Iminium-linked hyperporphyrin covalent organic framework mediates type I photodynamic therapy via a photoredox process

Domain: biology
Published: 2026-03-28
Editorial outcome: major_revision

Abstract
Most photosensitizers (PSs) mediate type II photodynamic therapy (PDT) via energy transfer to produce singlet oxygen. However, this mechanism is oxygen-dependent and less effective in hypoxic tumors. Type I PDT, which generates radical reactive oxygen species such as superoxide through electron transfer, is more hypoxia-tolerant, yet molecular design strategies remain limited. Herein, we report an iminium-linked hyperporphyrin covalent organic framework (IH-COF) that facilitates efficient type I PDT via a photoredox process. In a one-pot synthesis, trimethyloxonium tetrafluoroborate simultaneously quaternizes imine bonds to introduce electron acceptors and protonates porphyrins, red-shifting the Q-band to 725 nm via the hyperporphyrin effect. Mechanistic studies reveal that photoinduced electron transfer from hyperporphyrin units to iminium ions generates α-amino radicals, which reduce oxygen to superoxide while regenerating iminium ions. The oxidized hyperporphyrins are then reduced by biomolecules such as 1,4-dihydronicotinamide adenine dinucleotide, sustaining the photocatalytic cycle. Consequently, IH-COF exhibits excellent PDT performance under both normoxic and hypoxic conditions and elicits potent antitumor efficacy in colorectal and triple-negative breast cancer models in female mice. This study highlights the potential of COFs as versatile and biocompatible platforms for synergistic photomedicine applications.

Introduction
Photodynamic therapy (PDT) represents a promising cancer treatment modality with many advantages including minimal invasiveness, low side effects, and spatiotemporal control over conventional cancer treatments such as surgery, chemotherapy and radiotherapy.1-4 PDT generates cytotoxic reactive oxygen species (ROS) by exciting a photosensitizer (PS) with light to specifically induce cell death in the irradiated tissue. The majority of PSs mediate type II PDT by generating singlet oxygen (1O ) through energy transfer (EnT) from an excited PS (3PS*) to molecular oxygen (O ).5-10 Due to its strong dependence on local O concentration, the therapeutic efficacy of type II PDT is compromised in typically hypoxic solid tumors.11-13 In rare cases, excited PSs can undergo electron transfer (ET) to O or water to generate radical ROS such as superoxide radical (O •−) or hydroxyl radical (•OH) in type I PDT processes which are less O concentration-dependent.14-16 The formed O •− can participate in additional catalytic cascades to generate highly cytotoxic •OH species while regenerating O .17-19 Despite significant efforts on designing molecular PSs to mediate type I PDT processes,15, 17 the progress has been hindered by the relatively sluggish ET process compared to the efficient EnT from 3PS* to O . Furthermore, molecular PSs with high redox activities and hence faster ET processes to O (such as bacteriochlorins) tend to be photochemically unstable and cannot be developed as clinically relevant PSs for type I PDT.20 New design strategies are needed for the development of PSs to mediate efficient type I PDT.

Constructed by reticulating organic molecules via covalent linkages, covalent organic frameworks (COFs) have emerged as a novel class of crystalline porous organic materials.21-27 COFs have been extensively studied for potential applications across many fields including gas sorption and separation,28-34 catalysis,35-43 and energy storage.44-46 With excellent molecular tunability, biocompatibility, high chemical stability, and light-harvesting ability, COFs have recently been explored for biological applications,47-49 including PDT.50-53 Their intrinsic porosity facilitates molecular O₂ diffusion and mass transport, promoting more effective interactions between PS and O to enhance PDT efficacy.50, 54-55 However, existing COFs mediate PDT via type II processes due to rapid EnT from the excited PS units to O .

Photoredox catalysis has experienced a renaissance in recent years and provides a powerful tool to mediate photoinduced ET between excited photosensitizers and organic substrates.56 In particular, electrophilic iminium ions have been widely used in photoredox catalysis through single-electron reduction.57 We have recently demonstrated enhanced photoredox catalysis in COFs by facilitating ET processes through the proximity between excited PSs and organic substrates.35 We hypothesized that iminium-linked COFs could provide a unique strategy to facilitate type I PDT via facile photoinduced reduction of iminium ions to α-amino radicals and ET from α-amino radicals to O molecules.

Herein, we report the design of an iminium-linked hyperporphyrin COF (IH-COF) to not only red-shift and enhance Q-band absorption in the tissue-transparent near-infrared window58 but also facilitate type I PDT via a photoredox process. IH-COF was synthesized by simultaneous methylation and protonation of an imine-linked porphyrin COF (Im-COF) with trimethyloxonium tetrafluoroborate (Me OBF ). In this process, the imine units were methylated to form iminium ions and the porphyrins were protonated to generate hyperporphyrins which exhibit a significant bathochromic shift with the maximum absorption at 725 nm. Upon 725 nm irradiation, ET from an excited hyperporphyrin to an iminium ion generated an α-amino radical intermediate which underwent facile ET to O to form O •−, thus efficiently mediating type I PDT. In vitro experiments under 725 nm irradiation demonstrated high cytotoxicity of IH-COF through a type I PDT process under both normoxic and hypoxic conditions. IH-COF mediated type I PDT elicited potent antitumor efficacy in subcutaneous tumor model of murine colon CT26 cancer and orthotopic tumor model of murine 4T1 triple-negative breast cancer.

Results
Design principle of IH-COF for type I PDT
In PDT, the 3PS* can return to its ground state through a type II EnT or a type I ET pathway (Fig. 1). Generally, the energy gap ∆E between the ground state of a PS and the 3PS* is significantly larger than the energy difference of 0.977 eV (1270 nm) between O and 1O to favor type II PDT. As hyperporphyrins are known to exhibit a significant bathochromic shift in their Q-band absorptions, their decreased ∆E values can slow down EnT from their 3PS* states to O . The hyperporphyrins are formed by the charge delocalization between the protonated porphyrin and the N atom of an imine.59 We further envisioned that IH-COF would promote efficient ET from the 3PS* states of hyperporphyrins to iminium electron acceptors, leading to efficient O •− generation and type I PDT.

