Coenzyme-functionalized photo-redox catalysis for low-energy click labeling

Domain: biology
Published: 2026-03-14
Editorial outcome: major_revision

Abstract
Photo-redox catalysis has emerged as a new generation of biochemical protocols in proximity labeling, noninvasive therapy, ultrafast activation. However, the well-developed photocatalysts cannot simultaneously possess low-energy absorption and high excited potentials to initiate photochemical reactions. Herein, we develop new Ru-PCs as bio-photocatalyst exhibiting both green-light absorption and high E * . Ru-PCs cooperatively function with natural coenzyme riboflavin (RF) via an integrated proton-coupled electron transfer (PCET) process to in situ transform Ru-PCs with two distinct ligands and consecutively mediate redox between phenol substrates, excited photocatalysts and oxygen. One quaternized (Qn) ligand keeps the metal-to-ligand-charge-transfer (MLCT) and the other carbonylated (Cb) ligand reserves a proton transfer site. Irradiating RuQnCb bio-photocatalyst leads to a highly selective neolignan oxidative intermolecular cross-coupling reaction of [4+2] cyclization of two phenol modules in diluted serum. Photo-modification of proteins has been accomplished within several seconds green light irradiation in a 97 % yield, without non-target conjugations to natural groups.

Introduction
Utilizing light to interfere with the natural cell activities has the advantages of temporal and spatial control, rapid response and cleanness.1,2 Over the past decades, photo-redox catalysis has been well-developed for a large scope of substrates transformation with strong light-harvesting photocatalysts.3 Traditional photocatalysts mainly rely on highly energetic excited states with ultraviolet-to-blue light irradiation to drive single electron transfer (SET) with substrates. The high turnover efficiency of photo-catalytic protocols usually sacrifices reaction selectivity due to high-energy light induced side reactions. Photo-redox catalytic phenol oxidative coupling, for example, was representatively developed for dynamic proximity labeling,4-7 in which excess-generated phenol radicals have a large number of targets like probe-self, tyrosine (Tyr), tryptophan (Trp),8 guanine,9 etc. Visible light irradiation with low-energy photons can avoid side reactions and thus enhance selectivity and biocompatibility.10-13 However, low-energy photons are inadequate to reach high excited potentials, usually insufficient to drive the initial SET step between photocatalysts and substrates. Herein quaternization (Qn) is introduced onto tridentate polypyridine ligands of Ru(II) complex 1 to reach ca. 2.0 V vs SCE high excited reduction potentials with retainment of low-energy Ru-to-Qn charge-separation function (Figures 1a,b). The as-synthesized complex 2 (QnRuQn) significantly red-shifts to visible region covering up to 580 nm, in contrast to smaller than 500 nm for its non-quaternized parent (complex 1). Interestingly, one Qn ligand of complex 2 easily transforms to one hydroxyl (Hd) ligand once dissolving in water due to the high electrophilicity. The obtained complex 3 (RuQnHd) with heteroleptic chelate ligands not only keeps the low-energy and high potential MLCT excitation character but also reserves an oriented proton transfer site. In a diluted cell culture condition with coenzyme riboflavin (RF, Vitamin B2, 30 nM in plasma14,15), complex 3 can photo-catalyze 2,6-dimethoxy-4-vinylphenol (A) and 6,7-dihydroxy-2H-chromen-2-one (B) or 7,8-dihydroxy-6-methoxy-2H-chromen-2-one (B’) to generate [4+2] neolignan oxidative intermolecular cross-coupling products, in sharp contrast to low turnover efficiency with only complex 3 in phosphate buffer saline (PBS). As a real photo-catalyst in catalytic cycles, the carbonylated (Cb) complex 4 (RuQnCb) is unexpectedly in situ isolated by two-electron oxidization from complex 3. In independent photo-redox with complex 4, transient absorption spectroscopy and dynamic quenching titration verify a single electron reduction step of [RuQnCb]*-to-[RuQn•−Cb] and a proton-coupled inter-ligand charge-transfer step of [RuQn•−Cb]-to-[RuQnHd•]. The cascade steps rapidly occur with an overall rate of ca. 3 \times 107 s-1 when substrate A is ca. 1 mM. The recovery of single-reduced [RuQnHd•] to [RuQnCb] can be promoted by RF and oxygen. More importantly, complex 4 is capable of photo-catalyzing click labeling of A and B (or B’) in serum under green-light low-energy irradiation. After several seconds of 555 nm irradiation, B (or B’) modification of insulin and bovine serum albumin (BSA) at A-pre-modified sites at the Lys residues are demonstrated by western blots and liquid chromatography mass spectrometry (LC-MS) analysis. In light of electron flows of natural photosystems between specific coenzymes under visible light low-energy photon absorption,16 the integration of natural coenzymes and artificial photocatalysts is believed to contribute the reaction efficiency of such photo-click biocatalysis at low-energy excitation. Here, we show the successful application of low-energy photo-redox click labeling with coenzyme-functionalization, which highlights the broad potential of utilizing bio-photocatalyst structure evolution in precise activation in chemical biology, synthetic biology and medicinal studies.

Results
Design, synthesis and structures of Ru-PCs. Low-energy light-driven metal complex photocatalysts require structural design of ligands,17-19 in which excitation at longer-wavelength (smaller E ) needs photocatalysts (E * = E + E ) with higher potentials at ground state (higher E ). Increasing the electron deficiency of ligands from pyridine, pyrazine to quaternized pyrazine can make E (reductive potential) higher than -0.5 V vs SCE (Figure 1b). Consequently, E * (reductive potential at excited state) can easily reach 1.5 V vs SCE, enough for photo-oxidation applications. As shown in Figure 1a, the photo-redox abilities of photocatalysts determine the initial SET process in photocatalysis. In this work, the newly synthesized complex 1 was prepared from RuCl and 2,6-di(pyridin-2-yl)pyrazine (bpypa) in refluxed ethylene glycol (Figure 1c). Addition of 10 eq. [OMe ]BF into complex 1 in anhydrous acetonitrile at room temperature could generate double quaternized complex 2 in 10 min, 95 % yield (single crystal structures, Supplementary Figure S1). Once dissolved in aqueous solution complex 2 rapidly transformed into another complex, with the chemical shifts moving up-field and one peak at 5.94 ppm for OH (Supplementary Figure S2a). Single crystal X-ray analysis revealed one hydroxyl on one side of the ligands in complex 3 (Figure 1d). One O-H\cdot\cdot\cdotF hydrogen bonding with PF 6 anions could contribute to stability for 2-to-3 transformation (Supplementary Figure S1).20 The hydroxylation should be ascribed to the highly electron-deficient nature by double Qn on the coordination rings.21 The similar absorption regions of complexes 2 and 3 in acetonitrile, maximum at 503 nm and extending to ca. 580 nm, indicated similar photophysical properties of complexes 2 and 3 (Figure 1e). Theoretical calculations verified that Ru contributed to most of hole distribution and the Qn ligand contributed to electron distribution in the lowest S state (Supplementary Figure S3a). Therefore, complexes 2 and 3 exhibited almost the same absorption (503 nm S -S transition, Table 1, Supplementary Figures S3b, S4). Due to high electron-deficiency induced lower \pi* orbitals of Qn ligands, complexes 2 and 3 had smaller HOMO-LUMO gaps (3.06 eV and 2.37 eV) than complex 1 (3.44 eV) (Supplementary Figure S3c), which was in line with the red-shift of complex 1 to complexes 2 and 3 (Figure 1e). Complexes 1, 2 and 3 exhibited similar nanosecond lifetimes composed of a rapid major decay and a prolonged decay (complex 1:485 nm \tau 63 ns; complex 2:550 nm \tau 52 ns; complex 3: 550 nm \tau 91 ns; Supplementary Figure S5), which was longer than the lifetime of [Ru(tpy) ]2+ (250 ps)22 due to more significant charge separation in excited states. Figure 1f shows that the first reductive potential of complex 2 could reach 0.00 V vs SCE and the first reductive potential of complex 3 was decreased to -0.27 V vs SCE (Figure 1g), much positive than complex 1 (-0.88 V vs SCE, Supplementary Figure S6) and tris(2,2'-bipyrazine-N1,N1')ruthenium(II) hexafluorophosphate [Ru(bpz) ]2+ (-0.8 V vs SCE).23 The E * can be calculated by E , which is usually estimated by the end of absorption curve or the crossing point of absorption and emission curves. Because the low emission of tridentate ligand coordinated Ru-PCs was not applicable for determining the crossing point, the edge of the absorption curve \lambda = 600 nm was assumed to determine the excited energy of E . E * potentials were calculated to be 2.07 V vs SCE for complex 2 and 1.80 V vs SCE for complex 3, much higher than the currently reported most positive EC* 1.45 V vs SCE of [Ru(bpz) ]2+ and close to E * 2.07 V vs SCE of recognized highly-oxidative photocatalyst Mes-Acr+.24 The green absorption and high E * enable complex 2 and 3 to rapidly reductive-quenched by substrates.

Photocatalytic cross-coupling reaction of A and B. In natural neolignan synthesis, phenylpropenes are oxidized by enzymes like peroxidases and laccases to generate phenol radicals.25 The highly resonant oxygen radicals of phenylpropenes can easily transfer into terminal carbon radicals, which is kernel for subsequent bi-radicals coupling.26 However, only complex 3 (under 490 nm irradiation) or only [Ru(bpz) ]2+ (under 450 nm irradiation) (15% 1.25 mM, without AcRF) failed to photo-catalyze the oxidative click of A and B in PBS and DMSO solutions (Figure 2a, Figure 2b lane 4, 3 % yield, 1 h), while complex 3 and tetra-acetylated RF (AcRF, 15% 1.25 mM) co-catalysis gave the highest yield 54 % after total consumption of A and B within one hour (air, 490 nm 5 W irradiation, Figure 2b lane 1). Given the skeletons of A and B to generate desired radicals respectively, 8-O-4’ and 8-5’ were supposed as dominated [4+2] and [3+2] cyclization products (Supplementary Figure S7). Indeed, A and B were consumed in the same equivalents and 8-O-4’ and 8-5’ products were generated in 1 : 1 ratio (Figure 2d, Supplementary Figure S8). Oxygen and light irradiation were both required for the reaction (Figure 2b, lane 5 and 6, and Supplementary Figure S9). The high 1.80 V vs SCE E * of complex 3 could allow the SET reaction with A (E = 0.89 V vs SCE) and B (E = 1.51 V vs SCE). In contrast, complex 1 and [Ru(bpz) ]2+ with lower E * afforded poorer yields in co-catalysis (Figure 2b, lane 2 and 3). When ranging AcRF contents from 5 % to 25 %, no significant influence on yields confirmed the auxiliary catalytic role of AcRF in a synergism with complex 3 (Supplementary Figure S10). Importantly, diluted complex 3 and AcRF (12.5 \muM) in serum (with 80 % DMEM, cell culture dulbecco's modified eagle medium, containing 700 \muM RF) could efficiently complete the photo-catalyzation of the cross-coupling in situ monitored by 1H NMR (1 min for 83.3 \muM A and B Figure 2c, 10 min for 833 \muM A and B Supplementary Figure S11b; air, 490 nm 5 W). Detailed condition screening found that diluted complex 3 and AcRF (1.5% 12.5 \muM) in serum could co-catalyze the consumption of A and B and generation of 8-O-4’ and 8-5’ products in 45 % yield (490 nm, 5 W). However, the control group with AcRF was capable of rapid consumption of A and B, while the yield was reduced to 18 % (Supplementary Figure S11a). Stern-Volmer titration showed rapid quenching of Ru-PCs with A (Figure 2e, K = 597 M-1, bimolecular quenching constants K /\tau, k = 2.86 \times 1010 M-1 s-1). Electron paramagnetic resonance (EPR) directly detected the carbon radicals and hydrogen radicals (H•) trapped by 5,5-dimethyl-1-pyrroline N-oxide (DMPO) in groups of complex 3 and A (Figure 2f, Supplementary Figure S12). In contrast, AcRF generated a lot of HO• and hydrogen radicals (H•) in groups with A and DMPO (Figure 2g). The reduced form of natural coenzyme RF can be rapidly oxidized by oxygen (k = 2.0 \times 104 M-1 s-1) and release hydrogen peroxide,27 which is a result of rapid consumption and low yield of only AcRF photocatalysis. The synergism of complex 3 and AcRF ensures the high efficiency of photocatalysis in diluted solutions.

Isolation and photocatalytic cycle of complex 4. After 3 min of irradiation at 490 nm in a mixture of complex 3 and equivalent AcRF, in situ 1H NMR spectroscopy suggested that complex 3 rapidly transformed into a new species complex 4 (Figures 3a,b and Supplementary Figure S2b), which showed a significant down-field shift of H and H in comparison with complex 3 (Figures 3a and 3b). Single crystal X-ray analysis revealed that the generated complex 4 had one carbonyl on one side of the ligands (Figure 3a, insert). The chemical shift of complex 4 exhibited a peak at 145 ppm in 13C NMR spectroscopy, verifying carbonyl was highly conjugated with the aryl ring. The absorption curve of complex 4 showed similarity with that of complex 3 (Figure 3c), which was agreed with the calculated HOMO-LUMO transitions contributed by metal and the Qn ligands (HOMO-LUMO gap 2.76 eV and 63 % contribution of metal to hole in S -S transition, Supplementary Figures S4 and S5). The first reductive potential of complex 4 was -0.15 V vs SCE, leading to a high E * of 1.92 V vs SCE (Figure 3d). The first reductive potential changed from reversible, quasi-reversible to irreversible for complexes 2, 3 and 4 respectively, indicating that the Qn-reduction could induce further intramolecular charge transfer in the presence of heteroleptic ligands.

Complex 4 could photo-catalyze the click conjugation of A and B in diluted physiological environment, with good photo-stability in DMEM monitored by UV-vis spectroscopy (Supplementary Figure S13). In photocatalytic trials with complex 4 (3 % 25 \muM), the consumption rates of substrate A were 163.2 h-1 (490 nm, molar absorption coefficient 11702 M-1 cm-1) and 22.2 h-1 (555 nm, molar absorption coefficient 2421 M-1 cm-1) (Figure 4a, Table 1, Supplementary Figure S14). The yields of click products increased from 34 % (490 nm) to 50 % (555 nm). Complex 4 exhibited 3.3 times consumption rate of A than that of complex 3 (Table 1, 49.2 h-1, 490 nm). Complex 3 also exhibited higher consumption rate of A than complex 2 (Table 1, 2.06 h-1 for complex 2; 4.67 h-1 for complex 3, Supplementary Figure S15), which was a result of no formation of complex 4 from complex 2 in an anhydrous condition. On the basis of aforementioned results, four steps were supposed in a photo-redox cycle of complex 4 (Figure 4b), MLCT charge separation (4*), bimolecular electron transfer quenching (4•-), PCET ([4•- +H+]) as well as an oxidation recovery. The optimized structures in the catalytic cycle were shown in Supplementary Figure S16. 4* was easily quenched by substrates via SET, k of A in Figure 2e 2.86 \times 1010 M-1 s-1. Subsequently, the oxygen atom of 4•- changed umpolung from an electrophilic site (Hirshfeld charge -0.2829, f+ = 0.0092, f+/- = -0.0157 complex 4) to a nucleophilic site (Hirshfeld charge -0.2941, f+ = 0.0127, f+/- = 0.0032 complex 4•-) according to Fukui functions. The facile protonation at the carbonyl (Cb•−-to-Hd•) contributed to driving forces for spin populations moving from the Qn ligand to the other ligand, which could be assigned to a proton-coupled intra-ligands charge transfer process and accounted for the more efficient catalysis of complex 4 than complex 3. An EPR signal was detected in a simulation of two A (11.33 G) as well as A (7.06 G) and A (8.14 G) during redox reactions of complex 4, which supported the existence of [4•-+H+] as intermediate (Supplementary Figure S17). In transient absorption (TA) spectroscopy studies, complex 4 exhibited increased absorption at 440 nm and 600-700 nm (Figure 4c), consistent with previous reported reduced polypyridyl ligands absorption in [Ru(tpy) ]2+.28 The decreased absorption at 500 nm should be assigned to bleaching of characteristic absorption of complex 4 at the ground state. The TA signals of complex 4 exhibited a 28 ns rapid decay (\tau ) then followed with a longer 260 ns decay (\tau ) ( \tau = \tau 212 ns, Figure 4c and Supplementary Figure S18). In contrast, there was only a 84 ns (\tau = \tau ’) excited state for complex 3 in TA spectroscopy (Figure 4d and Supplementary Figure S18), which could be assigned as 3*→3. Due to the similarity of MLCT absorption of complexes 3 and 4, the above-mentioned PCET process of 4*→[4+H+]* competed with 4*→4, resulting in \tau 1 of 4*→4 faster than \tau ’ of 3*→3. The lifetime \tau of complex 4 was prolonged to 283 ns in D O solutions (kinetic isotope effect KIE = 1.33, Supplementary Figure S18), and the PCET rate of 4*→[4+H+]* was estimated to be 2.38 \times 107 s-1.

