Solid-phase assembly of backbone amide-protected peptide segments: an efficient and reliable strategy for the synthesis of small proteins

Abstract

In the preceding paper we showed that epimerization of the C-terminal residue of a fully protected peptide segment was minimized when activation (and subsequent coupling) was performed using 1-hydroxybenzotriazole-catalysed reaction with diisopropylcarbodiimide in dichloromethane (DCM). Good solubility of the segment in DCM was ensured by the incorporation of appropriately placed N-(2-acetoxy-4-methoxybenzyl)(AcHmb) backbone amide protection into the fully protected peptide. This low-epimerization protocol, combined with our previously described straightforward purification of AcHmb-substituted fully protected segments, provides an efficient, reliable and flexible strategy for the solid-phase assembly of small proteins. We describe here the preparation of HIV-1_Brutat[1–72, Cys(Acm)^{22,25,27,30,31,34,37}] protein through the sequential solid-phase assembly of five backbone-protected segments (11–17 residues). Individual addition of each segment was very efficient, achieving > 95% acylation using a two-fold excess with reaction for 6 h. The target 72-mer was readily purified and isolated in 38.4% overall yield.