Synthesis and characterization of COFs
IH-COF was synthesized by the polymerization of 5,10,15,20-tetrakis(4-aminophenyl)porphyrin (TAPP) and terephthalaldehyde (TPA) with the presence of Me OBF in acetonitrile (Fig. 2a). The protonation of porphyrins and methylation of imines were confirmed at the molecular level using model monomers (Supplementary S2). The imine moieties remained incompletely methylated in IH-COF to enable the hyperporphyrin effect. However, these un-methylated imine bonds are still present as iminium ions according to the resonance structures in Fig. 1. For comparison, Im-COF and methylated Im-COF (Me-CSOF) were synthesized using the previously established methods (Fig. 2b, Supplementary Figs. 1-5).53, 60

Fourier-transform infrared (FT-IR) spectroscopy was used to probe the construction of IH-COF (Fig. 2c). The appearance of C=N+ bands at 1630 c m-1 and B-F bands at 1058 cm-1 supported the formation of iminium linkages. Solid-state 13C cross-polarization magic-angle-spinning NMR spectrum of IH-COF showed the carbons of C=N+ at 163 ppm and −CH at 57 ppm, consistent with the methylation of imine groups (Supplementary Fig. 7). X-ray photoelectron spectroscopy (XPS) further supported the successful synthesis of IH-COF. Two signals at 400.2 eV and 402.3 eV were attributed to N 1s signals for the iminium C=N+ and hyperporphyrinic C-N moieties, respectively (Fig. 2d). The non-methylated imine could not be distinguished from the iminium due to its quaternization resulting from charge delocalization. These N 1s signals shifted to higher energies over those of Im-COF (Supplementary Fig. 8a, b), which is consistent with electron-deficient iminium and hyperporphyrinic moieties of IH-COF. IH-COF showed significantly higher intensity of C 1s signal at 288 eV over Im-COF due to the methylation of imine bonds (Supplementary Fig. 8c, d). The synthesis of IH-COF was further supported by the appearance of B 1s and F 1s signals in the XPS spectrum (Supplementary Fig. 8e, f).

Powder X-ray diffraction (PXRD) studies showed that IH-COF nanoparticles exhibited low crystallinity but their main peaks appeared at the same 2q values as crystalline bulk IH-COF samples synthesized under solvothermal conditions (Fig. 2e). Transmission electron microscopy (TEM) revealed nearly spherical morphology for IH-COF (Fig. 2f). Dynamic light scattering (DLS) measurement gave a particle size of 191 nm for IH-COF in water (Fig. 2g), which was consistent with the particle size observed by TEM. DLS results suggested the nanoparticles of Im-COF and Me-COF have also been successfully obtained. IH-COF showed a zeta potential of +24 mV, which is significantly higher than that of Im-COF at -30 mV (Fig. 2h). This highly negative potential might be due to the formation of neutral complexes for coordinated metal centers or effective charge screening by counterions.61-62 Stability tests indicated that the COFs were stable under various physiologically relevant conditions (Supplementary Fig. 1S1).

Type I ROS generation by IH-COF and mechanistic study
Compared to Me-COF and Im-COF, UV-vis absorption spectrum of IH-COF exhibited a significant red-shift of Q band to 725 nm due to the protonation-induced hyperporphyrin effect (Fig. 3a).59 Consistent with this, the band gap decreased from 1.76 eV for Im-COF to 1.24 eV for IH-COF (Supplementary Fig. 12). This red-shift from charge delocalization decreased the energy gap ∆E in IH-COF, which is conducive to suppressing the EnT pathway for type II PDT.

ROS generation by IH-COF, Me-COF and Im-CO F was evaluated under 725/660 nm irradiation to match their Q band maxima. Total ROS generation was first detected by the 2',7'-dichlorodihydrofluorescein diacetate (DCEFH-DA) assay. After 10 minutes of light irradiation, IH-COF generated 6.0- and 2.2-fold more total ROS than Im-COF and Me-COF, respectively (Fig. 3b). Next, 1O and O •− generation were measured using singlet oxygen sensor green (SOSG) and dihydrorhodamine 123 (DHR 123) probes, respectively. Im-COF generated the most 1O through the energy transfer pathway while IH-COF generated a negligible amount of 1O (Fig. 3c). Interestingly, IH-COF generated 3.4- and 2.3-fold more O •− than Im-COF and Me-COF, respectively (Fig. 3d). These results demonstrate efficient generation of O •− by IH-COF to mediate type I PDT. However, IH-COF did not generate •OH under irradiation (Supplementary Fig. 13). The presence of superoxide dismutase (SOD) drastically quenched the fluorescence of DHR 123 (Supplementary Fig. 14), confirming that O •− is the predominant ROS in this system.

Several experiments were conducted to uncover the mechanism of photoinduced O •− generation by IH-COF. Fluorescence quenching experiment demonstrated an efficient photoinduced ET process between TAPP and an iminium ion with a Stern-Völmer constant K of 6.6 × 104 M-1. In contrast, no photoinduced ET process was observed between TAPP and an imine (Fig. 4a). In the presence of TAPP and triethanolamine, irradiation of a mixture of the model compound diphenyl-iminium ion (M2) and TEMPO at 660 nm generated the TEMPO adduct of α-amino radical (Fig. 4b, Supplementary Fig. 15). The α-amino radical can be oxidized by O to form O •− and regenerate the iminium ion. The formation of O •− was verified by the characteristic sextet feature of the DMPO-O •− adduct in the electron paramagnetic resonance (EPR) spectrum of an irradiated mixture of IH-COF and DMPO in air (Fig. 4c). Cyclic voltammetry (CV) measurements of M2 showed a significant current intensity increase from 0.026 under N to 0.328 mA under O at -1.03 V (vs. Ag/AgCl) (Fig. 4d), suggesting electrocatalytic O reduction to O •− by M2. This reduction potential of iminium at -1.03 V is more negative than the reduction potential of O (-0.33 V), indicating the ability of the α-amino radical to undergo ET with O to form O •−. The reaction between α-amino radical and O may also occur through a peroxide intermediate, which was supported by free energy changes (Supplementary Figs. 27-31). Under N atmosphere, the CV curve of M2 exhibited a small irreversible peak, assignable to dimerization of the α-amino radical (Fig. 4e).63 However, this deactivation pathway of the α-amino radical will be shut down in IH-COF due to the isolation of two radicals in the framework.

