a-Chymotrypsin-catalyzed peptide synthesis using N-protected D-amino acid carbamoylmethyl esters as acyl donors Sayed Mohiuddin Abdus Salam, Ken-ichi Kagawa & Katsuhiro Kawashiro* Department of Chemical Science and Technology, Faculty of Engineering, The University of Tokushima, 770-8506, Minamijosanjima, Tokushima, Japan *Author for correspondence (Fax: 81-88-655-7025; E-mail: kawasiro@chem.tokushima-u.ac.jp) Received 13 April 2005; Revisions requested 28 April 2005; Revisions received 7 June 2005; Accepted 10 June 2005 Key words: D-amino acid, carbamoylmethyl ester, a-chymotrypsin, peptide synthesis Abstract a-Chymotrypsin-catalyzed peptide synthesis was carried out between an N-protected D-amino acid ester and an L-amino acid amide (acyl donor, 10 mM; acyl acceptor, 50 mM; enzyme, 2 mg ml )1 ; pH 8). By using a highly reactive carbamoylmethyl (Cam) ester as acyl donor, the D-amino acid was incorporated into the N-terminus of the resulting dipeptide amide. N-Protected dipeptide amides bearing D-amino acids such as D-Phe, D-Leu and D-Ala at their N-terminus were synthesized in high yields (up to 80%) in 1–3 h. Introduction The advantages of enzymatic peptide synthesis are free from racemization, minimum activation and mild reaction (Wong & Whitesides 1994; Bordusa, 2002). With these advantages, however, comes the disadvantage that the substrates of proteases are generally limited to natural L-ami- no acids, consistent with normal recognition properties of proteases. Particularly, proteases are specific to L-amino acids as the P 1 residue (the acyl donor), although the P 1 ¢ residue (the acyl acceptor) is more flexible and both L- and D- amino acids are acceptable. The enzymatic incor- poration of D-amino acids into peptides is of interest because of their prevalence in various biologically active compounds. The enantioselectivity of serine protease catal- ysis can be altered by the activation of acyl do- nor and the reaction medium (Broos et al. 1995; Kawashiro et al. 1996). The enantioselectivity of subtilisin, for example, changed dramatically upon a transition from aqueous solution to anhydrous tert-amyl alcohol (Margolin et al. 1987). These authors were the first to incorporate D-amino acids by using N-acetyl-D-amino acid 2- chloroethyl esters as acyl donors and L -amino acid amides as acyl acceptors. We have observed that porcine pancreatic lipase also can incorpo- rate D-Ala into the N-terminus of the resulting dipeptide amide even in aqueous DMSO (50%, v/v) (Kawashiro et al. 1993). Recently, Miyazawa et al. (2001) found that a-chymotrypsin-catalyzed incorporation of D-Ala to the N-terminus of the resulting dipeptide amide can take place by the use of the carbamoylmethyl (Cam) ester as acyl donor in acetonitrile with a low water content. These findings suggest that the activation of ester moiety of acyl donor plays an important role for broadening the enantioselectivity of an enzyme. However, these peptide syntheses suffer from a prolonged reaction time due to the presence of an anhydrous organic solvent (Margolin et al. 1987) or a rather low yield due to unfavorable hydrolysis of acyl donor (Kawashiro et al. 1993; Miyazawa et al. 2001). The present communication describes the fac- ile enzymatic incorporation of a D-amino acid into the N-terminus of the resulting dipeptide in aqueous media. Biotechnology Letters (2005) 27: 1199–1203 Ó Springer 2005 DOI 10.1007/s10529-005-0018-8