Rational structural design of aromatic Azo photoactive small molecules for biomedical applications

Abstract

Aromatic Azo molecules, including azobenzenes (Ph–N=N–Ph) and heteroaryl Azo (Het–N=N–Ph or Het–N=N–Het), have emerged as versatile and high-performing photoactive switches for biomedical applications. Despite decades of extensive research on aromatic Azo as molecular photoswitches, the full translational potential of these small molecules remains underexploited. This review systematically outlines structural design strategies for aromatic Azo, spanning from functional substituent engineering to π-conjugation modulation, to fine-tune its photophysical properties. We summarize state-of-the-art synthetic methodologies for crafting multifunctional aromatic Azo frameworks, contrast the distinct isomerization mechanisms of azobenzenes versus heteroaryl Azo derivatives, and highlight the latest biomedical application advances, including biological imaging and detection, drug delivery, photopharmacology, phototherapy, miscellanea photo responsive biomaterials and constructs, and control in chemical biology. Furthermore, we discuss clinical translation challenges and opportunities in this field, proposing innovative strategies to address critical issues. This review aims to substantially advance the burgeoning field of aromatic Azo photoactive small molecules for biomedical applications.

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Junjie Ding

Junjie Ding received his BSc in materials science and engineering from Nanjing Tech University (NanjingTech) in 2024, and then pursued his Master's degree at the Institute of Flexible Electronics (IFE), Xiamen University (XMU). His research interests focus on the fluorescence of black hole molecules.

Ze Huang

Ze Huang received his PhD in organic chemistry from Anhui University under the supervision of Prof. Jiaxiang Yang in 2023. Now, he is a postdoctoral fellow in Prof. Lin Li's lab at the Institute of Flexible Electronics (IFE), Xiamen University (XMU). His research interest focuses on the mitochondria targeted fluorescent probes.

Bin Fang

Bin Fang obtained his MS in Chemistry (2019) from Anhui University (AHU) and his PhD in materials science (2024) from Northwestern Polytechnical University (NPU). He is currently pursuing postdoctoral research under the co-mentorship of Prof. Lin Li and Academician Prof. Wei Huang at the Institute of Flexible Electronics (IFE), Xiamen University (XMU). His research focuses on optical materials for mitochondrial diseases.

Lin Li

Lin Li received his BSc and PhD in Chemistry from Anhui University (AHU) in 2004 and 2009, respectively. After that, he obtained postdoctoral training as a research fellow at the National University of Singapore (NUS) in YAO Shao Q.'s lab. In 2014, he became a full professor in Nanjing Tech University (NanjingTech). He took up his current position as a professor at Xiamen University (XMU) in 2023. His research group mainly focuses on mitochondrial anti-aging.

Wei Huang

Wei Huang received his PhD from Peking University (1992). He began his postdoctoral research at the National University of Singapore (NUS, 1993); moved to Fudan University where he founded the Institute of Advanced Materials (IAM, 2002); was appointed as the Deputy President of Nanjing University of Posts and Telecommunications (NJUPT, 2006); and was elected as Academician of the Chinese Academy of Sciences (CAS) in 2011. He assumed his duty as the President of Nanjing Tech University (NanjingTech, 2012) and was appointed as the Deputy President & Provost of Northwestern Polytechnical University (NPU, 2017). His research interests include organic/flexible electronics, nanomaterials, and nanotechnology.

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1. Introduction

Azobenzene (AZB), the prototype of aromatic Azo molecules, was first synthesized by Mitscherlich¹ in 1834, and underwent pivotal evolution following the 1861 discovery of aniline yellow,^2,3 marking a transition from plant-derived dyes (Fig. 1). Over time, structural modifications to AZB have yielded a diverse range of colors, from Yellow 12 to Trypan blue, with adjustable light absorption properties enabling applications in industries such as textiles, leather treatment, and food additives. Domagk synthesized the prontosil in 1935,⁴ the first antibiotic derived from an Azo compound, which revolutionized medical practice. Hartley's discovery of the trans (E) and cis (Z) isomeric states⁵ of Azo compounds in 1937, with the E isomer being more stable and interconvertible via light or heat exposure, further advanced the field.⁵ Over time, aromatic Azo compounds, as great achievements of the human chemical industry, have progressed from simple dyes to sophisticated molecules capable of manipulating material properties,⁶ such as photoswitches,⁷ histology dyes,⁸ antibiotics,⁹ and molecular machines¹⁰ due to the reversible E–Z photoisomerization¹¹ and tunable chemical and physical properties.¹² Erlanger's demonstration of the correlation between E → Z isomerization and visual signal transduction highlighted their potential in photopharmacology.¹³ Heteroaryl Azo molecules (Het–N=N–Ph/Het), a pivotal subclass of aromatic Azo compounds, are renowned for their photochromic properties, significant geometric shifts between E and Z isomers, tunable photoswitchability, and broad UV-vis absorption spectra,^14,15 as high-performance supplements to azobenzenes (Ph–N=N–Ph) which offer less broader structural diversity.¹⁶

Fig. 1 The developmental evolution and main scientific achievements of aromatic Azo molecules.

The photoactivation properties of aromatic Azo compounds are evaluated through four different parameters. Foremost among these parameters lies the synergistic relationship between the maximum absorption wavelength (λ_max) and isomerization quantum yield (QY), where the latter parameter reflects the characteristics of the transformation attributed to the irradiation and excited-state relaxation dynamics. The λ_max of aromatic Azo is largely modulated by substituents on the aryl rings, which offer enhanced structural diversity and unique spectral properties.^17,18 This variability encompasses the locations of absorption peaks, as detailed in the data for several representative aromatic Azo (Fig. 2).¹⁹ Spectral engineering of aromatic Azo derivatives achieves tunable light absorption band modulation (400–800 nm), permitting optical control within biological transparency windows via visible or near-IR (NIR) irradiation.^20,21 This photoresponsiveness underpins emerging photopharmacological applications^22,23 where spatiotemporal precision is paramount.^24,25 The relative thermal stability of E and Z isomers constitutes the second critical performance parameter, exhibiting significant variations across different systems. Certain azoswitches display rapid spontaneous E form regeneration,²⁶ whereas others maintain metastable Z form for extended durations.^27,28 The lifetime of Z form plays a critical role in multidisciplinary implementations, including materials science²⁹ and medicine.²³ Beyond these fields, its functional persistence directly governs performance in innovative technical fields spanning from logic gates,³⁰ data storage,³¹ or real-time information transfer.³² Experimental characterization of Z isomer stability, such as lifetimes (τ) or half-lives (t_1/2), requires solution-phase measurements, with solvent properties critically influencing thermal stability of the Z form.¹⁴ The third characteristic property of photoresponsive molecular switches lies in the steady-state relative abundance between E and Z isomers under specific illumination parameters or ambient darkness. This thermodynamic metric, mathematically formalized as an equilibrium constant or a ratio, defines the photostationary distribution (PSD) at the photostationary state (PSS).¹⁹ The fourth defining performance parameter, termed cyclability/fatigue, quantitatively measures a photoswitching system's ability to maintain functional fidelity across repeated photochemical cycles.¹¹ Aromatic Azo compounds demonstrate negligible functional deterioration during extended irradiation protocols, with a phenomenon attributed to the absence of any side reactions.³³

Fig. 2 Absorption spectra of Azobenzene (a), mono-heteroaryl molecules (b), and bis-heteroaryl molecules (c).

Aromatic Azo molecules interconvert their E and Z isomers via light³⁴ or thermal energy,¹⁹ either through a rotational isomerism around the Azo-bond or an inversion mechanism,³⁵ to achieve non-radiative deactivation³⁶ of the excited electronic states, S₁ and S₂.³⁷ Typically, UV irradiation induces a shift from the thermodynamically stable E configuration to the metastable Z configuration,^38,39 accompanied by a hypsochromic shift in the π–π* absorption band and enhanced n–π* transition (Fig. 3a).⁴⁰ This photoreactivity enables the incorporation of aromatic Azo structures into pharmaceuticals, allowing light-mediated modulation of drug activity,^41,42 with significant potential in photopharmacology.^24,25 Systematic analysis of literature on aromatic Azo fluorescent probes reveals two main distinct probe design strategies: (i) aromatic Azo dyes are employed as a pro-fluorophore defined as an Azo fluorophore which activated by reductase or chemical reducing agent (Fig. 3b), or (ii) aromatic Azo dyes serve as a quencher (non-fluorescent energy acceptors) in the context of Förster resonance energy transfer (FRET)-based probes (Fig. 3c).³⁷ For the first approach, the N=N bond's reactivity with sodium dithionite renders aromatic Azo compounds suitable as cleavable linkers in chemical biology.⁴³ Aromatic Azo molecules, serving as hypoxia-sensitive probes, are valuable for in vivo hypoxia imaging⁴⁴ and targeted cancer therapies.⁴⁵ Under hypoxic conditions, aromatic Azo molecules, acting as fluorescent molecular switches,⁴⁶ undergo reduction, triggering fluorescence and enabling applications in photodynamic therapy (PDT) and chemotherapy.⁴⁷ The azoreductase-catalyzed reduction of Azo compounds to aromatic amines proceeds via one-electron transfer, forming a radical-anion intermediate sensitive to oxygen concentration (Fig. 3b). In hypoxia (p(O₂) = 0.1–5%), the intermediate stabilizes and further reduces to hydrazine, ultimately cleaving to yield aromatic amines.⁴⁸ For the second approach, aromatic Azo molecules are typically weak or non-fluorescent due to the non-radiative deactivation of excited states via E–Z isomerization. Consequently, when conjugated with fluorophores,⁴⁹ aromatic Azo enable efficient FRET,^37,50 facilitating energy transfer between molecules. Aromatic Azo-based FRET activatable probes bearing an enzyme cleavable substrate are used to detect biologically relevant hydrolytic enzymes (mainly proteases) in complex physiological milieus (Fig. 3c).⁵¹

Fig. 3 Photochemical properties and main applications in biomedicine of aromatic Azo molecules. (a) Photoisomerization of heteroaryl Azo molecules and its applications. (b) The mechanism is widely recognized for the degradation of Azo dyes mediated by AzoR and its applications. (c) Design of aromatic Azo compounds as acceptors in quencher systems for FRET-based detection platforms.

We will summarize recent advances of aromatic Azo molecules, provide a comprehensive discussion of their structure–property relationships, design strategies, biomedical applications, and highlight avenues for future improvement. It may be valuable to develop versatile and high-performing aromatic azoswitches not only to broaden practical applications in biomedical use and smart materials to expand the application scenarios, but also to deepen the understanding of the fundamental photoisomerization mechanisms of aromatic Azo for a broad range of scientists within different disciplinary backgrounds, which may spark new ideas, collaborations, and applications.

2. Photophysical properties and photoisomerization

2.1. Explicating photoisomerization mechanisms

Traditionally, the reversible E–Z isomerization property of AZB⁵² was attributed to rotation or inversion mechanisms.⁵³ However, pure rotation or inversion represents extreme simplifications and does not fully capture the complexity of the process. A deep understanding of the photophysical processes is crucial for elucidating the photoisomerization pathway of aromatic Azo.

2.1.1. Photophysical process. The photophysical process of aromatic Azo initiates with the absorption of ultraviolet or visible light, exciting electrons from the ground state (S₀) to higher energy states (S₁ or S₂). Specifically, electronic transitions primarily occur via n–π* and π–π* excitations. The n–π* transition, involving lower energy photons, exhibits a longer excited-state lifetime and higher quantum efficiency. In contrast, the higher-energy π–π* transition, coupled with enhanced vibrational modes, results in a shorter lifetime and lower efficiency. Post-excitation, the molecule enters an excited state and rapidly undergoes vibrational relaxation and internal conversion of the potential energy surface. During this process, the molecule slides along the excited-state potential energy surface (S₁ or S₂) and gradually approaches the conical intersection region (Fig. 4a).⁵⁴ This exploration of the excited-state potential energy surface is driven by two key molecular motions: the torsion of one phenyl ring around the central N=N bond and the bending oscillations of the two C–N=N angles.⁵⁵

Fig. 4 Photophysical process of aromatic Azo molecules. (a) Potential energy surface evaluations of azobenzene were performed at fixed θ_CNNC torsional angles, while all remaining molecular degrees of freedom were permitted to relax. Reprinted from ref. 40 with permission. Copyright, 2021, Springer Nature Limited. (b) Statistical overview of dominant photochemical pathways and their corresponding trajectory proportions for Z-AZB following excitation to the nπ* (yellow labels) and ππ* (green labels) electronic states. Reprinted from ref. 71 with permission. Copyright 2021, Royal Society of Chemistry. (c) Chemical structures of DNAB, red Azo dye (SRG) and two dispersed blue dyes (DB366, DB165). (d) and (e) Three isomerization mechanisms Ar–N=N–Ar undergoes: inversion, rotation, and tautomerism. (f) Electron-donating five-membered heterocyclic systems containing N-methyl substituents stabilize T-shaped Z-conformers through orthogonal aryl group orientations that facilitate C–H⋯π bonding. This geometric preference, combined with effective π–orbital delocalization between the heteroaromatic framework and the Azo linkage, contributes to the Z form being more stable than in the case of Ph–N=N–Ph. (g) Azo-based molecular switches functionalized with electron-rich and electron-deficient moieties operate as push–pull systems. The Azo group exhibits a single N–N bond character, where rotation around the N–N axis mitigates steric hindrance in the Z isomer through rapid photoisomerization to the thermodynamically favored E conformation. (h) Heteroaromatic Azo derivatives containing ionizable protons in protic environments undergo dynamic tautomeric equilibration with hydrazone forms characterized by freely rotatable single N–N bonds. The Z-configured isomers exhibit limited kinetic stability due to rapid structural interconversion through s-Z/s-E hydrazone intermediates, ultimately favoring the E isomer. Notably, in systems such as 3-indolyl-azo-phenyl derivatives, this tautomerization process is mediated by solvent-bridged proton transfer mechanisms. (d–h) All reprinted from ref. 19 with permission. Copyright, 2019, Springer Nature Limited.