The holes on the hyperporphyrin after photoinduced ET may be reduced by endogenous reducing agents such as 1,4-dihydronicotinamide adenine dinucleotide (NADH) under physiological conditions, which is also beneficial to promoting cancer cell death.64 Upon irradiating a NADH solution in the presence of IH-COF, the absorption of NADH at 339 nm significantly decreased whereas the absorption of IH-COF remained, suggesting the efficient oxidation of NADH to NAD+ and excellent photostability of IH-COF (Fig. 4f). The UV-vis results under dark conditions demonstrated that light is essential for the oxidation of NADH (Supplementary Fig. 16). The positive correlation between O •− production and NADH concentration further supports that NADH plays a role in regeneration of PS (Supplementary Fig. 17). Besides NADH, this redox reaction can also occur between glutathione and IH-COF (Supplementary Fig. 18). Triplet quenching experiments revealed photoinduced ET from an S1 excited state of the hyperporphyrin to the iminium group, which was further supported by a natural transitional orbital (NTO) analysis (Fig. 4g, Supplementary Fig. 19). The apparent quantum yield for O •− formation was estimated to be 1.1% (Supplementary Fig. 20). Ultrafast transient absorption spectroscopy was performed to analyze excited state dynamics, which supports an ET process from the initial photoexcited state occurring within < 10 ps. The lifetime of the subsequent ET state depends on oxygen concentration implying reaction with dissolved oxygen (Supplementary Figs. 21-26). Based on these findings, we propose the mechanism in Fig. 4h for IH-COF mediated type I PDT in biological systems. Under 725 nm irradiation, the photoexcited hyperporphyrin [H-Por]* transfers one electron to the iminium linkage to generate the α-amino radical. The α-amino radical reduces O to form O •− and regenerates the iminium ion. The resulting [H-Por]•+ is reduced by biomolecules such as NADH to regenerate [H-Por], thus completing the photoredox cycle.

In vitro PDT performance
The strong ROS generation by IH-COF prompted us to evaluate its in vitro cytotoxicity on cancer cells. [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)- 2H-tetrazolium (MTS) assays revealed that IH-COF plus 725 nm light irradiation (denoted IH-COF(+)) exhibited an IC value of 2.62 μM in CT26 cells under normoxia (21% O ). This IC value was much lower than those of Me-COF (IC =13.1 μM) and Im-COF (IC ＞40.0 μM) under 660 nm irradiation (Fig. 5a). The COFs did not show appreciable dark toxicity in CT26 cells, but the monomer TAPP showed a strong dark toxicity with an IC of 1.82 μM, indicating that the COF framework structure reduces the dark toxicity of photosensitizers (Supplementary Fig. 32). Under hypoxic conditions (0.5% O ), IH-COF showed an IC value of 6.2 μM in CT26 cells, while IC values of Me-COF and Im-COF further increased to 32.7 μM and >80.0 μM, respectively (Fig. 5b). These results indicate that IH-COF shows strong type I PDT performance in vitro under both normoxic and hypoxic conditions. The presence of an appropriate amount of exogenous NADH enhanced the cytotoxicity, which supports the proposed mechanism (Supplementary Fig. 33).

IH-COF showed higher cellular uptake than Me-COF and Im-COF, indicating its superior intracellular delivery efficiency (Supplementary Fig. 34). We next used confocal laser scanning microscopy (CLSM) and flow cytometry to assess photoinduced ROS production by COFs in CT26 cells. DCFH-DA assays indicated that IH-COF(+) induced the strongest total ROS accumulation in CT26 cells, with 1.9-fold and 8.0-fold higher total ROS signals than those of Me-COF(+) and Im-COF(+), respectively (Figs. 5c, d). Consistent with O •− generation efficiencies in test tubes, flow cytometry showed that IH-COF(+) generated 2.1-fold and 1.5-fold more O •− in CT26 cells than Im-COF(+) and Me-COF(+), respectively (Fig. 5e, Supplementary Fig. 35). These results show that IH-COF(+) elicits potent anticancer activity by mediating efficient type Ⅰ PDT.

The positive charge of IH-COF inspired us to investigate its mitochondrial targeting ability.65 We tagged COFs by chemical conjugation with FITC and labeled mitochondria with MitoTracker Red CMXROS. After 8 hours of incubation, IH-COF was abundantly enriched in mitochondria with a Pearson’s correlation coefficient (R2) of 0.79, while the R2 value of Im-COF to mitochondria was only 0.59. This result indicates a strong mitochondrial targeting ability of IH-COF (Supplementary Fig. 36). Given the short lifetime and limited diffusion distance of ROS, mitochondrial-targeting ability of IH-COF is expected to accumulate ROS in mitochondria for mitochondrial disruption.66 IH-COF(+) induced the most significant depolarization of the mitochondrial membrane potential, as evidenced by the enhanced monomer signals in the JC-1 assay (Supplementary Fig. 37). Co-localization studies of cytochrome c with mitochondria showed that a large amount of cytochrome c was released into the cytoplasm after IH-COF(+) treatment, with an R2 value of 0.53. In comparison, the R2 values were 0.86, 0.80, and 0.69 for PBS(+), Im-COF(+), and Me-COF(+), respectively. More cytochrome release induced by IH-COF(+) further activated the apoptotic pathway (Supplementary Fig. 38). Mitochondrial dysfunction causes oxidative stress, disruption of ion balance, inflammation, and activation of mitochondria apoptosis-related proteins, all of which can enhance apoptotic cell death.67 As a result, IH-COF(+) induced the strongest cell apoptosis (72.7%) which was markedly higher than those of Im-COF(+) at 12.9% and Me-COF(+) at 19.5% (Fig. 5f, Supplementary Figs. 39-40). Taken together, IH-COF(+) elicits strong anticancer effects via efficiently mediating type I PDT and disrupting mitochondria.

In vivo antitumor effects
To prolong the blood circulation time and increase tumor uptake of COFs, we PEGylated the COFs with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino-(polyethylene glycol)-2000] before conducting animal experiments (Supplementary Figs. 9-10). PEGylated IH-COF showed a half-life of 2.4 h, an AUC of 371.8 mg/L·h, and a C of 66.15 mg/L. Tumor uptake experiments revealed that IH-COF reached its maximum accumulation in the tumor at 12 h after intravenous injection (Supplementary Fig. 41). The in vivo antitumor efficacy was evaluated in subcutaneous CT26 murine colon cancer model and 4T1 murine triple-negative breast cancer model in BALB/c mice. When the tumor volume reached ~100 mm3, mice were injected intravenously with PEGylated COFs at 0.9 μmol porphyrin equivalent. The tumors were irradiated with a 660 nm or 725 nm LED at 12 h post-injection. Im-COF(+) moderately inhibited tumor growth with tumor growth inhibition index (TGI) of 46.5% and 23.1% on CT26 and 4T1 tumor-bearing mice, respectively. Me-COF(+) treatment was more effective with TGI values of 67.1% and 56.0% on CT26 and 4T1 tumor-bearing mice, respectively. Remarkably, IH-COF(+) showed the most potent tumor growth inhibition with TGI values of 93.1% and 80.6% on CT26 and 4T1 tumor-bearing mice, respectively (Figs. 6b-e, Supplementary Fig. 43). IH-COF without light irradiation did not show any antitumor effect (Supplementary Fig. 44), indicating that the strong antitumor activity of IH-COF(+) was due to the efficient type I PDT effect.