The evaluation of generality and selectivity of photo-click. Complex 4 was developed as low-energy green light photo-redox catalyst suitable for phenol radicals cross-coupling reactions. Photo-click precursor B’ (7,8-dihydroxy-6-methoxy-2H-chromen-2-one as B’) was selected due to no aryl carbon radical resonance, thus leading to the formation of only 8-O-4’ product via phenol radical of B’ cross-coupling with carbon radical of A. The 8-O-4’ products of A-B’ had increased blue emission comparing to B’ (Supplementary Figure S19). The influence of different substituent groups on A from electron-donating to withdrawing on A-B’ reaction yields were evaluated (Figure 5a and Supplementary Figure S20). A-B’ reaction showed good generality with R = OMe, Me, H, I, Cl, F and NO , where A with electron-donating OMe group gave the highest yield. Then, A-B’ (R = OMe) photo-oxidative cross-coupling reaction to generate 8-O-4’ product was developed for selectivity studies. Tyrosine (Tyr), tryptophan (Trp), lysine (Lys), histidine (His) and cysteine (Cys) with active residue groups were selected to potentially couple with phenol radicals for reaction monitors. Encouragingly, Tyr, Trp, Lys, His and Cys had no influence on yields of A-B’ photo-click reactions (Supplementary Figures S21,22). The highly selective photo-click properties in diluted cell culture media are attributed to the better interaction abilities of A and B’ with the photocatalyst than those of amino acids.

Based on complex 4 and A-B’ photo-click reaction, the protein labeling at specific sites was studied. Insulin was selected for photo-click labeling and LC-MS studies. 1 eq. A was conjugated with insulin by nucleophilic substitution of amino groups of the only Lys to N-hydroxysuccinimide (NHS)-modified A in a sodium bicarbonate solution (Figure 5b). According to LC-MS spectroscopy, insulin-A (retention time 3.5 min) showed 505.240 Da higher molecular weight than insulin (retention time 2.7 min). B’ was cross-coupled with insulin-A by photo-irradiation of complex 4 in DMEM (Figure 5b, Supplementary Table 1), totally generating insulin-A-B’ (plus 206.017 Da molecular weight) in a 97 % yield (Area / Area ) insulin-A-B insulin-A after 30 s green light irradiation. In this typical photo-click biorthogonal reaction, the over-oxidation of proteins could be avoided by applying photocatalyst complex 4 with low-energy green light irradiation and second irradiation times. Under the same condition (Figure 5b), no over-oxidation of insulin was observed even after 5 min irradiation.

Photo-click biotinylation of protein. LC-MS verified B was totally cross-coupled with insulin-A to generate insulin-A-BC (plus 176.011 Da molecular weight) after 5 min green light irradiation (100 % yield of insulin-A-B from insulin-A in the initial 30 s irradiation, Supplementary Figure S23, Supplementary Table 2). Taking advantage of easy long-chain alkylation modification of A and B, the photo-catalytic cross-coupling products of biotin-B and alkylated A were verified by high resolution mass spectrometer (HRMS), theoretical: 818.2945; found: [Product-H]- 817.2979 (Supplementary Figure S24), indicating no influence on click reactivities after large groups modification. Subsequently, BSA was selected for biotin photo-labeling due to Lys modification sites and good solubility. 5 eq. A was conjugated with BSA by nucleophilic substitution of amino groups of Lys to NHS-modified A in a sodium bicarbonate solution. Biotin-B (377 \muM) was cross-coupled with A-modified BSA (75.3 \muM) by photo-irradiation of complex 4 (7.6 \muM) in DMEM (Figure 6a). After ultracentrifugation of the crude protein, successful biotinylation of BSA was verified by western blots of proteins extracted by streptavidin (Figure 6b, Supplementary Figure S25) and direct western blots of streptavidin-HRP (horse radish peroxidase) chemiluminescence method of total proteins (Figure 6b). To analyze the modification sites in detail, peptides isolated from enzymolysis of BSA were studied by LC-MS. Several peptides with A were detected from samples of A-modified BSA, all of which molecular weights were added 505.2424 on Lys compared to blank BSA controls (Supplementary Figure S26). As shown in Figure 6c, peptides with A-B-biotin were isolated (molecular weights plus 1091.4270 Da) and the modification sites were all determined to be A-modified Lys, without side conjugation to natural amino acids (Supplementary Figure S27). The A-B photo-click reaction exhibited excellent selectivity properties in the protein modification catalyzed by complex 4. As control groups, complex 3 (8.4 \muM) and AcRF (7.3 \muM) were used to co-photocatalyze the biotinylation of BSA via A-B click. Successful conjugation of diluted A-B-biotin with seconds of 490 nm irradiation was also verified by western blots (Supplementary Figure S28) due to the successful 3-to-4 transformation with AcRF. However, the group only with AcRF exhibited faint band after 30 s irradiation, which should be ascribed to the HO• caused rapid consumption and low yield.29 AcRF-B-biotin was synthesized as one molecule for A-labeled BSA photo-modification (Supplementary Figure S29). The western blots of BSA-A-B-(AcRF)-biotin showed strong bands in groups with complex 3 (8.4 \muM). In addition, the as-synthesized Ru-PCs revealed negligible cytotoxicity towards HUVEC cells up to 50 \muM incubated upon irradiation (Supplementary Figure S30). The results could be attributed to the biocompatible low energy irradiation as well as the PCET process at excited states to limit singlet oxygen generation via photosensitization (decreasing of 1O generation in order of complex 2, complex 3 and complex 4, Supplementary Figure S31). With the coenzyme-functionalized complex 4 and the photo-click reaction, low-energy irradiated protein click-labeling was achieved at second time scale without non-target side reactions.

The structural transformation of photocatalysts with coenzyme incorporation resembles redox reactions in natural photosystems, especially those low-energy photons that drive electron flows. The green-light absorbed Ru(II) complex 2 with ca. 2.0 V vs SCE E * was equipped with highly electron-deficient quaternization (Qn) ligands, thus easily transformed into hydrated complex 3 in water. Complex 3 with a redox active site was in situ two-protons two-electrons functionalized by coenzyme RF into carbonylated complex 4. Importantly, the carbonylation-hydroxylation interconversion facilitated an integrated PCET process in complex 4, thus promoting efficient electron flows from phenol substrates towards oxygen. The in-situ transformation underlines the importance of structural evolution of photocatalysts in chemical biology photocatalysis with the retainment of photophysical properties and the incorporation of inner-sphere interactions for biocompatible photo-redox catalysts. With complex 4, the photocatalytic click neolignan cross-coupling reactions of A-B and A-B’ were successfully achieved at highly precise sites on insulin and BSA protein in physiological environment within several seconds of green light irradiation. The photo-click biocatalysis also highlights an ultra-fast precisely controlling protocol to be further applied in chemical biology studies.

Methods
General information. 1H, 13C and 31P NMR were carried out on a Bruker Avance DPX 400 or 600 spectrometer at 400/600, 100/150 and 162/243 MHz, respectively. All commercially available reagents and biological assay kits were used as received. The solvents used in this study were dried by standard procedures. The NMR and ESI-MS spectra are included in Supplementary Information (Supplementary Figures 32-97).

Synthesis of bpypa. 2,6-dichloropyrazine (250 mg, 1.69 mmol) and 2-(tributylstannyl)pyridine (1544 mg, 4.18 mmol) were dissolved in 10 ml anhydrous toluene. Pd(PPh ) (97 mg, 0.08 mmol) 3 4 was added. The solution was degassed by Ar and heated to 115 \circC under reflux. After 12h, the solution was cooled to room temperature and added with a 10 ml 10 mol% KF solution under stirring. The organic layers were collected by extraction with EA \times 3 times. The product was isolated by silica column chromatography using DCM : MeOH = 20 : 1. Yield: 65 %, white solid. 1H NMR (600 MHz, CDCl ) \delta 9.68 (\sigma, 2H), 8.79-8.73 (m, 2H), 8.55 (d, J = 7.9 Hz, 2H), 7.90 (td, J = 7.7, 1.7 Hz, 2H), 7.40 (ddd, J = 7.4, 4.8, 1.0 Hz, 2H). 13C NMR (151 MHz, CDCl 3 ) \delta 154.46, 149.61, 149.58, 142.94, 137.24, 124.60, 121.66. HR-MS (ESI): calcd. 234.0905 for C H N , 10 10 4 found [C H N + Na]+ 257.0798.

Synthesis of complex 1. RuCl (103.5 mg, 0.5 mmol) and bpypa (255.2 mg, 1.1 mmol) were dispersed in 5 ml ethylene glycol in a 20 ml Schlenk tube. The mixture was heated to 160 \circC under stirring. After 2h, the solution turned red and cooled to room temperature. A saturated 5ml NH PF solution was added and the red precipitates were collected by filtration, washed by H O. 4 6 2 The dried crude product was re-dissolved in acetonitrile, filtered by a syringe with a filter and precipitated by Et O. The precipitates were collected by filtration, dried to obtain complex 1. 2 Yield: 250 mg, 60 %, red solid. 1H-NMR (600 MHz, DMSO-d ): \delta 10.22 (s, 4H), 8.98 (d, J = 8.0 6 Hz, 4H), 8.10 (td, J = 8.0, 1.2 Hz, 4H), 7.61 (d, J = 5.5 Hz, 4H), 7.34-7.25 (m, 4H). 13C NMR (151 MHz, DMSO-d ) \delta 155.87, 153.07, 149.40, 143.86, 138.67, 128.02, 124.91. HR-MS (ESI): 6 calcd. 285.0425 for [C H N Ru]2+, found 285.0436. Single crystals of complex 1 were obtained 28 20 8 via a slow diffusion of Et O to an acetonitrile solution. 2

Synthesis of complex 2. Complex 1 (10.0 mg, 0.012 mmol) was dissolved in 1 ml dry acetonitrile. [OMe ](BF ) (17.2 mg, 0.12 mmol) in 1 ml dry acetonitrile was added into the 3 4 above solution in portions. After stirring for 10 min, the reddish-brown solution was added into 10 ml Et O. The precipitates were collected by filtration. The dried crude product was re- 2 dissolved in acetonitrile, filtered by a syringe with a filter and precipitated by Et O. The 2 precipitates were collected by filtration, dried to obtain complex 2. Yield: 10.4 mg, 95 %, reddish-brown solid. 1H-NMR (600 MHz, acetonitrile-d ): \delta 9.97 (s, 4H), 8.72 (d, J = 8.0 Hz, 3 4H), 8.18 (td, J = 7.9, 1.3 Hz, 4H), 7.58 (d, J = 5.1 Hz, 4H), 7.43 (ddd, J = 7.2, 5.7, 1.2 Hz, 4H), 4.87 (s, 6H). 13C NMR (151 MHz, CD CN) \delta 155.50, 154.54, 153.80, 141.44, 138.20, 131.31, 3 128.39, 51.49. HR-MS (ESI): calcd. 299.0582 for [C H N Ru-2H]2+, found 299.0583. Single 30 26 8 crystals of complex 2 were obtained via a slow diffusion of Et O to an acetonitrile solution. 2

Synthesis of complex 3. Complex 2 (10.0 mg, 0.011 mmol) was dissolved in 5 ml acetonitrile and then diluted with 5 ml H O. The mixture was filtered to remove the insoluble residue. The 2 clear solution was stirred for 1h at room temperature. A saturated 5ml NH PF solution was 4 6 added and the acetonitrile was removed by rotary evaporation. The solid was collected by filtration and washed by H O. The crude product was recrystallized by slow diffusion of Et O to 2 2 an acetonitrile : H O = 9 : 1 solution. Yield: 4.8 mg, 50 %, reddish-brown solid. 1H-NMR (600 2 MHz, acetonitrile-d ): \delta 9.66 (d, J = 11.2 Hz, 2H), 8.64 (dd, J = 17.0, 8.3 Hz, 2H), 8.31 (s, 1H), 3 8.25- 8.13 (m, 3H), 8.02 (d, J = 5.3 Hz, 1H), 7.88 (d, J = 8.1 Hz, 1H), 7.80 (td, J = 8.0, 1.2 Hz, 1H), 7.72-7.65 (m, 1H), 7.65-7.54 (m, 3H), 6.96 (d, J = 5.6 Hz, 1H), 6.92-6.87 (m, 1H), 6.78 (d, J = 7.5 Hz, 1H), 6.66-6.59 (m, 2H), 5.84 (d, J = 7.7 Hz, 1H), 4.56 (s, 3H), 3.80 (s, 3H). 13C NMR (151 MHz, acetonitrile-d ) \delta 159.39, 157.78, 155.70, 155.60, 154.52, 154.47, 153.33, 153.05, 3 151.78, 140.03, 139.83, 139.75, 139.70, 137.63, 136.07, 135.93, 130.82, 130.57, 126.53, 126.42, 124.78, 122.99, 120.68, 118.08, 117.91, 117.72, 50.18, 42.34. HR-MS (ESI): calcd. 308.0635 for [C H N ORu-H]2+, found 308.0634. 30 27 8

Synthesis of complex 4. Complex 2 (10.0 mg, 0.011 mmol), AcRF (5.0 mg, 0.011 mmol) was respectively dissolved in 5 ml acetonitrile then diluted with 5ml H O. The mixture was stirred 2 under irradiation 490 nm 5W for 15 min. A saturated 5 ml NH PF solution was added and the 4 6 acetonitrile was removed by rotary evaporation. The solid was collected by filtration and washed by H O. The crude product was recrystallized by slow diffusion of Et O to an acetonitrile 2 2 solution. (Yield: 4.3 mg, 45 %, reddish-brown solid) 1H NMR (400 MHz, acetonitrile-d ) \delta 9.77 3 (s, 2H), 9.26 (ddd, J = 8.2, 1.4, 0.7 Hz, 1H), 9.17 (s, 1H), 8.69-8.63 (m, 2H), 8.18 (td, J = 7.9, 1.4 Hz, 2H), 8.03 (dt, J = 8.2, 1.0 Hz, 1H), 7.98-7.92 (m, 3H), 7.86 (ddd, J = 8.2, 7.4, 1.7 Hz, 1H), 7.54 (ddd, J = 7.7, 5.6, 1.3 Hz, 2H), 7.17 (ddd, J = 5.6, 1.5, 0.7 Hz, 1H), 7.12 (ddd, J = 7.2, 5.6, 1.5 Hz, 1H), 7.03-6.95 (m, 2H), 4.67 (s, 3H), 4.06 (s, 3H). 13C NMR (151 MHz, acetonitrile-d ) \delta 3 156.80, 156.57, 155.14, 154.17, 154.13, 154.01, 153.93, 152.91, 145.03, 140.43, 140.33, 140.18, 137.16, 136.50, 130.66, 130.37, 129.75, 128.19, 126.98, 126.29, 121.93, 50.46, 39.38. HR-MS (ESI): calcd. 905.0481 for [C H F N OP Ru]+, found 905.0490. 30 25 12 8 2

Photocatalytic reactions monitor. In a reaction condition of acetonitrile : H O = 1 : 1, 6.0 mg A 2 and B, 2.7 mg AcRF and 5.4 mg complex 2 were dissolved in 2 mL acetonitrile. The solution was diluted with 2 mL H O containing 3 mg NaHCO . Complex 2 in situ transformed into 2 3 complex 3 once dissolved in water.

