. Can. J. Chem. 65, 619 (1 987).Pure mixed anhydrides R10CO-Xxx-O-COO~2 (Xxx = NHCHRCO) have been obtained by reaction of N-alkoxycarbonylamino acids RIOCO-Xxx-OH with alkyl chloroformate R 2 0~0 C 1 (R2 = methyl, ethyl, isobutyl, menthyl) in the presence of N-methylmorpholine or N-methylpiperidine (NMP) in dichloromethane at -YC, followed by washing the mixtures with aqueous solutions and removal of the solvent. R'OCO-XXX-0-COOR~ (R2 = ethyl, tert-butyl) have been obtained by reaction of R'OCO-XXX-0-.+ HNMP with the corresponding dialkyl dicarbonates followed by the same work-up. Mixed anhydride generation is much slower when triethylamine is used as base. The initial reaction in the decomposition ofmixed anhydrides is cyclization to the 2-alkoxy-5(4H)-oxazolone (R1O-oxazolone) followed by release of alcohol R~O H .Symmetrical anhydride ( R ' O C O -X X X )~~ and ester R'OCO-Xxx-OR2 are also formed. The ease of cyclization depends on R2, and is in the order methylsulfonylethyl (Ms) >> methyl/ethyl/isobutyl >> menthyl. RIOCO-Xxx-0-COOMS preparations contained a substantial amount of R1O-oxazolone. Attempts to use this reaction as a preparative procedure for obtaining RIO-oxazolones failed because they are too sensitive to hydrolysis and react too quickly with the product, forming the symmetrical anhydride. The effect of various parameters on the amount of urethane generated by aminolysis at the carbonate moiety of R'OCO-Xxx-0-COOR~ was examined without the uncertainty that any urethane arose from arninolysis of unconsumed chloroformate.
A study of the factors contributing to urethane formation during the coupling of N-alkoxycarbonylamino acids with an amino acid ester by the mixed carboxylic–carbonic acid anhydride method has been carried out, using nuclear magnetic resonance spectroscopy and high-performance liquid chromatography for the quantitation of products. Urethane formation is associated primarily with reactions of activated hindered residues such as isoleucine and N-methylamino acids. Of prime importance in dictating the amount of urethane generated is the tertiary amine/solvent combination. N-Methylpiperidine/dichloromethane is the best combination for minimizing urethane formation, N-methylmorpholine/tetrahydrofuran is a good combination, while triethylamine/dichloromethane is a particularly bad one. In dimethylformamide, the differences between these amines are marginal. Aqueous dimethylformamide is a good solvent for mixed anhydride generation and coupling. A small excess of substrate reduces the amount of urethane. Less racemization accompanies the coupling of peptide acids in tetrahydrofuran than in halogen-containing solvents, N-methylpiperidine being the superior base in these solvents, but not in dimethylformamide. Racemization is reduced by one half when menthyl chloroformate instead of isobutyl chloroformate is used in the couplings.
Four series of diastereomeric peptide pairs (X-Lys, Lys-Y, Gly-X-Lys, and Gly-Lys-Y; where X and Y = Ala, Leu, Val, Ile, Phe, and Pro) have been synthesized by conventional procedures using benzyl-based protecting groups. Chromatographic separation of each pair, except Gly-Lys-Pro, has been achieved by elution from a 15-cm Aminex A-5 resin column using pH 5.5 buffer for tripeptides, pH 6.5 buffer for dipeptides, and pH 7.5 buffer for phenylalanyl peptides. The isomers have been identified by chromatography of the L-L isomers. Examples of the use of the model peptides for optical purity determinations and as tests for racemization are presented. The series Gly-X-Lys allows comparison of the tendencies to racemize of residues X during couplings. The separations of other peptides where Y = MeVal and X = N- and O-methylhydroxyamino acids are also described.
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