Development of Biological-Event Manipulators Triggered by Light-Activated Compounds

Abstract

Photoresponsive molecular tools have become powerful platforms for manipulating biological functions with high spatiotemporal precision. In this review, we highlight recent advances in the development of light-activated compounds that interact with key signaling molecules and microenvironments. Inspired by various chemical reactions triggered by light-matter interactions, this review covers three representative systems: photoactivatable peroxynitrite (ONOO⁻) generators, visible-light-driven nitric oxide (NO) releasers, and optochemical oxygen (O₂) scavengers. ONOO⁻, a reactive nitrogen species formed from NO and superoxide (O₂⁻), plays a critical role in protein nitration and cellular oxidative stress. By designing molecules that generate both NO and O₂⁻ upon light exposure, efficient ONOO⁻ release was achieved and used to induce nitration reactions. For NO manipulation, the authors developed a class of photoresponsive releasers that utilize photoinduced electron transfer (PeT) to enable blue-to-red light-triggered NO release. These photoresponsive releasers allowed optical control of vasodilation both ex vivo and in vivo, which forms the basis of a minimally invasive approach to modulate blood flow. In addition, a light-responsive O₂ scavenger was developed to induce localized hypoxia in cell cultures. The light-responsive O₂ scavenger enabled optical regulation of the hypoxia-responsive pathway and activation of the transient receptor potential ankyrin 1 (TRPA1) calcium channel, which underscores the utility of this approach. Together, these studies illustrate how rational molecular design, combined with precise photochemical control, can create innovative systems for probing and directing biological events. These technologies are valuable as both a basic research tool and for potential future therapeutic applications.

1. Introduction

Photomanipulation technologies in life science involve using light-responsive molecules to modify biological systems. This approach is not only capable of controlling specific biological events with high spatiotemporal resolution but is also attracting attention as a new therapeutic approach with minimal side effects. Examples include optogenetics using rhodopsin and photoimmunotherapy using photoresponsive phthalocyanine.^1,2) In addition, caged compounds, which can release bioactive molecules in response to light, represent a versatile means of controlling the activity of specific bioactive molecules.³⁾ Although various photoresponsive molecules have been developed, it is still challenging to control highly reactive or poorly controllable molecular species, such as gaseous transmitters or reactive oxygen species. The present author has developed chemical tools to optically manipulate bioactive species that are difficult to control but essential for maintaining homeostasis. This review summarizes our recent efforts to develop photocontrollable compounds that donate reactive nitrogen species and optochemical oxygen (O₂) scavengers. We also discuss biological events that can be optically controlled using these photoresponsive compounds.

2. Development of Peroxynitrite Generators

Peroxynitrite (ONOO⁻) is a reactive nitrogen species that displays potent oxidizing and nitrating abilities, which is formed by the reaction between nitric oxide (NO) and superoxide radical anion (O₂⁻) under a diffusion control process (k = 6.7 × 10⁹ M⁻¹ s⁻¹).^4,5) While ONOO⁻ is associated with the pathogenesis of various diseases, the nitrated molecules, such as 8-nitro-cGMP, are associated with essential cell signaling pathways.^6–8) As such, compounds that are able to control the release of ONOO⁻ in a spatiotemporal manner can act as powerful tools to reveal the biological significance of ONOO⁻ or nitrated molecules. The most widely used ONOO⁻ generator is 3-(4-morpholinyl)sydnonimine hydrochloride (SIN-1), which spontaneously produces equimolar amounts of O₂⁻ and NO under physiological conditions, leading to the formation of ONOO⁻.⁹⁾

Inspired by the mechanism of SIN-1 (Fig. 1a), we designed a photocontrollable ONOO⁻ generator, DiP-DNB (1, Fig. 1b), which releases NO and O₂⁻ stoichiometrically. We have previously reported photoinduced NO release from 2,6-dimethylnitrobenzene derivatives exposed to UV irradiation.^10–14) In this reaction, we believe a phenoxyl radical is formed upon release of NO. Based on this insight, DiP-DNB (1) was expected to convert to a semiquinone-like form with subsequent release of NO, which in turn would generate O₂⁻ from O₂ by one-electron reduction.¹⁵⁾ As such, this compound was expected to act as a photocontrollable ONOO⁻ generator.¹⁶⁾ However, although DiP-DNB (1) underwent the anticipated light-induced degradation, ONOO⁻ was involved in a nucleophilic attack on the degradation product stilbenequinone (2), which diminished the levels of ONOO⁻.

