<i>In situ</i> neutralization in Boc‐chemistry solid phase peptide synthesis

TLDR

Boc‑chemistry solid phase peptide synthesis on polystyrene resins has been streamlined by simple, effective protocols for both manual and machine‑assisted synthesis. The method employs in situ neutralization during coupling, high‑concentration Boc‑amino acid‑OBt esters with base, 100 % TFA for rapid Boc removal, a 30‑second DMF flow wash, 10‑minute coupling steps, yielding 15‑minute manual and 19‑minute machine‑assisted cycles (≈75 residues/day). The protocol shows no racemization, eliminates key side reactions, markedly improves chain‑assembly efficiency for difficult sequences, and has successfully produced HIV‑1 protease fragments and the full‑length protease.

Abstract

Simple, effective protocols have been developed for manual and machine‐assisted Boc‐chemistry solid phase peptide synthesis on polystyrene resins. These use in situ neutralization [i.e. neutralization simultaneous with coupling], high concentrations (&gt; 0.2 M) of Boc‐amino acid‐OBt esters plus base for rapid coupling, 100% TFA for rapid Boc group removal, and a single short (30 s) DMF flow wash between deprotection/coupling and between coupling/deprotection. Single 10 min coupling times were used throughout. Overall cycle times were 15 min for manual and 19 min for machine‐assisted synthesis (75 residues per day). No racemization was detected in the base‐catalyzed coupling step. Several side reactions were studied, and eliminated. These included: pyrrolidonecarboxylic acid formation from Gln in hot TFA‐DMF; chain‐termination by reaction with excess HBTU; and, chain termination by acetylation (from HOAc in commercial Boc‐amino acids). The in situ neutralization protocols gave a significant increase in the efficiency of chain assembly, especially for “difficult” sequences arising from sequence‐dependent peptide chain aggregation in standard (neutralization prior to coupling) Boc‐chemistry SPPS protocols or in Fmoc‐chemistry SPPS. Reported syntheses include HIV‐1 protease(1–50,Cys.amide), HIV‐1 protease(53–99), and the full length HIV‐1 protease(1–99).