Aromatic Azo isomerization proceeds through an inversion-assisted torsional mechanism, in which the central C–N=N–C bond undergoes torsional rotation aided by movements of the C–N=N. This “pedal motion” mechanism involves stationary phenyl rings while the central C–N=N–C segment rotates and moves through them in a motion resembling a pedal stroke. Although the phenyl rings eventually align with the plane, their motion lags behind the central unit.⁵⁶ Phenyl rings remain slightly out of the plane and continue twisting afterward, even when the isomerization concludes and the central C–N=N–C dihedral angle stabilizes. Additionally, the “hula-twist” model explains the simultaneous twisting of adjacent single and double bonds during Z → E isomerization.⁵⁷

Nonadiabatic transitions are pivotal in the photophysical process. As the molecule nears the S₁/S₀ conical intersection, the electron transitions from the excited state back to the ground state, and molecular geometry adjusts accordingly, completing the photochemical isomerization. The excitation wavelength critically influences this process: high-energy π–π* excitation often generates excessive vibrational energy, leading to excited-state decomposition, whereas low-energy n–π* excitation tends to guide the molecule back to the ground state via a more efficient photochemical path. Persico et al. employed a hybrid quantum-classical methodology employing surface hopping dynamics and semi-empirical potential energy surfaces to investigate the E → Z isomerization of heteroaryl Azo, which include excitation to nπ*(S₁) and ππ*(S₂) states after excitation.⁵⁸ They found that for the E → Z isomerization after π–π* excitation, the S₁/S₀ crossing occurred at a reduced time along the C–N=N–C torsional coordinate pathway relative to processes following n–π* excitation, revealing distinct behaviors for E → Z isomerization.

2.1.2. Photophysical properties. The photoisomerization performance parameters of aromatic Azo dyes, particularly the photostationary distribution (PSD) and thermal isomerization half-life (t_1/2), are critical determinants of their biological applicability. PSD refers to the ratio of Z or E isomers at photoequilibrium under specific wavelength irradiation. This parameter dictates the maximum achievable concentration of the bioactive isomer, directly impacting the precision and signal-to-noise ratio of photoswitching in biological systems. Substituent electronic effects enable directional tuning of PSD. Push–pull electronic systems enhance the Z isomer ratio by strengthening ICT, which broadens visible-light absorption. For example, pyridine-thiazole Azo compounds achieve a PSD of 94% under purple light,⁵⁹ surpassing traditional azobenzenes (PSD < 75%). The significant spectral overlap between the n → π* bands of E and Z isomers frequently prevents complete E/Z photoreversion. Sufficient spectral separation of n → π* bands of E and Z isomers by structural modifications enables high-efficiency bidirectional photoconversion using visible light. A representative system, developed by Siewertsen et al., employed an azobenzene derivative covalently bridged at ortho-positions via an ethylene linker. This modification achieves ≈100 nm separation of the n → π* transitions, facilitating mostly photoconversions under visible light irradiation.⁶⁰ A high PSD value is essential for effective photoregulation in biological systems. In photopharmacology, near-complete isomerization (PSD > 90%) is required to maximize target activation/inhibition efficiency. For example, pyrazole-thiazole Azo derivatives achieve a PSD of 92% under 400 nm irradiation, significantly outperforming conventional azobenzene (PSD < 75%).⁶¹ This high efficiency renders them suitable for constructing photoantagonists for G protein-coupled receptors (GPCRs). Neuro-optogenetic tools require wavelengths matching tissue penetration (500–650 nm). π-Conjugation extension in heteroaromatic rings red-shifts absorption peaks, achieving PSD = 90% with λ > 500 nm, overcoming the conversion bottleneck of conventional switches in the visible region.⁶² The thermal half-life denotes the time required for the Z isomer to revert thermally to the E isomer. Its duration critically impacts functional stability and applicability. Matthew J. Fuchter identified compounds with Z isomer half-lives ranging from seconds to hours, to days and to years, and variable absorption characteristics, all through tuning of the heteraromatic ring.⁶³ Extended t_1/2 enhances stability but must align with biological timescales. For instance, arylazopyrazoles are employed in applications including in supramolecular complexes and as photopharmacological agents due to their half-lives of days at 25 °C.^64,65 Short t_1/2 is vital for rapid processes. Electron-withdrawing groups accelerate thermal relaxation, reducing t_1/2 to milliseconds, which suits rapid neuronal photostimulation.

The wavelength-dependent QY of aromatic Azo is also a critical parameter of its properties. Extensive theoretical studies predominantly focused on E → Z isomerization due to its pivotal role in photochromic molecules.⁶⁶ Excitation at ∼450 nm into the nπ*(S₁) state yields a QY (Φ_E–Z) of ∼0.25, while excitation at ∼320 nm to the ππ*(S₂) state results in a lower QY of ∼0.12.⁶⁷ This difference arises from the additional vibrational energy in ππ*(S₂) excitation compared to nπ*(S₁) excitation, according to the prevailing theory, which promotes S₁/S₀ decay at higher energies. Specifically, the added vibrational energy, especially in the asymmetric bending mode of the C–N=N linkage, enables transitions through higher-energy regions of the S₁/S₀ conical intersection seam than those accessed via nπ*(S₁) excitation. These high-energy regions correspond to dihedral angles nearer to 180°, occurring at a reduced time along the C–N=N–C torsional coordinate.^66,68 This favors E isomer formation, reducing E → Z isomerization efficiency.

The Z → E isomerization remains comparatively underexplored, with poor isomerization dynamics under both the nπ*(S₁) and ππ*(S₂) excitations. Zhu et al. developed a surface-hopping algorithm incorporating adiabatic electronic energies and gradients, followed by conducted ab initio SA-CASSCF(6,6) trajectory surface-hopping calculations for Z → E isomerization.^69,70 They reported QYs of Φ_Z–E = 0.39 for n–π* and Φ_Z–E = 0.3–0.45 for π–π* excitation. Their method accurately replicated the experimental QY trends for E → Z isomerization, yielding Φ_E–Z = 0.33 for n–π* excitation and Φ_E–Z = 0.11–0.13 for π–π* excitation. These results indicate that the QYs for Z → E isomerization is generally higher than those for E → Z. Morgane Vache attributed this QY reduction to a potential well on the S₂ surface near C–N=N–C = −90° accessible after both n–π* and π–π* excitations by employing advanced ab initio techniques. Two factors associated with this potential well led to QY reduction: (i) a subset of photochromes, which would exclusively form E-AZB after n–π* excitation, became trapped in the well following π–π* excitation, and (ii) photochromes excited to the ππ* state exhibited a higher propensity to revert to Z-AZB upon relaxation than those populated in the nπ* state.⁷¹ This review systematically summarized the dominant pathways followed by Z-AZB following nπ* and ππ* excitations, as computationally characterized in the study, along with the fraction of photochromes for each pathway (Fig. 4b).⁷¹

Recent studies suggest that push–pull substitution influences QY by altering the balance between torsional motion (phenyl rotation around N=N) and C–N=N bending.⁷² These motions form an extensive S₁/S₀ crossing seam, with the visited region determining the QY of photoisomerization. In push–pull aromatic Azo, reduced π–π* excitation energy compared to the parent compound decreases symmetric C–N=N oscillations, enhancing energy transfer to the torsional coordinate, which is more productive for the photoisomerization process.⁷² Garavelli's group compared push–pull azobenzene (DNAB) to red Azo dye (SRG) and two dispersed blue dyes (DB366, DB165) with varying hydrogen bond strengths (methyl versus traditional H-bonds) (Fig. 4c).⁷³ DNAB's push–pull substitution reduces S₂ state lifetimes and symmetric bending, promoting torsional activation and higher QY. SRG's keto tautomer eliminates bending deactivation, thereby potentially enhancing QY.

Conversely, dispersed blue dyes exhibit lower QYs due to intramolecular hydrogen bonding, which restricts isomerization, particularly in DB165, where strong hydrogen bonding significantly inhibits QY. These findings highlight the intricate interplay between electronic excitation, vibrational dynamics, and molecular structure in determining the photoisomerization QY of aromatic Azo, offering insights for optimizing photochromic performance.

The PSD, t_1/2, and QY collectively form the “performance triangle” for aromatic Azo-based biomaterials where PSD determines response speed, t_1/2 regulates action duration and QY ensures energy efficiency. Through substituent engineering (e.g., fluorine atoms lowering rotational energy barriers, π-conjugation tuning absorption wavelengths) and environmental adaptation (e.g., pH/oxygen concentration regulation), these three parameters can be synergistically optimized. This advances precision in light-controlled drug delivery, neuro-modulatory interfaces, and smart diagnostic-therapeutic platforms.

2.1.3. Common mechanistic features. Advances in heteroaryl azoswitches design have yielded mono-/bis-heteroaryl Azo molecules that outperform conventional azobenzene derivatives in terms of molecular design, photochromic properties, and functional applications like photopharmacology,^22,23 leveraging their distinct photophysical properties.¹⁹ Nevertheless, the potential of heteroaryl Azo remains largely unexplored. Heteroaryl Azo can enable frontier applications from photonic devices to bio-interfaces and energy storage systems^40,74 through advanced molecule properties,⁷⁵ such as UV absorption spectrum red shift, Z isomer thermal robustness, near-stoichiometric bidirectional photoswitching,⁷⁶ and high QY.

Current understanding of light-driven E → Z isomerization in heteroaryl azoswitches lacks the mechanistic clarity established for the Ph–N=N–Ph prototype, though current hypotheses suggest similar isomerization mechanisms (Fig. 4a). While comprehensive QY measurements of the E → Z photoreaction remains incomplete for heteroaryl azoswitches, existing data reveal reduced absorption cross-sections when the photoisomerization process originates from the higher-energy S₂ excited state compared to the lower-lying S₁ state. This deviation from Kasha's rule mirrors observations in Ph–N=N–Ph systems,^17,76–80 with theoretical modelling identifying critical S₁ → S₀ CI accessible exclusively after population of S₂ state, that facilitate ground state (S₀) recovery rather than geometric isomerization.^81–84 Detailed mechanistic analyses propose a rotation-inversion pathway along the S₁ potential surface, culminating in conical intersection-mediated conversion between the E isomer's nπ* excited state and the Z isomer's ground state.^81–83,85 Notably, this pathway selectivity demonstrates significant substituent effects: E-azoarylimidazoles predominantly undergo bending-mediated transitions to the Z ground state, while their N-methylated derivatives exhibit distinct relaxation patterns involving S₁ state twisting and S₂ state bending.⁸⁶

Thermally driven Z → E isomerization in metastable systems has attracted extensive research attention, with documented lifetimes providing crucial context for system evaluation (Fig. 5–8).^14,32 This reaction in heteroaryl Azo adheres to established AZB canonical mechanisms, operating through three primary pathways: inversion, rotation, and tautomerism (Fig. 4d). Comparative analysis reveals that inversion-mediated processes yield the thermodynamically favored Z isomer compared to rotational or tautomeric pathways, primarily due to reduced steric strain. Substituent selection and solvent environment critically dictate mechanistic preference in Ar–N=N–Ar derivatives, with heteroatom incorporation significantly modifying these tendencies.³² While comprehensive theoretical frameworks for thermal switching of heteroaryl azoswitches remain under development, empirical evidence demonstrates that heteroarene substitution markedly enhances Z form stability relative to conventional arene-based azobenzenes.

Fig. 5 The platform of Ph–N=N–Ph is used to control photophysical and photochemical properties. The yellow arrows indicate spectral red-shifts, while the green one indicates spectral blue-shifts. [a] = MeCN, [b] = water, and [c] = CH₂Cl₂.

Fig. 6 The push–pull systems of hybrid Ph–N=N–Ph are used to control photophysical and photochemical properties. The yellow arrows indicate spectral red-shifts, while the green one indicates spectral blue-shifts. [a] = MeCN, [b] = DMSO, and [c] = EtOH.

Fig. 7 The platform of Ph–N=N-Net is used to control photophysical and photochemical properties by using electron-rich heterocycles or p-electron-donating substituents. The yellow arrows indicate spectral red-shifts, while the green one indicate spectral blue-shifts. [a] = MeCN, and [b] = DMSO.

Fig. 8 The platform of Net₁-N=N-Net₁ is used to control photophysical and photochemical properties. (a) Thiazolyl combined with azopyrazole to synthesize a series of substitutions of nonsymmetric bis-heteroaryl molecules. (b) A family of azobispyrazoles was synthesized by altering the linkage position of pyrazole rings to the Azo group. The yellow arrows indicate spectral red-shifts, while the green ones indicate spectral blue-shifts. [a] = MeCN, [b] = water, and [c] = EtOH.