Fig. 1. Reaction Mechanisms of ONOO⁻ Generators

(a) Spontaneous ONOO⁻ generation mechanism of SIN-1; (b) Plausible photoinduced ONOO⁻ release mechanism from DiP-DNB (1).

In order to develop a more efficient photocontrollable ONOO⁻ generator, we focused on N-nitrosoaniline derivatives that efficiently release NO.¹⁷⁾ N-Nitrosoaniline derivatives can release NO by N–N bond homolysis in response to UV light irradiation to form an aminyl radical product. Based on this mechanism, we designed a novel photocontrollable ONOO⁻ generator, P-NAP¹⁸⁾ (3, Fig. 2a). P-NAP (3) can form semiquinoneimine after photoinduced NO release, which can react with O₂ to form O₂⁻. To block the undesired reaction of ONOO⁻ with the quinone decomposition product and to accelerate the one-electron reduction of O₂ by semiquinoneimine, four methyl groups were introduced on the structure of P-NAP (3). For confirmation of photoinduced ONOO⁻ generation from P-NAP (3), we employed HKGreen-3, a ONOO⁻-specific fluorogenic probe¹⁹⁾ (Fig. 2b). While the fluorescence was strongly increased upon photoirradiation (330–380 nm) in the presence of P-NAP (3), it was suppressed under an argon atmosphere or under aerobic conditions in the presence of superoxide dismutase, suggesting O₂⁻ generation from O₂ was required for ONOO⁻ formation. Furthermore, tyrosine nitration, a specific reaction of ONOO⁻, was also induced by P-NAP (3) with UV irradiation, which did not occur with DiP-DNB (1, Fig. 2c). Using the fluorogenic probe HK-Green3A to detect intracellular ONOO⁻, it was confirmed that ONOO⁻ generation could be controlled by UV irradiation in a cell culture system (Fig. 2d).

Fig. 2. Photochemical ONOO⁻ Generation and Detection from P-NAP (*3*)

(a) A plausible mechanism of ONOO⁻ generation from P-NAP (3). (b) Detection of ONOO⁻ generation from P-NAP (3) using a ONOO⁻ fluorescence probe, HK-Green3. (c) Detection of tyrosine nitration induced by photodecomposition of P-NAP (3). A chromatogram of ethyl N-acetyl-3-nitrotyrosinate (Ac-nitroTyr-Et, upper) and that of a solution containing ethyl N-acetyltyrosine (Ac-Tyr-Et) and P-NAP (3) after irradiation. (d) Fluorescence images of ONOO⁻ generation from P-NAP (3) in HCT-116 cells in the presence of a cell-applicable ONOO⁻ fluorescence probe. Adapted with permission from Ieda et al. “Photocontrollable Peroxynitrite Generator Based on N-Methyl-N-nitrosoaminophenol for Cellular Application” J. Am. Chem. Soc. 134, 2563–2568 (2012). Copyright 2012 American Chemical Society.

In summary, we have developed a photocontrollable ONOO⁻ generator, P-NAP (3), which does not suffer from the disadvantages associated with DiP-DNB (1). P-NAP (3) will be a useful tool for controllable ONOO⁻ generation in biological research.