The excised CT26 tumors were sliced for hematoxylin and eosin (H&E) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining analysis. Compared to Im-COF(+) and Me-COF(+) groups, IH-COF(+)-treated mice exhibited the most pronounced nuclear shrinkage and the highest percentage of apoptosis in tumor cells (Fig. 6f). No weight loss or organ toxicity was observed during the treatment period for CT26 tumor-bearing mice, indicating that these treatments did not cause general toxicity (Supplementary Fig. 43). Hemolysis assays showed that IH-COF, Im-COF, and Me-COF caused minimal red blood cell lysis, with hemolysis rates remaining well below the 10% safety threshold even at concentrations up to 320 μM (Supplementary Fig. 42a), indicating good hemocompatibility. In addition, liver function markers (aspartate aminotransferase and alanine aminotransferase) showed no increase at 24 or 48 hours after administration (Supplementary Fig. 42b, c), indicating that IH-COF does not induce liver toxicity. Together, these results highlight the excellent biocompatibility and biosafety of IH-COF.

In this work, we developed a strategy to design efficient COF-based PSs for type I PDT by linking hyperporphyrins with iminium ions. The quaternization of imine bonds introduced strong electron acceptors while the protonation of porphyrins red-shifted the Q band to 725 nm via the hyperporphyrin effect in IH-COF. Under 725 nm irradiation, the photoexcited hyperporphyrin [H-Por]* transfers one electron to the iminium linkage to generate the α-amino radical. The α-amino radical reduces O to form O •− and regenerates the iminium ion. The oxidized hyperporphyrin is reduced by biomolecules such as NADH to regenerate the hyperporphyrin. This photoredox cycle mediates radical ROS generation for efficient type I PDT. As a result, IH-COF mediated PDT exhibited higher in vitro cytotoxicity over Im-COF and Me-COF controls under both normoxic and hypoxic conditions and exhibited strong in vivo antitumor efficacy in subcutaneous tumor model of murine colon CT26 cancer and orthotopic tumor model of murine 4T1 triple-negative breast cancer. This work not only demonstrates an efficient strategy for designing efficient COF PSs for type I PDT through the incorporation of photoredox chemistry and hyperporphyrin effect but also highlights the potential of COFs as a biocompatible platform to incorporate multiple synergistic functionalities for other biomedical applications.

Methods
The murine colorectal carcinoma cell line CT26 and the murine triple-negative breast cancer cell line 4T1 were obtained from the American Type Culture Collection (ATCC, Rockville, MD). Cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin G sodium, and 100 μg/mL streptomycin sulfate, in a humidified incubator at 37 °C with 5% CO . For in vivo studies, female BALB/c mice aged 6–8 weeks were utilized. All animal procedures were conducted in accordance with a protocol approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Chicago (#72408), in compliance with PHS Assurance #D16-00322 (A3523-01). Animals were maintained at a density of 4–5 mice per cage, under controlled environmental conditions (~25°C, regulated humidity, and a 12-hour light/dark cycle). Histological analyses were performed with assistance from the Human Tissue Resource Center at the University of Chicago.

Synthesis of IH-COF
A solution of terephthalaldehyde (18.8 mg, 0.14 mmol) in CH CN (10 mL) was added to a solution of TAPP (47.4 mg, 0.07 mmol) in CH CN (15 mL) in the presence of glacial acetic acid (2 mL). The dispersion was vigorously stirred (1100 rpm) at room temperature for 15 h. Then, a solution of (CH ) OBF (41.4 mg, 0.28 mmol) in 1 mL CH CN was added, and the mixture was stirred for 2 additional hours. IH-COF nanoparticles were collected by centrifugation at 24000 ×g for 15 min, washed with CH CN three times, and then re-dispersed in CH CN by sonication for 1 h. The suspension was centrifugated at 5000 ×g for 15 min to obtain IH-COF nanoparticles of desired sizes in the supernatant. Yield: 20 mg (33%).

Bulk IH-COF powder was synthesized under solvothermal conditions. Specifically, a mixture of TAPP (27.0 mg, 0.04 mmol), terephthalaldehyde (11.2 mg, 0.08 mmol), and (CH ) OBF (24.0 mg, 0.16 mmol) in a mixture of ethanol (0.5 mL) and mesitylene (0.5 mL) was heated at 120 oC for 3 days in the presence of CH COOH (72 μL). The solid was collected by filtration and dried to afford bulk IH-COF as a black powder (30 mg, 79 % yield).

Synthesis of Im-COF and Me-COF
A solution of terephthalaldehyde (18.8 mg, 0.14 mmol) in CH CN (10 mL) was added to a solution of TAPP (47.4 mg, 0.07 mmol) in CH CN (15 mL) in the presence of glacial acetic acid (2 mL). The dispersion was vigorously stirred (1100 rpm) at room temperature for 18 h. Im-COF nanoparticles were collected by centrifugation at 24000 ×g, washed with CH CN three times, and then re-dispersed in CH CN by sonication for 1 h. The suspension was centrifugated at 5000 ×g to obtain Im-COF nanoparticles of desired sizes in the supernatant. Yield: 32 mg (52%). To obtain Me-COF nanoparticles, a mixture of TAPP (47.4 mg, 0.07 mmol), terephthalaldehyde (18.8 mg, 0.14 mmol), CH I (5 mL) and glacial acetic acid (2 mL) in 25 mL CH CN was stirred at 1100 rpm at room temperature for 20 h. Me-COF nanoparticles were collected by centrifugation at 24000 ×g for 15 min, washed with CH CN three times, and then re-dispersed in CH CN by sonication for 1 h. The suspension was centrifugated at 5000 ×g for 15 min to obtain Me-COF nanoparticles of desired sizes in the supernatant. Yield: 37 mg (60%). Bulk Im-COF and Me-COF powders were synthesized following the same procedure to IH-COF except for the use of different methylation reagent.

