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. 2025 Jun 19;5(7):3058–3069. doi: 10.1021/jacsau.4c01161

Singzyme: A Single–Molecule Enzyme–Like Photocatalyst Powered by the Super Cage Effect

Zhiqing Long 1, Kaixuan Wang 1, Ke Luo 1, Maijun Zhu 1, Qing He 1,*
PMCID: PMC12308452  PMID: 40747068

Abstract

Photodynamic therapy (PDT) represents a powerful approach for treating cancers through light–induced reactive oxygen species (ROS) generation; however, its efficacy is often constrained by limitations in photosensitivity, ROS yield, and tumor microenvironment adaptation. Addressing these challenges requires highly engineered photosensitizers capable of enhanced light absorption, stable catalytic performance, and selective redox modulation. Herein, we introduce a structurally and functionally single–molecule enzyme–like photocatalyst (superphane 1), a supramolecular photosensitizer based singzyme designed through a “super cage effect” or “superphane effect” that promotes enhanced photosensitivity, superior ROS generation, photocatalytic oxidation and multi–enzyme mimetic activities. Its unique structure, featuring precisely and densely arranged phenothiazine units, maximizes light absorption and optimizes electron transfer dynamics, resulting in an excellent singlet oxygen (1O2) yield of 70% under 450 nm irradiation, significantly surpassing the yields of 42% for cage 2, 39% for macrocycle 3, and 24% for monomer 4. Functionally, singzyme 1 exhibits excellent catalytic efficiency to NADH (K cat/K m = 2.43 × 104 L mol–1 min–1) and catalyzes oxidation of NADH and GSH, mimicking NADH oxidase and GSH oxidase activity. This selectively depletes NADH and GSH, inducing an intracellular “oxygen burst” effect. This redox imbalance inhibits mitochondrial oxidative phosphorylation (OXPHOS), reduces ATP production, and promotes apoptosis and ferroptosis pathways. With stable activity across acidic and neutral pH environments typical of tumors, singzyme 1 provides a versatile, biocompatible platform for precision cancer therapy. This study establishes a superphane–based singzyme with precise structure and multi–enzyme mimetic functionality, paving the way for single–molecule enzyme mimicry.

Keywords: singzyme, superphane, ROS, photocatalyst, covalent organic cage

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Introduction

Enzymes are nature’s most efficient catalysts, responsible for orchestrating a vast range of biochemical reactions with exceptional specificity, speed, and selectivity under mild physiological conditions. , However, despite their remarkable capabilities, natural enzymes and their derivatives are often limited by poor stability outside of their native environments and narrow substrate scope, restricting their application in industrial processes. , To address these limitations, considerable research has focused on creating artificial enzymes, or enzyme mimetics, designed to replicate the catalytic precision of natural enzymes while providing enhanced stability and flexibility. , Chemzymes, for example, are small–molecular–weight models that mimic natural enzyme active sites, offering promise in diverse chemical transformations. − Additionally, artificial enzymes based on supramolecular scaffolds have made notable progress by leveraging host–guest chemistry, dynamic covalent interactions, and molecular recognition to improve reaction specificity. − However, despite these advances, their catalytic efficiency and selectivity still fall short of their natural counterparts, requiring ongoing research to bridge the gap.

Nanozymes, as a rising category of artificial enzymes, have gained significant attention due to their tunable catalytic activity, robustness, and broad functional versatility. − Compared to natural enzymes, nanozymes demonstrate superior stability and customizable properties, making them ideal for applications in biosensing, environmental remediation, and therapeutics. , To further achieve superior catalytic performance, strategies such as integrating single active sites or photosensitizers into porous organic polymers, metal–organic frameworks (MOFs), , or covalent organic frameworks (COFs) , have been employed (Figure a–c). These integrations leverage the so–called “polymeric effect”, boosting both efficiency and selectivity of catalytic functions, thereby addressing some of the limitations inherent in natural enzyme systems. − Despite promising advancements, systems like nanozymes and polymeric frameworks face inherent limitations, including imprecise structural definition, primarily heterogeneous catalysis, and unclear catalytic mechanisms, which restrict their ability to match the precision and efficiency of natural enzymes. Therefore, the development of structurally and functionally precise enzyme mimics featuring well–defined nanoscale three–dimensional (supra)­molecular architectures, which we designate as “singzymes”, remains both essential and challenging.

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Typical strategies for achieving enzyme–like properties by integrating active sites into: (a) polymers; (b) MOFs; (c) COFs. (d) Superphane–based singzymes (reported in this work) and the top view of the spherical sulfur oxygenase reductase (note: the image of the enzyme has been redrawn using the PDB file downloaded from the Protein Data Bank (PDB code: 6M35).

Over the past decades, molecular cages, with finely tunable cavities and well–defined porous structures, have emerged as highly versatile platforms for sensing, separation, drug delivery and catalysis. − In contrast to conventional porous materials for enzyme mimics, molecular cages offer exceptional structural precision and adaptability, enabling the stabilization of transition states and the creation of chiral microenvironments, much like natural enzymes. − For example, metal–organic cages (MOCs) present distinct advantages, including high surface areas, multiple catalytic metal centers and the capacity for selective guest encapsulation; however, their stability under harsh conditions and challenges in scalable synthesis pose limitations. − Covalent organic cages (COCs), on the other hand, exhibit robust stability due to their covalent frameworks and feature customizable pore sizes and functional groups that allow for highly selective encapsulation and catalytic activity. − Despite significant advancements in enzyme mimics using molecular cages, their structural and functional complexity stays far from natural enzymes. Consequently, ongoing efforts are needed to develop novel cage systems with enhanced stability, structural precision, and catalytic activity to meet the stringent selectivity and reactivity demands of natural enzymes.