Heteroaryl Azo compounds exhibit distinct structural-property relationships: electron-donating N-methylated five-membered heterocycles confer exceptional extremely long lives to Z isomers which is critical to long-acting controlled drug delivery systems. These metastable configurations undergo E form conversion via inversion kinetics considerably slower than the Ph–N=N–Ph benchmark (Fig. 4f).¹⁷ Structural analysis suggests this enhanced thermal stability stems from T-shaped molecular geometries facilitating intramolecular C–H⋯π interactions within the azaheterocyclic system, a spatial arrangement sterically prohibited in bulkier Ph–N=N–Ph analogues.⁷⁷ Moreover, Azo systems combining electron-deficient pyridine/pyrimidine moieties with electron-rich aryl groups display accelerated rotational isomerization which realize fast photoresponse, meeting the requirements for real-time optogenetic modulation. This dynamic behavior originates from a synergistic push–pull electronic effect: electron-donating substituents (–OR/–NR₂) on the aryl group cooperate with electron-withdrawing heterocycles (Fig. 4g).^14,87 In particular, distinct resonance configurations are observed, where charge-neutral coexist alongside zwitterionic resonance structures. The latter exhibit rapid rotational freedom about the single N–N bond, thereby enabling efficient isomerization processes.^88,89 Crucially, these resonance effects operate in both E and Z isomers, with the Z isomer exhibiting pronounced kinetic lability to give the more stable E isomer. Quaternary nitrogen functionalization (e.g., methylation) or protonation amplifies this effect through cationic charge localization, dramatically increasing electron-withdrawing capacity.⁸⁹

Diverging from resonance phenomena, heterocyclic NH-containing Azo exhibit dynamic equilibration with hydrazone tautomers, a process analogous to –OH/–NH₂ substituted azobenzene systems (Fig. 4e). Structural analysis reveals hydrazone tautomers possess a N–N single bond whose rotational freedom drives rapid s-E ↔ s-Z conformational switching.⁸¹ This coupled tautomerization-rotation process is widely hypothesized as the primary driver of Z isomer lability in NH-azoswitches.^17,77 Experimental validation currently exists solely for azoimidazole⁷⁸ and azoindole⁸¹ systems in proton-donating solvents, where isomerization kinetics exhibit dual dependency on the concentration of the protic media and azoswitches,^76,81 with a computational study verifying that H⁺ transfer events mediate the isomerization pathway for azoindoles (Fig. 4h).⁸¹

2.2. Azo-structural related photophysical properties

Due to their tunable photophysical and photochemical properties, aromatic derivatives of AZB⁹⁰ are widely utilized in organic dyes, molecular photoswitches, and therapeutic agents.¹² Compared to AZB, heteroaryl Azo molecules are renowned for their photochromic properties, significant geometric shifts between E and Z isomers, tunable photoswitchability, broad UV-vis absorption spectra, and high QY.^14,15,91 The reversible E–Z isomerization drives advances in light-responsive materials crucial for photopharmacology, photovoltaic actuators, and solar energy storage.¹² The efficacy of heteroaryl Azo is largely modulated by substituents on the aryl rings, which offer enhanced structural diversity and unique spectral properties.^17,18 Mono-heteroaryl molecules, such as 3-indole and 2-pyrrole, exhibit red-shifted π–π* bands compared to AZB (λ_π–π* = 317 nm), while bis-heteroaryl molecules, including symmetric azoheteroarenes (Het–N=N–Het), which incorporate 2-imidazole, 5-pyrazole, and benzazoles, extend π conjugation through the incorporation of two electron-rich or π-conjugated systems (Fig. 2).⁹² This section explores structural modifications of heteroaryl Azo molecules, examining substitutions’ impact on photophysical properties.

2.2.1. Azobenzene. Electron-donating substituents on the phenyl ring elicit red-shifts in the absorption wavelength of AZB. Compared to unsubstituted AZB (λ_π–π* = 317 nm), AZB derivatives substituted with –OH, –NMe₂, –OMe, or –NEt₂ groups display red-shifted π–π* bands (λ_π–π* = 335–479 nm), primarily through enhanced conjugation due to increased electron density on the azobenzene ring. Here, the synthesis of a series of ortho-substituted AZB derivatives is summarized. ortho-Substituted 4,4′-diacetamido azobenzenes (λ_π–π* = 370 nm) show significant redshifts, influenced by steric and electronic effects (Fig. 5). The presence of a six-membered ring (molecule 5viii) introduces steric interactions that lead to loss of sp² character on the N atom, whereas a five-membered ring (molecule 5ix) produces enhanced N delocalization. Pyrrolidine substituents induce the longest wavelength absorption due to intramolecular charge transfer (ICT) from a typical π* orbital.⁹³

Tetra-ortho-fluoro azobenzene with para-ester groups exhibits long-wavelength photoswitching, with its tetra-ortho-methoxy derivative showing a red-shifted π–π* band.⁹⁴ In molecule 5xii (Fig. 5), esters as electron-withdrawing groups (EWGs) synergize with ortho-fluorine atoms to yield optimal photochromic performance, including a 50 nm separation of the n–π* bands, enabling efficient E–Z photoconversions.⁶² Recent advances reveal that chlorine substitution (Cl₄-TOAB) further optimizes performance: The larger atomic radius of Cl induces greater steric distortion, red-shifting n–π* absorption of E-isomer to 457 nm, enhancing thermal stability (t_1/2(Z) = 38 days in DMSO), and enabling red-light switching at 625 nm. Chalcogen substituents (S/Se) amplify the bathochromic shift via intramolecular chalcogen bonding: (MeS)₄-TOAB absorbs at 514 nm, while (MeSe)₄-TOAB extends to 534 nm. Para-substitutions also modulate photophysics synergistically. Electron-donating groups (EDGs) (e.g., –NMe₂) coupled with ortho-chalcogens achieve ICT-driven shifts beyond 500 nm, whereas electron-withdrawing groups reduce thermal half-lives.⁹⁵

The pyridine ring, as an electron-deficient heterocycle, is widely used in push–pull systems. Phenylazopyridine modified with EDGs (para-NH₂, para-NMe₂, para-NEt₂) red-shifts the π–π* band to 420–450 nm like molecules 6i–6iii, while altering the N atom's position in phenylazopyridine has minimal impact (Fig. 6). This push–pull mechanism is further enhanced by the introduction of stronger electron-withdrawing pyridinium salts. Azopyridinium salts with para-NMe₂ substitution (molecule 6ix) exhibit remarkable red-shifts to 552 and 560 nm alongside exceptionally short half-lives of 329 ns and 368 ns, respectively.⁸⁸ Notably, solvent polarity and protic character significantly modulate these thermal half-lives. The half-life of the molecule 6ix in EtOH is 329 ns, while in water it is 310 ns, possibly due to water's extraordinary hydrogen-bonding capacity and high polarity, which significantly lowers the energy barrier.

2.2.2. Mono-heteroaryl Azo molecules. Mono-heteroaryl Azo molecules represent a versatile molecular framework, enabling visible-light photoswitching through the incorporation of electron-rich heterocycles or p-EDGs substituents on the phenyl ring.⁹⁶ Compared to arylazopyrazole of 4-pyrazole (λ_π–π* = 328 nm), other arylazopyrazoles exhibit red-shifted π–π* λ_max due to their superior π-donating ability to the electron-withdrawing Azo group, consistent with π-deficient azopyridines.¹⁷ The π system in mono-heteroaryl Azo molecules can be further enhanced by introducing EDGs at the phenyl ring's para position, leveraging p–π conjugation to extend electron delocalization (Fig. 7). The red-shift magnitude correlates with the substituent's electron-donating strength, quantified by the electrophilic substituent constants σ_p+. For example, unsubstituted arylazopyrrole shows a π–π* absorption band at λ_π–π* = 328 nm, while introducing groups like –OCOMe, –OMe, –NH₂, –NHMe, or –NMe₂ consistently shifts it to longer wavelengths like molecules 7i–7v.

Ortho-amino substitutions in azobenzenes have enabled the development of visible-light-sensitive heteroaryl Azo molecules. Currently, various ortho-amino-substituted Azo heteroarenes have been synthesized, incorporating heterocycles such as six-membered piperidine rings, para-substituted pyrrolidine, or amino groups like primary amines and dimethylamino groups (Fig. 7).⁹⁷ For instance, arylazopyrrole with six-membered piperidine (molecule 7x) exhibits a π–π* absorption band at 376 nm, while para-pyrrolidine (molecule 7xii) induces a red-shift to 458 nm. This shift arises from the pyrrolidine's enhanced conjugation effect, driven by the increased sp² character of the pyrrolidine-N atom.⁹² These design strategies highlight the potential for tailoring heteroaryl Azo molecules for specific photophysical properties.

2.2.3. Bis-heteroaryl Azo molecules. Bis-heteroaryl Azo molecules achieve extended π conjugation through synergistic pairing of heterocycles (electron-rich or extended-π), demonstrating superior performance over mono-heteroaryl Azo. Substitutions at the heterocycle complement the effect of modifying the heterocycle type. Thiazolylazopyrazoles, introduced by Li's group, utilize an electron-rich thiazole ring for visible-light responsiveness and a pyrazole ring for ortho-substitutions (Fig. 8a). The parent thiazolylazopyrazole exhibits a π–π* band at 366 nm, red-shifted by 38 nm compared to phenylazopyrazole. Methyl substitutions (molecule 8i) minimally affect the spectrum but reduce the half-life from 2.1 days to 1.1 hours.¹⁵ Carbonyl substitutions (molecule 8iv) red-shift the π–π* peak to 371 nm, enhance Z isomer n–π* absorption intensity via planar distortion, and achieve ∼100% E → Z photoconversion with a maximum Z isomer half-life of 3.9 days. Interestingly, carboxyl substitutions (molecule 8vi) shorten the Z isomer half-life to 0.9 hours despite red-shifting the π–π* peak to 392 nm, attributed to strong intramolecular hydrogen bonding modulating thermal relaxation kinetics.

Symmetric Azo heteroarenes (Net₁-N=N-Net₁), which also play a critical role, such as azobispyrazoles, have been synthesized by altering pyrazole linkage positions. A new family of azobispyrazoles has been introduced by altering the linkage position of pyrazole rings to the Azo group (Fig. 8b). Azobis(5-pyrazole), with electron-rich 5-pyrazole units on both ends, acts as a visible-light photoswitch, achieving 85% E → Z conversion under 400 nm light, albeit with a short thermal half-life of a few hours. In contrast, azobis(2-imidazole) (molecule 8viii) exhibits a π–π* band at 440 nm, a significant red-shift compared to azobis(5-pyrazole) (356 nm), enhancing visible-light addressability.⁹⁸ Furthermore, azobenzazoles, featuring benzothiazole, benzoxazole, or benzimidazole (molecule 8x–8xii), show π–π* λ_max at 421, 430, and 439 nm, respectively, with E → Z conversions of 73–84% under 415–448 nm light but suffer from thermal half-lives under 1 hour. Overall, extending conjugation in bis-heteroaryl Azo molecules effectively shifts the π–π* band into the visible-light region.⁹⁹

Compared to symmetric counterparts, nonsymmetric bis-heteroaryl structures (Het₁–N=N–Het₂) integrate the advantages of distinct heterocycles, enabling innovative visible-light switches. For instance, molecule 8ix (Fig. 8b) combines electron-deficient benzothiazole with electron-rich bithiophenylpyrrole to establish a push–pull system, achieving a π–π* λ_max of 544 nm and a thermal half-life of 70 μs.⁹⁹

2.2.4. Theory-guided molecular design. In recent years, machine learning (ML) methodologies have demonstrated significant potential for advancing photoswitchable material design. Axelrod et al. achieved high-precision prediction of photoisomerization QY in aromatic Azo derivatives by developing a diabatic artificial neural network (DANN) based on diabatic states. Trained on excitation energy surfaces across 630 000 configurations, this model maintains exceptional predictive correlation for unseen molecules.¹⁰⁰ Subsequently, the same research team extended the ML framework to thermodynamic parameter prediction. By incorporating intersystem crossing mechanisms to correct systematic biases in conventional transition state theory, their transfer learning model screened a virtual library of 19 000 compounds, revealing strong correlations between substituent spatial distributions and activation entropy.¹⁰¹

In quantitative structure–property relationship (QSPR) studies, Byadi et al. developed a descriptor-based predictive model using structural features. Comparative analysis of eight molecular descriptors identified fragment count descriptors as optimal for predicting key properties: maximum absorption wavelength (λ_max) and thermal half-life (t_1/2). This work pioneers dual-parameter prediction solely from 2D structural information, offering a novel paradigm for rational design of visible-light-driven azophotoswitches.¹⁰² Notably, Konrad et al. synthesized a bistable azobenzene derivative with deep redshift properties through computationally guided design. Combining C–H activation chlorination and X-ray crystallographic analysis, they confirmed that ortho-fluoro/chloro mixed substitution simultaneously enables: significant absorption redshift, near-quantitative photostationary state ratio and enhanced thermal stability. This molecule is activatable under 660 nm deep-red light within the biological optical window, providing a new tool for photopharmacological applications.¹⁰³

3. Design and synthesis of biomedical oriented aromatic Azo molecules

3.1. Preferred synthetic strategies for aromatic Azo scaffolds

Aromatic Azo molecules are classified into linear and bridged (diazocine) categories based on the E–Z isomers' relative stability. Here, we will outline efficient approaches to synthesize aromatic Azo molecules and highlight some recent examples (Fig. 9).

Fig. 9 Synthesis methods of aromatic Azo-based small molecules. Ar^1/2 = Ph/Het, EDG = electron-donating group, M = metal, X = O, S, N, etc.

