AMIDATION OF GROWTH-HORMONE RELEASING-FACTOR (1-29) BY SERINE CARBOXYPEPTIDASE CATALYZED TRANSPEPTIDATION

The applicability of serine carboxypeptidase catalysed transpeptidation reactions, using amino acid amides as nucleophiles, for C-terminal amidation of peptides has been investigated. With the aim of converting an unamidated precursor into GRF(1-29)-NH2, an interesting biologically active derivative of growth hormone releasing factor, a number of model reactions were initially investigated. In such a transpeptidation reaction. where the C-terminal amino acid is replaced by the amino acid amide, used as nucleophile, the C-terminal amino acid residue of the substrate can be chosen freely since it functions as leaving group and does not constitute part of the product. Since the C-terminal sequence of GRF(1-29)-NH2 is-Met-Ser-Arg-NH2 the model reactions Bz-Met-Ser-X-OH (X = Ala, Leu, Arg) + H-Arg-NH2 --> Bz-Met-Ser-Arg-NH2 + H-X-OH were first studied. With carboxypeptidase Y and X = Ala or Leu the amidated product could be obtained of 98 % and 41 %, respectively. With carboxypeptidase W-II and X = Arg a yield of no more than 72 % could be obtained. The choice of Ala as leaving group in combination with carboxypeptidase Y therefore appeared optimal. With the longer peptide Bz-Leu-Gln-Asp-Ile-Met-Ser-Ala-OH the amidated product could be obtained in a yield of 78 %, using carboxypeptidase Y, the only other product being Bz-Leu-Gln-Asp-Ile-Met-Ser-OH, formed due to the competing hydrolysis reaction. The full length peptide GRF(1-28)-Ala-OH was synthesized by the continuous flow polyamide solid-phase method. With this peptide the yield of amidation was 87 % and the remaining products were GRF (1-28)-OH and GRF(1-28)-Arg-Arg-NH2. However, these can be separated from the desired product by ion exchange chromatography. The results demonstrate that it is feasible to use the generally stable and easily accessible serine carboxypeptidase for C-terminal amidation of peptides, e.g. produced by fermentation of genetically manipulated microorganisms.