In this work, we report the design and synthesis of a photosensitized superphane 1a unique covalent organic cage architecture characterized by two parallel benzene rings linked by six bridging units (Figure d). This superphane consisting of six photosensitive phenothiazine moieties served as a singzyme and exhibited significantly enhanced light absorption, superior separation and transfer efficiency of photogenerated electrons, and markedly reduced recombination of photogenerated carriers when compared to phenothiazine–based control compounds, e.g. cage 2, macrocycle 3 and monomer 4 (Figure a). We attribute this exceptional photosensitivity to a “super cage effect” or “superphane effect”, an enhancement mechanism complementary to the established polymeric and macrocyclic effects. The three-dimensional nanoscale cavities of superphane 1, densely functionalized with phenothiazine units, impart enzyme-like behavior that enables highly efficient and selective photocatalytic oxidation of organic substrates. Under intermittent light exposure, superphane 1 mimics both NADH oxidase and glutathione (GSH) oxidase activities. This dual function facilitates the targeted oxidation of NADHa key metabolic cofactorand GSH, which is typically overexpressed in cancer cells, thereby generating reactive oxygen species (ROS) and inducing oxidative stress. These events collectively trigger cancer cell death via synergistic apoptotic and ferroptotic pathways, establishing superphane 1 as a multifunctional molecular platform with significant potential in photocatalytic therapy and redox-based anticancer strategies.

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(a) The structure of superphane 1 and control compounds cage 2, macrocycle 3, monomer 4; (b) The synthetic routes for superphane 1, cage 2 and macrocycle 3. (c) Single crystal structure of superphane 1. The C8 chains and residue solvent molecules were omitted for clarity.

Results and Discussion

Design, Synthesis and Characterization

In nature, many enzymes achieve precise molecular structures and catalytic function through near–enclosed or fully enclosed chambers, formed by the synergistic assembly of protein monomers or subunits. Inspired by these architectures, we aimed to precisely design nanoscale single–molecule enzyme–like structures by incorporating functional phenothiazine units into superphane frameworksmolecular constructs featuring two parallel benzene rings connected by six bridges. − This design, which we term “singzyme” resulted in superphane 1, where each bridge is functionalized with a phenothiazine moiety, with an added C8 carbon chain to enhance compound solubility (Figure a). For comparison, we synthesized a series of controls: a cage (2) with three phenothiazine–bearing linkages, a macrocycle (3) containing two phenothiazine units, and a monomer (4) with a single phenothiazine unit.

The synthesis began with the direct alkylation of phenothiazine using 1–bromooctane and NaH as a base for deprotonation, yielding intermediate 11 with a 95% yield. Subsequent bromination of 11 introduced two bromine atoms ortho to the nitrogen, resulting in 10 with a 98% yield (Figure b and Scheme S4). Next, a Suzuki–Miyaura cross–coupling reaction between 10 and a formyl–substituted boronic acid provided the key intermediate 5 in an 85% yield (Scheme S5). With intermediate 5 in hand, superphane 1 was synthesized with a 65% yield through a [6 + 2]–type imine condensation reaction with hexakis–amine (6) in a DCM/MeOH mixture (1/1, v/v), followed by NaBH4 reduction (Figure b and Schemes S6 and S7). Similarly, condensation of dialdehyde 5 with tri–amine (7), diamine (8), or benzylamine (9) in a DCM/MeOH mixture (1/1, v/v), followed by in–situ NaBH4 reduction, yielded targeted cage 2, macrocycle 3, and monomer 4, respectively (Schemes S8–S10). The structures of key intermediate 5, superphane 1, cage 2, macrocycle 3, and monomer 4 were thoroughly characterized by standard spectroscopic methods, including 1H NMR, 13C NMR spectroscopy, and high–resolution mass spectrometry (for details, see Supporting Information).

The structure of superphane 1 was further validated through X–ray crystallography. Single crystals suitable for analysis were obtained by slow diffusion of methanol into a toluene solution of superphane 1. As anticipated, the resulting crystal structure revealed a lantern–like architecture, with two benzene rings connected by six phenothiazine–bearing bridges (Figures c and S1). Remarkably, the structure resembles a spherical enzyme, featuring a core dimension of approximately 2.0 nm in height and 1.6 nm in diameter.

Optical and Electrochemical Properties

Considering the dense arrangement of photosensitive phenothiazine units surrounding the chamber of superphane 1, we next investigated its photochemical and electrochemical properties. As shown in Figure a, the UV–Vis diffuse reflectance spectrum of superphane 1 exhibits a absorption band between 300–500 nm. In contrast, as the number of phenothiazine units decreases from superphane 1 to monomer 4, a progressive blueshift in the absorption edge is observed (545 nm for superphane 1, 510 nm for cage 2, 495 nm for macrocycle 3, and 482 nm for monomer 1). This trend indicates that superphane 1 has the highest light–capturing capacity. Additionally, the solid–state colors transition from brown–yellow in superphane 1 to light yellow in monomer 4, with varying luminescent colors under 365 nm light (inset, Figure a). Interestingly, the UV–Vis spectra of these compounds in chloroform reveal similar absorption features with peaks around 280 and 340 nm, further underscoring their substantial visible–light absorption potential (Figure S2a).