Constructing Azo groups is the essential step in synthesizing linear heteroaryl Azo molecules. An effective strategy emphasizes short reaction durations, high yields, and diverse coupling partners. Classic approaches include Azo coupling reactions, as well as the Mills and Wallach reactions.¹⁰⁴ Griess, a 19th-century Brewer, developed a versatile synthesis method for aromatic Azo compounds, starting with the diazotization of aromatic amines and subsequent coupling with electron-rich aromatic nucleophiles (route i in Fig. 9).¹⁰⁵ The Mills reaction (route ii) features a nucleophilic attack on aromatic nitroso derivatives in an acidic environment, followed by dehydration to yield the final product in good overall yield.¹⁰⁶ A typical approach employs aromatic nitroso derivatives with anilines in glacial AcOH. To expand the substrate range of these methods, diazonium salts have been coupled with metalated arenes (route iii). Feringa and co-workers applied this method to create difficult-to-access tetra-ortho-substituted red-shifted azobenzenes from ortho-lithiation of aryl nucleophiles.¹⁰⁷ The Wallach reaction (route v) transforms azoxybenzenes into either 4-hydroxy-substituted Azo derivatives or the 2-hydroxy derivative using strong acids.¹⁰⁸ Due to the Wallach rearrangement, the reaction forms a mixture of heteroaryl Azo compounds.¹⁰⁴ Moreover, there are many synthetic methods of recent development like catalytic C–H functionalization. Konrad et al. developed a Pd(OAc)₂-catalyzed C–H activation that installs four chlorine atoms to azobenzene scaffolds in a single step (56–76% yield), overcoming the steric limitations and low yields (<26%) of traditional Azo-coupling or oxidative dimerization methods.¹⁰⁹ Building upon this foundation, Ruiz-Soriano et al. introduced a dual-catalytic method involving Pd(PPh₃)₄/Cu(OTf)₂ for ortho-tetrabromination of azobenzenes (yielding 67–99%), followed by CuI-catalyzed methoxylation with methyl formate/NaOMe (yielding 60–99%). This technique addresses limitations of polyhalogenated substrates and symmetry issues in oxidative dimerization, achieving excellent yields.¹¹⁰

Although numerous synthetic methods for aromatic Azo compounds exist, the diazotization reaction remains the most widely utilized approach for synthesizing aromatic Azo compounds because of its simplicity and ability to proceed in an aqueous medium. Oxidative coupling of aniline derivatives¹¹¹ (route iv) is another viable method for producing aromatic Azo molecules, including sodium perborate/acetic acid, H₂O₂/Na₂WO₄, AgO, Ag₂O, or Ag₂CO₃. In 2008, Corma et al. reported that gold-supported TiO₂ nanoparticles efficiently catalyze the oxidation of anilines with O₂ as the oxidant, yielding highly selective and high-performing Azo derivatives.¹¹² Subsequently, Zhang et al. introduced a single-electron-based oxidation process for aniline, employing air/O₂ and a Cu catalyst.¹¹³ The reduction of aromatic nitro derivatives (route vi) is also a common strategy, using reducing agents such as LiAlH₄, Zn/NaOH, NaBH₄, KOH, or Bi. Pahalagedara's groups developed a sea urchin-shaped Ni/graphene nanocomposite, which enables selective hydrazine-mediated reduction of nitroaromatics into heteroaryl Azo compounds with various substituents, offering high efficiency and easy catalyst recyclability.¹¹⁴

The presence of the ethylene bridge endows the bridged aromatic Azo with unique optical switching capabilities. However, conventional methods for synthesizing linear aromatic Azo often lack versatility, resulting in inconsistent yields. Bridged aromatic Azo synthesis typically employs oxidation coupling of –NO₂ (route vi) or reduction coupling of –NH₂ (route iv). Maier et al. introduced a new approach for cyclic azobenzenes using diarylamine as the starting material, with m-CPBA (dichloromethane, DCM) as the oxidant and a HOAc/DCM solvent mix, leading to the development of a new photomolecular switch.¹¹⁵ Jiang and co-workers developed an innovative method for creating heteroaryl Azo via pyrazol-5-amine iodination, using tert-butyl hydroperoxide (TBHP) as the oxidant and copper salts as catalysts.¹¹⁶ This process forms C–I and N=N bonds through simultaneous iodination and oxidation, and subsequent oxidative dehydrogenation mediates the formation of azopyrrole derivatives and their iodinated analogues, both incorporating diverse functional substituents. Subhajit et al. constructed a novel nine-membered macrocyclic compound containing sulfur molecules and methylene bridges produced through reductive cyclization of bis(nitrobenzyl)sulfane with glucose/NaOH.¹¹⁷ This molecule, characterized by both flexibility and rigidity, exhibited remarkable stability, maintaining its E isomer even when heated to 50 °C.

While classical Azo-coupling strategies have been widely employed for azobenzene synthesis, their application to heteroazoarenes faces inherent limitations. Traditional methods struggle with substrates bearing electron-deficient moieties and heterocycles due to poor regioselectivity and the requirement for strong EDGs on the coupling partner. Crucially, heteroaromatic systems often exhibit reduced nucleophilicity, further diminishing yields in conventional approaches. In contrast, Martin Oestreich et al. developed the palladium-catalyzed C(sp²)–N(sp²) cross-coupling strategy to overcome these constraints by employing silicon-masked diazenyl anions as versatile nucleophiles. As demonstrated in Fig. 10, heteroaryl bromides, including pyridyl (2a), thienyl (2b and 2c), benzofuranyl (2e), benzothiophenyl (2f), and quinolinyl (2g) derivatives, efficiently coupled with aryl-substituted diazenyl pronucleophiles to afford non-symmetric heteroazoarenes (3a–3h) in 57–89% isolated yields. This method accommodates sterically congested and electron-poor heterocycles without competitive denitrogenation, a significant advancement over prior systems requiring large excesses of reagents or specific substitution patterns.¹¹⁸ Notably, heterocycles particularly electron-deficient heterocycles, exhibit diminished nucleophilicity compared to their aryl counterparts, which elevates the energy barrier for the transmetalation step and consequently reduces reaction yields. Furthermore, coordination of heteroatoms (S/N) to the catalytic center may induce catalyst deactivation. This necessitates more demanding reaction conditions, ultimately compromising overall efficiency.

Fig. 10 Palladium-catalyzed cross-coupling of functionalized silylated aryldiazenes 1 and various heteroaryl bromides 2a–h. All reactions were performed on a 0.20 mmol scale. Yields are of isolated products after purification by flash chromatography on silica gel.

Moreover, tailored Azo building blocks exhibit exceptional versatility and synthetic utility. Carreño et al. reported a mild and efficient protocol for synthesizing asymmetric azobenzenes from quinone bisketals and arylhydrazines. This method employs catalytic ceric ammonium nitrate (CAN) under ambient conditions to facilitate N=N bond formation, circumventing the need for strongly acidic or basic environments. This approach complements classical diazotization strategies and highlights valuable synthetic utility for functionalized Azo molecules that have been used in various systems.¹¹⁹ For instance, John et al. leveraged this strategy to synthesize a series of red-shifted azobenzene amino acids. Their route involved oxidation of tyrosine derivatives to quinonoidal spirolactones, followed by CAN-catalyzed coupling with fluorinated phenylhydrazines. This methodology overcame the pH sensitivity of traditional diazotization, enabling a 91% yield of tetra-ortho-fluoro-substituted azobenzene-alanine. The amino acid was successfully site-specifically incorporated into superfolder green fluorescent protein (sfGFP) via an orthogonal tRNA/PylRS pair, providing a novel tool for visible-light-mediated protein regulation.¹²⁰ Carreño's team further exploited the regioselectivity of quinone bisketals to synthesize chiral azobenzenes bearing a p-tolylsulfinyl group. By strategically controlling steric bias in the quinone bisketal substrate, they achieved precise positioning of the sulfoxide moiety relative to the Azo bond. This design enabled conformational switching via photoinduced isomerization, with the sulfinyl group acting as a chiral director.¹²¹ Guisán-Ceinos et al. extended this synthetic approach to BODIPY fluorophores, developing 3-azo-conjugated BODIPY probes. These non-fluorescent compounds served as turn-on biosensors, where reductive cleavage of the Azo bond restored BODIPY fluorescence, allowing visualization of hypoxia-like conditions in live cells.¹²²

3.2. Design of multifunctional aromatic Azo molecules

Based on the foundational studies of traditional aromatic Azo compounds, recent research has expanded into the design and synthesis of advanced Azo dyes as dark quenchers, addressing critical needs in fluorescence-based sensing and imaging. Simultaneously, the development of NIR-responsive aromatic Azo systems has opened new avenues for applications in PDT and drug delivery, broadening the biomedical applications.

3.2.1. Design and synthesis of aromatic Azo dyes as quenchers. The development of quenchers containing one or two aromatic Azo groups gained significant attention from (bio)organic chemists in the early 2000s. This interest was catalyzed by Biosearch Technologies (now part of the LGC group), which introduced the first bioconjugatable polyaromatic Azo-based quenchers, branded as Black Hole Quenchers (BHQs). These quenchers, activated as N-hydroxy succinimidyl (NHS) esters, are highly robust, demonstrating excellent coupling efficiency and a high extinction coefficient. As depicted in Fig. 11, BHQ-1, BHQ-2, BHQ-3, and BBQ-650 exhibit broad absorption spectra. The preparation of Azo-based quenchers typically involves a conventional azocoupling reaction.³⁷ The Azo-coupling reaction proceeds via interaction of a diazonium species derived from an aniline precursor with π-excessive N,N-dialkylaniline derivatives, following a mechanism similar to that of the electrophilic aromatic substitution (S_EAr) reaction (Fig. 11a). To easily functionalize Azo dyes, the preferred synthetic approach employs an unsymmetrical mono N-substituted tertiary aniline, featuring a short alkyl chain terminated with either a protected or free functional group.

Fig. 11 (a) Typical synthetic method of Azo-based quenchers. (b) BHQ-based bioorthogonal conjugation-capable analogues are chemically accessible and functionally tailored for biolabeling. QR = Quenching range. The λ_abs were determined in [a] = MeCN; [b] = DMSO.

DABCYL is utilized as quenchers as one of the earliest fluorophores (Fig. 11b). Bombarda et al. enhanced its hydrophilicity by incorporating three hydroxy groups, resulting in a probe with improved bioavailability, making it suitable for high-throughput enzymatic assays.¹²³ In 2013, Jing and Cornish introduced the synthesis of primary amine-modified BHQ-1 via conventional Azo coupling.¹²⁴ BHQ-1 exhibits a maximum absorption wavelength of 535 nm, which surpasses that of DABCYL and overlaps directly with the emission peak of FAM fluorophores. Building on this strategy, the Burkart group developed a primary alcohol derivative of BHQ-2, whose absorption spans 550–650 nm.¹²⁵ BHQ-2 has become one of the most popular quenchers in the BHQ family, effectively quenching fluorophores like FAM, Cy3, Cy5, and ROX. Around the same time, the properties of BHQ-3, a quencher for far-red or NIR cyanine fluorophores, were re-evaluated, revealing superior FRET quenching efficiency compared to DABCYL. Among these, BBQ-650 stands out with its broad absorption spectrum (550–750 nm, abs max 650 nm). This quencher is ideal for long-wavelength fluorophores due to its higher chemical stability and extended quenching range compared to BHQ-2 and BHQ-3. Tinnefeld employed a nanoswitch constructed on a DNA origami platform to detect the activity of anti-digoxin antibodies.¹²⁶ This nanoswitch features DNA strands functionalized with a dye-quencher pair, ATTO 647 N and BBQ-650, which forms a dye-quencher pair. When the antibody binds to its corresponding antigen, the DNA strands unfold, causing an enhanced fluorescence signal that facilitates precise detection.

Based on the unique physicochemical properties of Azo-based quenchers, including their high photostability, tunable absorption spectra, and efficient energy transfer capabilities, recent advancements in the synthesis and functionalization of Azo-based quenchers have expanded their application scope, promoting their use in PDT, chemical biosensing, and other fields. Costa and co-workers developed a simple method for synthesizing a BHQ-3 derivative via an Azo-coupling reaction between Methylene Violet 3RAX and a tertiary aniline with a pendant primary amine.¹²⁷ This derivative was specifically designed for conjugation with peptides to facilitate its application in FRET-based protease activity assays. Similarly, Xue introduced an innovative bioorthogonal method that employs an antidote containing a BHQ-3 linked to a bicyclo[6.1.0]non-4-yne (BCN) group and a tetrazine-substituted boron dipyrromethene-based photosensitizer, aimed at minimizing skin damage during light irradiation.¹²⁸ Using tumor-bearing nude mice as models, it was demonstrated that administering this antidote post-PDT effectively deactivates residual photosensitizer activity. BHQ-3 interacts with the photosensitizing unit through a rapid click reaction, neutralizing photodynamic effects.

The design of aromatic Azo compounds as efficient quenching moieties has been significantly advanced by pioneering work leveraging their hypoxia-selective bioreduction. Recent studies demonstrated the unique potential of azobenzene derivatives to serve as hypoxia-activated fluorescent probes. Nagano and colleagues developed hypoxia-sensitive NIR fluorescent probes, where an Azo bond conjugated to a cyanine fluorophore acts as a quencher via FRET under normoxia. Critically, the Azo linkage is selectively cleaved by reductase activity in hypoxic environments, triggering a substantial fluorescence increase. This enabled the first real-time, in vivo fluorescence imaging of acute ischemia in organs like the liver and kidney in live mice.¹²⁹ Building upon the concept of Azo-based hypoxia activation, Urano et al. extended the strategy to PDT. They engineered a seleno-rosamine photosensitizer (AzoSeR) where the Azo group critically quenches both fluorescence and singlet oxygen (¹O₂) generation by inhibiting intersystem crossing (ISC). Hypoxia-induced reductive cleavage of the Azo bond restores the ISC capability and ¹O₂ production of the core sensitizer (SeR), allowing selective ablation of cells under clinically relevant mild hypoxia, while sparing normoxic cells.¹³⁰ These studies established that Azo groups can effectively quench fluorescence and photosensitization under normoxic conditions, and be selectively reduced in hypoxic environments, thereby enabling highly specific imaging or therapeutic uses in PDT.

3.2.2. Further design strategies for biomedical use of aromatic Azo molecules. Traditional azobenzenes face challenges in biomedical applications due to their reliance on UV light for photoswitching, which poses risks to tissues and cells. To address this, heteroaryl Azo compounds responsive to red and NIR light are being developed. In Section 2.1, we discussed methods to enhance substituent patterns and their chemical properties to achieve longer absorption wavelengths in heteroaryl Azo compounds. Approaches include para- and ortho-substitution on benzene rings, designing push–pull electronic systems, and adding electron-rich heterocycles to expand π-conjugation. These strategies have successfully created aromatic Azo compounds capable of one-photon photoswitching with visible-to-NIR light, achieving high photoconversion rates and thermally stable Z isomers.²¹ Additionally, the current strategies for tuning the photoisomerization wavelength into the visible and NIR regions also involve two distinct approaches: one utilizes upconversion nanoparticles (UCNPs) to convert long-wavelength light into higher-energy emissions, while the other employs two-photon absorption mechanisms in aromatic Azo molecules, which enable efficient light absorption at extended wavelengths through simultaneous photon excitation (Fig. 12a).

Fig. 12 (a) Methodological developments in constructing aromatic Azo derivatives with red-light-activated photoresponsivity. (b) Illustration of the fabrication of UCNP-BDP-azo as a hypoxia-sensitive NIR fluorescent nanoprobe, designed for biocompatible 808 nm NIR light activation. Reprinted from ref. 132 with permission. Copyright 2025, Royal Society of Chemistry. (c) Mesoporous silica-based nanoplatforms functionalized with Azo-chromophores A and two-photon-excitable fluorophores F. This nanoarchitecture, termed MAF nanoimpellers, enables a two-photon (760 nm) activated release of drug molecules through FRET coupled with reversible azoisomerization. Reprinted from ref. 137 with permission. Copyright 2013, Wiley-VCH.

