Extended Data Fig. 3: Screening oxidative cyclization reagents for biocompatible tryptophan conjugation.

a) Reaction scheme for Trp-CLiC with tryptophan, where the cycloadduct is the desired product, while the hydroxyl tryptophan is the side product. b) Model of a Trp-CLiC reaction between 50 μM Ac-Trp-OMe and 60 μM oxaziridine for 10 min at room temperature in the co-solvent (PBS/MeOH =1:1). The cycloadduct and the hydroxyl tryptophan (Trp oxidized) are the proposed products. The reactions were monitored by detecting the relative quantification of peak intensity with LC-MS at 254 nm. Taking the reaction of Ac-Trp-OMe with Ox-W1 and Ox-W18 for example, the absorption peaks close to 3.1 min, 4.3 min and 6 min belong to the hydroxyl tryptophan, Ac-Trp-OMe starting material, and cycloadduct, respectively. The yield can thus be calculated by the relative peak integration of the LC-MS trace. Ox-W18 shows >91% efficacy to produce the desired cycloadduct. c) The reactivity of N-sulfonyl oxaziridine reagent Ox-W18 was also tested on other representative amino acids. d) The traceless reversibility of Cys oxidation. LC-MS trace was applied to monitor the reaction between 100 μM Fmoc-Cys-OH and 110 μM Ox-W18 for 10 min at room temperature in the co-solvent (PBS/MeOH =1:1). All Cys sites were oxidized and could be reduced upon the addition of 150 μM TCEP. e) The traceless reversibility of Met oxidation. Fmoc-Met-OH would be oxidized to form methionine sulfoxide (Met(O)). MsrA and MsrB could catalyse the reduction of S/R-Met(O), respectively. About 50% Met(O) could be reduced with high efficiency in vitro by MsrA, which catalyses the reduction of S-Met(O). We anticipate that use of both MsrA and MsrB proteins could reduce the two stereoisomers.