Communication Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
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Visible Light-Initiated Bioorthogonal Photoclick Cycloaddition Jinbo Li,†,§ Hao Kong,†,§ Lei Huang,†,§ Bo Cheng,‡ Ke Qin,‡ Mengmeng Zheng,† Zheng Yan,† and Yan Zhang*,† †
State Key Laboratory of Analytical Chemistry for Life Sciences, Jiangsu Key Laboratory of Advanced Organic Materials, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China ‡ College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China
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rare. Lin et al. have modified tetrazoles with specific substitution groups such as oligothiophenes or naphthalenes to make them activatable with a 405 nm laser11 or two-photon excitation.12 However, the nitrile imine intermediates generated therein were not stable enough to suppress nucleophilic additions by water or thiol groups, since only sterically shielded diaryltetrazoles were reported to generate nitrile imines resistant to side reactions.13 It is therefore still a challenge to develop a visible light-initiated bioorthogonal reaction that is able to proceed smoothly in complex biological environments without interference from nucleophilic additions. Here, we report a new bioorthogonal photoclick cycloaddition that is readily triggered by visible light from a handheld LED lamp (Figure 1A). This bioorthogonal [4+2]
ABSTRACT: Here we report a visible light-triggered, catalyst free bioorthogonal reaction that proceeds via a distinct pathway from reported bioorthogonal reactions. The prototype of this bioorthogonal reaction was the photocycloaddition of 9,10-phenanthrenequinone with electron-rich alkenes to form fluorogenic [4+2] cycloadducts. The bioorthogonal photoclick cycloaddition was readily initiated using a conventional visible light source such as a hand-held LED lamp. The reaction proceeded rapidly under biocompatible conditions, without observable competition from side reactions such as nucleophilic additions by water or common nucleophilic species. The bioorthogonal functionality in this reaction did not cross react with various alkynes and electron-deficient alkenes such as monomethyl fumarate. We demonstrated orthogonal labeling of two proteins using this reaction together with a strain promoting azide−alkyne click reaction or the UV-triggered reaction of tetrazole with monomethyl fumarate. The application of this reaction in the temporal and spatial labeling of live cells was also demonstrated.
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iocompatible reactions controlled by photoirradiation are highly in demand in optochemical biology.1 Commonly used bioorthogonal cycloaddition reactions, including Cu(I)catalyzed or strain-promoted azide−alkyne cycloaddition,2 Staudinger ligation,3 or the cycloaddition of alkenes with tetrazines,4 have greatly facilitated the labeling of various biomacromolecules,5 but not in a temporally and spatially resolved manner. Light irradiation is an ideal way to realize temporal and spatial control.1b,6 Photoclick chemistry using UV light to in situ generate reactive nitrile imine, 7 quinonemethide8 or strained alkyne9 has provided the possibility to control the following cycloaddition reaction with temporal or spatial resolution. However, nucleophilic thiol or water additions to the highly reactive nitrile imine or quinomethide intermediates can be quite competitive with the cycloaddition reaction in complex biological environments.10 It is desirable, then, to develop a photoinduced bioorthogonal reaction via new reactive species with reasonable reactivity to cycloaddition and resistance to nucleophilic thiol or water additions. Visible light has less phototoxicity to biological systems than UV, but bioorthogonal reactions induced by visible light are © XXXX American Chemical Society
Figure 1. Visible light-triggered bioorthogonal photoclick cycloaddition of PQ and VE. (A) Reaction scheme; (B) UV and fluorescence spectra of the substrate and product; (C) pictures of the reaction mixture before and after LED light irradiation. Ex, 365 nm.