PEGylation of COF nanoparticles
COF nanoparticles (30 mg) as synthesized above were dispersed in 20 mL water, followed by the addition of 40 mg DSPE-PEG2K. The dispersion was stirred at 1100 rpm at room temperature for 24 h. PEGylated COF nanoparticles were collected by centrifugation at 24000 ×g for 15 min and washed with water three times in quantitative yields.

ROS detection
Total ROS was detected by the 2',7'-dichlorodihydrofluorescein-diacetate (DCFH-DA) assay. COF dispersions (total porphyrin: 2.3 μM) in water were added to freshly prepared DCFH-DA solutions and the final concentration of DCFH-DA was 10 μM. 150 μL of each COF suspension was added to 96-well plates (n = 3). Next, the whole plate was illuminated by light (100 mW/cm2) for different periods of time (0, 2, 4, 6, 8, 10 min). The fluorescence signal of DCFH (Ex. 485 nm; Em. 520 nm) was collected with the HTX microplate reader immediately after light irradiation. A 725 nm LED was used for IH-COF and a 660 nm LED was used for Im-COF and Me-COF. Singlet oxygen was detected by SOSG assay and superoxide radical was detected by DHR 123 assay using the same procedure as DCFH assay.

α-Amino radical trapping experiment
In a glove box, a vial (10 mL) was charged with the diphenyl iminium iodide M2 (16.2 mg, 0.05 mmol), TEMPO (15.6 mg, 0.1 mmol), TAPP (6.8 mg, 0.01 mmol), triethanolamine (13 μL, 0.1 mmol), and CH CN (5 mL). The resulting mixture was stirred at room temperature under 660 nm LED irradiation overnight. After filtration, the filtrate was subjected to LC-MS analysis.

Cyclic voltammetry
The working electrode was polished with alpha alumina paste to remove surface adsorbates. Subsequent sonication of the working and counter electrodes in H O was performed to remove alumina residue and provide additional cleaning. Before each measurement, the solution was saturated with N or O for 10 min and an N atmosphere was maintained throughout the test for the N saturated condition. The concentration of analytes was 1mM and tetrabutylammonium hexafluorophosphate (NBu PF ) solution in CH CN (100 mM) was used as the supporting electrolyte. The scan rates were set at 100 mV/s for all measurements.

Photocatalytic NADH oxidation
A mixture of NADH (1 mL, 200 μM in water) and IH-COF (1 mL, total porphyrin: 10 μM) was illuminated with a 725 nm LED. After different periods of time (0, 2, 4, 6, 8, 10 min), the UV-vis spectra were collected.

