Influence of Nε-Protecting Groups on the Protease-Catalyzed Oligomerization of L-Lysine Methyl Ester

The direct oligomerization of L-lys-OMe by bromelain catalysis gave oligo(L-lys) with DPavg similar to 3.6 and dispersity similar to 1.1. Since higher chain length oligo(L-lys) with lower dispersity values and one reactive amine for selective conjugation would be beneficial, we explored protease-catalyzed oligomerization of N-epsilon-protected L-lys monomers where N-epsilon-groups included tert-butoxycarbonyl (Boc) or benzyloxycarbonyl (Z) groups. By using N-epsilon-protected L-lys monomers, oligopeptide side-chains are hydrophobic-neutral which should dramatically alter enzyme kinetic and binding constants relative to nonprotected L-lys. Schechter and Berger's conceptual model guided our choice of papain as the protease catalyst. Papain-catalyzed oligomerization of N-epsilon-Boc-L-Lys-OMe gave products with DPavg values that were pH dependent and varied from 4.7 +/- 0.2 to 7.5 +/- 0.1. Similarly, oligo(N-epsilon-Z-L-Lys) synthesis was pH dependent, and DPavg values varied from 4.3 +/- 0.2 to 5 +/- 0.2. Oligo(N-epsilon-Boc/Z-L-Lys) that precipitates from reaction media had a low dispersity (similar to 1.01). The relatively smaller N-epsilon-Boc group should increase propagating chain solubility enabling oligopeptides to reach higher DPavg values prior to their precipitation. Since papain-catalyzed oligomerizations of N-epsilon-Boc/Z-L-Lys proceeded slowly at 0.54 mg/mL, higher enzyme concentrations were studied. By increasing the enzyme concentration in oligomerizations from 0.54 to 1.62 mg/mL for 3 h reactions, the %-yield and DPavg of oligo(N-epsilon-Z-L-lys) increased from 24 +/- 0 to 88 +/- 2 and 4.1 +/- 0.7 to 5.7 +/- 0.1, respectively. Furthermore, at 1.89 mg/mL papain, the %-yield of oligo(N-epsilon-Z-L-lys) increased with time reaching 91% in 2 h. Acetonitrile at 20%-by-volume was a useful cosolvent that increased the oligopeptide yield and DPavg relative to reactions run in pure buffer.