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Optical and electrochemical properties of superphane 1, cage 2, macrocycle 3, and monomer 4. (a) UV–Vis diffuse reflectance spectra (inset: optical properties of the four compounds in question under sunlight and 365 nm irradiation); (b) Tauc plot for optical band gap estimation; (c) photoluminescence spectra (excitation at 380 nm, slit width: 5 nm/5 nm); (d) electrochemical impedance spectroscopy (EIS) Nyquist plot; (e) photocurrent measurements under visible light irradiation (450 nm LED, 6 W); (f) schematic representation of the optical band gap vs normal hydrogen electrode (NHE); (g) ESR spectra with DMPO as a spin–trap agent; (h) ESR spectra with TEMP as a spin–trap agent under visible light irradiation (450 nm, 6 W), ([DMPO] = [TEMP] = 50 mM, [1] = 1 mM, [2] = 2 mM, [3] = 3 mM, [4] = 6 mM); (i) cyclic voltammograms recorded in dry CHCl3 with 0.1 M Bu4NPF6 as the supporting electrolyte at a scan rate of 100 mV s–1.

Subsequently, the optical band gap (E g) of superphane 1, estimated from the Tauc plot, is approximately 2.45 eVsignificantly narrower than that of cage 2 (2.58 eV), macrocycle 3 (2.62 eV), and monomer 4 (2.69 eV) (Figure b), indicating enhanced electron delocalization. Photoluminescence (PL) spectra (Figure c) reveal that as the number of phenothiazine units increases from monomer 4 to superphane 1, there is a decrease in fluorescence intensity, along with a gradual redshift in PL emission from 480 to 515 nm and spectral broadening. Similar redshifts, intensity changes, and spectral broadening were also observed in chloroform (Figure S2b). The pronounced redshift and broadening in superphane 1 are likely due to increased through–space conjugation and enhanced electron interactions within its unique structure. Collectively, these observations indicate superphane 1’s propensity for photoinduced electron transfer via intersystem crossing (ISC), a process advantageous for generating reactive oxygen species (ROS) for biological applications.

To further assess the electrochemical properties of superphane 1, electrochemical impedance spectroscopy (EIS) and photocurrent testing were conducted. As shown in Figure d, the EIS spectra demonstrated that superphane 1 exhibited the lowest resistance compared to cage 2, macrocycle 3, and monomer 4, following the order superphane 1 < cage 2 < macrocycle 3 < monomer 4. This exceptionally low resistance observed for 1 indicates significantly reduced charge transfer resistance and enhanced charge separation efficiency, both crucial for effective carrier separation and migration in photocatalysis. Consistent with this, the instantaneous photocurrent responses showed that superphane 1 displayed the strongest photocurrent, with an intensity 32 times that of monomer 4, 10 times that of macrocycle 3, and 6 times that of cage 2 (Figure e). This marked difference, despite the structural similarity, in particular, between superphane 1 and cage 2, suggests a unique “super cage effect” or “superphane effect”. Notably, superphane 1 maintained a stable photocurrent response over six cycles of irradiation (450 nm, 6 W), indicating continuous and stable electron–hole generation under light exposure. These results establish superphane 1 as the most efficient photocatalyst among the compounds tested, attributed to its superior charge transfer efficiency and electron–hole separation facilitated by the “super cage effect”.

The n–type semiconductor properties of superphane 1, as well as cage 2, macrocycle 3, and monomer 4, were confirmed through Mott–Schottky curve analysis (Figures f and S3). Specifically, the conduction band (CB) positions of these compounds closely align with their flat–band potentials. After calibration against the normal hydrogen electrode (NHE), the conduction potentials were determined to be −0.47 V for superphane 1, −0.52 V for cage 2, −0.53 V for macrocycle 3, and −0.55 V for monomer 4. Using the formula E VB = E CB – E g, the estimated valence potentials were calculated as approximately 1.98 V for superphane 1, compared to 2.06 V for cage 2, 2.09 V for macrocycle 3, and 2.14 V for monomer 4. These findings indicate that all four compounds are capable of reducing O2 to form superoxide radicals (O2 •‑).

To further evaluate the reactive oxygen species (ROS) generation capabilities of these photosensitizers, electron spin resonance (ESR) spectroscopy was performed using DMPO (5–dimethyl–1–pyrrolidine–N–oxide) as a trapping agent for superoxide radicals (O2 •‑) and TEMP (2,2′,6′,6’–tetramethylpiperidine) for singlet oxygen (1O2) (Figure g,h). In chloroform solutions containing superphane 1 (1.0 mM), cage 2 (2.0 mM), macrocycle 3 (3.0 mM), or monomer 4 (6.0 mM), 5 min of illumination produced characteristic peaks for both DMPO–O2 •‑ and TEMPO, compared to the blank, confirming the generation of the respective ROS. Quantitatively, ROS generation rates followed the order: superphane 1 > cage 2 > macrocycle 3 > monomer 4, indicating that superphane 1 exhibits the highest generation rate of both O2 •‑ and 1O2 under light exposure facilitated by “super cage effect”.

