The Structural Requirements for an Inverse Substrate for Enzymatic Peptide Synthesis: Position Isomers of Guanidinonaphthyl Esters as the Acyl Donor Component

peptide synthesis. Enzymatic peptide synthesis is more advantageous than chemical synthesis since the enzymatic method is stereoselective and is secure from racemization. There are, however, several problems to be solved with this method. Secondary hydrolysis of the resulting peptide may arise from the inherent nature of the protease. Moreover, the method is limited to the use of amino acid derivatives which meet the enzymatic specificity as the coupling component. In our previous work, it was shown that p-amidinoand pguanidinophenyl esters behave as specific substrates for trypsin and trypsin-like enzymes. In these compounds the site-specific group for the enzyme, charged amidinium or guanidinum group, is not included in the acyl moiety but in the leaving group. Thus a new term, “inverse substrate”, was proposed for these esters. The esters provided a novel method for the specific introduction of an acyl group for a wide variety of acyl groups into a trypsin active site, and such acyl enzymes have proven to be useful for the peptide coupling reaction. p-(Guanidinomethyl)phenyl esters have proved especially useful for the enzymatic coupling of hindered a-amino acids. The combination of the use of m(guanidinomethyl)phenyl esters and Streptomyces griseus (SG) trypsin was also relevant for the coupling of some amino acids. Thus the design of a variety of inverse substrates can be one of the effective approaches for the development of trypsin-catalyzed peptide synthesis. In this paper, we report the preparation of four new position isomers of guanidinonaphthyl esters (Fig. 1), their kinetic properties toward spontaneous and enzymatic hydrolysis, and their application to enzymatic peptide coupling. Chemistry Aminonaphthol includes fourteen position isomers. Among them, four position isomers (4-amino-1naphthol (1), 5-amino-1-naphthol (6), 5-amino-2-naphthol (10), 6-amino-1-naphthol (14)) were selected as the starting material for the preparation of four groups of guanidinonaphthyl esters (Fig. 1). The synthetic procedure for each guanidinonaphthyl ester is shown in Chart 1. Aminonaphthol was reacted with 1-[N9,N 0-bis(Z)amidino]pyrazole. Pure [N9,N 0bis(Z)guanidino]naphthol was obtained in 58—98% yields. The physical and spectral data of the [N9,N 0-bis(Z)guanidino]naphthols are listed in Table 1. Condensation of Nblocked guanidinonaphthol with N-Boc-amino acids by using DCC and DMAP in a mixture of dioxane and CH2Cl2 was successful. Reaction yields of the esters were 75—92%, as shown in Table 2. The next deprotection step was carried out by catalytic hydrogenation to give N-Boc amino acid guanidinonaphthyl esters as the TsOH salts essentially in quantita104 Chem. Pharm. Bull. 47(1) 104—110 (1999) Vol. 47, No. 1