The model reactions were conducted with N αacetyl-L-lysine methyl-N-amide (K') as substrate (Figure ). Methylamide amino acids have been used as model compounds for site-specific modification as well as computational calculations to simulate lysine incorporation in proteins via the two adjacent peptide bonds. The model reactions were carried out at a concentration of 0.1 M in phosphate buffered saline (PBS, pH = 7.4) and monitored through 1 H NMR spectroscopy (Figure ). Regarding starting compound 3b, H NMR signals of the methine proton at the stereocenter (δ = 4.56 ppm, green) as well as the adjacent methylene group (δ = 3.34 ppm, yellow) are indicative for chemical modifications on the heterocycle (Figure ). Reacting 3b with K' resulted in an upfield shift of the methine proton to δ = 4.11 ppm (green), while the signal of β-methylene group remained unchanged (Figure ). The latter finding pointed towards the formation of the cyclized neutral product 4 as opposed to a ring-opened thiol-containing species that was initially anticipated. The existence of K'-substituted adduct 4 was confirmed by mass spectrometry dispaying singly charged ions (4+H) + and (4+Na) + (Figure ). It therefore appears as the aminolysis of 3b is followed by a nucleophilic attack of the liberated thiol on the amidinium species (Figure ). Subsequent elimination of ammonium chloride yields the re-cyclized product with an intact neutrally charged N-substituted iminothiolactone structure. Similar observations have been made with the original Traut's reagent by others. Complementary to K', 3b is converted with monoamino ethanol (MEA, an organic amine) to deliver hydroxyethyl-substituted 5. Comparison of both model reactions revealed slightly slower reaction kinetics when MEA was used (Figure ). Detailed 1 H NMR assignments for both 4 and 5 are found in the supporting information (Figure -29 and S38-42).