cycloaddition is based on the excitation of the 9,10phenanthrenequinone (PQ) functionality that has an absorption shoulder peak in the visible region (Figure 1B). Under biocompatible conditions, the excited PQ* reacted rapidly and selectively with the electron-rich vinyl ether (VE) functionality and generated fluorogenic [4+2] cycloadducts with the phenanthrodioxine (PDO) framework (Figure 1C). Electron-rich alkenes such as VE14 and vinyl thiolether15 were bioorthogonal functionalities that were reported to react Received: August 1, 2018 Published: October 23, 2018 A
DOI: 10.1021/jacs.8b08175 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
Communication
Journal of the American Chemical Society with tetrazines or the precursors of ortho-quinolinone quinonemethide, respectively. Compared to electron deficient alkenes such as monomethyl fumarate (MF), VE was not a good substrate for the tetrazole-ene photoclick reaction.16 Inspired by our previous work on the organic photoreactions of electron-rich alkenes with various substrates,17 we screened reactions of the commercially available 2-(vinyloxy)ethan-1-ol that contained VE functionality with several CO containing compounds in 1:1 CH3CN/PBS upon visible light irradiation (Table S1). To our delight, we found that PQ reacted readily with VE in aqueous solution upon irradiation with a hand-held LED lamp emitting white light and rapidly generated the PDO product with fluorescence turn on (Figures S1 and S2). Moreover, HPLC monitoring on PQ in CH3CN/PBS with the presence of a variety of nucleophilic biomolecules under the same photoirradiation condition for 10 min or in the dark for 24 h showed no conversion of PQ (Figure S3). Together with the high stability of PDO product (Figure S4), these results indicate that we have found a new visible light-triggered bioorthogonal reaction. The excitation of PQ to PQ* in CH3CN was known to induce electron transfer to oxazoles and form various biradical intermediates.17a PQ has an absorption shoulder peak of λmax around 430 nm with extinction coefficients (εmax) of approximately 1472 cm−1 M−1 (Figure 1B), which made it possible to use mild visible light to excite PQ to its excited state PQ*. With the presence of VE as an electron rich CC functionality, electron transfer happened between PQ* and VE and led to the formation of a 1,6-biradical intermediate, which rapidly proceeded via intramolecular radical recombination to form [4+2] cycloadducts (Figure S5). The transient PQ* preferred to go back to its ground state if there was no electron rich CC for the electron transfer process, which explains the resistance of PQ to various nucleophilic additions. We tested the kinetics of PQ-VE cycloaddition initiated by different LED light sources including the LED illumination systems suited for fluorescence microscopy and hand-held LED lamps emitting white light. The rate constants of the reactions initiated by different LED light sources ranged from 0.28 to 2.76 M−1 s−1 (Table S2). Because the hand-held LED lamp with focused white light beam was easy to handle and it induced the fastest photocycloaddition process, we used it as the visible light source in the following experiments. One major motivation for us to develop a new bioorthogonal reaction is to address the demand of concurrent labeling of various biomolecules with a set of bioorthogonal reactions whose substrates do not cross react with each other.18 Because alkynes and alkenes were the common substrates in the majority of the bioorthogonal reactions,1a,4a,19 we examined the reactions of PQ with various substrates containing an alkene or alkyne functional group (Figure 2A). It is apparent that the electron property of the alkene is essential to the rate of the reaction. Vinyl acetate (VA), MF and 1hexenol (HE) all failed to react with PQ under visible light irradiation. Strained alkene trans-COE showed reactivity toward PQ but at a much lower rate compared to the reaction rate of VE. It is noteworthy that PQ did not show obvious reactivity with various alkynes including an electron-rich alkyne ethoxyethyne (EE), a terminal alkyne pent-4-ynoic acid (PA) and a strained alkyne in the cyclooctyne derivative (COY) upon visible light irradiation (Figure 2B). These results indicate the potential of using this reaction concurrently with the azide−alkyne click reaction or UV-triggered tetrazole-ene
Figure 2. Reactivity of PQ with various bioorthogonal functionalities under visible light irradiation. (A) Structure of the substrates bearing bioorthogonal functionalities; (B) conversion of PQ in its mixture with different substrates in 1:1 CH3CN/PBS upon visible light irradiation for 0, 1, 2, 3, 4, or 5 min.
