Alanine racemization

The alanine racemization catalyzed by alanine racemase is considered to be initiated by the transaldimination (Fig. 8.5).26) In this step, PLP bound to the active-site lysine residue forms the external Schiff base with a substrate alanine (Fig. 8.5, 1). The following a-proton abstraction produces the resonance-stabilized carbanion intermediates (Fig. 8.5, 2). If the reprotonation occurs on the opposite face of the substrate-PLP complex on which the proton-abstraction proceeds, the antipodal aldimine is formed (Fig. 8.5,3). The subsequent hydrolysis of the aldimine complex gives the isomerized alanine and PLP-form racemase. The random return of hydrogen to the carbanion intermediate is the distinguishing feature that differentiates racemization from reactions catalyzed by other pyridoxal enzymes such as transaminases. Transaminases catalyze the transfer of amino group between amino acid and keto acid, and the reaction is initiated by the transaldimination, followed by the a-proton abstraction from the substrate-PLP aldimine to form a resonance-stabilized carbanion. This step is common to racemases and transaminases. However, in the transamination the abstracted proton is then tranferred to C4 carbon of PLP in a highly stereospecific manner The re-protonation occurs on the same face of the PLP-substrate aldimine on which the a-proton is abstracted. With only a few exceptions,27,28) each step of pyridoxal enzymes-catalyzed reaction proceeds on only one side of the planar PLP-substrate complex. However, in the amino acid racemase... 

Fig. 8.6 Partial reaction profile of alanine racemization by B. stearothermophilus. Dotted lines indicate kinetically insignificant steps. (Reproduced with permission from Faraci and Walsh, Biochemistry, 27, 3264(1988)).

Both pure L- and D-amino acids can be made using hydantoinase enzymes. These enzymes catalyze the stereoselective hydrolysis of racemic hydantoins such as (50) which is used for the production of D-alanine (15) (58). 

DL-Alanine is the first amino acid which was synthesi2ed chemically (138). Glycine and DL-methionine have also been suppHed by this method (20). However, amino acids formed by the chemical method are racemic, and it is necessary to resolve the mixture to get the L- or D-form amino acid which is usually demanded. 

It is mentioned in Section II, C, 1 that iV -benzoyl-D-alanine cyclizes in 100% sulfuric acid to. give an azlactone which racemizes very slowly. ... 

The improvements in resolution achieved in each deconvolution step are shown in Figure 3-3. While the initial library could only afford a modest separation of DNB-glutamic acid, the library with proline in position 4 also separated DNP derivatives of alanine and aspartic acid, and further improvement in both resolution and the number of separable racemates was observed for peptides with hydrophobic amino acid residues in position 3. However, the most dramatic improvement and best selectivity were found for c(Arg-Lys-Tyr-Pro-Tyr-(3-Ala) (Scheme 3-2a) with the tyrosine residue at position 5 with a resolution factor as high as 28 observed for the separation of DNP-glutamic acid enantiomers. 

Figure A8.18 A racemic mixture of a-hydroxyacids (like L, D-lactate) can be transformed via the corresponding a-ketoacid (pyruvate) to the desired L-amino acid (L-alanine) with cofactor recycling.

Photodriven reactions of Fischer carbenes with alcohols produces esters, the expected product from nucleophilic addition to ketenes. Hydroxycarbene complexes, generated in situ by protonation of the corresponding ate complex, produced a-hydroxyesters in modest yield (Table 15) [103]. Ketals,presumably formed by thermal decomposition of the carbenes, were major by-products. The discovery that amides were readily converted to aminocarbene complexes [104] resulted in an efficient approach to a-amino acids by photodriven reaction of these aminocarbenes with alcohols (Table 16) [105,106]. a-Alkylation of the (methyl)(dibenzylamino)carbene complex followed by photolysis produced a range of racemic alanine derivatives (Eq. 26). With chiral oxazolidine carbene complexes optically active amino acid derivatives were available (Eq. 27). Since both enantiomers of the optically active chromium aminocarbene are equally available, both the natural S and unnatural R amino acid derivatives are equally... 

The phosphotriesterase from Pseudomonas diminuta was shown to catalyze the enantioselective hydrolysis of several racemic phosphates (21), the Sp isomer reacting faster than the Rp compound [65,66]. Further improvements using directed evolution were achieved by first carrying out a restricted alanine-scan [67] (i.e. at predetermined amino acid positions alanine was introduced). Whenever an effect on activity/ enantioselectivity was observed, the position was defined as a hot spot. Subsequently, randomization at several hot spots was performed, which led to the identification of several highly (S)- or (R)-selective mutants [66]. A similar procedure was applied to the generation of mutant phosphotriesterases as catalysts in the kinetic resolution of racemic phosphonates [68]. 

When the substrate is availabT in either the d- or 1-racemic form, it is preferable to use the appropriate isomer rather than its mixture In a case of transaminase assays for GOT and GPT activity, for example, the initial assays used the d-1 amino acid as substrate, and a marked improvement in activity and linearity was found by Henry and co-workers when they used 1-aspartate or 1-alanine, respectively (28) ... 