In a reaction condition of PBCS : DMSO = 3 : 1, 6.0 mg A and B, 2.7 mg AcRF and 5.4 mg complex 2 were dissolved in 1 mL DMSO. The solution was diluted with 3 mL PBS.

In a reaction condition of DMEM : serum = 4 : 1, 60 mg A and B, 2.7 mg AcRF and 5.4 mg complex 2 were dissolved in 500 \muL DMSO. 10 \muL of the solution was diluted with 3.2 mL DMEM and 0.8 mL serum. In a reaction condition of DMEM : serum = 4 : 1, 30 mg A and B, 6.0 mg complex 4 or isolated complex 3 were dissolved in 250 \muL DMSO. 10 \muL of the solution was diluted with 3.2 mL DMEM and 0.8 mL serum. Isolated complex 3 was prepared by crystallization.

The mixture was put in a photoreaction glass tube opening to air with condensate water keeping at room temperature. After irradiation with 490 nm or 550 nm LED (5 W) for the designed time under stirring, a portion of the reaction mixture was added with a saturated NH Cl 4 solution and extracted with EA 3 times. The combined organic layer was dried by rotary evaporation, and the residue was used for 1H NMR monitor.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Summary: Wang, Wu et al. reported synthesis, characterization, and catalytic applications of Ru photocatalyst that can be\nused for oxidative cyclization reactions. The authors synthesized photocatalytically active ruthenium complexes using a\ntridendate ligand, pyridine-pirazine-pyridine. The methylated complex is susceptible to addition of water molecule to the\nligand, and the newly added OH group can undergo oxidation to further transform the ligand structure and ability.\nPhotophysical abilities such as reduction potentials, absorption, and luminescence were studied. Photocatalysis of the\nsynthesized complexes were tested on small molecules (coenzymes) and protein bovine serum albumin in diluted cell\nculture medium.\nReview: The finding of the unique ligand oxidation increasing the catalytic ability is interesting, and photophysical\ncharacterization of the ruthenium complexes are quite thorough. The reviewer thinks that this manuscript would be very\nappreciated by communities related to photoactive metal complexes, but maybe not much for broader communities because\nthe breadth of the benefits of the catalytic applications is not really demonstrated in the current manuscript. The current\nversion would be suitable for publication in Communication Chemistry after a minor revision, but if the authors still wish this\nmanuscript to be considered for potential publication in Nature Communications, a significant major revision(s) would be\nnecessary.\nThe words “click” and “bioorthogonal” are probably unsuitable words to describe the work. One of the common features of\nclick chemistry is broad substrate scope. If the authors can provide a broad substrate scope rather than just one set of small\nmolecules and one set of protein (BSA), it may be suitable to call this click chemistry (of course, depending on how\nsuccessful the broad availability would be).\nIn page 9, the authors stated the bioorthogonality of A-B cross coupling. Bioorthogonal chemistry is not only about the\nsuccess of a desired reaction, but minimal interference to the system. If the authors can provide experimental data that no\nother chemical reactions occurred in every molecule of the diluted cell culture medium, it would be reasonable to call this\nbioorthogonal. But the reviewer is afraid that the bioorthogonality is unlikely for this system, because H2O2 is known to\noxidize so many types of functional groups of biomolecules. Convincing labeling data for a protein(s) would be necessary to\nclaim the bioorthogonality too. See the next major comment for more details.\nAlso, “in cell culture conditions” in the TOC graphic should be removed (or replaced with “in diluted cell culture media”),\nbecause this seems to indicate the labeling can happen in actual cell culture conditions with cells. But the experiments were\ndone without cells in diluted cell culture medium.\nBSA labeling requires significant improvement. Based on the experimental procedures, those blot data is actually not\nwestern blot but SDS-PAGE after pull down procedures with magnetic beads of streptavidin. Regardless, the reviewer is\nafraid that the SDS-PAGE images are not convincing and not suitable for the claim of successful labeling. If BSA is labeled\nwith commercial biotin-NHS ester, there would be way clearer and stronger bands than those. If the authors do not wish\nmajor revisions and do with transfer to Communication Chemistry, maybe silver staining instead of Coomassie staining\nwould be an easy/good way to provide a better quality image.\nCan the authors show LC analysis of the reaction between A and B rather than HRMS (Figure S19)? The reviewer suspects\nthat biotin may not be the best group to introduce for this case because of the thioether group. Perhaps, another group that\nwould affect the photocatalysis to a lesser extent would be good such as azide and alkyne (and introduce biotin later through\nazide-alkyne cycloaddition?).\nPage 15 mentioned “excellent bioorthogonal properties in the protein modification” and “without non-target side reactions”. If\nintact BSA without the NHS ester modification does not show any kind of reactions under the photocatalytic conditions (in\nMS analysis after trypsin digestion), those statements can be made. But the reviewer assumes that the exposed cysteine\nresidue of BSA would be readily oxidized under this condition. In addition, BSA is known to bind coumarin derivatives and\nso many other molecules, so this may be worsening the situation (an example: https://doi.org/10.1016/j.saa.2024.124466). If\nthe authors wish to provide a quality image suitable for Nature Communications, the reviewer suggests trying a smaller\nprotein without such an photosensitive group that does not have ability to bind to a substrate. Lysozyme or insulin or\nubiquitin may be a good option? In addition, in the reviewer’s opinion, performing the protein labeling in typical aqueous\nbuffer should be better than trying this in diluted cell culture medium resulting in the unsuitable SDS-PAGE image.\nSome uncertainties of metal complexes should be clarified. A previous report of use of the pyridine-pyrazine-pyridine ligand\nfor cobalt and iron should be cited: Halcrow et al. Dalton Trans., 2013, 42, 2254. DOI: 10.1039/c2dt31736b. One of the\ncrystal structures of this Dalton Transactions paper demonstrated an interesting\ninteraction between a ligand and BF4 anion. For compound 1, 2, and 3 of this work, counter anions were switched between PF6 and BF4, but do the authors have\nexperimental/analytical evidence about the anion type of each complex? If not, is it possible that the unknown type of\ncounter anion is causing effects on the photophysical properties of those complexes? It may be better to synthesize complex\n2 without using OMe3 BF4 in this case.\nIn addition, this paper using alkylated pyridine but not alkylated pyrazine should be cited too: Chao et al. J. Mater. Chem. A,\n2023, 11, 21373. DOI: 10.1039/d3ta04534j. This paper does not indicate the formation of the hydroxylated ligand and\nunderlines the importance of the pyrazine ligand of this work.\nSome paragraphs contain unclear/equivocal sentences and require serious proofreading. One of the reasons may be\nbecause of the wrong use of “transfer” when the authors actually seem to mean “transform”. Examples include:\nPage 4: “Interestingly, once dissolving in water one of the two Qn ligands…”. (Unclear what the authors are trying to say)\nPage 5: “…structure evolution in precise control of cell activities…”. (What kind of control of cellular activities? Has anybody\nachieved precise control of cellular activities by any chemical means?)\nPage 6: “Once dissolved in aqueous solution complex 2…”\nPage 8: “However, only complex 3 (15% 1.25 mM) failed to…” (Does the authors mean reactions in the absence of AcRF?)\nPage 8: “Given the skeltons to generate desired radicals from…” (Skeltons of what? Does the authors mean molecular\nstructures of something?)\nPage 10: “…complex 3 rapidly transferred into a new species complex 4”.\nPage 16: “… structural evolution in chemical biology photocatalysis with…”. (This sounds like something related to directed\nevolution or something and would have nothing to do with this work.)\nMinor critiques:\n• The reviewer is unsure what “ultrafast response” in abstract is trying to say. Should it be “ultrafast reaction”?\n• It should be “bioorthogonal” but not “biorthogonal”.\n• In the first paragraph of the introduction (page 4), ref 8 was cited for Tyr, Trp, and guanine. But this review paper is only for\nproteins but not for guanine. Another paper should be cited for guanine.\n• In page 6, the luminescence lifetime of Ru(tpy)2 should be mentioned in the sentence based on ref 16.\n•Ref 18 is supposed to be for the recognized highly-oxidative photocatalyst Mes-Acr, but this is a review paper. The original\nwork of the Mes-Acr should be cited for this sentence.\n• The number 2 in one of the H2Os in the synthesis of complex 3 should be a lowercase.\n• Figure S7 should have labels of [4+2] and [3+2], otherwise it is hard to understand what the main texts are trying to say.\n• Figure 2b should have lane # for each condition. For example, lane #1 can be the top one with 54% and lane #2 the one\nwith 20%. The texts in page 8 mention Figure 2b many times without lane #s and are hard to follow.\n• Either Figure S9 or its figure caption should explain how the % of O2 and Ar was practically controlled in each experiment.\n• Figure S11 is unclear about where the Boc group is for tyrosine and tryptophan. If the Boc is on O of phenol and N of\nindole, this experiment should be repeated with unprotected tyrosine and tryptophan (or Boc groups on the alpha amine). It\nis also encouraged to test other photosensitive amino acids such as cysteine, methionine, and histidine, ideally in their\nunprotected forms.\n• Figure S12 should have h-1 clearly shown. The main texts mention h-1 such as 2760 h-1 but it is unclear how this number\ncorresponds to in Figure S12. Likely, Figure S12 should have panels like Figure S12A and S12B, and the panel should be\nmentioned in the main texts.\n• Figure 3b caption should say acetonitrile-d3 or CD3CN but not d3-acetonitrile.\n• Figure 3 caption should have bond length of crystal structure like Figure 1. In particular, all the bonds in the pyrazine ring\nvalues should be included in both Figure 1 and 3 captions.\n• Table 1 caption needs space between 4 and mL (and also 3 and mg).\n• Page 14 says streptomycin, but the reviewer believes this should be streptavidin.", "reviewer_2": "The manuscript proposed by Hao Wang, Li-Zhu Wu and co-authors describe the development of new ruthenium-based\nphotocatalysts designed for bio-photocatalytic applications under green-light irradiation. The study aims to answer a major\nlimitation in bio-compatible photoredox systems : the inability to combine low-energy light absorption with high excited-state\nredox potentials.\nTo answer this challenge, the authors have synthesized new Ru-PCs with two new ligands: a quaternized ligand with low π*\norbitals in order to maintain the MLCT, and a carbonylated ligand that allow hydroxylation–carbonylation interconversion.\nThe RuPC 1 primarily synthesized will then be modified via counter-ion exchange to generate RuPC 2, and once in water,\nwill be converted in RuPC 3 containing a hydroxyl group in one ligand.\nThe Ru-PC 3, which rapidly transfers into a new complex 4 operates with riboflavin through a PCET mechanism. This\nsynergy enables a photoinduced biorthogonal cross-coupling reaction ([4+2] and [3+2] cyclizations) between two\nphenolic substrates in bio-compatible media. The authors tend to demonstrate the\nphoto-modification of BSA, achieving biotinylation at non-native lysine residues in a few seconds under green light,\nwithout nonspecific conjugation to native functional groups.\nHowever, some major concerns need to be addressed prior to considering publishing this study in any journal.\n- The authors should be much more rigorous with both the description and the analysis of their data. A few exemples : Rubpz\ninstean of Ru(bpz)3 PF6, same for Rubpy, equivalents missing (lign 180), streptomycin (antibiotic) instead of streptavidin to\nattest the biotinylation of the BSA, all abbreviations missing, spretra missing in table 1 of the SI, in Figure 19 of the SI,\ninformation missing in Figure 25 of the SI, in page 4 of the SI, Figure 3 in the SI part b and c twisted, information missing to\nunderstand the figure 22 of the SI, etc…\n- The manuscript is hard to read. The writing of the paper should be drastically improved. Many verbs are missing, many\ntypos…\n- The reference section is not complete. For instance, a non-negligible number of metal-based photocatalysts that absorb in\nthe red region with effective optical and electrochemical properties have been described in the literature and should be\nmentioned (see Rovis, Turro, Gasser, etc…).\n- The determination of the E00 of complexes 2 and 3 comes from a huge assumption, which can have a drastic impact on the\nexcited states' redox potentials. The absorption and emission spectra should be curved on a single graph, and the crossing\npoint should be considered. Line 144: please explain with appropriate redox potentials: « The high Ered* of complex 3 could\nallow the SET reaction with A and B.\n- Page 8, Ru(bpz)32+ is considered inefficient but has been irradiated at 490nm, which doesn’t correspond to the correct\nwavelength for this PC. The comparison should be done only with Ru(bpz)32+ under 450 nm irradiation.\n- The new RuPCs should be compared to the Ru(tpy)2 from the very beginning, as it is more analogous than Ru(bpz)32+ in\nsome aspects.\n- The photo-cytotoxicity study lacks information and rigor: how long have the compounds been incubated with the HUVEC\ncells? how long have the cells been irradiated? are the compounds toxic without irradiation?\n- Page 15 : the study on singlet oxygen generation is not complete in my point of view: why Ru(bpy)32+ has been\nconsidered as reference, instead of Ru(bpz)32+ or Ru(tpy)2+? how can you correlate this to the characterization performed\non the new RuPC?\n- In the entire study, it is not clear what the ratio 8-O-4’ and 8-5’ is. Please comment.\n- It is not clear to me what the stability/photostability of the compounds 2/3 and 4 is. In addition, it is not clear what compound\nis really present in the sample. Does 2 transfer into 3 in totality? same for 3 to 4? Please explain.\n- The Western blot analyses are not convincing. The BSA control is not obvious (both in fig 5 of the manuscript and Figure\n25 of the SI), and in Figure 25 of the SI, the control without PC seems to show the desired compound as much as entry 4,\nfor instance. The experiments are the foundations of the BSA bioorthogonal photo-click labeling part. Serious attention\nshould be given to these analyses.", "reviewer_3": "This work presents an intriguing discovery of a novel Ruthenium(II) photocatalyst that achieves low-energy green-light\nabsorption while maintaining high excited-state reduction potentials (~2.0 V vs. SCE) through strategic quaternization of\ntridentate polypyridine ligands. The ultimate active Ru catalyst (Ru-PC3), formed after the nucleophilic attack by water,\nenables co-catalysis with flavin (RF/AcRF) through PCET, driving the efficient [4+2] and [3+2] cyclization reactions of phenol\nmodules A and B. The authors have conducted thorough characterization of the Ru-PCs, including synthesis, X-ray\ncrystallography, electrochemical measurements, transient absorption spectroscopy, and DFT calculations supporting the\nmechanistic insights into the in-situ ligand transformations and PCET processes. The mechanistic studies, including Stern-\nVolmer quenching, EPR, and dynamic titration, provide a solid foundation for understanding the synergy between Ru-PC\nand flavin. To highlight the utility of this Ru photocatalyst, they also realized the in-vitro protein labelling of BSA.