A series of 24 peptides Z-Gly-Xaa(R)-OH where Xaa = 15 different residues and R = H, NH2, tBu, Bzl, Trt, Mtr, and StBu were coupled with valine benzyl ester in dimethylforniamide or dichloromethane at + 5". The accompanying racemization was determined by analysis of the epimeric products by normal phase highperformance liquid chromatography (HPLC) for Xaa(R) = Met, Cys(StBu) and Lys(Z) and by reversed-phase HPLC after removal of benzyl-based protecting groups for Xaa(R) = Ser(tBu), Thr(tBu) and Arg(Mtr). The coupling methods examined included mixed anhydride (MxAn) at -5 O , and N,N -dicyclohexylcarbodiimide (DCC), benzotriazol-1-yl-tris(dimethy1amino)phosphonium hexafluorophosphate (BOP) and O-benzotriazol-1-yl-N,N,N' ,N' -tetramethyluroniumhexafluorophosphate (HBTU) in the presence of 1-hydroxybenzotriazole (HOBt). Very few couplings gave stereochemically pure products. The order of sensitivity to racemization of residues depended on the method of coupling and the solvent. It varied most when comparing MxAn to HOBt-assisted reactions; it varied moderately when comparing HOBt-assisted reactions. There was less variation in comparing BOP and HBTU reactions that are initiated by the same mechanism. Leu, Nle, Phe, Asn, Lys(2) and Asp(0Bzl) are identified as the residues least sensitive to racemization. DCC-HOBt generally led to less epimerization than the other methods.

N‐tert‐Butoxycarbonylamino acids (Boc‐Xaa‐OH) were coupled with p‐nitrophenol (HONp) in dichloromethane using N,N′‐dicyclohexylcarbodiimide (DCC) and N‐ethyl‐N′(3‐dimethylaminopropyl)carbodiimide hydrochloride (EDC), and the products were identified and quantitated by high‐performance liquid chromatography. Boc‐Xaa‐OH with Xaa = Val was coupled also with pentafluorophenol (HOPf) and hydroxy‐containing additives (HOR), and the products were similarly determined as the methylamides. EDC‐mediated reactions of Boc‐Xaa‐OH gave 8–25% of Boc‐Xaa‐Xaa‐OR as well as Boc‐Xaa‐OR for R = Ph, Np, Pf, benzotriazole (Bt) and 5‐norbornene‐endo‐2,3‐dicarboxamide; DCC‐mediated couplings, 5–7% for R = Np and Bt. No dimer was formed in couplings with N‐hydroxysuccinimide or 3‐hydroxy‐4‐oxo‐3,4‐dihydro‐1,2,3‐benzotriazine. Dimerization was eliminated from DCC‐mediated reactions by the addition of 1 equiv. of N‐methylmorpholine, from the EDC‐mediated reactions by carrying them out in pyridine. Dimerization is attributed to formation of the intermediate 2‐alkoxy‐5(4H)‐oxazolone that undergoes fragmentation to the N‐carboxyanhydride, which reacts with the alcohol giving amino acid ester. Ester produces dimer by aminolysis of the O‐acylisourea. Decomposition (1.4%) was also detected by analysis for H‐Val‐Phe‐OMe in DCC‐mediated reactions of Boc‐valine with H‐Phe‐OMe, and was demonstrated to be caused by the hydrochloride of the ester salt that had been neutralized with N‐methylmorpholine. Decomposition was eliminated by the addition of 5%, of pyridine, which also had the beneficial effect of suppressing N‐acylurea formation.

Generation of a mixed anhydride using ethyl or isopropyl chloroformate and N‐methylmorpholine in dichloromethane at room temperature, followed by addition of excess ring‐substituted phenol or N‐substituted hydroxylamine and a catalytic amount of tertiary amine, provides an efficient synthesis of activated esters.

N‐9‐Fluorenylmethoxycarbonyl‐ (Fmoc) amino‐acid chlorides have been prepared by reaction of hydrogen chloride on purified mixed Fmoc‐amino acid‐monoalkyl carbonic acid anhydrides in dichloromethane. The products partially undergo subsequent conversion to the corresponding esters due to the presence of the liberated alcohol, the extent depending on the nature of the alkyl group. Esterification occurred to 5–20% when the alkyl group was isopropyl. Anhydrides of monoisopropenyl carbonic acid which liberate acetone instead of an alcohol gave products uncontaminated with ester. The three components in a reaction mixture could be determined as the reaction progressed by normal phase high‐performance liquid chromatography of aliquots, which had been freed of excess hydrogen chloride, on a μPorasil (underivatized silica) column using tert‐butanol‐hexane (1.5:98.5) as solvent.

The asymmetric induction occurring during aminolysis by an amino acid benzyl ester of the 5(4H)‐oxazolones obtained from N‐acyl‐dl‐valine for acyl = formyl, acetyl, benzoyl, trifluoroacetyl and A′‐benzyloxycarbonyl‐Xaa where Xaa = Gly, Ala and Leu in dichloromethane and dimethylformamide at + 5° and – 5° was determined by analysis of the epimeric products by high‐performance liquid chromatography after removal of protecting groups by hydrogenolysis. The influence of the side‐chain of the activated residue on induction was assessed by examining aminolysis of the 5(4H)‐oxazolones from N‐benzyloxycar‐bonyl giycyl‐Xaa‐OH for Xaa = Ala, Leu, Val, and Phe. The contribution of induction to the epimeric content of products obtained from couplings mediated by N,N′‐dicyclohexylcarbodiimide in the presence and absence of 1‐hydroxybenzotriazole, and by the mixed‐anhydride method, were calculated. The induction was affected at varying levels by the nature of the N‐acyl group, the side‐chain of Xaa, the nature of the aminolyzing nucleophile, the nature of the solvent, and the temperature, with diastereomeric excesses reaching – 32 and +53. The influence of the side‐chain of Xaa on the induction was different in the two solvents. For the N‐acyl series, the epimeric content of products did not always correctly reflect the relative tendencies of the derivatives to racemize. The order for epimeric content of the products also depended on the method of coupling.