FORMATION OF N-SUBSTITUTED 2-IMINOTHIOLANES WHEN AMINO-GROUPS IN PROTEINS AND PEPTIDES ARE MODIFIED BY 2-IMINOTHIOLANE

The reagent 2-iminothiolane (2-IT) is used to introduce thiol groups i nto proteins and peptides by reactions of their amino groups. In this study, we report that the thiol adduct initially formed by the reactio n of an amine with 2-IT (a 4-mercaptobutyramidine) is unstable and dec ays by a first order process to a nonthiol product (an N-substituted 2 -iminothiolane) with the loss of ammonia. The thiol adducts derived fr om amines of low pK(a) values (similar to 8; e.g., alpha-amino groups in peptides) decay more rapidly than those derived from amines of high pK(a) values (similar to 9.5; e.g., benzylamine, ethanolamine, lysine residues in proteins), with half-lives at pH 8 ranging from 0.3 to 3 h at 23 degrees C, and from 1 to 44 h at 0 degrees C. In the case of r eactions of peptides with 2-IT, the substituents at the alpha-carbon a lso influence the decay of the initial thiol adducts. The decay of the initial thiol adduct to an N-substituted 2-iminothiolane was confirme d for the reaction between benzylamine and 2-IT by the isolation of N- benzyl-2-iminothiolane and its characterization by elemental analysis and mass spectrometry. The decay of the initial 4-mercaptobutyramidine is prevented if the thiol group is capped, e.g., in the form of a dis ulfide group, or if the solution is acidified (pH 3 to 4). Immediate c apping of the thiol is, therefore, recommended when using 2-IT in the formation of bioconjugates. For amines of high pK(a), the N-substitute d 2-iminothiolane product can be cleaved by hydroxylamine, resulting i nitially in a thiol which then decays to N-hydroxy-2-iminothiolane reg enerating the original amine. For amines of low pK(a), the N-substitut ed 2-iminothiolane product can be hydrolyzed at pH 5 to generate a sta ble thiol with an amide functionality (an N-substituted 4-mercaptobuty ramide), (C) 1996 Academic Press, Inc.