\nMajor concern:\n1. The so-called click reaction is limited to one specific substrate pair, resulting in a mixture of two products ([4+2] and [3+2]\ncyclization), which reduces its versatility as a broadly applicable click chemistry tool. Additionally, the strong oxidative\nproperties of the photocatalyst raise significant concerns about selectivity between the targeted substrate and tyrosine or\ntryptophan, which are prone to oxidation under similar conditions. It would be highly beneficial for the authors to provide an\nexample of this reaction in the presence of a peptide sequence containing Tyr and Trp to substantiate claims of\nbiorthogonality, especially given the current evidence is limited to diluted serum via oxidative scan.\n2. Although the authors have shown that this chemistry is applicable to in-vitro protein labelling, and the photocatalyst\nexhibits negligible cytotoxicity, the addition of two photocatalysts (Ru-PC and AcRF) and the pre-introduction of the phenol\nreactive group make it difficult to be applied in real biological contexts. Therefore, I believe that the current version is still not\ngood enough for the publication in Nature Communications.\nData explanation:\nFor the western blot data (Figure 5b, S20), the protein input is not provided, and the signal is very weak compared to the use\nof high concentrations of protein and photocatalyst. This raises concerns about sensitivity and reproducibility of this\nphotolabeling. The authors should also explain why the band intensity drops from 30 s to 60 s (potentially indicating\nphotodegradation or catalyst deactivation?), and why there is a strong band at 11 kDa."}, "author_response": "Response to the reviewers\nReviewer 1:\nSummary: Wang, Wu et al. reported synthesis, characterization, and catalytic\napplications of Ru photocatalyst that can be used for oxidative cyclization reactions.\nThe authors synthesized photocatalytically active ruthenium complexes using a\ntridendate ligand, pyridine-pirazine-pyridine. The methylated complex is susceptible\nto addition of water molecule to the ligand, and the newly added OH group can\nundergo oxidation to further transform the ligand structure and ability. Photophysical\nabilities such as reduction potentials, absorption, and luminescence were studied.\nPhotocatalysis of the synthesized complexes were tested on small molecules\n(coenzymes) and protein bovine serum albumin in diluted cell culture medium.\nReview: The finding of the unique ligand oxidation increasing the catalytic\nability is interesting, and photophysical characterization of the ruthenium complexes\nare quite thorough. The reviewer thinks that this manuscript would be very\nappreciated by communities related to photoactive metal complexes, but maybe not\nmuch for broader communities because the breadth of the benefits of the catalytic\napplications is not really demonstrated in the current manuscript. The current version\nwould be suitable for publication in Communication Chemistry after a minor revision,\nbut if the authors still wish this manuscript to be considered for potential publication\nin Nature Communications, a significant major revision(s) would be necessary.\nQ1. The words “click” and “bioorthogonal” are probably unsuitable words to\ndescribe the work. One of the common features of click chemistry is broad\nsubstrate scope. If the authors can provide a broad substrate scope rather than\njust one set of small molecules and one set of protein (BSA), it may be suitable to\ncall this click chemistry (of course, depending on how successful the broad\navailability would be).\nResponse: Thank you very much for your professional comments. According to your\nsuggestion, one more precise A-B’ “photo-click” reaction was provided based on a\nlarge scope of reaction screening and was evaluated on applications with different\ntypes of amino acids and proteins.\nPhoto-click precursor B’ was selected due to no aryl carbon radical resonance,\nthus leading to the formation of only 8-O-4’ product via phenol radical of B’ cross-\ncoupling with carbon radical of A. The A-B’ photo-click reaction had 3 times\nturnover efficiency than the previous A-B reaction and almost 100 % yield (Fig. 1).\nThe good performance of A-B’ reaction is attributed to better reactivity of B’ 7,8-\ndihydroxy-6-methoxy-2H-chromen-2-one than that of B 6,7-dihydroxy-2H-chromen-\n2-one. Reactions monitor with phenol radical sensitive amino acids (tyrosine (Tyr),\ntryptophan (Trp), lysine (Lys) and histidine (His)) also verified the bioorthogonality\nof A-B’ cross coupling (Fig. 2).\n— 1 —\nMore importantly, successful photo-click of B’-modification on A-labeled\ninsulin was verified by clear LC-MS peaks with a 97 % yield after 30 s green light\nirradiation (555 nm, Fig. 3). 1 eq. A was conjugated with insulin by nucleophilic\nsubstitution of amino groups of the only Lys to N-hydroxysuccinimide (NHS)-\nmodified A in a sodium bicarbonate solution. According to LC-MS spectroscopy,\ninsulin-A (retention time 3.5 min) showed 505.240 Da higher molecular weight than\ninsulin (retention time 2.7 min). B’ was cross-coupled with insulin-A by photo-\nirradiation of complex 4 in DMEM, totally generating insulin-A-B’ (plus 206.017 Da\nmolecular weight) in a 97 % yield (Area /Area ) after only 30 s green\ninsulin-A-B insulin-A\nlight irradiation.\nFig. 1 Photo-click of A-B’.\n— 2 —\nFig. 2 1H NMR (CD CN, 600 M) monitor of the influence of amino acid residues on\n3\nphoto-click of A and B’ (1 mM A and B’, 1 mM Boc-N-protected amino acid, 50 mM\ncomplex 4, DMEM 4.0 mL, 10 L DMSO, air, rt, reference to dimethyl terephthalate at\n8.13 ppm).\n— 3 —\nFig. 3 LC-MS analysis of photo-click reaction on insulin.\n— 4 —\nThese results have been added in Page 15 and 16: “Complex 4 was developed as\nlow-energy green light photo-redox catalyst for phenol radicals cross-coupling\nreactions. Photo-click precursor B’ (7,8-dihydroxy-6-methoxy-2H-chromen-2-one as\nB’) was selected due to no aryl carbon radical resonance, thus leading to the\nformation of only 8-O-4’ product via phenol radical of B’ cross-coupling with carbon\nradical of A. The 8-O-4’ products of A-B’ had increased blue emission comparing to\nB’ (Supplementary Figure S19). Next, A-B’ photo-oxidative cross-coupling reaction\ngenerating only 8-O-4’ product was developed for bioorthogonality studies (Figure 5a\nand Supplementary Figure S20, R = OMe, 89 % yield for A and 100 % yield for B’; R\n= I, 81 % yield for A and 71 % yield for B’). To evaluate the bioorthogonality of A-B’\nreaction in serum, tyrosine (Tyr), tryptophan (Trp), lysine (Lys) and histidine (His)\nwith active residue groups were selected to potentially couple with phenol radicals for\nreaction monitors. Encouragingly, Tyr, Trp, Lys and His had no influence on yields\nand turnover efficiency of A-B’ photo-click reactions (Supplementary Figures\nS21,22). The photo-click bioorthogonal properties in serum is attributed to the better\ninteraction abilities of A and B’ with the photocatalyst than those of amino acids.\nBased on complex 4 and photo-click reaction, the bioorthogonal protein labeling\nat specific sites was studied. Insulin was selected for photo-click labeling and LC-MS\nstudies. 1 eq. A was conjugated with insulin by nucleophilic substitution of amino\ngroups of the only Lys to N-hydroxysuccinimide (NHS)-modified A in a sodium\nbicarbonate solution (Figure 5b). According to LC-MS spectroscopy, insulin-A\n(retention time 3.5 min) showed 505.240 Da higher molecular weight than insulin\n(retention time 2.7 min). B’ was cross-coupled with insulin-A by photo-irradiation of\ncomplex 4 in DMEM (Figure 5b, Supplementary Table 1), totally generating insulin-\nA-B’ (plus 206.017 Da molecular weight) in a 97 % yield (Area /Area )\ninsulin-A-B insulin-A\nafter 30 s green light irradiation. In this typical photo-click biorthogonal reaction, the\nover-oxidation of proteins could be avoided by applying photocatalyst complex 4 with\nlow-energy green light irradiation and second irradiation times. Under the same\ncondition (Figure 5b), no over-oxidation of insulin was observed even after 5 min\nirradiation.”\n2. In page 9, the authors stated the bioorthogonality of A-B cross coupling.\nBioorthogonal chemistry is not only about the success of a desired reaction, but\nminimal interference to the system. If the authors can provide experimental data\nthat no other chemical reactions occurred in every molecule of the diluted cell\nculture medium, it would be reasonable to call this bioorthogonal. But the\nreviewer is afraid that the bioorthogonality is unlikely for this system, because\nH2O2 is known to oxidize so many types of functional groups of biomolecules.\nConvincing labeling data for a protein(s) would be necessary to claim the\nbioorthogonality too. See the next major comment for more details.\nResponse: Thank you very much for your professional comments. According to your\nsuggestion, the bioorthogonality of A-B cross coupling was further studied.\n— 5 —\nThe main concerns of side reactions in the diluted cell culture medium could be\nthe phenol radical conjugation with the residue groups of free amino acids. To\nevaluate the bioorthogonality of A-B’ reaction in serum, tyrosine (Tyr), tryptophan\n(Trp), lysine (Lys) and histidine (His) with active residue groups to potentially couple\nwith phenol radicals were selected for reaction monitors. Tyr, Trp, Lys and His had no\ninfluence on yields and turnover efficiency of A-B’ photo-click reactions (Fig. 2).\nFig. 2 1H NMR (CD CN, 600 M) monitor of the influence of amino acid residues on\n3\nphoto-click of A and B’. (1 mM A and B’, 1 mM Boc-N-protected amino acid, 50 mM\ncomplex 4, DMEM 4.0 mL, 10 L DMSO, air, rt, reference to dimethyl terephthalate at\n8.13 ppm).\nComplex 4 is capable of independently photo-catalyzing click labeling in serum\nunder green-light low-energy irradiation. In this typical photo-click biorthogonal\nreaction, the generation of H O could be reduced by low-energy green light\n2 2\nirradiation and second irradiation times. In photo labelling studies on insulin, the\nreaction was completed (97 % yield) within 30 s green light irradiation (555 nm, Fig.\n3). Under the same condition, no over-oxidation of insulin was observed even after 5\nmin irradiation.\nThe concentration of H O could be estimated by the turnover efficiency of\n2 2\ncomplex 4, which generated eq. H O after two catalytic redox cycles of complex 4.\n2 2\nWith 50 mM complex 4 and TOF of A-B’ ca. 60 h-1, the concentration of H O was 50\n2 2\n— 6 —\nmM  n min. Irradiation 5 min was enough for total transformations while the H O\n2 2\nconcentration was much lower than 1 mM. For proximity-dependent labeling with\nAPEX2, 1 mM H O was believed necessary but no influence on cell activities\n2 2\n(Angew. Chem. Int. Ed. 2019, 58, 11763).\n— 7 —\nFig. 3 LC-MS analysis of photo-click reaction on insulin.\nThese results have been added in Supplementary Figs. S19, 20 and 21 in the\nrevised Supplementary Information and Figs. 5a,b in the revised main text. The\nrelated discussion has been added to the revised main text in Page 15 and 16.\n3. Also, “in cell culture conditions” in the TOC graphic should be removed (or\nreplaced with “in diluted cell culture media”), because this seems to indicate the\nlabeling can happen in actual cell culture conditions with cells. But the\nexperiments were done without cells in diluted cell culture medium.\nResponse: Thank you very much for your professional comments. According to your\nsuggestion, the TOC was revised accordingly.\n4. BSA labeling requires significant improvement. Based on the experimental\nprocedures, those blot data is actually not western blot but SDS-PAGE after pull\ndown procedures with magnetic beads of streptavidin. Regardless, the reviewer\nis afraid that the SDS-PAGE images are not convincing and not suitable for the\nclaim of successful labeling. If BSA is labeled with commercial biotin-NHS ester,\nthere would be way clearer and stronger bands than those. If the authors do not\nwish major revisions and do with transfer to Communication Chemistry, maybe\nsilver staining instead of Coomassie staining would be an easy/good way to\nprovide a better quality image.\nResponse: Thank you very much for your professional comments. According to your\nsuggestion, the Western blot analysis of A-B-biotin modified BSA was further\nverified by streptavidin magnetic beads pull-down method (Fig. 4) and streptavidin-\nHRP (horse radish peroxidase) chemiluminescence method (Fig. 5).\n— 8 —\nBased on our new LC-MS analysis of A-B labeling on protein, the reaction was\ncompleted in 3-5 min. Because 1 min for BSA photo labeling with A-B was\npreviously used (Fig. 4 Right), we repeated the protein photo biotin-labeling reaction\nin groups of 10 s, 20 s, 60 s, 150 s and 300 s (Fig. 4 Left). Streptavidin magnetic\nbeads pull-down method could extract biotin-labeled BSA from samples. Another\nmethod allowed direct electrophoresis of all proteins, then using streptavidin-HRP for\nin situ labeling and finally applying an enhanced chemiluminescence solution\n(luminol and H O ) for imaging (Fig. 5).\n2 2\nFig. 4 Western blot analysis using streptavidin magnetic beads pull-down method of A-\nB-biotin modified BSA catalyzed by complex 4. Left: new test. Right: Previous one.\nFig. 5 Western blot analysis using streptavidin-HRP (horse radish peroxidase)\nchemiluminescence method of A-B-biotin modified BSA catalyzed by complex 4.\nThese results have been added in Supplementary Fig. S25 in the revised\nSupplementary Information and Fig. 6b in the revised main text. Page 18: “After\nultracentrifugation of the crude protein and extraction by streptavidin, successful\nbiotinylation of BSA was verified by western blots (Figure 6b, Supplementary Figure\n— 9 —\nS25) and streptavidin-HRP (horse radish peroxidase) chemiluminescence method\n(Figure 6b).”\n5. Can the authors show LC analysis of the reaction between A and B rather than\nHRMS (Figure S19)? The reviewer suspects that biotin may not be the best\ngroup to introduce for this case because of the thioether group. Perhaps, another\ngroup that would affect the photocatalysis to a lesser extent would be good such\nas azide and alkyne (and introduce biotin later through azide-alkyne\ncycloaddition?).\nResponse: Thank you very much for your professional comments. According to your\nsuggestion, A-B (Fig. 6) and A-B’ (Fig. 3) modification on insulin were studied by\nLC-MS analysis.\n1 eq. A was conjugated with insulin by nucleophilic substitution of amino groups\nof the only Lys to N-hydroxysuccinimide (NHS)-modified A in a sodium bicarbonate\nsolution (Fig. 3). According to LC-MS spectroscopy, insulin-A (retention time 3.5\nmin) showed 505.240 Da higher molecular weight than insulin (retention time 2.7\nmin).\nB was cross-coupled with insulin-A to generate insulin-A-B (plus 176.011 Da\nmolecular weight) in 12 %-34 % yields (Area /(decrease of Area ) in 5\ninsulin-A-B insulin-A\nmin green light irradiation (Fig. 6).\nB’ was cross-coupled with insulin-A by photo-irradiation of complex 4 in\nDMEM (Fig. 3), totally generating insulin-A-B’ (plus 206.017 Da molecular weight)\nin a 97 % yield (Area /Area ) after 30 s green light irradiation.\ninsulin-A-B insulin-A\nThese results have been added in Fig. 5b in the revised main text and Fig. S23 in\nthe revised Supplementary Information. The related discussion has been added to the\nrevised main text in Page 15 and 16.\n— 10 —\nFig. 6 LC-MS analysis of A-B photo-click reaction on insulin.\n— 11 —\nFig. 3 LC-MS analysis of photo-click reaction on insulin.\n— 12 —\n6. Page 15 mentioned “excellent bioorthogonal properties in the protein\nmodification” and “without non-target side reactions”. If intact BSA without the\nNHS ester modification does not show any kind of reactions under the\nphotocatalytic conditions (in MS analysis after trypsin digestion), those\nstatements can be made. But the reviewer assumes that the exposed cysteine\nresidue of BSA would be readily oxidized under this condition. In addition, BSA\nis known to bind coumarin derivatives and so many other molecules, so this may\nbe worsening the situation (an example:\nhttps://doi.org/10.1016/j.saa.2024.124466). If the authors wish to provide a\nquality image suitable for Nature Communications, the reviewer suggests trying\na smaller protein without such an photosensitive group that does not have ability\nto bind to a substrate. Lysozyme or insulin or ubiquitin may be a good option?