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INTRODUCTION

Nuclear magnetic resonance (NMR) spectroscopy is one of the most common methods used to determine enantiopurity and assign the absolute configuration of chiral compounds. The strategy that has been most exploited, as first recognized by Raban and Mislow in 1965 [1], is to use an enantiopure chiral reagent to distinguish a pair of enantiomers through the formation of nonequivalent diastereomeric complexes. With the diastereomeric complexes, the resonances of enantiotopic nuclei become anisochronous and may split into two resonances, one for the (R)-derivative and one for the (S)-derivative of the analyte. The area of the two resonances can be used to determine enantiopurity. The enantiopure probe molecule functions as either a chiral derivatizing agent (CDA) or a chiral solvating agent (CSA). Furthermore, the association of an enantiopure compound with a prochiral molecule with nuclei that are enantiotopic by internal comparison (e.g. the methyl groups of 2-propanol) renders these nuclei nonequivalent such that distinct resonances are often observed in the NMR spectrum. Classifying chiral metal compounds as either CDAs or CSAs is sometimes difficult. What is important is whether the analyte molecule undergoes fast or slow exchange with the metal center. Strategies based on different packing orders for a pair of enantiomers, such as it occurs in liquid crystals or solid-state systems, have also been used for chiral analysis in NMR spectroscopy.

1.1. CHIRAL DERIVATIZING AGENTS

CDAs form a covalent bond with a reactive moiety of the analyte. Many CDAs are available for the analysis of carboxylic acids, alcohols, and amines, although strategies for preparing derivatives of many other functional groups will be described as well throughout the text. There are two potential concerns with the application of CDAs when determining enantiopurity. One is the possibility of kinetic resolution, which involves a situation where one enantiomer reacts faster with the CDA than the other. If the reagents are not allowed to react for a long enough time, the proportion of the two diastereomers will not be equivalent to the proportion of the two enantiomers in the original mixture. Kinetic resolution is significant when determining enantiopurity, but it is not significant if the CDA is being used to assign the absolute configuration of an enantiopure analyte such as a natural product.

A second concern with CDAs is that no racemization occurs during the derivatization reaction. This can be significant whether it happens to the analyte or the CDA. With some CDAs for which unacceptable levels of racemization did occur, further study was undertaken to develop reaction conditions that minimize or eliminate racemization. When pertinent, these studies are described in the text.

A general understanding is that CDAs used for determining the enantiopurity of an analyte should be 100% enantiopure. A method for using CDAs that are less than 100% enantiopure has been described. The enantiopurity of the reagent must first be accurately measured using an appropriate method. A set of equations is provided in the report to determine the enantiopurity of an unknown from the known purity of the chiral reagent [2].

Many CDAs incorporate moieties, such as aryl rings, that produce specific and predictable perturbations in the chemical shifts of the resonances of the analyte. In such cases, the changes in chemical shifts in the spectrum of an enantiopure analyte in the derivatives with the (R)- and (S)-enantiomers of the CDA can be used to assign absolute configuration. In other situations, moieties on the analyte may cause specific and predictable perturbations of the chemical shifts of resonances of the CDA. If so, these can be used to assign absolute configuration as well.

Another procedure that is often used with CDAs or CSAs is to look for the presence of specific trends in the chemical shifts that correlate with the absolute configuration of the analyte. The assumption is that if the trends are consistent among a series of compounds with known configurations, then they will be consistent for an unknown analyte with a similar structure. Empirical trends such as these have been observed in many situations and are described where appropriate throughout the text.