The electrochemical properties of these four photosensitizers in dry chloroform solution were further examined via cyclic voltammetry (CV). As illustrated in Figure i, each photosensitizer displayed similar reversible oxidation waves, with the oxidation onset potential progressively shifting lower from monomer 4 to superphane 1 (0.8 V for monomer 4, 0.75 V for macrocycle 3, 0.65 V for cage 2, and 0.35 V for superphane 1). This decrease in oxidation potential indicates an increased ease of oxidation, particularly for superphane 1. Based on these oxidation onsets, the HOMO levels of superphane 1, cage 2, macrocycle 3, and monomer 4 were calculated to be −4.56, −4.86, −4.97, and −5.03 eV, respectively, with corresponding LUMO levels at – 2.11, −2.28, −2.35, and −2.34 eV (Table S1). These results indicate that the “super cage effect” significantly impacts both the HOMO and LUMO energy levels of the photosensitizers. In addition, we confirmed that the gap of the four compounds is the same trend by theoretical calculations (Figure S4).

Oxidative Coupling of Benzylamine and Thioether Oxidation Enhanced by “Super Cage Effect”

Encouraged by the photochemical properties of these photosensitizers, inter alia superphane 1, we next explored their catalytic efficiency in visible–light–driven oxidative transformations, viz. Benzylamine oxidative coupling and thioether oxidation (Figure a,b). Specifically, to systematically evaluate the role of the “super cage effect” or “superphane effect” in catalytic efficiency, we conducted comparative studies on the four molecular architecturessuperphane 1, cage 2, macrocycle 3, and monomer 4using benzylamine as a model substrate in n–hexane. After 12 h of irradiation at room temperature, only superphane 1 exhibited near–quantitative (>99%) benzylamine conversion, while the other three compounds showed significantly reduced activity (Figures c and S5). The turnover number (TON) of superphane 1 was remarkably enhanced, being 3.57–fold, 10.52–fold, and 33.33–fold higher than that of cage 2, macrocycle 3, and monomer 4, respectively, underscoring the significant influence of the “super cage effect” in optimizing photocatalytic efficiency. Furthermore, superphane 1 achieved this exceptional performance without any detectable by–product formation, demonstrating outstanding selectivity (>99%) toward the coupling product.

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Performance of four compounds in catalyzing organic oxidation reactions. (a) Photocatalytic oxidative coupling reactions of benzylamines by superphane 1; (b) photocatalytic selective oxidation of thioethers by superphane 1; (c) performance comparison of four compounds in the organic catalytic reactions of benzylamine oxidation coupling and thioether oxidation.

To elucidate the mechanistic aspects underlying this enhanced catalysis, we conducted a series of controlled inhibition experiments with various scavengers and sacrificial agents (Figures S6 and S7). The introduction of NaN3 (1O2 scavenger), p–benzoquinone (O2 •– scavenger), AgNO3 (electron scavenger), and KI (hole scavenger) all resulted in diminished catalytic yield. Notably, O2 •– was identified as the primary reactive oxygen species (ROS) driving the reaction, while 1O2 played a minor role. These results further corroborate the mechanistic framework in which photocatalyst, light source, and molecular oxygen are indispensable components of the reaction pathway. Expanding beyond the model substrate, we investigated the substrate scope of oxidative coupling using superphane 1 under optimized conditions (Figure a). A variety of benzylamine derivatives bearing electron–withdrawing (−F, −CF3, −OH) and electron–donating (−CH3, −OCH3) substituents at the para–position were examined. Superphane 1 exhibited broad substrate compatibility and consistently high catalytic activity across all derivatives. Notably, substrates with electron–donating groups displayed significantly shorter reaction times, highlighting the influence of electronic effects on reaction kinetics. The remarkable reusability of superphane 1 was further confirmed through cyclic performance evaluations. Even after five consecutive cycles, the catalytic activity remained unchanged, as evidenced by the stable product yield (Figure S8a). Moreover, 1H NMR analysis confirmed that the structural integrity of superphane 1 was fully retained post–reaction, reinforcing its durability as a molecular photocatalyst (Figure S8b).

More evidence for supporting the “super cage effect” came from photocatalytic oxidation of thioether. The photooxidation of organic sulfides is widely recognized as a model reaction for evaluating O2 activation and singlet oxygen (1O2) generation efficiency. , As illustrated in Figure b, under a 1 atm O2 atmosphere and irradiation with a 450 nm LED light source for 3 h, superphane 1 (0.1% mmol) efficiently catalyzed the oxidation of thioanisole (0.5 mmol) to sulfoxide, demonstrating its potential for pollutant degradation. In sharp contrast, the catalytic efficiency of cage 2, macrocycle 3, and monomer 4 followed the same decreasing trend observed in benzylamine oxidative coupling, with all three compounds significantly less effective than superphane 1 (Figure c). To further elucidate the key factors governing this reaction, we conducted a series of controlled experiments. Negligible product formation was observed under dark conditions, in the absence of a catalyst, or under an N2 atmosphere, confirming the essential roles of light, molecular oxygen, and photocatalyst activation (Figure S7). These studies also identified 1O2 as the primary reactive oxygen species (ROS) driving the reaction.

