Enzymatic semisynthesis of a superpotent analog of human growth hormone-releasing factor.
A superpotent analog of human growth hormone-releasing factor, [desNH2Tyr1,D-Ala2,Ala15]-GRF(1-29)-NH2 (4), was prepared from the precursor, [Ala15,29]-GRF(4-29)-OH (1), by a two-step enzymatic semisynthesis. The amidated C-terminus, essential for high biological potency, was obtained via a carboxypeptidase Y-catalyzed exchange of Ala29-OH for Arg29-NH2 to produce [Ala15]-GRF(4-29)-NH2 (2). The N-terminal desNH2Tyr-D-Ala moiety, which greatly increases in vivo duration of action, was then incorporated by V8 protease-catalyzed condensation of segment 2 with desNH2Tyr-D-Ala-Asp(OH)-OR [R = CH3CH2- (3a) or 4-NO2C6H4CH2-(3b)]. The main focus of this report was to develop conditions to use the V8 protease-catalyzed coupling while avoiding a competing cleavage of the proteolytically-sensitive Asp25-Ile26 bond in GRF. Conversion of 2 to 4 in couplings employing the alpha-ethyl ester of the acyl component 3a was limited to about 60% by competing proteolysis at Asp25-Ile26. This system was adequate for preparing, isolating, and fully characterizing the target analog 4 and identifying the side products. The 4-nitrobenzyl ester 3b proved to be a superior substrate, resulting in 90% conversion of 2 to 4 with no detectable loss to proteolysis and requiring significantly lesser amounts of catalyst. These results demonstrate that enzymatic semisynthesis of a biologically-active peptide amide which contains unnatural amino acids at the N-terminus can be achieved from a biosynthetic precursor in good yield and purity.
Bongers
J
J
Department of Peptide Research, Roche Research Center, Hoffmann-La Roche Inc., Nutley, New Jersey 07110.
Lambros
T
T
Liu
W
W
Ahmad
M
M
Campbell
R M
RM
Felix
A M
AM
Heimer
E P
EP
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis; D012697:Serine Endopeptidases / Q000737:chemistry; D017337:Sermorelin / Q000031:analogs & derivatives* / Q000737:chemistry
D010446:Peptide Fragments; C077568:somatotropin releasing hormone (1-29) NH2, desNH2Tyr(1)-Ala(2,15)-; D017337:Sermorelin; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase