Application of tryptic condensation strategy for the synthesis of alpha-melanocyte stimulating hormone.
The use of trypsin in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) paired with N,N-dimethylformamide (DMF) has been proposed for the enzymatic condensation of peptides. The tryptic condensation strategy was applied to the synthesis of the 13-peptide, alpha-melanocyte stimulating hormone (alpha-MSH), which contains two susceptible points to trypsin, in a low-water-containing solvent system, 4% H2O in HFIP/DMF (1/1, v/v). The N-terminal 8-peptide segment (S1) ending with Arg and C-terminal 5-peptide with the side-chain protections at Lys and Trp (S2) were prepared by the stepwise coupling on p-nitrobenzophenone oxime resin and by a conventional method, respectively. Both segments were condensed by the aid of trypsin. The acid component was converted into the partially protected alpha-MSH at as high as 95% conversion determined by reversed-phase HPLC. When the side-chain of Lys at the 11-position was not protected, alpha-MSH was obtained only in 35% yield, and was contaminated with products of secondary hydrolysis. Although the Lys11-Pro12 sequence was very poorly susceptible to trypsin, the side-chain of Lys had to be protected in order to be inert to trypsin under the synthetic conditions. HFIP is demonstrated as a good solvent with DMF to allow the efficient tryptic condensation of peptides. The strategy increased the value of the enzymatic condensation as practical method by avoidance of secondary hydrolysis, high dissolution of peptides and retention of activity of enzyme.
Department of Applied Chemistry, Faculty of Engineering, Kyushu Institute of Technology, Kitakyushu, Japan.
H
H
Mihara
M
M
Xu
N
N
Nishino
T
T
Fujimoto
D000433:1-Propanol / Q000737:chemistry; D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry; D004126:Dimethylformamide / Q000737:chemistry; D006868:Hydrolysis; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry; D020005:Propanols; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism*; D000521:alpha-MSH / Q000138:chemical synthesis*; D006845:Hydrocarbons, Fluorinated
D000433:1-Propanol; D001120:Arginine; D004126:Dimethylformamide; D008239:Lysine; D010446:Peptide Fragments; D020005:Propanols; D014357:Trypsin; D000521:alpha-MSH; C001337:hexafluoroisopropanol; D006845:Hydrocarbons, Fluorinated