are explained on the basis ofthe conformational composition and structure of the9-AMA esters (see Figure 46 in Section 3.1.3). In the(R)-9-AMA ester of the alcohol of Figure 46, substitu-ent L1 is located under the shielding cone of theanthryl ring of the reagent in the sp conformer,whereas in conformer ap, it is not shielded. Thereverse holds for substituent L2 (shielded in theminor conformer ap and unaffected in sp) and, as aresult, when one compares the spectrum of an alcoholin Figure 182a with that of its (R)-9-AMA derivative,

the signals for substituent L1 are shifted to a greaterextent than those in L2 (Figure 182b).

In this way, the signals of the alcohol that arestrongly shifted upfield upon esterification with (R)-9-AMA correspond to the protons that are located onthe same side as the anthryl group in the sp con-former, i.e., the right-hand side of the asymmetriccenter in the projection shown (substituent L1 inFigure 182c). In turn, the protons that are slightlyshielded (as they are not affected by the aryl group)are located on the left-hand side (substituent L2,Figure 182c). The reverse holds for the (S)-AMAesters and also when the configuration of the alcoholis opposite.

Attempts to use auxiliary reagents other than9-AMA for assignment of the configuration wereunsuccessful. Thus, when the (R)- and (S)-MPAauxiliaries were tested with a wide range of sub-strates, the ∆δAR and ∆δAS obtained were very smalland negative in some cases. As a consequence, theidentification of substituent L1 or L2 by associationwith the shielded and nonshielded protons is notalways possible; therefore, MPA cannot be recom-mended for this approach.

Similarly, 1- and 2-naphthylmethoxyacetic acids (1-NMA and 2-NMA, respectively) were assayed withlinear secondary alcohols and did not give satisfac-tory results, although a limited approach based oncertain empirical increment parameters has beenproposed.111

4.1.2. Glycosidation Shifts

4.1.2.1. Glycosidation Shifts in 13C NMR. Amethod for determining the absolute configurationof a secondary hydroxyl group based on characteristicglycosidation-induced shifts in 13C NMR spectroscopywas reported previously in the development of NMRmethods for configurational assignment.55a,b Thegeneral strategy consists of several steps that include(a) measurement of the 13C NMR spectrum of thesecondary alcohol (in pyridine-d5), (b) synthesis of thecorresponding â-D- or R-D-glucopyranosides (Figure183), (c) measurement of the 13C NMR spectrum ofeither the â-D- or R-D-glucopyranoside, (d) calculationof the glucosidation-induced shift variations for theanomeric carbon and for the R- and â-carbons of theaglycone using the relations ∆δ(anomeric carbon, 1′)

Figure 185. (a) Correlation model for secondary alcohols as â-D-glucopyranosides. (b) δD - δROH values for dimethyl D-and L-malate (in ppm, at room temperature, CDCl3).

Figure 186. Alcohols of known configuration studied asâ-D-glucopyranosides.


) δ(alcoholic glucoside) - δ(methyl glucoside) and∆δ(R- and â-carbons) ) δ(alcoholic glucoside) - δ-(alcohol), and (e) the absolute configuration of thealcohol is then determined by comparison of the 13CNMR chemical-shift changes with the glucosidationshifts of a series of secondary alcohols of knownabsolute configuration, classified according to theirsteric hindrance and degree of substitution in the Catoms next to the chiral center.

A caution note alerts the readers about the ap-plication of this procedure to sterically crowdedalcohols.

4.1.2.2. Tetra-O-acetyl-â-glucosylates in 1HNMR. The tetra-O-acetylglucosidation-induced 1HNMR shifts of a series of secondary alcohols were alsofound highly characteristic of their absolute configura-tion.55c Particularly important for the diagnosticswere the protons of the aglycone attached to the Catoms at the â-position (see Figure 184), and accord-ingly, the tetra-O-acetylglucosidation-induced 1H NMR

shifts were calculated as follows:

As in the 13C NMR method, comparison of the valuesobtained with those of secondary alcohols of knownconfiguration and different steric hindrance is neces-sary for assignment.

The same caution note that was mentioned previ-ously applies when this procedure is intended for usewith highly crowded alcohols.

4.1.2.3. Tetra-O-benzoyl-â-glucosylates in 1HNMR. Another procedure based on the comparisonof the NMR spectrum of the substrate with that of a

Figure 187. (a) Structure of the sp and ap conformers of an (R)-MPA ester. (b) Chemical shifts of substituents L1 and L2in the sp and ap conformers. (c) Average chemical shifts of L1 and L2 at room temperature (T1). (d) Average chemical shiftsof L1 and L2 at low temperature (T2).

Figure 188. Distribution of shielded and deshielded areas in the (R)- and (S)-MPA esters of (-)-menthol and selected∆δT1T2 values.

∆δ(anomeric proton, 1′)) δ(alcoholic glc-Ac4) -

δ(methyl glc-Ac4) for the sugar moietyand∆δ(R- and â-protons)

) δ(alcoholic glc-Ac4) -δ(alcohol) for the aglycone unit


single derivative of an auxiliary reagent, and closelyrelated to the previous methods, has been describedby Trujillo et al.56 for the assignment of the config-uration of alcohols.

In this procedure, the chiral alcohol is convertedto the corresponding tetra-O-benzoyl-â-glucoside byreaction with commercial 2,3,4,6-tetra-O-benzoyl-R-D-glucopyranosyl bromide in the presence of silvertrifluoromethanesulfonate and 1,1,3,3-tetramethyl-urea. The 1H NMR spectrum of the product is thencompared with that of the free alcohol. The observeddifferences between the two spectra are caused bythe anisotropic effect of the benzoyl groups and the

glycosylation-induced NMR shifts. This effect isquantified as the difference between the chemicalshifts in the D-glucopyranoside derivative and thefree alcohol (∆δ ) δD - δROH). The protons anti tothe endocyclic glucopyranoside oxygen (O-5) areshielded due to the effect of the benzoyl group at C-2(negative ∆δ), whereas protons syn to that O atomshow positive ∆δ values, because of their proximityto O-5, as shown in the model of Figure 185a. Figure185b shows experimental ∆δ values that fit the modelfor an enantiomeric pair of alcohols.

Other alcohols of known absolute configurationthat have been used to assess the viability of thismethodology are shown in Figure 186. In some cases,the ∆δ signs are opposite to those expected (forinstance, in compounds 186.1, 186.5, 186.11, and186.13); however, the authors state that the config-uration can still be deduced if all the other protonsare considered. However, in case of doubt, theyrecommend that a double-derivatization procedure beused (formation of both the D- and L-benzoylgluco-pyranosides) and the larger δD - δL values consid-ered (Section 3.1.4.7) rather than δD - δROH.Attempts to assign the configuration of the alcoholusing the δD - δROH shift of the H atom at thechiral center (C1′) alone did not give successfulresults.

4.1.3. Low-Temperature NMR of MPA Esters

As mentioned previously, reducing the temperatureof the equilibrium should lead to an increase in thepopulation of the most stable conformer. In the case

Figure 189. Secondary alcohols of known configurationstudied through the low-temperature method.

Figure 190. Diagram to deduce the absolute configuration of an alcohol from the experimental ∆δT1T2 signs of either its(R)- or (S)-MPA ester.


of MPA esters, this is the sp conformer and, therefore,greater shielding of the substituent located on thesame side as the Ph group in the (R)-MPA ester isexpected. The practical application of this approachrequires the preparation of a single MPA ester (eitherthe R or the S derivative) and comparison of two 1HNMR spectra taken at two sufficiently differenttemperatures.112 The differences in chemical shiftsare now measured as ∆δT1T2 (the chemical shift at thehigher temperature T1 minus that at the lowertemperature T2) and obey the scheme shown inFigure 187, where, at lower temperature, the signalfor substituent L2 moves to lower field and that ofsubstituent L1 to higher field. The sign of this

parameter is positive for substituent L1 (the grouplocated on the same side as the Ph ring) and negativefor substituent L2 (the group located on the otherside) and provides the configurational information.

Several alcohols of known absolute stereochemistryhave been used to demonstrate the general ap-plicability of this phenomenon. Figure 188 shows theshielded and deshielded areas in the MPA esters of(-)-menthol. Other alcohols that have been tested areshown in Figure 189. From a practical standpoint, itseems that temperatures lower than -70 °C are notnecessary, and, therefore, the commonly used CDCl3

or a CS2/CD2Cl2 (4:1) mixture can be used as theNMR solvent. The scheme shown in Figure 190

Figure 191. Partial spectra of the (R)-MPA ester of diacetone-D-glucose at (a) 303, (b) 223, and (c) 193 K in CS2/CD2Cl2.

Figure 192. Plot of the proportional increase of ∆δRS with decreasing probe temperature T (in K) for the Me(9′) group of(-)-menthyl esters of arylmethoxyacetic acids.


outlines the steps that should be followed for theapplication of this methodology using either the (R)-or (S)-MPA derivative.

Figure 191 shows the actual spectra of the (R)-MPAester of diacetone-D-glucose, taken at 303, 223, and193 K, and illustrates the upfield and downfield shiftsof the protons situated on the shielded and non-shielded areas around the chiral center.

Curiously, when the usually more efficient reagent9-AMA was used instead of MPA, no improvementin the ∆δT1T2 values was observed and this is relatedto the different sp/ap ratio present in the two

reagents.29,30 In fact, the study of the sensitivity ofchemical shifts, relative to changes in the tempera-ture, for the esters of (-)-menthol with severalreagents (Figure 192) shows that the ∆δRS values ofMA, MPA, and 2-NMA esters increase steadily atlower temperatures whereas those of the 9-AMAesters remain practically constant. This is becausein the first case there is a progressive shift of theequilibrium toward the most stable sp conformerwhereas, in the 9-AMA esters, the sp/ap ratio is atroom-temperature already shifted toward the sp andthere is little place for further displacement.

4.1.4. Complexation of MPA Esters with Barium(II)

A second procedure devised for the controlled shiftof the conformational equilibrium in AMAA deriva-tives consists of the addition of a metal salt that iscapable of stabilizing a certain conformer by com-plexation. Approaches for secondary alcohols113 andprimary amines are described in the literature. Inboth cases, MPA was used as the auxiliary reagentand barium(II) as the chelating agent.

The conformational equilibrium for the MPA estersof chiral alcohols is outlined in Figure 193. Theaddition of the Ba2+ salt (i.e., Ba(ClO4)2) to the NMRtube containing either the (R)- or (S)-MPA ester inMeCN-d3 causes the spectra to change in a way thatis practically identical to that observed in the low-temperature experiments, indicating that the con-formational changes introduced are similar (i.e.,there is an increase in the population of the spconformer). In fact, in addition to 1H NMR evidence,13C NMR and CD experiments, together with high-level ab initio Hartree-Fock (HF) and density func-tional theory (DFT) calculations, also prove that theBa2+ atom becomes strongly bonded to the O atomsof the CdO and MeO groups, therefore leading to anincrease in the stability and population of the sp form(see Figure 194).

Figure 193. Formation of Ba2+ complexes of the (R)-MPAester of a chiral secondary alcohol. The population of thesp form increases by formation of the most stable sp-Ba2+

complex, so the signals for substituent L1 shift to higherfield.

Figure 194. HF optimized geometries for the (a) sp-Ba2+ and (b) ap-Ba2+ conformers of the (R)-MPA ester of 2-propanol.The sp-Ba2+ geometry is more stable than that of ap-Ba2+, by 13.9 kcal/mol.


Several other ions have been investigated but theresults are not as good; even the Mg2+ ion, which iswell-known for its superior ability to complex withoxygen donors, produces lower shifts than the Ba2+

ion. The reasons for this apparent incoherence werehighlighted by ab initio studies, which showed that,in these cases, powerful solvent-cation interactionsare operative and, thus, make these metals lesssuitable, because of strong solvation by the solventMeCN. The importance of the Ba2+/MeCN-d3 pair istherefore a key aspect in this method.

From the standpoint of the application of thisprocedure to the assignment of absolute configura-tion, only one derivative should be prepared (witheither the R or S auxiliary) and two NMR spectrashould be taken: one of the derivative alone and theother after the addition of Ba2+ ions. The shiftsproduced are expressed as ∆δBa ) δ(MPA ester) -δ(MPA ester + Ba2+) and should be positive for oneof the substituents and negative for the other. Figure195 shows the values obtained for some representa-tive examples of known absolute configuration thathave been used to test the coherence, reliability, andscope of this approach.

The scheme shown in Figure 196 illustrates graphi-cally the steps that should be followed for theassignment of the absolute configuration of a second-ary alcohol by this procedure.

In regard to the limitations of this method, thereis an obvious drawback in terms of those substratesin which substituents L1/L2 contain functional groupsthat are able to complex with the Ba2+ ion. In thesecases, there is no guarantee that the general confor-mational scheme shown in Figure 193 is still opera-tive, and, therefore, safe assignment of the substratecannot be assured. Examples where competitivechelation occurs because of the presence of extracarbonyl groups (i.e., 195.18 and 195.19 in Figure195) have been described.113

The actual spectra of the (R)-MPA ester of di-acetone-D-glucose before and after the addition ofBa2+ ions are shown in Figure 197. Comparison withthe spectra for the same compound taken at differenttemperatures (Figure 191) illustrates the similarityin the shifts generated by both procedures becauseof the increase in the population of the most stablesp conformers.

4.2. Application to r-Chiral Primary Amines:Complexation of MPA Amides with Barium(II)

In a similar way, the absolute configuration ofR-chiral primary amines can be deduced from theNMR complexation effect produced by the additionof a Ba2+ salt on a single MPA amide, prepared fromeither (R)- or (S)-MPA.77

In this case, the summarized conformational equi-librium is shown in Figure 198 for an (R)-MPAamide. The most stable conformer, in the absence of

any chelating agent, is the ap form, where the L2

substituent is shielded by the phenyl ring of thereagent. Upon addition of a Ba2+ salt (e.g., Ba(ClO4)2)to the NMR tube containing the MPA amide (MeCN-d3 as the solvent), changes in the NMR chemicalshifts are observed, indicating that the equilibriumis being shifted to the sp form (L1 is now the shieldedgroup). The changes in the chemical shifts of L1 andL2 are expressed as ∆δBa ) δ(MPA amide + Ba2+) -δ(MPA amide) and are positive for one substituentand negative for the other. (See Figure 199.)

The scheme shown in Figure 200 illustrates graphi-cally the steps that should be followed for theassignment of the absolute configuration of a primary

Figure 195. Selected ∆δBa values (in ppm) obtained fromthe 1H NMR spectra of the (R)- and (S)-MPA esters ofstructurally representative chiral esters.


amine by this method. Figure 201 shows the changesproduced in the spectra of the (R)-MPA amide ofL-valine methyl ester upon the addition of Ba(ClO4)2.

As discussed previously for MPA esters, aminesthat have functional groups in substituents L1/L2 thatare able to complex with the Ba2+ ion should not beassigned by this method. 4-Oxo-R-amino acids, suchas 202.1 (see Figure 202), are good examples thatshow the effect of such competitive chelation.114

5. New Trends5.1. Hyphenated High-Pressure LiquidChromatography (HPLC) NMR−MS Techniquesfor Automatization and Microscale Analysis

The development of hyphenated techniques forrapid separation of mixtures and spectroscopic analy-sis of its components is a topic of great interest,particularly when the amount of sample is limitedand the need for automatization is high. This is thecase for high-pressure liquid chromatography nuclearmagnetic resonance-mass spectroscopy (HPLC NMR-MS), which has been shown to be extremely usefulin certain pharmaceutical and medical applications.

Quite recently, Riguera et al. reported115 that thederivatization of mixtures of enantiomeric secondaryalcohols with 9-AMA, followed by tandem HPLCNMR, allows the separation of the enantiomers, the

evaluation of their enantiomeric excess, and theassignment of their absolute configuration at themicroscale level and in a single operation.

Two different cases can be distinguished:(a) If the absolute configuration of each enantiomer

in a mixture (including racemic ones) is to be deter-mined, the mixture is derivatized with either (R)- or(S)-9-AMA, according to standard procedures. Themixture of 9-AMA esters is submitted to HPLC NMRand comparison of the 1H NMR spectra of the twoHPLC peaks allows the assignment of the configu-ration based on the shielding/deshielding effectscaused in substituents L1/L2 by the auxiliary reagentof known configuration (see Figure 203 and Section3.1.3).

(b) If assignment of the absolute configuration ofa single enantiomer is the objective, then the alcoholis derivatized with a 3:1 mixture of (R)- and (S)-9-AMA. The unequal mixture of 9-AMA esters issubjected to HPLC NMR and the spectra of bothpeaks are compared, taking into consideration thatthe major component of the mixture corresponds tothe (R)-ester and the minor component correspondsto the (S)-ester (see Figure 204).

In both cases, ,1 mg of the alcohol is needed forderivatization and no more than 10-15 µg of themixture of 9-AMA esters, dissolved in acetonitrile-

Figure 196. Diagram to deduce the absolute configuration of a chiral secondary alcohol from the ∆δBa experimentalvalues of one ester (either the (R)- or the (S)-MPA derivative).


d3, are injected onto a standard reverse phase columneluted with acetonitrile-d3/deuterium oxide/formicacid mixtures.

The generality of this approach, including thereliability of the NMR data obtained in acetonitrile-d3/deuterium oxide/formic acid mixtures instead ofdeuteriochloroform, was demonstrated by its success-ful application to the secondary alcohols of knownabsolute configuration (205.1-205.18, shown in Fig-ure 205, including saturated, unsaturated, aromatic,acyclic, and cyclic compounds).

The authors also mention that if actual isolationof the 9-AMA esters of the enantiomeric alcohols isneeded for preparative purposes or else, higherloading can be obtained when the HPLC separationis performed with normal-phase HPLC and provides

experimental conditions and many examples of suchseparation.

The number and structural variety of the alcoholsthat have been used to validate the procedure indi-cate beyond doubt that this methodology is quitegeneral for secondary alcohols and that it can be usedin the analytical and semipreparative scales. Thesimplification involved in the use of the same auxil-iary reagent for both the HPLC separation and theNMR configurational assignment, and its implemen-tation in directly coupled HPLC NMR should beextremely useful for the automatization of manyprocesses involving optically active compounds. HPLCNMR has been recently used to purify and analyzethe two MTPA esters of a very small sample of anatural product.116

Figure 197. Partial 1H NMR spectra (250 MHz) for the (R)-MPA ester of diacetone-D-glucose (a) before and (b) after theaddition of Ba2+.

Figure 198. Main conformers present in the equilibrium of (R)-MPA amides in the absence and presence of a barium(II)salt.


5.2. Resin-Bound Auxiliaries: The “Mix andShake” Method

In the search for new ways to simplify the configu-rational assignment by NMR, a new procedure thatallows determining the absolute configuration in justfew minutes and without any type of separation,workup, or manipulation being needed has beenrecently presented.117 In this approach, the requiredderivative/s are prepared by simply mixing a solidmatrix-bound auxiliary reagent (i.e., MPA, MTPA, orBPG) with the chiral substrate (i.e., a primary amineor a secondary alcohol) directly in the NMR tube andthe NMR spectra of the resulting derivatives areobtained without any further manipulation after afew minutes.

The key for the success of this methodology lies inthe linking of the auxiliary reagent to the solidmatrix through a mixed anhydride group, whichallows the auxiliary reagent moiety to act as anelectrophile toward the chiral substrate and permitsthe solid resin to be the leaving group, liberating theester or amide derivative into the solution (see Figure206).

Resins containing either a single enantiomer ormixtures of both enantiomers of MPA, MTPA, andBPG, linked through anhydride groups, were pre-pared from commercial carboxypolystyrene and usedto derivatize alcohols and amines. The reaction isperformed directly in the NMR tube, in Cl3CD, andtakes from 5 min (with primary amines) to a fewhours (with secondary alcohols), depending on thesubstrate. After that time, the spectra are recordedas usual, the resin that remains in the tube causesno interference at all, and the assignment of absoluteconfiguration is conducted from the ∆δRS signs usingthe corresponding configurational models (see Section3 of this review).

The resulting spectrum is sufficiently clean to allowassignment of the absolute stereochemistry at the

Figure 199. Selected ∆δBa values (given in ppm) obtainedfrom the 1H NMR spectra of the (R)- and (S)-MPA amidesof structurally representative chiral amines.

Figure 200. Diagram to deduce the absolute configuration of a chiral primary amine from the experimental ∆δBa signs,from either the (R)- or (S)-MPA amide.


microscale level (<0.5 mg of substrate). In fact, if a1:2 (R)/(S)-MPA resin is used, the assignment can beperformed in a single operation and by taking justone spectrum because the different ratios of the NMRsignals of the two diasteromeric MPA derivatives

(those of the (R)-MPA derivative are half the size ofthose for the (S)-derivative) allow the upfield/down-field shifts (∆δRS) to be measured.

Another successful adaptation that makes theusual double derivatization (with (R)- and (S)-MPA)and recording of two spectra unnecessary is themethod based on the complexation of MPA estersor amides with the Ba2+ ion (see Section 4 of thisreview). This approach can also be performed directlyin the NMR tube with a resin-bound MPA (singleenantiomer) in CD3CN, although formation of the

Figure 201. Partial 1H NMR spectra of the (R)-MPA amide of L-valine methyl ester in the presence (upper) and absence(bottom) of barium perchlorate. The observed shifts are highlighted.

Figure 202.

Figure 203. Enantioresolution and assignment of the absolute configuration of the enantiomers of a chiral secondaryalcohol by tamden HPLC NMR of its (R)-9-AMA esters (the assignment is the opposite if (S)-9-AMA is chosen as thereagent).


derivative in this solvent takes longer time (∼90 min)than in Cl3CD.

A precedent of this methodology can be found inthe preparation of a ROMPgel support of activatedMosher ester (MTPA) as a means to performing thepurification free synthesis of MTPA amides by theBarrett group.118

6. Concluding Remarks: A Critical Assessment

The interest of researchers on the use and discov-ery of procedures for the determination of absoluteconfiguration of organic compounds by NMR isevidently originated by the economy and simplicityof the methods and the wide availability of NMRequipments in all laboratories. These advantagesexplain the very large number of papers publishedon this topic that are either devoted to the proposalof new chiral-derivatizing agents (CDAs) or mention-ing their application to the configurational assign-ment of a certain substrate. It is among thoseworking in the field of natural productssplaguedwith small samples difficult to crystallizeswhere theapplication of these techniques is more appreciatedand the highest number of papers is found, the sameas for those working in the synthesis of chiralcompounds.

Nevertheless, the fact that, using these NMRmethods, the absolute configuration is determined insolution and that conformational and other structuralinformation related to the through-space anisotropiceffect of the reagent is also obtained, wide theirapplications to other fields.

There are also many publications that have pro-posed new CDAs; however, researchers should beaware that the reliability of the NMR assignmentdepends very much on the number and structuralvariety of the substrates of known absolute configu-ration that have been used as test compounds tovalidate the procedure/reagent. Consequently, of themany reagents and procedures described in thisreview, only a few can be considered to producereliable assignments, because, in many cases, thenumber of test compounds is so small that no generalbehavior can be deduced from the data available.

In the past few years, the experimental data hasbeen frequently complemented with theoretical work(conformational analysis, energy minimization, shield-ing-effect calculations) and this certainly providedsupplementary support to the method/reagent in-

Figure 204. Assignment of the absolute configuration of a chiral secondary alcohol by derivatization with a (R)-9-AMA/(S)-9-AMA 3:1 mixture followed by tamden HPLC NMR of the diasteromeric esters.

Figure 205. Chiral alcohols studied by this method.


volved; however, these calculations also showed thatthe differences in energy between the derivatives arefrequently so small that calculations can in no wayreplace the use of a collection of compounds of knownabsolute configuration and representative structuresas a test compounds.

In regard to the substrates investigated, most ofthe efforts have been directed to monofunctionalcompounds. Secondary alcohols and primary amineshave been the most frequently investigated, andalthough other functional groups were also studied,those remain the most amenable to assignment byNMR procedures. Quite recently, an extension of theprocedure to diols has been described, which willsurely open the way to the assignment by NMR ofother polyfunctional compounds.

Although many procedures that are based on theuse of NMR of nucleus different from 1H (i.e., 19F,13C) have been described and recommended on thebasis of the simplified NMR spectra obtained, the lowsensitivity of those nuclei to the anisotropic effectsproduced by the CDA originate very small shifts thatovercome that advantage. Nevertheless, the simpli-fication of the spectra renders those reagents veryuseful when the enantiomeric excess of a mixture(but not the configurational assignment of eachenantiomer), is the objective.

Other than papers directed to widen the scope offunctional groups amenable to be studied by theNMR procedure, in the past few years, some effortshave been described to simplify the method andreduce the amount of sample. In the classical ap-proach, the chiral substrate should be transformedto two diasteroisomeric derivatives and their NMRcompared. Simplified methods have been describedfor assignment of the configuration of alcohols andamines that require the preparation of only onederivative and represent evident practical advan-tages, particularly when only a very small amount

of substrate sample is available. In a paper recentlypublished, the enantioresolution of secondary alco-hols (either an unequal or racemic mixture of enan-tiomers) and the assignment of the absolute config-uration of the enantiomers is achieved in a singleoperation and with just a few micrograms of sub-strate by tandem HPLC NMR of the 9-AMA deriva-tives. In another publication by the same group, thesimplification of the methodology is attained usingsolid matrix-bound auxiliaries that allow the deriva-tization to happen in the NMR tube and within a fewminutes. In this way, the NMR spectra can beobtained after just mixing the resin and the substratewithout other operations being necessary.

In summary, NMR procedures for assignment ofthe absolute configuration have evolved in a fewdecades from the original “Mosher’s method” to amature field, where a “menu” containing a largenumber of CDAs is available for the study of differentsubstrates using a variety of approaches. New excit-ing advances will undoubtedly emerge in the future.

7. AcknowledgmentThe authors are grateful to Ministerio de Ciencia

y Tecnologıa and Xunta de Galicia for financialsupport (currently through Grant Nos. BQU2002-01195 and PGIDT02BTF20902PR) and to their co-workers, who have contributed to many of the articlesdescribed herein.

8. References(1) (a) Nakanishi, K.; Berova, N.; Woody, R. W. Circular Dichro-

ism: Principles and Applications; VCH Publishers: New York,1994. (b) Polavarapu, P. L. Chirality 2002, 14, 768. (c) Wang,F.; Wang, Y.; Polavarapu, P. L.; Li, T.; Drabowicz, J.; Pi-etrusiewicz, K. M.; Zygo, K. J. Org. Chem. 2002, 67, 6539. (d)Constante, J.; Hecht, L.; Polavarapu, P. L.; Collet, A.; Barron,L. D. Angew. Chem., Int. Ed. Engl. 1997, 36, 885.

(2) Weisman, G. R. Nuclear Magnetic Resonance Analysis UsingChiral Solvating Agents. In Asymmetric Synthesis; Morrison, J.D., Ed.; Academic Press: New York, 1983; Vol. 1, p 153.

Figure 206. MPA, MTPA, and BPG resins used in this “mix and shake” methodology.


(3) (a) Parker, D. Chem. Rev. 1991, 91, 1441. (b) Rothchild, R.Enantiomer 2000, 5, 457. (c) Finn, M. G. Chirality 2002, 14, 534.

(4) (a) Rinaldi, P. L. Prog. Nucl. Magn. Spectrosc. 1982, 15, 291. (b)Yamaguchi, S. Nuclear Magnetic Resonance Analysis UsingChiral Derivatives. In Asymmetric Synthesis; Morrison, J. D.,Ed.; Academic Press: New York, 1983; Vol. 1, p 125.

(5) Fraser, R. R. Nuclear Magnetic Resonance Analysis Using ChiralShifts Reagents. In Asymmetric Synthesis; Morrison, J. D., Ed.;Academic Press: New York, 1983; Vol. 1, p 173.

(6) (a) Seco, J. M.; Quinoa, E.; Riguera, R. Tetrahedron: Asymmetry2001, 12, 2915. (b) Uray, G. In Houben-Weyl Methods inOrganic Chemistry; Helchen, G., Hoffmann, R. W., Mulzer, J.;Schaumann, E., Eds.; Thieme: Stuttgart, New York, 1996; Vol.1, p 253. (c) Eliel, E. L.; Wilen, S. H.; Mander, L. N. Stereochem-istry of Organic Compounds; Wiley-Interscience: New York,1994; p 221.

(7) (a) Raban, N.; Mislow, K. Tetrahedron Lett. 1965, 4249. (b)Raban, N.; Mislow, K. Topics in Stereochemistry; Allinger, N.L., Eliel, E. L., Eds.; Wiley-Interscience: New York, 1967; Vol2, p 199. (c) Jacobus, J.; Raban, N.; Mislow, K. J. Org. Chem.1968, 33, 1142.

(8) (a) Dale. J. A.; Mosher, H. S. J. Am. Chem. Soc. 1968, 90, 3732.(b) Dale, J. A.; Dull, D. L.; Mosher, H. S. J. Org. Chem. 1969,34, 2453. (c) Dale, J. A.; Mosher, H. S. J. Am. Chem. Soc. 1973,95, 512. (d) Sullivan, G. R.; Dale, J. A.; Mosher, H. S. J. Org.Chem. 1973, 38, 2143.

(9) (a) Joshi, B. S.; Pelletier, S. W. Heterocycles 1999, 51, 183. (b)Joshi, B. S.; Newton, M. G.; Lee, D. W.; Barber, A. D.; Pelletier,S. W. Tetrahedron Asymmetry 1996, 7, 25.

(10) Kusumi, T.; Ohtani, I. Determination of the Absolute Configu-ration of Biologically Active Compounds by the Modified Mosh-er’s Method. In The Biology-Chemistry Interface; Cooper, R.,Snyder, J. K., Eds.; Marcel Dekker: New York, 1999; pp 103.

(11) Pehk, T.; Lippmaa, E.; Loop, M.; Paju, A.; Borer, B. C.; Taylor,R. J. K. Tetrahedron: Asymmetry 1983, 4, (7), 1572.

(12) (a) For compound 9.1, see ref 8a. (b) For compounds 9.2-9.6,see Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am.Chem. Soc. 1991, 113, 4092. (c) For compound 9.7, see Rieser,M. J.; Hui, Y.; Rupprecht, J. K.; Kozlowski, J. F.; Wood, K. V.;McLaughlin, J. L.; Hanson, P. R.; Zhuang, Z.; Hoye, T. R. J. Am.Chem. Soc. 1992, 114, 10203.

(13) Seco, J. M.; Quinoa, E.; Riguera, R. Tetrahedron: Asymmetry2000, 11, 2781.

(14) (a) Inouye, Y.; Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa,H. Chem. Lett. 1990, 2073. (b) Ohtani, I.; Kusumi, T.; Kashman,Y.; Kakisawa, H. J. Org. Chem. 1991, 56, 1296.

(15) Kusumi, T.; Fujita, Y.; Ohtani, I.; Kakisawa, H. TatrahedronLett. 1991, 32, (25), 2923.

(16) (a) Ohtani, I.; Kusumi, T.; Ishitsuka, M. O.; Kakisawa, H.Tetrahedron Lett. 1989, 30, (24), 3147. (b) Kusumi, T.; Hamada,T.; Ishitsuka, M. O.; Othani, I.; Kakisawa, H. J. Org. Chem.1992, 57, 1033.

(17) (a) Ohtani, I.; Hotta, K.; Ichikawa, Y.; Isobe, M. Chem. Lett. 1995,513. (b) Kubo, I.; Jamlamadaka, V.; Kamikawa, T.; Takahashi,K.; Kusumi, T. Chem. Lett. 1996, 441.

(18) Banskota, A. H.; Tezuka, Y.; Tran, K. Q.; Tanaka, K.; Saiki, I.;Kadota, Sh. J. Nat. Prod. 2000, 63, 57.

(19) (a) For compound 15.1, see Suzuki, M.; Takahashi, Y.; Matsuo,Y.; Guiry, M. D.; Masuda, M. Tetrahedron 1997, 53, 4271. (b)For compound 15.2, see Ninomiya, M.; Hirohara, H.; Onishi, J.;Kusumi, T. J. Org. Chem. 1999, 64, 5436. (c) For compound 15.3,see Amico, V.; Piatelli, M.; Bizzini, M.; Neri, P. J. Nat. Prod.1997, 60, 1088. (d) For compound 15.4, see Shi, X.; Attygalle,A. B.; Liwo, A.; Hao, M. H.; Meinwald, J.; Dharmaratne, H. R.W.; Wanigasekera, A. P. J. Org. Chem. 1998, 63, 1233. (e) Forcompound 15.5, see Reese, M. T.; Gulavita, N. K.; Nakao, Y.;Hamann, M. T.; Yoshida, W. Y.; Coval, S. J.; Scheuer, P. J. J.Am. Chem. Soc. 1996, 118, 11081.

(20) (a) For compound 16.1, see Tsuda, M.; Endo, T.; Kobayashi, J.J. Org. Chem. 2000, 65, 1349. (b) For compound 16.2, seeKobayashi, J.; Shimbo, K.; Sato, M.; Shiro, M.; Tsuda, M. Org.Lett. 2000, 2, 2805. (c) For compound 16.3, see Zhang, Y. J.;Tanaka, T.; Iwamoto, Y.; Yang, C. R.; Kouno, I. Tetrahedron Lett.2000, 41, 1781. (d) For compound 16.4, see Bode, H. B.; Zeeck,A. J. Chem. Soc., Perkin Trans. 1, 2000, 323. (e) For compounds16.5 and 16.6, see Jansen, R.; Kunze, B.; Reichenbach, H.; Hofle,G. Eur. J. Org. Chem. 2000, 913.

(21) (a) For compound 17.1, see Kim, J. W.; Shin, K.; Furihata, K.;Hayakawa, Y.; Seto, H. J. Org. Chem. 1999, 64, 153. (b) Forcompound 17.2, see Takada, N.; Suenaga, K.; Zheng, S.; Cheng,H.; Uemura, D. Chem. Lett. 1999, 1025. (c) For compound 17.3,see Tsuda, M.; Endo, T.; Kobayashi, J. Tetrahedron 1999, 55,14565. (d) For compound 17.4, see Ohta, T.; Uwai, K.; Kikuchi,R.; Nozoe, S.; Oshima, Y.; Sasaki, K.; Yoshizaki, F. Tetrahedron1999, 55, 12087. (e) For compounds 17.5 and 17.6, see Gavagnin,M.; Napoli, A.; Cimino, G.; Iken, K.; Avila, C.; Garcıa, F. J.Tetrahedron: Asymmetry 1999, 10, 2647. (f) For compound 17.7,see Takada, N.; Sato, H.; Suenaga, K.; Arimoto, H.; Yamada,K.; Ueda, K.; Uemura, D. Tetrahedron Lett. 1999, 40, 6309. (g)

For compound 17.8, see Cheng, X. C.; Jensen, P. R.; Fenical, W.J. Nat. Prod. 1999, 62, 605.

(22) (a) For compound 18.1, see Shigemori, H.; Tenna, M.; Shimazaki,K.; Kobayashi, J. J. Nat. Prod. 1998, 61, 696. (b) For compounds18.2 and 18.4, see Golakoti, T.; Ogino, J.; Heltzel, C. E.; Husebo,T. L.; Jensen, C. M.; Larsen, L. K.; Patterson, G. M. L.; Moore,R. E.; Mooberry, S. L.; Corbett, T. H.; Valeriote, F. A. J. Am.Chem. Soc. 1995, 117, 12030. (c) For compound 18.3, see Dromer,P. G.; Smith, A. B. Tetrahedron Lett. 1992, 33, 1717. (d) Forcompound 18.5, see Tanahashi, T.; Takenaka, Y.; Nagakura, N.;Nishi, T. J. Nat. Prod. 1999, 62, 1311. (e) For compound 18.6,see Harrigan, G. G.; Luesch, H.; Yoshida, W. Y.; Moore, R. E.;Nagle, D. G.; Biggs, J.; Park, P. U.; Paul, V. J. Nat. Prod. 1999,62, 464. (f) For compound 18.7, see Moore, B. S.; Chen, J. L.;Patterson, M. L.; Moore, R. E. Tetrahedron 1992, 48, 3001.

(23) Kobayashi, J.; Tsuda, M.; Cheng, J.; Ishibashi, M.; Takikawa,H.; Mori, K.; Tetrahedron Lett. 1996, 37, 6775.

(24) Tomoda, H.; Nishida, H.; Kim, Y. K.; Obata, R.; Sunazuka, T.;Omura, S. J. Am. Chem. Soc. 1994, 116, 12097.

(25) (a) For compound 19.4, see Sinz, A.; Matusch, R.; Kampchen,T.; Fiedler, W.; Schmidt, J.; Santisuk, T.; Wangcharoentrakul,S.; Chaichana, S.; Reutrakul, V. Helv. Chim. Acta 1998, 81, 1608.(b) For compound 19.5, see Shimada, H.; Nishioka, S.; Singh,S.; Sahai, M.; Fujimoto, Y. Tetrahedron Lett. 1994, 35, 3961.

(26) (a) Takagi, Y.; Nakatani, T.; Itoh, T.; Oshiki, T. Tetrahedron Lett.2000, 41, 7889. (b) Kelly, D. R. Tetrahedron: Asymmetry 1999,10, 2927.

(27) Latypov, Sh.; Seco, J. M.; Quinoa, E.; Riguera, R. J. Org. Chem.1996, 61, 8569.

(28) (a) Trost, B. M.; Belletire, J. L.; Goldleski, P. G.; McDougal, P.G.; Balkovec, J. M.; Baldwin, J. J.; Christy, M.; Ponticello, G.S.; Varga, S. L.; Springer, J. P. J. Org. Chem. 1986, 51, 2370.(b) Trost, B. M.; Curran, D. P. Tetrahedron Lett. 1981, 22, (49),4929. (c) Trost, B. M.; O’Krongly, D. O.; Belletire, J. L. J. Am.Chem. Soc. 1980, 102, 7595.

(29) Latypov, Sh. K.; Seco, J. M.; Quinoa, E.; Riguera, R. J. Org.Chem. 1995, 60, 504.

