A bioorthogonal ligation enabled by click cycloaddition of o-quinolinone quinone methide and vinyl thioether

Article abstract of DOI:10.1021/ja401989p

There is an increasing interest in the use of bioorthogonal ligation to advance biomedical research through selective labeling of biomolecules in living systems. Accordingly, discovering new reactions to expand the toolbox of bioorthogonal chemistry is of particular interest to chemical biologists. Herein we report a new bioorthogonal ligation enabled by click hetero-Diels-Alder (HDA) cycloaddition of in situ-generated o-quinolinone quinone methides and vinyl thioethers. This reaction is highly selective and proceeds smoothly under aqueous conditions. The functionalized vinyl thioethers are small and chemically stable in vivo, making them suitable for use as bioorthogonal chemical reporters that can be effectively coupled to various biomolecules. We utilized this bioorthogonal ligation for site-specific labeling of proteins as well as imaging of bioactive small molecules inside live cells.

Full text of DOI:10.1021/ja401989p

Communication
pubs.acs.org/JACS
A Bioorthogonal Ligation Enabled by Click Cycloaddition of
o‑Quinolinone Quinone Methide and Vinyl Thioether
Qiang Li,†,‡,∥ Ting Dong,†,‡,∥ Xiaohui Liu,§ and Xiaoguang Lei*,‡,§
†Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100730, China
‡National Institute of Biological Sciences (NIBS), Beijing 102206, China
§College of Pharmaceutical Science and Technology, Tianjin University, Tianjin 300072, China
S
* Supporting Information
use of strong acid or base. oQMs have been broadly utilized in
natural product synthesis15 but rarely applied to bioorthogonal
ligation to date, presumably because of the required harsh
reaction conditions, which would be detrimental to the cell or
organism. Arumugam and Popik16 have pioneered the develop-
ment of photochemically generated o-naphthoquinone me-
thides (oNQMs), which undergo facile cycloadditions with
dienophiles such as vinyl ethers or enamines (Scheme 1A).
However, the requirement of UV light might hinder their
applications to living systems.
ABSTRACT: There is an increasing interest in the use of
bioorthogonal ligation to advance biomedical research
through selective labeling of biomolecules in living
systems. Accordingly, discovering new reactions to expand
the toolbox of bioorthogonal chemistry is of particular
interest to chemical biologists. Herein we report a new
bioorthogonal ligation enabled by click hetero-Diels−
Alder (HDA) cycloaddition of in situ-generated o-
quinolinone quinone methides and vinyl thioethers. This
reaction is highly selective and proceeds smoothly under
aqueous conditions. The functionalized vinyl thioethers
are small and chemically stable in vivo, making them
suitable for use as bioorthogonal chemical reporters that
can be effectively coupled to various biomolecules. We
utilized this bioorthogonal ligation for site-specific labeling
of proteins as well as imaging of bioactive small molecules
inside live cells.
Scheme 1. Development of Bioorthogonal Ligations Using
oQMs
ioorthogonal ligations have been widely used to facilitate
B
studies of diverse and complex biological processes.1 As
such reactions should meet the stringent requirements of click
chemistry,2 including high reactivity and selectivity of functional
groups, good stability in aqueous solutions, and excellent
biocompatibility and high reaction rate under physiological
conditions, only a handful of bioorthogonal ligations have been
developed and broadly utilized to date. The select remarkable
works include nucleophilic addition of hydrazine or alkoxy-
amine to ketone or aldehyde,3 Staudinger ligation,4 Cu(I)-
catalyzed azide−alkyne cycloaddition (AAC),5 Cu-free strain-
promoted AAC (SPAAC),6 reactions involving alkenes such as
1,3-dipolar7 and Diels−Alder cycloadditions,8−11 quadricyclane
ligation,12 and Pictet−Spengler ligation.13 Although we have
witnessed the significant advance of this field over the past
decade, the discovery of new reactions, especially ones
compatible with AAC, which is still the gold standard for
bioorthogonal ligations,1b in order to expand the toolbox of
bioorthogonal chemistry remains a pressing task, yet it is
challenging because of the aforementioned stringent require-
ments for ideal bioorthogonal ligations.
