Orthogonal solid-phase synthesis of human gastrin-I under mild conditions.
An approach to the solid-phase segment condensation synthesis of the 17-peptide amide human gastrin-I has been developed. N alpha-amino and side-chain protection were provided by 9-fluorenylmethyloxycarbonyl (Fmoc) and tert.-butyl groups, and a series of anchors cleavable under mild conditions were used. The N-terminal pentapeptide pGlu-Gly-Pro-Trp-Leu-OH was prepared using a p-alkoxybenzyl ester linkage made by a preformed handle strategy. Cleavage, in 65% yield, was with the new Reagent M: CF3COOH-CH2Cl2-beta-mercaptoethanol--anisole (70:30:2:1), which was optimized to preserve the labile tryptophan residue. A new preformed handle procedure expedited solid-phase synthesis of the protected "middle" hexapeptide, Fmoc-(Glu(OtBu]5-Ala-OH, anchored as an o-nitrobenzyl ester. Chains were not lost during this assembly, and final photolytic cleavage (350 nm) in toluene--CF3CH2OH (4:1) occurred in 59% yield. Both protected intermediates were purified by simple gel filtration, whereupon they were shown to be pure by analytical HPLC, and gave satisfactory NMR and FABMS spectra. Last, the C-terminal hexapeptide, Tyr(tBu)-Gly-Trp-Met-Asp(OtBu)-Phe, was assembled on a tris(alkoxy)benzylamide "PAL" support. For the polymer-supported segment condensation, the middle and N-terminal pieces were added respectively in greater than 98% and 89% yields (judged by amino acid analysis and solid-phase sequencing), by overnight couplings in N,N-dimethylformamide (DMF) mediated by benzotriazolyl N-oxytrisdimethylaminophosphonium hexafluorophosphate (BOP) in the presence of 1-hydroxybenzotriazole (HOBt) and N-methylmorpholine (NMM). Racemization was 4% and 11% respectively at Ala and Leu. Cleavage with Reagent M followed by reversed-phase chromatography gave pure gastrin-I in an overall 30% isolated yield. These results compare favorably with those from a stepwise assembly.
Department of Chemistry, University of Minnesota, Minneapolis.
N
N
Kneib-Cordonier
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F
Albericio
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G
Barany
D000595:Amino Acid Sequence; D055598:Chemical Phenomena; D002621:Chemistry; D005755:Gastrins / Q000138:chemical synthesis*; D006801:Humans; D007202:Indicators and Reagents; D008969:Molecular Sequence Data
D005755:Gastrins; D007202:Indicators and Reagents; C036227:gastrin I