Sequence-assisted peptide synthesis (SAPS).

Abstract

In solid-phase peptide synthesis (SPPS) the growing peptide chain may undergo chain aggregation which can cause serious synthetic problems. A number of investigations concerning this problem have been reported in the chemical literature. During a study of such "difficult sequences" using the Fmoc-protection strategy, we have observed that peptide-chain aggregation may be significantly reduced when certain amino acid sequences are incorporated C-terminally (1). Thus, synthesis of the difficult poly-alanine, (Ala)n, sequence (n < or = 20) has been investigated with (Lys(Boc))m (m < or = 6) and (Glu(tBu))m (m > or = 6) as pre-sequences. With m > or = 3, peptides are obtained as single, homogeneous products while a complex mixture of deletion peptides and corresponding Fmoc-protected peptides is formed (n > or = 6) without the pre-sequence. A mixed pre-sequence, (Lys(Boc)Glu(tBu))3, has a similar favorable effect on the synthetic results, but the positive effect seems confined to a rather narrow framework of amino acids and side-chain protecting groups in the pre-sequence as discussed in the article. Among other reputedly difficult sequences the synthesis of H-(Thr-Val)5-OH, H-Val-Asn-Val-Asn-Val-Gln-Val-Gln-Val-Asp-OH, the Acyl Carrier Protein (65-74) and the human insulin B-chain has been investigated. In all cases introduction of a pre-sequence gives rise to satisfactory synthetic results. In the latter case, the lysine pre-sequence may be cleaved enzymatically to give the desB30 insulin B-chain. NIR-FT Raman studies of the synthesis of the poly-alanine, (Ala)n, sequences have shown that the pre-sequence (Lys(Boc))6 shifts the conformation of the growing peptide chain from a beta-structure (n > or = 6) to a random coil conformation (1c). This result is in agreement with the general observation that SPPS proceeds optimally under random coil conditions.