To improve the efficiency and robustness of oQM chemistry
in order to make it more suitable for bioorthogonal ligation, we
envisioned that both reaction partners for the HDA cyclo-
addition should be optimized. Conceivably, the introduction of
more electronegative heteroatoms such as nitrogen into the
oQM precursor should improve its reactivity for the HDA
cycloaddition as well as its hydrophilicity. We also considered
that vinyl thioethers (VTs), which are small, chemically stable,
and able to act as more electron-rich dienophiles, should be
o-Quinone methides (oQMs) are highly reactive and versatile
synthetic intermediates that can undergo rapid and selective
hetero-Diels−Alder (HDA) cycloadditions with electron-rich
dienophiles.14 The traditional methods for generation of oQMs
include oxidation, high temperature, photoirradiation, and the
Received: February 25, 2013
© XXXX American Chemical Society
A
dx.doi.org/10.1021/ja401989p | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
a
Scheme 2. Synthesis of Biotinylated and Fluoresceinated oQQM Precursors
a
Reagents and conditions: (a) FeSO4·7H2O/H2O2, CH3OH, conc. H2SO4, 50 °C, 3 h, 70% brsm. (b) 37% HCHO(aq), 2 M NaOH, 1.5 h. (c) 2,2-
Dimethoxypropane, PPTS (cat.), DMF, 80 °C, 5.5 h, 32% over two steps. (d) I2, imidazole, PPh3, DCM, 3 h, 95%. (e) NaN3, DMF, 12 h, quant. (f)
5% Pd/C, H2, MeOH, 12 h, quant. (g) 11, TEA, DMF, 2 h, 88%. (h) 0.5 M HCl/THF, 48 h, 95%. (i) FITC isomer, DIPA, DMF, 16 h, 95%. (j) 0.8
M HCl/THF, 48 h, 90%.
better bioorthogonal chemical reporters. Accordingly, we
prepared 8-(hydroxymethyl)-2-methylquinolin-7-ol (1)17 as
an o-quinolinone quinone methide (oQQM) precursor and 2-
(vinylthio)ethanol (2)18 as a dienophile, which enabled us to
investigate the HDA cycloaddition further (Scheme 1B). To
our delight, we observed that under physiological conditions
(H2O, 37 °C, 24 h), 1 and 2 could undergo a click HDA
cycloaddition to furnish 3 in 92% isolated yield. Interestingly,
the oQQM could be generated smoothly without the need for a
catalyst or UV light. The second-order rate constant for this
and 2 were combined with (azidomethyl)benzene (19) and
5,6-didehydro-11,12-dihydrodibenzo[a,e]cyclooctene (20) in
H2O/CH3CN at 37 °C, we observed that the two ligations
proceeded simultaneously without interfering with each other
(Figure S8).17
Having established the model reaction for the oQQM−VT
ligation, we sought to prepare both biotin and fluorescein-
labeled oQQM precursors for further applications to biological
systems (Scheme 2). The synthesis commenced with direct,
regioselective C−H functionalization of readily available 2-
methylquinolin-7-ol (4) to afford 5. Under basic conditions, 5
reacted with formaldehyde to generate 6, which was
subsequently protected with 2,2-dimethoxypropane, affording
7 in 32% yield over two steps. Primary alcohol 7 was
transformed to iodide 8, which was further substituted with an
azido group to furnish 9. Azide 9 was reduced with H2 in the
presence of Pd/C (5%) to afford a quantitative yield of primary
amine 10, which could be further coupled with different tags.
Treatment of 10 with the readily available biotin tetrafluor-
ophenyl ester (biotin-TFP, 11) smoothly generated amide 12,
which was further deprotected under acidic conditions (HCl/
THF) to obtain the desired biotinylated oQQM precursor 13.
In addition, coupling of 10 with commercially available
fluorescein isothiocyanate (FITC) isomer followed by depro-
tection provided the fluoresceinated compound 15.
With 13 and 15 in hand, we first conducted selective protein
labeling studies in vitro (Figure 1). Using N-succinimidyl 2-
(vinylthio)ethyl carbonate (16), we coupled the free lysine
residues of bovine serum albumin (BSA) with the required VT
reporter (Figure 1A). The VT-modified BSA protein was
subsequently incubated with either biotinylated 13 or
fluoresceinated 15, and the resulting products were then
assayed by Western blots using biotin antibody or in-gel
fluorescence measurements, respectively. Robust biotinylation
was observed for BSA modified with the VT reporter (Figure
1B). In addition, we were delighted to find that fluorescein
reaction was estimated to be (1.5
0.1) × 10−3 M−1 s−1
(Figure S1 in the Supporting Information),17 which is
comparable to those for the widely used Staudinger ligation
(k = 2.5 × 10−3 M−1 s−1)19 and first-generation SPAAC (k = 1.2
× 10−3 M−1 s−1).6f
The stability of the VT reporter under physiological
conditions, especially in the presence of oxidants or reactive
amino acids, was examined next (Figures S2 and S3). As a
result, 2 was shown to be inert toward various oxidants such as
O2 and H2O2 (200 nM) as well as either cysteine (20 mM) or
lysine (20 mM) in D2O at 37 °C for 24 h.17 Notably, we also
found that 2 was stable at pH 4−7.4 (Figure S4). Concerned
that the oQQM would undergo Michael addition with the free
thiol functionality of cysteine residues, we also investigated the
reaction of 1 and 2 in the presence of cysteine (Figure S5).
