Amidation of growth hormone releasing factor (1–29) by serine carboxypeptidase catalysed 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 carboxy‐peptidase for C‐terminal amidation of peptides, e.g. produced by fermentation of genetically manipulated microorganisms.