CONVERGENT SOLID-PHASE PEPTIDE-SYNTHESIS - AN EFFICIENT APPROACH TO THE SYNTHESIS OF HIGHLY REPETITIVE PROTEIN DOMAINS

Convergent solid phase peptide synthesis is a straightforward approach to the synthesis of protein domains with repetitive structures. The peptide H-(Val-His-Leu-Pro-Pro-Pro)(8)-OH corresponding to the N-terminal domain of gamma-zein has been synthesized using the protected segment Fmoc-(Val-His(Trt)-Leu-Pro-Pro-Pro)-OH as synthetic precursor. Solid phase repetitive coupling of the segment was achieved effectively using a synthetic protocol centered on the use of the new 7-aza-1-hydroxybenzotriazole-based reagents HATU and HOAt. The protected peptide segment Fmoc-Val-His(Trt)-Leu-Pro-Pro-Pro-OH was synthesized on a solid-phase using a combination of the base-labile Fmoc group for the alpha-amino protection, the acid-labile trityl group for the protection of the side-chain of histidine, and the highly acid-labile 4-(4-(hydroxymethyl)-3-methoxyphenoxy)butyric acid (HMPB) as handle, which allows the cleavage of the protected segment from the resin by treatment with 1% TFA in CH2Cl2 with complete retention of the amino acid side-chain protecting groups. This approach also allows the same peptide sequence at different levels of protection to be obtained by varying the cleavage program used and demonstrates that the versatile HMPB handle is completely compatible with the use of the trityl group for protection of the side-chain of histidine. The use of a small quantity of pyridine in the purification of this protected peptide by RPMPLC does not affect the Fmoc group and avoids premature imidazole detritylation. Finally, the general synthetic strategy described in this paper avoids the severe problems previously reported on the synthesis of these highly repetitive structures as demonstrated by a careful analysis of the target compounds by cation-exchange HPLC and electrospray MS.