A novel cyclic GRF analog, cyclo(Asp8‐Lys12)‐[Asp8,Ala15]‐GRF(1‐29)‐NH2, i.e. cyclo8.12[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 cyclo8.12[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 cyclo8.12[Asp8,Ala15]‐GRF(1‐29)‐NH2 contains a long α‐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 α‐helical conformation. The high biological activity of cyclo8.12[N‐MeTyr1,d‐Ala2,Asp8,Ala15]‐GRF(1‐29)‐NH2 may be explained on the basis of retention of a preferred bioactive conformation.
The utility of repetitive nonhydrolytic base cleavage of α‐amino protective groups in solid phase peptide synthesis is shown by a preparation of the model tetrapeptide leucyl‐alanyl‐glycyl‐valine on a p‐benzyloxybenzyl ester polystyrene–1% divinylbenzene resin support. Nα‐9‐Fluorenylmethyloxycarbonyl (Fmoc: Carpino & Han, 1970, 1972) amino acids were coupled by the symmetrical anhydride procedure, followed by Fmoc group cleavage using 50% piperidine in methylene chloride. Quantitative removal of the Fmoc‐tetrapeptide from the solid support was effected by treatment with 55% trifluoroacetic acid in methylene chloride. Homogeneous free tetrapeptide was obtained in 87% overall yield. The procedure is proposed to offer advantages over present solid phase methods which use acidolysis for repetitive α‐amino group deblocking.
The utility of repetitive nonhydrolytic base cleavage of a-amino protective groups in solid phase peptide synthesis is shown by a preparation o f the model tetrapeptide leucyl-alanyl-glycyl-valine on a p-benzyloxybenzyl ester polystyrene-I % divinylbenzene resin support. NOL-9-Fluorenylmethyloxycarbonyl (Fmoc: Carpino & Han, 1970 amino acids were coupled by the symmetrical anhydride procedure, followed by Fmoc group cleavage using 50% piperidine in methylene chloride. Quantitative removal of the Fmoc-tetrapeptide from the solid support was effected by treatment with 5S% trifluoroacetic acid in methylene chloride. Homogeneous free tetrapeptide was obtained in 87% overall yield. The procedure is proposed to offer advantages over present solid phase methods which use acidolysis for repetitive a-amino group deblocking.Key words: p-benzyloxybenzylester resin support; Na-9-fluorenylmethyloxycarbonylam~~ acids; nonhydrolytic base cleavage; solid phase synthesis; trifluoroacetic acid cleavage of peptide from resin.
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