REVERSIBLE ENZYMATIC PROTECTION OF THE ALPHA-AMINO GROUP OF AMINO-ACID DERIVATIVES USING AN AMINOPEPTIDASE-A(1)

In previous papers we have reported that an aminopeptidase A (EC 3.4.1 1.7) purified from Staphylococcus chromogenes was able to catalyse the introduction Of L-malic acid at the N-terminus of Tyr and Phe derivat ives. We now show that this enzyme can be used for selective alpha-ami no protection of derivatives of probably all amino acids, except Gly a nd Pro, by the malyl group. The following L-malyl derivatives were syn thesized in thermodynamically controlled reactions with yields ranging from 4 to 47%: L-malyl-Tyr-OEt, -ALA-OMe, -Ser-OEt, -Lys-OEt, -Phe-OM e, -Met-NH2, -Glu-MH2, Arg-NH2, -Tye-NH2, -Val-NH2, -Ala-Phe and -Ala- Phe-NH2 (OEt and OMe are ethyl and methyl esters respectively). The re actions were monitored by reverse-phase h.p.l.c.; the products were qu antified by amino acid analysis, and their structure was confirmed by m.s. No synthesis was obtained with Gly and Pro derivatives as nucleop hiles. The effects of pH, temperature, enzyme concentration, nucleophi le concentration, reaction time and addition of an organic co-solvent were studied. An important shift towards synthesis was obtained by car rying out the reactions at 55-degrees-C in the presence of 55% organic co-solvent Triglyme (2,4,8,11-tetraoxadodecane) [8-60-fold increase i n K(syn) ([product] [acyl-donor]-1 [nucleophile]-1)].