(30) Seco, J. M.; Quinoa, E.; Riguera, R. Tetrahedron 1997, 53, 8541.(31) (a) For compound 33.1, see Zhou, B. N.; Baj, N. J.; Glass, T. E.;

Malone, S.; Werkhoven, M. C. M.; Troon, F.; Wisse, J. H.;Kingston, D. G. I. J. Nat. Prod. 1997, 60, 1287. (b) For compound33.2, see Harrison, B.; Crews, P. J. Nat. Prod. 1998, 61, 1033.(c) For compound 33.3, see Gupta, S.; Krasnoff, S. B.; Renwick,J. A. A.; Roberts, D. W. J. Org. Chem. 1993, 58, 1062. (d) Forcompound 33.4, see Adamczeski, M.; Quinoa, E.; Crews, P. J.Org. Chem. 1990, 55, 240. (e) For compound 33.5, see Ulubelen,A.; Goren, N.; Jiang, T. Y.; Scott, L.; Ramomojy, M. T.; Snyder,J. K. Magnetic Resonance in Chemistry 1995, 33, 900. (f) Forcompound 33.6, see Wang, G. Y. S.; Crews, P. Tetrahedron Lett.1996, 37, (45), 8145. (g) For compound 33.7, see Wilson, K. E.;Burk, R. M.; Biftu, T.; Ball, R. G.; Hoogstenn, K. J. Org. Chem.1992, 57, 7151. (h) For compound 33.8, see Gupta, S.; Pieser,G.; Nakajima, T.; Hwang, Y. S.; Tetrahedron Lett. 1994, 35, 6009.(i) For compound 33.9, see Alvi, K. A.; Rodrıguez, J.; Diaz, M.C.; Moretti, R.; Wilhelm, R. S.; Lee, R. H.; Slate, D. L.; Crews,P. J. Org. Chem. 1993, 58, 4871. (j) For compound 33.10, seeVazquez, M. J.; Quinoa, E.; Riguera, R.; Martın, A. S.; Darias,J. Liebigs Ann. Chem. 1993, 1257. (k) For compound 33.11, seeGalinis, D. L.; Wiemer, D. F. J. Org. Chem. 1993, 58, 7804.

(32) Fernandez, E.; Ley, S. V. Chemtracts 1999, 12, 539.(33) Seco, J. M.; Latypov, Sh. K.; Quinoa, E.; Riguera, R. Tetrahedron

Lett. 1994, 35, 2921.(34) (a) Chatainer, I.; Lebreton, J.; Durand, D.; Guingant, A.;

Villieras, J. Tetrahedron Lett. 1998, 39, 1759. (b) Parve, O.;Aidnik, M.; Lille, U.; Martin, I.; Vallikivi, I.; Vares, L.; Pehk, T.Tetrahedron: Asymmetry 1998, 9, 885.

(35) Seco, J. M.; Quinoa, E.; Riguera, R. Tetrahedron 1999, 55, 569.(36) (a) Kusumi, T.; Takahashi, H.; Fukushima, T.; Asakawa, Y.;

Hashimoto, T.; Kan, Y.; Inouye, Y. Tetrahedron Lett. 1994, 35,(25), 4397. (b) Kusumi, T.; Takahashi, H.; Hashimoto, T.; Kan,Y.; Asakawa, Y. Chem. Lett. 1994, 1093. (c) Kimura, M.; Kunoki,A.; Sugai, T. Tetrahedron: Asymmetry 2002, 13, 1059.

(37) (a) Kouda, K.; Ooi, T.; Kaya, K.; Kusumi, T. Tetrahedron Lett.1996, 37, (35), 6347. (b) Kouda, K.; Kusumi, T.; Ping, X.; Kan,Y.; Hashimoto, T.; Asakawa, Y. Tetrahedron Lett. 1996, 37, (26),4541.

(38) Seco, J. M.; Latypov, Sh. K.; Quinoa, E.; Riguera, R. Tetrahe-dron: Asymmetry 1995, 6, 107.

(39) Latypov, Sh. K.; Galiullina, N. F.; Aganov, A. V.; Kataev, V. E.;Riguera, R. Tetrahedron 2001, 57, 2231.

(40) Lenis, L. A.; Ferreiro, M. J.; Debitus, C.; Jimenez, C.; Quinoa,E.; Riguera, R. Tetrahedron 1998, 54, 5385.

(41) (a) For details on compounds 48.1-48.13, see ref 30. (b) Fordetails on compounds 49.1-49.5, see refs 30 and 37b. (c) Fordetails on compounds 49.6 and 49.7, see ref 36a. (d) For detailson compound 49.8, see Watanabe, K.; Tsuda, Y.; Yamane, Y.;Takahashi, H.; Iguchi, K.; Naoki, H.; Fujita, T.; Van Soest, R.W. M. Tetrahedron Lett. 2000, 41, 9271. (e) For details oncompounds 49.9 and 49.10, see ref 37a.


(42) (a) For details on compounds 51.1-51.3, see Abad, J. L.; Fabrias,G.; Camps, F. J. Org. Chem. 2000, 65, 8582. (b) For details oncompounds 51.4 and 51.5, see ref 37b.

(43) Horeau, A. Tetrahedron Lett. 1961, 506.(44) (a) Helmchen, G.; Ott, R.; Sauber, K. Tetrahedron Lett. 1972,

3873. (b) Helmchen, G. Tetrahedron Lett. 1974, 1527.(45) (a) Helmchen, G.; Volter, H.; Schule, W. Tetrahedron Lett. 1977,

1417. (b) Helmchen, G.; Schmierer, R. Angew. Chem., Int. Ed.Engl. 1976, 15, 703.

(46) (a) Niederer, D.; Tamm, C.; Zurcher, W. Tetrahedron Lett. 1992,33, (28), 3997. (b) Augustiniak, H.; Irschik, H.; Reichenbach, H.;Hofle, G. Liebigs Ann. 1996, 1657. (c) Oh, H.; Gloer, J. B.;Shearer, C. A. J. Nat. Prod. 1999, 62, 497. (d) Tang, Y. Q.;Sattler, I.; Grabley, S.; Feng, X. Z.; Thiericke, R. Nat. Prod. Lett.2000, 14, 341.

(47) Arnone, A.; Bernardi, R.; Blasco, F.; Cardillo, R.; Resnati, G.;Gerus, I. I.; Kukhar, V. P. Tetrahedron 1998, 54, 2809.

(48) Nadkarni, P. J.; Sawant, M. S.; Trivedi, G. K. TetrahedronAsymmetry 1995, 6, (8), 2001.

(49) (a) Harada, N.; Watanabe, M.; Kuwahara, S.; Sugio, A.; Kasai,Y.; Ichikawa, A. Tetrahedron: Asymmetry 2000, 11, 1249. (b)Fujita, T.; Kuwahara, S.; Watanabe, M.; Harada, N. Enantiomer2002, 7, 219. (c) Ichikawa, A.; Hiradate, S.; Sugio, A.; Kuwahara,S.; Watanabe, M.; Harada, N. Tetrahedron: Asymmetry 1999,10, 4075. (d) Ichikawa, A.; Hiradate, S.; Sugio, A.; Kuwahara,S.; Watanabe, M.; Harada, N. Tetrahedron: Asymmetry 2000,11, 2669. (e) Ichikawa, A.; Ono, H.; Hiradate, S.; Watanabe, M.;Harada, N. Tetrahedron: Asymmetry 2002, 13, 1167.

(50) (a) Goto, J.; Hasegawa, M.; Nakamura, S.; Shimada, K.; Nam-bara, T. J. Chromatogr. 1978, 152, 413. (b) Goto, J.; Hasegawa,M.; Nakamura, S.; Shimada, K.; Nambara, T. Chem. Pharm.Bull. 1977, 25, 847.

(51) (a) Fukushi, Y.; Yajima, C.; Mizutani, J. Tetrahedron Lett. 1994,35, (4), 599. (b) Fuhushi, Y.; Yajima, C.; Mizutani, J. TetrahedronLett. 1994, 35, (50), 9417. (c) Latypov, Sh.; Aganov, A. V.; Tahara,S.; Fukushi, Y. Tetrahedron 1999, 55, 7305.

(52) Harada, K.; Shimizu, Y.; Kawakami, A.; Fuji, K. TetrahedronLett. 1999, 40, 9081.

(53) Latypov, S. K.; Riguera, R.; Smith, M. B.; Polivkove, J. J. Org.Chem. 1998, 63, 8682.

(54) Oshikawa, T.; Yamashita, M.; Kumagai, S.; Seo, K.; Kobayashi,J. J. Chem. Soc., Chem. Commun. 1995, 435.

(55) (a) Seo, S.; Tomita, Y.; Tori, K.; Yoshimura, Y. J. Am. Chem.Soc. 1978, 100, 3331. (b) Seo, S.; Tomita, Y.; Tori, K.; Yoshimura,Y. J. Am. Chem. Soc. 1980, 102, 2512. (c) Faghih, R.; Fontaine,C.; Horibe, I.; Imamura, P. M.; Lukacs, G.; Olesker, A.; Seo, S.J. Org. Chem. 1985, 50, 4918.

(56) Trujillo, M.; Morales, E. Q.; Vazquez, J. J. Org. Chem. 1994,59, 6637.

(57) Kobayashi, M. Tetrahedron 1997, 53, 5973.(58) (a) Noe, C. E. Chem. Ber. 1982, 115, 1591. (b) Noe, C. E.;

Knollmuller, M.; Oberhauser, B.; Steinbauer, G.; Wagner, W.Chem. Ber. 1986, 119, 729. (c) Noe, C. E.; Knollmuller, M.;Steinbauer, G.; Jangg, E.; Vollenkle, H. Chem. Ber. 1988, 121,1231.

(59) Takeuchi, Y.; Itoh, N.; Satoh, T.; Koizumi, T.; Yamaguchi, K. J.Org. Chem. 1993, 58, 1812.

(60) (a) Apparu, M.; Tiba, Y. B.; Leo, P. M.; Hamman, S.; Coulom-beau, C. Tetrahedron: Asymmetry 2000, 11, 2885. (b) Barrelle,M.; Boyer, L.; Fong, J. C.; Hamman, S. Tetrahedron: Asymmetry1996, 7, 1961. (c) Barrelle, M.; Hamman, S. J. Chem. Res.,Synop. 1990, 100. (d) Heumann, A.; Faure, R. J. Org. Chem.1993, 58, 1276. (e) Takahashi, T.; Fukuishima, A.; Tanaka, Y.;Takeuchi, Y.; Kabuto, K.; Kabuto, C. Chem. Commun. 2000, 788.

(61) Weibel, D. B.; Walker, T. R.; Schroeder, F. C.; Meinwald, J. Org.Lett. 2000, 2, 2381.

(62) (a) Yasuhara, F.; Yamaguchi, S. Tetrahedron Lett. 1977, 4085.(b) Yamaguchi, S.; Yashuara, F.; Kabuto, K. Tetrahedron 1976,32, 1363. (c) Yamaguchi, S.; Yashuara, F. Tetrahedron Lett.1977, 89.

(63) (a) Sugimoto, Y.; Tsuyuki, T.; Moriyama, Y.; Takahashi. T. Bull.Chem. Soc. Jpn. 1980, 53, 3723. (b) D’Auria, M. V.; Minale, L.;Pizza, C.; Riccio, R. Zollo, F. Gazz. Chim. Ital. 1984, 114, 469.(c) Bruno, I.; Minale, L.; Riccio, R.; La Barre, S.; Laurent, D.Gazz. Chim. Ital. 1990, 120, 449. (d) Tachibana, K.; Sakaitani,M.; Nakanishi, K. Tetrahedron 1985, 41, 1027.

(64) D’Auria, M. V.; De Riccardis, F.; Minale, L.; Riccio, R, J. Chem.Soc., Perkin Trans. 1, 1990, 2889.

(65) Finamore, E.; Minale, L.; Riccio, R. Rinaldo, G.; Zollo, F. J. Org.Chem. 1991, 56, 1146.

(66) (a) De Riccardis, F.; Minale, L.; Riccio, R.; Giovannitti, B.; Iorizzi,M.; Debitus, C. Gazz. Chim. Ital. 1993, 123, 79. (b) De Rosa, S.;Milone, A.; Crispino, A.; Jaklin, A.; De Giulio, A. J. Nat. Prod.1997, 60, 462. (c) Shigemori, H.; Sato, Y.; Kagata, T.; Kobayashi,J. J. Nat. Prod. 1999, 62, 372. (d) Tsuda, M.; Endo, T.;Kobayashi, J. J. Org. Chem. 2000, 65, 1349. (e) Kubota, T.;Tsuda, M.; Kobayashi, J. J. Org. Chem. 2002, 67, 1651.

(67) Ramon, D. J.; Guillena, G.; Seebach, D. Helv. Chim. Acta 1996,79, 875.

(68) Ferreiro, M. J.; Latypov, Sh. K.; Quinoa, E.; Riguera, R.Tetrahedron: Asymmetry 1996, 7, 2195.

(69) Latypov, Sh. K.; Ferreiro, M. J.; Quinoa, E.; Riguera, R. J. Am.Chem. Soc. 1998, 120, 4771.

(70) Ciminiello, P.; Dell’Aversano, C.; Fattorrusso, E.; Forino, M.;Magno, S.; Tetrahedron 2001, 57, 8189.

(71) Izumi, S.; Moriyoshi, H.; Hirata, T. Bull. Chem. Soc. Jpn. 1994,67, 2600.

(72) Takahashi, H.; Kato, N.; Iwashima, M.; Iguchi, K. Chem. Lett.1999, 1181.

(73) Kobayashi, M.; Tetrahedron 1998, 10987.(74) Kusumi, T.; Fukushima, T.; Ohtani, I.; Kakisawa, H. Tetrahe-

dron Lett. 1991, 32, 2939.(75) Seco, J. M.; Latypov, Sh. K.; Quinoa, E.; Riguera, R. J. Org.

Chem. 1997, 62, 7569.(76) (a) Latypov, Sh. K.; Seco, J. M.; Quinoa, E.; Riguera, R. J. Org.

Chem. 1995, 60, 1538. (b) Trost, B. M.; Bunt, R. C.; Pulley, S.R. J. Org. Chem. 1994, 59, 4202.

(77) Lopez, B.; Quinoa, E.; Riguera, R. J. Am. Chem. Soc. 1999, 121,9724.

(78) Seco, J. M.; Quinoa, E.; Riguera, R. J. Org. Chem. 1999, 64, 4669.(79) Pirkle, W. H.; Simmons, K. A. J. Org. Chem. 1981, 46, 3239.(80) (a) Polh, L. R.; Trager, W. F. J. Med. Chem. 1973, 16, 475. (b)

Valente, E. J.; Polh, L. R.; Trager, W. F. J. Org. Chem. 1980,45, 543.

(81) Harada, K.; Shimizu, Y.; Fujii, K. Tetrahedron Lett. 1998, 39,6245.

(82) Chinchilla, R.; Falvello, L. R.; Najera, C. J. Org. Chem. 1996,61, 7285.

(83) Chinchilla, R.; Falvello, L. R.; Najera, C. Yus, M. Tetrahedron:Asymmetry 1995, 6, 1877.

(84) Fujiwara, T.; Omata, K.; Kabuto, K.; Kabuto, C.; Takahashi, T.;Segawa, M.; Takeuchi, Y. Chem. Commun. 2001, 2694.

(85) (a) Hoye, T. R.; Rener, M. K. J. Org. Chem. 1996, 61, 8489. (b)Hoye, T. R.; Rener, M. K. J. Org. Chem. 1996, 61, 2056.

(86) (a) Stewart, W. E.; Siddall, T. H. Chem. Rev. 1970, 70, 517. (b)Jacobus, J.; Jones, T. B. J. Am. Chem. Soc. 1970, 92, 4583.

(87) (a) Ferreiro, M. J.; Latypov, S. K.; Quinoa, E.; Riguera, R.Tetrahedron: Asymmetry 1997, 8, 1015. (b) Ferreiro, M. J.;Latypov, S. K.; Quinoa, E.; Riguera, R. J. Org. Chem. 2000, 65,2658.

(88) Tyrrell, E.; Tsang, M. W. H.; Skinner, G. A.; Fawcett, J.Tetrahedron 1996, 52, 9841.

(89) (a) Nagai, Y.; Kusumi, T. Tetrahedron Lett. 1995, 36, 1853. (b)Yabuuchi, T.; Kusumi, T. J. Org. Chem. 2000, 65, 397.

(90) Yabuuchi, T.; Ooi, T.; Kusumi, T. Chirality 1997, 9, 550.(91) (a) Cafieri, F.; Fattorusso, E.; Taglialatela-Scafati, O. Tetrahe-

dron 1999, 55, 7045. (b) Singh, S. B.; Zink, D.; Polishook, J.;Valentino, D.; Shafiee, A.; Silverman, K.; Felock, P.; Teran, A.;Vilella, D.; Hazuda, D. J.; Lingham, R. B. Tetrahedron Lett.1999, 40, 8775. (c) Ciminiello, P.; Fattorusso, E.; Forino, M.;Poletti, R.; Viviani, R. Eur. J. Org. Chem. 2000, 291.

(92) (a) Sasaki, K.; Satake, M.; Yasumoto, T. Biosci., Biotechnol.,Biochem. 1997, 61, 1783. (b) Yabuuchi, T.; Kusumi, T. J. Org.Chem. 2000, 65, 397.

(93) Singh, I. P.; Milligan, K. M.; Gerwick, W. H. J. Nat. Prod. 1999,62, 1333.

(94) Yabuuchi, T.; Kusumi, T. J. Org. Chem. 2000, 65, 397.(95) Sata, N. U.; Wada, S.; Matsunaga, S.; Watabe, S.; van Soest, R.

W. M.; Fusetani, N. J. Org. Chem. 1999, 64, 2331.(96) Fukushi, Y.; Shigematsu, K.; Mizutani, J.; Tahara, S. Tetrahe-

dron Lett. 1996, 37, 4737.(97) Hoye, T. R.; Koltun, D. O. J. Am. Chem. Soc. 1998, 120, 4638.(98) Hoye, T. R.; Hamad, A.-S. S.; Koltun, D. O.; Tennakoon, M. A.

Tetrahedron Lett. 2000, 41, 2289.(99) (a) Toki, M.; Ooi, T.; Kusumi, T. J. Nat. Prod. 1999, 62, 1504.

(b) Morohashi, A.; Satake, M.; Nagai, H.; Oshima, Y.; Yasumoto,T. Tetrahedron 2000, 56, 8995.

(100) Yabuuchi, T.; Kusumi, T. J. Am. Chem. Soc. 1999, 121, 10646.(101) Kobayashi, M.; Aoki, S.; Kitagawa, I. Tetrahedron Lett. 1994,

35, 1243.(102) (a) Duret, P.; Waechter, A.; Figadere, B.; Hocquemiller, R.; Cave,

A. J. Org. Chem. 1998, 63, 4717. (b) Alali, F.; Zeng, L.; Zhang,Y.; Ye, Q.; Hopp, D. C.; Schewedler, J. T.; McLaughlin, J. L.Bioorg. Med. Chem. 1997, 5, 549. (c) Jiang, Z.; Yu, D.-Q. J. Nat.Prod. 1997, 60, 122.

(103) Lee, J.; Li, J.-H.; Oya, S.; Snyder, J. K. J. Org. Chem. 1992, 57,5301.

(104) Rodriguez, J.; Riguera, R.; Debitus, C. J. Org. Chem. 1992, 57,4624.

(105) (a) Seco, J. M.; Martino, M.; Quinoa, E.; Riguera, R. Org. Lett.2000, 2, 3261. (b) Authors’ laboratory; unpublished results.

(106) Woo, M. H.; Cho, K. Y.; Zhang, Y.; Zeng, L.; Gu, Z.-M.;McLaughlin, J. L. J. Nat. Prod. 1995, 58, 1533.

(107) (a) Ichikawa, A.; Takahashi, H.; Ooi, T.; Kusumi, T. Biosci.,Biotechnol., Biochem. 1997, 61, 881. (b) Ichikawa, A. Enantiomer1997, 2, 327.

(108) Ichikawa, A. Enantiomer 1998, 3, 255.


