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. cyclo^8.12[Asp⁸,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 (～2h) using the BOP reagent. Substitution of Ala¹² with d -Ala² and/or NH₂-terminal replacement (desNH₂-Tyr¹ or N-MeTyr¹) in the cyclo^8.12[Asp⁸,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 cyclo^8.12[Asp⁸,Ala¹⁵]-GRF(1-29)-NH₂ contains a long α-helical segment even in aqueous solution. A series of cyclo^8.12 stereoisomers containing d -Asp⁸ and/or d -Lys¹² were prepared and also found to be highly potent and to retain significant α-helical conformation. The high biological activity of cyclo^8.12[N-MeTyr¹,d -Ala²,Asp⁸,Ala¹⁵]-GRF(1-29)-NH₂ may be explained on the basis of retention of a preferred bioactive conformation.