To explore the substrate scope of thioether oxidation, we introduced electron–withdrawing (−F) and electron–donating (−CH3, −OCH3) substituents at the para–position. Interestingly, in contrast to benzylamine oxidative coupling, where electron–donating groups accelerated the reaction, the oxidation of fluorinated thioethers proceeded more rapidly, with reaction times shortened to 3 h. In contrast, substrates bearing electron–donating groups required an extended reaction time of 6 h. Despite these kinetic differences, all tested thioethers achieved near–quantitative conversion and excellent selectivity (up to 99%). Based on our experimental observations and literature reports, − we propose a plausible reaction mechanism (Figure S9). Upon light irradiation, superphane 1 undergoes photoexcitation, generating electron–hole pairs. The excited electrons (e–) can engage in electron transfer to O2, forming superoxide radicals (O2 •–), or undergo energy transfer via intersystem crossing, producing 1O2. Concurrently, the highly oxidizing holes (h+) facilitate electron abstraction from substrates such as benzylamines and thioethers, generating radical intermediates. These intermediates subsequently react with ROS to yield imine and persulfoxide intermediates, which undergo further transformations to afford the final products. Collectively, these findings establish the “super cage effect” as a powerful tool for enhancing photocatalytic efficiency.

Singzyme: the Enzyme–Like Catalytic Properties

Building on its exceptional ability to generate O2 •– and 1O2 under 450 nm irradiation, superphane 1 also exhibits the formation of a highly oxidizing radical species, superphane 1* (Figure S10). This observation prompted us to explore its broader catalytic potential beyond conventional photocatalysis. NADH, a crucial coenzyme in biological redox processes, plays a pivotal role in regulating tumor–related signaling pathways and cellular antioxidant mechanisms. − In oxidase–catalyzed reactions, NADH provides electrons and protons, undergoing conversion to NAD+, while NAD+ can accept electrons and protons, being reduced back to NADH. Disruption in the NADH/NAD+ balance can lead to intracellular disturbances, ultimately resulting in cell death. Importantly, NADH oxidase (NOX), a key enzyme that catalyzes the oxidation of NADH to NAD+, holds significant relevance in the mitochondrial respiratory chain, underscoring its critical function in cellular redox homeostasis. ,

Motivated by the optical and electrochemical and photocatalytic properties of superphane 1 endowed by the “super cage effect”, we then investigated its potential to mimic NOX activity. A series of spectral experiments were conducted with superphane 1 and control compoundscage 2, macrocycle 3, and monomer 4. Notably, protonation with hydrochloric acid enhanced the water solubility of these hydrophobic photosensitizers, particularly superphane 1, without compromising their ROS (O2 •‑ and 1O2) generation efficiency (Figures S11 and S12). Much to our surprise, a singlet oxygen (1O2) yield of 70% was achieved for singzyme 1 under 450 nm irradiation, significantly surpassing the yields of 42% for cage 2, 39% for macrocycle 3, and 24% for monomer 4 (Figure S12), Which is at a relatively high level. In PBS buffer solution (10 mM, pH 7.4), NADH shows two absorption peaks at 260 and 340 nm. Under 450 nm irradiation at an intensity of 10 mW·cm–2, NADH’s absorption remained stable in the absence of a catalyst (Figure S13c). In contrast, the addition of superphane 1 led to a gradual decrease in the 340 nm peak over time, accompanied by a slight increase at 260 nm, indicating NADH oxidation. Remarkably, superphane 1 achieved a high turnover frequency (TOF) of 11.250 min–1 for NADH oxidation, significantly surpassing the TOF values of cage 2 (3.984 min–1), macrocycle 3 (2.574 min–1), and monomer 4 (0.715 min–1). The TOF value is seemingly 6.72 times higher than that of the widely explored transition metal–containing photocatalysts (1.673 min–1). These findings confirm superphane 1’s efficient NADH oxidation capability, demonstrating excellent NOX–like activity and justifying its selection for further experiments based on its superior catalytic performance.

During the course of our NOX mimic research, we observed that NADH oxidation could proceed solely under irradiation, while remaining virtually inactive in the absence of light (Figure a). To explore this further, we analyzed common products generated by natural NOX, such as H2O2 and NAD+. Upon adding alcohol dehydrogenase (ADH) and alcohol to the oxidized system, the UV absorption at 340 nm was restored to 90% within 10 min, confirming that the primary product of the reaction was enzymatically active NAD+ rather than (NAD)2 (Figure b). This transformation was directly validated by 1H NMR spectroscopy (Figure S14). During NADH oxidation, O2 can be reduced either to H2O via a 4–electron pathway or to H2O2 via a 2–electron pathway. Using a colorimetric method,H2O2 production was detected (Figure S16a). When horseradish peroxidase (HRP) and 3,3′,5,5′–tetramethylbenzidine (TMB) were added to the NADH and superphane 1 system, a characteristic TMBox absorption peak at 652 nm was observed, absent in the NADH–only control and weaker in the superphane–only control. This suggests that H2O2 is generated via electron transfer during NADH oxidation. Additional evidence of superphane 1’s notable oxidation properties was provided by kinetic experiments, where the Michaelis–Menten curve revealed a K m of 70.5 μM, indicating a strong affinity for NADH compare to nature NOX (192.9 μM, Figure S15), and a V max of 17.1 μM min–1, effectively balancing the typical trade–off commonly observed in NOX mimics (Figure c,d and Table S2). The catalytic constant (K cat) of superphane 1 and native NOX were 1.71 min–1 and 221.4 min–1, respectively. The catalytic efficiencies (K cat/K m) with values of 2.43 × 104 and 114.77 × 104 L mol–1 min–1, respectively. These data suggest that superphane 1 might replace natural enzyme, achieve high efficiency of enzyme catalytic reaction. Moreover, superphane 1 retained 99% catalytic activity for NADH oxidation over five cycles, demonstrating its exceptional stability and sustained catalytic performance (Figure S16b).

