Light-Triggered Click Chemistry

Abstract

The merging of click chemistry with discrete photochemical processes has led to the creation of a new class of click reactions, collectively known as photoclick chemistry. These light-triggered click reactions allow the synthesis of diverse organic structures in a rapid and precise manner under mild conditions. Because light offers unparalleled spatiotemporal control over the generation of the reactive intermediates, photoclick chemistry has become an indispensable tool for a wide range of spatially-addressable applications, including surface functionalization, polymer conjugation and crosslinking, and biomolecular labeling in the native cellular environment. Over the last decade, a growing number of photoclick reactions have been developed, especially those based on the 1,3-dipolar cycloadditions and Diels-Alder reactions owing to their excellent reaction kinetics, selectivity and biocompatibility. This review summarizes the recent advances in the development of photoclick reactions and their applications in chemical biology and materials science. A particular emphasis is placed on the historical contexts and mechanistic insights into each of the selected reactions. The in-depth discussion presented here should stimulate further development of the field, including the design of new photoactivation modalities, the continuous expansion of λ-orthogonal tandem photoclick chemistry, and the innovative use of these unique tools in bioconjugation and nanomaterial synthesis.

Graphical Abstract

1. Introduction

In nature, many fundamental life processes, such as photosynthesis, use sunlight as energy to drive complex biochemical cascade reactions.^1,2 Inspired by nature, chemists have successfully harnessed light to trigger key chemical transformations in their efforts to synthesize complex molecules and biomolecular conjugates.^3–5 In contrast to the thermal process, the light-triggered reactions often display high yield and selectivity without the need for acids, bases, or transition metals for substrate activation. Thus, the photochemical strategy can considerably reduce the number of steps needed in the total synthesis of complex natural products.³ Notably, aside from triggering the initial reaction, the light may not alter the subsequent chemical transformations (vide infra). From the application perspective, the use of light endows spatial and temporal control over the reaction, which makes them highly valuable in the realms of chemical biology and materials science.^5–9 The use of light as an external stimulus also allows chemical reactions to operate at the single-cell or even subcellular levels.

In 2001, Sharpless introduced the concept of click chemistry for facile synthesis of functional molecules from modular building blocks.¹⁰ A set of efficiency criteria were put forward for a reaction to be classified as “click reaction”: 1) the reaction must be operationally simple and have a broad substrate scope; 2) the reaction must be fast, high yielding, and selective; 3) the reaction must generate no or benign byproducts, which can be removed using non-chromatographic techniques; and 4) the reaction must be carried out under ambient conditions in non-hazardous solvents or neat. Many photochemical reactions satisfy these stringent criteria when suitable privileged substrates are used. The merging of click chemistry with benign photochemical processes has led to the creation of a new class of light-triggered click reactions, also known as photoclick chemistry, enabling synthesis of diverse molecular structures, conjugates, and networks in a spatiotemporally controlled manner in complex systems.^11,12

A growing number of photoclick reactions have been reported in the literature over the last decade. The prominent examples include the photoinduced tetrazole-alkene cycloaddition,¹³ the light-triggered hetero-Diels–Alder reactions,¹⁴ the light-triggered azide–alkyne cycloadditions,¹⁵ the photoinduced sydnone–alkene/alkyne cycloaddition,¹⁶ the photoinduced azirine–alkene cycloaddition,¹⁷ and the light-triggered oxime ligation reactions¹⁸ (Scheme 1).

Scheme 1:

Representative examples of photoclick reactions

The photoclick reactions begin with the absorption of photons to generate the reactive species. Based on how light is harnessed, the photoclick chemistry can be classified into three types (Scheme 2). Type I involves irreversible photo-generation of a reactive intermediate with varying stability. In general, a precursor is ruptured under photochemical conditions to release N₂, CO₂, or a photo-protecting group and generate the reactive intermediate, which is then intercepted selectively by a cognate reaction partner (Scheme 2a). The light-triggered tetrazole–alkene cycloaddition, sydnone–alkene/alkyne cycloadditions, oxime ligation, and azirine–alkene cycloaddition depicted in Scheme 1 represent this type. Notably, the long half-lives of reactive intermediates may negatively affect the spatial control of the process. Type II involves photo-isomerization to generate a highly unstable reactive intermediate, which can either revert back to the starting materials or proceed to react with its partner to form a covalent adduct (Scheme 2b). The examples of this type include photoinduced hetero-Diels–Alder reactions based on o-naphthoquinone methide and o-quinodimethane. Type III reactions require a catalyst whose activation is mediated by light, e.g., light-triggered azide–alkyne cycloaddition and tetrazine ligation (Scheme 2c). Collectively, these photoclick reactions offer potent tools to many research fields, including biomolecular conjugation, drug discovery, polymer synthesis, lithography, and material sciences. Since several excellent reviews on thiol-ene photoclick reactions have appeared in the literature,^19–22 in this Review, we decided to focus on primarily the cycloaddition-based light-triggered click reactions and their myriad applications in chemical biology and materials sciences, with emphases on the historical context and mechanistic studies.

Scheme 2:

Three types of photoclick reactions: a) type I; b) type II; c) type III

2. Tetrazole Photoclick Chemistry

2.1. History and Synthesis of Tetrazoles

Tetrazoles are an essential class of heterocycles comprised of a five-member ring with four nitrogen and one carbon atoms.²³ Unlike pentazoles, tetrazoles play a crucial role in drug design because they are stable and bioisosteric to carboxylic acids. In 1885, J. A. Bladin reported the first synthesis of tetrazole by reacting dicyanophenylhydrazine with nitrous acid.²⁴ Later, Hantzsch and Vagt reported [3+2]-cycloaddition of azides with nitriles,²⁵ a method used widely for the synthesis of tetrazoles (Scheme 3a). Because of the widespread use of 2,5-diaryltetrazoles in bioorthogonal ligation and polymer synthesis, several synthetic approaches have been reported.^26,27 For example, Kakehi and coworkers reported the facile synthesis of 2,5-disubstituted tetrazoles by reacting phenylsulfonyl hydrazide with arene diazonium salts (Scheme 3b).²⁸ The advantages of this method include the use of readily available starting materials and good yields. However, this method employs hazardous reagents and suffers from laborious purification steps. Alternatively, the transition metal-catalyzed N²-arylation of 5-substituted tetrazoles with diverse arylating reagents such diaryliodonium salts and aryl boronic acids have been developed.^29–35 Among these approaches, the Cu-catalyzed regioselective arylation of 5-substituted tetrazoles provides rapid access to 2,5-disubstituted tetrazoles under mild conditions (Scheme 3c).³²

Scheme 3:

Synthesis of tetrazoles

2.2. Generation and Structure of Nitrile Imines

In 1960, Huisgen and coworkers discovered the unique reactivity of tetrazole moiety in a photo-triggered 1,3-dipolar cycloaddition reaction of 2,5-diphenyltetrazole 9 with methyl crotonate.^36–38 Irradiation of 2,5-diphenyltetrazole with a mercury lamp led to the formation of a pair of pyrazoline regioisomers in a 3:1 ratio (Scheme 4a). A two-step reaction mechanism was proposed: upon absorption of light, the tetrazole undergoes fast cycloreversion to generate the reactive nitrile imine 10 with the extrusion of N₂; this nitrile imine then undergoes fast 1,3-dipolar cycloaddition with methyl crotonate to afford the pyrazoline product. The in situ generated nitrile imine species was confirmed by the reaction of a phenylhydrazonyl chloride with an alkene dipolarophile in the presence of a base via 1,3-dehydrochlorination, giving rise to the same cycloadduct. Following these initial studies, the reactivity of nitrile imines was explored in 1,3-dipolar cycloaddition reactions with various dipolarophiles as well as in intramolecular ring-closure reactions.^39–41

Scheme 4:

Early reports of nitrile imine-mediated 1,3-dipolar cycloaddition reactions

Later, Holm and coworkers carried out a detailed study to investigate the formation of nitrile imines using spectroscopic techniques, including UV–vis, infrared, and MS-based fragmentation involving the ¹⁵N-labeled tetrazoles.⁴² The experimental studies revealed that 2,5-diaryltetrazoles underwent efficient ring rupture upon 290 nm UV irradiation with a quantum yield of 0.5~0.9 and that the electronic properties of substituents had minimal effect on ring rupture. Very recently, Barner-Kowollik and coworkers studied the wavelength-dependence and mechanism of the tetrazole-alkene cycloaddition reaction by using a combination of computational and experimental studies.^43,44

Nitrile imines could also be generated by other means. Broggini and coworkers reported their generation from the hydrazonyl chlorides under aqueous conditions and their subsequent reactions with the various dipolarophiles (Scheme 4b).⁴⁵ The rate of cycloaddition was accelerated in aqueous media due to the hydrophobic effect. In the absence of dipolarophiles, tetrazine products were formed via dimerization of the nitrile imine. Recently, Carell and coworkers exploited the hydrazonyl chlorides for bioorthogonal protein modification in aqueous media.⁴⁶ The drawback of using hydrazonyl chloride as the nitrile imine precursor is that the hydrazonyl chloride is prone to hydrolysis in aqueous media.⁴⁷ In contrast, tetrazoles exhibit excellent stability under aqueous conditions and provide rapid on-demand access to nitrile imines via photoirradiation.

In principle, the non-stabilized nitrile imines can adopt four possible electronic structures, including propargylic, allenic, 1,3-dipolar, and carbenic forms (Scheme 5a). A photo-crystallographic study of the tetrazole-Zn coordination complex at 90 K revealed a bent geometry for the in situ generated nitrile imine (Scheme 5b).⁴⁸ A separate water-quenching study confirmed that the twisted geometry represents the 1,3-dipolar form, a primary electronic structure likely responsible for its outstanding reactivity toward alkene dipolarophiles in aqueous media.

Scheme 5:

a) Electronic structures of the nitrile imine; b) direct observation of the bent nitrile imine 1,3-dipolar structure in the solid-state. Adapted from ref 48. Copyright 2009 American Chemical Society.

