Solution synthesis and biological activity of human pleiotrophin, a novel heparin-binding neurotrophic factor consisting of 136 amino acid residues with five disulfide bonds

Human pleiotrophin (hPTN), a novel heparin-binding neurotrophic factor cons isting of 136 amino acid residues with five intramoleular disulfide bonds, was synthesized by solution procedure in order to demonstrate the utility o f our strategy using our newly developed solvent system, a mixture of trifl uoroethanol (TFE) and dichloromethane (DCM) or chloroform (CHL). The final protected peptide was synthesized by coupling two larger protected intermed iates, Boc-(1-64)-OH and H-(6 5-136)-OBzl, in CHL/TFE (3:1; v/v) using 1-et hyl-3(3-dimethylam inopropyl)-carbodiimide (EDO in the presence of 3,4-dihy dro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBt). After removal of all prote cting groups using the HF procedure followed by treatment with Hg(OAc)(2), the fully deprotected peptide was subjected to an oxidative folding reactio n. The product was confirmed as having the correct disulfide structure by e xamining the cystine peptides obtained by enzymatic digestions, and as poss essing the same biological activities as those of the natural product. The N- and C-terminal half domains (1-64 and 65-136) were also synthesized, and measurement of their biological activities indicated that the C-terminal h alf domain displays almost all the activities of the full-length molecule, whereas the N-terminal half domain shows almost no activity. From these res ults, we were able to confirm that the C-terminal half domain is responsibl e for the expression of biological activities in the same manner as human m idkine (hMK), another heparin-binding neurotrophic growth factor.