Data availability
All data supporting the findings of this study are available from the corresponding author.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Zhou et al. designed porphyrin-based COFs with enhanced NIR absorption at the Q band and investigated the type-I photosensitizing capability. Interestingly, the photoexcitation of COFs could induce generation of α-amino radicals via photoinduced electron transfer from hyperporphyrin units to iminium ions. The mechanistical study revealed that the reduction of O2 into superoxide ion was attributed to the single electron transfer from O2 to the α-amino radicals. This approach would benefit new understanding of carbon-radical based type-I PDT against hypoxic cancers.\nThe major weakness is the questionable mechanism about the origin of superoxide. The authors proposed a straightforward SET-based photoredox mechanism (Fig 4g) which might be acceptable. However, the SET between carbon-centered free radical and O2 is not common, lacking sufficient theoretical calculations to strengthen their argument. In the presence of O2, carbon radical (R*) is prone to form peroxide radical (ROO*) and further ROOH. This pathway can also lead to superoxide production. I’d suggest the authors investigate more about the mechanism which is one of the most valuable parts of the paper. Besides, I’d mention another question with Fig 4g. The authors proposed SET between NADH and [H-Por]*+ to interpret the regeneration of [H-Por]. If this hypothesis is true, however, NAD* radical rather than NAD+ should be generated. Is there evidence supporting Fig 4f that NADH was transformed into NAD+?\nI have additional questions:\n1. The zeta potential of Im-COF was very negative (-30 mV). Why?\n2. It would be better if the authors could investigate/discuss the impact of oxygen carrying capacity of porous COF photosensitizers on superoxide generation.\n3. NADH plays a role in regeneration of [H-Por]. Was superoxide production positively correlated with NADH concentration?\n4. Regarding the effect of type-I PDT, the authors discussed mitochondrial dysfunction with evidence of cytochrome c release (under normoxia). Under hypoxia where O2 supply (e.g., 0.5% O2) was seriously limited, how did the photosensitizer function in the cells? Was PDT effect dependent on NADH? Clarification of the essential role of COF photosensitizers in the inhibition of hypoxic tumors would enhance the advantages of this work.", "reviewer_2": "In this study, a hyperporphyrin covalent organic framework (IH-COF) based on imine ion linkage was developed for efficiently mediating type I photodynamic therapy (PDT). It is interesting to combine imine ions with superporphyrin in COF to realize efficient type I PDT through photoredox cycle, which would solve the hypoxia limitation problem of conventional PDT. However, the characterization of the COF structure seems incomplete, the mechanism for O2•− generation should be deeply understood, and the biological experiment is insufficient. Therefore, this work is not recommended to publish in this high-level journal at the current state. The following issues should be addressed.\n1. For UV-vis absorption, the spectra shown in Fig. 3a and Supplementary Fig. 4 are different. UV-vis absorption spectrum in Supplementary Fig. 4 is more red-shift than that in Fig. 3a. A careful explanation should be provided, and even some experiment or theoretical calculations are necessary.\n2. In Fig. 3c and d, the PL spectra for detections of singlet oxygen and superoxide radical should be provided. And the intensities of which wavelength should be provided. The experimental information is seriously lack, which make the current data difficult to understand.\n3. For PXRD study, the authors only gave the patterns for IH-COF, how about other COFs, such as Im-COF and Me-COF? In Fig. 2e, the crystallinity of IH-COF seems very low, which is crucial for materials performance and the next biological applications. The assignment of PXRD peaks has not been done and the high-resolution transmission electron microscopy (HRTEM) should be utilized to clearly characterize for understanding the crystallinity of COFs. How about the stacking mode for these COFs? Overall, the characterization of the COF structure is very simple and incomplete.\n4. The 13C cross-polarization magic-angle-spinning (CP/MAS) NMR spectrum was strongly suggested to verify the characteristic resonance signals for confirm whether the imine bonds and the methylation form in COFs.\n5. For the electron transfer to generate O2•−, the mechanism seems very rough, and it is necessary to provide insights into the excited state dynamics. How about the excited state dynamics? Does it from the triplet state or singlet state? It is recommended that the authors give more experimental and theoretical data.\n6. The authors directly mentioned that redox reaction between COFs and NADH, how about the redox reactions occur between COFs and other endogenous agents is very interesting to explore.\n7. In Fig. 3b, c, d and Fig. 5c, the titles for y axles are not suitable for understanding, it is suggested to change according to the reported literatures. Incomplete data expression in Fig. 5f, there is no information about the x and y axles.\n8. The authors does not provide the details about the cellular uptake of the COFs.\n9. There is no statistical analysis in Fig. 6b and d.\n10. The influence of light and COF selves on the tumor treatment should be provided for careful control.\n11. The stability data of COFs in different physiological environments (e.g., PBS, serum-containing medium, acidic/alkaline conditions) need to be supplemented to confirm its structural integrity for in vitro and vivo applications.\n12. Additional tests are needed on the quantum yields and lifetimes of COFs.\n13. In vivo experiments were conducted by intravenous injection, but no data related to the targeting of the material were provided. It is suggested to add relevant experiments to support the maximum enrichment of the material in the tumor for another 12 h after intravenous injection and to confirm the metabolism time of the material in the organism;\n14. Some of the images in the Supplementary Information are of low resolution (e.g., Supplementary Fig. 7) and need to be optimized to ensure the readability of the data.\n15. Lack of comparative experiments with existing type I PDT photosensitizers, which should be added to highlight the superiority of IH-COF.\n16. Which cell death mechanisms are induced by these COFs, apoptosis or ferroptosis? It is recommended to clarify by necessary experimental data.", "reviewer_3": "In this manuscript, Lin et al demonstrate an iminium-linked hyperporphyrin covalent organic framework (IH-COF) as type I PS for PDT. However, this manuscript does not match the requirement of publication due to the following concerns.\n1. This manuscript lacks innovation. Similar structure has been reported in established literature (doi.org/10.1002/ange.202418319) and it’s hard to find the advantage of IH-COFs in this manuscript compared to Ptp-Fe in the previous study (doi.org/10.1021/jacs.4c03519) and other type I PSs that have been reported.\n2. Type I PDT is particularly promising strategy to generate superoxide (O2•-) and hydroxyl radicals (•OH) and hydrogen peroxide (H2O2), without substantial reliance on O2. However, authors completely ignored the important role of •OH or H2O2 type I PDT in the absence of sufficient experimental evidence. What are the considerations behind doing this?\n3. The mass spectrometry results in Figure 4b are quite strange and completely different from the common characterization results, is it really the data came from mass spectrometric analyses?\n4. The mechanism of type I PDT generation by the whole IH-COF was not investigated sufficiently. Authors only determined the CV curves of the M2 unit through cyclic voltammetry.\n5. Nanoparticles with positively charged surfaces or too large particle size may not be able to keep stable in vivo long circulation and the EPR effect. What are the basic properties of PEGylated IH-COF nanoparticles, including surface charge, size, morphology, stability, etc?\n6. There is no evidence for distribution in mice of IH-COF. Why choose to treat at 12 hours after PEGylated IH-COF administration?\n7. It would be desirable for the author to complement experiments to verify that IH-COF was able to perform photoredox cycle to achieve efficient type I PDT in vitro and in vivo.\n8. The biological safety needs to be further explored. It is not convincing to demonstrate the good biological safety of COF materials only by H&E staining of major organs. The description “the potential of COFs as a biocompatible platform to incorporate multiple synergistic functionalities for other biomedical applications” is doubtful. Firstly, PEGylation of COF nanoparticles was used for drug delivery in vivo. In addition, what is the metabolic pathway of IH-COF? Will IH-COF remain in the mice after treatment?