2.3. Mechanism of Nitrile Imine-Mediated Cycloaddition

Liu and coworkers studied the mechanism of the nitrile imine-mediated cycloaddition with alkenes.⁴⁹ In this work, they used hydrazonyl chlorides as the nitrile imine precursor to study the kinetics of the reactions. They found that the pH and the chloride concentrations have significant effects on reaction rate; the rate was higher under basic pH and in the absence of chloride ion, which they attributed to faster deprotonation and de-chlorination, respectively. Based on these results, they proposed a mechanism in which the generation of the nitrile imine from the hydrazonyl chloride is the rate-determining step of the overall reaction (Scheme 6).

Scheme 6:

A theoretical framework to account for the nitrile imine generation and all the nitrile imine mediated reactions including 1,3-dipolar cycloaddition (highlighted in blue)

Irrespective of how the nitrile imine is produced, scheme 6 shows a theoretical framework to consider the nitrile imine-mediated cycloaddition reactions as well as potential side reactions. Unlike other 1,3-dipoles such as azide and nitrones, nitrile imines are generally much less stable in an aqueous medium. In the absence of a suitable dipolarophile, they would react with water or other strong nucleophiles such as thiols to form the stable adducts, e.g., compounds 21 and 25 in Scheme 6.^50–53 Thus, to ensure that the 1,3-dipolar cycloaddition predominates among all the competing pathways, the nitrile imine and alkene reactivities need to be optimally tuned to increase the cycloaddition reaction rate while suppressing the competing ones.⁵⁴

2.4. Enhancing Tetrazole Reactivity and Selectivity

In 2007, Lin and coworkers reported an efficient synthesis of diverse pyrazolines 29 via the photoinduced 1,3-dipolar tetrazole–alkene cycloaddition (Scheme 7).⁵⁵ A 302-nm handheld UV lamp commonly used for thin-layer chromatography was found to be sufficient for the tetrazole ring-rupture. The transformation displayed excellent functional group tolerance, outstanding regioselectivity, and high yield (Scheme 7). Remarkably, the reactions could be performed in protic solvents, including ethanol and EtOH/H₂O (7:1) mixture. This report offers the first glimpse into the desirable characteristics of the photoinduced tetrazole–alkene cycloaddition as a photoclick reaction, including its operational simplicity, functional group tolerance, mild reaction conditions tolerating water as a cosolvent.

Scheme 7:

Photoinduced 1,3-dipolar cycloaddition for the synthesis of pyrazolines

To probe how substituents affect the rate of the tetrazole photoclick chemistry, Lin and coworkers prepared a series of 2,5-diaryltetrazoles carrying the electron-withdrawing or electron-donating groups on the phenyl rings and determined the second-order rate constants, k₂, of the photoinduced cycloaddition reactions with 4-penten-1-ol (Scheme 8).⁵⁶ A plot of calculated HOMO energies, E_HOMO, of the corresponding nitrile imine versus log(rate of reaction) displayed a linear relationship, indicating that the rate enhancement is principally a result of HOMO-lifting effect, which decreases the HOMO (dipole)−LUMO (dipolarophile) energy gap in the transition state and thus accelerates the cycloaddition reaction.⁵⁷

Scheme 8:

Plot of E_HOMO vs. log(rate) reveals HOMO-lifting effect in tetrazole photoclick chemistry. Reproduced with permission from ref 56. Copyright 2009 John Wiley & Sons,Inc.

One of the most effective strategies to accelerate the click reactions is through substrate activation via ring strain, particularly for applications in biological systems. For tetrazole photoclick chemistry, the strained substrates offer faster cycloadditions without concurrent increases in the side reactions (Scheme 6). To this end, Lin and coworkers synthesized a series of conformationally constrained macrocyclic tetrazoles by placing a bridge between the two flanking phenyl rings (Scheme 9a).⁵⁸ Upon photoirradiation, macrocyclic tetrazoles produced the cyclic nitrile imines with reduced conformational flexibility, which lowers distortion energy in the cycloaddition reaction.^59,60 Compared to the acyclic analog, macrocyclic tetrazole 34 displayed enhanced reactivity in labeling a norbornene‐modified lysozyme in PBS (Scheme 9b).

Scheme 9:

a) Design of macrocyclic tetrazoles; b) Application of macrocyclic tetrazole in protein labeling

For enhanced selectivity for cycloaddition, An et al. prepared a series of sterically shielded tetrazoles containing structural pendants at the ortho positions of the C-aryl ring (Scheme 10a).⁵⁴ Among them, the di(ortho-2′-N-Boc-pyrrole)-substituted tetrazole 43 displayed an exquisite selectivity for the cycloaddition (Scheme 10b). The DFT calculations and structural studies revealed that N-Boc-pyrrole groups at ortho-positions of the C⁵-aryl ring block nucleophilic addition, and as a result, increase the stability of nitrile imines with a half-life of 102 s in the aqueous medium. Moreover, the HOMO energy of the nitrile imine can be modulated through the substituent effect of the N-aryl group (Scheme 10c).⁶¹ Owing to its excellent selectivity and fast kinetics, the sterically shielded tetrazole 43 was later successfully used to label a strained alkene-encoded membrane protein in live mammalian cells (vide infra).

Scheme 10:

Sterically shielded tetrazoles for bioorthogonal cycloaddition reaction

2.5. Tuning Photoactivation Wavelength

Because a prolonged exposure of 302-nm UV light causes photodamage to cells, visible or NIR light-triggered tetrazole photoclick chemistry is more desirable in cellular applications. To this end, Lin and coworkers synthesized the substituted diaryltetrazoles that undergo ring rupture upon 365-nm photoirradiation (Scheme 11).⁶² The substituents on the N-phenyl ring were found to be critical in determining absorption maxima; the presence of NH₂, NMe_2, and styryl groups led to a shift of absorption maxima to the longer-wavelength region and higher absorption coefficient at 365 nm (Scheme 11a). Notably, these substrates displayed similar reactivity in the photoinduced 1,3-dipolar cycloadditions with alkenes in organic solvents as well as in aqueous buffer. In addressing the photobleaching associated with the amino-containing pyrazolines products, a scaffold-hopping strategy was reported in the design of long-wavelength photoactivable tetrazoles consisting of chromophores such as naphthalene and coumarin (Scheme 11b).⁶³ Specifically, the naphthalene derived tetrazole 49 showed excellent reactivity in the photoinduced cycloaddition reaction with the alkene dipolarophiles under 365-nm photoirradiation.

Scheme 11:

Tetrazole photoclick reactions under 365-nm photoirradiation

In 2013, An et al. reported the design of laser-activatable tetrazoles with improved biocompatibility.³³ A series of oligothiophene based tetrazoles were synthesized through Cu-catalyzed N-arylation of substituted 5-(thiophen-2-yl)tetrazoles with phenyl(thiophen-2-yl)iodonium salts (Scheme 12a). Among them, tetrazole 53b showed excellent reactivity in the photoinduced cycloaddition reaction with second-order rate constants approaching 619 ± 108 M⁻¹ s⁻¹ (Scheme 12b). Notably, the quantum yield for 405 nm laser triggered tetrazole ring rupture was determined to be 0.16, significantly higher than those of 365 nm photoactivable tetrazoles (Φ = 0.006 ~ 0.04).⁶² A water-soluble tetrazole 53f was then used to image microtubules in CHO cells in a spatially controlled manner. Additionally, the emission wavelength of oligothiophene pyrazoline products was tuned by introducing extended π-conjugation at the C⁵- position of the tetrazole (Scheme 12c).⁶⁴ The pyrazoline products displayed solvent-dependent red fluorescence with emission maxima in the range of 575~644 nm, which could be useful in probing the polarity change in biological systems.

Scheme 12:

a) Synthesis of oligothiophene-based tetrazoles; b) Kinetic characterization of the oligothiophene-based tetrazoles in 405 nm laser-triggered photoclick chemistry; c) Laser-activatable tetrazoles with the extended π-system

In 2015, the Barner-Kowollik group reported a visible light-triggered tetrazole photoclick chemistry by using a pyrene chromophore (Scheme 13).⁶⁵ The pyrene aryl tetrazole (PAT) 57 was synthesized in two steps from readily available starting materials. The pyrene-tetrazole displayed excellent reactivity towards a variety of dipolarophiles. The utility of this tetrazole was demonstrated in small-molecule ligation, design of block copolymers, and polymer end-group modification. The same group also reported an up-converting nanoparticles (UCNPs) assisted, NIR light-induced tetrazole photoclick chemistry (Scheme 13).⁶⁶ In this process, UCNPs (yttrium, tantalum, and ytterbium nanoparticles) were irradiated with the 974-nm NIR light to convert PAT to the nitrile imine, which then reacts with dipolarophiles. However, the cytotoxicity of the pyrene chromophore could potentially limit the broad use of these approaches in biological applications.

Scheme 13:

Visible or NIR light-induced cycloaddition based on Pyrene Aryl Tetrazole (PAT)

The two-photon excitation (2PE) induced process provides excellent spatiotemporal control over the single-photon process owing to the decreased light scattering of near-infrared (NIR) light and improved three-dimensional localization of excitation. Yu et al. reported 2PE-triggered tetrazole photoclick chemistry by taking advantage of strong two-photon absorption of naphthalene.⁶⁷ A femtosecond 700 nm NIR pulsed laser was used to generate the nitrile imines. A series of tetrazoles containing the auxochromic and oligo(ethylene glycol) groups at β-position of naphthalene were synthesized and tested in the 2PE-induced cycloaddition reaction (Scheme 14). The 2PE-triggered cycloaddition reaction of tetrazole 65 with acrylamide followed zero-order kinetics (k₀ = 0.067 ± 0.001 μM/min), indicating that the photoinduced tetrazole ring rupture is the rate-determining step. The two-photon absorption cross-section of tetrazole 65 was determined to be 12 GM (1 GM = 10⁻⁵⁰ cm⁴ s/photon), and the cycloaddition reaction cross-section was 3.8 GM. The utility of two-photon-triggered, fluorogenic photoclick reaction was demonstrated in a spatially controlled microscopic imaging of microtubules in live mammalian cells. The ability to generate nitrile imine species from photoactivable tetrazoles at different wavelengths enables the use of tetrazole photoclick reaction in conjunction with other photoclick reactions.^68–71 Recently, several λ-orthogonal photoligation strategies were presented for surface functionalization, in which the photoactivable tetrazoles selectively activated in the presence of other photoactivable functionalities that undergo photoclick reactions.^69,70

