Enzymic synthesis of galactopyranosyl- l-serine derivatives using galactosidases

Abstract

Although the physiological role of the carbohydrate moieties in biologically active glycoproteins is still a subject of investigation, it is known that sugars extend the biological half-life of these molecules. Furthermore, the attachment of a sugar residue to a peptide sequence could improve the affinity of the molecule for its receptor. For this reason, several glycosylated enkephalins have been synthesisedlm3. Chemical syntheses of glycopeptides are well developed”. However, because carbohydrates contain multiple hydroxyl groups of similar reactivity, the chemical methods involved in regioselective synthesis require numerous protection and deprotection steps. In addition, stereospecific reactions that give the desired (r or /I) anomer are often difficult. Many enzymes are now available commercially and their routine use in synthesis5 is becoming accepted. Most glycosidases can transfer the glycosyl moiety of a substrate to acceptors other than water and, although they are less selective than glycosyl transferases, they have been used in the synthesis of glycosides’.‘. For example, D-Dgalactosidase catalyses transgalactosidation with monosaccharides, oligosaccharides, alkanols8~‘0, and phenols”. The reactions of E. c&p-D-galactosidase in the presence of various acceptors have been investigated, as have the structural requirements and reactivity”. Alkyl galactosides, lactose, and raffinose can be used as glycosyl donors with various primary alcohols as acceptors for the synthesis of dior tri-saccharide glycosides”-“. We now report on the use of lactose and P-D-galactosidase for the formation of 3-O-/?-D-galactopyranosyl-L-serine @‘-Gal-Ser). p-D-Galactosidase does not induce the synthesis of /I-Gal-Ser from lactose and serine, and no condensation occurs with the N-urethane derivative of the amino acid or the amino acid ester. Both the amino and the carboxyl groups of the amino acid have to be protected for transgalactosidation to take place. The choice of the amino blocking group is important. The best yields (15%) for transgalactosidation were observed with serine methyl esters N-protected by a tertbutoxycarbonyl group. With N-benzyloxycarbonylserine methyl ester, the yield was