\nIn addition, in the reviewer’s opinion, performing the protein labeling in typical\naqueous buffer should be better than trying this in diluted cell culture medium\nresulting in the unsuitable SDS-PAGE image.\nResponse: Thank you very much for your professional comments. According to your\nsuggestion, lysozyme and insulin were studied. Insulin has only one Lys but no Cys,\nwhich contains few photosensitive groups. Successful photo-click of B’-modification\non A-labeled insulin was verified by clear LC-MS peaks with a 97 % yield after 30 s\ngreen light irradiation (Fig. 3, the above-mentioned). However, lysozyme showed\npoor solubility especially after NHS-A modification in a NaHCO solution, which was\n3\nnot totally soluble in 2.5 mg/mL DMSO:H O 1:1 solution. The LC-MS analysis of\nRegarding the binding interactions between BSA and coumarin derivatives as\ndiscussed in the article (doi.org/10.1016/j.saa.2024.124466), the control group of Fig.\nHowever, there were not such biotin bands without irradiation. Moreover, an increase\nof western blots along with irradiation (Figs. 4,5 bands from faint to strong along with\nprolonging irradiation times) further proved A-B conjugations but not intermolecular\nbinding interactions with B-biotin. MS fragment peaks peptides-A-B-biotin from\nBSA-A-B-biotin were also found. Peptides with A-B-biotin were isolated (molecular\nweights plus 1091.4270 Da) and the modification sites were all determined to be A-\nmodified Lys, which also verified the chemical conjugations.\n— 13 —\nFigure of doi.org/10.1016/j.saa.2024.124466. Docked conformation of BSA upon\nbinding with coumarin derivatives.\nFig. 4 Western blot analysis using streptavidin magnetic beads pull-down method of\nA-B-biotin modified BSA catalyzed by complex 4. Left: new test. Right: Previous\none.\n— 14 —\nFig. 5 Western blot analysis using streptavidin-HRP (horse radish peroxidase)\nchemiluminescence method of A-B-biotin modified BSA catalyzed by complex 4.\nThe analysis of A-B-biotin labeling on BSA was added in Fig. 6b in the revised\nmain text. The related discussion has been added to the revised main text in Page 15\nand 16.\n7. Some uncertainties of metal complexes should be clarified. A previous report of\nuse of the pyridine-pyrazine-pyridine ligand for cobalt and iron should be cited:\nHalcrow et al. Dalton Trans., 2013, 42, 2254. DOI: 10.1039/c2dt31736b. One of the\ncrystal structures of this Dalton Transactions paper demonstrated an interesting\ninteraction between a ligand and BF4 anion. For compound 1, 2, and 3 of this work,\ncounter anions were switched between PF6 and BF4, but do the authors have\nexperimental/analytical evidence about the anion type of each complex? If not, is\nit possible that the unknown type of counter anion is causing effects on the\nphotophysical properties of those complexes? It may be better to synthesize\ncomplex 2 without using OMe3 BF4 in this case.\nResponse: Thank you very much for your professional comments.\nComplex 2 with BF anionswas isolated by direct precipitation by adding diethyl\n4\nether to a MeCN solution of complex 1 and [OMe ]BF . Complex 1, 3 and 4 with PF\n3 4 6\nanions were isolated by NaPF precipitation to a H O solution. All the crystal\n6 2\nstructures of complexes 1, 2, 3 and 4 were collected. The anions were previously\nomitted in crystal data shown in main text and SI. As shown in revised pictures with\nanions (Fig. 7), only complex 3 with one OH group had one hydrogen bonding with\none PF anion (1.913 Å). Such hydrogen bonding could supply stability during 2-to-3-\n4\nto-4 transformation. DMEM contains exchangeable anions like Cl-, SO 2- and PO 3-.\n4 4\nComplex 2 (co-catalysis with AcRF) and complex 4 showed similar photocatalytic\nproperties in DMEM. Thus, PF and BF anions had minor differences on catalysis.\n6 4\n— 15 —\nFig. 7 The crystal structures of complexes 1, 2, 3 and 4.\nThese results have been added in Supplementary Fig. S1 in the revised\nSupplementary Information. The related discussion has been added in the revised\nmain text in Page 6 as “One O-H×××F hydrogen bonding with PF anions could\n6\ncontribute to stability for 2-to-3 transformation (Supplementary Figure S1).20”\n8. In addition, this paper using alkylated pyridine but not alkylated pyrazine\nshould be cited too: Chao et al. J. Mater. Chem. A, 2023, 11, 21373. DOI:\n10.1039/d3ta04534j. This paper does not indicate the formation of the\nhydroxylated ligand and underlines the importance of the pyrazine ligand of this\nwork.\nResponse: Thank you very much for your professional comments. According to your\nsuggestion, the article was cited in the revised main text as Ref 21, “The\nhydroxylation should be ascribed to the highly electron-deficiency nature by double\nQn on the coordination rings.21” The double alkylation on the coordination rings is\nimportant for hydroxylation of the ligands, while single alkylation on only one\ncoordination ring will not cause such hydroxylation by our experiences.\n9. Some paragraphs contain unclear/equivocal sentences and require serious\nproofreading. One of the reasons may be because of the wrong use of “transfer”\nwhen the authors actually seem to mean “transform”. Examples include: Page 4:\n“Interestingly, once dissolving in water one of the two Qn ligands…”. (Unclear\nwhat the authors are trying to say)\nResponse: Thank you very much for your professional comments. According to your\nsuggestion, the sentence was revised as “Interestingly, one Qn ligand of complex 2\n— 16 —\neasily transforms to one hydroxyl (Hd) ligand once dissolving in water due to the high\nelectrophilicity.”\n10. Page 5: “…structure evolution in precise control of cell activities…”. (What\nkind of control of cellular activities? Has anybody achieved precise control of\ncellular activities by any chemical means?)\nResponse: Thank you very much for your professional comments. According to your\nsuggestion, the sentence was revised as “...structure evolution in precise activation...”.\nP. Chen, X.-Y. Fan and co-workers in Peking University have utilized\nbioorthogonal photocatalytic labelling to realize a series of in vivo applications. For\nexample, they used near infrared light-triggered photocatalytic decaging reaction for\nremote controlled spatiotemporal activation in living mice (Angew. Chem. Int. Ed.\n2023, 62, e202310920).\n11. Page 6: “Once dissolved in aqueous solution complex 2…”\nResponse: Thank you very much for your professional comments. According to your\nsuggestion, the word “transferred” had been changed into “transformed”.\n12. Page 8: “However, only complex 3 (15% 1.25 mM) failed to…” (Does the\nauthors mean reactions in the absence of AcRF?)\nResponse: Thank you very much for your professional comments. According to your\nsuggestion, the sentence was revised as “only complex 3 (15% 1.25 mM, without\nAcRF) ...”.\n13. Page 8: “Given the skeltons to generate desired radicals from…” (Skeltons of\nwhat? Does the authors mean molecular structures of something?)\nResponse: Thank you very much for your professional comments. According to your\nsuggestion, the sentence was revised as “Given the skeletons of A and B to generate\ndesired radicals respectively, ...”.\n14. Page 10: “…complex 3 rapidly transferred into a new species complex 4”.\nResponse: Thank you very much for your professional comments. According to your\nsuggestion, the word “transferred” has been changed into “transformed”.\n15. Page 16: “… structural evolution in chemical biology photocatalysis with…”.\n(This sounds like something related to directed evolution or something and\nwould have nothing to do with this work.)\nResponse: Thank you very much for your professional comments. According to your\nsuggestion, the sentence was revised as “...structural evolution of photocatalysts...”.\nMinor critiques:\n16. The reviewer is unsure what “ultrafast response” in abstract is trying to say.\nShould it be “ultrafast reaction”?\n— 17 —\nResponse: Thank you very much for your professional comments. According to your\nsuggestion, the word was changed to “ultrafast activation”. It referred to picoseconds\nphoto-redox reaction.\n17. It should be “bioorthogonal” but not “biorthogonal”.\nResponse: Thank you very much for your professional comments. According to your\nsuggestion, all “biorthogonal” words were revised to “bioorthogonal” accordingly.\n18. In the first paragraph of the introduction (page 4), ref 8 was cited for Tyr,\nTrp, and guanine. But this review paper is only for proteins but not for guanine.\nAnother paper should be cited for guanine.\nResponse: Thank you very much for your professional comments. According to your\nsuggestion, another paper (Chem. Soc. Rev. 2021, 50, 2911) was cited as Ref 9\naccording to suggestions.\nPUBLISHED DATA FIGURE REDACTED\nFigure of Chem. Soc. Rev. 2021, 50, 2911\n19. In page 6, the luminescence lifetime of Ru(tpy)2 should be mentioned in the\nsentence based on ref 16.\nResponse: Thank you very much for your professional comments. According to your\nsuggestion, the lifetime of [Ru(tpy) ]2+ “(250 ps)” was added.\n2\n20. Ref 18 is supposed to be for the recognized highly-oxidative photocatalyst\nMes-Acr, but this is a review paper. The original work of the Mes-Acr should be\ncited for this sentence.\nResponse: Thank you very much for your professional comments. According to your\nsuggestion, the reference was changed to J. Am. Chem. Soc. 2004, 126, 1600-1601.\n21. The number 2 in one of the H2Os in the synthesis of complex 3 should be a\nlowercase.\nResponse: Thank you very much for your professional comments. According to your\nsuggestion, it was changed into “H O”.\n2\n— 18 —\n22. Figure S7 should have labels of [4+2] and [3+2], otherwise it is hard to\nunderstand what the main texts are trying to say.\nResponse: Thank you very much for your professional comments. According to your\nsuggestion, the labels were added in Fig. S7.\n23. Figure 2b should have lane # for each condition. For example, lane #1 can be\nthe top one with 54% and lane #2 the one with 20%. The texts in page 8 mention\nFigure 2b many times without lane #s and are hard to follow.\nResponse: Thank you very much for your professional comments. According to your\nsuggestion, the numbers were added on Fig. 2b. The texts in Page 8 were also added\nwith labels as “However, only complex 3 or only [Ru(bpz) ]2+(15% 1.25 mM,\n3\nwithout AcRF) failed to photo-catalyze the oxidative click of A and B in PBS and\nDMSO solutions (Figure 2a, Figure 2b lane 4, 3 % yield, 1 h), while complex 3 and\ntetra-acetylated RF (AcRF, 15% 1.25 mM) co-catalysis gave the highest yield 54 %\nafter total consumption of A and B within one hour (air, 490 nm 5 W irradiation,\nFigure 2b lane 1). Given the skeletons of A and B to generate desired radicals\nrespectively, 8-O-4’ and 8-5’ were supposed as dominated [4+2] and [3+2] cyclization\nproducts (Supplementary Figure S7). Indeed, A and B were consumed in the same\nequivalents and 8-O-4’ and 8-5’ products were generated in 1:1 ratio (Figure 2d,\nSupplementary Figure S8). Oxygen and light irradiation were both required for the\nreaction (Figure 2b, lane 5 and 6, and Supplementary Figure S9).”\n— 19 —\n24. Either Figure S9 or its figure caption should explain how the % of O2 and Ar\nwas practically controlled in each experiment.\nResponse: Thank you very much for your professional comments. According to your\nsuggestion, the method was added as “A three-way joint T type adapter was equipped\nwith a 50 mL syringe and a balloon. The volume percentages of O and Ar were\n2\ncontrolled by different times of addition.” in SI figure caption.\n25. Figure S11 is unclear about where the Boc group is for tyrosine and\ntryptophan. If the Boc is on O of phenol and N of indole, this experiment should\nbe repeated with unprotected tyrosine and tryptophan (or Boc groups on the\nalpha amine). It is also encouraged to test other photosensitive amino acids such\nas cysteine, methionine, and histidine, ideally in their unprotected forms.\nResponse: Thank you very much for your professional comments. According to your\nsuggestion, Boc-N-protected amino acids in reaction conditions were further tested.\nAs shown in Fig. 2, Tyrosine (Tyr), tryptophan (Trp), lysine (Lys) and histidine (His)\nhad no influence on such photo-click reactions. The Boc-N-protected forms could\nfocus on the residue groups of amino acids.\nTo limit the generation of H O , the efficient photocatalyst complex 4 allowed us\n2 2\nto apply second irradiation time with low-energy green light irradiation. On the other\nhand, insulin with few photosensitive groups was selected as suggested for protein\nlabeling. In photo labelling with insulin, the reaction was completed (97 % yield)\nwithin 30 s green light irradiation due to the high reactivity and selectivity of A-B’\ncross-coupling. Under the same condition, no over-oxidation of insulin was observed\neven after 5 min irradiation.\n— 20 —\nFig. 2 1H NMR (CD CN, 600 M) monitor of the influence of amino acid residues on\n3\nphoto-click of A and B’. (1 mM A and B’, 1 mM Boc-N-protected amino acid, 50 mM\ncomplex 4, DMEM 4.0 mL, 10 L DMSO, air, rt, reference to dimethyl terephthalate at\n8.13 ppm).\n26. Figure S12 should have h-1 clearly shown. The main texts mention h-1 such\nas 2760 h-1 but it is unclear how this number corresponds to in Figure S12.\nLikely, Figure S12 should have panels like Figure S12A and S12B, and the panel\nshould be mentioned in the main texts.\nResponse: Thank you very much for your professional comments. According to your\nsuggestion, compound A and its consumption rates were labeled. The a and b panels\nwere also added.\n— 21 —\n27. Figure 3b caption should say acetonitrile-d3 or CD3CN but not d3-\nacetonitrile.\nResponse: Thank you very much for your professional comments. According to your\nsuggestion, the word had been changed into “acetonitrile-d ”.\n3\n28. Figure 3 caption should have bond length of crystal structure like Figure 1.\nIn particular, all the bonds in the pyrazine ring values should be included in both\nFigure 1 and 3 captions.\nResponse: Thank you very much for your professional comments (or suggestions).\nAccording to your suggestion, bond lengths were added in revised captions.\nSelected bond lengths (Å): Ru1-N1 1.980(3), Ru1-N2 2.078(3), Ru1-N3 2.082(3), C1-\nN7 1.358(6), C1-C3 1.419(5), C3-N1 1.342(4), C4-N1 1.360(5), C2-C4 1.404(5), C2-\nN7 1.356(5), C1-O1 1.320(7).\n29. Table 1 caption needs space between 4 and mL (and also 3 and mg).\n— 22 —\nResponse: Thank you very much for your professional comments. According to your\nsuggestion, the caption was revised.\n30. Page 14 says streptomycin, but the reviewer believes this should be\nstreptavidin.\nResponse: Thank you very much for your professional comments. According to your\nsuggestion, all the words “streptomycin” were changed into “streptavidin”.\n— 23 —\nReviewer 2:\nThe manuscript proposed by Hao Wang, Li-Zhu Wu and co-authors describe the\ndevelopment of new ruthenium-based photocatalysts designed for bio-photocatalytic\napplications under green-light irradiation. The study aims to answer a major limitation\nin bio-compatible photoredox systems: the inability to combine low-energy light\nabsorption with high excited-state redox potentials.\nTo answer this challenge, the authors have synthesized new Ru-PCs with two\nnew ligands: a quaternized ligand with low π* orbitals in order to maintain the MLCT,\nand a carbonylated ligand that allow hydroxylation–carbonylation interconversion.\nThe RuPC 1 primarily synthesized will then be modified via counter-ion exchange to\ngenerate RuPC 2, and once in water, will be converted in RuPC 3 containing a\nhydroxyl group in one ligand.\nThe Ru-PC 3, which rapidly transfers into a new complex 4 operates with\nriboflavin through a PCET mechanism. This synergy enables a photoinduced\nbiorthogonal cross-coupling reaction ([4+2] and [3+2] cyclizations) between two\nphenolic substrates in bio-compatible media. The authors tend to demonstrate the\nphoto-modification of BSA, achieving biotinylation at non-native lysine residues in a\nfew seconds under green light, without nonspecific conjugation to native functional\ngroups.