Figure show corresponding graphs for the single modification of 3b. Surprisingly, the reaction of K' with 3b displays the slowest overall kinetics when compared to the other single modifications (Figure , Figure ). The conversion reaches 89% over the course of 24 h, showing little difference between the two buffer conditions. Compared to the model reaction at a concentration of 0.1 M (84% conversion after 1 h) slower kinetics are expected due to reduced reactant concentrations. Surprisingly, the single modifications involving peptides proceeded faster. Reaction of 3b with KFRGDS towards adduct 6 (Figure ) reaches 78% and 67% conversion after 4 h in NaPi and PBS, respectively, reaching a plateau at 91% (Figure ). Mass spectrometry shows the doubly charged species (6+2H) 2+ = 432.480 Da. When 3b is converted with GRGDS to form adduct 7, a similar reaction rate is observed (Figure , Figure ). After 4 h, 78% conversion is reached in NaPi, while reaction in PBS shows 65%, both plateauing at approximately 91%. The mass spectrum shows the singly charged species (7+H) + = 645.500 Da and doubly charged species (7+2H) 2+ = 323.292 Da (Figure inset graph, Figure ). Single modification of 3b with KLVFF to deliver product 8 reaches 85% conversion within 4 h reaction time (Figure , Figure ). The conversion saturates at 98% and the influence of pH on the reaction kinetics are negligible. The product species is detected as singly and double charged species, (8+H) + = 807.690 Da and (8+2H) 2+ = 404.530 Da (Figure inset graph, Figure ). Overall the influence of the buffer onto the kinetics of the single modification remain limited and the re-cyclization of the ring-opened intermediate appears to be the rate determining step. The double modifications proceed in presence of NMM as thiol trapping agent. Transformation of 3b to 4* is completed within 4 h at slightly basic pH (NaPi, Figure , Figure ). The reaction in PBS reaches 75% conversion at equal duration (Figure ). Double modifiation using KFRGDS towards 6* reaches 97% and 78% conversion within the first 4 h using NaPi and PBS, respectively (Figure ). When GRGDS is reacted with 3b to give 7*, 4 h reaction time result in conversions of 97% and 87% in NaPi and PBS, respectively (Figure , Figure -73). The mass spectrum reveals the correct monoisotopic mass for doubly charged species (7*+H) 2+ = 387.430 Da (Figure inset graph, Figure ). Finally, modification of 3b with peptide KLVFF to deliver 8* is completed within 4 h in NaPi (97%, Figure , Figure ). The kinetic data for PBS shows slightly slower conversion, reaching 88% after 4 h reaction time (Figure ). Product formation was confirmed via mass spectrometry displaying the species (8*+H) 2+ = 468.570 Da (Figure , Figure ). As opposed to the single modification, the usage of the slighlty basic NaPi buffer significantly enhances the overall reaction kinetics. In presence of NMM, the previously rate limiting re-cyclization is successfully suppressed and the much faster thiol-maleimide addition takes place. In conclusion, both the single and double modification of 3b could be successfully carried out using near-stoichiometric amounts of various peptides at low concentrations and benign reaction conditions to selectively yield respective adducts 4, 6-8 and 4*, 6*-8*. The presented kinetic data confirms the highly efficient nature of the ITL ligation, confirming 3b's capacity for the covalent attachement of chosen peptide sequences.