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In–vitro enzyme–like activity of superphane 1. (a) Photocontrollable NADH (100 μM) oxidation activity of superphane 1 (10 μM); (b) NADH (100 μM) consumption and recovery catalyzed by superphane 1 (10 μM) and ADH (10 μL, 1 mg/mL), respectively; (c) steady–state kinetic analysis and (d) Lineweaver–Burk plots of NADH oxidase–like activity of superphane 1. (NADH 25–250 μM, superphane 1: 10 μM, irradiation: 5 min, error bars are presented as ± S.D, n = 3); (e) Cyt c reductase–like activity of superphane 1 (NADH: 100 μM, Cyt c 10 μM; superphane 10 μM); (f) UV–Vis absorption spectra of different solutions after 30 min of reaction; (g) steady–state kinetic analysis and (h) Lineweaver–Burk plots of Cyt c reductase–like activity of superphane 1, (NADH: 100 μM, Cyt c 2.5–100 μM, superphane 1: 10 μM, irradiation: 5 min, error bars are presented as ± S.D, n = 3); (i) GSH (50 μM) oxidation activity of superphane 1 (5 μM) (DTNB 112 μM); (j) steady–state kinetic analysis of GSH oxidase–like activity of superphane 1 (GSH: 10–60 μM, superphane: 5 μM, irradiation: 5 min, DTNB: 112 μM); (k) oxidase–like activity of superphane 1 (TMB: 100–1200 μM, superphane 1: 10 μM incubated under light for 5 min). ; (l) steady–state kinetic analysis of the oxidase–like activity of superphane 1, (GSH: 100–1200 μM, superphane 1: 10 μM, irradiation: 5 min, error bars are presented as ± S.D, n = 3). The above spectral experiments were all tested in PBS buffers (10 mM, pH 7.4).

In the mitochondrial respiratory chain, NOX transfers electrons not only from NADH to O2 but also to Cyt c, exhibiting Cyt c reductase–like activity. As shown in Figure e, when the NADH and superphane 1 system was irradiated, Cyt c reduction occurred alongside NADH oxidation: the NADH absorption peak at 340 nm diminished, the Cyt c absorption at 550 nm increased, and the soret band red-shifted from 409 to 412 nm, indicating conversion of iron in Cyt c to its ferrous form. In contrast, superphane 1 alone caused only slight spectral changes, and NADH alone induced none, confirming that superphane 1 mediates electron transfer from NADH to Cyt c (Figure f). From the enzyme kinetics, a K m of 40.1 μM and V max of 3.5 μM min–1 were derived from the Michaelis–Menten curve (Figure g,h).

Glutathione (GSH) is essential in regulating cellular oxidative stress. With superphane 1’s potent ROS–generating capability under irradiation, it emerges as a promising candidate for down–regulating intracellular GSH, potentially rebalancing redox states to aid in cancer therapy. We thus evaluated the GSH oxidase–like activity of superphane 1. Using DTNB (dithio–nitrobenzene) as a chromogenic agent, a notable color change in the solution of superphane 1 and GSHfrom bright yellow to colorlesswas observed within 30 min of irradiation (Figure i). Furthermore, increasing superphane 1 concentrations significantly enhanced GSH depletion under 450 nm laser irradiation, demonstrating its ability to trigger marked GSH depletion and sensitize tumor cells to ROS–induced damage. The K m and V max values were determined to be 15.6 μM and 4.3 μM min–1, respectively, indicating strong oxidase–like activity, fully in line with the TMB colorimetric assays (Figure j). In addition, through two controlled experiments in Figure S16c,d, we demonstrated that the spectral changes were due to the GSH oxidase–like activity of superphane 1, excluding any other side reactions.

In an extensive TMB/TMBox reaction model study, we observed that the absorption intensity at 652 nm increased with rising TMB concentrations (Figure k). Enzyme kinetics experiments generated a Michaelis–Menten curve for superphane 1, with K m and V max values of 452.4 μM and 16 μM min–1, respectively (Figure l). Considering the slightly acidic nature of the tumor microenvironment, we further examined the pH influence on superphane 1’s enzyme–like activity. As illustrated in Figure S17, its enzyme–like functions, particularly NADH oxidase–like, GSH oxidase–like and oxidation activities, were enhanced under acidic conditions, while Cyt c reductase activity was optimized at neutral pH. These findings underscore superphane 1’s pH–dependent enzyme–like characteristics, making it particularly suited for the tumor microenvironment.

