Acylamino acids racemization

Exercise 25-21 How could an optically pure W-acylamino acid racemize and lead to racemic W-acylpeptides as the result of a peptide coupling reaction wherein the carboxyl group of the amino acid was converted to an anhydride group (Review Section 25-5A.)... 

Enzymatic hydrolysis of A/-acylamino acids by amino acylase and amino acid esters by Hpase or carboxy esterase (70) is one kind of kinetic resolution. Kinetic resolution is found in chemical synthesis such as by epoxidation of racemic allyl alcohol and asymmetric hydrogenation (71). New routes for amino acid manufacturing are anticipated. 

FMF Chen, NL Benoiton. Racemization of acylamino acids and the carboxy-terminal residue of peptides, hit J Pept Prot Res 31, 396. 

W Williams, GT Young. Amino acids and peptides. XXXV. Effect of solvent on the rates of racemization and coupling of acylamino acid p-nitrophenyl esters. Base strengths of amines in organic solvents, and related investigations. J Chem Soc Perkin Trans 1 1194, 1972. 

Saturated 5(4//)-oxazolones are easily obtained from //-acylamino acids in the presence of a cyclization agent and have been used extensively in coupling reactions as synthetic equivalents of a-amino acids in the synthesis of peptides. In this context, tautomeric equilibrium can be a significant problem due to the racemization associated with the isomerization. For example, trifluoroacetylation of tryptophan in ether affords the 5(4//)-oxazolone 5 without racemization. However, upon dissolution in acetonitrile, 5 completely racemizes. Further, upon heating, an aqueous dioxane solution of 5 cleanly isomerizes to the isomeric 5(2//)-oxazolone 6 (Scheme 7.2). 

In addition, the use of an enantioselective or diastereoselective hydrolysis of racemic oxazolones offers another possibility to obtain new synthetic amino acids. Similarly, alcoholysis of 5(4H)-oxazolones gives the corresponding A -acylamino acid esters. 

In the heterocyclic series, racemic 3-(fur-2-yl)alanine has been prepared from furfural using this approach. In addition, (3-(pyrid-3-yl)alanine, ° p-(quinol-3-yl)alanine, ° a p-(benzofuranyl)alanine derivative, 2-amino-3-(2,2 -bipyridi-nyl)propanoic acid, and some interesting derivatives of histidines—in particular 1-alkylhistidines with amphiphilic properties have all been synthesized using this methodology. The complete reaction sequence starting from an aldehyde and an A-acylamino acid derivative is shown in Scheme 7.150. 

The favorable effect of the enamide function on asymmetric induction is indicated not only by the result with compound I, but also by later results summarized in Table I, where optical purities in the range of 70 to 80% were generally obtained for various derivatives of alanine, phenylalanine, tyrosine, and 3,4-dihydroxyphenylalanine (DOPA). The Paris group found that the Rh-(-)-DIOP catalyst yielded the unnatural R or d -amino acid derivatives, whereas l-amino acid derivatives could be obtained with a (+)-DIOP catalyst. Since the optical purity of the IV-acylamino acids can often be considerably increased by a single recrystallization (fractionation of pure enantiomer from racemate) and the IV-acetyl group can be removed by acid hydrolysis, this scheme provides an excellent asymmetric synthesis route to several amino acids. 

The use of homochiral complexation agents to separate the fluorine-19 chemical shifts of enantiomers containing fluorine has also been examined. Addition of the supported dipeptide 9 to a solution of the racemic iV-acylamino acid ester 10 in carbon tetrachloride leads to the appearance of two CF3 peaks corresponding to the two enantiomers. The separation is obviously concentration-dependent, but peak separation was sufficient for mixtures of enantiomers to give enantiomeric excesses comparable with those determined by gc analysis55. 

Active Esters. The synthesis of reactive esters of acylamino acids and their use in lengthening a peptide chain by reaction at the amino end is a justifiably popular method racemization is avoided, yields are good, and purification of the products is relatively easy. Esters of N-hydroxy-succinimide (l), N-hydroxypiperidine (ll) and 8-hydroxyquinoline (III) have received particular attention during I966. 

