Synthesis, biological activity, and conformation of cyclic growth hormone releasing factor analogs

Growth hormone-releasing factor, GRF(1-44)-NH2, has been shown to stimulate growth hormone release in vitro and in vivo in humans and a variety of animal species. Preliminary clinical studies have shown that GRF(1-44)-NH2 can be used for the treatment of growth hormone deficient children. Structure-activity studies have shown that the shortened analog, GRF (1-29)-NH2, retains the full intrinsic activity with only slightly reduced potency. Replacement analogs of GRF(1-29)-NH2 have resulted in peptides with increased potency which are being used for performance enhancement applications in domestic livestock (pigs and lactating cows). Conformational analysis (molecular dynamics calculations based on NOE-derived distance constraints) has been carried out to correlate bioactivity with conformation. These studies have led to the design and synthesis of i-(i+4) cyclic analogs of GRF(1-29)-NH2. A solid phase method for the synthesis of the cyclic peptides was developed using an orthogonal protection scheme. This included conventional N alpha-Boc-benzyl protection and the incorporation of side-chain 9-fluorenylmethyl (-OFm) ester protection of Asp and N epsilon-Fmoc protection of Lys. Following specific deprotection of Asp(OFm) and Lys(Fmoc) using piperidine, solid phase side chain to side chain lactamization was carried out most efficiently with the BOP reagent. The high biological activity of the resultant cyclo [Asp, Ala]-GRF(1-29)-NH2 system may be explained on the basis of retention of a preferred bioactive conformation which includes substantial alpha-helicity in H2O between residues 7-17 and 21-15. The alpha-helical conformation of these cyclo peptides was only minimally disrupted with either stereoisomeric or isosteric replacements and the corresponding peptides retained substantial biological activity. The cyclic peptides were also more stable to dipeptidylpeptidase degradation than the linear analogs.