Labels mass-labeled enantiomers

Besides the measure of the enantiomeric purity of chiral products, the use of mass-labelled enantiomers (or pseudo-enantiomers) in conjunction with the ESI-MS has been also set up to determine selectivity factors. 

The 8,9- and 10,11-dihydrodiols formed in the metabolism of BA and DMBA respectively are all highly enriched (>90%) in R,R enantiomers (Table III). Labeling experiments using molecular oxygen-18 in the in vitro metabolism of the respective parent compounds and subsequent mass spectral analyses of dihydrodiol metabolites and their acid-catalyzed dehydration products indicated that microsomal epoxide hydrolase-catalyzed hydration reactions occurred exclusively at the nonbenzylic carbons of the metabolically formed epoxide intermediates (unpublished results). These findings indicate that the 8,9- and 10,11-epoxide intermediates, formed in the metabolism of BA and DMBA respectively, contain predominantly the 8R,9S and 10S,11R enantiomer, respectively. These stereoselective epoxidation reactions are relatively insensitive to the cytochrome P-450 isozyme contents of different rat liver microsomal preparations (Table III). 

Therefore, directed evolution was applied to solve these problems. To screen for enantioselectivity, the Miilheim MS-based high-throughput ee assay (92,93) (Section III.C) was applied (46). In this case, the necessary isotope labeling focused on the use of in the pseudo-meso compound N-(J )-17 (see Section III.C for a detailed discussion). An (5)-selective nitrilase leads preferentially to N-(5)-18, whereas an 7 -selective variant results in the picw o-enantiomer (J )-18. They differ by one mass unit and can therefore be distinguished by MS, both qualitatively and quantitatively (by integration of the relevant peaks). 

Conversion of racemic mixtures with one of the enantiomers carrying a labeling group. This approach is well suited for screening by mass spectrometry (Sect. 4.2). 

Reetz and coworkers developed a highly efficient method for screening of enantioselectivity of asymmetrically catalyzed reactions of chiral or prochiral substrates using ESI-MS [60]. This method is based on the use of isotopically labeled substrates in the form of pseudo-enantiomers or pseudo-prochiral compounds. Pseudo-enantiomers are chiral compounds which are characterized by different absolute configurations and one of them is isotopically labeled. With these labeled compounds two different stereochemical processes are possible. The first is a kinetic separation of a racemic mixture, the second the asymmetric conversion of prochiral substrates with enantiotopic groups. The conversion can be monitored by measuring the relative amounts of substrates or products by electrospray mass spectrometry. Since only small amounts of sample are required for this method, reactions are easily carried out in microtiter plates. The combination of MS and the use of pseudo-enantiomers can be used for the investigation of different kinds of asymmetric conversion as shown in Fig. 3 [60]. 

M. Sawada et al., Chiral recognition in host-guest complexation determined by the enantiomer-labeled guest method using fast atom bombardment mass spectrometry. J. Am. Chem. Soc. 117, 7726-7736 (1995)... 

M. Sawada et al., Chiral amino acid recognition detected by electrospray ionization (ESI) and fast atom bombardment (FAB) mass spectrometry (MS) coupled with the enantiomer-labelled (EL) guest method. J. Chem. Soc. Perkin Trans. 2, 701-710 (1998)... 

Amelung, W., and Brodowski, S. (2002). In vitro quantification of hydrolysis-induced racemization of amino acid enantiomers in environmental samples using deuterium labeling and electron-impact ionization mass spectrometry. Anal. Client. 74, 3239-3246. 

In order to measure the parameter r, we applied a chemical mass-spectrometric method. The racemates were constructed by mixing equimolar amounts of fully protiated enantiomer (-)-(/ ) (2) and deuterium-labelled ( + )-(5) (3). 

One of the critical aspects of this approach is that two different experiments have to be performed between which the particular instrument conditions must be carefully kept constant in order not to affect the intensity ratios. This problem can be overcome by the enantiomer-labeled guest method [47]. It is based on the mass spectrometric examination of one enantiomer of the host with a pseudo-racemic mixture of the guest. In order to be able to detect both diastereomers separately, one enantiomer of the guest must be isotopically labeled, usually with deuterium. In the same experiment, both diastereotopic complexes are formed and their intensities can be compared directly. However, the stereochemical effect might additionally be superimposed by an unknown isotope effect. A way to separate stereochemical and isotope effects is to perform the same experiment with the second host enantiomer [4B]. In one experiment both stereochemical and isotope effects disfavor the same complex and thus work in the same direction. In the other experiment, they partly cancel each other. If both experiments have been performed, one can use the two experimental values for the intensity ratios of both diastero-meric complexes to deconvolute both effects [49]. 

Crown ethers have been used for improving the detection of fullerenes by electrospray mass spectrometry [13]. Sawada et al. have developed a FABMS methodology for the determination of chiral recognition of amino acid esters by crown ethers [14]. This method requires that the racemic mixture of the guests contains one enantiomer in its isotoplcally labeled form. Mass spectral analysis of the molecular ion peaks for H + and H + G(5) allows a direct comparison of their relative abundances, where H and G are host and guest, respectively. 

Ethyl cellulose (EC) was purchased from Aldrich (viscosity 4 cP, 5 % in toluene/ethanol 80 20, extent of labeling 48% ethoxyl). Rosemary oil was provided from Etol, Celje, Slovenia. R(+)limonene (95% sum of enantiomers) was used from Fluka. All other chemicals were of analytical reagent grade. Pure cotton vrith a mass of 140 g/m was used after it was first desized, scoured, bleached and mercerized on continuous production equipment. 

Horeau published in 1982 a pioneering work on the use of mass spectrometry to evaluate the extent of KR in the esterification of racemic alcohols by a chiral anhydride. The principle was to label by deuterium one of the enantiomers of the alcohol and use it to prepare a pseudo-racemic mixture of the alcohol [102]. Mass spectrometry revealed which enantiomer reacted preferentially with the chiral reagent... 

Reetz et al. [103] extended this approach. He reported the principle of a method based on electrospray ionization mass spectrometry (ESI-MS) enabling the determination of enantioselectivity of catalytic or stoichiometric asymmetric reactions. The method involves the reaction of a chiral reagent or catalyst with an equimolecular mixture of pseudo enantiomers, one isotopically labelled and the other non-labelled, simulating a racemate. 

A KR process on 180/181 would produce true enantiomers 182 and 183, together with labelled and non-labelled achiral products 184 and 185. The ratios of the total intensities of 180/181 and 184/185 in the mass spectra allow for the determination of the enantioselectivity. 

B. Enantiomer-Labeled Method Synthesize one isotopically labeled guest enantiomer mix 1 1 with the second unlabeled enantiomer and add a small amount of enantiopure host record ESI mass spectrum and compare the intensities of both diastereomeric complexes directly to separate isotope and stereochemical effects, the other pseudoracemate needs to be subjected to the same experiment. 

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