Photo-Triggered Click Chemistry for Biological Applications
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In the last decade and a half, numerous bioorthogonal reactions have been developed with a goal to study biological processes in their native environment, i.e., in living cells and animals. Among them, the photo-triggered reactions offer several unique advantages including operational simplicity with the use of light rather than toxic metal catalysts and ligands, and exceptional spatiotemporal control through the application of an appropriate light source with pre-selected wavelength, light intensity and exposure time. While the photoinduced reactions have been studied extensively in materials research, e.g., on macromolecular surface, the adaptation of these reactions for chemical biology applications is still in its infancy. In this chapter, we review the recent efforts in the discovery and optimization the photo-triggered bioorthogonal reactions, with a focus on those that have shown broad utility in biological systems. We discuss in each cases the chemical and mechanistic background, the kinetics of the reactions and the biological applicability together with the limiting factors.
KeywordsBioorthogonal reaction Photo-triggered reaction Photoclick Tetrazole Nitrile imine Azirine Cyclopropenone o-Naphthoquinone methide o-Quinodimethanes Hetero Diels–Alder reaction
Cu-catalyzed azide–alkyne cycloaddition
Highest occupied molecular orbital
Lowest unoccupied molecular orbital
Phosphate buffered saline
Strain-promoted azide–alkyne cycloaddition
Unnatural amino acid
To study biological pathways in living system, it is often necessary to fluorescently label the biomolecules involved in the pathway so that their movement and function can be visualized directly inside a living cell. To this end, click chemistry  and many other bioorthogonal reactions have been developed in the last decade and a half, and they all share the following features: (1) the reactants do not interfere with the native biochemical processes inside cells; (2) the reaction rate is comparable to that of a biological process, and (3) the reactants are stable and nontoxic in living cells and animals [2, 3]. Major efforts have been devoted to achieve a faster reaction rate, to improve the physicochemical properties of the reactants such as size, solubility and membrane permeability, and to demonstrate the utilities of these reactions in biological systems. Amongst these reactions, the photo-triggered reactions represent a special class as they generally exhibit a finer control of the reaction initiation and duration, together with greater resolution in space and time. These controls are achieved through the use of an appropriate light source with pre-selected wavelength, light intensity and exposure time. Because the photo-triggered reactions proceed without the need of metal catalysts and ligands, they are generally less toxic to cells than the metal-catalyzed reactions.
Photoinduced 1,3-Dipolar Cycloaddition Reaction Between Tetrazoles and Alkenes
Theoretical Background and Reaction Mechanism
In addition to the photo-generation, Carell and coworkers showed that the hydrazonyl chloride (13) could also serve as a precursor to the reactive nitrile imine (17a) in neutral aqueous buffer . Intrigued by these results, Liu and coworkers conducted a mechanistic study of the hydrazonyl chloride-mediated cycloaddition by examining the effects of pH and chloride concentration on the rates of nitrile imine formation and subsequent cycloaddition reactions . They concluded that the cycloaddition reaction is most favorable at basic pH and in the absence of the chloride ion, and that the cycloaddition rate approaches 3.4 × 104 M−1 s−1 after factoring in the various equilibria, which makes this cycloaddition one of the fastest click reactions. A plausible mechanism unifying the two nitrile imine generation pathways is shown in Scheme 4, in which water or HCl addition products serve as reservoirs for the unstable nitrile imine prior to the cycloaddition reaction.
Synthesis of Tetrazoles
Effects of Substrates on Photoactivation Wavelength and Spectral Properties of Pyrazoline
Since 302-nm UV light may pose considerable phototoxicity to cells , for wider use of the tetrazole-based cycloaddition reaction in biological systems it is imperative to shift the photoactivation wavelength to the long-wavelength region. To this end, a series of substituted diaryltetrazoles (33) were synthesized (Scheme 6b) , and their absorption maxima and absorption coefficients at 365 nm (wavelength of the long-wavelength hand-held UV lamp) were determined in MeOH/H2O (1:1). It was found that the electron-donating NH2 and NMe2 groups yielded the largest shift in absorption maxima (to 310 and 336 nm, respectively) along with increased absorption coefficients at 365 nm (0.35 × 104 and 1.87 × 104 M−1 cm−1, respectively).
Site-Specific Labeling of Proteins Via the Photoinduced Tetrazole–Alkene Cycloaddition
In the very first application of the tetrazole-based cycloaddition chemistry to proteins , a carboxylic acid functionalized tetrazole was coupled to a tripeptide (RGG) and the kinetics of the cycloaddition reaction between the tetrazole-modified peptide and acrylamide was investigated under the 302-nm photoirradiation condition. The photolysis of the tetrazole-modified peptide to its corresponding nitrile imine was extremely rapid with a first-order rate constant to be 0.14 s−1; the subsequent cycloaddition with acrylamide proceeded with a second-order rate constant, k 2, of 11.0 M−1 s−1. In the next step, the surface Lys residues of lysozyme were modified with a water-soluble tetrazole succinimide (60), and the resulting tetrazole-modified lysozyme was irradiated with 302 nm light for 2 min in the presence of acrylamide (Scheme 8a). The LC–MS analysis indicated that the conversion of the tetrazole-modified lysozyme to the pyrazoline adduct was very specific with an estimated yield around 90 %. We also prepared a tetrazole-containing enhanced green fluorescent protein, EGFP-Tet, using the intein-based chemical ligation strategy (Scheme 8a). The photo-triggered cycloaddition reaction between EGFP-Tet and N-hexadecylmethacrylamide proceeded in bacterial lysate with ~52 % conversion based on LC–MS analysis. The application of the tetrazole-modified EGFP was also used to probe the effect of lipidation on protein localization in live cells without the use of lipidation enzymes .
