Comparison of enzymatic semisyntheses of peptide amides: human growth hormone releasing factor and analogs.
Enzymatic semisyntheses of growth hormone releasing factor (GRF), a 44-residue peptide amide hormone, from C-terminal acid precursors, are compared. A recombinant alpha-amidating enzyme was used to convert the glycine-extended precursor, GRF(1-44)-Gly-OH, to GRF(1-44)-NH2 in an essentially quantitative fashion. Trypsin was used to convert the precursors, GRF(1-43)-OH and GRF(1-44)-OH, to GRF(1-44)-NH2 (60 and 15% conversion, respectively) in a 75% v:v N,N'-dimethylacetamide solution containing a large excess of leucine amide. Carboxypeptidase Y catalyzed transpeptidations of the precursors, GRF(1-44)-OH and [Ala44]-GRF(1-44)-OH, to GRF(1-44)-NH2 in aqueous leucine amide solutions were also attempted. The trypsin catalyzed direct amidation of [Ala15]-GRF(1-29)-OH in concentrated ammonium acetate/ammonia buffer (95% 1,4-butanediol cosolvent) to form the superactive analog, [Ala15]-GRF(1-29)-NH2 (ca. 25% conversion at equilibrium), is also described.
Bongers
J
J
Department of Peptide Research, Hoffmann-La Roche Inc., Nutley, NJ 07110.
Offord
R E
RE
Felix
A M
AM
Lambros
T
T
Liu
W
W
Ahmad
M
M
Campbell
R M
RM
Heimer
E P
EP
D000595:Amino Acid Sequence; D002268:Carboxypeptidases / Q000737:chemistry; D013007:Growth Hormone-Releasing Hormone / Q000031:analogs & derivatives* / Q000138:chemical synthesis* / Q000737:chemistry; D006801:Humans; D006899:Mixed Function Oxygenases / Q000737:chemistry; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D011498:Protein Precursors / Q000138:chemical synthesis / Q000737:chemistry; D014357:Trypsin / Q000737:chemistry
D009097:Multienzyme Complexes; D011498:Protein Precursors; D013007:Growth Hormone-Releasing Hormone; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D002268:Carboxypeptidases; C022633:serine carboxypeptidase; D014357:Trypsin