(109) Konno, K.; Fujishima, T.; Liu, Z.; Takayama, H. Chirality 2002,13, 72.

(110) (a) Riccio, R.; Santaniello, M.; Greco, O. S.; Minale, L. J. Chem.Soc., Perkin Trans. 1 1989, 823. (b) De Riccardis, F.; Izzo, I.;Iorizzi, M.; Palagiano, E.; Minale, L.; Riccio, R. J. Nat. Prod.1996, 59, 386. (c) Ukiya, M.; Akihisa, T.; Motohashi, S.; Ya-sukawa, K.; Kimura, Y.; Kasahara, Y.; Takido, M.; Tokutake,N. Chem. Pharm. Bull. 2000, 48, 1187.

(111) Yamase, H.; Ooi, T.; Kusumi, T. Tetrahedron Lett. 1998, 39, 8113.(112) (a) Latypov, S. K.; Seco, J. M.; Quinoa, E.; Riguera, R. J. Am.

Chem. Soc. 1998, 120, 877. (b) Williamson, R. T.; Boulanger, A.;Vulpanovici, A.; Roberts, M. A.; Gerwick, W. H. J. Org. Chem.2002, 67, 7927.

(113) Garcıa, R.; Seco, J. M.; Vazquez, S. A.; Quinoa, E.; Riguera, R.J. Org. Chem. 2002, 67, 4579.

(114) Earle, M. A.; Hultin, P. G. Tetrahedron Lett. 2000, 41, 7855.(115) Seco, J. M.; Tseng, L. H.; Godejohann, M.; Quinoa, E.; Riguera,

R. Tetrahedron: Asymmetry 2002, 13, 2149.(116) Guilet, D.; Guntern, A.; Ioset, J.-R.; Queiroz, E. F.; Ndjoko, K.;

Foggin, C. M.; Hostettmann, K. J. Nat. Prod. 2003, 66, 17.(117) Porto, S.; Duran, J.; Seco, J. M.; Quinoa, E.; Riguera, R. Org.

Lett. 2003, 5, 2979.(118) Arnauld, T.; Barrett, A. G. M.; Hopkins, B. T.; Zecri, F.

Tetrahedron Lett. 2001, 42, 8215.

CR000665J


Use of (S)-BINOL as NMR Chiral Solvating Agent for theEnantiodiscrimination of Omeprazole and its Analogs

JORDI REDONDO,* ANNA CAPDEVILA, AND ISABEL LATORRE

Department of Research and Development, Esteve-Quımica S.A., Barcelona, Spain

ABSTRACT The application of (S)-1,10-binaphthyl-2,20-diol as NMR chiral solvatingagent (CSA) for omeprazole, and three of its analogs (lanso-, panto-, and rabe-prazole)was investigated. The formation of diastereomeric host–guest complexes in solutionbetween the CSA and the racemic substrates produced sufficient NMR signal splittingfor the determination of enantiomeric excesses by 1H- or 19F-NMR spectroscopy. Usingof hydrophobic deuterated solvents was mandatory for obtaining good enantiodiscrimi-nation, thus suggesting the importance of intermolecular hydrogen bonds in the stabili-zation of the complexes. The method was applied to the fast quantification of the enantio-meric purity of in-process samples of S-omeprazole. Chirality 00:000–000, 2009. VVC 2009

Wiley-Liss, Inc.

KEY WORDS: esomeprazole; lansoprazole; pantoprazole; rabeprazole; host-guest;enantiorecognition; enantiomeric purity

INTRODUCTION

Since the recommendations of the US Food and DrugAdministration (FDA) and other regulatory drug agenciesconcerning the development of chiral drugs,1–3 it is a com-mon and challenging issue in the process research of opti-cally pure Active Pharmaceutical Ingredients (APIs) tofind fast and reliable analytical methods to follow andquantify the enantiomeric enrichment (or the degree ofasymmetric induction) when a resolution (or an enantiose-lective synthesis) is applied.

Among the assortment of analytical techniques, and de-spite its inherent low sensitivity, NMR spectroscopy isprobably the most useful in terms of speed and minimalsample preparation, two key points when considering theusually fast milestones involved in the discovery and de-velopment of drugs. In this sense, the use of enantiomeri-cally pure chiral solvating agents (CSA),4–6 chiral derivatiz-ing agents7,8 and, more recently, chiral liquid crystal sol-vents,9 constitute a well established toolbox for the NMRenantiodiscrimination of optically active molecules.

The potent proton-pump inhibitor (PPI) omeprazole(5(6)-methoxy-2-((4-methoxy-3,5-dimethylpyridin-2-yl)methylsulfinyl)-1H-benzo[d]imidazole, 1, Fig. 1)y has beenuntil recent times one of the most used and marketed ra-cemic drugs worldwide.10,11 The stereogenic center of theomeprazole molecule is located on the sulfur atom, and ithas been reported that the S enantiomer (esomeprazole,(2)-(S)-1) shows higher bioavailability than the racematedue to a difference in the rates of metabolism of both enan-tiomers.12–14 Esomeprazole, as its magnesium salt, wasapproved in 2001 and is also a blockbuster anti-acid drug.

Although several chromatographic methods for the chi-ral analysis of omeprazole and its analogs have been pub-lished yet,15–19 no information about NMR as a chiral tech-nique for this family of drugs can be found in the litera-ture. This is probably due to the high degradation rate ofthese compounds in water, which hinders the use ofhydrosoluble CSAs like cyclodextrins or chiral organicacids/bases; their low affinity with chiral lanthanidereagents, and the lack of functional groups capable ofreacting easily with Mosher reagents. As a part of ourinvestigation on chemical processes for the preparation ofesomeprazole, we found that the chiral host (S)-1,10-binaphthyl-2,20-diol ((S)-BINOL, 2, Fig. 1), which is alsoused for the resolution of the omeprazole racemate in labo-ratory20 and industrial scale,21 acts as a convenient NMRCSA for the rapid and accurate quantification of low levelsof the minor enantiomer. It has been previously reportedthat enantiopure BINOL is a good NMR chiral shift rea-gent for sulfoxides,22 selenoxides,23 and sulfinimines,24

and the enantiomers of BINOL and some of its derivativeshave also been found to be excellent compounds for thechemical resolution of chiral sulfoxides.25 More in general,(R)-BINOL has been recently applied as NMR CSA forsome nonsulfoxide important drugs.26

In this work, we report the results of the application ofthis NMR method to the enantiodiscrimination of omepra-zole and three structurally related analogs (lansoprazole,

yOmeprazole presents tautomerism in the benzimidazole group. There-fore, both tautomers must be considered when using the systematic no-menclature. For simplicity, only one tautomer is represented in Figure 1.

Additional Supporting Information may be found in the online version ofthis article.*Correspondence to: Jordi Redondo, Department of Research and Devel-opment, Esteve-Quımica S.A., Caracas street 17–19, Barcelona E-08030,Spain. E-mail: [email protected] for publication 1 September 2008; Accepted 17 June 2009DOI: 10.1002/chir.20766Published online in Wiley InterScience(www.interscience.wiley.com).

CHIRALITY 00:000–000 (2009)

VVC 2009 Wiley-Liss, Inc.

3, pantoprazole, 4, and rabeprazole, 5 (Fig. 2)) in differentdeuterated solvents. Furthermore, some considerationsconcerning the host–guest interaction between (S)-BINOLand the S-enantiomer of 1 are presented in the light of theNMR results.

MATERIALS AND METHODS

The PPIs 1, (S)-1, 3, 4, and 5 were obtained from ourlaboratory using proprietary synthetic processes,21,27,28

and were characterized by 1H-, 19F-, and 13C-NMR as wellas by mass spectrometry. (S)-BINOL (2) was purchasedfrom Sigma-Aldrich. The absolute configuration of (S)-1and the optical purity of the sample were determined bymeans of the chiral tests described in the European Phar-macopoeia.29 All NMR measurements were obtained witha Varian Mercury spectrometer operating at 400.1 MHzfor 1H and 376.4 MHz for 19F. The host-guest complexeswere prepared by dissolving equimolecular quantities of 2and the PPI in the deuterated solvents at ambient tempera-ture (the concentrations were around 40 mM). The assign-ment of the 1H-NMR spectra of compounds 1-5 wasaccomplished by means of standard 1D and 2D NMR tech-niques, namely 2D COSY (1 scan per FID; 200 incre-ments) and selective 1H {1H} NOE experiments (mixingtime: 800 ms, 256 scans).

RESULTS AND DISCUSSIONEnantiodiscrimination Detected by 1H-and 19F-NMR

The power of enantiodiscrimination of a NMR chiral sol-vent agent relies on the formation of a pair of diastereo-meric complexes coexisting in solution. If the lifetime ofsuch complexes is long enough on the NMR time scale,splitting could be observed for several resonances of themolecular species, thus permitting the identification andquantification of the substrate single enantiomers. Hydro-gen bonds, p–p interactions, and van der Waals forces arethe common factors invoked for explaining the formationof host–guest complexes in solution. Moreover, the effectof the solvent must be regarded as one of the importantissues in the stabilization of the diastereomeric pairs and,therefore, in the enantiodiscrimination ability of the CSA.

In this sense, it was found that, among the common deu-terated solvents used in NMR spectroscopy, only thehydrophobic ones (namely, CDCl3, CD2Cl2, and toluene-d8) afforded good enantiodiscrimination of the studied

PPIs. Conversely, no signal splitting at all was observedwhen using hydrophilic solvents (CD3OD, acetonitrile-d3,acetone-d6, and DMSO-d6), thus suggesting the impor-tance of the intermolecular H bonds in the formation ofthe complexes, since hydrophobic solvents must help,through a solvophobic effect, to establish such H bondsbetween host and guest, whereas water-miscible solventsmust difficult them (significantly, the addition of only onedrop of DMSO-d6 to a sample prepared in any of thehydrophobic deuterated solvents completely removes theenantiodiscrimination effect).

Table 1 shows that in the presence of one equivalent of(S)-BINOL using CD2Cl2 as solvent, most of resonances ofomeprazole are split at room temperature as a conse-quence of the coexistence of two diastereomeric host-guest complexes in solution. It is noteworthy that the im-portant nonequivalence of H13 (DDd: 0.084 ppm) that per-mits the easy and reliable quantification of the enantio-meric purity (Fig. 3). The complexation also produced achange in the chemical shift of the methylene H8AB pro-tons as well as a considerable broadening of their signals.Similar signal splitting was found when CDCl3 or toluene-d8 was used. Unfortunately, no information could beextracted from the signals of omeprazole H4 and H7 pro-tons, due to the extreme broadness caused by the tautom-erism of the benzimidazole group at room temperature.§

Splitting was also observed for several 1H signals ofcompounds 3, 4, and 5 in the presence of (S)-BINOL. Inthose cases, not only H13 but also the pyridine proton H12,not present in omeprazole, showed an appreciable none-quivalence induced by the enantiodiscrimination phenom-enon (Fig. 4 and Table 1). In the case of compound 5, the

Fig. 2. Molecular structures of lansoprazole (3), pantoprazole (4) andrabeprazole (5).

Fig. 1. Molecular structures of omeprazole (1) and (S)-BINOL (2).

§Claramunt et al30 have reported that both tautomers of omeprazole canbe observed by NMR at 195 K in THF-d8, with predominance of the 6-methoxy tautomer (63%). At room temperature, however, the 1H-NMRspectrum of omeprazole consists of single signals revealing the weightedaverage of the two tautomeric situations.

2 REDONDO ET AL.

Chirality DOI 10.1002/chir

lack of splitting in the signals from the side chain protonsH16, H17, and H18 suggests that this part of the molecule isfar away from the binding sites involved in the enantiore-cognition process (in fact, only H15, which are the closestchain protons to the pyridine ring, manifest some degreeof splitting).

The effect of the enantiorecognition was also evident inthe 19F-NMR spectrum of the CHF2 group of compound 4,whose original doublet (JF-H: 74 Hz) slightly split in thepresence of (S)-BINOL (Fig. 5). Interestingly, no extrasplitting was observed in the 19F-NMR signal of the tri-fluoromethyl group of compound 3 (a triplet with JF-H: 7.5Hz), probably due to the high mobility of the OCH2CF3

chain and its relatively far distance from the sites of inter-action with (S)-BINOL.

Clearly, among the solvents studied, toluene-d8 was theless favorable due to the relatively low solubility of com-pounds 3 and 4 (for those compounds, the host/guestmolar ratios achieved in toluene solution were 5.4:1 and1.3:1, respectively, as determined by integration of the cor-responding 1H signals).

In addition to the induced proton nonequivalence, theformation of the host-guest complex produced changes inthe chemical shifts of the involved molecular species. In

TABLE 1. Chemical shift nonequivalence (DDd, ppm) for protons of compounds 1, 3, 4, and 5 in presence of (S)-BINOL indifferent deuterated solvents at 228C (signals from protons H4 and H7 not reported due to their broadness by effect of the

benzimidazole tautomerism)

Compound Host/guest ratio H6 H5 H8A H8B H12 H13 H15 H16 H14 H17 H18

1 (CD2Cl2) 1:1 0.000 – 0.042 0.018 – 0.084 0.033 0.005 0.017 0.004 –1 (CDCl3) 1:1 0.000 – 0.000 0.014 – 0.055 0.017 0.000 0.007 0.000 –1 (tol-d8) 1.5:1 bd – 0.007 0.000 – 0.040 0.020 0.014 0.022 0.004 –3 (CD2Cl2) 1:1 bd bd 0.008 0.031 0.027 0.054 0.000 – 0.009 – –3 (CDCl3) 1:1 bd bd 0.000 0.025 0.016 0.050 0.000 – 0.005 – –3 (tol-d8) 4:1 bd bd 0.032 0.000 0.000 0.016 bd – 0.006 – –4 (CD2Cl2) 1:1 – bd 0.011 0.019 0.028 0.072 0.003 0.005 0.000 – –4 (CDCl3) 1:1 – 0.022 0.014 0.020 0.025 0.075 0.000 0.008 0.000 – –4 (tol-d8) 1.5:1 – bd 0.000 0.000 0.015 0.054 0.006 0.009 0.000 – –5 (CD2Cl2) 1:1 bd bd 0.000 0.021 0.035 0.060 0.007 0.000 0.012 0.000 0.0005 (CDCl3) 1:1 bd bd 0.015 0.005 0.028 0.060 0.005 0.000 0.008 0.000 0.0005 (tol-d8) 1:1 bd bd 0.000 0.007 0.015 0.015 bd 0.000 0.015 0.000 0.000

In all cases, H8A refers to as the downfield H8 proton and H8B to the upfield H8 proton.bd, broad signal.

Fig. 3. Partial 1H-NMR spectrum of A: omeprazole in CDCl3; B: anequimolecular mixture of omeprazole and (S)-BINOL in CDCl3. Theresonances of H13 omeprazole enantiomers are indicated by arrows. Aster-isks indicate (S)-BINOL signals.

Fig. 4. Partial 1H-NMR (CD2Cl2) spectra of A: 3/(S)-BINOL 1:1; B: 4/(S)BINOL 1:1; C: 5/(S)-BINOL 1:1. The H12 and H13 doublets from eachenantiomer are indicated by ~ and l, respectively.

3BINOL AS NMR CSA FOR OMEPRAZOLE AND ANALOGS


this sense, it was found that, among the resonances ofomeprazole, those of the pyridine H13 and the H8AB pro-tons displayed the more notable upfield shifts (low d val-ues) in the presence of the CSA, thus suggesting an im-portant interaction of this part of the molecule with the ar-omatic groups of (S)-BINOL. A similar behavior was foundfor the corresponding protons of compounds 3, 4, and 5(important upfield shifts for H13 and H8AB protons, me-dium shifts for H12 protons, slight shifts for methyl andother aliphatic protons, see Table 2). On the other hand,most of the (S)-BINOL protons also showed upfield shiftswhen forming the complex, an effect that could be attrib-uted to magnetic anisotropy from the heteroaromaticgroups of the guest molecules and/or to conformationalchanges of the (S)-BINOL molecule around its chiral axisinduced by the molecular recognition phenomenon.

As expected, when a molar excess of (S)-BINOL wasapplied to the substrate, a shift of the complexation equi-librium toward the associated species occurred and, conse-quently, an increase of the signal separation was observed.So, when the host:guest molar ratio reached 5:1 in CDCl3,almost all the visible proton signals of omeprazole weresplit. In this sample, the CH2 resonance can be describedas a pair of semioverlapped AB system signals (JA-B:13.5 Hz), one for each diastereomeric complex (Fig. 6). Aclear pattern of four doublets was also observed for com-pounds 3, 4, and 5, even at host/guest ratios around 1:1(Fig. 7, as an example).

Solution Stoichiometry of the Complex( S)-BINOL/Esomeprazole

Regarding the structural aspects of the complexes, it isimportant to know the stoichiometry of the supramolecularhost–guest species in solution. One of the usual ways toget this information is constructing a Job’s plot.31 Thismethod, also known as the Continuous VariationMethod,32 consists of plotting the increment in the chemi-cal shift (Dd) of the protons of one of the components ver-sus the mole fraction of such component. Provided noother secondary processes are present (for example,

TABLE 2. 1H chemical shift increments (Dd) induced oncompounds 1, 3, 4, and 5 by complexation with one

equivalent of (S)-BINOL, in CDCl3 at 228C

Compound Assignment Dd (ppm)

1 H13 (R) 20.105H13 (S) 20.147H8A 20.063H8B 20.061H15 (R) 20.025H15 (S) 20.037

3 H13 (d) 20.142H13 (u) 20.192H12 (d) 20.075H12 (u) 20.095H8A 20.103H8B (d) 20.081H8B (u) 20.105H15 20.032H14 (d) 20.031H14 (u) 20.036

4 H13 (d) 20.118H13 (u) 20.194H12 (d) 20.047H12 (u) 20.072H8A (d) 20.101H8A (u) 20.114H8B (d) 20.082H8B (u) 20.101

5 H13 (d) 20.137H13 (u) 20.196H4 1 H7 20.080H12 (d) 20.048H12 (u) 20.077H8A (d) 20.116H8A (u) 20.132H8B (d) 20.118H8B (u) 20.123

For simplicity, increments below 20.025 not reported.Abbreviations: (d), downfield signal; (u), upfield signal.

Fig. 5. 19F-NMR (CD2Cl2) of compound 4 in absence (A) and pres-ence (B) of 1 equivalent of (S)-BINOL.

Fig. 6. Partial 1H-NMR (CDCl3) of (S)-BINOL/omeprazole, molar ratio5:1. The inset shows the split signal from omeprazole H8A, H8B protons.

4 REDONDO ET AL.


molecular aggregation due to excessive concentration ofthe solutes), the maximum of the obtained parabolic curveindicates the average solution stoichiometry of the com-plexes. In our case, nine samples with a host 1 guest con-stant concentration (43.4 mM) were prepared in CDCl3,and variable amounts of esomeprazole (99% ee) wereadded. The chemical shift changes induced for severalprotons of esomeprazole (H8A, H8B, H13) were followed at258C, and in all cases similar curves were obtained show-ing maxima at an approximate mole fraction of 0.5, consist-ent with a 1:1 solution stoichiometry for the complex (S)-BINOL/esomeprazole (Fig. 8).

