Seeking Citius—Photochemical Access of Reactive Intermediates for Faster Bioorthogonal Reactions

Abstract

Fast bioorthogonal reactions are sought after because of their superior performance in labeling low-abundance biomolecules in native cellular environments. An attractive strategy to increase reaction kinetics is to access the reactive intermediates through photochemical activation. To this end, significant progress was made in the last few years in harnessing two highly reactive intermediates—nitrile imine and tetrazine—generated through photoinduced ring rupture and catalytic photooxidation, respectively. The efficient capture of these reactive intermediates by their cognate reaction partners has enabled bioorthogonal fluorescent labeling of biomolecules in live cells.

Graphical Abstract

An attractive strategy to increase reaction kinetics in bioorthogonal chemistry development is to access highly reactive intermediates in situ. This article discusses efficient photochemical generation of two reactive intermediates—nitrile imine and tetrazine—from their stable and unreactive precursors and their subsequent use in rapid bioorthogonal labeling of proteins in live cells.

Photo-triggered bioorthogonal reactions

Bioorthogonal reactions enable selective labeling of biomolecules containing unique chemical groups in their native environment, thereby permitting visualization and functional studies of proteins, glycans, lipids, and nucleic acids in living systems.^[1] Because many biomolecules exist in low abundance in cells and high reagent concentrations may cause cytotoxicity, bioorthogonal ligation reaction must possess the highest rates possible to ensure good product formation in a reasonable time.^[2] Two general approaches have been successfully developed in accelerating bioorthogonal ligation reactions in aqueous media. One involves using a metal catalyst to lower the transition state energy. A prominent example is click chemistry in which the formation of dinuclear copper(I) acetylide decreases the activation barrier by approximately 3–6 kcal mol⁻¹.^[3] The other resolves around substrate activation, i.e., raising the ground state energies of reactants through electronic or ring strain. The excellent examples include trans-cyclooctyne in copper-free click chemistry^[4] and trans-cyclooctene in tetrazine ligation.^[5] A significant concern about the activated substrates is that they become chemically unstable, increasing the extent of side reactions with biological nucleophiles and electrophiles abundant in living systems.

One way to get around this reactivity dilemma is to access highly reactive intermediates through photochemical reactions such that their concentrations remain low to minimize the competing side reactions. This type of photo-triggered click reactions^[6] has enabled fast and selective chemical modification of biomolecules with an added benefit of spatiotemporal control invaluable to many biological processes. In general, the precursor substrate is inactive towards its reaction partner and native functionalities in biological systems, but upon photoirradiation generates a reactive intermediate that undergoes bioorthogonal ligation reaction with the biomolecule carrying a chemical reporter (Figure 1). To this end, some prominent photochemical precursors include tetrazoles,^[7] sydnones,^[8] azirines,^[9] and quinones.^[10] Besides bioorthogonal labeling, photogenerated reactive intermediates such as quinone methides also permit the profiling of protein–protein and protein–DNA complexes^[11] as well as mitochondrial proteomes in living cells.^[12] Herein, we focus our discussion on recent advances in tetrazole photoclick chemistry and photo-triggered tetrazine ligation because of their superior reaction kinetics and greater compatibility with biological systems.

Figure 1.

Photochemical access of a reactive intermediate for bioorthogonal ligation reaction with biomolecules containing a chemical reporter (Y) in discrete subcellular compartments or on cell surface.

Tetrazole photoclick chemistry

Among the various light-triggered bioorthogonal reactions, tetrazole photoclick reactions stand out owing to their fast reaction kinetics and ease of operation. In 2008, Lin and coworkers reported the first example of photoclick reactions between tetrazoles and alkenes and their use in site-specific protein labeling in vitro^[7a] and in living cells.^[7b] Genetically encoded strained alkenes such as cyclopropene^[13] and spirohexene^[14] were subsequently developed for efficient reactions with the photochemically generated, highly reactive nitrile imines, enabling visualization of proteins in their native environment.^[15] In parallel, tetrazoles photoactivatable by either violet 405-nm laser^[16] or two-photon femtosecond near-infrared laser^[17] were developed for the spatially controlled photoclick reactions in live cells. A unique feature of the tetrazole photoclick chemistry is that fluorescent pyrazoline adducts are generated after cycloaddition with alkene dipolarophiles, making it possible to monitor the reaction directly by fluorescence in biological systems.