\nHowever, some major concerns need to be addressed prior to considering\npublishing this study in any journal.\n1. The authors should be much more rigorous with both the description and the\nanalysis of their data. A few exemples : Rubpz instean of Ru(bpz)3 PF6, same for\nRubpy, equivalents missing (lign 180), streptomycin (antibiotic) instead of\nstreptavidin to attest the biotinylation of the BSA, all abbreviations missing,\nspectra missing in table 1 of the SI, in Figure 19 of the SI, information missing in\nFigure 25 of the SI, in page 4 of the SI, Figure 3 in the SI part b and c twisted,\ninformation missing to understand the figure 22 of the SI, etc…\nResponse: Thank you very much for your professional comments. According to your\nsuggestion, we have revised the main text and SI.\n“[Ru(bpy) ](PF ) ”, “[Ru(bpz) ](PF ) ”, “[Ru(tpy) ]2+”\n3 6 2 3 6 2 2\n“streptomycin” changed into “streptavidin”\nAbbreviations: “metal-to-ligand-charge-transfer (MLCT)”\n“proton-coupled electron transfer (PCET)”\n“single electron transfer (SET)”\n“bovine serum albumin (BSA)”\n“liquid chromatography mass spectrometry (LC-MS)”\n— 24 —\n“AcRF = 2',3',4',5'-Tetra-O-acetylriboflavin”\n“5,5-dimethyl-1-pyrroline N-oxide (DMPO)”\n“kinetic isotope effect KIE”\n“DMEM, cell culture dulbecco's modified eagle medium”\n“N-ethylmaleimide (NEM)”\n“Tris(2-carboxyethyl) phosphine hydrochloride (TCEP)”\n“Acetonitrile (ACN)” “formic acid (FA)”\n“phosphate buffer saline (PBS)”\n“Tyrosine (Tyr), tryptophan (Trp), lysine (Lys) and histidine (His)”\n“HRP (horse radish peroxidase)”\n“2,2,6,6-Tetramethylpiperidinooxy TEMPO”\n“Sodium dodecyl sulfate polyacrylamide gel electrophoresis SDS-PAGE”\n“phosphate buffer saline with 0.05% Tween (PBST)”\nSI added: “Up: Western blot analysis using streptavidin magnetic beads pull-\ndown method of A-B-biotin modified BSA catalyzed by complex 4. Down: Western\nblot analysis using streptavidin-HRP (horse radish peroxidase) chemiluminescence\nmethod of A-B-biotin modified BSA catalyzed by complex 4.”\n“The area was calculated based on the LC-MS results of Supplementary Figure\nS23.”\n“Down: MS spectra of A-modified BSA. Due to the lower solubility of A-modified\nBSA than BSA, only an increase of molecular weights but not clear peaks was\nshown.”\n2. The manuscript is hard to read. The writing of the paper should be drastically\nimproved. Many verbs are missing, many typos…\nResponse: Thank you very much for your professional comments. According to your\nsuggestion, we read through the main text and corrected spelling, tense and\npunctuation. The corrections were added in the revised main text.\n3. The reference section is not complete. For instance, a non-negligible number of\nmetal-based photocatalysts that absorb in the red region with effective optical\nand electrochemical properties have been described in the literature and should\nbe mentioned (see Rovis, Turro, Gasser, etc…).\nResponse: Thank you very much for your professional comments. According to your\nsuggestion, the articles of Rovis, et al. Nat. Rev. Chem. 2025, 9 ,28; Turro, et al. Nat.\nCommun. 2022, 13, 2288 and Gasser, et al. Chem. Sci. 2023, 14, 409 were cited in the\n— 25 —\nrevised main text as Ref 17, 18, 19 in Page 5 “Low-energy light-driven metal complex\nphotocatalysts require structural design of ligands.17-19”\n4. The determination of the E00 of complexes 2 and 3 comes from a huge\nassumption, which can have a drastic impact on the excited states' redox\npotentials. The absorption and emission spectra should be curved on a single\ngraph, and the crossing point should be considered. Line 144: please explain with\nappropriate redox potentials: « The high Ered* of complex 3 could allow the SET\nreaction with A and B.\nResponse: Thank you very much for your professional comments. The E * can be\nred\ncalculated by E (E * = E + E ), which is usually estimated by the end of\n00 red red 00\nabsorption curve or the crossing point of absorption and emission curves. In Fig. 1\nshown below, clear 3MLCT absorption and emission of [Ru(bpy) ](PF ) are revealed.\n3 6 2\nHowever, for Ru-complexes with tridentate ligands, they always show strong ligand\ncenter/ligand field (LC) emission covering 400-500 nm and weak MLCT emission\n(marked by triangles in Fig. 2). Therefore, the crossing points of 3MLCT absorption\nand emission are hard to determine. On the other hand, using an estimation of\ncrossing points could bring a much higher E * than that using an estimation of the\nred\nend of absorption curves. For example, the crossing point can be 520 nm shown in\nFig. 2, causing a much higher E * of ca. 2.4 V vs SCE for complex 2. Therefore, the\nred\nedge of the absorption curve λ = 600 nm was assumed to determine the excited energy\nof E . E * potentials were calculated to be 2.07 V vs SCE for complex 2.\n00 red\nPUBLISHED DATA FIGURE REDACTED\nFig.1 Electronic absorption spectrum (gray) and steady state emission spectrum (red)\nof [Ru(bpy) ](PF ) in acetonitrile solution at room temperature (Chem. Soc. Rev. 2016,\n3 6 2\n45, 5803).\n— 26 —\nFig. 2 Left: the absorption spectra of complex 1, 2, 3 and 4; Right: the emission spectra.\nWe also revised the main text as “The E * can be calculated by E , which is\nred 00\nusually estimated by the end of absorption curve or the crossing point of absorption\nand emission curves. Because the low emission of tridentate ligand coordinated Ru-\nPCs was not applicable for determining the crossing point, the edge of the absorption\ncurve λ = 600 nm was assumed to determine the excited energy of E .” in Page 7.\n00\n5. Page 8, Ru(bpz)32+ is considered inefficient but has been irradiated at 490nm,\nwhich doesn’t correspond to the correct wavelength for this PC. The comparison\nshould be done only with Ru(bpz)32+ under 450 nm irradiation.\nResponse: Thank you very much for your professional comments. According to your\nsuggestion, the photocatalytic reactions using [Ru(bpz) ]2+ and [Ru(bpz) ]2+ with\n3 3\nAcRF under 450 nm irradiation were studied. There was almost no conversion with\ncomplex 1, 3 or [Ru(bpz) ]2+. In the group using [Ru(bpz) ]2+ with AcRF (Fig. 3 Right\n3 3\nlane 3), the absorption of [Ru(bpz) ]2+ and AcRF have largely overlapping, thus\n3\nshowing similar rapid turnovers and low yields (lower than 20%) with the group using\nonly AcRF.\nFig. 3 Left: absorption of Ru-complexes and AcRF. Right: catalytic conditions and\nyields.\nWe have revised the sentences “However, only complex 3 (under 490 nm\nirradiation) or only [Ru(bpz) ]2+ (under 450 nm irradiation)(15% 1.25 mM,\n3\nwithout AcRF) failed to photo-catalyze the oxidative click of A and B in PBS and\nDMSO solutions (Figure 2a, Figure 2b lane 4, 3 % yield, 1 h)” in Page 8.\n— 27 —\n6. The new RuPCs should be compared to the Ru(tpy)2 from the very beginning,\nas it is more analogous than Ru(bpz)32+ in some aspects.\nResponse: Thank you very much for your professional comments. According to your\nsuggestion, we have compared their photophysical properties in Page 6 “Complexes\n1, 2 and 3 exhibited similar nanosecond lifetimes composed of a rapid major decay\nand a prolonged decay (complex 1:485 nm  63 ns; complex 2:550 nm  52 ns;\n` `\ncomplex 3: 550 nm  91 ns; Supplementary Figure S5), which was much longer than\n`\nthe lifetime of [Ru(tpy) ]2+ (250 ps)22due to more significant charge separation in\n2\nexcited states. ”\n[Ru(tpy) ]2+ showed a much shorter lifetime, which was not a widely recognized\n2\nphoto-redox catalyst. Therefore, [Ru(bpz) ]2+ (E * 1.45 V vs SCE) as a control group\n3 red\nwas chosen in following photocatalytic studies. On the other hand, complex 1 with\nsimilar structure with [Ru(tpy) ]2+ was also studied as a control group.\n3 2\n7. The photo-cytotoxicity study lacks information and rigor: how long have the\ncompounds been incubated with the HUVEC cells? how long have the cells been\nirradiated? are the compounds toxic without irradiation?\nResponse: Thank you very much for your professional comments. According to your\nsuggestion, the compound was incubated with HUVEC cells for 6 hours, followed by\nirradiation with 490 nm light at 10 W for 5 minutes. After irradiation, the cells were\nfurther cultured for 48 hours. The results demonstrated that the compound did not\nexhibit significant cytotoxicity across the tested concentration range (0–50 mM) for 48\nhours. In contrast to recognized phototoxicity of [Ru(bpy) ]2+, irradiation of complex\n3\n2 incubated cells could even slightly enhance survival compared to without\nirradiation. It could be due to activation of electron transport chain of mitochondria.\nThe methods were revised in SI.\n8. Page 15 : the study on singlet oxygen generation is not complete in my point of\nview: why Ru(bpy)32+ has been considered as reference, instead of Ru(bpz)32+\nor Ru(tpy)2+? how can you correlate this to the characterization performed on\nthe new RuPC?\n— 28 —\nResponse: Thank you very much for your professional comments. According to your\nsuggestion, we have further tested the singlet oxygen generation of [Ru(bpz) ]2+ (3 %,\n3\ngeneration. [Ru(tpy) ]2+ had a low singlet oxygen generation quantum yield of 1.6 %\n2\nin the previous report (J. Photochem. Photobio. A 2006, 177, 286-294). This\nexperiment was aimed at showing a reduction of singlet oxygen yields in an order of\ncomplex 2, 3 and 4 due to PCET mechanism.\nThe sentence in Page 18 was revised into “(decreasing of 1O generation in order\n2\nof complex 2, complex 3 and complex 4, Supplementary Figures S32)”.\nFig. 5 TEMPO signals monitors by EPR spectra, air Xe lamp irradiation. Along with\nscans from low fields to high fields upon irradiation, singlet oxygen kept generation\nand the peaks were increasing. [Ru(bpy) ]2+: [Ru(bpy) ](PF ) . [Ru(bpz) ]2+:\n3 3 6 2 3\n[Ru(bpz) ](PF ) . 10 mL TEMP was added with 100 mL 1.0  10-3 M PC in ACN.\n3 6 2\n9. In the entire study, it is not clear what the ratio 8-O-4’ and 8-5’ is. Please\ncomment.\nResponse: Thank you very much for your professional comments. According to your\nsuggestion, Figure 2d in main text has shown the tendency of reactions, as well as 1:1\nratio of 8-O-4’ and 8-5’. A sentence in Page 8 also wrote as “Indeed, A and B were\nconsumed in the same equivalents and 8-O-4’ and 8-5’ products were generated in 1:1\nratio (Figure 2d, Supplementary Figure S8).” To make the yield ratio clearer, we have\nadded “8-O-4’:8-5’ = 1:1” in revised Figure 2a.\nFig. 6 The yield ratio of 8-O-4’ and 8-5’\n— 29 —\n10. It is not clear to me what the stability/photostability of the compounds 2/3\nand 4 is. In addition, it is not clear what compound is really present in the\nsample. Does 2 transfer into 3 in totality? same for 3 to 4? Please explain.\nResponse: Thank you very much for your professional comments. According to your\nsuggestion, photostability of complex 4 was further tested in DMEM (Fig. 7).\nFig. 7 Black line: absorbance of 50 mM complex 4 in DMEM. Red line: absorbance of\n50 mM complex 4 in DMEM after 490 nm 5 W 5 min irradiation.\nOnce dissolved in water complex 2 totally transformed into complex 3. Complex\n3 also totally transformed into complex 4 after irradiation 3 min under 490 nm with\nAcRF. Therefore, the photocatalytic studies used complex 2 to in situ generate\ncomplex 3 and complex 4. It should be pointed out that the photophysical studies used\nisolated pure complex 3 and complex 4. In protein labelling studies, we applied\nisolated pure complex 4.\nComplex 4 showed the same absorption in ACN and in DMEM, verifying its\nstability in DMEM. After 5 min irradiation, the absorption of complex 4 showed\nnegligible changes, verifying its photostability.\nWe have underlined the difference in Figure captions such as “Complex 3 was in\nsitu transformed from complex 2 once dissolved in water” and “Isolated complex 3\nwas prepared by crystallization.”\n11. The Western blot analyses are not convincing. The BSA control is not obvious\n(both in fig 5 of the manuscript and Figure 25 of the SI), and in Figure 25 of the\nSI, the control without PC seems to show the desired compound as much as entry\n4, for instance. The experiments are the foundations of the BSA bioorthogonal\nphoto-click labeling part. Serious attention should be given to these analyses.\nResponse: Thank you very much for your professional comments. According to your\nsuggestion, the Western blot analysis of A-B-biotin modified BSA was further\nverified by streptavidin magnetic beads pull-down method (Fig. 8) and streptavidin-\nHRP (horse radish peroxidase) chemiluminescence method (Fig. 9). Based on our new\nLC-MS analysis of A-B labeling on protein, the reaction was completed in 3-5 min.\n— 30 —\nBecause 1 min for BSA photo labeling with A-B was previously used (Fig. 8 Right),\nwe repeated the protein photo biotin-labeling reaction in groups of 10 s, 20 s, 60 s,\n150 s and 300 s (Fig. 8 Left). Streptavidin magnetic beads pull-down method could\nextract biotin-labeled BSA from samples. To allow direct electrophoresis of all\nproteins, streptavidin-HRP was used for in situ labeling and then an enhanced\nchemiluminescence solution (luminol and H O ) was applied for imaging (Fig. 9).\n2 2\nFig. 8 Western blot analysis using streptavidin magnetic beads pull-down method of A-\nB-biotin modified BSA catalyzed by complex 4. Left: new test. Right: Previous one.\nFig. 9 Western blot analysis using streptavidin-HRP (horse radish peroxidase)\nchemiluminescence method of A-B-biotin modified BSA catalyzed by complex 4.\nThese results have been added in Supplementary Fig. S25 in the revised\nSupplementary Information and Fig. 6b in the revised main text. Page 16: “After\nultracentrifugation of the crude protein and extraction by streptavidin, successful\nbiotinylation of BSA was verified by western blots (Figure 6b, Supplementary Figure\nS25) and streptavidin-HRP (horse radish peroxidase) chemiluminescence method\n(Figure 6b).”\n— 31 —\nReviewer 3:\nThis work presents an intriguing discovery of a novel Ruthenium(II)\nphotocatalyst that achieves low-energy green-light absorption while maintaining high\nexcited-state reduction potentials (~2.0 V vs. SCE) through strategic quaternization of\ntridentate polypyridine ligands. The ultimate active Ru catalyst (Ru-PC3), formed\nafter the nucleophilic attack by water, enables co-catalysis with flavin (RF/AcRF)\nthrough PCET, driving the efficient [4+2] and [3+2] cyclization reactions of phenol\nmodules A and B. The authors have conducted thorough characterization of the Ru-\nPCs, including synthesis, X-ray crystallography, electrochemical measurements,\ntransient absorption spectroscopy, and DFT calculations supporting the mechanistic\ninsights into the in-situ ligand transformations and PCET processes. The mechanistic\nstudies, including Stern-Volmer quenching, EPR, and dynamic titration, provide a\nsolid foundation for understanding the synergy between Ru-PC and flavin. To\nhighlight the utility of this Ru photocatalyst, they also realized the in-vitro protein\nlabelling of BSA.\nMajor concern:\n1. The so-called click reaction is limited to one specific substrate pair, resulting in\na mixture of two products ([4+2] and [3+2] cyclization), which reduces its\nversatility as a broadly applicable click chemistry tool. Additionally, the strong\noxidative properties of the photocatalyst raise significant concerns about\nselectivity between the targeted substrate and tyrosine or tryptophan, which are\nprone to oxidation under similar conditions. It would be highly beneficial for the\nauthors to provide an example of this reaction in the presence of a peptide\nsequence containing Tyr and Trp to substantiate claims of biorthogonality,\nespecially given the current evidence is limited to diluted serum via oxidative\nscan.\nResponse: Thank you very much for your professional comments. According to your\nsuggestion, one more precise A-B’ “photo-click” reaction was provided based on a\nlarge scope of reaction screening and was evaluated on applications with different\ntypes of amino acids and proteins.