Scheme 14:

Design of naphthalene-tetrazoles for two-photon triggered photoclick chemistry

2.6. Dipolarophiles Other Than Alkenes

The tetrazole photoclick chemistry is not limited to alkenes; other dipolarophiles such as alkynes and quinones could also participate in the cycloaddition reactions.^72–74 Bochet and coworkers reported the synthesis of pyrazoles via 1,3‐dipolar cycloaddition between the photogenerated nitrile imines and the alkynes (Scheme 15a).⁷² Later, Yu and coworkers tuned the reactivity of nitrile imine by installing CF₃ group at ortho-positions of C⁵-phenyl ring of the tetrazole for highly selective cycloaddition reaction with BCN (Scheme 15b).⁷³ The exquisite selectivity in light-triggered tetrazole-alkyne cycloaddition was attributed to the electrostatic shielding effect of the CF₃ groups. Exceptionally robust kinetics was observed for the tetrazole-BCN cycloaddition reaction, with k₂ values approaching 10⁵ M⁻¹ s⁻¹. In the absence of BCN, the oligothiophene moiety on the N²-position of the tetrazole structure quenched the reactive nitrile imine to form the product 73. The tetrazole-BCN photoclick reaction was later employed in protein bioconjugation under visible light. Separately, Ribagorda and coworkers reported the synthesis of pyrazoline‐fused quinones via cycloaddition of tetrazoles with quinones (Scheme 15c).⁷⁴

Scheme 15:

Tetrazole photoclick reaction with various dipolarophiles

2.7. Applications in Bioorthogonal Protein Labeling

Over the last decade and a half, bioorthogonal chemistry has emerged as a powerful tool for visualizing multiple biomolecules including lipids, proteins, glycans and nucleic acids in their native cellular environment as well as manipulating biological processes in living systems.^75–77 In general, bioorthogonal chemistry comprises of two steps: 1) site-specific introduction of a chemical reporter into the target biomolecule; 2) bioorthogonal reaction between the chemical reporter and its reaction partner. Among a growing number of bioorthogonal reactions, tetrazole photoclick chemistry has generated a strong interest owing to its fast kinetics and spatiotemporal control.

In 2008, Lin and coworkers reported the first use of tetrazole photoclick chemistry for bioorthogonal protein modifications.⁷⁸ The initial study of the reaction between a tetrazole-modified peptide and acrylamide in PBS supports a two-step reaction mechanism involving the photoinduced generation of the nitrile imine followed by its subsequent cycloaddition with acrylamide. The second step is the rate-determining step that proceeds with a second-order rate constant of 11.0 M⁻¹ s⁻¹ (Scheme 16a). The selectivity of the reaction was verified through residue-specific modification of a tetrazole-modified lysozyme by acrylamide in PBS under a brief 302-nm photoirradiation (Scheme 16b). For site-specific protein modification, a tetrazole-containing enhanced green fluorescent protein (EGFP) prepared through intein-mediated chemical ligation and reacted with N-hexadecyl methacrylamide to produce the fluorescent EGFP-pyrazoline adduct after 1 min 302-nm photoirradiation (Scheme 16c).

Scheme 16:

Photoclick chemistry mediated bioorthogonal labeling of a) a tetrazole-modified peptide; b) a tetrazole-modified lysozyme; and c) a tetrazole-containing EGFP

In 2008, the Lin group demonstrated the suitability of tetrazole photoclick chemistry for fluorescent labeling of an O-allyl-tyrosine encoded Z-domain protein in E. coli cells (Scheme 17).⁷⁹ While light-triggered tetrazole ring rupture was rapid, the subsequent cycloaddition with O-allylphenyl ether dipolarophile was slow (k₂ = 0.00202 M⁻¹ s⁻¹) because of a large dipole HOMO-dipolarophile LUMO energy gap. Nonetheless, BL21(DE3) cells expressing the O-allyl-tyrosine-encoded Z-domain (alkene-Z) were selectively labeled by tetrazole 78 after photoirradiation at 302 nm for 4 min followed by overnight incubation (Scheme 17). Adding the electron-donating groups on the phenyl rings increases HOMO energies of the photogenerated nitrile imines, leading to faster reactions.⁵⁶ In particular, 2-(p-methoxyphenyl)-5-phenyltetrazole 79 displayed robust reactivity toward O-allylphenyl ether with k₂ value of 0.79 M⁻¹ s⁻¹, about 200-fold faster than tetrazole 78. With this improvement, tetrazole 79 allowed fluorescent labeling of the O-allyl-tyrosine encoded Z-domain protein in E. coli in one minute.⁵⁶

Scheme 17:

Fluorescent labeling of alkene-encoded proteins in E. coli via tetrazole photoclick chemistry. Reproduced from ref 79. Copyright 2008 American Chemical Society.

To encode tetrazoles at any position in a protein structure for site-specific modification via photoclick chemistry, Lin and coworkers designed a series of photoreactive tetrazole amino acids in which the amino acid moiety was attached to either C⁵ or N²-aryl ring (Scheme 18a).⁸⁰ One of the amino acids, p-tetrazole-phenylalanine (p-Tpa, 82c), was incorporated site-specifically into myoglobin using the amber codon suppression technique with an M. jannaschii amber suppressor MjTyrRS mutant. Furthermore, p-Tpa served as a chemical reporter for fluorescent labeling of myoglobin via tetrazole photoclick chemistry (Scheme 18b).⁸¹ Because p-Tpa lacks C-aryl ring, it exhibited slower reaction kinetics (k₂ = 0.082 M⁻¹ s⁻¹ towards dimethyl fumarate) and required 254 nm UV light for photoactivation.⁸¹

Scheme 18:

a) Synthesis of photoreactive tetrazole amino acids; b) Selective fluorescent labeling of the p-Tpa-encoded myoglobin via tetrazole photoclick chemistry. Adapted from ref 81. Copyright 2010 American Chemical Society.

Since tetrazole amino acids are large, it is challenging to identify specific aminoacyl-tRNA synthetases to charge them into proteins for structural and functional studies. Therefore, it is preferable to encode their smaller reaction partners—alkenes—into proteins to minimize perturbation to protein structure. An early example of using tetrazole photoclick chemistry to image proteins in mammalian cells involved the metabolic incorporation of a methionine surrogate, homoallylglycine (HAG, 86).⁸² The advantage of this metabolic approach is its simplicity: no genetic manipulation is required for the introduction of the alkene chemical reporter. Indeed, β-galactosidase pre-tagged with HAG showed selective labeling by tetrazoles in both cell lysates and living mammalian cells. Because the adducts from tetrazole photoclick chemistry are fluorescent, HAG served as a robust chemical reporter for spatiotemporally controlled, fluorescent imaging of the newly synthesized proteins in HeLa cells.

Many synthetic alkene amino acids suitable for tetrazole photoclick chemistry have been incorporated site-specifically into proteins using the amber codon suppression technique (Scheme 19).^83,84 Whereas the earlier O-allyltyrosine 87 was a viable substrate for tetrazole photoclick chemistry in bacteria; it is not suitable for mammalian systems because the MjTyrRS/tRNA_CUA pair are not orthogonal to the mammalian synthetase/tRNA pairs. Because acrylamide is an excellent substrate for photoclick chemistry, the Liu and Wang groups independently reported the genetic encoding of an acrylamide-modified lysine (AcrK, 88) for fluorescent protein labeling via tetrazole photoclick chemistry (Scheme 20a).^85,86 The advantages of using AcrK as a chemical reporter include its fast kinetics and minimum perturbation to the protein structure due to its small size. When AcrK was incorporated into FtsZ proteins in bacterial and mammalian cells, it allowed fluorescent labeling of FtsZ directly in intact cells (Scheme 20a).⁸⁶ To improve fluorescence turn-on efficiency, Guo and coworkers reported the genetic encoding of styrene 89 as a dipolarophile for tetrazole photoclick chemistry (Scheme 20b).⁸⁷ The utility of this styrene-based alkene reporter was demonstrated by labeling the intracellular stress response protein HdeA in live cells (Scheme 20b).

Scheme 19:

Genetically encoded alkene amino acids for bioorthogonal protein labeling

Scheme 20:

Bioorthogonal protein labeling in live cells via tetrazole photoclick chemistry with a genetically encoded a) acrylamide or b) styrene-containing amino acid

While electron-deficient alkene amino acids are excellent reaction partners for tetrazoles, they are susceptible to Michael addition by thiols in biological systems. To avoid this problem, several groups have turned attention to the strained alkenes due to their excellent reactivity in the nitrile imine-mediated cycloaddition reactions and inertness in biological systems. For example, the Carell group reported the genetic encoding of a norbornene-modified Lys (NorK, 90) into human polymerase j, which was further modified with hydrazonyl chlorides.⁴⁶ Similarly, the Lin group developed a cyclopropene amino acid (CpK, 91) for fluorescent labeling of proteins inside mammalian cells via tetrazole photoclick chemistry (Scheme 21).⁸⁸ Compared to acrylamide and norbornene, 3,3-disubstituted cyclopropene 95d exhibited faster kinetics owing to the partial release of the cyclopropene ring strain after the cycloaddition reaction (Scheme 21a). When CpK was incorporated site-specifically into EGFP in HEK293 cells, it directed rapid and selective labeling of EGFP by a 365-nm photoactivatable tetrazole 97 based on confocal microscopy (Scheme 21b).

Scheme 21:

A cyclopropene amino acid (CpK) for bioorthogonal labeling of proteins: a) kinetic studies; b) selective labeling of CpK‐encoded EGFP in HEK293 cell via tetrazole photoclick chemistry. Adapted and reproduced with permission from ref 88. Copyright 2009 John Wiley & Sons,Inc.

To minimize steric repulsion between the C³-substituent adjacent to the cyclopropene π-bond and the incoming nitrile imine in the transition state, Yu et al. reported the design of a highly reactive, yet stable spiro[2.3]hex-1-ene (Sph, 99) for superfast photoclick reaction.⁸⁹ The crystal structure of Sph shows that the cyclobutane ring in Sph pulls C³-substituents away from π-faces of the cyclopropene ring, resulting in a decreased bond angle of 92.3° compared to 113.5° for Cp 98 (Scheme 22a).

