Use of thiol acids in peptide segment coupling in non-aqueous solvents

Methods for preparing protected peptide thiol acids by solution and solid-phase procedures have been developed. The coupling of boc-Gly-Ala-SH to polymer-bound Leu was effected with dicyclohexylcarbodiimide (DCC), 2,4-dinitrofluorobenzene (FDNB), N-bromosuccinimide (NBS), and heat. In each case, the resulting H-Gly-Ala-leu-OH was analyzed for the content of H-Gly-D-Ala-Leu-OH (Izumiya test). The sensitivity of the test (0.1% epimer) was increased to 0.003% by developing a double chromatographic procedure involving partition chromatography prior to ion-exchange chromatography. The amounts of DL epimer found in the H-Gly-Ala-Leu-OH for the various tests were (solvent and temperature in parentheses): DCC (CH2Cl2, 0 degrees), 0.012%; DCC (DMF, 24 degrees),0.04%; FDNB (DMF, 24 degrees), 0.53%; NBS (DMF, 24 degrees), 0.46%, heat (DMF, 60 degrees), 0.86%. Under the same conditions conventional couplings of Boc-Gly-Ala-OH gave: DCC (DMF, 24 degrees), 16.5%; DCC/1-hydroxybenzotriazole (DMF, 24 degrees), 0.13%. In a competitive coupling of equimolar amounts of Boc-Gly-Ala-SH and Boc-Gly-Ala-OH with DCC in DMF the level of DL epimer was 0.21%, indicating that the thiol acid can be selectively coupled in the presence of carboxyl groups. In a variation of the test Boc-Gly-Leu-SH coupled with DCC (CH2Cl2, 0 degrees) to resin bound Ala with an epimer level of 0.05%.