Rare-earth-doped nanoparticles (RENPs) exhibit good biocompatibility and offer versatile surface functionalization options, aiding the biodistribution and delivery of organic chromophores used as biosensors.¹³¹ When integrated with aromatic Azo molecules, these materials, functioning as UCNPs, enable red-shifting of the photoisomerization wavelength into the visible-NIR spectrum without altering their structure. UCNPs, often composed of a NaYF₄ matrix doped with lanthanide ions, have facilitated the photoisomerization of photoactive aromatic Azo compounds within micelles, liposomes, silica, and nanocapsules to promote chemotherapeutic drug release. Ortgies and co-workers introduced an innovative hypoxia-sensitive nanoprobe by linking Azo-BODIPY dyes to UCNPs with a core/shell structure of NaGdF₄:2%Yb³⁺,3%Nd⁴⁺,0.2%Tm³⁺/NaYF₄.¹³² The Azo dye, UCNP-BDP-Azo, was synthesized through nucleophilic substitution using 3-mercaptopropionic acid and attached to UCNPs via EDC/NHS-mediated coupling, enabling efficient NIR excitation (808 nm) and visible-range emission through upconversion (Fig. 12b). Despite its potential, this approach faced limitations for in vivo use, including proximity requirements between UCNPs and aromatic Azo, shallow tissue penetration, overheating risks, and low QY. Furthermore, concerns about UCNP toxicity and their biological clearance remain underexplored.¹³³

Two-photon absorption also enables the NIR photoswitches of aromatic Azo molecules by absorbing two photons, each carrying half the energy required for single-photon excitation, in a nearly simultaneous process.¹³⁴ Typically, two-photon excitation of photoswitches is achieved indirectly through a molecular fragment with a high nonlinear absorption cross-section, capable of resonance energy transfer to the photoswitches. After two-photon excitation with low-energy light, the singlet-excited donor transfers its energy to the photoswitches, usually via FRET, activating the singlet-excited state of the photoswitches and triggering isomerization.¹³⁵ An efficient photoswitch using indirect two-photon excitation, commonly within the 750–800 nm wavelength range, has been demonstrated for aromatic Azo compounds. These molecules can undergo both direct two-photon excitation¹³⁶ and indirect excitation by incorporating a two-photon-absorbing antenna to induce isomerization.¹⁰⁷ For example, Croissant et al. employed AZB nanoimpellers to trap anticancer agents inside mesoporous SiO₂ nanoparticles as E isomers, where indirect two-photon excitation (760 nm) caused E–Z isomerization, leading to cancer cell death in vitro (Fig. 12c).¹³⁷ Moreover, Peón developed a two-photon responsive Cy-SAP system that achieved NIR photoswitching (860 nm) of azaaryl Azo compounds.¹³⁸ This system comprises IR780 cyanine dye as the two-photon absorption antenna and stilbenyl-azopyrrole (SAP) as the isomerization actuator. Upon excitation, energy transfers from the antenna's excited state to the SAP unit, driving the E–Z isomerization of the Azo compound. The efficiency of this design stems from the energy transfer mechanism and the antenna's long-lived excited state, enabling effective NIR photoisomerization.

In conclusion, strategies for achieving visible-to-NIR photoisomerization of aromatic Azo compounds are shown below: (i) upconversion nanoparticles (UCNPs), (ii) two-photon absorption, and (iii) direct chemical substitution. UCNPs allow low-energy NIR excitation by converting it to high-energy emissions capable of triggering photoisomerization. They offer deep tissue penetration but involve synthetic complexity, low upconversion efficiency, and unresolved biocompatibility concerns. Two-photon absorption strategies enable spatially controlled NIR activation without nanoparticles, typically via antenna-assisted energy transfer. However, they require femtosecond lasers and most Azo compounds have inherently low two-photon cross-sections. In contrast, chemical modification approaches, such as push–pull substitution and π-conjugation extension, achieve red-shifted absorption intrinsically. These strategies avoid the need for auxiliary materials and offer greater simplicity and biocompatibility, but are limited in how far the absorption can be extended into the NIR range. Therefore, the choice of strategy depends on the intended biomedical context, balancing excitation wavelength, delivery complexity, and biological safety.

4. Photoactive aromatic Azo small molecules for biomedical applications

Due to its capability for precise spatiotemporal manipulation, light serves as an optimal non-invasive regulatory tool, particularly in modulating photoswitchable aromatic Azo systems with exceptional temporal and spatial precision.¹³⁹ Moreover, hypoxia microenvironments serve as a biologically relevant control strategy, as hypoxia-sensitive aromatic Azo compounds undergo an irreversible reductive cleavage of the Azo bond,¹⁴⁰ releasing the fluorophore or drug, demonstrating microenvironment-specific activation with high selectivity. In photopharmacology, aromatic Azo compounds serve as reversible photoswitches, enabling spatiotemporal control of drug activity through E/Z isomerization. This mechanism allows precise target modulation with minimal off-target effects.¹⁴¹ In phototherapy, they act as irreversible triggers for microenvironment-specific drug release or photothermal conversion via excited-state energy transfer, generating cytotoxic reactive oxygen species (ROS) or hyperthermia. Aromatic Azo molecules serve as molecular hinges for photoswitchable drugs while functioning as tunable photosensitizers in therapeutic contexts. For biomedical applications, aromatic Azo molecular design strategies can be categorized as follows: (i) direct use of functionalized Azo small molecules (e.g., Azo-based kinase inhibitors) achieves precise targeting but faces bioavailability challenges due to rapid clearance or poor solubility. (ii) Nanomaterial-integrated systems (e.g., Azo-COFs for controlled drug release) utilize porous frameworks to enhance drug payload, tumor targeting, and photoisomerization efficiency through confinement effects. However, they introduce complexities in biocompatibility and reproducibility.

4.1. Biological imaging and detection

Photoluminescence (PL)-enabled bioimaging offers real-time signal acquisition, enhanced detection sensitivity, and superior spatial resolution.¹⁴² Therefore, aromatic Azo compounds have garnered significant attention in the field of biological imaging due to their unique photophysical properties.¹⁹ Here, we summarize the recent advancements in the application of aromatic Azo-based probes for bioimaging and biosensing, focusing on three primary mechanisms: photoisomerization-driven structural switching, Azo bond cleavage for stimulus-responsive detection, and FRET-based signal amplification.

Aromatic Azo derivatives serve as essential units in photoactive platforms, due to their reversible E/Z photoisomerization under light irradiation, which is accompanied by fluorescence modulation (Fig. 13a).³⁹ For instance, Huang et al. developed a Azo-fluorescent switch (AzoPJ) activated by visible light, featuring a julolidine donor and pyrazole acceptor bridged by an Azo unit.¹⁴³ The E–Z photoisomerization of AzoPJ under 440/535 nm irradiation induced reversible fluorescence changes (507 nm emission) due to the suppression of twisted intramolecular charge transfer (TICT). To assess the fluorescence imaging potential of AzoPJ@NPs in biological systems, Huang et al. selected Rhizoctonia solani, a globally pathogenic fungus causing severe crop diseases. Confocal laser scanning microscope (CLSM) analysis revealed green fluorescence emission from AzoPJ@NPs within the fungal hyphae, which notably decreased upon 440 nm irradiation, demonstrating their intracellular fluorescence activity (Fig. 13b).

Fig. 13 Biological imaging applications of aromatic Azo molecules. (a) Schematic of fluorescence changes induced by photoisomerization of aromatic Azo; (b) (i) structure, photoisomerization and molecular design of AzoPJ in which the TICT process is inhibited; (ii) fluorescence spectra of AzoPJ upon emission at 440 and 535 nm (λ_ex  =  350 nm); (iii) CLSM visualization of Rhizoctonia solani treated with AzoPJ nanoparticles under dark and 440 nm light exposure conditions, capturing dynamic morphological changes via fluorescence imaging; (iv) comparative analysis of fluorescence emission profiles in Rhizoctonia solani treated with AzoPJ nanoparticles under dark versus 440 nm light-exposed conditions. Reprinted from ref. 143 with permission. Copyright 2024, Springer Nature. (c) Schematic of the cleavage of aromatic Azo. (d) (i) schematic characterization of the SDT-triggered generation of Si@NPs using Py-N=N-RC as the substrate; (ii) the corresponding fluorometric signal changes. Reprinted from ref. 146 with permission. Copyright 2024, American Chemical Society. (e) (i) Reversible hypoxia sensing mechanism of HDSF; (ii) fluorescence-based visualization of intermittent hypoxia dynamics during ischemia-reperfusion injury models through bilateral hindlimb intramuscular delivery of HDSF. Reprinted from ref. 149 with permission. Copyright 2021, Springer Nature. (f) Schematic of two types of FRET. (i) A quencher quenches a fluorophore; (ii) a quencher quenches multiple fluorophores. (g) Conceptual illustration of a peptide substrate-based fluorescent probe employing an EDANS/DABCYL FRET pair for the detection of 3CL-Pro. Reprinted from ref. 152 with permission. Copyright 2021, American Chemical Society. (h) (i) Structural diagram of 3MBP5. (ii) and (iii) The fluorescence spectra of 3MBP5 with M^pro or PL^pro showed significant fluorescence enhancement; (iv) CLSM images of HEK 293T cells incubated with PR8 (influenza virus protein), M^pro, and PL^pro plasmids. Reprinted from ref. 154 with permission. Copyright 2021, Wiley-VCH.

Azo-based probes can also respond to specific chemical reactions occurring in unique microenvironments, such as those containing enzymes, metal ions, or hypoxia, thereby serving as biomarkers for cancer diagnosis.¹⁴⁴ For example, the Azo group can undergo reduction or cleavage under hypoxic conditions, enabling the development of hypoxia-responsive fluorescent probes (Fig. 13c). Hypoxia, characterized by low oxygen levels in tissues, is a hallmark of various diseases, including solid tumors and inflammatory disorders.¹⁴⁵ Nsanzamahoro et al. developed a hypoxia-responsive probe (Py-N=N-RC) that undergoes Azo bond cleavage via azoreductase or sodium dithionite (SDT).¹⁴⁶ This reaction released 2,4-dihydroxyaniline (DHA), which reacts with 3-[2-(2-aminoethylamino)-ethylamino]propyltrimethoxysilane (AEEA) to generate yellow fluorescent silicon nanoparticles (Si@NPs) (Fig. 13d). The probe demonstrated high selectivity for hypoxia over competing analytes (e.g., ROS, metal ions). However, most reported organic fluorescent hypoxia probes were irreversible,¹⁴⁷ even though reversible NIR luminescent probes are essential for long-term in vivo tracking of cycling hypoxia.¹⁴⁸ Therefore, Zhang et al. engineered a reversible NIR probe (HDSF) featuring a 3,5-ditrifluoromethylbenzene group conjugated to a xanthene/cyanine fluorophore.¹⁴⁹ The electron -withdrawing –CF₃ groups stabilized the phenylhydrazine intermediate during hypoxia, enabling reversible Azo reduction and oxidation (Fig. 13e). This probe exhibited a 6-fold fluorescence enhancement at 705 nm under hypoxia (0.1% O₂) and successfully visualized cycling hypoxia in zebrafish embryos and murine ischemia-reperfusion models.

Fluorescent probes often employ FRET as a key mechanism for detecting biochemical processes, where aromatic Azo-based quenchers serve as versatile acceptors due to their broad absorption range and efficient FRET capabilities. These compounds effectively quench single or multiple fluorophores through FRET, making them important components in bioimaging probes (Fig. 13f).¹⁵⁰ Despite advances, the COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a significant global health challenge.¹⁵¹ In a pioneering study, Kuzikov et al. developed a probe by attaching EDANS and DABCYL to the peptide KTSAVLQSGFRKM, which serves as a specific substrate for 3CL-Pro, the primary protease of SARS-CoV-2.¹⁵² The presence of active 3CL-Pro triggered the restoration of EDANS fluorescence (Fig. 13g), enabling the identification of MG-132 as a potent inhibitor of 3CL-Pro. This discovery provides critical insights for the design of targeted COVID-19 therapeutics. Papain-like protease (PL^pro) and main protease (M^pro) are essential for SARS-CoV-2 replication, making them prime targets for antiviral therapeutics.¹⁵³ Jokerst et al. engineered a FRET-based dual-color fluorescent probe, 3MBP5, comprising five components: Cy3 fluorophore, a M^pro-responsive peptide (SAVLQ/SGFRKMA), BHQ-2 quencher, a PL^pro-responsive peptide (RLRGG/K), and Cy5 fluorophore.¹⁵⁴ The fluorescence intensity of 3MBP5 increased progressively with higher concentrations of M^pro and PL^pro under varying excitation wavelengths. CLSM experiments in plasmid-transfected HEK 293T cells confirmed that 3MBP5 can simultaneously detect M^pro and PL^pro activity in SARS-CoV-2-infected cells (Fig. 13h).

In addition to fluorescence imaging, magnetic resonance imaging (MRI) is widely employed in clinical diagnosis and prognosis due to its non-invasiveness, high spatial resolution, and deep tissue penetration.¹⁵⁵ However, its limited specificity and sensitivity constrain applications in precision medicine.¹⁵⁶ To enhance the signal-to-noise ratio in tumor MRI, Tan et al. integrated tumor microenvironment-specific enzymes with magnetic resonance tuning (MRET). An MRET-based probe with an Azo reductase-activated “switch-ON” mechanism was developed by assembling superparamagnetic nanoparticles, Gd-DOTA, and functional nucleic acids.¹⁵⁷ This intelligently designed probe selectively targeted tumors and was activated exclusively within the tumor microenvironment, enabling precise in situ imaging of small tumors. The proposed strategy has promise for the early detection of deep-seated malignancies, improved prognostic assessment, and enhanced patient compliance.

