Formation of N-substituted 2-iminothiolanes when amino groups in proteins and peptides are modified by...

The reagent 2-iminothiolane (2-IT) is used to introduce thiol groups into proteins and peptides by reactions of their amino groups. In this study, we report that the thiol adduct initially formed by the reaction of an amine with 2-IT (a 4-mercaptobutyramidine) is unstable and decays by a first-order process to a nonthiol product (an N-substituted 2-iminothiolane) with the loss of ammonia. The thiol adducts derived from amines of low pKa values (approximately 8; e.g., alpha-amino groups in peptides) decay more rapidly than those derived from amines of high pKa values ( similar 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 reactions of peptides with 2-IT, the substituents at the alpha-carbon also influence the decay of the initial thiol adducts. The decay of the initial thiol adduct to an N-substituted 2-iminothiolane was confirmed 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 disulfide group, or if the solution is acidified (pH 3 to 4). Immediate capping of the thiol is, therefore, recommended when using 2-IT in the formation of bioconjugates. For amines of high pKa, the N-substituted 2-iminothiolane product can be cleaved by hydroxylamine, resulting initially in a thiol which then decays to N-hydroxy-2-iminothiolane regenerating the original amine. For amines of low pKa, the N-substituted 2-iminothiolane product can be hydrolyzed at pH 5 to generate a stable thiol with an amide functionality (an N-substituted 4-mercaptobutyramide).