3. Development of Photocontrollable Nitric Oxide Releasers Based on Photoinduced Electron Transfer

NO, an endogenously synthesized signaling molecule derived from L-arginine, plays important roles in vasodilation, neurotransmission, and immune modulation.^20–23) NO is a reactive gaseous species that readily interacts with biomolecules under physiological conditions. As such, NO-releasing compounds (NO releasers) are utilized for NO research as well as for the treatment of hypertension and angina pectoris.²⁴⁾ Among them, photocontrollable NO releasers (caged NOs) enable precise spatiotemporal control of NO release, making them valuable tools for cardiovascular research and potential therapeutic applications.^17,25–28) Conventional caged NOs require UV irradiation in the range of 300–350 nm or are composed of transition-metal-nitrosyl complexes, which can readily damage cells and tissues. Thus, non-metal caged NOs that are controllable by longer wavelength light are desirable for wider biological applications. However, it is difficult to deliver sufficient energy to cleave chemical bonds with longer wavelength light, such as visible light. To address this dilemma, we focused on the reaction mechanism of BNN-3 (4, Fig. 3a), a caged NO reported by Namiki et al.¹⁷⁾ BNN-3 (4) releases two equivalents of NO in response to UV irradiation. It has been suggested that while the first NO release is triggered using UV light energy to break the N–N bond, the second NO release from the aminyl radical takes place spontaneously as a result of the formation of a stable quinone. We hypothesized that a visible-light-controllable NO releaser could be developed if such an oxidation state is accessible via visible light irradiation. To achieve this redox state, we chose to employ a photoinduced electron transfer (PeT) system.²⁹⁾ It has been reported that certain dyes bearing electron-rich or electron-poor substituents can undergo photoinduced single-electron transfer between the dye and the substituent. This phenomenon, when integrated with the reaction mechanism of BNN-3 (4), was considered to offer a conceptual basis for the development of a new molecular design strategy for caged NOs, as depicted in Fig. 3b. That is, upon visible light irradiation, the light-harvesting dye (antenna moiety) becomes photoexcited, promoting single-electron transfer from N-nitrosoaminophenol to the excited dye. Oxidation of N-nitrosoaminophenol facilitates NO release, accompanied by the formation of a stable quinone structure. Thus, visible light serves as a trigger for NO liberation.³⁰⁾

Fig. 3. Photoinduced NO Release Mechanisms and Design Concept

(a) Previously proposed mechanism of NO release from BNN-3 (4). (b) Concept for visible-light-controllable NO releasers based on a photoinduced electron transfer mechanism.

As a prototype, we designed and synthesized a blue-light-controllable compound, NOBL-1 (5, Fig. 4a), which comprises BODIPY as an antenna moiety tethered to N-nitrosoaminophenol. Given the poor water-solubility of BODIPY dyes, a carboxy group was introduced onto the NO-releasing moiety to enhance its hydrophilicity. Blue-light-responsive NO release was confirmed using the ESR spin-trapping method. Specifically, the Fe²⁺-N-methyl-D-glucamine dithiocarbamate complex (Fe-MGD), which can react with NO to form an NO-Fe-MGD complex, shows three typical signals in the ESR spectrum³¹⁾ (Fig. 4b). Furthermore, NO release was also confirmed by a fluorescence method using DAR-4M, which is a NO-responsive fluorescence probe. The results of these investigations verified that NO release was temporally controllable³²⁾ (Fig. 4c). Even in cellular experiments, the NO release was controlled by light irradiation. Using a bleaching function of the confocal microscope in which the excitation beam is injected into a galvanometric scanning system, spatial control of NO release can be achieved at a resolution level of within a few cells. It is well known that NO induces potent transient vasodilation activity resulting in activation of soluble guanylyl cyclase (sGC).²¹⁾ As shown in Fig. 4e, vasodilation of a rat aorta strip was successfully controlled through the combined use of NOBL-1 (5) and a blue-light source.

Fig. 4. Photochemical NO Release from NOBL-1 (*5*) and Its Application in Photovasodilation

(a) Structure of a blue-light-controllable NO releaser, NOBL-1 (5). (b) ESR spectrum of a solution containing NOBL-1 (5), and Fe-MGD in phosphate buffer (1% DMF) after photoirradiation with blue light (470–500 nm). (c) Fluorescence measurement for detection of NO generation from NOBL-1 (5) in phosphate buffer using DAR-4M. The fluorescence intensity was determined at 575 nm with excitation at 560 nm. Irradiation light was not attenuated or was attenuated to 50, 20, or 0%. (d) Fluorescence imaging of NO release from NOBL-1 (5) in HEK293 cells in the presence of DAR-4M AM. The cells were irradiated inside the indicated circle using a confocal microscope equipped with an argon laser. (e) Changes in tension of rat aorta ex vivo induced by blue-light-mediated NO release from NOBL-1 (5). L-NAME: L-nitroarginine methyl ester, NA: noradrenaline. Adapted with permission from Ieda et al. “Photomanipulation of Vasodilation with a Blue-Light-Controllable Nitric Oxide Releaser” J. Am. Chem. Soc. 136, 2563–2568 (2014). Copyright 2014 American Chemical Society.