Some attempts were made to achieve more structural in-formation via 2D NOESY and ROESY experiments on the(S)-BINOL/esomeprazole complex but, unfortunately, no

cross correlation peaks were observed between host andguest protons, probably due to the weakness of the supra-molecular association. The only reliable intermolecularNOE contact was found between H3

0 of (S)-BINOL and theselectively irradiated H8AB esomeprazole protons, usingthe standard 1D NOESY sequence (Fig. 9). Although thisunique result, of course, does not permit the inference ofany preferred host–guest geometry, at least it shows thetransient proximity of both species and probably reflects

Fig. 7. Partial 1H-NMR (CD2Cl2) spectrum of (S)-BINOL/pantoprazole1:1 complex showing the split system of H8AB. The symbol ~ indicatessignals from one enantiomer and l from the other.

Fig. 8. Continuous variation plots of H8A, H8B, and H13 protons of eso-meprazole ((S) 2 1) complexed with (S)-BINOL.

Fig. 9. Intermolecular NOE on H03 of (S)-BINOL after irradiation of H8AB protons of esomeprazole (intramolecular NOEs on esomeprazole H13 and

H14 protons are shown).



the important role of the intermolecular H bonds in theformation of the supramolecular association, since H3

0 isthe proton adjacent to the OH group in (S)-BINOL.

Determination of the Enantiomeric Excess

The use of NMR as a quantitative tool for the determina-tion of the enantiomeric purity of optically enriched PPIswas checked by applying (S)-BINOL to samples of esome-prazole ((S)-1) containing different amounts of its R-enan-tiomer, and recording the corresponding 1H-NMR spectrawith sufficient number of scans (typically 64) to achievegood signal-to-noise levels. Under such conditions, a rea-sonable limit of detection was established to be at least0.5% of the minor enantiomer of omeprazole (Fig. 10),since it was possible to observe and integrate easily thecorresponding signal of H13 in a sample containing (S)-BINOL and esomeprazole ‘‘99.1% ee’’ (enrichment deter-mined by UV-detected chiral chromatography). Thismethod permits a fast, routine in-process control of opti-cally enriched samples obtained throughout the chemicalresolution of omeprazole.

ACKNOWLEDGMENTS

The authors thank Anna Gracia for her assistance in theliterature search. They also acknowledge Antonio Bautistafor his help in the preparation of this manuscript.

LITERATURE CITED

1. Food and Drug Administration (FDA). Available at: http://www.fda.gov/cder/guidance/stereo.htm, 5/1/1992.

2. Branch S. Subramanian G., editors. International regulation of chiraldrugs. Chiral separation techniques. A practical approach. Weinheim:Wiley-VCH; 2001. 319–342.

3. Shindo H, Caldwell J. Development of chiral drugs in Japan: anupdate on regulatory and industrial opinion. Chirality 1995;7:349–352.

4. Pirkle WH, Hoover DJ. NMR chiral solvating agents. Top Stereochem1982;13:263–327.

5. Weisman GR. Nuclear magnetic resonance analysis using chiral sol-vating agents. Asymm Synth 1983;1:153–171.

6. Wenzel TJ. Lanthanide chiral solvating agent couples as chiral NMRshift reagents. Trends Org Chem 2000;8:51–64.

7. Yamaguchi S. Nuclear magnetic resonance analysis using chiral deriv-atives. Asymm Synth 1983;1:125–152.

8. Wenzel TJ, Wilcox JD. Chiral reagents for the determination of enan-tiomeric excess and absolute configuration using NMR spectroscopy.Chirality 2003;15:256–270.

9. Courtieu J, Lesot P, Meddour A, Merlet D, Aroulanda C. Grant DM,Harris R., editors. Chiral liquid crystal NMR: a tool for enantiomericanalysis. Encyclopedia of nuclear magnetic resonance, New York:John Wiley & Sons; 2002. p 497–505.

10. Lindberg P, Brandstrom A, Wallmark B, Mattsson H, Rikner L, Hoff-man KJ. Omeprazole: the first proton pump inhibitor. Med Res Rev1990;10:1–54.

11. Shi S, Klotz U. Proton pump inhibitors: an update of their clinical useand pharmacokinetics. Eur J Clin Pharmacol 2008;64:935–951.

12. Lindberg P, Keeling D, Fryklund J, Andersson T, Lundborg P, Carl-son E. Esomeprazole-enhanced bio-availability, specificity for the pro-ton pump and inhibition of acid secretion. Aliment Pharmacol Ther2003;17:481–488.

13. Andersson T, Weidolf L. Stereoselective disposition of proton pumpinhibitors. Clin Drug Investig 2008;28:263–279.

14. Olbe L, Carlsson E, Lindberg P. A proton-pump inhibitor expedition:the case histories of omeprazole and esomeprazole. Nat Rev Drug Dis-cov 2003;2:132–139.

15. Cirilli R, Ferretti R, Gallinella B, De Santis E, Zanitti L, La Torre F.High-performance liquid chromatography enantioseparation of protonpump inhibitors using the immobilized amylose-based Chiralpak IAchiral stationary phase in normal-phase, polar organic and reversed-phase conditions. J Chromatogr A 2008;1177:105–113.

16. Jenkins AL, Hedgepeth WA. Analysis of chiral pharmaceuticals usingHPLC with CD detection. Chirality 2005;17(Suppl):S24–S29.

17. Matlin SA, Tiritan ME, Cass QB, Boyd DR. Enantiomeric resolutionof chiral sulfoxides on polysaccharide phases by HPLC. Chirality1996;8:147–152.

18. Berzas-Nevado JJ, Castaneda-Penalvo G, Rodrıguez-Dorado RM.Method development and validation for the separation and de-termination of omeprazole enantiomers in pharmaceutical prepara-tions by capillary electrophoresis. Anal Chim Acta 2005;533:127–133.

19. Toribio L, Alonso C, Del Nozal MJ, Bernal JL, Jimenez JJ. Enantio-meric separation of chiral sulfoxides by supercritical fluid chromatog-raphy. J Sep Sci 2006;29:1363–1372.

20. Deng J, Chi Y, Fu F, Cui X, Yu K, Zhu J, Jiang Y. Resolution of ome-prazole by inclusion complexation with a chiral host BINOL. Tetrahe-dron: Asymmetry 2000;11:1729–1732.

21. Coppi L, Berenguer R, Gasanz Y, Medrano J. Process for the prepara-tion of optically active derivatives of 2-(2-pyridylmethylsulfinyl)-benz-imidazole via inclusion complex with 1,10 -binaphthalene-2,20 -diol, PCTInt. Appl.WO 2006094904 A1. 2006.

22. Toda F, Mori K, Okada J, Node M, Itoh A, Oomine K, Fuji K. Newchiral shift reagents, optically active 2,20-dihydroxy-1,10-binaphtyl and1,6-di(o-chlorophenyl)-1,6-diphenylhexa-2,4-diyne-1,6-diol. Chem Lett1988;131–134.

23. Drabowicz J, Duddeck H. 1H-NMR spectral non-equivalence of sulph-oxide enantiomers in the presence of 2,20-dihydroxy-1,10-binaphthyl.Sulfur Lett 1989;10:37–40.

24. Ardej-Jakubisiak M, Kawecki R. NMR method for determination ofenantiomeric purity of sulfinimines. Tetrahedron: Asymmetry 2008;19:2645–2647.

25. Liao J, Sun X, Cui X, Yu K, Zhu J, Deng J. Facile optical resolution oftert-butanethiosulfinate by molecular complexation with (R)-BINOLand study of chiral discrimination of the diastereomeric complexes.Chem Eur J 2003;9:2611–2615.

Fig. 10. Partial 1H-NMR spectrum of optically enriched omeprazole(99.1 ee in the S-enantiomer) complexed with one equivalent of (S)-BINOL, in CDCl3. The arrows indicate H13 proton signals from each enan-tiomer.

6 REDONDO ET AL.


26. Salsbury JS, Isbester PK. Quantitative 1H-NMR method for the routinespectroscopic determination of enantiomeric purity of active pharma-ceutical ingredients fenfluramine, sertraline and paroxetine. MagnReson Chem 2005;43:910–917.

27. Coppi L, Berenguer R. Method for obtaining derivatives of [[substi-tuted-pyridyl)methyl]thio]benzimidazole, useful as intermediates foromeprazole and related antiulcer agents, PCT Int. Appl. WO2001079194 A1. 2001.

28. Berenguer R, Campon J, Coppi L. Method for oxidizing a thioethergroup into a sulfoxide group in benzimidazole derivatives, PCT Int.Appl. WO 2001068594 A1. 2004.

29. Anon. Esomeprazole magnesium trihydrate monograph. EuropeanPharmacopoeia, 6th edition, Available at: http://online.pheur.org/entry.htm, 2009.

30. Claramunt RM, Lopez C, Alkorta I, Elguero J, Yang R, Schulman S.The tautomerism of omeprazole in solution: a 1H- and 13C-NMR study.Magn Reson Chem 2004;42:712–714.

31. Job P. Formation and stability of inorganic complexes in solution. AnnChim Applicata 1928;9:113–125.

32. Djedaini F, Lin SZ, Perly B, Wouessidjewe D. High-field nuclear mag-netic resonance techniques for the investigation of a beta-cyclodextrin:indomethacin inclusion complex. J Pharm Sci 1990;79:643–646.



Chem. Educator 2004, 9, 359�363 359

The Mosher Method of Determining Enantiomeric Ratios: A Microscale NMR Experiment for Organic Chemistry

Steve Lee

School of Science and Mathematics, Roosevelt University, Chicago and Schaumburg, IL, [email protected]

Received August 4, 2004. Accepted September 16, 2004.

Abstract: Mosher�s established technique of measuring the enantiomeric ratio of a chiral compound has been adapted to be suitable for an undergraduate organic chemistry experiment. In this technique a chiral alcohol or amine reacts with the Mosher acyl chloride, α-methoxy-α-(trifluoromethyl) phenylacetyl chloride (MTPA-Cl), to yield the corresponding diastereomeric ester or amide. The ester or amide products are analyzed by 1H and/or 19F NMR spectroscopy, and signals for diastereotopic groups on the products are integrated to determine the ratio of the diastereomeric products and consequently the ratio of enantiomeric substrates. This experiment has the benefits of helping students (1) apply advanced topics in stereochemistry, such as stereoheterotopic ligands and kinetic resolution; (2) learn a quantitative application of NMR spectroscopy, including 19F NMR; and (3) be introduced to microscale techniques. Furthermore, even though this is a microscale NMR technique, it can be conducted on a low-field (60- or 90-MHz) NMR spectrometer with satisfactory results.

Introduction

The most commonly used methods for determining the enantiomeric ratio of a sample of a chiral compound involve (1) chiral stationary phase HPLC or GC, (2) chiral shift reagents with NMR spectroscopy, (3) optical rotation, or (4) derivatization into diastereomers with subsequent analysis [1]. Within this last method Mosher has provided a chiral derivatizing agent that has proven to become an industry standard [2, 3]. This method has been updated and adapted into an experiment that is pedagogically beneficial for organic chemistry students as it relates to stereochemistry, 1H and 19F NMR spectroscopy, and microscale techniques. With this technique students are able to analyze a sample of a chiral compound to determine its enantiomeric ratio and the configuration of the major enantiomer. This technique can either be used as its own experiment or attached to an asymmetric synthesis or resolution, for example, the resolution of α-methylbenzylamine [4], which is a common organic laboratory experiment. The topics and skills for this experiment are suitable for sophomore-level organic chemistry courses or upper-level advanced courses. It has been used at Roosevelt University in our Advanced Organic Chemistry Laboratory course for several years.

The Mosher Method

A pair of enantiomers is indistinguishable by NMR spectroscopy; however, if the two enantiomers are reacted with a chiral compound the products can become diastereomeric with diastereotopic groups that can be distinguished by NMR. This feature is used by Mosher in his technique of determining the enantiomeric ratio of a sample of a chiral compound, and it is illustrated in Scheme 1.

The method begins with an enantiopure sample of the chiral derivatizing agent α-methoxy-α-(trifluoromethyl)phenylacetyl chloride (MTPA-Cl, 1), which is sometimes referred to as the Mosher acyl chloride. Either the R or S enantiomer of 1 can be

used, and so the S enantiomer is arbitrarily shown in this scheme. The enantiomeric substrate to be analyzed must contain either a hydroxyl or amino functional group to permit reaction with the acyl chloride. In the generic example shown in Scheme 1, (S)-1 reacts with enantiomeric alcohols (2) to yield their corresponding Mosher esters (3). The products are now diastereomeric with diastereotopic groups that can thus be distinguished and have their relative amounts measured by NMR spectroscopy.

The racemization of the starting materials or products during the reaction is not mechanistically reasonable in most cases due to the mild reaction conditions; however, if the products are solid at ambient temperature, differential crystallization could lead to an inaccurate determination of the enantiomeric ratio and, thus, care must be taken whenever dealing with solid products.

HO

HO RS

O

ClF3C

OCH3Ph

O

OF3C

OCH3Ph

O

OF3C

OCH3Ph

RS

(S)-1 Mosher esters

+

(R,R)-3

(R,S)-3

2

pyridine

RR

RR

Scheme 1 An aside on the change of nomenclature of the

configurations shown in Scheme 1 should be noted. The S configuration of 1 is shown with the relative priorities for the substituents on the stereogenic carbon being (1) �OCH3, (2) �COCl, (3) �CF3, and (4) �Ph, but when (S)-1 is converted to the Mosher ester 3, the relative priorities for the substituents on the same carbon change to (1) �OCH3, (2) �CF3, (3) �CO2R, and (4) �Ph, now with an R configuration. Thus, although the name of the configuration changes, the absolute configuration is retained.

© 2004 The Chemical Educator, S1430-4171(04)06843-X, Published on Web 11/16/2004, 10.1333/s00897040843a, 960359sl.pdf

360 Chem. Educator, Vol. 9, No. 6, 2004 Steve Lee

Control Test for the Possibility of Kinetic Resolution

In Mosher�s method the principle reaction might undergo a kinetic resolution and so must be investigated for this possibility in a control test. Because the reaction of the two enantiomeric substrates with the Mosher acyl chloride leads to diastereomeric products, it must be confirmed that the ratio of diastereomeric products accurately reflects the ratio of the original enantiomers. A kinetic resolution would invalidate the Mosher method for that particular substrate. A kinetic resolution involves resolving a mixture of enantiomers by reacting them with a chiral, nonracemic agent and not allowing the two reactions to go to completion. The different reaction rates of the two enantiomers lead to one diastereomeric product being formed faster than the other, thereby resolving the two enantiomers. A kinetic resolution is not desired in the Mosher method because this would invalidate the method for that particular substrate; however, the effect of the different rates is minimized by using an excess of the Mosher acyl chloride and a sufficiently long reaction time to allow both reactions to proceed to completion.

The possibility for a kinetic resolution can be considered in an example with sec-butanol (4) as shown in Scheme 2. The reactions of (S)-1 with (R)-4 and (S)-4 lead to diastereomeric products. Because a pair of enantiomers is reacting with a chiral reagent to form diastereomeric products, it is likely that the two reactions will proceed with different rates (i.e., kRR will likely be different than kRS). This difference in rates could potentially be significant enough to alter the product ratio in which case the enantiomeric substrates would be kinetically resolved; however, for this method to be valid, this difference in rates must not be significant enough to alter the product ratio. In other words, the product ratio of (R,R)-8 to (R,S)-8 needs to accurately reflect the ratio of (R)-4 to (S)-4 in order for this method to validly determine the enantiomeric ratio of 4 in the original sample.

O

ClF3C

OCH3Ph

HO

H

O

F3C

OCH3Ph

O

OF3C

OCH3Ph

H

(S)-4

(R,R)-8

(R,S)-8

kRR

kRS

kRR ≠ kRS

(S)-1

+

HO

H

O

H

(R)-4

Scheme 2 It must, therefore, be tested whether the difference in the

rates of the two reactions (i.e., kRR and kRS) is significant enough to change the product ratio of (R,R)-8 to (R,S)-8. This possibility of kinetic resolution can be tested by running a control experiment with a racemic sample of the enantiomeric substrate. If a racemic sample of the substrate reacts with the Mosher acyl chloride to yield a 50:50 mixture of the two diastereomeric products, this would confirm that our method is valid so that the ratio of diastereomeric products accurately reflects the initial ratio of enantiomeric substrates. On the other hand, if the racemic sample of the substrate reacts to yield an unequal mixture of diastereomeric products, this would indicate that the substrate is not suitable for this method. For this reason, the Mosher method has generally been limited to sterically-unhindered, primary and secondary alcohols and

amines because of the slower rates with sterically bulky substrates; however to help minimize the effect of the different rates upon the product ratio, the reaction conditions are designed to include an excess of the Mosher acyl chloride and a sufficient reaction time to ensure a complete reaction for both diastereomeric reactions.

The Laboratory Experiment

The chemical reagents in this experiment are all commercially available, for example, from the Sigma-Aldrich Company; however, because the Mosher acyl chloride is expensive, the corresponding acid can instead be purchased at a lower cost and converted with thionyl chloride. The procedure involves a 50-hour reflux, and so unfortunately it is not conducive to most student laboratory formats. This would have to be conducted by the instructor or laboratory personnel [2].

Regarding alcohol and amine substrates, four compounds have been tested and found to be suitable for this technique. Their NMR data and configurations have been reported previously [2] and are updated here. These compounds have the added benefit of having both their racemic and enantiopure forms commercially available so that samples of varying enantioenrichments can conveniently be prepared. The compounds are sec-butanol (4), α-trifluoromethylbenzyl alcohol (5), methyl mandelate (6), and α-methylbenzylamine (7). Obviously, countless other alcohols or amines can be tested with this technique.

HO

CH3H

PhHO

CF3H

PhHO

CO2CH3H

4 5 6

PhH2N

CH3H

7

The overall procedure for the experiment is logistically simple. In the first laboratory period students start their reactions by adding their reagents into a conical vial, which is left in their laboratory drawers at ambient temperature [5]. In the following laboratory period students carry out the work-up, with no purification required, and analyze their product samples by NMR spectroscopy [6]. The recommended reaction time is 12 hours at ambient temperature, but the reaction mixture can be kept at ambient temperature for one week without a noticeable degradation of the product.

Students can be organized into pairs or groups. One student could carry out the control test using a racemic sample of the substrate while the other student(s) in the group use an enantioenriched sample of the same substrate. Students then compare each other�s NMR spectra with the literature NMR data to fully assign their spectra, determine the enantiomeric ratio, and determine the configuration of the major enantiomer.

A sample experiment is shown in Scheme 3. In this case, (S)-1 reacts with (R)-5 and (S)-5 to yield the corresponding esters (R,R)-9 and (R,S)-9. The 1H NMR spectra in Figure 1 show the signals for the methoxy hydrogens in 9. (These signals are split into quartets due to long distance 1H-19F coupling.) In the control reaction, a racemic sample of 5 was used. The 1H NMR spectrum of its product sample (Figure 1a) shows that the diastereomeric products are in nearly equal amounts (51.9% to 48.1%), which indicates that the enantiomeric substrates are not kinetically resolved. The slight deviation from the expected ratio of 50:50 can be explained by the use of a sample of (S)-1 that was not completely enantiopure. It was labeled with 98% e.e. as determined by GLC according to the commercial source. From the second reaction starting with enantioenriched 5, the relative integration in its NMR spectrum (Figure 1b) indicates a diastereomeric ratio of 64.2% to 35.8%, or 28.4% d.e. for (R,R)-9 and consequently 28.4% e.e. (R) for 5 (d.e. = diastereomeric excess = % major diastereomer � % minor diastereomer; e.e. = enantiomeric excess = % major enantiomer � % minor enantiomer). This value is close to that (25% e.e.) based upon a mass analysis of racemic and enantiopure samples of 5 used to prepare the enantioenriched sample.