photoclick reaction for simultaneous labeling of different biomolecules without any interference. The efficiency of the visible light-triggered cycloaddition reaction on biomacromolecules was examined with the model protein bovine serum albumin modified with a vinyl ether functionality (BSA-VE) (Figure 3A). Because the formation of
Figure 3. Visible light-initiated photoclick cycloaddition on bovine serum albumin modified with VE (BSA-VE). (A) Schematic illustration; (B) dose-dependent labeling of BSA-VE with 10 min of irradiation by LED lamp on samples of BSA or BSA-VE mixed with the indicated amount of PQ; (C) time-dependent labeling of BSA-VE with 300 μM PQ under LED lamp irradiation for indicated time. Fluorescence signals were acquired using EB channel. Coomassie staining was used to assess BSA loading.
the photocycloadducts PDO was accompanied by fluorescence turn-on, the efficiency of the reaction was examined by the fluorescence intensity of the resulted protein mixture containing BSA-PDO. The BSA-VE was mixed with different amounts of PQ, and the mixtures were irradiated with the hand-held LED lamp for 10 min. The resulting protein mixtures were then subject to an in-gel fluorescence assay. The results showed that VE on BSA reacted readily with PQ upon visible light irradiation in a dose-dependent manner (Figure 3B). The amount of BSA-VE that was further modified with PDO through the photocycloaddition was also dependent on the irradiation time (Figure 3C). Furthermore, the specific labeling of BSA with PQ-VE photocycloaddition was also confirmed by LC-MS/MS analysis (Figure S6). These results suggest the possibility of labeling proteins with temporal resolution through visible light irradiation. We then used this visible light-initiated PQ-VE ligation concurrently with other bioorthogonal reactions for simultaneous labeling of different proteins. As described above, PQ and VE did not cross react with azides or strained alkynes (Figure 2). Meanwhile, the PQ-VE photocycloaddition and the photoclick reaction of tetrazole-MF can be orthogonal by adjusting the wavelength of the light source to initiate the two reactions (Figure S7). With BSA (∼66 kDa) pretagged with VE (BSA-VE) and lysozyme (Lyso, ∼14 kDa) pretagged with B
DOI: 10.1021/jacs.8b08175 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
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Journal of the American Chemical Society azide or MF (Lyso-azide or Lyso-MF), we demonstrated the orthogonal labeling of the two proteins (Figure 4).
Figure 5. Temporal and spatial labeling of live cells by visible light irradiation. (A) Schematic illustration; (B) colocalization of cetuximab on the surface of A549 cells with pretagged FITC and VE that was further labeled with PQ-TAMRA through the PQ-VE photocycloaddition; (C) spatial and (D) time-dependent labeling of cetuximab on the surface of A549 cells with pretagged VE that was further labeled with PQ-TAMRA through the PQ-VE photocycloaddition. Scale bar: 50 μm. Figure 4. Simultaneous and orthogonal labeling of BSA and lysozyme proteins through the orthogonal bioorthogonal reactions. (A) Schematic illustration; (B) orthogonal fluorescent labeling of BSAVE and Lyso-MF through the reaction with PQ-TAMRA and tetrazole, respectively, under indicated conditions; (C) orthogonal fluorescent labeling of BSA-VE and Lyso-azide through the reaction with PQ-TAMRA and SA-Cy5, respectively, under indicated conditions. Fluorescence signals were acquired using IVIS Lumina XR III small animal imaging system.