Scheme 22:

Design of spiro[2.3]hex-1-ene for superfast photoclick chemistry: a) crystal structure of Sph vs. Cp; b) genetic encoding of SphK into sfGFP; c) bioorthogonal labeling of GCGR in live cells. Adapted and reproduced from ref 54. Copyright 2018 American Chemical Society.

Kinetic analysis revealed that Sph is about 15 times more reactive than cyclopropene in the reaction with tetrazole 79. When the spiro[2.3]hex-1-ene modified lysine (SphK, 92) was incorporated site-specifically into superfolder GFP (sfGFP), it enabled rapid modification of sfGFP by a water-soluble tetrazole 100 with the second-order rate constant of 10,420 M^–1 s^–1 in Cl^–-free phosphate buffer (Scheme 22b). SphK was then used in rapid fluorescent labeling of GCGR, a member of class B G protein-coupled receptors, in live HEK 293T cells with the sterically shielded tetrazole 101 (Scheme 22c).⁵⁴ Two nitrogen-containing spiroalkenes, azaspiro[2.3]hex-1-ene and azaspiro[2.4]hept-1-ene, were also synthesized, which show improved water solubility and reactivity in tetrazole photoclick chemistry with k₂ values as high as 33,200 M^–1 s^–1 in phosphate buffer/ACN (1:1) mixed solvent.⁹⁰

2.8. Applications in Nucleic Acid Modification

The tetrazole photoclick chemistry was also used in post-synthetic modifications of nucleic acids.⁹¹ Wagenknecht and coworkers reported the synthesis of a series of 2′-deoxyuridine building blocks containing diaryltetrazoles 102 as photoactivable groups (Scheme 23a).^92,93 The rapid photo-triggered modification of DNA was achieved by photoirradiation of the samples in aqueous medium at 365 or 405 nm (outside the absorption region of nucleic acids), with second-order rate constants up to 89 M^–1 s^–1. Later, Zhou and coworkers exploited an intramolecular photoclick reaction for fluorescent detection of DNA base variation (Scheme 23b).⁹⁴ Since the alkene moiety can be readily incorporated into nucleosides, the Rentmeister and Zhang groups designed the RNA and DNA building blocks bearing O-allyl group for post-synthetic modifications via the tetrazole photoclick chemistry.^95,96

Scheme 23:

a) Tetrazole photoclick chemistry for post-synthetic modifications of DNA; b) Intramolecular photoclick reaction for fluorescent detection of DNA base variation

2.9. Chemosensors

Because tetrazole photoclick chemistry produces fluorescent cycloadducts, Yu et al. exploited this unique property and designed the photoactivatable fluorescent probes based on an intramolecular photoclick chemistry.⁹⁷ In a proof-of-concept study, they prepared a series of taxoid-tetrazoles conjugates by linking 7-β-alanyltaxol core with a tetrazole unit containing an O-allyl overhang at N-phenyl ring 104 (Scheme 24). Upon photoirradiation, the conjugates proceeded through an intramolecular photoclick reaction to form fluorescent pyrazoline adducts with fluorescence turn-on ratios as high as 112-fold for taxoid pyrazoline 105d. The utility of this type of photoactivable fluorescent probes was demonstrated in spatially controlled fluorescent labeling of microtubules in live CHO cells.

Scheme 24:

Design of photoactivatable turn-on fluorescent microtubule probes based on an intramolecular photoclick chemistry

Because fumarate is an excellent alkene substrate and the cycloadduct is fluorescent, Meier and coworkers designed a tetrazole probe 106 to detect the oncometabolite fumarate (Scheme 25a).⁴⁷ Owing to the efficient photochemical generation of nitriles imines, this approach provides improved sensitivity compared to the previously reported hydrazonyl chloride probes.⁹⁸ Similarly, An et al. developed a BODIPY‐linked bithiophene‐tetrazole 109 as an off-on fluorescence probe for the detection of hydrogen peroxide inside HeLa cells.⁹⁹ A drastic decrease of BODIPY fluorescence was observed for the meta-linked BODIPY-tetrazole after reaction with dimethyl fumarate. The BODIPY fluorescence was recovered after treatment with hydrogen peroxide (Scheme 25b). This unique fluorescence off-on was exploited for the detection of hydrogen peroxide inside HeLa cells using a water-soluble BODIPY-tetrazole 109. Recently, Wittman and coworkers reported visualization of complex carbohydrates based on the reaction between the tetrazole and an acrylamide modified mannosamine.¹⁰⁰ Separately, Binco and coworkers designed a novel profluorescent nitroxide 113 via a tetrazole photoclick reaction to monitor the redox or radical processes (Scheme 25c).¹⁰¹

Scheme 25:

a) Photoinducible detection of the oncometabolite fumarate; b) Design of a BODIPY‐tetrazole based fluorescence reporter of hydrogen peroxide; c) Design of redox/radical sensing molecules

2.10. Applications in Materials Science

The ability to provide spatiotemporal control makes the tetrazole photoclick chemistry a powerful tool in materials science applications, including surface functionalization, polymer synthesis and cross-linking, and fullerene conjugation.^7,102,103 In 2011, Barner-Kowollik and coworkers demonstrated the utility of this photoclick reaction in surface functionalization by ligating a maleimide containing polymer chain to the tetrazole-functionalized silicon wafer surface (Scheme 26).¹⁰⁴ Later, they ligated poly(dopamine) (PDA) films with antifouling poly(MeOEGMA) brush polymers to control cell adhesion using tetrazole photoclick chemistry.¹⁰⁵ Similarly, Nallani and Liedberg reported the conjugation of the tetrazole-functionalized horseradish peroxidase with methacrylate-terminated ABA block copolymers.¹⁰⁶ Following these studies, numerous reports of nitrile imine-mediated surface patterning appeared.^7,107–115

Scheme 26:

Preparation of a poly(methyl methacrylate) grafted silicon wafer

In 2014, the Barner-Kowollik group reported a facile approach for photolytic preparation of fluorescent polymers by employing polymerization of non-fluorescent photoreactive monomers based on tetrazole photoclick chemistry (Scheme 27a).^116,117 Later, they applied the photoclick reaction for the synthesis of extended linear polymers.¹¹⁸ In this work, the selective reaction of a tetrazole-modified nitrile-butadiene rubber (NBR) 116 with a bis-maleimide linker 117 provided access to nitrile rubber of high molecular weight (Scheme 27b). Notably, tetrazole photoclick chemistry was used together with other photoclick reactions such as photo-enol chemistry for the synthesis of copolymers.⁷¹

Scheme 27:

a) Tetrazole-alkene based polymerization using a monomer of the A-B type; b) Light-induced ligation of NBR building blocks to obtain high molecular weight nitrile rubber; c) Reagent employed for cross-linking of a butadiene polymer

The photoinduced cross-linking of polymers plays a vital role in tuning material properties such as solubility, viscosity, and optical properties. To this end, tetrazole photoclick chemistry has proven to be a valuable tool for polymer cross-linking owing to the precise control over photogeneration of the nitrile imines. Darkow and coworkers demonstrated a UV light-induced tetrazole-alkene reaction mediated cross-linking in membrane polymers.¹¹⁹ Recently, the Barner-Kowollik group presented sunlight-induced cross-linking of 1,2-polybutadienes under ambient conditions employing a difunctional tetrazole 119 (Scheme 27c).¹²⁰ Several reaction parameters, including reaction time and concentration and light source, were optimized. They expanded the utility of tetrazoles as cross-linking agents to prepare fluorescent cellulose network,¹²¹ as well as patterned surface and microparticles by direct laser writing.¹²² Moreover, the quantification of ligation points in polystyrene networks was carried out by monitoring the fluorescence intensity of the cross-linked product.¹²³ On the other hand, single-chain nanoparticles (SCNPs) prepared via intramolecular cross-linking of single polymeric chains have found applications in chemosensors, catalysis, and drug delivery. In this regard, the intramolecular cycloaddition reactions between tetrazoles and alkene-functionalized polystyrenes were employed in the synthesis of SCNP.^124,125

Fullerenes (C₆₀) are used widely in material and energy sciences due to their high electron affinity and excellent electron transfer properties. They are also known to undergo 1, 3-dipolar cycloaddition reactions with nitrile imines. Several examples of fullerene functionalization based on the cycloaddition chemistry have been reported.¹²⁶ Barner-Kowollik and coworkers developed an efficient method for fullerene functionalization based on tetrazole photoclick chemistry (Scheme 28).^127,128 The linear polymers such as tetrazole-functionalized polyethyleneglycol and poly(tert-butyl)acrylate were conjugated to fullerene to form the hybrid networks under UV irradiation.

Scheme 28:

Tetrazole photoclick chemistry for fullerene conjugation

2.11. Miscellaneous Applications

The intramolecular photoclick chemistry was also exploited for the synthesis of stapled peptides.¹²⁹ For example, Madden et al. used Balaram’s 3₁₀-helix as a peptide helix model and appended the tetrazole and alkene groups at the side chain of i and i + 4 residues, respectively (Scheme 29). The resulting peptides were subjected to 302-nm photoirradiation to trigger the photoclick chemistry mediated peptide stapling. Peptide precursors with the lysine sidechain provided higher yields than those with the ornithine sidechain. Both tetrazole reactivity and alkene rigidity are crucial in determining macrocyclization yield (Scheme 29). This intramolecular photoclick chemistry was further extended to the design of the potent stapled peptide-based dual inhibitors of Mdm2/Mdmx.¹³⁰

Scheme 29:

Synthesis of stapled peptides via intramolecular photoclick chemistry

By taking advantage of the spatiotemporal control, Zhong and coworkers employed the tetrazole photoclick chemistry to prepare hydrogels with unique features such as tunable gelation and high specificity (Scheme 30a).¹³¹ These PEG-based hydrogels were used for the controlled release of therapeutic proteins. Similarly, Zhang and coworkers exploited the intramolecular photoclick chemistry to regulate self-assembly of supramolecular hydrogels (Scheme 30b).¹³² The formation of the pyrazoline products led to the photodegradation of supramolecular hydrogels and the release of encapsulated biological materials such as cells and proteins.