The tetrazole-based cycloaddition chemistry was subsequently applied to protein labeling in bacteria cells via a genetically encoded O-allyl-tyrosine (O -allyl-Tyr, Scheme 8c) . In this study, the reactions between tetrazole derivatives and allyl phenyl ether were first investigated. The formation of nitrile imine was found to be fast (<2 min) but the cycloaddition steps was slow (k 2 = 0.00202 M−1 s−1 with allyl phenyl ether in 1:1 PBS/MeCN; 75-fold slower than acrylamide). In-gel fluorescence-based screen identified 4-(methoxycarbonyl)phenyl substituted tetrazole in the C5-position from a panel of tetrazole compounds as a suitable reagent for selective labeling of O-allyl-tyrosine-encoded Z-domain protein, which was then used to image the same protein in E. coli cells. Since the reaction rate is inversely related to the energy gap between HOMOdipole and LUMOdipolarophile in the nitrile imine–alkene cycloaddition , it was envisioned that raising the HOMO energy of the nitrile imine should lead to reaction acceleration. Thus, a series of substituted diaryltetrazoles were synthesized and their reaction rates toward 4-penten-1-ol were measured . It was found that the electron-donating substituents on the N2-phenyl ring such as –NH2 and –OMe groups significantly raise the HOMO energies of the photo-generated nitrile imine dipoles, with second-order rate constants reaching as high as 0.79 M−1 s−1 in 1:1 PBS/MeCN. Furthermore, a methoxy-substituted diphenyltetrazole was shown to label the O-allyl-tyrosine-encoded Z-domain proteins in E. coli in <1 min.
To overcome slow reaction kinetics observed with the genetically encoded system, two complementary efforts were untaken. One approach was to incorporate the tetrazole moiety into the amino acid side chain and evolve orthogonal aminoacyl-tRNA synthetase/tRNA pairs to charge the resulting tetrazole amino acids into proteins site-specifically. To this end, we synthesized a series of tetrazole modified unnatural amino acids (Scheme 8b), and tested their reactivity in the photo-triggered cycloaddition reaction with methyl methacrylate in PBS/MeCN (1:1) . While four out of six tetrazole amino acids gave excellent reaction yields, only p-tetrazole-phenylalanine (p -Tpa) was incorporated into myoglobin site-specifically using an evolved synthetase via the amber codon suppression . A drawback of this genetically encodable tetrazole amino acid is that a 254-nm UV light is needed for triggering the reaction because of the shortened π-conjugation system. In addition, the cycloaddition reaction with dimethyl fumarate proceeded rather sluggishly, with a measured k 2 value of 0.082 ± 0.011 M−1 s−1 in 1:1 PBS/MeCN.
The second approach focused on the genetic encoding of the reactive alkene-containing amino acids, which are in general smaller than the tetrazole amino acids and thus easier to derive the specific orthogonal aminoacyl-tRNA synthetase/tRNA pairs (Scheme 8c). To increase alkene reactivity in the photoclick chemistry without unwanted side reactions such as Michael addition, ring strain has been successfully exploited. In 2010, we showed that norbornene served as an excellent substrate in the photo-triggered cycloaddition reaction with the macrocyclic tetrazoles . Later, Carell and co-workers showed that a norbornene-modified Lys (68) can be genetically encoded into His6-tagged human polymerase κ, and that efficient labeling of polymerase κ can be accomplished with the nitrile imines generated from either the hydrazonyl chloride precursor or the corresponding tetrazole . The utility of the norbornene modified amino acids was also demonstrated in the tetrazine ligation in eukaryotic cells by other research groups [46, 47, 48]. Smaller strained alkenes such as cyclopropene have also been developed for the photoclick chemistry. Cyclopropene has a ring strain of 54.1 kcal mol−1  versus 21.6 kcal mol−1 for norbornene ; after cycloaddition, the cyclopropane product has a decreased strain of 28.7 kcal mol−1 . Based on these considerations, we synthesized a cyclopropene modified Lys, CpK, and showed that CpK can be site-specifically incorporated into proteins using the amber codon suppression strategy . Fast cycloaddition kinetics was observed with the 3,3-disubstituated cyclopropene in 1:1 PBS/MeCN; the second-order rate constant was determined to be 58 ± 16 M−1 s−1 (for comparison: k 2 = 46 ± 9 M−1 s−1 for acryl amide, 32 ± 12 M−1 s−1 for norbornene, and 0.95 M−1 s−1 for allyl phenyl ether). Importantly, CpK was shown to direct site-specific modification of green fluorescent protein inside HEK293T cells via the tetrazole-based photoclick chemistry. Notably, the cyclopropene moiety has also been used as a reaction partner in tetrazine ligation [53, 54], with its reactivity preference between the tetrazine and tetrazole chemistries depending on the substitution pattern . Realizing the cyclopropene reactivity can be further increased by reducing the steric hindrance at position 3, we fused a cyclobutane ring with the cyclopropene to generate spiro[2.3]hex-1-ene (Sph) . Sph reacted with methoxy-diphenyltetrazole in CD3CN 17-times faster than the 3,3-disubstitued cyclopropene. A spiro[2.3]hex-1-ene modified lysine (SphK) was synthesized and shown to be successfully incorporated into the superfolder GFP (sfGFP) using the wild-type PylRS/tRNA pair. To our delight, the SphK-encoded sfGFP displayed a fast reaction kinetics with a water-soluble tetrazole in phosphate buffer in the photo-triggered cycloaddition reaction with a measured k 2 value of 1.0 × 104 M−1 s−1, comparable to the typical tetrazine-trans-cyclooctene cycloaddition reaction (k 2 = 2.2 × 104 M−1 s−1) .
