In this manuscript, we advanced current TL ligation chemistry by introducing highly reactive γ-functional iminiumthiolactone derivatives. Compared to conventional TLs, ITLs were exhibited innate water solubility and increased reactivity towards amine-containing substrates. We synthesized various γ-functional ITL derivatives and performed orthogonal modifications on the double bond of 3b. The same compound was used to study model reactions and reaction kinetics. Lysine derivative K' was used as substrate to elucidate 3b's reactivity and reaction mechanism. Similar to the Traut's reagent, 3b could undergo single or double modification forming either a re-cyclized N-substituted ITL or a positively charged amidinium species. Depending on the substrates present, the reaction trajectory of 3b could be precisely controlled to engage in either of the two pathways. Further to this, K' as well as three different peptides were separately converted with 3b in two different buffers at a concentration that was 10 times lower than the one used in the initial model reactions. Using these benign reaction conditions, the single modification was rate limited by the re-cyclization step, while all double modifications were much faster and a slightly basic environment (NaPi buffer, pH = 8.0) accelerated the reaction kinetics further. Finally using 3b, we performed single and double modifications on the protein lysozyme C. At low concentrations of 50 µM, conversion of the protein were limited. Nonetheless, a clear tendency to form the singly-substituted species in both modification cases was observed (confirming the clean process of our modification strategy). Additionally, it was also shown that with higher quantities of the reagents both conversions and degree of modifications could be tuned. We believe that using γfunctional ITLs as ligation strategy may find wider application in the fields of biohybrid materials, polymer chemistry and multicomponent reactions, given the original non-substituted Traut's reagent has already proven to be a reliable tool in these areas. As such, we aim to explore further reagent derivatives and potential ways to apply this chemistry to macromolecular systems.