Traditionally, solid-phase synthesis has relied on polystyrene-based resins for the synthesis of all kinds of peptides. However, due to their high hydrophobicity, these resins have certain limitations, particularly in the synthesis of complex peptides, and in such cases, poly(ethylene glycol) (PEG)-based resins are often found to give superior results. Another powerful strategy for expediting the assembly of complex peptides is to employ pseudoproline dipeptides. These derivatives disrupt the interactions among chains that are usually the cause of poor coupling yields in aggregated sequences. Here we report on an efficient stepwise solid-phase synthesis of RANTES (1-68) by combining the advantages of the totally PEG-based ChemMatrix resin and pseudoproline dipeptides.
A study of the factors contributing to urethane formation during the coupling of N-alkoxycarbonylamino acids with an amino acid ester by the mixed carboxylic–carbonic acid anhydride method has been carried out, using nuclear magnetic resonance spectroscopy and high-performance liquid chromatography for the quantitation of products. Urethane formation is associated primarily with reactions of activated hindered residues such as isoleucine and N-methylamino acids. Of prime importance in dictating the amount of urethane generated is the tertiary amine/solvent combination. N-Methylpiperidine/dichloromethane is the best combination for minimizing urethane formation, N-methylmorpholine/tetrahydrofuran is a good combination, while triethylamine/dichloromethane is a particularly bad one. In dimethylformamide, the differences between these amines are marginal. Aqueous dimethylformamide is a good solvent for mixed anhydride generation and coupling. A small excess of substrate reduces the amount of urethane. Less racemization accompanies the coupling of peptide acids in tetrahydrofuran than in halogen-containing solvents, N-methylpiperidine being the superior base in these solvents, but not in dimethylformamide. Racemization is reduced by one half when menthyl chloroformate instead of isobutyl chloroformate is used in the couplings.
Side-chain protected peptidyl amides, peptidyl tert-butyl esters, and the novel peptidyl 4-[ N-(oxyacety1)-p-alaninamidel-benzyl ester (HMP-p-Ala-NH2 ester) have been synthesised in excellent yields o n N-Fmoc-9-amino-xanthen-3-yloxymethyl polystyrene resin and selectively cleaved f r o m the solid support using 1% trifluoroacetic acid in dichloromethane within 7-8 min.
The mixture of N‐alkoxycarbonyl‐ or N‐acylamino acid substrate and corresponding N‐methylated product is analyzed by HPLC on a μBondapak‐C18 column with acidic aqueous methanol as eluent and u.v.‐absorbance monitoring.
The separations by reversed phase high-performance liquid chromatography on a pBondapak-C,, column of 53 epimeric N-substituted di-, tri-and tetrapeptide acids and esters have been attempted, with success in three quarters of the cases. Substituents include acetyl, benzoyl, benzyloxycarbonyl, tert.-butoxycarbonyl and 9fluorenylmethoxycarbonyl. The series N-benzyloxycarbonylglycyl-Xxx-valine ethyl ester with Xxx = alanyl, valyl, leucyl and phenylalanyl, is recommended for use in studies on racemization. Results on racemization attending the coupling of an amino acid ester as compared with a di-and tripeptide ester vary with the coupling method.
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