On the other hand, Liu and co-workers demonstrated the accelerated photoclick chemistry with proteins using the electron-deficient acrylamide modified lysine (AcrK) . The basis for faster kinetics with acrylamide is its lower LUMO energy compared to allyl phenyl ether. The AcrK-encoded sfGFP was successfully labeled with the hydrazonyl chloride precursor. The utility of AcrK was further demonstrated with fluorescent labeling of an overexpressed membrane protein OmpX in E. coli. Independently, Wang and co-workers evolved an orthogonal tRNA/aminoacyl-tRNA synthetase pair that allowed selective incorporation of AcrK into bacterial tubulin-like cytoskeleton protein FtsZ . The utility of AcrK in the tetrazole-based photoclick chemistry was demonstrated with the purified proteins in vitro as well as in E. coli and mammalian cells. Additionally, AcrK was incorporated into GFP-TAG-mCherry-HA protein in Arabidopsis thaliana, a commonly used plant model.
1,3-Dipolar Cycloaddition Reaction Between Azirines and Alkenes
Very recently, Blinco and Barner-Kowollik have successfully shifted the photoactivation wavelength into the visible region (>390 nm) . By replacing the phenyl group with the pyrene group on the azirine ring, they showed that the photo-triggered cycloaddition proceeded in <1 min under ambient conditions with the electron-deficient alkenes such as fumarates, maleimide, acrylates, and activated acetylenes. Reactions were initiated using a photoreactor equipped with an LED light (410–420 nm, 3 W). The utility of this modified reaction was demonstrated through efficient conjugation of PEG chains to the pyrene fluorophore.
Photoinduced Hetero Diels–Alder Reactions
Diels–Alder (DA) reaction is one of the most useful chemical transformations. This reaction typically proceeds slowly in the absence of a catalyst, but can be accelerated with the use of Lewis acids . It is characterized by high yield under various conditions, has less sensitivity against solvents, and does not produce byproducts. It is characterized by high yield under various conditions, insensitive toward solvent polarity, and little byproducts. Indeed, the tetrazine/trans-cyclooctene based inverse electron-demand DA reaction has become the most popular bioorthogonal reaction in the literature because of its fast reaction kinetics —second-order rate constant as high as 2.8 × 106 M−1 s−1 has been reported . So far, two photo-triggered hetero-dienes have been exploited in the DA reactions for biological use, namely, o-quinone methides and hydroxy-o-quinodimethanes.
Lei and coworkers modified this structure to allow a reaction with vinyl thioethers (VT) to form a stable covalent bond . The biological utility was demonstrated by labeling a VT-modified BSA and imaging a VT-modified taxol in live HeLa cells. Introducing a N into the aromatic system eliminate the need for irradiation, therefore losing the advantage provided by photoinitiation.
Strain-Promoted Azide–Alkyne Cycloaddition
Copper-catalyzed azide–alkyne cycloaddition (CuAAC) is one of the most powerful click reactions. The only disadvantage is that the copper is toxic to certain cells . Despite efforts to make the copper complexes more biocompatible [91, 92], the breakthrough was achieved by the Bertozzi group  through harnessing the ring strain present in cyclooctyne to accelerate the reaction. A variety of cyclooctynes and one cycloheptyne have subsequently been reported [94, 95].
Optimized parameters for the photo-triggered click reactions
Rate constant (k 2) (solvent)
405, 700 nm (2PE)
3.4 × 104 M−1 s−1 (1:1 PB/MeCN)
0.0379 M−1 s−1 (1:1 PBS/MeCN)
300, 350 nm
4–6 × 104 M−1 s−1 (1:1 PB:MeCN)
o-Methyl phenyl ketone/aldehyde-alkene
7.6 × 10−2 M−1 s−1 (MeOH)
Work on the tetrazole-based photoclick chemistry in QL lab was supported by the National Institutes of Health (GM 085092). AH thanks the Rosztoczy Foundation (to A.H.) for a scholarship.
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