An improved procedure for N- to C-directed (inverse) solid-phase peptide synthesis

A method for solid-phase peptide synthesis in the N- to C-direction that de livers good coupling yields and a low degree of epimerization is reported. The optimized method involves the coupling, without preactivation, of the r esin-bound C-terminal amino acid with excess amounts of amino acid tri-tert -butoxysilyl (Sil) esters, using HATU as coupling reagent and 2,4,6-trimeth ylpyridine (TMP, collidine) as a base. For the amino acids investigated, th e degree of epimerization was typically 5%, except for Ser(t-Bu) which was more easily epimerized (ca. 20%). Five tripeptides (AA(1)-AA(2)-AA(3)) with different properties were used as representative model peptides in the dev elopment of the synthetic method: Asp-Leu-Glu, Leu-Ala-Phe, Glu-Asp-Val, As p-Ser-Ile, and Asp-D-Glu-Leu. The study used different combinations of HATU and TBTU as activating agents, N,N-diisopropylethylamine (DIEA) and TMP as bases, DMF and dichloromethane as solvents, and cupric chloride as an epim erization suppressant. The epimerization of AA(2) in the coupling of AA(3) was further reduced in the presence of cupric chloride. However, the use of this reagent also resulted in a decrease in loading onto the resin and sig nificant cleavage between AAI and AA2. Experiments indicated that the obser ved suppressing effect of cupric chloride on epimerization in the present s ystem merely seemed to be a result of a base-induced cleavage of the oxazol one system, the key intermediate in the epimerization process. Consequently , the cleavages were most pronounced in slow couplings. An improved synthes is of fully characterized amino acid tri-tert-butoxysilyl (Sil) ester hydro chloride building blocks is presented. The amino acid Sil esters were found to be stable as hydrochlorides but not as free bases. Although only a few peptides have been used in this study, we believe that the facile procedure devised herein should provide an attractive alternative for the solid-phas e:synthesis of short (six residues or less) C-terminally modified peptides, e.g., in library format.