Visible Light-Initiated Bioorthogonal Photoclick Cycloaddition

Article abstract of DOI:10.1021/jacs.8b08175

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.

Full text of DOI:10.1021/jacs.8b08175

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Communication
Visible Light-Initiated Bioorthogonal Photoclick Cycloaddition
Jinbo Li, Hao Kong, Lei Huang, Bo Cheng, Ke Qin, Mengmeng Zheng, Zheng Yan, and Yan Zhang
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 23 Oct 2018
Downloaded from http://pubs.acs.org on October 23, 2018
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Visible Light-Initiated Bioorthogonal Photoclick Cycloaddition
Jinbo Li,1,‡ Hao Kong,1,‡ Lei Huang,1,‡ Bo Cheng,2 Ke Qin,2 Mengmeng Zheng,1 Zheng Yan1 and Yan
Zhang1,*
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1State 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
2College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China
Supporting Information Placeholder
since only sterically shielded diaryltetrazoles were reported to
ABSTRACT: Here we report a visible-light-triggered, catalyst
free bioorthogonal reaction that proceeds via a distinct pathway
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.
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 biorthogonal photoclick
cycloaddition was readily initiated using a conventional visible
light source such as 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.
Here, we report a new biorthogonal photoclick cycloaddition that
is readily triggered by visible light from a hand-held LED lamp
(Figure 1A). This bioorthogonal [4+2] cycloaddition is based on
the excitation of the 9,10-phenanthrenequinone (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).
Biocompatible reactions controlled by photoirradiation are
highly in demand in optochemical biology.1 Commonly used
biorthogonal 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.
Figure 1. Visible-light-triggered biorthogonal 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.
Electron-rich alkenes such as VE14 and vinyl thiolether15 were
bioorthogonal functionalities that were reported to react 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 C=O 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
Visible light has less phototoxicity to biological systems than
UV, but bioorthogonal reactions induced by visible light are rare.
Lin et al. have modified tetrazoles with specific substitution groups
such as oligothiophenes or naphthalenes to make them activatable
with a 405nm 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,
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generated the PDO product with fluorescence turn on (Figures S1-
using this reaction concurrently with the azide-alkyne click
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S2). Moreover, HPLC monitoring on PQ in CH3CN/PBS with the
presence of a variety of active biomolecules under the same
photoirradiation conditions for 10 minutes or in the dark for 24
hours showed no conversion of PQ (Figure S3). These results
indicated that we have found a new bioorthogonal reaction
triggered by visible light to form a reactive species with high
reactivity to cycloaddition and resistance to various nucleophilic
additions. The stability test of the PDO product (Figure S4)
indicated that the photo-cycloadduct was stable enough for labeling
purposes in complex biological systems.
reaction or UV-triggered tetrazole-ene 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 the
photocycloadducts PDO was accompanied with 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
minutes. 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.
9
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 C=C 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 C=C 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).
Since 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.
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Figure 3. The visible light-initiated photoclick cycloaddition on bovine
serum albumin modified with VE (BSA-VE). (A) Schematic illustration of
the BSA modification; (B) Dose-dependent labeling of BSA-VE with 10
minutes 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 from the resulted BSA-PDO were indicative of the
labeling efficiency and were acquired using EB channel. Coomassie
staining was used to assess BSA loading.
Figure 2. Reactivity of PQ with various biorthogonal functionalities under
visible light irradiation. (A) Structure of the substrates bearing biorthogonal
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 minutes.
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) pre-tagged with VE (BSA-VE) and
lysozyme protein (Lyso, ~14 kDa) pre-tagged with azide or MF
(Lyso-azide or Lyso-MF), we demonstrated the orthogonal
labeling of the two proteins (Figure 4).
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 1-hexenol (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
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 pre-tagged
either with MF (Lyso-MF) or with azide (Lyso-azide). Photoclick
reactions of tetrazole with Lyso-MF upon mild UV irradiation
generate pyrazoline fluorophores that emit green fluorescence
within 2 minutes. 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 (Figures
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4B and S8A). Visible light irradiation of the mixture containing
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BSA-VE, Lyso-azide, PQ-TAMRA and SA-Cy5 gave Cy5
labeled Lyso and TAMRA labeled BSA without cross-labeling
(Figures 4C and S8B).
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Figure 5. Temporal and spatial labeling of live cells by visible light
irradiation. (A) Schematic illustration of live cell labeling with PQ-VE
photocycloaddition; (B) Co-localization of cetuximab on the surface of
A549 cells with pre-tagged 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
pre-tagged 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 of the orthogonal labeling strategy; (B) Orthogonal fluorescent
labeling of BSA-VE 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. The
concentrations of the proteins were 2 mg/mL. The concentrations of PQ-
TAMRA, tetrazole and SA-Cy5 were 300 µM. Fluorescence signals from
TAMRA, pyrazoline and Cy5 were acquired using IVIS Lumina XR III
small animal imaging system.
In summary, we developed a visible light-triggered, catalyst-free
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.
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-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 should be able to be
labeled with red fluorescence from TAMRA. Figure 5B showed
the cells with cetuximab-VE/ FITC incubated with PQ-TAMRA
before and after visible light irradiation. Upon visible light
irradiation, co-localized green fluorescence and red fluorescence
were observed on the cell surface. Meanwhile, control group using
PQ-TAMRA to incubate with A549 cells without cetuximab-VE
treatment showed no fluorescence signal after visible light
irradiation (Figure S11).These results together confirmed the
successful labeling of live cells through the PQ-VE
photocycloaddition.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website.
Experimental details, supporting Figures S1-S11 and Tables S1-S2.
AUTHOR INFORMATION
Corresponding Author
*njuzy@nju.edu.cn
Author Contributions
‡These authors contributed equally.
The visible light-triggered PQ-VE photocycloaddition making 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 able to be labeled with
different amounts of the fluorescent dye 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 light-triggered bioorthogonal reaction.
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
We would like to acknowledge the financial support from National
Natural Science Foundation of China (91753116, 21572102,
21672103), Ministry of Science and Technology of China
(2018YFA0507100) and the Fundamental Research Funds for the
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9. (a) Arumugam, S.; Popik, V. V., Sequential "click" - "photo-click"
Central Universities (020514380137, 020514380133). We also
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Technology and Prof. Xing Chen and Prof. Peng Chen at Peking
University for insightful discussions on this work.
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