In general, longer-wavelength visible light offers improved tissue penetration and reduced phototoxicity, thereby enhancing biocompatibility for biological applications. To further improve the biological utility of the PeT-type caged NOs, we designed and synthesized NO-Rosa1 (6, Fig. 5), in which the BODIPY antenna of NOBL-1 (5) was replaced with a rosamine chromophore that absorbs green light.³³⁾ It was demonstrated that NO-Rosa1 (6) can release NO in response to irradiation by green light, confirming that the excitation wavelength of PeT-type caged NOs can be effectively red-shifted depending on the photochemical characteristics of the antenna moiety. To simplify the synthetic strategy, the carboxy group was removed and both the structure of the antenna moiety and its substituent position relative to the NO-releasing moiety were changed.^34,35) For NO-Rosa5 (8), the distance between the antenna and the NO-releasing moiety is shorter than for NO-Rosa3 (7). Photochemical evaluation of these compounds revealed that NO-Rosa5 (8) exhibited high NO release efficiency. These results suggest that the efficiency of PeT is modulated by the spatial proximity between the antenna and the NO-releasing moiety. This factor directly influences the NO release efficiency. Guided by the structure–activity relationship findings, we developed a red-light-responsive PeT-type caged NO, NORD-1 (9).³⁶⁾ Various substituents were introduced onto the linker moiety anchoring the NO-releasing moiety and the antenna moiety. While some substituents had minimal impact on NO release efficiency, amino groups were found to reduce NO release^37–39) (8–15, Fig. 5). Noteworthy, alkoxy substituents did not adversely affect either NO release efficiency or vasorelaxation activity. These results suggest that derivatization via alkoxy-linked modifications at the linker site is a promising strategy for introducing functional groups, such as organ-targeting moieties, thereby facilitating future in vivo applications.

Fig. 5. Structures of PeT-Type Caged NOs Possessing Various Antenna Moieties (6–8) and Quantum Yields of NO Release for 9–16

Adapted from Ieda et al. “Structure–Activity Relationship of the Linker Moiety in Photoinduced Electron Transfer-Driven Nitric Oxide Releasers” Chem. Pharm. Bull. 73, 530–539 (2025), under CC BY-NC 4.0.

NORD-1 (9) enabled precise spatiotemporal control of NO release upon irradiation with red light. Exploiting the smooth muscle relaxation effect of NO, we demonstrated that intracavernous pressure in rats could be photochemically modulated in vivo (Fig. 6). This result represents the first example of blood flow regulation achieved by a small molecule in combination with optical control. Building on this, NORD-1 (9) holds significant promise as a novel therapeutic strategy for erectile dysfunction (ED).^40,41) In rat models, the combination of NORD-1 (9) and red light significantly improved erectile function in both neurogenic ED induced by cavernous nerve injury and diabetic ED induced by streptozotocin. Notably, this treatment enhanced intracavernosal pressure and promoted smooth muscle relaxation without affecting systemic blood pressure.

Fig. 6. In Vivo Photocontrol of Intracavernous Pressure Using NORD-1 (*9*)

(a) Experimental setup for photocontrolling intracavernous pressure (ICP) using the combination of NORD-1 (9) and a red light source. (b) Time-courses of blood pressure (BP) and ICP after injection of NORD-1 in heparized saline. (c) Statistical analysis of ICP changes induced by NORD-1 (9) and electrical stimulation. Mean arterial pressure (MAP) refers to the average value of BP. Data are expressed as mean ± standard deviation (S.D.) (shown as error bars, n = 4). Statistical significance was examined by application of a Bonferroni-type multiple t-test. * p < 0.01; ns: not significant. Adapted from Hotta et al. “The Effects of a Red-Light Controllable Nitric Oxide Donor, NORD-1 (9), on Erectile Dysfunction in Rats with Streptozotocin Induced Diabetes Mellitus” World J Mens Health. 43, 197–204 (2025), under CC BY-NC 4.0, and adapted with permission from Ieda et al. “Development of a Red-Light-Controllable Nitric Oxide Releaser to Control Smooth Muscle Relaxation in Vivo” ACS Chem. Biol. 15, 2958–2965 (2020). Copyright 2020 American Chemical Society.

