Practical approach to enzymatic resolution of Fmoc- and Boc-protected amino acid racemates

Abstract

Enantiomers of protected non-protein amino acids are useful substrates in synthesis of peptide analogs for structure-activity relationships studies. Introduction of such amino acids to naturally occuring sequences of peptides modifies their primary properties like resistance to hydrolysis by proteolytic enzymes or/and modifies capability of binding to the receptor. The availability of non protein amino acids is the critical point in a such approach. The stereoselective synthesis and resolution (enzymatic or chemical) of racemic amino acids are two methods, most commonly used for non-protein amino acid synthesis. Although the majority of methods is mostly focused on the preparation of free amino acids, N -protected amino acids are used in the peptide synthesis. In order to obtain the N-protected amino acids used directly in peptide synthesis we have developed a method of resolution of N-protected racemic amino acids by selective enzymatic hydrolysis of their esters. In our experiments Bocand Fmoc-protecting groups were used as the most interesting for further application in peptide synthesis. Amino acids with such N-protecting groups and their alkyl esters are very lipophylic therefore reaction should be carried out in water containing high concentration of organic solvents. We have optimized reaction conditions for hydrolysis of Fmocor Boc-aromatic amino acid methyl esters catalyzed with α-chymotrypsin in water/acetonitrile solutions. Enzyme can be succesfuly adsorbed on solid phase (silica gel) without visible desorption in optimal reaction conditions what makes possible to carry out this reaction as a continous process.