PQ-TAMRA to incubate with A549 cells without cetuximabVE treatment showed no fluorescence signal after visible light irradiation (Figure S11). These results together confirm the successful labeling of live cells through the PQ-VE photocycloaddition. The visible light-triggered PQ-VE photocycloaddition makes it possible for us to label cells with temporal and spatial resolution. As shown in Figure 5C, cells with cetuximab-VE was fluorescently labeled with spatial control simply by shielding part of the cells from LED lamp irradiation. Moreover, cells with cetuximab-VE upon incubation with same amount of PQ-TAMRA but with different exposure time to LED lamp were labeled with different amounts of TAMRA (Figure 5D). The capability of control chemical ligation in living systems with spatial and temporal resolution will open the door to various biological applications of this visible lighttriggered bioorthogonal reaction. In summary, we developed a visible light-triggered, catalystfree bioorthogonal reaction. The reaction was initiated by the excitation of PQ functionality that has a strong shoulder absorption peak in the visible region. The excited PQ* reacted rapidly and selectively with an electron-rich alkene, VE. Under the biocompatible conditions that allow fast photocycloaddition of PQ and VE, the two bioorthogonal functional groups were inert to alkynes, electron-deficient alkenes and various nucleophilic species. Utilizing this new visible light-controlled bioorthogonal reaction together with the azide−alkyne click reaction or the UV initiated tetrazole-MF photoclick reaction, we achieved concurrent and orthogonal labeling of two proteins. Live cell labeling with temporal and spatial control was demonstrated by using an LED lamp to control the ligation process of PQ and VE on cell surface. Temporal and spatial control of specific intracellular biomacromolecules and regulation on cell function by using this novel bioorthogonal reaction is now underway in our lab.
As shown in Figure 4A, the reaction of BSA-VE with fluorescently labeled PQ-TAMRA induced by an LED lamp was used for the labeling of BSA with TAMRA. Lyso was pretagged either with MF (Lyso-MF) or with azide (Lysoazide). Photoclick reactions of tetrazole with Lyso-MF upon mild UV irradiation generate pyrazoline fluorophores that emit green fluorescence within 2 min. In the mixture containing BSA-VE, Lyso-MF, PQ-TAMRA and tetrazole in PBS buffer, we were able to selectively label BSA or Lyso with red fluorescence from TAMRA or green fluorescence from pyrazoline, respectively, simply by irradiating the mixture with an LED lamp or mild UV light (Figure 4B and Figure S8A). Visible light irradiation of the mixture containing BSAVE, Lyso-azide, PQ-TAMRA and SA-Cy5 gave Cy5 labeled Lyso and TAMRA labeled BSA without cross-labeling (Figure 4C and Figure S8B). Finally, we explored the application of the PQ-VE photocycloaddition to live cell labeling. The compatibility of PQ, VE, PDO as well as LED irradiation with live cells was confirmed by a methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay (Figures S9 and S10). To introduce the VE functionality onto live cells, we modified the antibody cetuximab with VE and further installed cetuximab-VE on the surface of the EGFR positive cancer cell A549 through the specific binding of cetuximab to EGFR (Figure 5A). Upon visible light-induced ligation of PQ-TAMRA with the cetuximab-VE on the cell surface, the cells could be labeled with red fluorescence from TAMRA. Figure 5B shows the cells with cetuximab-VE/FITC incubated with PQ-TAMRA before and after visible light irradiation. Upon visible light irradiation, colocalized green fluorescence and red fluorescence were observed on the cell surface. Meanwhile, control group using
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ASSOCIATED CONTENT
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DOI: 10.1021/jacs.8b08175 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
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AUTHOR INFORMATION
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Yan Zhang: 0000-0002-8858-8894 Author Contributions §
These authors contributed equally.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We acknowledge the financial support from National Natural Science Foundation of China (91753116, 21572102, 21672103, 21877058), Ministry of Science and Technology of China (2018YFA0507100) and the Fundamental Research Funds for the Central Universities (020514380137, 020514380133, 020514380131). We also thank Prof. Yi Yang at East China University of Science and Technology and Prof. Xing Chen and Prof. Peng Chen at Peking University for insightful discussions on this work.
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