Kinetics and Mechanism of the Thiolytic Removal of the Dithiasuccinoyl (Dts) Amino Protecting Group

George Barany, R. B. Merrifield

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The dithiasuccinoyl (Dts) amino protecting group is removed by thiols through the intermediacy of open-chain carbamoyl disulfides. The elucidation of practical and effective conditions for carrying out the reductive deprotection was facilitated by a rapid, convenient, and quantitative method to directly measure starting materials, intermediates, and products on a standard amino acid analyzer. The apparent pseudo-first-order rate constants were determined as a function of thiol, base, and solvent composition and concentration. Both steps of the mechanism were first order in thiol. In anhydrous solutions, the rate of the second step, k^, varied directly with tertiary amine concentration, suggesting that the active species is an association complex of the thiol and the base. In contrast, a more complex explanation is required to account for the fact that plots of log k i against log [base] had a slope of only 0.7-0.8. The ratio of rates, = k2/k1, was generally between 0.1 and 5 for neutral monofunctional aliphatic thiols, but with bifunctional thiols, where the second step can proceed intramolecularly because a cyclic disulfide is formed, > 100 and consequently carbamoyl disulfide intermediates could not be observed. Intermediates were also never observed for thiophenol, the most acidic thiol tested, nor for 2-mercaptopyridine, a compound existing primarily as its thione tautomer. For these two cases, was estimated, by indirect means, as ≥105 and ≥109, respectively. The fastest overall rates were observed with thiols of intermediate acidity (pka = 8.0-9.5) in polar aprotic media of high dielectric constant. In aqueous solutions, the first step of the mechanism was rate limiting (~375 based on an independent measurement). The observed rates ka were directly proportional to the thiol anion concentration and the data for monofunctional thiols fit a Br^nsted correlation of thiol anion reactivity against pKa with slope /3nuc 0.9. The two steps in the mechanism of thiolytic deprotection of dithiasuccinoyl amines have strikingly different electronic requirements, meaning that the transition states are different. The driving force for the first step appears to be relief of the ring strain of the Dts heterocycle, while the rate of the second step correlates with the ease of ionization of the thiocarbamate leaving group. Suitable conditions for the quantitative removal of the Dts protecting group from any amino acid residue at 25 °C include (1) jS-mercaptoethanol (0.2 M)-triethylamine (0.5 M) in benzene for 5 min; (2) N'-methylmercaptoacetamide or dithiothreitol (0.1 M) in neat pyridine for 5 min; (3) N'-methylmercaptoacetamide or N'-acetyl-β-mercaptoethylamine (0.1 M)-A'-methylmorpholine (0.5 M) in acetonitrile for 1 min; (4) /3-mercaptoethanol (0.1 M) and 2-mercaptopyridine or thiophenol (1.1 equiv over Dts amine) in N',N'-dimethylformamide- pyridine (9:1) for 1 min; (5) N'-methylmercaptoacetamide (0.2 M) in N'.N'-dimethylformamide-acetic acid (9:1) for 2 min; (6) dithiothreitol (10 mM) in pH 7.0 phosphate buffer for 2 min. The reductive deprotection of the Dts group and of carbamoyl disulfide intermediates is much more facile than the reduction of acyclic aliphatic disulfides.

Original languageEnglish (US)
Pages (from-to)3084-3095
Number of pages12
JournalJournal of the American Chemical Society
Issue number9
StatePublished - Apr 1980


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