\n9. Please use a unified font when drawing and improve the clarity of the figures in manuscript and supplementary materials.\n10. Please confirm the accuracy of the IH-COF structure in Figure 2a. Should the protonated central -NH group of porphyrins be positively charged? This is inconsistent with its depiction in Figure 2a.\n11. There is no Figure e in Figure 3, the caption “In c-e” is incorrect.\n12. Figure 4g illustrates only a partial electron transfer mechanism within the IH-COF structure. It remains unclear whether electron transfer occurs between the incompletely methylated imine moieties and the porphyrin units. The authors are advised to supplement the electron transfer mechanism for the entire structural unit to ensure comprehensive analysis.\n13. What the red and black arrows in Figure 6b and 6d represent respectively?"}, "author_response": "We thank the reviewer for the constructive suggestion. Following the comment, we have performed DFT calculations to calculate free energy changes for two potential pathways of the reaction between α-amino radical and O (Figure R1). Compared to the one-step outer-sphere 2 electron transfer from α-amino radical to O , the formation of a peroxide intermediate is 2 thermodynamically more favorable by 0.5 kcal/mol. Subsequent heterolytic C-O bond cleavage is +0.5 kcal/mol uphill, resulting in the generation of superoxide radical and the iminium linkage. Thus, DFT calculations indicate that both pathways are feasible for the conversion of an α-amino radical to an iminium ion. Furthermore, we also carried out additional triplet quenching experiments and natural transitional orbital (NTO) analysis to further investigate the mechanism (Reviewer #2, Comment 5). The theoretical calculation and relevant description have been added to the revised manuscript (page 10, paragraph 1): “The reaction between α-amino radical and O 2 may also occur through a peroxide intermediate, which was supported by free energy changes (Supplementary Fig. 17).”\nRegarding the conversion of NADH to NAD+, this involves two 1e- oxidation steps 1 (NADH→NAD•→NAD+: Nat. Chem. 2019, 11, 1041-1048; Nat. Commun. 2022, 13, 6179). Specifically, the resultant NAD• from the SET process between NADH and [H-Por]•+ can be further oxidized by O •− or other oxidizing agents in biological environment to form NAD+ (Nat. 2 Chem. 2019, 11, 1041-1048). The transformation from NADH to NAD+ is supported by UV-vis absorption spectroscopy (Fig. 4f). Upon irradiating a NADH solution in the presence of IH-COF, the characteristic absorption of NADH at 339 nm significantly decreased whereas the absorption of NAD+ at 260 nm increased, suggesting the efficient oxidation of NADH to NAD+. For clarity, we have updated the photoredox cycle in Fig. 4h to indicate the presence of NAD•.\nFigure R1: Free energy changes calculated for two potential pathways of the reaction between α- amino radical and O . 2\nI have additional questions:\n1. The zeta potential of Im-COF was very negative (-30 mV). Why?\nResponse: We thank the reviewer’s comment. Im-COF has a very negative Zeta potential of -30 mV, which may be the coordination of metal ions to imine nitrogen atoms and balance of positively charged metal ions with anions in the outer shell of the particles.\n2. It would be better if the authors could investigate/discuss the impact of oxygen carrying capacity of porous COF photosensitizers on superoxide generation.\nResponse: We appreciate the reviewer for the comment. However, the oxygen carrying capacity 2 of COFs is unlikely to have a significant impact on PDT of COFs. Im-COF, Me-COF, and IH-COF do not have oxygen-affinity sites. More importantly, PDT is conducted at room temperature (in vitro) or 37 oC physiological condition (in vivo). Under these conditions, the oxygen carrying capacities of these COFs will not play a role. In fact, the dissolved oxygen in aqueous environments plays a more important role.\n3. NADH plays a role in regeneration of [H-Por]. Was superoxide production positively correlated with NADH concentration?\nResponse: We thank the reviewer for the helpful comment. As suggested, we conducted superoxide radical production experiments in the presence of different concentrations of NADH (Figure R2). After irradiation of 725 nm for 10 min, the fluorescence of DHR 123 increases with the increase of NADH concentration, suggesting that NADH promotes the generation of superoxide radical. The relevant information has been included to the revised manuscript (page 11, paragraph 1): “The positive correlation between O •− production and NADH concentration further 2 supports that NADH plays a role in regeneration of PS (Supplementary Fig. 14).”\nFigure R2. O •− production by IH-COF in the presence of different concentrations of NADH under 2 725 nm irradiation for 10 min.\n4. Regarding the effect of type-I PDT, the authors discussed mitochondrial dysfunction with evidence of cytochrome c release (under normoxia). Under hypoxia where O supply (e.g., 0.5% 2 O ) was seriously limited, how did the photosensitizer function in the cells? Was PDT effect 2 dependent on NADH? Clarification of the essential role of COF photosensitizers in the inhibition of hypoxic tumors would enhance the advantages of this work.\n[... Author responses to Reviewer 2 and 3 omitted for brevity as per instructions to provide the full structure]"}, {"round": 2, "reviewer_comments": {"reviewer_1": "Improvements have been made in the revised manuscript. I have a few questions:\n1. Regarding the mechanism shown in supplementary Fig. 17, activation energy values and transition states need to be seen.\n2. If oxygen carrying behavior is not considered, the pores of nonfluorescent COFs have no functions in the present design. Only the surface of COFs could contact oxygen molecules and NADH for generation of superoxide ions.\n3. In supplementary Fig. 21, images in separate FITC and MitoTracker channels should be provided. Scale bar information should be clearly indicated. In addition, preparation protocol for FITC-labeled COFs is missing. If the amino/imine groups are fully methylated in the COFs, which functionality is conjugated by FITC?\n4. Could the authors provide necessary concentration information in the caption of supplementary Figs. 15&16?\n5. How about the changes of absorption spectra of NADH/COFs solution in the dark conditions?\n6. The authors applied DHR123 for detection of superoxide ions (see supplementary Fig. 14). Is DHR123 superoxide specific? If not, alternative probes should be considered.\n7. Could the authors provide in vitro experimental evidence supporting the correlation between NADH levels and PDT effect?", "reviewer_2": "At present, the manuscript has made significant improvements. The authors have addressed some of the comments from the initial round of reviews; however, several concerns remain unresolved and require further clarification before publication: 1. it is still necessary to investigate the excited state dynamics for the electron transfer to understand the mechanism of O2•− generation by fs-TA technique. 2. Although the author clamed “Compared to previously reported type-I organic PDT agents, IH-COF efficiently absorbs in the tissue-penetrant near-infrared window and exhibits higher stability”, the stability has been not presented in table S5. Besides, 725 nm is generally located on the visible range, not NIR region.", "reviewer_3": "Comments have been addressed.", "reviewer_4": "This study presents an iminium-linked hyperporphyrin covalent organic framework designed for Type I photodynamic therapy. While the authors have addressed some of the concerns and suggestions raised in previous rounds of review, the overall quality of the discussion and the clarity of the presentation remain below the standards expected for publication in Nature Communications.\n1) The authors have presented a DFT study comparing two potential reaction pathways.\na. The basis set used in the study is reported as 6-311G+, which is both improperly notated and chemically insufficient for accurate DFT calculations involving molecular systems. First, diffuse functions (denoted by \"+\") must be placed before the \"G\" in Pople-style notation, and polarization functions (e.g., (d), (d,p)) are entirely missing. The correct and chemically meaningful form would typically be 6-311+G(d) or 6-311+G(d,p). The lack of polarization functions is particularly concerning the author concepts of computational modelling.\nb. The free energy diagram presented lacks transition states, which are crucial for understanding the reaction mechanism and comparing the activation barriers of different pathways. Without these key features, the reaction coordinate is incomplete.\nc. The reported free energy difference of 0.5 kcal/mol between the compared structures is very small and well within the typical error margin of DFT methods. Such a small difference cannot be considered definitive evidence for the preference of one structure or pathway over another. Additional benchmarking or validation with higher-level methods (e.g., CBS, CCSD(T), or alternative functionals with larger basis sets) would improve the reliability of their findings.