Based on the PeT-triggered N–N bond cleavage mechanism, we have successfully developed a novel class of visible-light-responsive NO releasers. These compounds offer precise spatiotemporal control over NO release. This approach would enable high-resolution investigation of NO-mediated physiological processes at the cellular level. Among PeT-type NO releasers, NORD-1 (9), which is responsive to tissue-penetrant red light, showed particular promise as a therapeutic agent capable of inducing localized vasodilation without causing systemic hypotension in vivo. These technologies are expected to serve both as powerful research tools for elucidating fundamental aspects of NO biology and as innovative therapeutic strategies for vascular dysfunction and blood flow-related diseases.

4. Development of an Optochemical O₂ Scavenger Enabling Spatiotemporally Regulated Hypoxia

O₂ is an essential molecule for life, serving as the foundation for respiration and energy metabolism that regulates diverse cellular processes such as survival, differentiation, and migration.^42–44) Consequently, living organisms possess the ability to sense and adapt to hypoxia, a potentially lethal condition.⁴⁵⁾ This adaptive capacity plays roles not only in cancer growth and metastasis but also in organ development and regeneration, making hypoxia research a field of great interest to a wide range of scientists.⁴⁶⁾ However, because O₂ is a gaseous molecule that easily permeates and diffuses through cell membranes, precise control of its concentration is challenging. In particular, the effects of controlled O₂ levels, gradients, and dynamic distribution changes on cells remain poorly understood. To address this issue, we reasoned that an optochemical O₂ scavenging system capable of inducing hypoxia in a spatiotemporal manner could serve as a powerful tool for hypoxia research.

To develop an optochemical O₂ scavenging system, we turned our attention to rhodamine derivatives in which the oxygen atom at the 10-position is substituted with a heavier chalcogen, such as selenium or tellurium (referred to as SeR and TeR, respectively, Fig. 7a).^47,48) Previous studies by the Detty and McCormick groups demonstrated that TeR possesses self-sensitizing properties. Specifically, TeR can act as a photosensitizer to produce singlet oxygen (¹O₂), which subsequently oxidizes the tellurium center to yield a tellurium oxide-containing rhodamine derivative (TeR-O, Fig. 7a). Notably, TeR-O is capable of oxidizing thiols into disulfides while simultaneously regenerating TeR. Based on these findings, we hypothesized that the TeR-catalyzed photooxidation of thiols could serve as a promising optochemical strategy for scavenging O₂. Rhodamine derivatives incorporating selenium (SeR) show similar reactivity, but exhibit reduced self-sensitization efficiency.⁴⁹⁾ This O₂ consumption reaction is believed to proceed via the generation of ¹O₂, raising concerns about potential cellular damage when applied in biological systems. To minimize the impact of ¹O₂ on cells while depleting O₂ from the extracellular environment, we designed and synthesized SeR-BCM (17) and TeR-BCM (18), derivatives of SeR and TeR bearing two carboxylic acid groups to reduce their cell membrane permeability (Fig. 7b).

Fig. 7. Design and Mechanism of Optochemical Oxygen Scavengers

(a) A plausible mechanism of O₂ consumption by rhodamine derivatives containing a selenium or tellurium atom. (b) Structures of SeR-BCM (17) and TeR-BCM (18).