A Microscale NMR Experiment for Organic Chemistry Chem. Educator, Vol. 9, No. 6, 2004 361


Table 1. Summary of NMR data for the diastereomeric products from reaction with (S)-1

Diastereomeric products Configuration of substrate Chemical shifts (ppm) CH3 H OCH3 CF3

a R 1.255 5.096 3.564 �72.00

O

H3C O

OCH3

CF3Ph

8

S 1.335 5.096 3.566 �72.00

R � 6.263 3.601 �76.17

OPh

F3C O

OCH3

CF3Ph

9

S � 6.342 3.479 �76.40

R 3.769 6.094 3.697 �72.20

OPh

H3CO2C O

OCH3

CF3Ph

10

S 3.740 6.115 3.552 �72.20

R 1.499 5.175 3.361 �69.25

NH

Ph

H3C O

OCH3

CF3Ph

11

S 1.539 5.175 3.403 �69.44

H

H

H

H

HO Ph

CF3H

O Ph

CF3H

(R)-5

(R,R)-9

a 19F NMR for the trifluoromethyl group in bold with CFCl3 as the internal reference set to 0 ppm.

(a) from racemic 5 (b) from enantioenriched 5

R,R R,S R,R R,S

Figure 1. Expanded regions of the 1H NMR (60 MHz) spectra for (R,R)-9 and (R,S)-9 for the methoxy hydrogens.

The assignment of the signals for the methoxy hydrogens in the diastereomeric products (δ 3.601 ppm for (R,R)-9 and δ 3.479 ppm for (R,S)-9) are based upon the chemical shifts reported by Mosher [2] and based upon the use of enantiopure (R)-5 (which was purchased from the Sigma-Aldrich Company) to provide the major enantiomer in the enantioenriched sample of 5. The consistency of these two independent factors also helps to confirm the accuracy of the assignment.

O

ClF3C

OCH3Ph

HO Ph

HF3C

O

F3C

OCH3Ph

O

OF3C

OCH3PhPh

HF3C

(S)-5

(R,S)-9(S)-1

+

Scheme 3 The NMR spectra shown in Figure 1 are from an upgraded Anasazi EFT 60-MHz NMR spectrometer. Although the signals are not completely separated at the baseline, the spectrum is still acceptable for determining the ratio of diastereomeric products for educational purposes. This shows that even though the technique is carried out on

a microscale with typical product yields of 20 to 50 mg, it is still suitable for a low-field NMR spectrometer. The product samples have also been analyzed with a higher-field 300-MHz instrument, which provides much better separations of the signals. In this example, the two signals are completely separated at 300 MHz.

A summary of the NMR data for all four diastereomeric products is given in Table 1. The reactions were carried out with the S enantiomer of 1. The table shows the chemical shifts for hydrogen or fluorine atoms in the diastereomeric products. With only a few exceptions, diastereotopic groups led to different chemical shifts. This data is an update and is consistent with that reported by Mosher [2]; however, in one case, Mosher reported the signals for the methine hydrogens in 10 to be identical for both diastereomers. Using a 300-MHz NMR spectrometer, however, these signals appear separately at 6.094 and 6.115 ppm. Complete 1H and 19F NMR data for these compounds are included in the experimental section.

This technique can be adapted into a laboratory experiment in a variety of ways. In a straightforward approach, students can be given a known substrate with an unknown enantiomeric ratio that is determined by the instructor. The students would then be required to carry out the reaction, analyze the product sample by 1H and/or 19F NMR, and determine the enantiomeric ratio and the major enantiomer.

Alternatively, the technique can be attached to another experiment involving an asymmetric synthesis or resolution. For example, the resolution of α-methylbenzylamine with tartaric acid is commonly

362 Chem. Educator, Vol. 9, No. 6, 2004 Steve Lee

used in organic chemistry courses [4]. The determination of the enantiomeric ratio of the resolved α-methylbenzylamine has been reported to be carried out by optical rotation [4a], chiral stationary phase HPLC [7], and chiral shift reagents [8]. This Mosher technique provides another means of determining the success of the students� resolution of α-methylbenzylamine, which has not been previously reported. The technique works particularly well with α-methylbenzylamine because the 19F NMR signals for the diastereotopic trifluoromethyl groups are very far apart providing a pedagogically interesting NMR experiment for observing another nucleus besides 1H or 13C [9].

Summary

The Mosher technique provides an alternative means of determining the enantiomeric ratio of chiral alcohols and amines, which can conveniently be used as a stand-alone organic experiment or attached to another experiment involving an asymmetric synthesis or resolution. If the configurations of the enantiomers have been reported, students can also determine the configuration of the major enantiomer. This technique is pedagogically beneficial as it introduces microscale techniques and integrates 1H and 19F NMR spectroscopy with advanced stereochemical topics that are not always sufficiently applied in organic chemistry courses.

Experimental Section

General Remarks. All chemicals were purchased from the Aldrich-Sigma Company. Chloroform was dried over calcium hydride, or its anhydrous form was purchased and used directly. Pyridine was dried over KOH and distilled over BaO, or its anhydrous form was purchased and used directly [10]. All other chemicals were purchased and used without further purification. The sample of α-methoxy-α-(trifluoromethyl)phenylacetyl chloride purchased from the Aldrich-Sigma Company is labeled as having 98% e.e. as determined by GLC [11]. 1H NMR spectra were taken on a Bruker Avance DPX 300-MHz (16 scans, data size = 32K) or Anasazi-upgraded Varian 360A (60 MHz, 4 scans, data size = 16K) FT-NMR spectrometer in deuterochloroform (CDCl3) with tetramethylsilane (TMS) as the internal standard. 19F NMR spectra (56.5 MHz, 4 scans, data size = 16K) were taken on an Anasazi-upgraded Varian 360A FT-NMR spectrometer in deuterochloroform (CDCl3) with trichlorofluoromethane (CCl3F) as the internal standard set to 0 ppm.

Safety and Hazards. All experiments should be performed in fumehoods with suitable eye and skin protection and with proper expert supervision. Any eye and skin contact, inhalation, or ingestion should be avoided with all reagents. Chloroform is a cancer suspect agent. Calcium hydride is a flammable solid and moisture-sensitive. Pyridine is flammable and an irritant. Potassium hydroxide is corrosive. Barium oxide is corrosive and moisture-sensitive. Diethyl ether is flammable and toxic and can form explosive peroxides. sec-Butanol (4) is a flammable liquid and irritant. α-Trifluoromethylbenzyl alcohol (5) is an irritant. α-Methylbenzylamine (7) is corrosive and toxic. α-Methoxy-α-(trifluoromethyl)phenylacetyl chloride (1) is corrosive and moisture-sensitive.

General Procedure for MTPA Derivatives. Into a cleaned and dried 3-mL conical vial were added in the following order: anhydrous pyridine (300 µL), enantiopure Mosher acyl chloride (35.4 mg, 26.2 µL, 0.140 mmol) [12], anhydrous chloroform [13] (300 µL), and the substrate (0.100 mmol). This was capped with a silicone-rubber seal, stirred gently, and then left at ambient temperature in the students� drawers until the following laboratory period, usually one week. The reaction solution was quenched with 1 mL H2O and taken up in 20 mL diethyl ether. The layers were separated, and the organic layer was washed with 5 mL each of 1 M HCl (aq), saturated Na2CO3 (aq), and saturated NaCl (aq). The organic layer was then dried (Na2SO4),

filtered, and concentrated�making sure that all traces of diethyl ether was removed [14]. Typical yields are approximately 70 to 90% with 20 to 50 mg of product. The entire residue was dissolved in 0.5 mL CDCl3 with TMS and filtered directly into an NMR tube. For 19F NMR analysis, a drop of CCl3F was added as the internal standard. Filtration was performed with a disposable glass pipette and a Kimwipe plug.

NMR Data. 3,3,3-Trifluoro-2-methoxy-2-phenylpropionic acid sec-butyl ester. [(R,R)-8]: 1H NMR (CDCl3, 300 MHz, δ) 7.544�7.261 (m, 5H), 5.132�5.056 (m, 1H), 3.564 (s, 1H), 1.551�1.737 (m, 2H), 1.255 (d, J = 6.2 Hz, 3H), 0.939 (t, J = 7.4 Hz, 3H). 19F NMR (CDCl3, 56.5 MHz, δ) �72.0 (m, 3F). [(R,S)-8]: 1H NMR (CDCl3, 300 MHz, δ) 7.544�7.261 (m, 5H), 5.132�5.056 (m, 1H), 3.566 (s, 1H), 1.737�1.551 (m, 2H), 1.335 (d, J = 6.2 Hz, 3H), 0.828 (t, J = 7.4 Hz, 3H). 19F NMR (CDCl3, 56.5 MHz, δ) �72.0 (m, 3F).

3,3,3-Trifluoro-2-methoxy-2-phenylpropionic acid 2,2,2-trifluoro-1-phenylethyl ester. [(R,R)-9]: 1H NMR (CDCl3, 300 MHz, δ) 7.527�7.250 (m, 10H), 6.263 (qt, J = 6.6 Hz, 1H), 3.601 (qt, J = 1.2 Hz, 3H). 19F NMR (CDCl3, 56.5 MHz, δ) �72.3 (m, 3F), �76.17 (d, J = 6.6 Hz, 3F). [(R,S)-9] 1H NMR (CDCl3, 300 MHz, δ) 7.527�7.250 (m, 10H), 6.342 (qt, J = 6.7 Hz, 1H), 3.479 (qt, J = 1.2 Hz, 3H). 19F NMR (CDCl3, 56.5 MHz, δ) �72.3 (m, 3F), �76.40 (d, J = 6.6 Hz, 3F).

3,3,3-Trifluoro-2-methoxy-2-phenylpropionic acid methoxycar-bonylphenylmethyl ester. [(R,R)-10]: 1H NMR (CDCl3, 300 MHz, δ) 7.435�7.370 (m, 10H), 6.094 (s, 1H), 3.769 (s, 3H), 3.697 (qt, J = 1.2 Hz, 3H). 19F NMR (CDCl3, 56.5 MHz, δ) �72.2 (m, 3F). [(R,S)-10]: 1H NMR (CDCl3, 300 MHz, δ) 7.435-7.370 (m, 10H), 6.115 (s, 1H), 3.740 (s, 3H), 3.552 (qt, J = 1.1 Hz, 3H). 19F NMR (CDCl3, 56.5 MHz, δ) �72.2 (m, 3F).

3,3,3-Trifluoro-2-methoxy-2-phenyl-N-(1-phenylethyl)-propanamide. [(R,R)-11]: 1H NMR (CDCl3, 300 MHz, δ) 7.533�7.254 (m, 10H), 7.228�7.014 (m, 1H), 5.201�5.151 (m, 1H), 3.361 (qt, J = 1.5 Hz, 3H), 1.499 (d, J = 6.9 Hz, 3H). 19F NMR (CDCl3, 56.5 MHz, δ) �69.25 (m, 3F). [(R,S)-11]: 1H NMR (CDCl3, 300 MHz, δ) 7.533�7.254 (m, 10H), 7.228�7.014 (m, 1H), 5.201�5.151 (m, 1H), 3.403 (qt, J = 1.5 Hz, 3H), 1.539 (d, J = 6.9 Hz, 3H). 19F NMR (CDCl3, 56.5 MHz, δ) �69.44 (m, 3F).

Acknowledgments. The author would like to acknowledge the National Science Foundation (DUE 0310353), the Max Goldenberg Foundation, and Roosevelt University for funding the Anasazi EFT system. He would also like to thank Professor Jeffrey Bjorklund and North Central College for the use of their Bruker Avance DPX 300-MHz NMR spectrometer.

Supporting Materials. A student handout, assignment, and experimental notes for the instructor are available in a Zip file (http://dx.doi.org/10.1333/s00897040843a).

References and Notes

1. For reviews of methods of determining enantiomeric ratios, see (a) Eliel, E. L.; Wilen, S. H. Stereochemistry of Organic Compounds; Wiley & Sons: New York, 1994; pp 214�274; (b) Rothchild, R. Chem. Educator [Online], 1996, S1430-4171(96)03036-11(3); DOI 10.1333/s00897960036a; (c) Rothchild, R. Enantiomer 2000, 5 (5), 457�471; (d) Shah, R. D.; Nafie, L. A. Curr. Opin. Drug Discov. Devel. 2001, 4, 764�775; (e) Finn, M. G. Chirality 2002, 14, 534�540; (f) Wenzel, T. J.; Wilcox, J. D. Chirality 2003, 15, 256�270.

2. Dale, J. A.; Dull, D. L.; Mosher, H. S. J. Org. Chem. 1969, 34, 2543�2549.

3. Dale, J. A.; Mosher, H. S. J. Am. Chem. Soc. 1973, 95, 512�519. 4. (a) Ault, A. J. Chem. Educ. 1965, 42, 269; (b) Krause, J. G. J. Chem.

Educ. 1994, 71, 596.


http://dx.doi.org/10.1333/s00897040843a

http://dx.doi.org/10.1333/s00897960036a

http://dx.doi.org/10.1333/s00897960036a

A Microscale NMR Experiment for Organic Chemistry Chem. Educator, Vol. 9, No. 6, 2004 363

5. This first part of the experiment where students simply combine the reagents is brief; so, the instructor may wish to arrange the laboratories so that students are finishing another experiment during the beginning of this laboratory period or use the time to introduce the Mosher method.

6. At Roosevelt University, the students analyze their samples by NMR on an Anasazi EFT upgraded 60-MHz spectrometer. Because they have used the instrument in previous courses and/or laboratories, they are able to obtain a spectrum quickly. The students use the spectrometer only to obtain a spectrum and then save and transfer the spectra files via disk or email for processing (i.e., to zoom, integrate, print, etc) later on our science or university computers using the NUTS software. This provides a rapid rotation of student use on the spectrometer while permitting students to access and analyze their spectra at a later time at their convenience. The author has also on occasion taken the class on a field trip to a nearby institution (North Central College) to use their 300-MHz FT-NMR.

7. Krumpolc, Miroslav. J. Chem. Educ. 1991, 68, A176�A178. 8. Viswanathan, Tito; Toland, Alan. J. Chem. Educ. 1995, 72, 945�946. 9. Another possible application involves the enzymatic resolution of α-

methylbenzylalcohol, which was recently reported as an organic chemistry experiment (Stetca, D.; Arends, I. W. C. E.; Hanefeld, U., J. Chem. Educ. 2002, 79, 1351�1352.). Hanefield and coworkers reported the determination of the enantiomeric excess by optical rotation; however, the enantiomeric excess could be determined with

the Mosher method. Mosher reports the use of this substrate with success [2].

10. The chloroform and pyridine were dried by the instructor after which the drying apparatus was shown and the drying technique was explained to the students. Other instructors may wish to have laboratory personnel dry these reagents or simply purchase anhydrous samples.

11. Two different samples of the α-methoxy-α-(trifluoromethyl)phenylacetyl chloride are commercially available from the Aldrich-Sigma Company with 98% e.e. or 99+% e.e., as determined by GLC.

12. Mosher [3] provides this additional note, which requires attention: �The formation of a precipitate at this point indicates that the pyridine was wet. A slight opalescence can be tolerated but a definite precipitate requires that the reaction can be abandoned and repeated with dry reagents.�

13. Mosher reports using CCl4 as the solvent, but due to its high costs, CHCl3 has been used as the solvent with acceptable results.

14. Even with thorough concentration on a high vacuum, diethyl ether consistently appeared in the 1H NMR spectrum; thus, after initial concentration, the residue was dissolved in some CHCl3 and concentrated again to ensure that all of the diethyl ether was removed. The NMR signals for diethyl ether appear adjacent to the signals for the products.


Mosher ester analysis for the determination ofabsolute configuration of stereogenic (chiral) carbinolcarbonsThomas R Hoye, Christopher S Jeffrey & Feng Shao

Department of Chemistry, University of Minnesota, 207 Pleasant Street, SE, Minneapolis, Minnesota 55455, USA. Correspondence should be addressed to T.R.H.([email protected]).

Published online 4 October 2007; doi:10.1038/nprot.2007.354

This protocol details the most commonly used nuclear magnetic resonance (NMR)-based method for deducing the configuration of

otherwise unknown stereogenic, secondary carbinol (alcohol) centers (R1R2CHOH (or the analogous amines where OH is replaced by

NH2)). This ‘Mosher ester analysis’ relies on the fact that the protons in diastereomeric a-methoxy-a-trifluoromethylphenylacetic acid

(MTPA) esters (i.e., those derived from conjugation of the carbinol under interrogation with MTPA) display different arrays of

chemical shifts (ds) in their 1H NMR spectra. The protocol consists of the following: (i) preparation of each of the diastereomeric

S- and R-MTPA esters and (ii) comparative (DdSR) analysis of the 1H NMR spectral data of these two esters. By analyzing the sign of

the difference in chemical shifts for a number of analogous pairs of protons (the set of DdSR values) in the diastereomeric esters

(or amides), the absolute configuration of the original carbinol (or amino) stereocenter can be reliably deduced. A typical Mosher

ester analysis requires approximately 4–6 h of active effort over a 1- to 2-d period.

INTRODUCTIONThe absolute configuration of a non-racemic sample of a chiralmolecule is a fundamental and essential property of that com-pound. This is especially so when the compound is of biologicalrelevance (e.g., a natural product or a synthetic compound underdevelopment as a possible therapeutic agent). For evidence, oneneeds to look no further than the infamous history of thedifferences in biological response to each of the two enantiomers(mirror image antipodes) of thalidomide1. Assignment of absoluteconfiguration to compounds where that important structural detailis not yet known is accomplished by a number of different methods.These include (i) correlation with compounds of known config-uration by synthetic interconversions and comparison of opticalrotation by polarimetric methods, (ii) approaches based on X-raycrystallographic, optical rotary dispersion, circular dichroism orexciton chirality methods and (iii) various empirical methodsbased on NMR spectroscopy2. Among the NMR-based methods,what is now commonly referred to as Mosher ester (or amide)analysis is the most frequently used.

In 1973, Dale and Mosher reported3 an NMR-based method,which has come to be known as Mosher ester analysis, for deducingthe absolute configuration of the stereogenic carbon center insecondary alcohols (i.e., R1R2CHOH, 1, wherein R1 a R2). Theyused a chiral, enantiomerically pure carboxylic acid to derivatize 1,the latter having an unknown configuration. Although variousacids have been studied in this role2, the discussion here will befocused on the most widely used a-methoxy-a-trifluoromethyl-phenylacetic acid (MTPA-OH, 2), commonly known as Mosher’sacid. By direct analogy, this method can also be applied to theanalysis of a-chiral amines (e.g., R1R2CHNH2 or R1R2CHNHR3;Mosher amide analysis3,4), but this analysis is required less com-monly, so the rest of the discussion in this protocol is confined toester derivatives of alcohol (carbinol) substrates.

The first step in the protocol for Mosher ester analysis for thedetermination of absolute configuration is shown in Figure 1. It

involves coupling of the hydroxyl group of the alcohol with, in twoseparate but analogous experiments, each enantiomer of Mosher’sacid (i.e., 2R and 2S). This acylation reaction is commonlyperformed using a carboxylic acid-activating agent such as N,N¢-dicyclohexylcarbodiimide (DCC). Alternatively, the acid chlorides3S and 3R (MTPA-Cl), derived from 2R and 2S, respectively, can beused directly as active acylating agents5,6. This results in theformation of each of the two diastereomeric Mosher esters 4Rand 4S, respectively. As diastereomers have different physical andspectroscopic properties, Dale and Mosher recognized that their 1H(proton) NMR spectra would differ. They proceeded to show thatby comparison of the different chemical shifts of resonancesobviously corresponding to protons residing within the substitu-ents R1 and R2 in 4 (or the 19F atoms of the CF3 group in the MTPAmoiety7), they could deduce the absolute configuration of thecarbinol center in the original alcohol 1.