An alternative, although much less-used, derivatizing strategy involves a self-coupling reaction of a chiral molecule. The self-coupling of two chiral molecules leads to the formation of a mixture of meso (R,S) and threo [(S,S)/(R,R)] derivatives. Assuming these species exhibit distinct resonances in the NMR spectrum, the areas of the different resonances depend on the enantiopurity of the analyte [3]. A recent example is a generalized procedure for determining the enantiopurity of 2-phenylpropionic acid and other profens. A stereospecific N,N´-dicyclohexylcarbodiimide coupling produces a statistical mixture of diastereoisomeric chiral ((R,R) and (S,S)) and meso ((R,S) and (S,R)) anhydrides. The ratio of the anhydrides in the 1H NMR spectrum can be related to the initial enantiopurity. The reaction can be done in an NMR tube in about 2 min. Because the coupling is stereo random, the reaction does not need to go to completion. The method is more accurate for samples with moderate-to-high enantiomeric excess than those closer to racemic proportions [4].

1.2. CHIRAL SOLVATING AGENTS

CSAs associate with the analyte through non-covalent interactions as shown in Eqs 1.1 and 1.2 for the (R) and (S) forms of an analyte (A). This can involve dipole-dipole, ion-pairing, and p-p interactions. Steric effects are also important in the recognition properties of many CSAs. The choice of solvent is an important parameter when using a particular CSA. Organic-soluble CSAs are often more effective in nonpolar solvents that cannot effectively solvate the polar groups of the CSA and analyte. Water-soluble CSAs, which are often organic macrocyclic compounds, usually rely on hydrophobic effects to promote the interaction or insertion of a hydrophobic portion of the analyte within the hydrophobic cavity of the CSA.

CSAs generally undergo fast exchange with analytes. With fast exchange, the NMR spectrum is a weighted average of the proportion of bound and unbound analyte. Resonances of the analyte double with the presence of chiral recognition. If slow exchange and enantiodifferentiation occur, and not all of the analyte is bound to the CSA, three resonances are observed for a particular nucleus in the NMR spectrum. One is for the unbound analyte. The other two are for the bound forms of the (R)- and (S)-isomers of the analyte. Sometimes the resonances of the analyte or CSA are broadened, which occurs if the system has an intermediate rate of exchange. In such cases, it may be possible to speed up the exchange to acceptable levels by warming the sample, while still retaining enantiodifferentiation.

CSAs are most often used to determine enantiopurity. There are instances, though, in which the interaction of the CSA with the analyte is understood with enough specificity to assign absolute configuration. Similar to the use of CDAs, the relative magnitudes of perturbations in chemical shifts with the (R) and (S) forms of the CSA are used in assigning absolute configuration. There are also other CSAs where empirical trends that correlate with absolute configuration are noted for compounds with similar structures. Unlike CDAs, when measuring enantiopurity with a CSA, it is not necessary to have 100% enantiopurity for the chiral reagent. What is needed is sufficient recognition to cause nonequivalence in the spectra of the enantiomers so that the resonances can be accurately integrated.

Chiral recognition with a CSA can occur from two mechanisms. One is that the CSA complexes with the (R)- and (S)-isomers of the analyte are diastereomers, and similar nuclei in the two analyte enantiomers may have different chemical shifts. The other is that the two enantiomers of the analyte often have different association constants (KR and KS for Eqs 1.1 and 1.2) with the CSA, such that the time-averaged solvation environments are different. In many cases, both mechanisms likely contribute to some extent to the nonequivalence that is observed in the NMR spectrum.

When using CSAs for enantiodifferentiation in NMR spectroscopy, it is usually best to record a series of spectra, often referred to as a titration, with increasing concentration of the CSA relative to the analyte. As the resonances shift position in the spectrum, they may often overlap with other resonances of the CSA or analyte. Recording a series of spectra better ensures that a spectrum with unobscured splitting of one of the resonances is observed. In some CSA-analyte pairs, resonances may exhibit broadening over one region of the titration because of an intermediate exchange rate, whereas broadening is not observed in other regions.

The spectra in Figure 1.1 illustrate many of the observations that may occur in the titration of an analyte with a CSA. The series of spectra in Figure 1.1 show the resonances for the diastereotopic methylene protons of...