Next, we evaluated the O₂-scavenging activity of SeR-BCM (17) and TeR-BCM (18) upon light irradiation (590 nm LED) in the presence of glutathione (GSH), which displays low levels of toxicity and is abundant in living cells, as a co-reductant using an O₂ electrode (Fig. 8a). SeR-BCM (17) exhibited higher O₂ consumption activity than TeR-BCM (18) while no O₂ consumption was observed in the absence of GSH. To investigate whether hypoxia could be optically controlled in living cultured cells, we prepared cells treated with a hypoxia-sensitive fluorescent probe (MAR), which emits green fluorescence under low-O₂ conditions⁵⁰⁾ (Fig. 8b). Upon addition of SeR-BCM (17) and GSH to these cells, followed by localized light irradiation, an increase in green fluorescence was observed only in the irradiated regions. This result suggests that hypoxic microenvironments can be generated in highly localized areas spanning only tens of micrometers. No fluorescence enhancement was detected when either SeR-BCM (17) or GSH was omitted. Furthermore, to examine whether the activity of hypoxia-responsive proteins could be optically regulated, we used HEK293T cells expressing transient receptor potential ankyrin 1 (TRPA1), a protein that allows calcium influx in response to hypoxia^51,52) (Fig. 8c). These cells were loaded with Fluo-4 AM, a calcium-sensitive fluorescent dye, and then treated with SeR-BCM (17) and GSH followed by light irradiation.⁵³⁾ A fluorescence increase indicating calcium influx was observed only in irradiated areas. This fluorescence response was abolished when the TRPA1 inhibitor AP-18 was added, or when SeR-BCM (17), GSH, or TRPA1 expression was absent. These findings demonstrate that SeR-BCM (17) enables optical control of the activity of hypoxia-responsive proteins.

Fig. 8. Optochemical Oxygen Scavenging and Its Application to Spatiotemporal Hypoxia Control

(a) O₂ consumption by SeR-BCM (17) or TeR-BCM (18) and GSH in sodium phosphate buffer at 37 °C with a 590 nm LED light source. (b) Imaging spatiotemporally controlled hypoxia with MAR in cellular conditions. The region inside the circle was irradiated with a 561 nm laser fitted on a confocal microscope. The increase in green fluorescence was monitored. The mean fluorescence change of cells inside the circle (F/F₀) is shown on the right hand side. Bars represent S.E. (n = 3). (c) Calcium ion imaging using Fluo4-AM in HEK293T cells expressing TRPA1. TRPA1-HEK293T cells in buffer containing SeR-BCM (17) and GSH were irradiated with a 561 nm LED laser fitted on a confocal microscope. The mean fluorescence change of cells inside the circle (F/F₀) is shown in the center graph. Bars represent S.E. (n = 3). The final values of F/F₀ are shown as the bar graph on the right hand side. The mean values were compared by Bonferroni multiple comparison (n = 3, ** p < 0.005). Adapted with permission from Ieda et al. “An Optochemical Oxygen Scavenger Enabling Spatiotemporal Control of Hypoxia” Angew. Chem. Int. Ed. 62, e202217585 (2023). Copyright 2023 Wiley-VCH.

In summary, SeR-BCM (17) facilitates precise spatiotemporal induction of hypoxia in living cells, offering a powerful platform for dissecting hypoxia-related cellular mechanisms. This optochemical system allows dynamic control of O₂ levels, which may be extended to in vivo applications and adapted for studying diverse O₂-sensitive biological events, ultimately contributing to fields such as cancer biology, developmental biology, and regenerative medicine.

5. Conclusion

The integration of molecular design with light-based activation has emerged as a powerful strategy for the precise regulation of biological events. As demonstrated across the studies discussed in this review, a diverse range of photochemical tools has been established to manipulate reactive nitrogen species and hypoxia in a spatiotemporally controlled fashion. These advances have led not only to fundamental insights into biological signaling but also to promising therapeutic applications. Despite these achievements, limitations such as inadequate light penetration, and the difficulty of concentrating these compounds in the target organs currently hinder the broad application of this technology. Addressing these challenges will require further optimization of photophysical properties, development of new delivery platforms, and the use of multiplexed control systems. These technologies are expected to empower researchers not only to observe but also to actively control dynamic biological processes in real time, with minimal invasiveness and maximum precision.

Acknowledgments

I express my sincere gratitude to Prof. Hidehiko Nakagawa at Nagoya City University for direction, continuous support, and encouragement. I sincerely thank my co-workers whose names appear in the reference section of this review. These works were partly supported by Global Facility Center (GFC), Hokkaido University. The work described in this review was supported by Grants from JSPS KAKENHI (Grant Numbers: 18K14873, 20K05752), as well as by the JST ACT-X Grant Number JPMJAX2011, the Tokyo Biochemical Research Foundation, Takeda Science Foundation, and Casio Science Promotion Foundation.

Conflict of Interest

The author declares no conflict of interest.

Notes

This review of the author’s work was written by the author upon receiving the 2025 Pharmaceutical Society of Japan Award for Young Scientists.

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