The photochemically generated nitrile imines are exceptional 1,3-dipoles for cycloadditions; however, when suitable dipolarophiles are absent, they are susceptible to nucleophilic addition due to nitrile’s electrophilic character.^[18] In harnessing nitrile imine’s 1,3-dipole reactivity for biomolecular labeling in living systems, a robust dipolarophile must be present along with careful tuning of tetrazole precursor structure to prevent the competing nucleophilic additions (Figure 2a). To this end, Lin and coworkers reported a bioinspired steric-shielding strategy in which tetrazoles carrying a pair of N-Boc-pyrrole groups at sites adjacent to the nitrile imine were designed (Figure 2b). Upon photoirradiation, the in situ generated nitrile imine displayed an extraordinarily long half-life of 102 s in an aqueous medium and excellent selectivity for the cycloaddition over the glutathione addition.^[19] Notably, when spiro[2.3]hex-1-ene (Sph) was used as a dipolarophile, the cycloaddition proceeded at a second-order rate constant of 2,800 M⁻¹ s ⁻¹ in phosphate buffer−acetonitrile (1:1). The utility of this sterically shielded nitrile imine was demonstrated through fluorescent labeling of glucagon receptors on live mammalian cells in 1 min with 500 nM tetrazole-Cy5.

Figure 2.

(a) Photochemical access of reactive nitrile imine intermediates for fast and selective 1,3-dipolar cycloaddition reactions with the strained alkene and alkyne. (b, c, and d) Designed tetrazole−Sph/BCN pairs and their major characteristics. (e) Bioorthogonal labeling of a GFP-fused glucagon receptor in live mammalian cells using a sulfonated tetrazole containing a Cy5 dye. The image 2e is reproduced with permission from Ref. 23. Copyright 2022 American Chemical Society.

The image was reproduced from reference 21 with permission from the American Chemical Society.

While bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN) has proven to be a versatile reagent in bioorthogonal ligation reactions due to its remarkable stability, fast kinetics, and synthetic accessibility,^[20] it has not been explored in tetrazole photoclick chemistry because of its relatively high LUMO energy.^[21] Two recent reports have dissipated this notion and shown its high potency instead in intercepting the exquisitely designed nitrile imines in biological systems. In one, Yu and coworkers designed diphenyltetrazoles carrying ortho-CF₃ groups at the C-phenyl ring to electronically shield the incoming nucleophile and prevent the competing nucleophilic additions (Figure 2c).^[22] High selectivity for the cycloaddition was achieved along with a calculated second-order rate constant of 9.7 × 10⁵ M⁻¹ s⁻¹ in CD₃CN for the optimized tetrazole through NMR-based competition experiments. In another, Lin and coworkers reported a series of sterically shielded, sulfonated tetrazoles and their cycloaddition reactions with BCN with second-order rate constants up to 3.92 × 10⁴ M⁻¹ s⁻¹ in phosphate buffer− acetonitrile (1:1) (Figure 2d).^[23] DFT calculations revealed that BCN approaches the photochemically generated nitrile imine end-on, allowing a closer interaction with the nitrile imine dipole without causing any steric repulsions. Compared to the N-Boc-pyrrole and CF₃ shielding groups, the N-sulfonated pyrroles also increase the water-solubility of the substituted tetrazoles. The N-sulfonated-tetrazole−BCN pair was then used for robust bioorthogonal fluorescent labeling of a glucagon receptor in as little as 15 seconds using only 200 nM of the tetrazole-Cy5 reagent on live mammalian cells (Figure 2e).