In the late 1980s, the Kakisawa group8–10 at Tsukuba described amajor development in the method, which has come to be known asthe modified (or advanced) Mosher ester analysis. Technologicaldevelopments had made access to more powerful (higher field),superconducting magnetic resonance spectrometers more com-monplace. Among other things, this meant that a greater portionof the individual protons could be routinely assigned in ‘small-molecule’ compounds (e.g., those having o2,000 amu) havingmoderate-to-high structural complexity. Today, it is not uncommon

p

uor

G g

n ih si l

bu

P eru ta

N 700 2©

nat

ure

pro

toco

ls/

moc.er

ut an.

ww

w//:ptt

h

H O

OOH

R-MTPA ester

R1

HO2C CF3

CF3CIOC

CF3

Ph

4R

2R

3S 3R

2S

1

OMe

Ph OMe

HO2C CF3

MeO Ph

Ph OMe

CF3CIOC

MeO Ph

R2

H O

O

S-MTPA ester

R1CF3

MeO

4S

Ph

R2H

R1R2

Figure 1 | Scheme for the synthesis of R- and S-Mosher esters 4 from the

generic carbinol 1.

NATURE PROTOCOLS | VOL.2 NO.10 | 2007 | 2451

PROTOCOL

to be able to confidently assign nearly allproton resonances in such molecules. Thethrust of the Kakisawa contribution was thatmany analogous pairs of protons residingwithin the R1 and R2 moieties in 4R versus4S (i.e., many data points for a single carbi-nol analyte) could be analyzed, resulting in amuch higher level of confidence in thededuced absolute configurations for amuch broader array of substrate carbinols1. The self-reinforcing nature of this analysisis an integral feature of this modified Moshermethod.

In addition to absolute configuration,Mosher ester derivatives can be used togain other types of stereochemical informa-tion. Mosher showed that one can deducethe ratio of two enantiomers in a given sample (i.e., enantiomericratio (er ¼ [major]:[minor]), sometimes expressed as the enantio-meric excess (%ee ¼ %major � %minor)) by measuring therelative intensities of analogous resonances (1H and/or 19F) ineach of the diastereomers 4R and 4S5. (Complementary measure-ment of this ratio is sometimes performed by gas or liquidchromatographic analysis.) While valuable11, this variant is notfurther discussed in this protocol, but it is mentioned here sinceone encounters language like ‘Mosher ester analysis was used todetermine the enantiomeric purity of this mixture’ in the literature.

The success of the Mosher ester method for deducing the absoluteconfiguration of a secondary alcohol relies upon the empiricallybased (and validated) conformational picture that is shown inFigure 2. Briefly, the important conformation for each diastereomeris taken to be the one in which the ester adopts the usual s-transarrangement about its O–CO bond (analogous to the major con-formation for acyclic, secondary amide C–N bonds), and both thetrifluoromethyl (CF3) substituent of the MTPA moiety and themethine proton of the secondary alcohol moiety are syn-coplanar(01 dihedral angle) with the carbonyl group. Thus, all of the under-lined atoms in the R1R2C(H)–O–C(¼O)–C–CF3 substructure ofeach Mosher ester are coplanar. Although this is not the onlyconformation populated in the ensemble of (rapidly interconverting)rotamers available to 4, it is the one that dominates the spectroscopicfeatures that distinguish the diastereomeric MTPA esters. Or, asMosher and coworkers stated so clearly, these conformations ‘‘areintended to represent a model which successfully correlates theknown results. These are not intended to represent the preferredground state conformation of the molecules under consideration.They may in fact measure an effective average of many conforma-tions or may represent a minor conformation which, however, exertsa proportionately large differential shielding of the [R1] and [R2]groups. Admittedly the success of the correlation tends to reinforcethe belief that these do indeed represent major conformations of themolecules in question, but it must still be borne in mind that thepossibility exists that this is a fortuitous array which happens to serveas an empirical correlation of the results’’7.

An aryl group (e.g., the phenyl substituent in each of the MTPAesters 4) is known to impose an anisotropic, magnetic shielding effecton protons residing above (or below) the plane of the aryl ring. Thisshielding results in a more upfield chemical shift for the affected(spatially proximal) proton(s) in the NMR spectrum. Inspection of the

dominant conformer for 4S versus that for 4R reveals a key distin-guishing feature. Protons residing within the R2 substructure of 4S arerelatively more shielded (and upfield in its spectrum) and, conversely,protons within the R1 substructure of 4R are relatively more shielded;this is the crux of the Mosher method3,7. Moreover, the ‘reach’ of thisanisotropic shielding effect extends a considerable distance within themolecule so that a fairly large number of (even remote) protons withineach R group are differentially shielded in the two diastereomers; this isthe crux of the modified (or advanced) Mosher method10. A con-sequence of all of the above is that the signs of the DdSR (defined, byconvention, to be dS� dR8,10) values for protons residing in R1 will bepositive and those in R2 will be negative. Thus, by knowing whichprotons have a positive versus a negative DdSR value, one can deducethe (sub)structures of R1 versus R2, which translates directly to theabsolute configuration of the secondary carbinol center in 1—theoriginal objective of the exercise.

Mosher ester (and amide) analysis, especially the modified version,is remarkably powerful and general. The most complementary alter-native, predicated on the same concepts, is the use of O-methylman-delate esters (or methoxyphenyl acetates), which was introduced byTrost et al.12. Although there are instances where the Mosher methodmust be applied with caution (e.g., for substrates with more than onecarbinol and/or amine functional groups), discussion of such subtle-ties is beyond the scope of this protocol. Interested readers areencouraged to consult the comprehensive review by the Rigueragroup2. Finally, note that some of the developers of more recentNMR-based methods similar to the Mosher analysis have elected touse the convention DdRS ¼ dR � dS. This is opposite to the DdSR

convention used universally in Mosher analyses. One will get to thecorrect answer either way, but it is essential that users unambiguouslystate which way the data are being interpreted and reported.

The protocol detailed below consists of two main components:(i) individually prepare (synthesize) each of the two diastereomericMTPA esters and then (ii) comparatively analyze the 1H NMRspectral data of each of the two diastereomeric MTPA esters. Wehave chosen (�)-menthol (5; Fig. 3) as a representative example ofthe universe of secondary carbinols 1. Of course, the absoluteconfiguration of (�)-menthol is known, but the synthesis andanalysis methodologies are unchanged by that fact. Given thesensitivity of 1H NMR spectroscopy, it is virtually never necessaryto carry out the preparation of the MTPA esters for a Mosheranalysis on a very large scale, even when the supply of carbinol 1 is

p

uor

G g

n ih si l

bu

P eru ta

N 700 2©

nat

ure

pro

toco

ls/

moc.er

ut an.

ww

w//:ptt

h

CF3

CF3

O

OH

OMeR1

R1

Ph

R2HO

MeOR1

R2CF3

O S

Diastereomer 4S

O CF3

PhMeO

R2R1

S

S

CF3O

OH

PhR1

R1

MeOHO

MeO R2

R2

R2

CF3

O

R

Diastereomer 4R

O

OMePh

R

R

Figure 2 | Commonly encountered representations of the conformations (rotational isomers) used for the

analysis of each of the diastereomeric MTPA esters 4S and 4R. The phenyl group shielding effect is

indicated by the gray arrows in each representation.

2452 | VOL.2 NO.10 | 2007 | NATURE PROTOCOLS

PROTOCOL

large. The more common situation is that 1 isprecious and in short supply (e.g., in the caseof initial structural studies of a newly isolatednatural product); if necessary, the method canbe carried out routinely on scales even below100 mg (o1 mmol). Accordingly, we haveprovided (in Step 1) three complementary,representative procedures for synthesis ofMTPA-menthyl esters 6 (Fig. 3) on differentscales and using different acylating agents.Namely, in option A we describe the preparation of 6S from R-(�)-MTPA-Cl (3R) on a 10 mg scale; in option B, 6S from R-(�)-MTPA-Cl (3R) on a 50 mg scale and in option C, 6R from R-(+)-MTPA-OH(2R) on a 10 mg scale. The interpretation of the data (described inSteps 2–7) is performed in an identical manner, regardless of how thepair of diastereomeric esters was prepared.

Throughout the protocol, it is essential that the individualsamples, spectra and data for each of the two diastereomers never

be confused/interchanged. For this reason, we recommend that thesequence of synthesis and NMR data collection and tabulation beperformed for each of the diastereomeric MTPA esters seriallyrather than in parallel. Although this may seem like an obviouspoint (as an interchange of the two samples will lead to theassignment of precisely the wrong absolute configuration!), experi-ence teaches that it is an easy issue over which uncertainty can arisein the mind of the experimenter.

MATERIALSREAGENTS.(R)-(+)-a-Methoxy-a-trifluoromethylphenylacetic acid (2R, R-(+)-MTPA-

OH; e.g., Acros).(S)-(�)-a-Methoxy-a-trifluoromethylphenylacetic acid (2S, S-(�)-MTPA-

OH; e.g., Aldrich).(S)-(+)-a-Methoxy-a-trifluoromethylphenylacetyl chloride (3S, S-(+)-

MTPA-Cl; prepared from R-(+)-MTPA-OH5,6,13, but this reagent is alsocommercially available; e.g., Fluka)

.(R)-(�)-a-Methoxy-a-trifluoromethylphenylacetyl chloride (3R, R-(�)-MTPA-Cl; prepared from S-(�)-MTPA-OH5,6,13, but this reagent is alsocommercially available; e.g., Lancaster)

.(�)-Menthol (5, (1R,2S,5R)-(�)-2-isopropyl-5-methylcyclohexanol; e.g., Aldrich)

.N,N¢-DCC (e.g., Acros)

.Pyridine (e.g., Aldrich)

.4-Dimethylaminopyridine (e.g., Aldrich)

.Chloroform-d (e.g., Cambridge Isotope Lab)

.Hexanes, ethyl acetate, diethyl ether, dichloromethane (CH2Cl2; standardreagent-grade laboratory solvents)

.Silica gel, bulk (typically 40- to 63-mm-diameter particle size, with nominal60 A pore size)

.Silica gel thin-layer chromatography (TLC) plates (SIL G/UV254, 0.25 mmlayer thickness; Machery Nagel, M805024)

.Ceric ammonium molybdate, phosphomolybdic acid, anisaldehydeor potassium permanganate (e.g., TCI America, Fisher Scientific orAldrich)EQUIPMENT.Screw-capped glass vial or other similar reaction vessel with a

relatively small headspace with respect to the volume of reactionsolvent

.Glass microliter syringes (Hamilton) or Wiretrols (Drummond) formeasuring small volumes of liquid

.Common glassware (round-bottomed flask, Erlenmeyer flask, disposablepipets, etc.)

.Magnetic stirrer and stir bar small enough to fit the reaction vial

.Rotary evaporator (Buchi)

.Balance (Mettler)

.Handheld UV lamp (UVP)

.Small chromatography column

.NMR sample tube (5 mm diameter; New Era)

.1H NMR spectrometer (Z200 MHz; Varian, Bruker)

PROCEDUREIndividually prepare (synthesize) and collect the 1H NMR spectrum for each of the two diastereomeric MTPA esters.1| Examples of three methods are provided as follows: (A) preparation of S-MTPA-menthyl ester 6S from (�)-menthol (5)and the R-acid chloride (3R, R-(�)-MTPA-Cl) on a 10 mg scale; (B) preparation of S-MTPA-menthyl ester 6S from (�)-menthol(5) and the R-acid chloride (3R, R-(�)-MTPA-Cl) on a 50 mg scale; and (C) preparation of R-MTPA-menthyl ester 6R from(�)-menthol (5), the R-Mosher acid (2R, R-(+)-MTPA-OH) and DCC on a 10 mg scale.(A) S-MTPA-menthyl ester 6S from (–)-menthol (5) and the R-acid chloride (3R, R-(–)-MTPA-Cl) on a 10 mg scale

(i) Fit a screw-capped 4 ml glass vial with a Teflon-coated magnetic stir bar.(ii) Transfer (�)-menthol (5, 10.0 mg, 64 mmol) and dry pyridine (16 ml, 0.20 mmol, 3.1 equiv.) to the vial.(iii) Dissolve the contents of the vial in anhydrous CH2Cl2 or chloroform (CHCl3) (1 ml, [5] ¼0.064 M). If a source of one of

these anhydrous solvents is not readily available, either can be dried using the same procedure described in the followingcritical step.m CRITICAL STEP If CHCl3 is selected as the solvent, it should first be passed through a column of fresh silica gel oralumina immediately before use to remove the ethanol (B0.75% (vol/vol)) that is present as a stabilizer in most commercialsources. For example, insert a small cotton plug into the neck of a disposable pipet and load the pipet with fresh silicagel (or alumina) to a height of approximately 5 cm. Pass several milliliters of CHCl3 through this column using pressureapplied from a pipet bulb.

p

uor

G g

n ih si l

bu

P eru ta

N 700 2©

nat

ure

pro

toco

ls/

moc.er

ut an.

ww

w//:ptt

h

O

O

R-MTPA ester

( or 3S )CF3

Ph

OH

6R 5

2R

OMe

Me Me

Me Me

O

O

S-MTPA ester

CF3

MeO

6S

Ph

Me

Me MeMe Me

( or 3R )

2S

Figure 3 | Scheme for the synthesis of R- and S-MTPA menthyl esters 6 from (�)-menthol (5).


PROTOCOL

(iv) Add the R-(�)-MTPA-Cl (3R, 23 ml, 0.12 mmol, 1.9 equiv.) to the mixture. This transfer can be performed using amicroliter syringe or Wiretrol.

(v) Cap the vial and stir at ambient temperature.(vi) Monitor the progress of the reaction by TLC analysis, by eluting with a solvent composed of hexanes and ethyl acetate in

a 4:1 ratio, and visualizing the plate by UV monitoring and/or staining with an appropriate TLC stain (e.g., ceric ammo-nium molybdate, phosphomolybdic acid, anisaldehyde or potassium permanganate). Because they are less polar, the MTPAesters will elute on silica gel more rapidly than the precursor carbinol (i.e., with a higher retention factor (Rf) on the TLCplate). For the case of menthol (5) to 6R, the former has an Rf of 0.46 and the latter 0.82. The choice of TLC elutionsolvent will vary depending on the polarity of the starting substrate under analysis.

(vii) After the reaction is complete (typically less than 2 h), partition the reaction mixture between diethyl ether (3 ml) andwater (1 ml).

(viii) Mix the layers thoroughly.(ix) Separate the layers and place the ether layer into a small Erlenmeyer flask.(x) Add 3 ml of ether to the aqueous layer that remains in the reaction vial and repeat Steps (viii) and (ix), adding this

ether layer to the original one in the Erlenmeyer flask.(xi) Repeat Step (x).(xii) Dry the combined ether extracts with a suitable solid drying agent (anhydrous solid Na2SO4 or MgSO4) in a 25- to 50-ml

Erlenmeyer flask. Typically, approximately 1 g of drying agent is used for the approximately 10 ml of combined organicextracts. Swirl the suspension occasionally over a period of approximately 5 (for MgSO4) or 30 (for Na2SO4) min. Ininstances where the substrate might contain acid-sensitive functionality, the use of Na2SO4 is recommended.

(xiii) Filter the ether, containing the Mosher ester, from this suspension into a round-bottomed flask (B25 ml).(xiv) Evaporate the volatiles using a rotary evaporator under reduced pressure using a 20–25 1C water bath.(xv) Purify the product by chromatography on silica gel (any of flash14, medium pressure liquid chromatography (MPLC), HPLC,

gravity column or prep-TLC plate methods can be used), eluting with hexanes/ethyl acetate (40:1) and monitoring thefractions using TLC (4:1, hexanes/ethyl acetate) (or another commonly available detection method like UV absorbance ordifferential refractive index).

(xvi) Combine the fractions containing the purified ester product 6S and remove the solvents under reduced pressure using a20–35 1C water bath.

(xvii) If necessary or desired, remove the remaining traces of ethyl acetate and hexane elution solvents from the purified productby attaching the sample flask to a high-vacuum source (e.g., 0.1–1 torr) at ambient temperature for approximately 1 h.

(xviii) Record the 1H NMR spectrum of 6S in deuterochloroform (CDCl3).(xix) Prepare the R-MTPA-menthyl ester 6R by repeating Steps (i–xvii) using S-(+)-MTPA-Cl in place of R-(�)-MTPA-Cl.(xx) Record the 1H NMR spectrum of 6R in CDCl3.

(B) S-MTPA-menthyl ester 6S from (–)-menthol (5) and the R-acid chloride (3R, R-(–)-MTPA-Cl) on a 50 lg scale(i) Fit a screw-capped 2 ml glass vial with a Teflon-coated magnetic stir bar.(ii) Transfer (�)-menthol (5, 50 mg, 0.32 mmol) and dry pyridine (1 ml, 12.5 mmol, 39 equiv.) to the vial.(iii) Dissolve the contents of the vial in anhydrous CDCl3 (100 ml, [5] ¼ 3.2 mM). Use of CDCl3 as the solvent allows one to

assay the reaction mixture directly by 1H NMR spectroscopy.(iv) Add the R-(�)-MTPA-Cl (3R, 1 ml, 5.2 mmol, 16 equiv.) to the vial, cap the vial and stir the contents at room temperature.(v) Perform Step 1A(vi).(vi) After the reaction is complete (typically less than 1 h), dilute the reaction mixture with dry CDCl3 (0.6 ml).(vii) Transfer the entire CDCl3 solution to a standard (5 mm diameter) NMR sample tube.(viii) Record a 1H NMR spectrum.(ix) Prepare the R-MTPA-menthyl ester 6R by repeating Steps 1B(i–vii), using S-(+)-MTPA-Cl in place of R-(�)-MTPA-Cl.(x) Record the 1H NMR spectrum of 6R in CDCl3.

(C) R-MTPA-menthyl ester 6R from (–)-menthol (5), the R-Mosher acid (2R, R-(+)-MTPA-OH) and DCC on a 10 mg scale(i) Fit a screw-capped 4 ml glass vial with a Teflon-coated magnetic stir bar.(ii) Transfer (�)-menthol (5, 10.0 mg, 64 mmol) and R-(+)-MTPA-OH acid (2R, 32 ml, 0.2 mmol, 3.1 equiv.) to the vial.(iii) Dissolve the contents of the vial in anhydrous CH2Cl2 or CHCl3 (1 ml, [5] ¼ 0.064 M). If a source of one of these anhydrous

solvents is not readily available, either can be dried using the same procedure described in the critical step above,following Step 1A(iii).

(iv) Add DCC (42 mg, 0.2 mmol, 3.1 equiv.) to the vial.! CAUTION DCC is an irritant and can lead to sensitization. Avoid any direct contact with the skin or inhalation of particulates.

(v) Add 4-dimethylaminopyridine (25 mg, 0.20 mmol, 3.1 equiv.), tightly cap the vial and stir the contents at roomtemperature.