\n2) The authors’ explanation for the highly negative zeta potential values (-30 mV), which they attribute to the coordination of metal ions to imine nitrogen atoms and the balancing of metal ion charges with anions in the outer shell, appears conceptually inconsistent. Metal coordination typically introduces localized positive charges, which would be expected to reduce the negative surface potential rather than increase it. Therefore, it is unlikely that metal coordination dominates the surface charge. It is more plausible that the coordinated metal centers form neutral complexes or that their charges are effectively screened by counterions, resulting in minimal contribution to the observed negative zeta potential.\n3) The authors did not clearly specify the theoretical methodology employed for the calculation of the NTOs. Specifically, it is unclear whether time-dependent DFT was used, and if so, which functional, basis set, and solvation model (if any) were applied.\n4) Could the authors clarify whether the light source used for irradiation covers the full solar spectrum or if optical filters were employed? It would be scientifically valuable to investigate the effects of irradiation under separate UV and visible light regions, as this could provide deeper insight into the wavelength-dependent behavior of the system.\n5) Since Type I photodynamic therapy depends on electron or hydrogen atom transfer from the excited photosensitizer to molecular oxygen or surrounding substrates to generate ROS, both the quantum yield and lifetime of the excited state are critical parameters for this mechanism. Therefore, these values should be clearly presented and discussed in the manuscript.\n6) In the presented N 1s XPS spectra deconvolution, the reported FWHM values for different components are inconsistent, with no clear justification. It is a standard practice in XPS fitting to apply identical or tightly constrained FWHM values to all components within the same spin-orbit or core level, unless there is compelling chemical or physical reasoning (such as differential broadening from highly disordered environments or strong shake-up features).\n7) The manuscript states that the COFs are dispersed in water, and absorption spectra are provided. However, it remains unclear whether the COFs are truly soluble or simply form colloidal suspensions. Please clarify the nature of the dispersion. Additionally, specify the concentration at which the dispersion remains stable and whether any aggregation or precipitation is observed over time.\n8) In the Stern–Völmer plots (Fig. 4a), please provide the R² values for the linear fits. Additionally, for M2, the data appear to exhibit two distinct linear regions with a transition occurring around 4.5 μM. Could the authors clarify this behavior and discuss the possible reasons for the change in slope?"}, "author_response": "Response: We appreciate the reviewer’s insightful comment. We have performed additional theoretical calculations for the proposed mechanism. In addition to the BELYP/6-311+G(d) method, we have also used other higher-level calculation methods (revDSD-PBEP86-D4/def2-TZVPP and DLPNO-CCSD(T)/def2-TZVPP) to evaluate the transition state and activation energy. Please see our detailed response to Reviewer #4, Comment 1.\n[... Author responses to Reviewer 2, 3, and 4 omitted for brevity]"}, {"round": 3, "reviewer_comments": {"reviewer_1": "The authors have improved calculation methods in the revised manuscript to show the transition states in the proposed mechanism involving superoxide generation. However, the calculation may not sufficiently support the hypothesis (Fig. 4h). The pi-conjugation of porphyrin unit to M1 or M2 likely significantly affect the transition states (or superoxide generation pathway) and free energies. Why not perform calculations in consideration of the porphyrin conjugation for better reliability?\nI also noticed the correlation between NADH and PDT effect (in response to previous question#7) is questionable. If exocellularly added NADH (0.1 mM, not exactly high concentration) suppresses the PDT effect, the hypothesis of NADH-dependent PDT in the living system lacks convincing support from experimental evidence. In addition, generally the level of NADH in cancerous cells is significantly elevated as compared to healthy cells. It would be problematic for a photosensitizer if NADH counteracts ROS generation and PDT efficacy. I’d suggest the authors repeat in vitro PDT experiments at larger concentration ranges of exocellularly added NADH.", "reviewer_2": "All comments have been well addressed.", "reviewer_4": "The authors have almost answered all the comments to the reviewers but still needs to address the following suggestions to be acceptable for publication.\n1) The authors state: “the pores of COFs play a crucial role in enhancing mass transfer and molecular diffusion… Thus, porous nanoparticles are critically important for optimal PDT performance.” While this is a reasonable hypothesis, the claim requires support through either appropriate references or experimental evidence. As currently presented, it reads as an assumption. I recommend the authors either cite relevant studies that substantiate this mechanism or include experimental data demonstrating the role of porosity in enhancing PDT efficacy. This statement also needs to be included into the manuscript.\n2) Although the authors have included the recommendation made by the reviewer about the highly negative zeta potential values as\n“This highly negative potential might be due to the coordinated metal centers form neutral complexes or that their charges are effectively screened by counterions”\nThe authors need to cite some references proving such statement"}, "author_response": "Response: We appreciate the reviewer’s insightful comment. As suggested, we performed DFT calculations on the fragment conjugated with the porphyrin ring using the B3LYP/6-311+G(d) basis set and incorporating the solvent effect of water. The resulting optimized structure displayed precisely one imaginary frequency, indicative of a first-order saddle point on the potential energy surface. To further corroborate the authenticity of this transition state (TS), we performed both frequency and intrinsic reaction coordinate (IRC) computations. The optimized TS geometry corresponding to the oxygen attack reaction and its specific vibrational mode are illustrated in Figure R1a-b, respectively. The relative Gibbs free energies of the reaction pathway are shown in Figure R2. The intermediate state (INT) is found to be energetically more favorable by -11.0 kcal/mol relative to the initial state (IS), while the final state (FS) is favorable by -9.5 kcal/mol. These results also support our hypothesis proposed in Fig. 4h. The relevant results have been provided in the revised supporting information (pages 23-24).\n[... Author responses to Reviewer 4 omitted for brevity]"}, {"round": 4, "reviewer_comments": {"reviewer_1": "The authors have supplemented additional calculation and experimental data in the revised manuscript. The result of exogenous NADH (to elevate endogenous NADH level) on phototoxicity is not so positive, but may reflect the competitive role of NADH in ROS generation and ROS compromisation. The current version of manuscript is suggested to be accepted. However, it would be pretty fine if the authors provide a discussion in the manuscript about the reason why the free energy of TS calculated for oxygen association is negative (relative to that of IS, see Supplementary Fig. S29&S31).", "reviewer_4": "The authors have sufficiently addressed the comments/recommendations made by the reviewers and thus acceptable in the present form."}, "author_response": "Response: We appreciate the reviewer’s insightful comment. The negative free energy difference of the transition state (TS) relative to the initial state (IS) for oxygen association (Supplementary Figs. 29 and 31) originates mainly from the solvation effect (water is considered in this manuscript). Specifically, when implicit solvent correction was included, the TS structure experienced stronger stabilization than the IS, leading to a slightly lower free energy of the TS. In contrast, when the same calculations were performed without the solvent effect, the TS was indeed found to be higher in energy than the IS, which is consistent with the expected energy profile (Figures R1-R2). We have added a discussion in the revised supporting information (page 23): “The calculated free energy of the TS for oxygen association is slightly lower than that of the IS. This feature mainly arises from the stabilization effect of the solvent model applied in the calculation. In the implicit solvent environment, the TS is stabilized more strongly than the separated IS species, which leads to an apparent negative free energy difference.”"}]

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