Scheme 30:

Tetrazole photoclick chemistry for the preparation of (a) hydrogels and (b) photodegradable supramolecular hydrogels. Adapted and Reproduced from ref 132. Copyright 2013 American Chemical Society.

3. Light-Triggered Azide–Alkyne Cycloadditions

3.1. Cu(I)-Catalyzed Azide–Alkyne Cycloadditions

The Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) is of paramount importance in synthetic chemistry and widely regarded as a prototypic click reaction.^77,133–137 The research groups of Meldal and Sharpless independently reported the reactions between azides and alkynes to produce 1,2,3-triazoles with high regioselectivity and excellent yields (Scheme 31a).^138,139 Owing to its robustness and versatility, it has been extensively employed in biomolecular ligation, combinatorial synthesis, medicinal chemistry, surface functionalization, and polymer synthesis. In most CuAACs, active Cu(I) catalysts are generated in situ from Cu(II) salts through 1) the use of a reducing agent, 2) electrochemical generation of Cu(I),^140,141 3) photochemical approaches,¹⁴² and 4) copper-containing nanoparticles.¹⁴³ Among these approaches, reducing agents such as sodium ascorbate, quinone, and hydroquinone are the most commonly used. The photochemical generation of Cu(I) catalysts provides spatial and temporal control over the process, which could be advantageous for some biological and material science applications. To this end, König and coworkers reported the first example of light-triggered CuAAC reaction through photoirradiation of riboflavin tetraacetate 120 in the presence of Et₃N (Scheme 31b).¹⁴² The resulting flavin reduces Cu(II) to Cu(I), which in turn catalyzes the reaction between the azide 123 and the alkyne 124.

Scheme 31:

Light-triggered CuAAC click reactions

The light-induced formation of Cu(I) species can be carried out by either direct or indirect photolysis of Cu(II) catalytic system. The direct photolysis involves the absorption of UV light by a ligand of Cu(II), which leads to a ligand metal charge transfer for the reduction of Cu(II) complexes (Scheme 31c). To this end, Yagci and coworkers generated Cu(I) species by UV/Vis irradiation of CuCl₂ in the presence of the PMDETA ligand (Scheme 31d).¹⁴⁴ Similarly, Schubert and coworkers reported a photoinduced CuAAC reaction based on copper(II) acetate salt.¹⁴⁵ For direct reduction, amine ligands play a crucial role in the light-triggered CuAAc reactions. In addition to the stabilization of Cu(I) species, ligands also improve the solubility of catalytic systems in organic solvents. Tertiary amines such as triethylamine, PMDETA, tetramethylethylenediamine, and hexamethylenetetramine were among the most effective ligands.

On the other hand, indirect photolysis involves the absorption of UV-visible light through a photoinitiator to generate radical intermediates, which reduces Cu(II) to Cu(I) (Scheme 31c). Usually, indirect reduction approaches are faster than direct irradiation of Cu(II) complexes in the generation of Cu(I) species. To this end, Bowman and coworkers reported the light-induced CuAAc via photoinitiated Cu(II) reduction using a CuSO₄·5H₂O as a catalyst and a cleavage type photoinitiator Irgacure 2959 for the synthesis of hydrogels (Scheme 32).¹⁴⁶ Upon light irradiation (400–500 nm), the photoinitiator generates the radical species, which efficiently reduces Cu(II) to Cu(I).

Scheme 32:

Substrates used in light-triggered CuAAc reaction for hydrogel formation

A wide range of photoinitiators with distinct UV/visible absorption characteristics are available for light-induced CuAAc click reactions, including DBMP, titanocene, TMDPO, camphorquinone/benzyl alcohol, and phenothiazine (Scheme 33).^147,148 Based on the mechanism for the generation of radical species, these photoinitiators can be classified into two types: Type I (unimolecular) and Type II (bimolecular). Although both types are capable of reducing Cu(II) to Cu(I), Type I photoinitiators are more efficient than Type II in CuAAC reactions. The difference was attributed to the relatively low quantum yield of the bimolecular reaction process in generating radical species (Scheme 33).

Scheme 33:

Photoinduced CuAAC click reaction using free radical photoinitiators

Over the years, a large number of copper catalysts including CuCl₂·2H₂O/sodium benzoate,¹⁴⁹ copper(II)(tris(2-aminoethyl)-amine)ketoprofenate,^150,151 copper(II)(N,N′-dimethylethylendiamine) ketoprofenate,¹⁵² copper(II)phenyl-2,4,6-trimethylbenzoylphosphinate,¹⁵³ and others^154,155 have been successfully used in light-triggered CuAAC click reactions. To avoid photodecomposition under UV light exposure, numerous visible light-triggered CuAAC reactions were reported.^156–159

In general, the efficiency of light-triggered CuAAC reactions depends on several factors, including irradiation time, light intensity, photoinitiator concentration. Studies have indicated that the photoinduced CuAAC reactions can proceed with the minimal amount of irradiation and the copper catalyst.¹⁴⁶ Importantly, the photoinduced CuAAC reactions provide temporal control over the process, as the reaction can be stopped at any time through bubbling of air into the system.¹⁵⁰ Upon introduction of air, re-oxidation of Cu(I) to Cu(II) occurs readily as the solution color changes to green. However, the inhibition of reaction is completely reversible; the reaction restarts by purging the solution with argon along with photoirradiation. Because of these features, the light-induced CuAAC reactions have been applied extensively to material sciences.^{102,103,146,160–169}

3.2. Strain-Promoted Azide–Alkyne Cycloadditions

Despite the advantages of CuAAC reactions, the toxicity of copper limits the widespread adoption of CuAAC in biological applications. Bertozzi and coworkers reported the strain-promoted azide–alkyne cycloaddition (SPAAC) involving cyclooctynes without the use of a copper catalyst.¹⁷⁰ Following this pioneering work, a variety of strained alkynes including fluorinated cyclooctynes, dibenzocyclooctynes, and thiacycloalkynes have been designed for accelerated copper-free azide-alkyne cycloaddition.^171–173 In 2009, Popik and coworkers reported a photoinduced, copper-free azide–alkyne cycloaddition reaction using cyclopropenones 139 as the photo-masked alkyne precursors (Scheme 34).¹⁵ These cyclopropenones were prepared in two steps: Friedel-Crafts alkylation of 137 with trichlorocyclopropenium cation followed by selective hydrolysis to generate the cyclopropenones 139 (Scheme 34a).^15,174,175 The cyclopropenones display excellent thermal stability and do not react with azides under ambient conditions in the dark. However, upon irradiation at 350 nm cyclopropenones 139 rapidly generate the corresponding dibenzocylcooctynes 140, which reacts with azides to form the cycloadducts 141 (Scheme 34b). The utility of this transformation was demonstrated through successful glycan labeling as well as surface immobilization. In these studies, the biotin-containing cyclopropenone 142 was used for cell labeling experiments, while the amino-terminated linker appended cyclopropenone 143 was used for immobilization on brush polymers (Scheme 34c).^15,176–178 The cyclopropenone-based photoinduced click chemistry was also applied to surface functionalization, hydrogel derivatization, functionalization of nanoparticles, and synthesis of hetero-bivalent agents.^178–183

Scheme 34:

a) synthesis of cyclopropenones; b) Light-induced strain-promoted cycloadditions between azides and alkynes; c) cyclopropenone substrates used in for cell labeling experiments and immobilization on brush polymers

The Popik group employed a SPAAC-based sequential click strategy for cross-linking two discrete azide-tagged substrates (Scheme 35).¹⁸⁴ The heterobifunctional linker 144 contains a cyclopropenone-masked dibenzocyclooctyne group on one end (photo-DIBO) and an azadibenzocyclooctyne (ADIBO) group on the other. This design allows sequential catalyst-free ligations with two different azide substrates because the photo-DIBO moiety reacts with an azide-containing compound only after photoirradiation.

Scheme 35:

Sequential photoinduced SPAAC reactions

The dibenzo[a,e]cyclooctadiyne (Sondheimer diyne, 148) represents a unique class of cross-linkers containing two strained triple bonds as reactive sites for double SPAAC reactions with two azides. This diyne has been successfully employed in cross-coupling numerous azide-functionalized partners from biomolecules to metal-organic frameworks.^185–188 However, the low stability and rapid decomposition of the DIBOD cross-linkers in aqueous solution limits its broader utility. To overcome this limitation, Popik and coworkers designed a novel photochemical precursor 147 for the DIBOD cross-linker, in which both alkynes are masked as cyclopropenones (Scheme 36a).¹⁸⁹ The photo-DIBOD 147 displayed high stability despite the presence of angle strain and an antiaromatic cyclooctatetraene core. Irradiation of photo-DIBOD with a 350 or 420 nm light in the presence of the azide resulted in the formation of a bis-triazole product. In this process, the addition of the second equivalent of azide to the diyne proceeds at a faster rate than that of the first azide addition. This rate difference in azide additions makes it challenging to achieve selective ligation with two different azides. To address this issue, the same group synthesized a mono-cyclopropenone-caged dibenzocyclooctadiynes (MC-DIBOD, 150).¹⁹⁰ MC-DIBOD allows sequential SPAAC cross-linking of two different azides (Scheme 36b). MC-DIBOD was prepared by using selective monodecarbonylation of photo-DIBOD 147 or mono-cyclopropanation of dibenzo[a,e]cyclooctadiyne (DIBOD, 148). Interestingly, the triazole-fused alkyne 152 displays excellent reactivity toward azides with k₂ values approaching 34 M⁻¹ s⁻¹, thus representing the fastest SPAAC reaction in organic solvents (Scheme 36b).