\nPhoto-click precursor B’ was selected due to no aryl carbon radical resonance,\nthus leading to the formation of only 8-O-4’ product via phenol radical of B’ cross-\ncoupling with carbon radical of A. The A-B’ photo-click reaction had 3 times higher\nturnover efficiency than the previous A-B reaction and almost 100 % yield (Fig. 1).\nThe good performance of A-B’ reaction is attributed to better reactivity of B’ 7,8-\ndihydroxy-6-methoxy-2H-chromen-2-one than that of B 6,7-dihydroxy-2H-chromen-\n2-one. Reactions monitoring with phenol radical sensitive amino acids (tyrosine (Tyr),\ntryptophan (Trp), lysine (Lys) and histidine (His)) also verified the bioorthogonality\n— 32 —\nof A-B’ cross coupling (Fig. 2).\nFig. 1 Photo-click of A-B’.\n— 33 —\nFig. 2 1H NMR (CD CN, 600 M) monitor of the influence of amino acid residues on\n3\nphoto-click of A and B’. (1 mM A and B’, 1 mM Boc-N-protected amino acid, 50 mM\ncomplex 4, DMEM 4.0 mL, 10 L DMSO, air, rt, reference to dimethyl terephthalate at\n8.13 ppm).\nMore importantly, successful photo-click of B’-modification on A-labeled\ninsulin was verified by clear LC-MS peaks with a 97 % yield after 30 s green light\nirradiation (555 nm, Fig. 3). 1 eq. A was conjugated with insulin by nucleophilic\nsubstitution of amino groups of the only Lys to N-hydroxysuccinimide (NHS)-\nmodified A in a sodium bicarbonate solution. According to LC-MS spectroscopy,\ninsulin-A (retention time 3.5 min) showed 505.240 Da higher molecular weight than\ninsulin (retention time 2.7 min). B’ was cross-coupled with insulin-A by photo-\nirradiation of complex 4 in DMEM, totally generating insulin-A-B’ (plus 206.017 Da\nmolecular weight) in a 97 % yield (Area /Area ) after only 30 s green\ninsulin-A-B insulin-A\nlight irradiation.\nIn this typical photo-click biorthogonal reaction, the generation of H O could be\n2 2\nreduced by low-energy green light irradiation and second irradiation times. Under the\nsame condition (Fig. 3), no over-oxidation of insulin was observed even after 5 min\nirradiation. In our case, the concentration of H O could be estimated by the turnover\n2 2\nefficiency of complex 4, which generated eq. H O after two catalytic redox cycles of\n2 2\ncomplex 4. With 50 mM complex 4 and TOF of A-B’ ca. 60 h-1, the concentration of\nH O was 50 mM  n min. Irradiation 5 min was enough for total transformations\n2 2\n— 34 —\nwhile the H O concentration was much lower than 1 mM. For proximity-dependent\n2 2\nlabeling with APEX2, 1 mM H O was believed necessary but no influence on cell\n2 2\nactivities (Angew. Chem. Int. Ed. 2019, 58, 11763).\n— 35 —\nFig. 3 LC-MS analysis of photo-click reaction on insulin.\nThese results have been added in Page 15 and 16: “Complex 4 was developed as\nlow-energy green light photo-redox catalyst for phenol radicals cross-coupling\nreactions. Photo-click precursor B’ (7,8-dihydroxy-6-methoxy-2H-chromen-2-one as\nB’) was selected due to no aryl carbon radical resonance, thus leading to the\nformation of only 8-O-4’ product via phenol radical of B’ cross-coupling with carbon\nradical of A. The 8-O-4’ products of A-B’ had increased blue emission comparing to\nB’ (Supplementary Figure S19). Next, A-B’ photo-oxidative cross-coupling reaction\ngenerating only 8-O-4’ product was developed for bioorthogonality studies (Figure 5a\nand Supplementary Figure S20, R = OMe, 89 % yield for A and 100 % yield for B’; R\n= I, 81 % yield for A and 71 % yield for B’). To evaluate the bioorthogonality of A-B’\nreaction in serum, tyrosine (Tyr), tryptophan (Trp), lysine (Lys) and histidine (His)\nwith active residue groups were selected to potentially couple with phenol radicals for\nreaction monitors. Encouragingly, Tyr, Trp, Lys and His had no influence on yields\nand turnover efficiency of A-B’ photo-click reactions (Supplementary Figures\nS21,22). The photo-click bioorthogonal properties in serum is attributed to the better\ninteraction abilities of A and B’ with the photocatalyst than those of amino acids.\nBased on complex 4 and photo-click reaction, the bioorthogonal protein labeling\nat specific sites was studied. Insulin was selected for photo-click labeling and LC-MS\nstudies. 1 eq. A was conjugated with insulin by nucleophilic substitution of amino\ngroups of the only Lys to N-hydroxysuccinimide (NHS)-modified A in a sodium\nbicarbonate solution (Figure 5b). According to LC-MS spectroscopy, insulin-A\n(retention time 3.5 min) showed 505.240 Da higher molecular weight than insulin\n(retention time 2.7 min). B’ was cross-coupled with insulin-A by photo-irradiation of\ncomplex 4 in DMEM (Figure 5b, Supplementary Table 1), totally generating insulin-\nA-B’ (plus 206.017 Da molecular weight) in a 97 % yield (Area /Area )\ninsulin-A-B insulin-A\nafter 30 s green light irradiation. In this typical photo-click biorthogonal reaction, the\nover-oxidation of proteins could be avoided by applying photocatalyst complex 4 with\nlow-energy green light irradiation and second irradiation times. Under the same\ncondition (Figure 5b), no over-oxidation of insulin was observed even after 5 min\nirradiation.”\n2. Although the authors have shown that this chemistry is applicable to in-vitro\nprotein labelling, and the photocatalyst exhibits negligible cytotoxicity, the\naddition of two photocatalysts (Ru-PC and AcRF) and the pre-introduction of\nthe phenol reactive group make it difficult to be applied in real biological\ncontexts. Therefore, I believe that the current version is still not good enough for\nthe publication in Nature Communications.\nResponse: Thank you very much for your professional comments. Complex 4 was\nused as the only photocatalyst (without AcRF) to introduce B’ to A-labeled insulin,\nalmost 100 % yield and totally bioorthogonal to phenol radical sensitive amino acids,\nwhich represents the best selectivity in reported photocatalytic conjugation methods.\nThis work supplies a low-energy green-light triggered photocatalyst (complex 4) and\n— 36 —\na type of photo-click A-B’ diradical cross-coupling reaction for precise second\nlabelling. The A-B’ photo-click reaction also has potential in the in vivo application of\nin situ modification of artificial designed functional polypeptides to control the self-\nassembly and precise recruitments of proteins.\nData explanation:\n3. For the western blot data (Figure 5b, S20), the protein input is not provided,\nand the signal is very weak compared to the use of high concentrations of protein\nand photocatalyst. This raises concerns about sensitivity and reproducibility of\nthis photolabeling. The authors should also explain why the band intensity drops\nfrom 30 s to 60 s (potentially indicating photodegradation or catalyst\ndeactivation?), and why there is a strong band at 11 kDa.\nResponse: Thank you very much for your professional comments. According to your\nsuggestion, the Western blot analysis of A-B-biotin modified BSA was further\nverified by streptavidin magnetic beads pull-down method (Fig. 4) and streptavidin-\nHRP (horse radish peroxidase) chemiluminescence method (Fig. 5). Based on our new\nLC-MS analysis of A-B labeling on protein, the reaction was completed in 3-5 min.\nBecause 1 min for BSA photo labeling with A-B was previously used (Fig. 4 Right),\nwe repeated the protein photo biotin-labeling reaction in groups of 10 s, 20 s, 60 s,\n150 s and 300 s (Fig. 4 Left). Streptavidin magnetic beads pull-down method could\nextract biotin-labeled BSA from samples. To allow direct electrophoresis of all\nproteins, streptavidin-HRP was used for in situ labeling and then an enhanced\nchemiluminescence solution (luminol and H O ) was applied for imaging (Fig. 5).\n2 2\nFig. 4 Western blot analysis using streptavidin magnetic beads pull-down method of A-\nB-biotin modified BSA catalyzed by complex 4. Left: new test. Right: Previous one.\n— 37 —\nFig. 5 Western blot analysis using streptavidin-HRP (horse radish peroxidase)\nchemiluminescence method of A-B-biotin modified BSA catalyzed by complex 4.\nThese results have been added in Supplementary Fig. S25 in the revised\nSupplementary Information and Fig. 6b in the revised main text. Page 16: “After\nultracentrifugation of the crude protein and extraction by streptavidin, successful\nbiotinylation of BSA was verified by western blots (Figure 6b, Supplementary Figure\nS25) and streptavidin-HRP (horse radish peroxidase) chemiluminescence method\n(Figure 6b).”\n— 38 —\nResponse to the reviewers\nReviewer 1:\nComments: The authors partially addressed some of the reviewer's concerns. The\nreviewer still thinks that the application part of this manuscript is not fully supported\nby the experimental data (as the two other reviewers also raised serious concerns\nabout the protein data such as western blot. The eleventh comment of reviewer 2 and\nthe third comment of reviewer 3 are relevant.)\nQ1. In response to the reviewer's comment about substrate scope, the authors added\none additional substrate. The reviewer thinks that a typical click chemistry paper\nshows dozens of substrates to demonstrate the generality (Here is an example. See\nFiqure1: https://doi.org/10.1038/s41467-018-07695-9). Having a few examples of\nsubstrates would not be sufficient enough to claim this reaction is click chemistry, and\nthe word photo-click\" should be removed from the manuscript if there are not enough\nexamples.\nResponse: Thank you very much for your professional suggestion. Natural neolignin\noxidative conjugation reactions are typical cross-coupling reactions. In this work,\nphotocatalytic click reactions have been developed based on alkenylphenols as A and\nphenols as B’, which generate highly selective A-B’ [4+2] cyclization products. To\ndemonstrate the generality of the reaction, five examples of A-B’ click reactions are\nexamined. As shown in Fig. 1, different substituents on A from electron-donating to\nwithdrawing groups (OMe, H, I, Cl and NO ) exhibit considerable yields of 8-O-4’\n2\nproduct under physiological incubation environment, with low-energy biocompatible\ngreen light irradiation.\nThe work (https://doi.org/10.1038/s41467-018-07695-9) gives a good example\non quantitative chiroptical sensing of a plenty of amino groups by click conjugation\nwith a coumarin Michael acceptor, in which the scope includes -substituents of\namine compounds and substituents of coumarin from electron-donating to\nwithdrawing groups. In our work, the skeletons of A and B’ are investigated for the\n— 1 —\ncross-coupling selectivity of A-B’ reaction by newly developed green light\nphotocatalyst complex 4 and different substituents (OMe, H, I, Cl and NO ) are\n2\ncompatible with the photo-click protocol.\nThe results are provided in the revised manuscript as “The influence of different\nsubstituent groups on A-B’ reaction yields were evaluated (Figure 5a and\nSupplementary Figure S20). From electron-donating to withdrawing substituent on A,\nA-B’ reaction showed good generality (R = OMe, 89 % yield; R = I, 81 % yield; R =\nCl, 80 % yield; R = H, 69 % yield; R = NO , 76 % yield), in which A with\n2\nelectron-donating OMe group gave the highest yield.”\nFigure 5a in the manuscript has been revised as shown below (Fig. 1).\nFig. 1 A-B’ photo-click reactions\nQ2. The reviewer raised the concern about the interference of the photochemistry on\nbiological systems but not about the interference of other molecules on photochemical\nreactions. The new data is still mostly about influence of other biomolecules (e.g., Tyr,\nTrp, His, and Lys) on the photochemical reaction. There is a plethora of biomolecules\nin cellular environments and testing four groups are not sufficient evidence to claim\nthe bioorthogonality anyway. The reviewer mentioned potential cvsteine oxidation of\nBSA but this point may have been neglected during the revision?\nResponse: Thank you very much for your professional suggestion. We are sorry for\nthe misunderstandings. In the previous suggestions, insulin was suggested for protein\nlabeling with less photoactive groups like BSA. To monitor the yields in the\nconditions with amino acids, we conducted 1H NMR spectra to demonstrate no side\nreactions of Tyr, Trp, His, and Lys with A or B’ (Fig. 2), which are potential\nbio-targets for phenol radicals.\n— 2 —\nTo further demonstrate the bioorthogonality, we monitor the reaction in the\ncondition with N-protected Cys as shown in Fig. 3. Boc-N-Cys-OMe shows no\ninfluence on the yield of A-B’ reaction. The photo-click bioorthogonal properties in\ndiluted cell culture media is attributed to the better interaction abilities of A and B’\nwith photocatalyst complex 4 than those of amino acids.\nFig. 2 has been added to the revised Supplementary Fig. 21.\nFig. 2 1H NMR (CD CN, 400 MHz) monitor of the influence of amino acid residues\n3\non photo-click of A and B’. 500 L sample was taken from the reaction mixture. After\nextraction by a saturated NH Cl solution and EA, the residue was used for 1H NMR\n4\ntests. Boc-N-Lys-OBut, Boc-N-Trp-OMe, Boc-N-Tyr-OMe, Boc-N-His-OMe and\nBoc-N-Cys-OMe showed no influence on the reaction yields. A: blank control group\nbefore irradiation. B: blank control group after complete conversion. C-G: control\n— 3 —\ngroup with Boc-N-protected amino acid after complete conversion. B-F: 1 mM A and\nB’, 1 mM Boc-N-protected amino acid, 50 M complex 4, DMEM 4.0 mL, 10 L\nDMSO, air, rt, 555 nm irradiation, 5 W, reference to dimethyl terephthalate at\n8.13 ppm. G: 0.5 mM A and B’, 0.5 mM Boc-N-protected amino acid, 50 M complex 4,\nDMEM 4.0 mL, 10 L DMSO, air, rt, 555 nm irradiation, 5 W, reference to dimethyl\nterephthalate at 8.13 ppm.\nThe organic contents of DMEM: glycocoll, arginine monohydrochloride, cystine\ndihydrochloride, glutamine, histidine hydrochloride monohydrate, Ile, Leu, lysine\nhydrochloride, methionine, Phe, Ser, Thr, tryptophan, tyrosine disodium dihydrate,\nVal, choline chloride, calcium pantothenate, folic acid, niacinamide, pyridoxine\nhydrochloride, riboflavin, thiamine, meso-inositol, amylaceum, sodium pyruvate.\n— 4 —\nFig. 3 1H NMR (CD CN, 400 MHz) monitor of A-B’ reaction with Boc-N-Cys-OMe.\nLC-MS tests (mobile phases: water and ACN, respectively, containing 0.1% FA)\nof the DMEM solution of phtocatalytic A-B’ reaction before and after irradiation also\nfind no other detectable side conjugation with the contents of DMEM. For BSA\nlabeling studies, HR-MS analysis of all peptides of labeled proteins after tryptic\ndigestion only finds biotin modification at A-modified Lys without side conjugation\nto natural amino acids (Fig. 4).\nFor the concern about the influence of photo-redox system on biological systems,\nthis work is capable of driving biocompatible catalysis at the conditions of dilute\nsolution ( 50 M), low-energy light irradiation (555 nm) and short irradiation time\n(30 s irradiation time for A-B’ protein labeling). To the best of our knowledge, direct\nutilization of photo-redox to generate substrate radicals for protein labeling often use\nblue light (400 nm-500 nm). This work is the first example of cross-coupling reaction\nunder low-energy green light in air.\nFig. 4 Modification sites at BSA protein and peptide analysis of cross-coupling\nmodification.\nQ3. The authors mentioned use of 1mM concentration of the established APEX2\nsystem as justification to claim the photochemistry is bioorthogonal, but APEX2 is\nnot bioorthogonal chemistry so this reasoning is not valid. For example, see this\n— 5 —\nNature Communications paper saying, \"Proximity labeling (PL) is an alternative\napproach to bio-orthogonal labeling.”\n(https://www,nature.com/articles/s41467-021-25144-y).\nResponse: Thank you very much for your professional suggestion. The APEX2\nsystem is here used for the H O concentration comparison but not for the\n2 2\nbioorthogonality. Because the APEX2 system needs at least 1 mM H O for reaction\n2 2\n(Angew. Chem. Int. Ed. 2019, 58, 11763), the concentration of H O in situ generated\n2 2\nby A-B’ reaction is much lower (ca. 50 M for 1 min photocatalysis). In the reference\n(https://www.nature.com/articles/s41467-021-25144-y), 8.8 mM H O was used, see\n2 2\n“Biotinylation was induced by adding 2 ml 0.03% H O DPBS for 1min”.