Synthesis, biological activity and conformational analysis of cyclic GRF analogs

A novel cyclic GRF analog, cyclo(Asp8-Lys12)-Asp8,Ala15-GRF(1-29)-NH2, i.e. cyclo8,12Asp8,Ala15-GRF(1-29)-NH2, was synthesized by the solid phase procedure and found to retain significant biological activity. Solid phase cyclization of Asp8 to Lys12 proceeded rapidly (approximately 2 h) using the BOP reagent. Substitution of Ala2 with D-Ala2 and/or NH2-terminal replacement (desNH2-Tyr1 or N-MeTyr1) in the cyclo8,12Asp8,Ala15-GRF(1-29)-NH2 system resulted in highly potent analogs that were also active in vivo. Conformational analysis (circular dichroism and molecular dynamics calculations based on NOE-derived distance constraints) demonstrated that cyclo8,12Asp8,Ala15-GRF(1-29)-NH2 contains a long alpha-helical segment even in aqueous solution. A series of cyclo8,12 stereoisomers containing D-Asp8 and/or D-Lys12 were prepared and also found to be highly potent and to retain significant alpha-helical conformation. The high biological activity of cyclo8,12N-MeTyr1,D-Ala2,Asp8,Ala15-GRF(1-29)- NH2 may be explained on the basis of retention of a preferred bioactive conformation.