OS 26] [R 4] [P 18] For dipeptide formation from the pentafluorophenyl ester of Boc-D-alanine and (S)-a-methylbenzylamine an extent of racemization of 5.6% was found [86]. This experiment also served to demonstrate monitoring of the racemization of an a-amino acid used in peptide synthesis. 

Jere et al. also reported on the stereoretentive C-H bond activation in aqueous phase catalytic hydrogenation of alanine (19). They demonstrated that hydrogenation of the carboxylic functionality is a stereo retentive process. Racemization occurs through a distinct process. 

New analyses of material from the interior of the Orgeuil and Ivona meteorites show the presence of P-alanine, glycine and y-amino-n-butyric acid as the main components (0.6-2.0ppm) traces of other amino acids were also detected. The amino acids were present as racemic mixtures, i.e., d/l = 1, so that an extraterrestrial origin can be assumed (Ehrenfreund et al., 2001). 

Ai-Stearoylamino acids and their methyl esters were synthesized from enantiomeric and racemic forms of tyrosine, serine, alanine, and tryptophan (Fig. 16). Analogs of these molecules were investigated initially over 30 years ago by Zeelen and Havinga, who found stereochemical differentiation in the monolayer HjA isotherms of these materials (Zeelen, 1956 Zeelen and Havinga, 1958). We have extended this study using more sensitive Langmuir balances, a wider array of dynamic and equilibrium techniques, and the A-stearoyl methyl esters of the amino acids (Harvey et al., 1989 Harvey and Arnett, 1989). 

Figure 17 shows the 11/A isotherms of racemic and enantiomeric films of the methyl esters of 7V-stearoyl-serine, -alanine, -tryptophan, and -tyrosine on clean water at 25°C. Although there appears to be little difference between the racemic and enantiomeric forms of the alanine surfactants, the N-stearoyl-tyrosine, -serine, and -tryptophan surfactants show clear enantiomeric discrimination in their WjA curves. This chiral molecular recognition is first evidenced in the lift-off areas of the curves for the racemic versus enantiomeric forms of the films (Table 2). As discussed previously, the lift-off area is the average molecular area at which a surface pressure above 0.1 dyn cm -1 is first registered. The packing order differences in these films, and hence their stereochemical differentiation, are apparently maintained throughout the compression/expansion cycles. 

Fig. 17 Surface pressure/area isotherms for the compression and expansion cycles of racemic (dashed line) and enantiomeric (solid line) stearoylserine (A), stearoyl-alanine (B), stearoyltryptophan (C), and stearoyltyrosine methyl esters (D) on a pure water subphase at 25°C carried out at a compression rate of 7.1 A2/molecule per minute. Arrows indicate the direction of compression and expansion.

The instability of these chiral monolayers may be a reflection of the relative stabilities of their bulk crystalline forms. When deposited on a clean water surface at 25°C, neither the racemic nor enantiomeric crystals of the tryptophan, tyrosine, or alanine methyl ester surfactants generate a detectable surface pressure, indicating that the most energetically favorable situation for the interfacial/crystal system is one in which the internal energy of the bulk crystal is lower than that of the film at the air-water interface. Only the racemic form of JV-stearoylserine methyl ester has a detectable equilibrium spreading pressure (2.6 0.3dyncm 1). Conversely, neither of its enantiomeric forms will spread spontaneously from the crystal at this temperature. 

This work demonstrated that AAR could give reasonable dates from smaller samples of bone than were necessary for radiocarbon, and had a time depth of at least 70 000 years, and possibly more if one of the more slowly racemizing amino acids such as alanine was used. The key paper came in 1974 (Bada et al., 1974), which published dates of between 6000 and 48 000 BP for various samples of human bone from the Californian coast (Table 8.1). The SDM (San Diego Museum) samples from site W-2 were from a shell midden near La Jolla excavated in 1926. Subsequently, it appears that 19 individual burials were recovered in a rescue operation from this site, known as La Jolla Shores SDM-16755 is thought to refer to more than one individual (La Jolla Shores I and II), with a third (La Jolla Shores III) identified as SDM-16740 (Taylor et al., 1985 Table 8.1). Site W-34 was located between Del Mar and Solano Beach, from a shell midden which had been largely destroyed by coastal... 

Extractions of aqueous solutions of racemic amino-acid ester salts with solutions of / -6/s(dinaphthyl)-22-crown-6 [284] in chloroform revealed the dependence of the enantiomeric distribution constant on the structure of the amino acid ester (Table 64). In order to limit the concentrations of complex in the aqueous phase, inorganic salts were added. In the case of tyrosine, serine and alanine no extraction of salt was observed obviously these salts form very hydrophilic complexes. The highest degree of chiral recognition was found with [284] and p-hydroxyphenylglycine methyl ester hexafluorophosphate [A(AG°)... 

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