Caution in the use of 2‐iminothiolane (Traut's reagent) as a cross‐linking agent for peptides. The formation ofN‐peptidyl‐2‐iminothiolanes with bombesin (BN) antagonist (d‐Trp⁶,Leu¹³‐ψ[CH₂NH]‐Phe¹⁴)BN₆₋₁₄andd‐Trp‐Gln‐Trp‐NH₂

Abstract

During a study aimed at generating a bispecific molecule between BN antagonist (D-Trp(6),Leu(13)-psi[CH(2)NH]-Phe(14))BN(6-14) (Antag1) and mAb22 (anti-FcgammaRI), we attempted to cross-link the two molecules by introducing a thiol group into Antag1 via 2-iminothiolane (2-IT, Traut's reagent). We found that reaction of Antag1 with 2-IT, when observed using HPLC, affords two products, but that the later eluting peptide is rapidly transformed into the earlier eluting peptide. To understand what was occurring we synthesized a model peptide, D-Trp-Gln-Trp-NH(2) (TP1), the N-terminal tripeptide of Antag1. Reaction of TP1 with 2-IT for 5 min gave products 1a and 3a; the concentration of 1a decreased with reaction time, whereas that of 3a increased. Thiol 1a, the expected Traut product, was identified by collecting it in a vial containing N-methylmaleimide and then isolating the resultant Michael addition product 2a, which was confirmed by mass spectrometry. Thiol 1a 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 regenerating TP1. Repeat reaction with Antag1 and 2-IT allowed us to isolate N-Antag1-2-iminothiolane (3b), FAB-MS (m/z) [1212.8 (M+H)(+)] and trap the normal Traut product 1b as its N-methylmaleimide Michael addition product 2b, ES-MS (m/z) [1340.8 (M+H)(+)]. Thiol 1b is also stable at acidic pH, but when neutralized is unstable and cyclizes, forming 3b and Antag1.

Stabilization of Glucagon by Trehalose Glycopolymer Nanogels

“…Initially, glucagon was modified with 2‐iminothiolane (2‐IT), resulting in the formation of an amidine group linked to a thiol ( Scheme A). While thiolation attempts were successful, we observed the formation of a cyclic nonthiol byproduct via liquid chromatography‐mass spectrometry (LCMS) that has also been reported in the literature (Figure S15, Supporting Information) . Because this byproduct forms after thiolation, we attempted to trap the thiol prior to cyclization using both PDS and PDSMA‐ co‐ TrMA, which was monitored using Ellman's assay (Figure S16, Supporting Information).…”

Morphogenesis Guided by 3D Patterning of Growth Factors in Biological Matrices

“…In order to decorate fluorescein-tagged avidin with thiol-bearing SAT peptides, we investigated several classical amine-thiol heterodifunctional cross-linkers with limited success. A few problems encountered were low substitution efficiency with maleimide-PEG-NHS 3.4 kDa (likely due to the large polymer chain crowding the NHS environment), lysis of linkages created by Traut's reagent in the presence of thiols at physiological pH, and retro-Michael addition from maleimide linkers, all known issues 35,36 . This motivated the synthesis of a short and highly reactive heterodifunctional cross-linker, vinylsulfone-(S)-glycolic acid-(N)-hydroxysuccinimide ester (VS-NHS, 5b), that is straightforward to synthesize (Fig.…”

Morphogenesis guided by 3D patterning of growth factors in biological matrices

“…In order to decorate fluorescein-tagged avidin with thiol-bearing SAT peptides, we investigated several classical amine-thiol heterodifunctional cross-linkers with limited success. A few problems encountered were low substitution efficiency with maleimide-PEG-NHS 3.4 kDa (likely due to the large polymer chain crowding the NHS environment), lysis of linkages created by Traut's reagent in the presence of thiols at physiological pH, and retro-Michael addition from maleimide linkers, all known issues 35,36 .…”