Optoacoustic (or photoacoustic) Imaging (PA) leverages non-invasiveness, micron-scale resolution, and deep-tissue penetration (>cm) for biomedical applications.^158,159 However, the limited photostability and quantitative performance of conventional molecular agents restrict their utility in precision medicine, such as enzyme activity monitoring.¹⁶⁰ To address this bottleneck, Müller et al. engineered novel PA chromophores AzHCy by merging aromatic Azo compounds with NIR-absorbing cyanine dyes.¹⁶¹ This molecular switch fusion strategy exploits the rigid π-conjugated system and ultrafast non-radiative relaxation of aromatic Azo to achieve accelerated excited-state decay (S₁ → S₀ < 10 ps). Validated via multispectral optoacoustic tomography (MSOT), the sulfonated derivative wsT-AzHCy maintained robust signal specificity in vivo for over 2 hours post-injection, demonstrating organ-distinct PA contrast. This “molecular switch integration” paradigm establishes a foundation for high-performance activatable probes, advancing tumor microenvironment imaging and precision diagnostics.

4.2. Drug release

To maximize therapeutic efficacy while minimizing systemic toxicity, precise control over drug delivery systems is paramount.¹⁶² These systems encompass diverse forms, including micro- and nanocapsules,¹⁶³ liposomes,¹⁶⁴ hydrogels,¹⁶⁵ and nanocarriers.^166,167 Among these, aromatic Azo-based systems stand out due to their dual responsiveness to light and hypoxia, offering spatiotemporal control for targeted therapeutic release at specific sites within the body.^47,168

Aromatic Azo compounds are particularly promising for light-responsive drug delivery systems due to their reversible photoisomerization properties, which function as molecular switches for controlled release.^75,169AzoPJ, developed by Huang et al., demonstrates dual functionality as both a photoisomerization-based imaging agent and a visible light-activated Azo-fluorescent switch for controlled drug delivery systems. Critically, the light-triggered E/Z isomerization of its aromatic Azo moiety enables precise spatiotemporal regulation of therapeutic release by inducing structural changes in the delivery vehicle.¹⁴³ Encapsulated within liposomes to form AzoPJ@NPs, this molecule triggered Azo-moiety-triggered disassembly of the liposomal carriers upon irradiation, as evidenced by a significant increase in particle size which facilitated drug release, providing a robust platform for light-controlled drug delivery. AzoPJ@NPs loaded with the antifungal agent flubeneteram (PEPA) achieved 40% drug release under 440 nm illumination (versus merely 3% in darkness) through this light-triggered liposomal disassembly mechanism (Fig. 14a). This study provides a novel strategy for designing imaging-guided light-controlled nano-delivery systems, highlighting the pivotal role of aromatic Azo photoswitches in advancing therapeutic precision and efficacy. Moreover, Chander et al. developed AzoPC which serves as a clinically translatable photoresponsive phospholipid that triggers light-regulated liposomal disassembly.¹⁷⁰ The light-triggered E/Z isomerization of its aromatic Azo moiety disturbs lipid bilayer packing (reducing molecular length by 4.4 Å, decreasing phase transition temperature by 3.6 °C), thereby acting as a “molecular wedge” to induce membrane destabilization. Encapsulated within DSPC-cholesterol liposomes, this molecule mediated light-triggered disassembly of lipid carriers upon 365 nm irradiation, as directly evidenced by cryo-EM showing vesicle rupture and drug expulsion. When loaded with doxorubicin, liposomes containing 10 mol% AzoPC achieved 65–70% drug release under pulsed UV light. This system was further upgraded to redAzoPC, achieving 70–75% release at 660 nm with maintained long circulation.

Fig. 14 Drug release applications of aromatic Azo molecules. (a) (i) Computational volumetrics of AzoPJ combined with spatial trajectory mapping for N-substituted groups upon N=N bond rotation; (ii) graphical depiction of the formation and photoactivated cargo release mechanism in AzoPJ-PEPA@NPs; (iii) light-triggered liberation of PEPA from AzoPJ-PEPA@NPs mediated through 440 nm activation, ambient daylight exposure, and ambient darkness. Reprinted from ref. 143 with permission. Copyright 2024, Springer Nature. (b) Schematic of drug release applications based on the cleavage of aromatic Azo. (c) (i) Light-driven activation of SA-azo-C in solution-phase environments; (ii) spectral changes of fluorescence emission during the progressing photocatalytic process shown in (i) (λ_ex = 405 nm). Reprinted from ref. 173 with permission. Copyright 2021 American Chemical Society. (d) Graphical workflow depicting the synthetic routes for TA-COF and TA-COF-P@CT. Reprinted from ref. 175 with permission. Copyright 2021, American Chemical Society. (e) Scheme of single molecular nanomedicine. Reprinted from ref. 176 with permission. Copyright 2024, American Chemical Society.

The hypoxic microenvironment facilitates selective Azo bond cleavage through elevated azoreductase activity or specific light conditions.¹⁷¹ This enzymatic or photolytic mechanism enables targeted drug release in hypoxic regions, such as tumors, by leveraging the unique biochemical milieu. Azo bonds serve as hypoxia-responsive linkers, ensuring precise drug activation while mitigating systemic toxicity (Fig. 14b).¹⁷²

Zhao et al. developed a red light-activated photocatalytic system utilizing methylene blue derivatives (NMB⁺) to cleave Azo bonds in both aqueous solutions and hypoxic cells.¹⁷³ The process involved forming a charge transfer complex between reduced NMB⁺ (LNMB) and Azo substrates, enabling electron transfer upon red light excitation. This mechanism triggered the photo-uncaging of fluorescent coumarin 120, as evidenced by a gradual increase in fluorescence over time (Fig. 14c). This approach enables precise drug release in hypoxic tumor environments, activating Azo prodrugs like olsalazine to release active drugs such as mesalazine, offering a novel bioorthogonal strategy for biomedical applications.

Covalent organic frameworks (COFs) with hypoxia-responsive properties have also been explored for advanced drug carriers.¹⁷⁴ Ge et al. synthesized an Azo bond-containing COF (TA-COF) for hypoxia -responsive drug delivery, encapsulating the photosensitizer chlorin e6 (Ce6) and the hypoxia-activated drug tirapazamine (TPZ).¹⁷⁵ Hypoxia triggered COF disintegration, releasing the drugs, while NIR light enhanced hypoxia by generating reactive oxygen species (ROS) viaCe6, promoting TPZ activation (Fig. 14d). This tandem mechanism significantly improves anticancer efficacy, as demonstrated in vitro and in vivo, enabling targeted drug release in hypoxic tumors with minimal off-target effects. Building on this concept, Yao et al. introduced the single-molecule nanomedicines (SMNMs) using macrocyclic carrier-drug conjugates for precise drug delivery.¹⁷⁶ By covalently linking chemotherapy drugs (SN38 or DOX) to a hypoxia-cleavable azocalix[4]arene carrier, the authors achieved self-included complexes that effectively encapsulate and shield the drugs (Fig. 14e). These SMNMs prevent premature drug leakage and off-target activation, significantly reducing side effects. Hypoxia-triggered drug release in tumor microenvironments enhances therapeutic efficacy, with in vivo studies demonstrating superior anticancer activity and reduced toxicity, positioning SMNMs as a promising platform for advanced drug delivery.

4.3. Photopharmacology

Photopharmacology represents a rapidly evolving therapeutic strategy, with its reversible modality demonstrating considerable promise as an investigative platform for pharmacochemical and biological systems.^177–179 The most common tools of reversible photopharmacology are E–Z photoswitches,¹⁸⁰ which undergo isomerization between the E and Z isomer upon irradiation with light.¹⁸¹ In particular, aromatic Azo molecules are the most frequently used switches for the reversible optical regulation of biological function, leveraging the inherent bioactive properties and hydrophilic characteristics of heterocyclic compounds.⁹² These compounds undergo reversible photoisomerization upon light irradiation, where light exposure at λ₁ triggers transient drug activation through photoisomerization to a thermodynamically unstable isomer, with activity decay occurring either spontaneously or via subsequent λ₂ light-triggered reversion (Fig. 15a).¹⁸² This modification enhances spatiotemporal precision in drug activity regulation and helps it function within physiological environments. The application of aromatic Azo compounds in photopharmacology can help to develop drugs with few side effects and low toxicity.¹⁸³

Fig. 15 Photopharmacological applications of aromatic Azo molecules. (a) Schematic representation of the principle of photopharmacology where photoisomerization of UV light and NIR light for aromatic Azo is included. Reprinted from ref. 182 with permission. Copyright 2023, Wiley-VCH. (b) (i) Photoinduced E/Z isomerization of azobenzene modulates agonist-potentiation efficacy and enables optical regulation of GABA_AR channel gating, while ΔT quantifies thermal Z to E relaxation kinetics under dark conditions; (ii) neuronal signaling must remain unperturbed under ambient darkness or green light exposure, while neuroinhibition should be elicited on demand through violet light activation within targeted neuroanatomical zones. Reprinted from ref. 186 with permission. Copyright 2024, American Chemical Society. (c) Phenylazobenzimidazole molecule as a visible-light-controlled molecular probe to activate the βarr2 pathway while not activating G protein pathways at CB₂R. Reprinted from ref. 187 with permission. Copyright 2023, Copyright 2023 Wiley-VCH. (d) (i) Chemical structures of photoisomers of HDACis; (ii) concentration-dependent cytotoxic profiles of HDAC inhibitors in HeLa cells were assessed via MTS viability assay following 48-hour exposure to dark or 550 nm light pre-illumination protocols. Reprinted from ref. 42 with permission. Copyright 2023, American Chemical Society. (e) (i) Chemical structures of Q-Azo4F-C in E or Z forms; (ii) antiproliferative activity of Q-Azo4F-C on HeLa using both E and Z-rich PSS, followed by evaluation post-photoconversion into the E-rich PSS. Reprinted from ref. 190 with permission. Copyright 2024, Wiley-VCH.

Photopharmacology leverages light-responsive molecules of aromatic Azo to achieve spatiotemporal control over biological processes,¹⁸⁴ offering a powerful tool for studying receptor dynamics and signalling pathways.¹⁸⁵ Recently, Maleeva et al. developed azocarnil, a novel Azo-based agonist-potentiator of Gamma aminobutyric acid type A receptor (GABA_AR), to achieve reversible neural inhibition in wild-type mice.¹⁸⁶Azocarnil incorporated a benzodiazepine-like scaffold with an Azo linker, enabling its activity to be controlled by visible violet light (400 nm) and deactivated with green light (505 nm). The compound exhibited a “E-ON” efficacy, with the E isomer selectively inhibiting neuronal currents in hippocampal neurons and spinal cord dorsal horns, reducing mechanical sensitivity in mice without systemic adverse effects (Fig. 15b). This study highlights the potential of aromatic Azo as a photopharmacological tool for treating conditions like muscle spasms, pain, and epilepsy. In another study, Steinmüller et al. explored the photopharmacological potential of benzimidazole Azo-arenes as selective cannabinoid 2 receptor (CB₂R) agonists.¹⁸⁷ They designed a series of benzimidazole derivatives with Azo linkages to enable visible light-induced isomerization, achieving selective activation of the β-arrestin2 (βarr2) pathway. This compound, a “E-ON” agonist, demonstrated βarr2-biased signaling without activating G protein pathways, as evidenced by CB₂R receptor internalization assays and ERK phosphorylation studies (Fig. 15c). The structural incorporation of the Azo bond into the benzimidazole scaffold allowed for precise control over receptor activation, with the E isomer exhibiting enhanced efficacy in βarr2 recruitment.

The lack of selectivity in anticancer drugs remains a significant limitation in chemotherapy. Light-activatable drugs, which can be precisely controlled using external light, offer a promising approach for localized drug action in tumors.¹⁸⁸ Josa-Culleré and Llebaria introduced a series of photoswitchable Azo-based histone deacetylase inhibitors (HDACis) that can be activated by visible light.⁴² The aromatic Azo moiety was incorporated into the linker region of HDAC inhibitors, which resulted in compounds that were >50-fold more active under illumination than in the dark during enzyme assays (Fig. 15d). Optimization of the AZB structure by introducing ortho-halogen atoms enabled activation under green light, which was more tissue-penetrable than ultraviolet light. Selected compounds reduced cell viability only under illumination in various cancer cell lines, demonstrating their potential as photoswitchable anticancer drugs.

G-quadruplex (G4) DNA structures are emerging as potential targets in cancer research due to their role in regulating key biological processes such as DNA replication and transcription.¹⁸⁹ Dudek et al. developed a fluorinated AZB photoswitch, Q-Azo4F-C, equipped with a quinoline unit and a positively charged carboxamide side chain, designed to target G4 structures in live cells.¹⁹⁰ The photochemical properties of Q-Azo4F-C allowed near-quantitative isomerization between E and Z states using visible light, making it suitable for in-cell applications. Dudek used visible light to explore regulating cytotoxicity through the state interconversion of the switch, which confirmed that the Z isomer significantly increased G4 levels in cancer cells, correlating with enhanced cytotoxicity (Fig. 15e). The study demonstrated reversible photoregulation of G4 structures and cytotoxicity in lung cancer cells, highlighting the potential of light-responsive G4 binders for precision cancer therapy.

Moreover, Alzheimer's disease (AD), a dementia subtype straining healthcare systems,¹⁹¹ targets Aβ aggregates¹⁹² whose photoactivatable modulation via light-sensitive aromatic Azo compounds enables spatiotemporally precise therapeutic intervention. Doran et al. engineered Aβ42 residues 25–27 with a photoswitchable β-hairpin mimetic (AMPP), where the E isomer replicated cytotoxic wild-type fibrillar assemblies, whereas the Z isomer induced nontoxic amorphous aggregation.¹⁹³ Photoisomerization-mediated bidirectional modulation of amyloidogenesis enabled dynamic interconversion between fibrillar and nonfibrillar Aβ states, offering spatiotemporal control over neurotoxic pathway manipulation. Recently, Qian et al. engineered photoisomerizable azobenzene clamps (LMTs) with terminal KLVFF (Aβ16-20) motifs, where Z isomer states enabled oligomer-selective capture via clamp-like spatial contraction, contrasting with E state inactivity.¹⁹⁴ In C. elegans models, Z-LMT1 demonstrated Aβ dimer-specific binding via biotin–streptavidin pulldown, conferring neuronal protection and ameliorating motility deficits through Aβ sequestration, highlighting the precision AD therapeutic potential of aromatic Azo via structural dynamism.