Scheme 36:

Double SPAAC of two azides with a) photo-DIBOD and b) MC-DIBOD

Recently, Spitale and coworkers reported the first cellular application of cyclopropenone-based light-triggered click chemistry (photo-SPAAC) to label nascent azide-modified RNAs (Scheme 37a).¹⁹¹ The cyclopropenone-caged oxa-dibenzocyclooctyne 155 displayed excellent stability even in the presence of high concentrations of thiols, and upon 350 nm irradiation, rapidly reacted with RNAs containing 2’-azidoadenosine (Scheme 37a). This light-triggered strategy enabled fluorescence imaging and enrichment of RNAs in subpopulations of cells. To enhance the utility of this reaction in biological systems, the Popik group reported the SPAAC click reaction using near-infrared (NIR) radiation (Scheme 37b).^192,193 The photoinduced decarbonylation of cyclopropenone was achieved by nonresonant two- and three-photon excitation. The utility of this multiphoton SPAAC was demonstrated in cellular labeling in intact tissue as well as 3D patterning of hydrogels using 159b. More recently, Kunishima and coworkers described a photocatalyst-promoted SPAAC reaction under blue light (450–500 nm) irradiation.¹⁹⁴

Scheme 37:

a) Photo-SPAAC rection for temporal labeling of nascent RNAs; b) MP-SPAAC click reactions

Bertozzi and coworkers reported photo-generation of dibenzoselenacycloheptyne from irradiation of 4-selenabicyclo[5.1.0]-octatrienone, a highly strained alkyne designed for copper-free click reaction.¹⁹⁵ However, these compounds displayed low stability and reactivity as they underwent a hydrogen atom transfer from the solvent rather than cycloaddition. Following this study, Klán and coworkers developed click reactions based on a highly strained seven-membered dibenzosilacyclohept-4-yne 161 and azides or 1,2,4,5-tetrazines.¹⁹⁶ This reagent was readily generated from photoirradiation of the cyclopropenone precursor and displayed high stability and reactivity in click reactions with azides and tetrazines (Scheme 38a). Later, they reported a bioorthogonal “catch and photo-release” strategy involving a sequential [3+2] azide-alkyne cycloaddition and a photo-Favorskii rearrangement (Scheme 38b).¹⁹⁷

Scheme 38:

a) Click reactions based on dibenzosilacyclohept-4-yne; b) Bioorthogonal “catch and photo-release” strategy based on light-triggered, strain-promoted cycloaddition

Separately, Schnarr and coworkers reported a photoinitiated benzyne click reaction using 2-(3-acetyl-3-methyltriaz-1-en-1-yl)benzoic acid 168 as a benzyne precursor (Scheme 39a).¹⁹⁸ The benzyne photo precursor 168 can be synthesized in four steps and is stable under ambient conditions. The reaction was compatible with a variety of functional groups and completed in less than 5 min. Theoretical studies on cycloaddition involving strained alkenes showed that only trans-cyclooctene is capable of rapid cycloaddition with azides at room temperature.¹⁹⁹ Encouraged by this result, Weaver and coworkers harnessed photochemical energy and developed a visible-light triggered cycloaddition of alkyl azide with benzofused cycloheptene 170 (Scheme 39b).²⁰⁰ The reaction involves a photocatalyst mediated light-induced isomerization of benzocycloheptene to the strained trans-cycloalkene, which then reacts rapidly with the azides. The reaction between the azide and benzocycloheptene occurs only in the presence of light and the photocatalyst 172, thus providing a temporal control to the reaction. This transformation displays broad functional group tolerance and was employed in fast bioconjugation of azide-functionalized insulin. The shortcomings of this reaction include precipitation of photocatalyst under higher aqueous concentration and requirement of long irradiation time, which limit its utility in biological systems.

Scheme 39:

a) Photo-triggered benzyne click reaction; b) Visible-light mediated [3 + 2]-cycloaddition of azides with alkenes

4. Light-Triggered Hetero-Diels–Alder Reactions

4.1. Hetero-Diels–Alder Reaction of Naphthoquinone Methides

The Diels–Alder reaction is a powerful reaction for building molecular complexity in the synthesis of natural products and molecules with biological significance.^201,202 Following the seminal work of Otto Diels and Kurt Alder in 1928, tremendous advancements have been made to improve the selectivity and yields of the reaction. The reaction rate can be accelerated by the use of lewis acids or in situ generated reactive dienes. The hetero-Diels–Alder reaction is a variant of Diels-Alder reaction in which hetero atom containing dienes or dienophiles are involved in the bond formation step. Light can be used to generate reactive hetero-dienes, e.g., o-quinone methides and hydroxy-o-quinodimethanes (photenols), from appropriate precursors. Since light-induced Diels–Alder reactions proceed under physiological conditions without requiring a catalyst or producing side products and allow spatiotemporal control of the process, they have been extensively exploited in biological applications and material chemistry.

Popik and coworkers reported the photoirradiation of 3-(hydroxymethyl)-2-naphthol (NQMP, 173) to generate reactive naphthoquinone-3-methides (oNQMs, 174), which undergoes facile hetero-Diels–Alder reaction with the electron-rich olefins to afford photostable benzochroman 175 in high yields (Scheme 40a).^14,203 The photoactivation of NQMP can be carried out using a low-pressure mercury lamp or fluorescent tube (300 nm or 350 nm) with high quantum yields (Φ₃₀₀ = 0.17 ± 0.02 for 173). The reactions produce the benzochroman products exclusively with second-order rate constants up to 4 × 10⁴ M⁻¹ s⁻¹; the unreacted oNQMs 174 undergo rapid hydration (k_H2O ~ 145 s⁻¹) to regenerate the starting material 173 (Scheme 40a). The distinctive features of this transformation are fast kinetics and the use of only 1.5 equivalents of alkene dienophiles. Notably, oNQMs react with vinyl ethers and enamines, but not other alkenes such as methyl acrylate, 2,5-dihydrofurans, dimethyl maleate, and methylcyclohexane (Scheme 40b). For unreactive alkenes, the hydration process predominates, resulting in regeneration of the starting material. Importantly, the product stability is dependent on the pH of the solution; for example, the use of enamines as an alkene partner results in the formation of 2-hydroxybenzochroman under neutral aqueous conditions (Scheme 40c). Later, the Popik group demonstrated the utility of this transformation in the light-directed surface derivatization and patterning.^204,205

Scheme 40:

Photoactivation of NQMP to generate oNQM for hetero-Diels-Alder cycloaddition

The in situ generated o-NQMs 174 can be intercepted by a variety of nucleophiles, including thiols and azide ions (Scheme 41a).²⁰⁶ The reaction between o-NQMs and thioethanolamine proceeds five times faster than the one with ethyl vinyl ether. The resulting thiol adducts are photolabile and can be converted into o-NQM during extended irradiation. This type of thiol–quinone methide photoclick chemistry has been exploited for various applications including selective and irreversible functionalization of proteins and patterned immobilization of biomolecules onto surfaces (Scheme 41b).^207,208

Scheme 41:

Thiol–quinone methide photoclick reactions

4.2. Hetero-Diels–Alder Reaction of o-Methyl Phenyl Ketones and Aldehydes

The o-quinodimethanes (oQDM) display remarkable activity in the Diels-Alder reaction and proven to be versatile intermediates for the synthesis of cyclic scaffolds.^209,210 In recent years, photo-triggered Diels–Alder reaction based on o-quinodimethanes have been exploited in various applications,²¹¹ including surface functionalization,^212,213 syntheses of sequence-defined macromolecules,²¹⁴ light-induced assembly of nanostructures,²¹⁵ 3D laser writing^216,217 and polymer-polymer conjugations.^218–222

In 2011, Barner-Kowollik and coworkers reported a light-triggered Diels–Alder reaction for polymer conjugation based on photoinduced generation of o-quinodimethanes (photoenols) from 2‐methylbenzophenones 183 (Scheme 42a).²²³ The reaction involves the formation of biradical 184, which undergoes rearrangement to form highly reactive E- and Z-photoenols. The E-isomer 185 diplays excellent reactivity with the activated dienophiles such as maleimides in conjugating polymeric building blocks. However, the Z-isomer 186 may undergo a [1,5]‐sigmatropic shift to deliver starting material (Scheme 42a). The efficiency and speed of the photoenol chemistry were demonstrated in the conjugation of polymeric building blocks containing o-methyl phenyl ketone and maleimide functionalities. The introduction of a hydrogen bond donor to the ortho position of the formyl group further enhanced the reactivity of photoenols (Scheme 42b).²¹² This enhancement is attributed to the presence of hydrogen bonding in stabilizing the intermediate, leading to its longer lifetime and thus the amount of Z-isomer formed 189.²²⁴ The Barner-Kowollik group designed a 2‐formyl‐3‐methyl phenoxy (FMP) substrate 188 as an efficient precursor of photoenol for light-triggered Diels–Alder reaction with a variety of dienophiles including the maleimide and acrylamide (Scheme 42b).²¹² The transformation is compatible with a wide range of solvents, including dichloromethane, acetonitrile, DMF, and water; however, irradiation time varies depending on the solvent.

Scheme 42:

Light-triggered hetero-Diels–Alder reaction of o-methyl phenyl ketones and aldehydes

Recently, Barner-Kowollik and coworkers studied wavelength-dependency of the photoinduced click reaction between o-QDMs and dienophiles to understand the mechanism and photophysical properties of the reaction.⁴³ To apply the photoenol chemistry to biological applications, they presented a visible light-induced Diels–Alder transformation based on reactive o-quinodimethane thioethers 191 and electron-deficient alkenes.²²⁵ The use of visible light for the generation of reactive dienes was achieved through a simple oxygen-to-sulfur switch within the o-methylbenzaldehyde (o-MBA) structure 191 (Scheme 42c), which lowers the energy gap for the π→π* transition in the formation of o-QDMs and contributes to the red-shift in absorption. The visible-light-induced [4+2] ligation proceeds quantitatively in aqueous solution as well as in organic solvents.

Since 2‐cyanopropyl dithiobenzoate (CPDB) based polymers (RAFT agents) and electron-deficient alkynes are compatible with the photoenol chemistry, hetero-Diels–Alder conjugation was used in the conjugation of the photoenol with the thioester terminus containing RAFT polymer under ambient conditions (Scheme 43a).²²⁰ The use of electron-deficient alkynes as dienophiles in Diels–Alder reaction with photocaged dienes 188 and 191 leads to the formation of pro-fluorescent Diels–Alder product 195 (Scheme 43b).²²⁶ Since the pro-fluorescent product 195 does not absorb light in the range of starting material, the photobleaching and other side reactions from competitive absorption were not observed. Interestingly, the pro-fluorescent product 195 was converted into fluorescent naphthalene 196 in the presence of a catalytic amount of acid via rapid E1 elimination (Scheme 43b). The controlled generation of the fluorescent product enables fluorometric evaluation of the ligation reaction.