The In–Vitro Enzyme–Like Catalytic Properties

Building on superphane 1’s demonstrated enzyme–like activities, we further investigated its in–vitro catalytic potential, focusing on its role in modulating cellular oxidative stress pathways. To assess biocompatibility, we first evaluated the cytotoxicity of these four phenothiazine–based photosensitizers, viz. Superphane 1, cage 2, macrocycle 3, and monomer 4, across four cell lines, viz. Hela cells, MCF–7 cells, Hepa 1–6 cells and 3T3 cells (Figure S18). Following 12–hour incubation with concentrations ranging from 0 to 64 μg/mL, cell survival rates remained above 90%, confirming the favorable biocompatibility of these photosensitizers. Moving forward, superphane 1’s cellular uptake efficiency was then assessed by incubating four cell lines with superphane 1 for 12 h and visualizing its fluorescence signal by confocal laser scanning microscopy (CLSM). As a result shown in Figure S19, bright green fluorescence observed within the cytoplasm confirmed efficient cellular uptake and biocompatibility.

Next, we focused on the Hepa 1–6 cell line to evaluate superphane 1’s potential to induce apoptosis. Hepa 1–6 cells were treated with protonated superphane 1 at varying concentrations for 12 h, irradiated with a 450 nm LED (10 mW·cm–2) for 10 min, then incubated for an additional 12 h. Cell viability, assessed using the CCK–8 assay (Figures a and S20), demonstrated significant apoptosis induction in the irradiated group compared to the non–irradiated control. Notably, superphane 1 exhibited an IC50 of 4.49 ± 0.53 μg/mL, significantly lower than that of cage 2 (19.98 ± 1.38 μg/mL), macrocycle 3 (34.48 ± 4.02 μg/mL), and monomer 4 (53.53 ± 6.05 μg/mL), highlighting its enhanced apoptotic efficacy under irradiation. These results underscore superphane 1’s promising potential for oxidative stress modulation and cancer therapy.

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In vitro therapeutic effects of superphane 1. (a) Cell viability of Hepa 1–6 cells with and without laser irradiation following superphane 1 treatment (0–64 μg/mL), error bars are presented as ± S.D, n = 6; (b) intracellular levels of NADH, ATP, and GSH in Hepa 1–6 cells treated with 15 μg mL–1 superphane 1, error bars are presented as ± S.D, n = 3; (c) Fluorescent images of intracellular ROS in Hepa 1–6 cells post–treatment with superphane 1 (15 μg mL–1), (− indicates without laser irradiation, while + indicates with laser irradiation); scale bar = 10 μm; (d) changes in mitochondrial membrane potential (ψm) in Hepa 1–6 cells following treatment superphane 1, scale bar = 10 μm; (e) CLSM of Calcein–AM/PI–stained Hepa 1–6 cells under different treatment conditions, scale bar = 100 μm; (f) illustration of the cell death mechanism induced by superphane 1 and a schematic of the “domino effect” triggered by NADH depletion catalyzed by superphane 1. Green channel: λex = 488 nm, λem = 500–560 nm. Red channel: λex = 561 nm, λem = 580–640 nm.

As shown in Figure b, intracellular NADH levels in Hepa 1–6 cells decreased by 66% after co–incubation with superphane 1, demonstrating its efficient catalytic activity in depleting NADH within cells. This depletion blocks cellular OXPHOS, leading to a reduction in mitochondrial membrane potential activity and a 50% drop in ATP levels. Additionally, the generated ROS and the catalytic oxidation of NADH, accompanied by H2O2 production, promote the oxidation of the overexpressed GSH to GSSG, resulting in a 77% decrease in GSH levels. As a control, we measured the changes in the levels of three indicators in normal 3T3 cells. As shown in Figure S21, the levels of the three indicators showed a slight decrease after treated with superphane 1 and laser irradiation. The changes in these three key indicators confirm that superphane 1 effectively disrupts the redox homeostasis of cancer cells, ultimately inducing apoptosis and ferroptosis, while exhibiting negligible side effects on normal cells.

Following the promising results of apoptosis induction in Hepa1–6 cells, we further investigated superphane 1’s impact on oxidative stress pathways, particularly under hypoxic conditions characteristic of most solid tumors. Hypoxia is commonly linked to poor prognosis, drug resistance, and metastasis, all of which compromise photodynamic therapy (PDT) effectiveness. Initial spectroscopic analyses confirmed that superphane 1 generates substantial ROS (1O2, O2 •‑), including H2O2, via NADH oxidation. This ROS surge disrupts the cell’s redox homeostasis, triggering an “oxygen burst” that drives cellular apoptosis. Using DCFH–DA (2,7–dichlorofluorescein diacetate) and DHE (dihydroethidium) staining, we detected significantly elevated intracellular ROS in cells treated with superphane 1 under 450 nm irradiation, as indicated by intense fluorescence compared to the control (Figure c).

