USE OF THIOL ACIDS IN PEPTIDE SEGMENT COUPLING IN NON‐AQUEOUS SOLVENTS

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Abstract
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°), 0.012%; DCC (DMF, 24°), 0.04%; FDNB (DMF, 24°), 0.53%; NBS (DMF, 24°), 0.46%; heat (DMF, 60°), 0.86%. Under the same conditions conventional couplings of Boc‐Gly‐Ala‐OH gave: DCC (DMF, 24°), 16.5%; DCC/1‐hydroxybenzotriazole (DMF, 24°), 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 (CH2 Cl2, 0°) to resin bound Ala with an epimer level of 0.05%.

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