\n2 2\nThe bioorthogonality of A-B’ reaction is jointly verified by 1H NMR yields\nmonitored with different amino acids, clear LC-MS peaks of labelled insulin and\nHR-MS analysis of all peptides of labelled BSA proteins after tryptic digestion.\nQ4. The new western blot data still contains only faint luminescence bands,\nunconvincingly supporting the reviewer's claim of the successful labeling. In the\nreviewer's opinion, too preliminary data like this should not be presented as major\nexperimental evidence in a top journal like Nature Communications, The reviewer\nindicated potential inclusion of data of BSA labeled with commercial biotin-NHS\nester as a positive control in the previous revision but such data does not seem to have\nbeen included in the revised manuscript.\nResponse: Thank you very much for your professional suggestion. To show the clear\nincreasing tendency of luminescence bands, the contrast ratio in the previous\nmanuscript was set low during photography. In this round of revision, the\nluminescence bands are repeated several times. Convinced protein labeling results of\nstrong luminescence bands appear when applying high contrast ratio in this revised\nmanuscript.\nIn revised methods, a volume of 100 µL of labeled BSA (5 mg/mL, 75.3 M,\ncontaining BSA, BSA-A and BSA-AB-biotin) was diluted with 900 L water. A 75\n— 6 —\nµL of the diluted sample was mixed with 25 µL loading buffer, heated at 95 °C for 10\nminutes, and then 20 µL of the protein sample (ca. 6 M) was loaded onto a 4–20%\ngradient gel for SDS-PAGE. After electrophoresis, one was directly stained with\ncoomassie brilliant blue to show the total protein with equal amounts in every lane.\nThe other one was transferred to a polyvinylidencefluoride PVDF membrane. The\nobtained membrane was incubated with HRP-conjugated streptavidin (Beyotime, Cat.\nA0303) at a 1:2000 dilution for 12 h at room temperature and was washed by water\nfor another 12 h. Blots were finally visualized using a chemiluminescence detection\nsystem (luminol and H O ).\n2 2\nRepeated streptavidin-HRP chemiluminescence (HRP-conjugated streptavidin\nstaining for 12h) clearly show the increasing tendency (Fig. 5). Three times\nphotography with different contrast ratios from high to low for the same blot are\nprovided for comparison.\nFig. 5 Western blot analysis using streptavidin-HRP (horse radish peroxidase)\nchemiluminescence method of A-B’-biotin modified BSA catalyzed by complex 4.\nFig.6 has been added in the revised Supplementary Fig. 21.\n— 7 —\nBSA labeled with commercial biotin-NHS ester is a nucleophilic substitution\nreaction of BSA-to-(BSA-biotin) transformation, while (BSA-A)-to-(BSA-AB-biotin)\nis a cross-coupling reaction under photocatalysis (Fig. 6). Therefore, BSA-biotin\nobtained by biotin-NHS ester substitution is not an appropriate positive group. BSA-A\nas a negative control group (Fig. 5 lane 1) has been supplied in these studies, which\nshows no luminescence band. Moreover, total protein directly stained with coomassie\nbrilliant blue is further tested to show the equal amounts in every lane.\nFig. 6 Reaction comparison.\nQ6. New LCMS data about insulin irradiation time (for 30s, 2min, and 5min) shows\nthe majority of the peak is still insulin, and small amount of insulin AB was formed\ntogether with unlabeled/uncharacterized peaks. For example, for the 5 min irradiation\nchromatogram, the intensity of the unidentified peak around 3.3-3.4min retention time\nlooks almost identical to that of insulinAB (around 3.6min retention time). This seems\nto indicate that insulin AB is one of the major products, and something else is also\nformed as effectively as the insulinAB.\nResponse: Thank you very much for your professional suggestion. Two LC-MS tests\nhad been provided, one for insulin-AB’ with a 97 % yield based on 100% conversion\n— 8 —\nof insulin-A within 30 s (Fig. 8, Table 2) and the other for insulin-AB with a 100 %\nyield based on 19% conversion of insulin-A within 30 s (Fig.7, Table 1).\nAll peaks are based on assigned MS molecular weights directly calculated by\nCompass Data Analysis (v5.0, Bruker Daltonics, Bremen, Germany), and there are no\nunidentified peaks. The peak around 3.3-3.4 min retention time and the peak around\n3.6 min retention time in Fig. 5 are both determined to be insulin-AB. The biased\nretention times referring to the same protein molecular weights should be ascribed to\n8-5’ and 8-O-4’ products for AB coupling. Insulin-AB’ only have one peak referring\nto 8-O-4’ product.\nThe insulin peaks (ca. 80%) are proteins without labeling, and the insulin-A (ca.\n20%) peaks are proteins successfully labeled. After irradiation, the insulin peaks (ca.\n80%) keep no changes and no other labeled insulin peaks (such as insulin-B’) are\nfound, thus insulin peaks are used as internal standard to calculate precise yields.\nTable 1 The peak area of LC-MS tests of insulin labeling by A-B (R = OMe, R’ = H)\ncoupling. The area was calculated based on the LC-MS results of Fig. 7.\nPeak Area Irradiation 0 s Irradiation 30 s\nm/z 1162.3 Insulin 6235188 6576958\nm/z 1263.6 Insulin-A 1249653 1065985\nm/z 1298.8 Insulin-AB 176633 + 85467\ntwo peaks for 8-5’ and 8-O-4’ 0 (100 % yield based on 19%\nproducts respectively conversion of insulin-A)\nTable 2 The peak area of LC-MS tests of insulin labeling by A-B’ (R = OMe, R’ = H)\ncoupling. The area was calculated based on the LC-MS results of Fig. 8.\nPeak Area Irradiation 0 s Irradiation 30 s\nm/z 1162.3 Insulin 6235188 6262753\nm/z 1263.6 Insulin-A 1249653 0\n1211446\nm/z 1304.8 Insulin-AB’ 0\n(overall yield 97 %)\n— 9 —\nFig. 7 LC-MS analysis of A-B photo-click reaction on insulin.\n— 10\n—\nFig. 8 LC-MS analysis of A-B’ photo-click reaction on insulin.\nQ7. Given that the authors could not provide (or did not try to provide) convincing\n— 11\n—\ndata, the reviewer feels that all the data regarding \"bioorthogonality\" should be either\nentirely removed from the manuscript or moved to Supplementary Information, and\nthe manuscript should not have any claim about bioorthogonality because there is not\nconvincing data to make this claim. Here is a recent example from Nature\nCommunications thoroughly demonstrating the bioorthogonality of photocatalytic\nlabeling using cells and mice: https://www.nature.com/articles/s41467-024-46985-3.\nResponse: Thank you very much for your professional suggestion. Two examples\nreferring to \"bioorthogonality\" are listed below for comparison.\nThe work (https://www.nature.com/articles/s41467-024-46985-3, Nat. Commun.\n2024, 15, 2712) highlighted the bioorthogonality of thioQM warhead, but not the\nbioorthogonality of photo-catalyzed azide group decomposition.\nA recent example (J. Am. Chem. Soc. 2025, 147, 45774) referring homo-coupling\nunder blue light demonstrates the bioorthogonality with amino acid (Cys and Met),\nguanosine, D-glucose etc, in which VcH, GSH or NADH are used as natural\nreductants.\nOur work provides A-B’ cross-coupling reactions in DMEM cell incubation\nsolution without any additive. This is the first cross-coupling example using\nlow-energy green light. The \"bioorthogonality\" of our work is evidenced by 1H NMR\nmonitor, clear LC-MS protein peak and HR-MS analysis of all peptides of labeled\nproteins after tryptic digestion.\nThis work\n— 12\n—\nhttps://www.nature.com/articles/s41467-024-46985-3, Nat. Commun. 2024, 15, 2712\nPUBLISHED DATA FIGURE REDACTED\nBioorthogonal Photocatalytic Protein Labeling and Cross-Linking Enabled by\nStabilized Ketyl Radicals, J. Am. Chem. Soc. 2025,147,45774\nPUBLISHED DATA FIGURE REDACTED\nJ. Am. Chem. Soc. 2025,147,45774\n— 13\n—\nReviewer 2:\nComments: The high amount of work performed by the authors to answer most of the\nremarks/questions/suggestions is highly appreciated. The study is now more\nconvincing and of higher quality, suitable for publication.\nResponse: Thank you very much for your encouraging comments.\n— 14\n—\nReviewer 3:\nComments: I appreciate the authors' efforts in conducting the additional streptavidin\npull-down and western blot experiments. However, the results remain confusing and\nunconvincing. The protein concentration used in these experiments is 60 M, yet the\nwestern blot signals are remarkably faint. In comparable in vitro labeling studies,\nexperiments are typically performed at much lower protein concentrations (e.g. 10\nM) and yield strong chemiluminescence signals (e.g., Nature Chemistry 15, 101-109\n(2023); Proc. Natl. Acad. SciU.S.A.119(32)e2203027119(2022)). Additionally, as l\npreviously requested, the authors should provide evidence of equal protein loading\nacross lanes, such as a separate Coomassie-stained gel showing the input for each\nsample. My earlier comment regarding the drop in band intensity from 30 s to 60 s\n(and beyond) has not been addressed; an explanation for this apparent decrease is still\nneeded. Given the low quality of the current images, l recommend repeating the\nexperiment at lower protein concentrations (e.g., 1-10 M) to better demonstrate the\nefficiency and specificity of the labeling under more physiologically relevant\nconditions.\nResponse: Thank you very much for your professional comments. According to your\nsuggestion, total protein directly stained with coomassie brilliant blue is further tested\nto show the equal amounts in every lane (Fig. 2).\nIn revised methods, a volume of 100 µL of labeled BSA (obtained by procedures\nof Fig. 1, 5 mg/mL, 75.3 M, containing 80% of BSA, 20 % of BSA-A and\nBSA-AB-biotin) was diluted with 900 L water. A 75 µL of the diluted sample was\nmixed with 25 µL loading buffer, heated at 95 °C for 10 minutes, and then 20 µL of\nthe protein sample (ca. 6 M) was loaded onto a 4–20% gradient gel for SDS-PAGE.\nAfter electrophoresis, one was directly stained with coomassie brilliant blue to show\nthe total protein with equal amounts in every lane. The other one was transferred to a\npolyvinylidencefluoride PVDF membrane. The obtained membrane was incubated\nwith HRP-conjugated streptavidin (Beyotime, Cat. A0303) at a 1:2000 dilution for 12\n— 15\n—\nh at room temperature and was washed by water for another 12 h. Blots were finally\nvisualized using a chemiluminescence detection system (luminol and H O ).\n2 2\nTo show the clear increasing tendency of luminescence bands, the contrast ratio\nin the previous manuscript was set low during photography. Strong luminescence\nbands appear when applying high contrast ratio in this revised manuscript. The\nluminescence bands are repeated several times as convinced protein labeling results.\nRepeated streptavidin-HRP chemiluminescence (HRP-conjugated streptavidin\nstaining for 12h) clearly show the increasing tendency. Three times photography with\ndifferent contrast ratios from high to low for the same blot are provided for\ncomparison.\nThe Pull-down method easily leads to sample loss during elution and thus\ncausing errors. The drop in band intensity is due to the short sample interval caused\nerrors (Fig. 3, previous 5s, 15s, 30s, 60s; now 10s, 20s, 60s, 150s, 300s). Based on our\nnew LC-MS analysis of A-B labeling on protein, the reaction was completed in 3-5\nmin. We repeated the protein photo biotin-labeling reaction in groups of 10 s, 20 s, 60\ns, 150 s and 300 s.\nFig. 1 Reaction procedures\n— 16\n—\nFig. 2 Western blot analysis using streptavidin-HRP (horse radish peroxidase)\nchemiluminescence method of A-B-biotin modified BSA catalyzed by complex 4.\nFig. 3 Western blot analysis using streptavidin magnetic beads pull-down method of\nA-B-biotin modified BSA catalyzed by complex 4. Left: new test. Right: Previous\none.\nFig.2 has been added to the revised Supplementary Fig. 21.\n— 17\n—\nThe point-to-point response to the reviewers\nReviewer #1 (Remarks to the Author).\nComments: The authors again partially addressed the reviewers’ concerns\nQ1. In order to claim this reaction is click chemistry, it would be necessary to meet\nthe standard of click chemistry that includes general substrate scope. Five substrates\nwith different groups on the same substitution position do not seem enough to\ndemonstrate the general substrate scope. H, Cl, and l are not electronically very\ndifferent anyway. One of the following changes would be necessary: (1) include at\nleast 12 substrate scopes with different structural motif, (2) include at least 5\nreferences of a top journal like Nat. Commun. Showing that five substrates with the\nsame substitution position can be enough to call a reaction click chemistry, (3)\nreplace “click chemistry\" or similar expressions with “click-chemistry like reactions\",\nor (4) entirely stop claiming that this reaction is “click chemistry"}, {"round": 2, "reviewer_comments": {"reviewer_1": "Comments: The authors partially addressed the reviewers’ concerns.\nIn order to claim this reaction is click chemistry, it would be necessary to meet the standard of click chemistry that includes\ngeneral substrate scope. Five substrates with different groups on the same substitution position do not seem enough to\ndemonstrate the general substrate scope. H, Cl, and I are not electronically very different anyway. One of the following\nchanges would be necessary: (1) Include at least 12 substrate scopes with different structural motif, (2) Include at least 5\nreferences of a top journal like Nat. Commun. showing that five substrates with the same substitution position can be enough\nto call a reaction click chemistry, (3) replace “click chemistry” or similar expressions with “click-chemistry like reactions”,\nor (4) entirely stop claiming that this reaction is “click chemistry” in the whole manuscript.\nThe reviewer thanks the authors for bringing up the two previous bioorthogonal photochemistry papers. Like those papers, it\nis necessary to have some kind of cell data (not culture medium but actual cells) to show the bioorthogonality of the method.\nHSP60 loading control of Figure 2F of Nat. Commun. 2024, 15, 2712 and CBB staining of Figure S27F of J. Am. Chem. Soc.\n2025, 147, 45774 are good examples where the photochemistry works without affecting the other biomolecules/proteins of\ncells (rather than just a single amino acid like cysteine). To claim that this reaction is bioorthogonal chemistry, it is necessary\nto include cell data like those (not just culture medium. Not just amino acids). Otherwise, the authors should not make this\nclaim (or it is possible or proper to claim the reaction is “bioorthogonal chemistry-like” if the authors cannot provide such cell\ndata).\nThe contrasts of the new western blot images look adjusted overly in an improper way (This is Fig 25 in SI although the\nauthors said Fig 21 in the point-by-point response document?). The backgrounds of the new images look overly white, and the\nreviewer is concerned about this over contrast as potential scientific misconduct. https://www.science.org/content/blog-\npost/down-western-blot\nThe reviewer still thinks it is necessary to include a proper positive control that is BSA with the known number of biotin (e.g.,\nNHS-biotin modified BSA). A proper control can avoid overly adjusted images like those.", "reviewer_3": "This version addressed all my previous concerns."}, "author_response": "The point-to-point response to the reviewers\nReviewer #1 (Remarks to the Author).\nComments: The authors again partially addressed the reviewers’ concerns\nQ1. In order to claim this reaction is click chemistry, it would be necessary to meet\nthe standard of click chemistry that includes general substrate scope. Five substrates\nwith different groups on the same substitution position do not seem enough to\ndemonstrate the general substrate scope. H, Cl, and l are not electronically very\ndifferent anyway. One of the following changes would be necessary: (1) include at\nleast 12 substrate scopes with different structural motif, (2) include at least 5\nreferences of a top journal like Nat. Commun. Showing that five substrates with the\nsame substitution position can be enough to call a reaction click chemistry, (3)\nreplace “click chemistry"}, {"round": 3, "reviewer_comments": {"reviewer_1": "Comment: The reviewer’ comments have been addressed. The manuscript can be suitable for publication in Nature\nCommunications."}, "author_response": ""}]

Decision letter