Scheme 43:

a) Light-induced hetero-Diels–Alder conjugation of photoenols with non-activated dithioester; b) Light-induced hetero-Diels–Alder reaction with alkynes

4.3. Hetero-Diels–Alder Reaction of 9,10-Phenanthrenequinone with Alkenes

In 2018, Zhang and coworkers reported visible light-triggered photoclick reaction involving [4+2]-cycloaddition between 9,10-phenanthrenequinone (PQ) 197 and electron-rich alkenes such as vinyl ethers (VE) to form fluorescent adducts 199 (dione–vinyl ether photocycloaddition).²²⁷ The reaction proceeds via photoinduced electron transfer (PeT) pathway involving excitation of PQ functionality to PQ*. The electron transfer between the excited PQ* and vinyl ether (VE) moiety leads to the formation of a 1,6-biradical intermediate 198, which undergoes intramolecular radical recombination to form [4+2] cycloadducts 199 (Scheme 44). This reaction displays high selectivity for the cycloaddition, and side reactions such as nucleophilic addition or cycloadditions with electron-deficient olefins were not observed. The authors demonstrated the utility of this photocycloaddition in orthogonal labeling of proteins together with strain-promoted azide-alkyne cycloaddition (SPAAC) or the tetrazole photoclick chemistry with monomethyl fumarate.

Scheme 44:

Visible light-triggered photoclick cycloaddition

4.4. Tetrazine Ligation

The inverse electron-demand Diels–Alder cycloaddition (iEDDAC) of tetrazines with strained alkenes or alkynes, also known as tetrazine ligation, has emerged as an ideal tool for biomolecular labeling.^228–230 Fox and co-workers reported a tetrazine ligation involving a cyclopropane-fused trans-cyclooctene with a second-order rate constant of 10⁶ M⁻¹ s⁻¹,^231,232 representing one of the fastest bioorthogonal reaction reported in the literature. Recently, light-induced tetrazine ligations (photo-iEDDAC) were developed based on in situ generations of one of the reaction partners.^233–236 For example, Fox and coworkers described a light-induced tetrazine ligation using visible light along with methylene blue photosensitizer for oxidation of dihydrotetrazine to tetrazine (Scheme 45a).²³³ Irradiation of dihydrotetrazine 200 with 660-nm light in the presence of methylene blue generates the reactive tetrazine 201, which then reacts with trans-cyclooctene to form cycloadduct 202 (Scheme 45a). The photocatalytic activation of tetrazine ligation has been used for the functionalization of polymeric materials. For example, Forsythe and coworkers used the light catalyzed tetrazine-norbornene iEDDAC reaction for polymer cross-linking to form the hydrogels.²³⁴ Recently, the photoactivation of cyclopropenones to generate to cycloalkynes was also adopted in the light-triggered tetrazine ligation for site-specific protein labeling in live cells (Scheme 45b).²³⁵ The cyclopropenone-caged BCN-based probe 204 displayed excellent reactivity towards tetrazine with rate constants of 50 M⁻¹ s⁻¹. Similarly, Laughlin and coworkers reported a photocaged approach in cyclopropene–tetrazine ligation by masking the cyclopropene reactivity with a photo-protecting group (Scheme 45c).^236,237 Here, a unique caged spirocyclopropene derivative 208 was designed in which the presence of a bulky photo-protecting group prevents the cycloaddition reaction. Upon irradiation, the protecting group is removed to release the unhindered cyclopropene 209, which readily reacts with the tetrazine 207.

Scheme 45:

a) Visible light-induced tetrazine ligation; b) Cyclopropenone‐caged bicyclononynes for light-triggered tetrazine ligation; c) Caged cyclopropenes for light-triggered tetrazine ligation

5. Light-Triggered Sydnone–Alkyne Cycloadditions

Sydnones are an important class of mesoionic heterocycles that have been studied extensively in synthetic chemistry due to their stability and facile synthesis.²³⁸ Generally, sydnones are depicted in the form of resonance structure 211, which is similar to the enolate structure. However, experimental studies suggested that the sydnones might not be aromatic; other representations such as 212 and 213 are in agreement with the spectral data (Scheme 46a).²³⁹ Sydnones are synthesized in two steps: nitrosylation of an amino acid derivative followed by cyclodehydration in the presence of an acid (Scheme 46b).²⁴⁰ Sydnones display excellent reactivity in 1,3-dipolar cycloadditions with a variety of dipolarophiles to form pyrazoles. The reaction proceeds through the formation of a bicyclic intermediate 218 that undergoes spontaneous CO₂ loss via a retro-Diels–Alder process to afford the pyrazole product 219 (Scheme 46c). Over the years, tremendous advancements have been made with this transformation.^238,241 Among them, copper-mediated sydnone–alkyne cycloaddition (CuSAC) is the most significant, enabling its use as a click reaction for bioconjugation.²⁴² Recently, Cu-free sydnone–alkyne cycloadditions were reported using the strained alkynes bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) and dibenzoazacyclooctyne with k₂ values of 0.054 M⁻¹ s⁻¹ and 1.46 M⁻¹ s⁻¹, respectively.^243,244 Surprisingly, the reaction of 4-fluorosydnones with the strained alkynes improved reaction kinetics tremendously, with the second-order rate constants reaching 10⁴ M⁻¹ s⁻¹.^245,246

Scheme 46:

a) Sydnone resonance structures; b) Synthesis of sydnones; c) Cycloaddition reaction between sydnones and alkynes

The generation of nitrile imines species upon photoirradiation of sydnones and their use in cycloaddition reactions have been well-documented in literature.^247–251 Inspired by their unique reactivity, Yu and coworkers reported the first photoinduced diarylsydnone–alkene cycloaddition reaction with high efficiency and excellent fluorescence turn-on (Scheme 47a).¹⁶ A series of diarylsydnones (DASyds) 220 were synthesized and screened in the photoinduced 1,3 dipolar cycloaddition reactions. Among them, diarylasydnone 220a displayed excellent reactivity in the fluorogenic photoclick reaction towards various alkenes, including TCO, norbornene, methyl methacrylate, diethyl fumarate, and 5-vinyl-2’-deoxyuridine (VdU). Notably, the competitive thermal cycloaddition was extremely slow as there were no cycloadducts observed in the dark for 24 hours. Selective photoinduced fluorogenic labeling of TCO-appended proteins was achieved by using 220a. Subsequently, they demonstrated fast photoclick reactions between the diarylsydnones and the strained alkynes (Scheme 47b).²⁵² Importantly, the reaction between 220d and BCN was achieved under 405-nm photo illumination with high efficiency and selectivity. The competing cycloaddition products derived from the diarylsydnone–alkyne cycloaddition (DASAC) pathway were also observed; however, this pathway proceeds at a much slower rate compared to the photoclick one (Scheme 47b). Significantly, the adjacent ortho-diaryl units on the diarylsydnones (DASyd) twisted around the sydnone core, which presents steric repulsion towards the incoming dienophile and hinders the DASAC pathway. Kinetic studies indicated that the decarbonylation of 4,5-diaryl-2-oxa-1,5-diazabicyclo[2.1.0]pentan-3-one 221 might be the rate-determining step in the transformation. The biocompatibility of this reaction was demonstrated through selective protein labeling on A549 cell surface. The same group also designed the photoactivatable β-diarylsydnone-L-alanines for the fluorogenic tracing of peptides in live cells via photoclick cyclization.²⁵³

Scheme 47:

Photo-triggered sydnone–alkene/alkyne cycloadditions

Recently, Yu and coworkers reported a visible-light accelerated bioorthogonal reaction of diarylsydnone 220 with the strained dibenzo[b,f][1,4,5]thiadiazepine (DBTD) (Scheme 48).²⁵⁴ In addition to the generation of nitrile imine species, visible light irradiation also induces the isomerization of DBTD from Z to E to confer ring-strain onto the macrocyclic azobenzene. Kinetic studies revealed that (E)-DBTD 229 reacts 6.6-fold faster than that of (Z)-DBTD 228. Notably, the cycloaddition between the N=N bond of (E)-DBTD 229 and the nitrile imine dipole was extremely fast, with k₂ of (1.6 ± 0.16) × 10⁵ M⁻¹ s⁻¹. The versatility of the DBTD reporter was demonstrated in selective fluorescence labeling of proteins.

Scheme 48:

DASyd-DBTD photoclick reaction

6. Light-Triggered Azirine–Alkene Cycloadditions

Upon photolysis, 2H-azirines 230 generate reactive nitrile ylides that can react with a variety of dienophiles to form cycloadducts 234 (Scheme 49a).^255–257 Lin and coworkers reported a photoinduced azirine–alkene cycloaddition, or azirine ligation, for efficient protein modification in an aqueous medium.¹⁷ The in situ generated nitrile ylides appear to be more reactive than the photogenerated nitrile imines and react with the electron-deficient alkenes such as dimethyl fumarate. The utility of azirine ligation was demonstrated in the selective functionalization of an azirine-modified lysozyme by PEG-modified fumarate (Scheme 49b). To expand the scope of azirine ligation, Barner-Kowollik and coworkers introduced a pyrene group on the azirine ring, leading to visible-light (>390 nm) triggered azirine ligation with the electron-deficient alkenes such as fumarates, acrylates, and maleimide under ambient conditions (Scheme 49c).²⁵⁸

Scheme 49:

Photoinduced azirine–alkene cycloadditions

7. Light-Triggered Oxime Ligation

In recent years, oxime ligation—the condensation of aldehydes with alkoxyamines—has been extensively studied owing to their high efficiency, selectivity, and operational simplicity.^259–261 The main advantages include the use of inexpensive starting materials with water being the only side product and compatibility with the aqueous medium, making this reaction attractive for biological and polymer applications. Light can be used as an external stimulus to generate one of the two reactants involved in an oxime click reaction. The Maynard and Yousaf groups reported the light-triggered oxime ligation reactions for immobilizing biomolecules based on the photochemical generation of the alkoxyamine species (Scheme 50a).^262,263 Owing to the inherent photochemical control, this reaction has been employed in micropatterning substrates.^18,264 Separately, Barner-Kowollik and coworkers reported a rapid generation of aldehydes through photo-deprotection of the o‐nitrobenzyl acetal derivatives at 370 nm (Scheme 50b).¹⁸ The utility of this light-triggered oxime reaction was demonstrated in surface patterning with a fluorophore as well as the GRGSGR peptide.

