This manuscript reports the development of a triggerable and fluorogenic bioorthogonal reaction probe by combining light-activated tetrazines with fluorogenic IEDDA. The authors designed an N-Voc photocaged tetrazine that, upon uncaging, reacts with BCN to generate a fluorogenic product. The authors conducted thorough and systematic structural characterization, including detailed NMR analysis of the products. Structural confirmation of the caged dihydrotetrazine was achieved through X-ray crystallography, validating the carbamate regioisomer formed. Overall, this work provides a valuable strategy for achieving spatiotemporal control of bioorthogonal reactions with an intrinsic fluorescent signal, and it represents a promising advance for both biological and materials science applications. However, some aspects still require revision:
o The fluorescent molecule constructed by the authors is based on a donor–acceptor (D–A) framework. Moreover, as shown in Figure 3, probe 15 may also undergo aggregation (evidenced by the decrease in fluorescence intensity when switching from organic solvents to water). This is not necessarily a drawback; in fact, it may indicate potential aggregation-induced emission (AIE) behavior. The authors need discuss this phenomenon and cite relevant literature: https://doi.org/10.1002/anie.202511705https://doi.org/10.1002/anie.202011544
2. The stability of probe 14 in solution (e.g., in aqueous buffer or cell culture medium) should be evaluated.
3. In Figures 2 and 4, the y-axis of the HPLC chromatograms presents absolute absorbance values at the corresponding wavelength, with relatively large scales. Since the spectra overlay multiple time points, it is recommended to present separate y-axes for clarity and to avoid potential confusion for readers.
4. In the NMR spectrum provided in Figure S1, the impurities should be labeled accordingly.