The reagent promotes the coupling in high yields of acylamino acids with amino acid esters in benzene, ethanol, or THF at room temperature. No racemization was detected in the supersensitive Young test, and Bz-Leu-Gly-OEt, an 33.5°, was synthesized in 95% yield. Activation of the carboxyl group involves the transient formation of a mixed carbonic anhydride.3... 

The enzymatic hydrolysis of N-acylamino acids has been known for a century and was first detected in aqueous kidney preparations 3. Based on the finding that this enzymatic hydrolysis proceeds enantiospecifically 2, Greenstein and coworkers developed a general and very attractive procedure for the resolution of a vast number of racemic N-acylated amino acids to the corresponding L-amino acids catalyzed by aminoacylase (E.C. 3.5.1.14) whereas the N-acetyl-D-amino acid does not react13 (Fig. 12.3-1). 

The acylase-catalyzed resolution of /V-acetyl-DL-amino acids is a key commercial process. The racemization of the remaining /V-acetyl-D-amino acid after separation of the L-amino acid must be performed, but adds complexity and cost. Therefore, in situ racemization with a racemase possessing specificity for N-acylamino acids without affecting the stereochemistry of the product L-amino acids is very desirable. The pentameric enzyme from Streptomyces sp. Y-53 specifically catalyzes the racemization of N-acylamino acids without acting on amino acids [182],... 

Peptide synthesis. The reagent effects coupling of acylamino acids with amino acid esters in DMF and triethylamine at -10 to 0° in 80-97% yields. Racemization according to the test of Young is less than 3%. The condensation involves the intermediate formation of an acyl azide, which has been isolated... 

Most of the enzymatic peptide forming reactions are strictly stereo-specific for L-amino acids so that racemization that often accompanies chemical coupUng of optically active amino acids does not occur. The formation of only the L-amino acid derivative out of a D,L-mixture, in an enzymatic formation e.g. of an anilide from a D,L-Z-amino acid ester and aniUne, makes proteolytic enzymes useful reagents for the resolution of racemic mixtures. This is supplementary to the enzymatic stereospecific deacylation of D,L-iV-acylamino acid mixtures where exclusively the L-derivative will be deacylated. 

Activation of the carboxyl group in itself is conducive to racemization. The otherwise chirally stable acylamino acids and peptides can lose chiral homogeneity once their free carboxyl group is converted into a reactive derivative. The electron-withdrawing effect of the activating group (X) renders the proton on the a-carbon atom slightly acidic and hence abstractable by base and chirality is, of course, lost in the carbanion. This simple mechanism, however, is operative only... 

There are considerable differences in the hydrolysis rates of different amino acids. If the rate is too low for practical purposes, then the chloroacetyl derivatives of the racemates can be applied as substrates instead of the acetyl derivatives. Of course, it is often worthwhile to recover the unchanged D-acylamino acid and hydrolyze it with aqueous acid to produce the D-enanthiomer of the amino acid. The unnatural D isomers are frequently used as building components in studies of structure-activity relationships, in the preparation of hormone analogs resistant to the action of proteolytic enzymes and in the synthesis of microbial peptides. 

Racemization during the synthesis of peptides is a complex problem. The diversity of possible courses followed in the process is compounded by the individuality of the amino acids. This was already shown on the example of S-alkyl-cysteine residues which lose chiral purity by a special mechanism even if protected by a urethane-type protecting group that prevents racemization in other acylamino acids. The opposite end of the scale is represented by proline... 

One especially interesting method for resolving amino acids is based on the use of enzymes called deacylases. These enzymes catalyze the hydrolysis of N-acylamino acids in living organisms. Since the active site of the enzyme is chiral, it hydrolyzes only A/ acylamino acids of the l configuration. When it is exposed to a racemic mixture of A/ acylamino acids, only the derivative of the L-amino acid is affected and the products, as a result, are separated easily ... 

In contrast to the above-mentioned amino acid resolution methods involving amino acid esters, -amides, or Af-acylamino acids where the natural L-enantiomer is preferably hydrolyzed from a racemic mixture, hydantoinases usually convert the opposite D-enantiomer [153-155], and L-hydantoinases are known to a lesser extent... 

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