Solid Phase Synthesis of Phosphopeptides Incorporating 2,2-Dimethyloxazolidine Pseudoproline Analogs: Evidence for trans Leu-Pro Peptide Bonds in Stat3 Inhibitors

Abstract

To answer the question of whether the conformation of the Leu-Pro bond is cis or trans in Ac-pTyr-Leu-Pro-Gln-Thr-Val-NH2 when complexed with the SH2 domain of Stat3, we substituted 2,2-dimethyloxazolidines derived from serine (Ser(ΨMe,Mepro)) and threonine (Thr(ΨMe,Mepro)) for proline. The 2,2-dimethyloxazolidine and 2,2-dimethylthiazolidine pseudoproline (ΨPro) analogs induce predominantly cis Xxx-ΨPro peptide bonds. As these ΨPro analogs are acid-labile, the phosphopeptides were synthesized using Fmoc-based SPPS using unprotected phosphotyrosine and 4-hydroxybenzoate as the linker that allowed release from the support by alkaline ammonolysis, conditions that kept the oxazolidine rings intact. Incorporation of Ser(ΨMe,Mepro) resulted in 69% cis Leu-ΨPro bond content in aqueous solution whereas that for Thr(ΨMe,Mepro) analog was 63%. Affinities for Stat3 were 3–5 fold lower than the lead compound and were inversely correlated with cis content. Thus we conclude that the Leu-Pro peptide bond is trans when the peptide is bound to Stat3.