Critically, the photopharmacological strategy extends beyond neurodegenerative diseases to antimicrobial therapy. Just-Baringo et al. recently developed a tetra-ortho-chloro-azobenzene amino acid (CEBA) that enables exclusive visible-light control of antibacterial activity.¹⁹⁵ Incorporated into linear tyrocidine A analogues, CEBA's E–Z isomerization under red light (650 nm) activates antimicrobial function against clinically relevant strains like Acinetobacter baumannii, while rapid reversion to inactive trans states occurs within minutes of sunlight exposure. This self-deactivation mechanism significantly reduces environmental antibiotic persistence and evolutionary pressure, a key advantage over UV-dependent systems. Structural optimization of peptide termini further yielded analogues with strain-selective activity. Moreover, azobenzene-quaternary ammonium salt (QAS)¹⁹⁶ conjugates, which have the advantages of easy chemical modification, simple synthesis, and fast photo-isomerization,¹⁹⁷ were developed as photo-switchable antibacterial agents¹⁹⁸ to address antibiotic resistance and environmental persistence.¹⁹⁹ By integrating light-responsive azobenzene with membrane-targeting QAS moieties, Zhang et al. synthesized Azo-QAS, which exhibited E and Z isomerization under 365 nm irradiation, enhancing aqueous solubility and antimicrobial potency through improved bacterial membrane interaction.²⁰⁰ The Z isomer's polarized structure facilitated QAS insertion into microbial membranes, disrupting lipid bilayers through electrostatic interactions and alkyl chain penetration. This photoregulated mechanism enabled reversible activation cycles without inducing resistance in Staphylococcus aureus or Escherichia coli during 30-day exposure.

The previously discussed techniques necessitate chemical alterations to the molecular framework, which significantly modify its core architecture. Such approaches render the optimization of photopharmacological ligands both time-consuming and molecule-specific. Methods of pursuing deuteration that can be broadly applied to any aromatic Azo-based system without the need for compound-specific engineering are, thus, needed.²⁰¹ In this context, Roβmann et al. introduced deuterated AZB photoswitches as a universal strategy to enhance the efficacy of photopharmacological agents, enabling precise optical regulation of ion channels and G protein-coupled receptors (GPCRs) within living cells.²⁰² Notably, deuteration augments AZB functionality while preserving the fundamental structure of the photopharmacological ligand.

4.4. Phototherapy

Aromatic Azo molecules have recently emerged as promising agents in PDT and photothermal therapy (PTT) due to their hypoxia-sensitive cleavage properties and structural versatility.²⁰³ Their unique Azo linkage (N=N) enables the conjugation of photosensitizers, photothermal agents, chemotherapeutic drugs, or targeting moieties, allowing targeted release via selective N=N bond cleavage in hypoxic environments (Fig. 16a). This dual-release mechanism, coupled with their adaptable chemical design, makes them highly attractive for precise cancer therapy and diverse biomedical applications.

Fig. 16 PDT/PTT applications of aromatic Azo molecules. (a) Strategies for connecting Azobenzene with photosensitizers in PDT/PTT. (b) Conceptual framework outlining the engineering strategy for oxygen-level-switchable type I photosensitizers with reversible hypoxia–normoxia responsiveness. Reprinted from ref. 206 with permission. Copyright 2023, Wiley-VCH. (c) Illustration of the synthesis of HRCOFs and HRCOFs-mediated synergistic PDT/PTT to heal bacterial infection wounds. Reprinted from ref. 210 with permission. Copyright 2024, American Chemical Society. (d) Design of the theranostic platform DHQ-Cl-Azo. Reprinted from ref. 213 with permission. Copyright 2022, Elsevier Inc. (e) Illustration of self-amplified immune therapy strategies. Reprinted from ref. 217 with permission. Copyright 2025, Wiley-VCH.

PDT, a non-invasive and highly effective cancer treatment, relies on reactive ROS generated by photosensitizers (PSs).²⁰⁴ However, conventional “always-ON” PSs often cause significant phototoxic side effects due to uncontrolled ROS generation.²⁰⁵ To overcome this limitation, Li et al. developed a hypoxia-responsive switchable PS, TPFN-AzoCF₃, combining a hypoxia–normoxia cycling unit (AzoCF₃) with a type I PS (TPFN).²⁰⁶ The arylazo group served as a reversible OFF–ON–OFF switch: under hypoxia, it converted to a hydrazine group to activate ROS production, while rapid E–Z isomerization under normoxia dissipated the excited-state energy of PSs for biosafety (Fig. 16b). This innovative design enables precise, on-demand ROS generation, minimizing side effects while enhancing therapeutic efficacy.

Bacterial infections, a major global health threat, have driven the exploration of advanced therapeutic strategies.²⁰⁷ PDT has emerged as a promising approach due to its non-invasiveness, broad-spectrum antimicrobial activity, and drug resistance mitigation.²⁰⁸ However, its efficacy is hindered by hypoxia and elevated glutathione (GSH) levels in wound microenvironments.²⁰⁹ To address this, Zhang et al. engineered hypoxia-responsive covalent organic frameworks (HRCOFs) integrating porphyrin photosensitizers and azobenzene groups.²¹⁰HRCOFs enabled both PDT and PTT, with the aromatic Azo moiety cleaved by overexpressed Azo reductase in wounds, ensuring biodegradability and biocompatibility. Under irradiation, HRCOFs generated singlet oxygen (660 nm) and thermal energy (808 nm), exhibiting potent synergistic antibacterial activity against Staphylococcus aureus and Escherichia coli (Fig. 16c). This dual-modal approach overcomes microenvironmental limitations, providing great value for promoting wound healing.

PDT has emerged as a promising approach in oncology,²¹¹ yet its efficacy is compromised by the hypoxic microenvironment of solid tumors.²¹² Kim et al. developed a theranostic molecule, DHQ-Cl-Azo, combining hypoxia-responsive chemistry and PDT capabilities for the cooperative treatment of solid tumors.²¹³ Constructed from a NIR rhodol-based fluorophore (DHQ-Cl) and a chemotherapeutic drug, nitrogen mustard linked by a hypoxia-sensitive Azo bond, DHQ-Cl-Azo released the fluorophore and chemotherapeutic drug upon reduction in hypoxic tumor regions. This dual-functional system enabled the PDT-mediated destruction of normoxic outer tumor cells while activating chemotherapy to eliminate hypoxic core cells, significantly improving therapeutic outcomes in murine models (Fig. 16d). By integrating imaging and hypoxia-sensitive dual-modality therapies into a single molecular platform, DHQ-Cl-Azo represents an innovative strategy to enhance PDT efficacy and address the challenges of solid tumor treatment.

Pyroptosis, a highly inflammatory form of programmed cell death (PCD), was identified in 1992 and characterized by membrane pore formation, cell swelling, and rupture.²¹⁴ Traditional pyroptosis of chemotherapeutics drugs suffers from drug resistance and side effects.²¹⁵ PDT-induced pyroptosis, though promising, often causes non-specific tissue damage due to “always-ON” photosensitizers.²¹⁶ To address these limitations, Zhang et al. designed HDIM, the first activatable pyroptosis-based photosensitizer immune-prodrug, combining an NIR photosensitizer (HDI-ER) and an immune checkpoint inhibitor drug (PD-1 inhibitor) via an Azo bond.²¹⁷ In hypoxic tumor microenvironments, the Azo bond of HDIM was cleaved, releasing HDI-ER and PD-1 inhibitors to monitor and initiate the tumor's immunotherapy (Fig. 16e). Under NIR irradiation, HDIM triggered both PDT-induced pyroptosis and immunotherapy, enhancing tumor immunogenicity and immune cell recruitment, exhibiting significantly superior tumor treatment.

4.5. Miscellanea photo responsive biomaterials and constructs

The pursuit of spatiotemporal precision in regulating biological systems, from subcellular processes to antimicrobial targeting, drives innovation in photoresponsive biomaterial design. Traditional approaches frequently lack the necessary accuracy to replicate native extracellular microenvironments, manipulate intracellular dynamics, or achieve targeted antimicrobial effects without inducing systemic toxicity and resistance development.^218–221 Aromatic Azo-based platforms address these limitations by exploiting reversible light-induced E–Z isomerization,^222,223 enabling on-demand spatiotemporal control over diverse biological interfaces.^224,225 This versatile photomechanical strategy facilitates heterogeneous constructs, spanning polymers, nanosheets, cyclodextrin complexes, and glycolipid analogs, with tailored functionalities. In cellular engineering, Azo systems precisely modulate morphology, membrane properties, and protein localization.²²⁶ Conversely, for antimicrobial applications, photoisomerization-triggered structural and dipole realignment disrupts bacterial membrane permeability,²²⁷ while Azo-drug conjugates allow spatially resolved antibiotic activation.^9,228 Recent advances extend to smart antibacterial surfaces²²⁹ and engineered large organic molecules^230,231 for combating biofilm-associated infections.²³² Collectively, these photo-responsive constructs bridge fundamental mechanisms with translational applications, demonstrating potential across biomaterial science and precision medicine.

4.5.1. Polymer. In cellular engineering applications where Azo systems modulate morphology and signaling,²²⁶ the extracellular matrix (ECM) offers a dynamic and reversible environment essential for morphogenesis, repair, and differentiation.²³³ Emulation of this intricate system through photoresponsive platforms can elucidate cellular mechanisms and enable precise control of cell behaviors. Sethi et al. engineered a photoswitchable mechanical DNA polymer with azobenzene groups to dynamically regulate the distance between cell adhesion peptides.²³⁴ The polymer, composed of a DNA scaffold with Azo-modified strands, underwent reversible conformational changes under UV (365 nm) and visible light (450 nm). UV light triggered Z isomerization, contracting the polymer and reducing peptide spacing, while visible light extended the polymer, increasing spacing (Fig. 17a). This photoresponsive modulation of the DNA polymer's mechanical properties induced reversible transitions between round and spindle-shaped cell morphologies, illustrating how heterogeneous biomaterials can recapitulate ECM-mediated cellular dynamics with molecular precision.

Fig. 17 Miscellanea photo responsive biomaterial and constructs. (a) Illustrative model of light-modulated mechano-DNA architectures demonstrating cellular morphological transitions through conformational switching of polymeric DNA assemblies. Reprinted from ref. 234 with permission. Copyright 2021, Wiley-VCH. (b) Schematic representation of selective antibacterial activity of photo-switchable Azo-MoS₂. Reprinted from ref. 240 with permission. Copyright 2023, Wiley-VCH. (c) Hypothetical model of photo-responsive host–guest complex 3a@β-CD. Reprinted from ref. 244 with permission. Copyright 2023, Wiley-VCH. (d) Multifunctional schematic representation of supramolecular polymer brush architectures, the chemical structures, and the photo-switching nature of the host–guest interaction. Reprinted from ref. 245 with permission. Copyright 2022, Royal Society of Chemistry.

Light-responsive nanomaterials provide innovative solutions to overcome the challenges of traditional antimicrobial agents, such as nonselectivity, drug resistance, and environmental toxicity, through precise spatiotemporal control. We will next focus on three representative architectures, nanosheets, cyclodextrin inclusion complexes, and glycolipid analogs which combine with aromatic Azo, to elucidate their design principles and antibacterial mechanisms.

4.5.2. Nanosheets. The lack of selectivity in conventional antimicrobial agents poses a critical challenge,²³⁵ often leading to microbial dysbiosis and accelerated resistance development.^236,237 Light-responsive nanomaterials provide advantages like precise spatiotemporal regulation, non-invasive and convenient operation, and user-friendly handling, which enhance their suitability for biomedical uses.^238,239 Sahoo et al. developed a photo-switchable 2D molybdenum disulfide (MoS₂) nanosheet functionalized with cationic azobenzene (Azo-MoS₂), enabling selective bactericidal activity against Gram-positive or Gram-negative bacteria via light-triggered isomerization.²⁴⁰ The trans-conformation (E-Azo-MoS₂) exhibited potent activity against Gram-negative pathogens through electrostatic interactions and intracellular ROS generation, while the cis-conformation (Z-Azo-MoS₂), activated by UV light, preferentially targeted Gram-positive bacteria via hydrophobic interactions and membrane disruption (Fig. 17b). In vivo evaluation using infected murine models demonstrated accelerated wound healing under Z-Azo-MoS₂ treatment, outperforming conventional antibiotics. This dual-mode, light-gated platform offers a reversible and strain-specific antimicrobial approach, addressing mixed infections while mitigating resistance risks.

4.5.3. Cyclodextrin. The indiscriminate use of conventional pesticides frequently induces bacterial resistance and environmental toxicity.^241,242 Light-responsive supramolecular systems also demonstrate superior spatiotemporal control for targeted antimicrobial therapy.²⁴³ Yang et al. developed a β-cyclodextrin-azobenzene inclusion complex (3a@β-CD) exhibiting photo-switchable antibacterial activity through reversible host–guest interactions (Fig. 17c).²⁴⁴ The E azobenzene derivative displayed enhanced biofilm inhibition against phytopathogens through structural isomerization under UV irradiation. β-CD encapsulation improved aqueous solubility and foliar adhesion while enabling controlled release of Z isomers with superior antimicrobial efficacy. This supramolecular system reduced Xanthomonas oryzae pathogenicity by 51.22–55.84% in vivo through biofilm disruption mechanisms, significantly outperforming commercial bactericides. Notably, the formulation maintained low cytotoxicity (IC₅₀ > 62 μM) towards non-target organisms, establishing an eco-compatible platform for resistance management in agricultural applications. Moreover, Zheng et al. engineered a photo-responsive polymer brush system for dynamic surface modulation (Fig. 17d).²⁴⁵ Under visible light (450 nm), E azobenzene maintains stable host–guest complexes with cyclodextrin, enabling simultaneous surface antifouling and contact-killing. UV irradiation (365 nm) triggers Z isomer-induced polymer desorption, releasing 85.1% surface-adhered bacteria. In addition, the Harada group engineered light-responsive hydrogels via cyclodextrin-azobenzene host–guest recognition. Tetra-armed poly(ethylene glycol) bearing azobenzene moieties (guest) formed dynamic networks with cyclodextrin hosts. UV-induced E → Z isomerization disrupted host–guest binding, causing hydrogel contraction. Visible-light-driven reversal restored crosslinking, enabling macroscopic shape-memory effects.²⁴⁶

4.5.4. Glycolipid analog. Mycobacterial infections, exemplified by Mycobacterium tuberculosis,²⁴⁷ present significant treatment challenges due to their complex cell envelope and antibiotic resistance mechanisms.²⁴⁸ While current antibiotics block critical biosynthetic pathways of the cell envelope, their indirect effects on bacterial membrane dynamics remain poorly understood.²⁴⁹ To address this, Kiessling et al. developed N-QTF, a fluorogenic probe utilizing an aromatic Azo-based quencher paired with a fluorophore.²⁵⁰ This design enables specific reporting of mycolyltransferase activity: enzymatic cleavage of the trehalose core by Ag85 complexes releases the fluorophore from the Azo-quencher, triggering fluorescence turn-on exclusively upon probe activation. The optimized glycolipid analog demonstrated enhanced hydrolytic stability compared to ester-linked predecessors, enabling real-time visualization of membrane remodelling under antibiotic stress. Notably, rifampicin (RIF)-treated mycobacteria exhibited unique fluorescent extracellular vesicles (EVs) containing immunomodulatory cAMP metabolites. These RIF-induced EVs significantly reduced macrophage cytokine production, revealing an unexpected antibiotic-mediated immune modulation pathway through altered EV composition.

