Influence of N_ε‑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 DP_avg ∼ 3.6 and dispersity ∼ 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_ε-protected l-lys monomers where N_ε-groups included <i>tert</i>-butoxycarbonyl (Boc) or benzyloxycarbonyl (Z) groups. By using N_ε-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_ε-Boc-l-Lys-OMe gave products with DP_avg values that were pH dependent and varied from 4.7 ± 0.2 to 7.5 ± 0.1. Similarly, oligo­(N_ε-Z-l-Lys) synthesis was pH dependent, and DP_avg values varied from 4.3 ± 0.2 to 5 ± 0.2. Oligo­(N_ε-Boc/Z-l-Lys) that precipitates from reaction media had a low dispersity (∼1.01). The relatively smaller N_ε-Boc group should increase propagating chain solubility enabling oligopeptides to reach higher DP_avg values prior to their precipitation. Since papain-catalyzed oligomerizations of N_ε-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 DP_avg of oligo­(N_ε-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_ε-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 DP_avg relative to reactions run in pure buffer