Light-induced tetrazine ligation

As the fastest bioorthogonal reaction, tetrazine ligation has been extensively exploited in chemical biology and material science.^[24] Among many advances made over the last decade,^[25] light-induced tetrazine ligation (Figure 3a) is very appealing for two reasons: 1) dihydrotetrazines (DHTz) are generally more stable than tetrazines in biological systems, allowing conditional access of reactive tetrazines via a photochemical oxidation mechanism; 2) it provides a spatiotemporal control over the oxidation reaction when DHTz is restricted to specific subcellular structures. The challenges for light-induced tetrazine ligation include 1) the need to identify biocompatible photocatalysts that can rapidly oxidize DHTz to tetrazines; 2) the selection of DHTz/tetrazine pairs that are of sufficient kinetic stability in aerobic aqueous solution under ambient light. Nevertheless, Fox and coworkers reported DHTz-1, whose conversion to tetrazine was accelerated by methylene blue under 660-nm light (Figure 3b).^[26] They also demonstrated selective modification of DHTz-fibers with Alexa-sTCO, a voracious reaction partner for the tetrazine with a second-order rate constant of 2.2 × 10⁴ M⁻¹ s⁻¹ in methanol. However, this photocatalytic system cannot be used in live cells because methylene blue is a potent photosensitizer for singlet oxygen generation and, thus, phototoxic.

Figure 3.

(a) Scheme for photocatalytic tetrazine ligations. (b, c, d) Designed DHTz/TCO pairs along with suitable photocatalysts. (e) Fluorescent labeling of GFP-fused proteins in various subcellular organelles using CABL with o-TCO-TAMRA. The image 3e was reproduced with permission from Ref. 28. Copyright 2022 American Chemical Society.

The image was reproduced from reference 21 with permission from the American Chemical Society.

To minimize phototoxicity, Fox and coworkers recently reported using common fluorophores such as silarhodamine (SiR) as photocatalysts.^[27] To improve DHTz stability against air oxidation under ambient light, they designed DHTz-2 in which an amino group was placed at the ortho position of the pyridine ring (Figure 3c). The utility of the SiR-based photocatalyst together with DHTz-2 was demonstrated through crosslinking the suitably functionalized hydrogels in mice. Unfortunately, DHTz-2 showed poor cell permeability, preventing its use inside live cells. More recently, Fox and coworkers reported the design of a cell-permeable DHTz-3, which can be efficiently oxidized into the tetrazine using fluorescein or SiR-based photocatalyst (Figure 3d).^[28] The photogenerated, highly reactive tetrazine reacts with trans-5-oxocene (o-TCO) with a second-order rate constant of 1.13 × 10⁶ M⁻¹ s⁻¹. Notably, the cytocompatibility of photocatalysis has been improved by limiting the extracellular production of singlet oxygen. The DHTz-3 tag was then incorporated into target proteins in mitochondria, nucleus, actin, and cytoplasm through HaloTag, and selective target labeling with TCO-TAMRA was accomplished using the catalytic activation through bioorthogonal chemistry with light (CABL) procedure (Figure 3e). Compared to traditional tetrazine ligation, this light-induced variant affords spatiotemporally controlled labeling and improved reaction efficiency by restoring the activity of a tetrazine moiety that may have partially reduced to the corresonding DHTz in subcellular compartments.

Conclusion and Future Directions

We summarized the exciting recent advances in tetrazole photoclick chemistry and light-induced tetrazine ligation. These photoinducible bioorthogonal reactions were successfully employed to label proteins in their native cellular environment. Notably, the specificity in tetrazole photoclick chemistry was achieved by placing the sterically or electronically shielding groups on the C-aryl ring of the tetrazole. The corresponding nitrile imines showed an extraordinarily long half-life in the aqueous medium. Importantly, these photogenerated reactive intermediates can be selectively captured by the strained spiroalkene and cycloalkyne that were readily incorporated into biomolecules in living systems. The utilities of this photoclick chemistry can be further expanded in the future through the development of cell-permeable tetrazole-fluorophore reagents that are activated by long-wavelength or two-photon light. On the other hand, light-induced tetrazine ligation offers a spatiotemporally controlled reaction tool to functionalize biomolecules inside living cells, owing to its fast reaction kinetics. The oxidation of DHTz to Tz was accomplished using a photocatalyst, which permits photoactivation wavelength to be tuned through the use of an appropriate organic dye. In the future, for more efficient photochemical access of the reactive tetrazines, it is desirable to identify an efficient biocompatible DHTz−Tz photocatalyst system that undergoes photoactivation in vivo in seconds. A major drawback of current photochemical activation conditions involves use of UV or visible light, which restricts their use to cultured cells and surfaces of tissue samples. For a wider use of the photochemical activation in bioorthogonal chemistry to probe biological systems in animals, it is imperative in the future to develop photochemical precursors that can be efficiently activated by tissue-penetrating infrared light.