Moreover, the synergistic interplay of photochromic molecules within supramolecular frameworks establishes a novel paradigm for functional materials development. Stefan Hecht recently proposed the “multi-switch cooperativity” concept, wherein precisely engineered spatial arrangements and interactions among multiple photoresponsive units enable orthogonal modulation, signal amplification, and dynamic reconfiguration, providing a new strategy for dealing with cooperative multi-azosystems or photoswitches embedded in supramolecular architectures.²⁵¹ Light can be used to steer the aggregation of nanoparticles through modulating their surface energy, and the Klajn group achieved wavelength-selective control through photoresponsive gold nanoparticles. Small nanoparticles functionalized with electron-rich aminoazobenzene underwent E → Z isomerization under 420 nm blue light, triggering solvophobic aggregation. Larger nanoparticles modified with pristine azobenzene responded independently to 365 nm UV light. Alternating irradiation enabled orthogonal assembly/disassembly cycles, establishing a hierarchical photoregulation platform for microenvironment engineering.²⁵²

4.6. Aromatic Azo-compounds for control in chemical biology

The precise manipulation of biomolecular functions, spanning peptides, proteins, and nucleic acids, demands molecular tools capable of high spatiotemporal resolution with minimal perturbation to native systems.^141,253 Aromatic Azo-compounds have emerged as pivotal photoresponsive platforms in chemical biology, leveraging reversible E–Z photoisomerization to dynamically regulate biological activities.^254,255 This photochemical switching mechanism offers unique advantages: rapid kinetics, exceptional fatigue resistance, and tunable spectral properties. Critical to their biological utility is the ability to engineer aromatic Azo for biocompatibility and functional precision. Strategic halogenation (e.g., tetra-ortho-fluoro substitution) red-shifts absorption spectra beyond UV,^256,257 mitigating phototoxicity while enhancing tissue penetration. Concurrently, modular synthetic approaches allow covalent integration into biomolecular scaffolds via click chemistry, unnatural amino acid incorporation, or intercalative binding, enabling targeted control like protein, peptide and nucleic acids.

4.6.1. Proteins. Artificial control of intracellular protein dynamics provides critical insights into complex biomolecular networks.²⁵⁸ Optogenetics and caged compound-based chemically induced dimerization (CID) systems enable spatiotemporal regulation of protein activity.²⁵⁹ Mashita et al. developed a photochromic CID system comprising two protein tags and a dimerizer (pcDH).²⁶⁰ The pcDH combined an Azo-based photochromic ligand for Escherichia coli dihydrofolate reductase (eDHFR) and a HaloTag ligand, enabling light-dependent protein dimerization to manipulate subcellular protein localization. Violet light (405 nm) induced Z isomerization, promoting pcDH binding to eDHFR and recruiting target proteins to specific subcellular compartments, while green light (555 nm) reverses the process (Fig. 18a). Applied to mitophagy regulation, this system achieved optical control of PTEN-induced kinase 1 (PINK1) trafficking, demonstrating spatiotemporal precision in probing protein-network dynamics.

Fig. 18 Aromatic Azo-compounds for control in chemical biology. (a) Conceptual representation of a light-responsive CID platform driven by ligand photoisomerization dynamics. Reprinted from ref. 260 with permission. Copyright 2024, Springer Nature America, Inc. (b) Schematic of Azo-PROTAC for Protein Knockdown. Reprinted from ref. 262 with permission. Copyright 2020, American Chemical Society. (c) Red-shifted azobenzenes as photo-switchable ion transporters. Schematic representation of the photo-switching of a supramolecular ion carrier. Reprinted from ref. 265 with permission. Copyright 2021, Royal Society of Chemistry. (d) Outline of the photocontrollable nucleosome targeting approach based on visible-light photoswitchable DNA binders and the structures of the studied molecules. Reprinted from ref. 266 with permission. Copyright 2019, Royal Society of Chemistry.

The reversible regulation of endogenous protein levels remains a significant challenge.²⁶¹ Jiang et al. developed Azo-based proteolysis-targeting chimeras (Azo-PROTACs) comprising an azobenzene linker bridging a ligand for the E3 ubiquitin ligase cereblon (lenalidomide) and a target protein ligand (dasatinib).²⁶² The aromatic Azo functioned as a photoswitch, with E and Z isomers modulating the spatial arrangement and activity of the PROTAC (Fig. 18b). UV light triggered E → Z isomerization, inducing protein degradation, while visible light reversed the process, enabling temporal control over protein knockdown. Azo-PROTAC combines potent protein knockdown and reversible photoswitchability, leveraging light-induced ON–OFF properties for precise protein regulation, offering a promising strategy for chemical biology applications. Moreover, Volarić et al. developed a tetra-ortho-fluoro-azobenzene photoswitch that enables visible-light regulation of pore-forming protein fragaceatoxin C (FraC) activity.²⁶³ Site-specific conjugation via nucleophilic aromatic substitution incorporated sulfonate groups for enhanced water solubility. The Z-isomer (activated by 530 nm green light) significantly boosted FraC-mediated cytolytic activity, while the E-form (430 nm blue light) suppressed activity. This light-gated control induced reversible cytotoxicity in human hypopharyngeal carcinoma cells, demonstrating precise modulation of toxic protein bioactivity.

4.6.2. Peptide. The dynamic regulation of peptide self-assembly and signaling pathways demands molecular tools capable of precise spatiotemporal resolution.²⁶⁴ Kerckhoffs et al. engineered tetra-ortho-halo azobenzene-squaramide conjugates that serve as visible-light-responsive switches for modulating peptide-mediated ion transport.²⁶⁵ These conjugates undergo reversible E–Z photoisomerization under green (530 nm)/red (625 nm) and blue light (450 nm), achieving high Z-isomer photostationary states (77–80%). The Z-isomer adopts a compact conformation where squaramide groups form intramolecular hydrogen bonds, creating a cooperative anion-binding pocket with enhanced chloride affinity (Fig. 18c). Molecular dynamics simulations confirm that this conformation stabilizes the complex at lipid bilayer interfaces, facilitating integration into peptide-based ion channels.

4.6.3. Nucleic acids. The dynamic modulation of DNA structure and chromatin accessibility is key to regulating genome functions and deciphering epigenetic mechanisms.²⁶³ Heinrich et al. engineered a novel photoswitchable ortho-fluoroazobenzene DNA binder F₄Azo-(PyDp)₂ that enables visible-light-regulated DNA interactions.²⁶⁶ These compounds undergo reversible E–Z photoisomerization under visible light (405/520 nm), with the E-isomer exhibiting stronger DNA-binding affinity than the Z-form. This conformational switch directly modulates intercalation strength into AT-rich sequences and alters nucleosome stability. Critically, the E-isomer disrupts nucleosome integrity, while the Z-form minimizes structural perturbation (Fig. 18d). This visible-light-responsive system provides a non-invasive tool for spatiotemporal control of chromatin dynamics, establishing a foundation for optoepigenetic applications in gene regulation.

5. Conclusion and prospects

Aromatic Azo photoactive small molecules have undergone remarkable progress in recent years, driven by their exceptional tunability in photophysical properties and light-responsive structural design. This review systematically discusses the rational design principles governing their photoisomerization mechanisms, synthetic strategies, and diverse biomedical applications, emphasizing their pivotal role as molecular bridges between chemical engineering and biomedicine. Heteroaryl Azo compounds exhibit distinct advantages over traditional azobenzenes, such as enhanced thermal robustness of the Z isomer, red-shifted absorption spectra,^61,94,96 and tailored bioactivity through heterocyclic functionalization. While significant advancements have been made in tuning photophysical properties and leveraging these molecules in biomedical applications, critical bottlenecks remain that limit their full translational potential like biocompatibility concerns.

Regarding this, the high biocompatibility of aromatic Azo dyes has enabled their widespread use in developing fluorescent imaging systems for detecting diverse biological analytes and enzymes.²⁶⁷ Further enhancing their biosafety, Azo compounds can be encapsulated into nanoparticles, which significantly improves their biocompatibility by reducing unintended interactions with biological matrices.¹⁴³ Additionally, aromatic Azo motifs can be integrated with β-cyclodextrin to construct smart photo-responsive supramolecular delivery systems which demonstrated minimal off-target toxicity toward non-target organisms and substantially enhanced both biocompatibility and bioavailability through controlled, stimulus-activated release mechanisms.²⁶⁸ Metabolic pathways of Azo compounds in biological systems involve three primary mechanisms: (i) photoisomerization: reversible E/Z transitions under light exposure, enabling spatiotemporal control of bioactivity. (ii) Enzymatic redox reactions: cytochrome P450 isoforms (CYP3A4/CYP2E1) catalyze oxidative cleavage to aromatic amines; Azoreductases utilize NAD(P)H for reductive degradation under anaerobic conditions. (iii) Microbial degradation: biohybrid systems enhance electron transfer and metabolic activity, accelerating Azo dye decolorization through synergistic bio-reduction.²⁶⁹ Rational molecular design and supramolecular engineering, such as introducing specific functional groups or construction of supramolecular systems, effectively optimize the biocompatibility and metabolic kinetics of aromatic Azo compounds. These strategies may expand their utility in biomedical and environmental applications.

Moving forward, several underexplored directions emerge (Fig. 19). (1) Expanding the diversity of aromatic Azo compounds through innovative synthetic methodologies, such as late-stage modifications,²⁷⁰ or enzyme-catalyzed cascade reactions,²⁷¹ could reveal unprecedented photophysical properties and enable the rational design of bioactivities with tailored specificity. Stereoselective synthesis may further achieve predictable regioselectivity. (2) The integration of computational tools, including machine learning-guided molecular dynamics simulations with quantum chemical calculations (e.g., TD-DFT,²⁷² CASSCF²⁷³), will expedite the rational design of molecules with tailored properties, such as extended NIR absorption, enhanced QY, or improved stability. Theoretical predictions with computational and machine learning approaches can effectively minimize experimental trial and error, thereby significantly reducing experimental expenditures. (3) Advancing biocompatible aromatic Azo systems, which are critical for clinical translation, demands addressing structure-toxicity relationships (STRs),²⁷⁴ screening of enzymatic degradation,²⁷⁵ and in vivo clearance mechanisms,²⁷⁶ minimizing off-target effects and long-term toxicity. (4) Hybridizing these molecules with nanotechnology²⁷⁷ (e.g., supramolecular Azo-nanocarriers with stimuli-triggered cargo release) or bioorthogonal probes¹⁷³ may enhance targeting precision, multifunctionality, and therapeutic efficacy. (5) The potential of aromatic Azo compounds as quenchers remains underexplored. Notably, their hypersensitive multiplexing capability, wherein a single quencher can simultaneously and effectively quench three or more fluorophores, may offer revolutionary solutions for molecular beacon design,²⁷⁸ dynamic bioimaging,²⁷⁹ or multi-target detection.¹⁵⁴ This approach demonstrates unique advantages in minimizing signal crosstalk and improving detection throughput. In addition, integrating aromatic Azo motifs into photoactive transition–metal complexes may serve as stimuli-responsive materials for optoelectronic switches and molecular machines because of reversible photoisomerization-driven luminescence switching and dynamic energy transfer modulation.²⁸⁰

Fig. 19 Outlook of aromatic Azo with several promising directions.

In conclusion, aromatic Azo photoactive small molecules stand at the forefront of photo-responsive biomaterials, offering opportunities in precision medicine and smart therapeutics. As the field evolves, its rational design and innovative application will continue to make critical contributions to the development of biomedical technologies, ultimately enhancing diagnostics, treatments, and patient outcomes. By embracing both fundamental exploration and translational innovation, this vibrant field may provide immense promise for biomedical applications of aromatic Azo photoactive small molecules.

Author contributions

B. F., L. L., and W. H. conceived and designed the review. J. J. D. and Z. H. wrote the initial draft. J. J. D., Z. H., D. T. Z., Y. W. Q., S. J. Z., and C. C. Z. reviewed, edited, and wrote the manuscript. B. F., L. L. and W. H. supervised the entire review. All the authors contributed to the discussion and manuscript preparation.

Conflicts of interest

The authors declare no competing financial interests.

Data availability

No primary research results, software or code have been included and no new data was generated or analyzed as part of this review.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (62288102 to W. H., 22577108 to L. L.), the Fujian Provincial Natural Science Foundation of China (2024J01060 to L. L.), the Postdoctoral Fellowship Program of CPSF (GZC20240889 to B. F.), the China Postdoctoral Science Foundation (2025M77286 to B. F.), the Startup Program of XMU (L. L.), and the Fundamental Research Funds for the Central Universities (W. H. and L. L.).

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Footnote

† These authors contributed equally to this work.

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