Caution in the use of 2-iminothiolane (Traut's reagent) as a cross-linkingagent for peptides. The formation of N-peptidyl-2-iminothiolanes with bombesin (BN) antagonist (D-Trp(6),Leu(13)-psi[CH2NH]-Phe(14))BN6-14 and D-Trp-Gln-Trp-NH2

During a study aimed at generating a bispecific molecule between BN antagon ist (D-Trp(6), Leu(13)-psi [CH2NH]-Phe(14))BN6-14 (Antag1) and mAb22 (anti- Fc gamma RI), we attempted to cross-link the two molecules by introducing a thiol group into Antag1 via 2-iminothiolane (2-IT, Traut's reagent). We fo und that reaction of Antag1 with 2-IT, when observed using HPLC, affords tw o products, but that the later eluting peptide is rapidly transformed into the earlier eluting peptide. To understand what was occurring we synthesize d a model peptide, D-Trp-Gln-Trp-NH2 (TP1), the N-terminal tripeptide of An tag1. Reaction of TP1 with 2-IT for 5 min gave products la and 3a; the conc entration of la decreased with reaction time, whereas that of 3a increased. Thiol la, the expected Traut product, was identified by collecting it in a vial containing N-methylmaleimide and then isolating the resultant Michael addition product Za, which was confirmed by mass spectrometry. Thiol la is stable at acidic pH, but is unstable at pH 7.8, cyclizes and loses NH3 to give N-TP1-2-iminothiolane (3a), ES-MS (m/z) [602.1 (M+H)(+)], as well as r egenerating TP1. Repeat reaction with Antag1 and 2-IT allowed us to isolate N-Antag1-2-iminothiolane (3b), FAR-MS (m/z) [1212.8 (M+H)(+)] and trap the normal Traut product Ib as its N-methylmaleimide Michael addition product 2b, ES-MS (m/z) [1340.8 (M+H)(+)]. Thiol Ib is also stable at acidic pH, bu t when neutralized is unstable and cyclizes, forming 3b and Antag1.