(vi) Perform Step 1A(vi).

p

uor

G g

n ih si l

bu

P eru ta

N 700 2©

nat

ure

pro

toco

ls/

moc.er

ut an.

ww

w//:ptt

h


PROTOCOL

(vii) After the reaction is complete (typically 12–24 h), filterthe white precipitate through a cotton plug. This solid islargely the unwanted by-product, N,N¢-dicyclohexylurea.

(viii) Purify the product as described in Steps 1A(xiv–xvii).(ix) Record the 1H NMR spectrum of 6R in CDCl3.(x) Prepare the S-MTPA-menthyl ester 6S by repeating

Steps (i–viii), using S-(�)-MTPA-OH (2S) in place ofR-(+)-MTPA-OH (2R).

(xi) Record the 1H NMR spectrum of 6S in CDCl3.

Comparatively analyze the 1H NMR spectral data of each ofthe two diastereomeric MTPA esters2| Unambiguously assign as many proton resonances aspossible in the 1H NMR spectrum for each of thediastereomeric esters 4S and 4R.

3| Determine the difference in chemical shifts (DdSR) foreach of the assignable analogous pairs of protons for theS- and R-MTPA esters according to the following convention:DdSR ¼ d(S-MTPA ester)�d(R-MTPA ester) (or DdSR ¼ dS �dR). Gather all of the resonances of positive and, then,negative DdSR values together. These DdSR data for the menthyl MTPA esters 6S versus 6R are presented in Table 1, listed in theorder from most positive to most negative.

4| Using the conformations shown in Figure 2 for the MTPA esters of generic secondary alcohols, decide which protons of theesters under study are part of the R1 substituent and which part of R2. More specifically, those protons that have positive DdSR

values reside within R1, whereas those with negative values ‘belong to’ R2. The structures shown in Figure 4 for the menthylesters 6S and 6R clearly show that the protons with positive DdSR values (i.e., those in R1) are all on one side (front) of theplane of the MTPA moiety, whereas those with negative values are all on the opposite (back) side of that plane.! CAUTION Occasionally, one of the proton resonances proves to be an exception to this trend. If so, it is usually for a protonwith a DdSR value that is relatively small in magnitude because this exceptional proton resides either near the plane of theMTPA ester moiety or quite remotely. For this reason, the secondary carbinol proton itself (i.e., H1 in 6) is typically ignored inthe analysis; an important corollary is that protons having the largest DdSR values are, qualitatively, weighted very heavily inthe analysis.

5| Use the Cahn Ingold Prelog convention15 to assign the configuration of the original carbinol center as R or S. C1 of(�)-menthol has the R configuration, as the substituent priority sequence is 1, OH; 2, C2; 3, C6; and 4, H.

6| Report the data. A typical ‘chemist-friendly’ format for reporting the synthesis and spectral properties of a Mosher ester ispresented in Box 1. Specifically, it describes the preparation of both the S- and R-MTPA esters of (�)-menthol (6S and 6R,respectively).

7| Finally, one particularly insidious aspect of the Mosher method relates to the Cahn Ingold Prelog convention nomenclaturementioned above and represents a potential pitfall. This problem is presented as a caveat in Box 2.? TROUBLESHOOTING

� TIMINGSteps 1A(i–iv): 15–20 minSteps 1A(v) and (vi): 1–3 hSteps 1A(vii–xiv): 20–40 minSteps 1A(xv) and (xvi): 20–60 minStep 1A(xvii): 5–30 minStep 1A(xviii): B3.5 hStep 1A(xix): 5–30 minSteps 1B(i–iv): 15–20 minStep 1B(v): 1–3 hSteps 1B(vi) and (vii): 2–5 minStep 1B(viii): 5–40 min

p

uor

G g

n ih si l

bu

P eru ta

N 700 2©

nat

ure

pro

toco

ls/

moc.er

ut an.

ww

w//:ptt

h

TABLE 1 | Dd (¼dS � dR) data for the S- and R-MTPA-menthyl Mosheresters 6S and 6R.

d S-ester(6S) (ppm)

d R-ester(6R) (ppm)

DdSR (¼dS � dR)

ppm Hz (500 MHz)

6ax 1.12 0.98 +0.14 +706eq 2.13 2.08 +0.05 +255Me 0.94 0.91 +0.03 +155ax 1.54 1.52 +0.02 +101ax 4.90 4.88 +0.02 +104ax 0.87 0.86 +0.01 +54eq 1.70 1.70 0.00 03ax 1.04 1.06 �0.02 �103eq 1.67 1.69 �0.02 �102ax 1.42 1.45 �0.03 �152¢Mea 0.67 0.77 �0.10 �502¢Meb 0.74 0.87 �0.13 �652¢ 1.56 1.88 �0.32 �160

For the menthyl MTPA esters 6S and 6R, all proton resonances are uniquely identifiable. However, thisneed not be the case, and, for many (most?) complex structures, not every proton in the compound isuniquely identifiable in its 1H NMR spectrum. In such cases, it is prudent to use only the subset ofanalogous proton pairs for which unambiguous assignments are known.

HH

H

H

H

H

H

H

H

H

H

O

OCF3 CF3

6S 6R

OMe

Me

Me

Me–10

–10

–160

–15

+10

+25

+70

+15

4

32 1

65

–50/–65

Me

OMTPA

∆�SR = �S – �R

Me

Me

HH

H

H

O

O

Me

Me

Me MeO

Figure 4 | Conformations used for the analysis of the menthyl esters 6S and 6R and the resulting DdSR

(¼dS � dR) values (in Hz) for each of the protons on the ‘front’ and ‘back’ sides of the menthyl moiety.


PROTOCOL

p

uor

G g

n ih si l

bu

P eru ta

N 700 2©

nat

ure

pro

toco

ls/

moc.er

ut an.

ww

w//:ptt

h

BOX 1 | EXAMPLE FORMAT FOR REPORTING RESULTS OF A MOSHER ESTER ANALYSIS

Preparation of the S- and R-MTPA-menthyl esters 6S and 6RTo a stirred solution of (�)-menthol (5, 10.0 mg, 64 mmol) and dry pyridine (16 ml, 0.20 mmol, 3.1 equiv.) in dry deuterochloroform (1 ml, [5]¼0.064 M) at room temperature, R-(�)-MTPA-Cl (3R, 23 ml, 0.12 mmol, 1.9 equiv.) was added. The reaction progress was monitored by thin-layerchromatography (TLC) on silica gel (4:1::Hex:EtOAc). After complete consumption of the menthol (B2 h), the reaction mixture was quenchedby the addition of water (B1 ml) and ether (B3 ml). The aqueous layer was extracted with two additional portions of ether (B3 ml), and thecombined organic layers were dried (Na2SO4), filtered and concentrated in vacuo. The crude product mixture was purified by silica-gelchromatography (MPLC, eluting with hexanes/ethyl acetate (40:1, Rf ¼ 0.18)) to give the S-MTPA-menthyl ester 6S (21.5 mg, 90%) as a whitesolid. 6S: 1H NMR (CDCl3) d 7.54 (m, 2H), 7.39 (m, 3H), 4.90 (ddd, 1H, J¼ 4.5, 11.0, 11.0 Hz), 3.59 (q, 3H, J ¼ 1.0 Hz), 2.13 (dddd, 1H, J ¼2.0, 3.5, 4.0, 11.5 Hz), 1.70 (dddd, 1H, J ¼ 3.5, 3.5, 3.5, 12.5 Hz), 1.67 (dddd, 1H, J ¼ 3.5, 3.5, 3.5, 13 Hz), 1.56 (dsept, 1H, J ¼ 3.0, 7.0),1.54 (ddddq, 1H, J¼ 3.5, 3.5, 12, 12, 7.0 Hz), 1.42 (dddd, 1H, J¼ 3.0, 3.0, 11.0, 12.5 Hz), 1.12 (ddd, 1H, J¼ 12, 12, 12 Hz), 1.04 (dddd, 1H,J ¼ 3.5, 12.5, 12.5, 12.5 Hz), 0.94 (d, 3H, J ¼ 7.0 Hz), 0.87 (dddd, 1H, J ¼ 4.0, 12.5, 13, 13 Hz), 0.74 (d, 3H, J ¼ 7.0 Hz), and 0.67 (d, 3H,J¼ 7.0 Hz). In an entirely analogous fashion, the R-MTPA-menthyl ester 6R was prepared using S-(+)-MTPA-Cl (3S). 6R: 1H NMR (CDCl3) d 7.52(m, 2H), 7.40 (m, 3H), 4.88 (ddd, 1H, J¼ 4.5, 11.0, 11.0 Hz), 3.53 (q, 3H, J¼ 1.0 Hz), 2.08 (dddd, 1H, J¼ 2.0, 3.5, 4.0, 12 Hz), 1.88 (dsept,1H, J¼ 3.0, 7.0), 1.70 (dddd, 1H, J¼ 3.5, 3.5, 3.5, 13 Hz), 1.69 (dddd, 1H, J¼ 3, 3, 3, 13 Hz), 1.52 (ddddq, 1H, J¼ 3.5, 3.5, 12, 12, 6.5 Hz),1.45 (dddd, 1H, J¼ 3.0, 3.0, 11.0, 12.5 Hz), 1.06 (dddd, 1H, J¼ 3.5, 13, 13, 13 Hz), 0.98 (ddd, 1H, J¼ 12, 12, 12 Hz), 0.91 (d, 3H, J¼ 6.5Hz), 0.87 (d, 3H, J ¼ 7.0 Hz), 0.86 (dddd, 1H, J ¼ 3.5, 12.5, 12.5, 12.5 Hz), and 0.77 (d, 3H, J ¼ 7.0 Hz).

BOX 2 | THE DIRTY LITTLE SECRET ABOUT THE ABSOLUTE CONFIGURATION OF THEMOSHER ACID CHLORIDE

Upon conversion of the MTPA acid (MTPA-OH) to the MTPA acid chloride (MTPA-Cl), there is a switch in the relative priority of two of the groups.Specifically, the trifluoromethyl group (CF3) is higher in priority than the carboxyl group (COOH) in the acid, but lower than the chlorocarbonylgroup (COCl) in the acid chloride. Thus, the R enantiomer of the Mosher acid (R-(+)-MTPA-OH, 2R) gives rise to the S enantiomer of the Mosheracid chloride (S-(+)-MTPA-Cl, 3S) (and vice versa for the S acid 2S to the R acid chloride 3R). This represents a somewhat rare instance in whichthe absolute configuration of a stereocenter changes as a result of a chemical reaction, even though none of the four bonds to the stereogeniccarbon was involved in the reaction.

CF3ClOC

Ph OMe

CF3HO2C

Ph OMe1

3CF3

Ph OMe

O

OR2

R1

3

R-(+)-MTPA-OHR Mosher acid

S-(+)-MTPA-ClS Mosher acid chloride

R Mosher ester

2

4 14

2 3

14

2

3S2R 4R

As was shown but not otherwise emphasized earlier in Figure 1, it follows that the R Mosher acid gives rise to the R Mosher ester, but that it isthe S Mosher acid chloride that gives rise to the R Mosher ester (since there is another change in relative priority: CF3 is lower than COCl buthigher than COOR). It is, of course, essential that every user of Mosher ester analysis considers this fact when performing the analysis.Otherwise, again, precisely the opposite (and wrong) absolute configuration will be assigned16. Somewhat ironically, this potential problem hasbeen exacerbated by the fact that the acid chloride became commercially available in the early 1990s. That is, earlier workers would oftenprepare the acid chloride from purchased, say, R acid, convert the derived acid chloride to the R Mosher ester and properly deduce the correctabsolute configuration, without even realizing that they were passing by way of the S acid chloride.

Finally, it is also important that users report the method by which they made their Mosher esters with absolute clarity, so that no doubt isleft in readers’ minds about the configuration of each MTPA ester. The experimental description in Box 1 represents one such unambiguouspresentation. If the publication venue does not lend itself to full experimental description, then an unambiguous statement like ‘‘esterificationof # with (S)- and (R) MTPA-Cl occurred only at the C-8 hydroxy group to give the (R)- and (S)-MTPA esters #a and #b, respectively’’17 is to behighly commended and recommended. All too often and in fact typically, authors are ambiguous on the issue of the precise origin of their R- andS-esters. For example, simple statements like ‘Mosher ester analysis led to the assignment of the R configurationy’ and ‘R- and S-MTPA esterswere prepared from reaction of carbinol X with the MTPA acid chlorides’ abound and leave doubt. This does not mean that the method wasmisused (i.e., that the user was unaware of the CIP switch), but it does (and should) erode the confidence that readers can have in theconclusions reached. The only instances in which it is clear that an error was made—and we have found a number of published examples ofthis—is where it is unambiguously stated that ‘the R-Mosher ester was prepared using R-MTPA-Cl’. Ironically, these cases are at least somewhatself-correcting, since the informed reader will know to reverse whatever conclusions were drawn from these analyses.

The bottom line is that Mosher ester analysis is an extremely reliable method for determining absolute configuration, provided that usersbe aware of the above pitfall and that they demonstrate that awareness by reporting the method of synthesis (origin) of each Mosher ester inan unambiguously stated manner so that others will have the full measure of confidence in the conclusions.


PROTOCOL

Step 1B(ix): B3.5 hStep 1B(x): 5–40 minSteps 1C(i–iv): 15–20 minSteps 1C(v) and (vi): 12–24 hStep 1C(vii): 5 minStep 1C(viii): 30–70 minStep 1C(ix): 5–30 minStep 1C(x): B24 hStep 1C(xi): 5–30 minStep 2: 15–90 minStep 3: 30–60 minStep 4: 20 minStep 5: 5 minStep 6: 1–2 h

? TROUBLESHOOTINGTroubleshooting advice regarding potential issues with the preparation, purification, and/or NMR data collection for esters 4Rand 4S can be found in Table 2.

ANTICIPATED RESULTSEach purified diastereomeric Mosher ester (4R and 4S) will be prepared and isolated in 50–80% yield from the startingcarbinol 1. 1H NMR spectroscopic data of sufficient quality for subsequent Dd analysis will be obtained for each ester. Afterthe prescribed DdSR analysis is performed, the absolute configuration of the carbinol 1 will have been deduced.

Published online at http://www.natureprotocols.comReprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions

1. Stephens, T.D. & Brynner, R. Dark Remedy: The Impact of Thalidomide and ItsRevival as a Vital Medicine (Perseus Publishing, Cambridge, MA, 2001).

2. Seco, J.M., Quinoa, E. & Riguera, R. The assignment of absolute configurationby NMR. Chem. Rev. 104, 17–117 (2004).

3. Dale, J.A. & Mosher, H.S. Nuclear magnetic resonance enantiomer reagents.Configurational correlations via nuclear magnetic resonance chemical shifts ofdiastereomeric mandelate, O-methylmandelate, and a-methoxy-a-trifluoro-methylphenylacetate (MTPA) esters. J. Am. Chem. Soc. 95, 512–519 (1973).

4. Hoye, T.R. & Renner, M.K. MTPA (Mosher) amides of cyclic secondary amines:conformational aspects and a useful method for assignment of amineconfiguration. J. Org. Chem. 61, 2056–2064 (1996).

5. Dale, J.A., Dull, D.L. & Mosher, H.S. a-Methoxy-a-trifluoromethylphenylaceticacid, a versatile reagent for the determination of enantiomeric composition of

alcohols and amines. J. Org. Chem. 34, 2543–2549 (1969).6. Ward, D.E. & Rhee, C.K. A simple method for the microscale preparation of

Mosher’s acid chloride. Tetrahedron Lett. 32, 7165–7166 (1991).7. Sullivan, G.R., Dale, J.A. & Mosher, H.S. Correlation of configuration and 19F

chemical shifts of a-methoxy-a-trifluoromethylphenylacetate derivatives. J. Org.

Chem. 38, 2143–2147 (1973).8. Ohtani, I., Kusumi, T., Ishitsuka, M.O. & Kakisawa, H. Absolute configurations of

marine diterpenes possessing a xenicane skeleton. An application of an advanced

Mosher’s method. Tetrahedron Lett. 30, 3147–3150 (1989).9. Ohtani, I., Kusumi, T., Kashman, Y. & Kakisawa, H. A new aspect of the high-field

NMR application of Mosher’s method. The absolute configuration of marinetriterpene sipholenol A. J. Org. Chem. 56, 1296–1298 (1991).

p

uor

G g

n ih si l

bu

P eru ta

N 700 2©

nat

ure

pro

toco

ls/

moc.er

ut an.

ww

w//:ptt

h

TABLE 2 | Troubleshooting table.

Problem Possible reason Solution

Poor conversion to product Moisture in reaction mixture that consumesMTPA-Cl or DCC-activated MTPA-OH

Use anhydrous solvents and oven-dried glassware

Slow reaction Reaction solution too dilute Make sure substrate alcohol and MTPA-OH orMTPA-Cl are present at Z0.05 M, and monitorreaction conversion carefully by TLC analysis

Product contamination, especially formicroscale

Leaching of impurities (e.g., plasticizers)from plastic labware by organic solvents

Use only glass reaction vessels, pipets, etc., andTeflon rather than plastic tubing

Product contamination by excess MTPA-Cl, again, especially for microscale

Need to use Z10 equiv. of MTPA-Cl becauseof limited availability of carbinol

Quench the reaction mixture Me2N(CH2)3NH2 beforeadding dilute HCl during workup3

Use stock solutions to accurately measure reagentquantities18

Overlapping resonances complicatingNMR interpretation

Overall molecular complexity and/or coinci-dental chemical shift similarity

Carry out 2D-NMR analysis (e.g., COSY or HMQC) tounambiguously assign overlapping resonances


PROTOCOL

10. Ohtani, I., Kusumi, T., Kashman, Y. & Kakisawa, H. High-field FT NMR applicationof Mosher’s method. The absolute configurations of marine terpenoids. J. Am.Chem. Soc. 113, 4092–4096 (1991).

11. Parker, D. NMR determination of enantiomeric purity. Chem. Rev. 91, 1441–1457(1991).

12. Trost, B.M. et al. On the use of the O-methylmandelate ester for establishment ofabsolute configuration of secondary alcohols. J. Org. Chem. 51, 2370–2374(1986).

13. Moon, S. et al. (+)-7S-Hydroxyxestospongin A from the marine spongeXestospongia sp. and absolute configuration of (+)-xestospongin D. J. Nat. Prod.65, 249–254 (2002).

14. Still, W.C., Kahn, M. & Mitra, A. Rapid chromatographic technique for preparativeseparations with moderate resolution. J. Org. Chem. 43, 2923–2925 (1978).

15. Cahn, R.S., Ingold, C.K. & Prelog, V. The specification of asymmetric configurationin organic chemistry. Experientia 12, 81–94 (1956).

16. Joshi, B.S. & Pelletier, S.W. A cautionary note on the use of commercial (R)-MTPA-Cl and (S)-MTPA-Cl in determination of absolute configuration by Mosher esteranalysis. Heterocycles 51, 183–184 (1999).

17. Garo, E. et al. Trichodermamides A and B, cytotoxic modified dipeptides from themarine-derived fungus Trichoderma virens. J. Nat. Prod. 66, 423–426 (2003).

18. Hoye, T.R. & Jeffrey, C.S. Student empowerment through ‘mini-microscale’reactions: the epoxidation of 1 mg of geraniol. J. Chem. Educ. 83, 919–920 (2006).

p

uor

G g

n ih si l

bu

P eru ta

N 700 2©

nat

ure

pro

toco

ls/

moc.er

ut an.

ww

w//:ptt

h


PROTOCOL

the assignment of absolute configuration by nmr · · 2012-03-12optical methods (e.g., circular...

Documents