Furthermore, superphane 1 exhibits NADH oxidase (NOX)–like activity, oxidizing NADH and thereby disrupting electron transfer within the mitochondrial respiratory chain. This obstruction impairs mitochondrial oxidative phosphorylation (OXPHOS) and reduces ATP production, leading to mitochondrial dysfunction and promoting apoptosis. To assess the mitochondrial transmembrane potential (ΔΨm), we used 5,5′,6,6’–tetrachloro–1,1′,3,3′–tetraethylbenzimidazolylcarbocyanine iodide (JC–1) as a fluorescent probe. In this assay, green fluorescence from JC–1 monomers indicates low–potential, depolarized mitochondria, while red fluorescence from JC–1 aggregates reflects healthy, high–potential, polarized mitochondria. As shown in Figure d, control cells displayed red fluorescence, indicative of intact mitochondrial membrane potential. However, upon irradiation of superphane 1–treated cells with a 450 nm laser, intense green fluorescence emerged, signaling a decrease in ΔΨm. With increasing superphane 1 concentration, the green–to–red fluorescence ratio further increased, indicating a concentration–dependent mitochondrial depolarization. These findings suggest that superphane 1–mediated NADH consumption induces a “domino effect” that leads to mitochondrial damage and apoptosis.

Finally, we evaluated the extracorporeal PDT effect of superphane 1 using Calcein/PI staining for cell viability (Figure e). In the absence of light, nearly all areas observed under CLSM displayed green fluorescence, indicating that cells remained viable with negligible toxicity from superphane 1. However, upon laser irradiation, regions with red fluorescence and blank areas expanded as the concentration of superphane 1 increased, signaling increased cell death. Notably, at a superphane 1 concentration of 15 μg/mL, almost all cells were non–viable, confirming the potent PDT efficacy of superphane 1 under light exposure.

The overall mechanism for multienzyme mimetic activities is depicted in Figure f, illustrating ATP production via oxidative phosphorylation (OXPHOS) within the inner mitochondrial membrane and the critical impact of redox homeostasis disruption. Given the promising photodynamic therapy (PDT) results, we introduced superphane 1 as an NADH oxidase (NOX) mimic, designed to compete with endogenous NOX enzymes for NADH oxidation. This competition led to a cascade of redox imbalance, effectively lowering intracellular NADH levels and impairing electron transport chain (ETC) function, thereby suppressing ATP synthesis and mitochondrial respiration. The resulting mitochondrial dysfunction inhibited OXPHOS, inducing energy depletion in tumor cells. Beyond its NOX-mimetic activity, superphane 1 also exhibited GSH oxidase–like properties, accelerating the oxidation of intracellular GSH and further exacerbating oxidative stress. This depletion of GSHone of the key cellular antioxidantscompromised the cell’s ability to neutralize reactive oxygen species (ROS), resulting in an intracellular oxidative burst. The sharp increase in ROS levels triggered extensive lipid peroxidation, a hallmark of ferroptosis, while also activating caspase-mediated apoptotic pathways. The convergence of these two distinct cell death mechanismsferroptosis and apoptosiscollectively enhances the cytotoxic impact of superphane 1 in tumor environments. These findings highlight superphane 1 as a potent multienzyme mimic capable of precisely modulating redox homeostasis and disrupting metabolic pathways. By leveraging its dual functionality as an NOX and GSH oxidase mimic, superphane 1 introduces a synergistic therapeutic strategy that selectively targets tumor cells through oxidative stress amplification, ATP depletion, and programmed cell death activation. This mechanistic versatility underscores its potential as a next-generation supramolecular therapeutic agent for cancer treatment.

Conclusion

In conclusion, this study introduces superphane 1 as a pioneering photosensitizer engineered through the “super–cage effect” strategy, which enhances photosensitivity, photocatalytic oxidation and confers multifaceted enzyme–mimetic properties for cancer therapy. Optimized at the molecular level, superphane 1 demonstrates significantly improved light absorption, superior photogenerated electron separation and transfer, and reduced carrier recombination under low–energy light (450 nm, 10 mW cm–2), resulting in high ROS yield and photocatalytic oxidation. The distinctive single-molecule architecture functions as a singzyme, exhibiting NADH oxidase- and GSH oxidase-like activities. This enables selective depletion of NADH and GSH, disrupting cellular redox homeostasis and inducing mitochondrial dysfunction. This “domino effect” initiates both apoptosis and ferroptosis pathways, with ROS accumulation, oxygen burst, and GSH depletion further downregulating GPX4 and accelerating tumor cell death. Importantly, singzyme 1’s pH–dependent, stable catalytic activity in acidic and neutral environments, characteristic of the tumor microenvironment, underscores its potential for precise and potent oncotherapy with minimal impact on healthy cells. These findings represent a significant breakthrough in supramolecular photodynamic therapy and enzyme–mimicking catalysis, establishing superphanes as versatile “singzymes” with exceptional capabilities in bioinspired catalysis, cancer treatment, and beyond. Furthermore, singzymes demonstrate broad potential in redox modulation and biochemical research, highlighting their transformative impact on advancing fundamental science and driving therapeutic innovation.

Supplementary Material

au4c01161_si_001.pdf (5.3MB, pdf)

Acknowledgments

This research was funded by the National Natural Science Foundation of China (22371068 to Q.H.), and Fundamental Research Funds for the Central Universities (Startup Funds to Q.H.). We also acknowledge the technical support of standard spectroscopic analysis (e.g., nuclear magnetic resonance and high–resolution mass spectrometry) at Analytical Instrumentation Center of Hunan University.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacsau.4c01161.

  • This material is available free of charge via the internet at experimental procedures, NMR spectroscopic studies, biological studies, HRMS, and X–ray structural data for superphane 1. X–ray crystallographic data for superphane 1 (CIF) (PDF)

The authors declare no competing financial interest.

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