Scheme 50:

Light-induced oxime ligation reactions

8. Miscellaneous Photochemical Reactions

Given the apparent advantages of photochemistry, several light-triggered reactions can be potentially turned into click reactions owing to the usefulness of the in situ generated reactive intermediates. In particular, photoinduced benzodioxinone-based ketene chemistry, photoinduced perfluorophenyl azide chemistry, and photoinduced [2+2] cycloaddition reactions have attracted significant interests in organic chemistry and material sciences.^11,102 Ketenes are a versatile class of intermediates with unique reactivity in synthetic chemistry and have proven to be highly reactive towards a variety of functional groups including amines, acids, alcohols, and unsaturated compounds.^265–267 Ketenes can be readily generated photochemically from irradiation of benzodioxinone 237 or dialkyl Meldrum’s acid and react with alcohols rapidly in the synthesis of linear and cross-linked polymers (Scheme 51a).^268–278 Similarly, the photoinduced perflurophenyl azide chemistry has been widely exploited in surface functionalization and nanomaterial synthesis owing to its fast kinetics, high efficiency, and easy preparation.²⁷⁹ Mechanistically, the reaction involves the photochemical generation of electron-deficient perfluorophenyl nitrene 241, which readily undergoes C-H or X-H insertion reactions (Scheme 51b). The utility of this reaction was demonstrated in the immobilization of carbohydrates to different nanomaterials such as silica wafer, gold substrates, and polymers.^280–284 Finally, the photoinduced reaction between a carbonyl compound and an alkene, commonly known as Paterno–Büchi reaction, is a photochemical reaction invaluable for the synthesis oxetanes and functionalization of polymers (Scheme 51c).^285–287 Similar to other click reactions, this reaction proceeds through a concerted mechanism with 100% atom economy. However, the reaction requires an excessive amount of the alkene reaction partner and proceeds rather slowly. Nevertheless, numerous applications based on [2+2]-photocycloaddition were well-documented in the literature.^287,288

Scheme 51:

Light-triggered a) ketene chemistry; b) perfluorophenyl azide chemistry; and c) [2+2] cycloaddition

9. Conclusions and Outlook

In this Review, we have discussed three types of light-triggered click reactions, including mainly 1,3-dipolar cycloadditions, hetero-Diels-Alder cycloadditions, and oxime ligation, and their growing impact on diverse research fields such as chemical biology and material sciences. The use of light as external stimuli to trigger click reactions not only enhances the efficiency of the click reactions but also offers rapid access to diverse scaffolds and molecular architectures under mild reaction conditions. Furthermore, the “on-demand” nature of light-induced reactions has added a spatiotemporal control to the click reactions for a variety of critical applications, including surface functionalization, polymer chemistry, and bioorthogonal chemistry. Importantly, the efficiency of photoclick reactions can be refined by adjusting wavelength, light intensity, and photoirradiation time.

The field of the light-triggered click chemistry continues to expand in the last decade as a growing number of photoclick reactions have been reported based on the photochemical generation of reactive species without the use of toxic metal catalysts or reagents. Among them, tetrazole photoclick reaction and hetero-Diels-Alder photoclick reactions have received substantial attention from the research community owing to their concerted reaction mechanism and rapid reaction rate. More recently, the use of light to introduce ring strain into the substrates represents an emerging area within the photoclick chemistry arena with a potential to open up new opportunities for bioconjugation and nanomaterial synthesis.^200,254,289

Since the majority of photoclick reactions are triggered by UV light, which may restrict their utility in biological systems, tremendous efforts have been devoted to the development of photo-triggered click reactions under visible and near-infrared (NIR) light. Significant advances have been made in recent years in tuning photoactivation wavelength, decreasing photoirradiation time, improving reactant stability in complex environments, and designing faster reactions. The availability of a sizeable photoclick reaction repertoire operating at varied but discrete wavelengths has made it possible to perform tandem λ-orthogonal photoclick reactions in materials science.⁷¹ With additional photoactivation modalities, it should be possible in the future to employ multiple photoclick reactions in multiplexed manipulation of biomolecules in a spatiotemporally controlled manner in their native cellular environments. With the advancements mentioned here and continuing expansion of the field, we envision that the powerful tools derived from the union of light with click chemistry will fuel a new age of molecular exploration in chemical biology and materials science in many years ahead.

ABBREVIATIONS

Ac

acetyl

ACN

acetonitrile

AcrK

N⁶-acryloyl-L-lysine

ADIBO

azadibenzocyclooctyne

AsphK

N⁶-((2-oxo-2-(5-azaspiro[2.3]hex-1-en-5-yl)ethoxy)carbonyl)-L-lysine

BCN

bicyclo[6.1.0]nonyne

Boc

tert-butoxycarbonyl

BODIPY

boron-dipyrromethene

Bn

benzyl

Bz

benzoyl

CpK

N⁶-(1-methylcycloprop-2-ene-1-carbonyl)-L-lysine

CQ

camphorquinone

CuAAc

copper-mediated azide alkyne cycloaddition

CuSAc

copper-mediated sydnone alkyne cycloaddition

CPDB

2‐cyanopropyl dithiobenzoate

DASAC

diarylsydnone-alkyne cycloaddition

DASyd

diarylsydnones

DBMP

2-benzyl-2-(dimethylamino)-4′-morpholinobutyrophenone

DBTD

dibenzo[b,f][1,4,5]thiadiazepine

DCM

dichloromethane

DIBOD

dibenzo[a,e]cyclooctadiyne

DIC

differential interference contrast

DIO

3,3-dioctadecyloxacarbocyanine

DMF

N,N-dimethylformamide

DNA

deoxyribonucleic acid

DIBO

dibenzocyclooctyne

DMSO

dimethylsulfoxide

EDG

electrondonating group

EWG

electron withdrawing group

EtOAc

ethyl acetate

E. coli

Escherichia coli

EGFP

enhanced green fluorescent protein

FT-IR

Fourier-transform infrared spectroscopy

GFP

green fluorescent protein

GPCRs

G protein-coupled receptors

HdeA

(histone-like nucleoid structuring)-dependent expression A

HEK

human embryonic kidney

HOMO

highest occupied molecular orbital

iEDDAC

inverse electron-demand Diels–Alder cycloaddition

LUMO

lowest unoccupied molecular orbital

LC-MS

liquid chromatography-mass spectrometry

Mdm2

mouse double minute 2 homolog

MC-DIBOD

mono-cyclopropenone-caged dibenzocyclooctadiynes

MjTyrRS

Methanocaldococcus jannaschii tyrosyl-tRNA synthetase

MP-SPAAC

multiphoton-triggered SPAAC

NMR

nuclear magnetic resonance

NorK

N⁶-((((1S,2R,4S)-bicyclo[2.2.1]hept-5-en-2-yl)methoxy)carbonyl)-L-lysine

Ph

phenyl

eq

equivalents

HAG

homoallylglycine

Me

methyl

NBR

nitrile-butadiene rubber

NIR

near-infrared

NQMP

3-(hydroxymethyl)-2-naphthol

o-MBA

o-methylbenzaldehyde

oNQMs

2-napthoquinone-3-methides

oQDM

o-quinodimethanes

PBS

phosphate-buffered salin

PBTA

poly(tertbutyl)acrylate

PDA

polydopamine

PEG

polyethylene glyco

2PE

two-photon excitation

PMDETA

N,N,N′,N′′,N′′-pentamethyldiethylenetriamine

poly(MeOEGMA)

poly[oligo(ethylene glycol)methyl ether methacrylate

PQ

9,10-phenanthrenequinone

RAFT

reversible addition–fragmentation chain transfer

RNA

ribonucleic acid

rt

room temperature

sfGFP

superfolder green fluorescent protein

SCNPs

single-chain nanoparticles

SPAAC

strain-promoted cycloaddition

Sph

spiro[2.3]hex-1-ene

SphK

N⁶-((spiro[2.3]hex-1-en-5-ylmethoxy)carbonyl)-L-lysine

StyrK

N⁶-(((4-vinylbenzyl)oxy)carbonyl)-L-lysine

Tet

tetrazole

Titanocene

dicyclopentadienyl bis[2,6-difluoro-3-(1-pyrrolyl)phenyl]titanium

TMEDA

tetramethylethanediamine

TMDPO

trimethylbenzoyl)diphenylphosphine oxide

TLC

thin layer chromatography

TCO

trans-cyclooctene

UV

ultraviolet

UCNPs

up-converting nanoparticles

VE

vinyl ethers

Biographies

Gangam Srikanth Kumar received a Master’s degree in Chemistry from Central University of Rajasthan in 2013 and a Ph.D. in organic chemistry from the Indian Institute of Science and Education Bhopal in 2018 under the guidance of Prof. Manmohan Kapur. His doctoral studies focused on the development and mechanistic insight of transition-metal catalyzed C-H functionalization methods for the synthesis of diverse heterocycles. After completing his Ph.D., he began his postdoctoral research in the group of Prof. Qing Lin at University at Buffalo, USA. He is currently working on the development and application of bioorthogonal methods based on the 1,3-dipolar cycloaddition reactions of nitrile imines.

Professor Qing Lin received his BS in Chemistry from the University of Science and Technology of China in 1994 and Ph.D. in Organic Chemistry from Yale University in 2000 under the direction of Professor Andrew Hamilton. After postdoctoral training in Professor Peter Schultz’s lab at Scripps Research and a brief stay in the industry, he joined the faculty of the State University of New York at Buffalo in 2005, where he is now Professor of Chemistry. His research interests include the development of (i) bioorthogonal reaction-based tools and their use in elucidating molecular mechanisms of the class B G protein-coupled receptor-mediated signaling, and (ii) enabling chemical technologies for the design of next-generation peptide and protein therapeutics.