Gratifyingly, because the Michael addition with the thiol group
is reversible and the HDA cycloaddition with the VT is
irreversible, the HDA cycloadduct 3 was observed exclusively in
H2O at 37 °C after 24 h.17 The reversibility of the thiol Michael
addition was supported by an experiment in which the thiol
Michael addition product further reacted with 2 to generate the
HDA cycloadduct 3 (Figure S6). Ultimately, we tested the
stability of thioacetal 3 under acidic conditions and found it to
be quite stable at pH 4.0−7.4 (Figure S7).
Next, we aimed to evaluate whether the oQQM−VT ligation
is compatible with the widely used SPAAC reaction. When 1
B
dx.doi.org/10.1021/ja401989p | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
defined intracellular structure to image.22 VT-labeled taxol (18)
was prepared smoothly through coupling of 16 to the
previously reported taxol derivative 7-β-alanyltaxol (17)23
(Scheme 3). Initial biological evaluations also confirmed that
18 retained taxol’s antitumor activity in Hela cells (Figure
S9).17
Scheme 3. Synthesis of VT-Labeled Taxol (18)
Figure 1. Labeling of VT-modified BSA with 13 or 15. (A)
Modification of free lysine residues on BSA with 16 and subsequent
labeling with 13 or 15. (B) Western blot analysis of biotin-labeled BSA
using biotin antibody. (C, D) Western blot analyses of (C) time-
dependent and (D) dose-dependent fluorescein labeling of BSA. In
B−D, protein loading was assessed by Coomassie staining.
labeling of VT-modified BSA proceeded effectively in an
excellent time- and dose-dependent manner with very little
background labeling of unmodified BSA (Figure 1C,D).
For live-cell imaging studies, Hela cells were first treated with
500 nM 18 for 2 h at 37 °C. After washing with medium three
times, the cells were exposed to medium containing 20 μM 15
for 12 h in an incubator, washed, and imaged on a confocal
microscope. We observed clearly stained intracellular structures
of tubule networks in the group treated with 18 (Figure 3).
Encouraged by these results, we then investigated the utility
of oQQM−VT ligation for live-cell imaging. To evaluate
whether the fluoresceinated oQQM precursor 15 would be
useful for intracellular studies, we examined its cell-membrane
permeability and cytotoxicity. Hela cells were first incubated
with 15 at different concentrations for 24 h, and the cells were
then washed thoroughly to ensure that little 15 was left around
the cell surface. We observed homogeneous fluorescence
staining inside the Hela cells using a confocal microscope
(Figure 2A). Furthermore, although compounds that sponta-
Figure 2. Studies of cell-membrane permeability and cytotoxicity of
fluoresceinated oQQM precursor 15. (A) Hela cells were treated with
dimethyl sulfoxide (DMSO) or 20 μM 15 for 24 h and then visualized
with green fluorescence. (B) 293T cells and (C) Hela cells were
treated with either DMSO or 15 at three different concentrations (10,
40, and 160 μM) for 24 h. The cell viability was determined by MTT
assay. The reported results are representative of three independent
experiments.
Figure 3. Imaging of VT-labeled taxol in live cells. Group treated with
VT-labeled taxol: live HeLa cells were stained with 15 (20 μM) at 37
°C for 12 h after treatment with 500 nM 18 for 2 h. Group treated
with taxol: live HeLa cells were stained by immunostaining with
antitubulin after treatment with 500 nM taxol for 2 h. Untreated
group: live HeLa cells were treated with 15 (20 μM) at 37 °C for 12 h
after treatment with DMSO for 2 h. All of the cells were treated with
the nuclear stain 4-amidinophenyl-6-indolecarbamidine dihydrochlor-
ide (DAPI) before being imaged on a confocal microscope.
neously produce reactive oQMs might be cytotoxic,20 no
detectable cytotoxicity of 15 was observed in either 293T or
Hela cells at high concentrations of up to 160 μM (Figure
2B,C).
Effective imaging of bioactive small molecules inside live cells
could significantly facilitate forward chemical genetics.21 We
therefore sought to demonstrate the capability of oQQM−VT
ligation for in vivo imaging of VT-labeled small molecules.
Taxol was selected as a showcase for this study because it has
been widely used in cancer chemotherapy through selective
targeting of microtubules, which could provide us a well-
This staining pattern was comparable to standard immunostain-
ing using antitubulin in the taxol-treated group. Control
experiments using 15 alone generated extremely low level
fluorescence background signals, providing additional evidence
that the fluorescence imaging was indeed generated by the
specific HDA cycloaddition. Collectively, these findings indicate
that oQQM−VT ligation is suitable for in vivo labeling
experiments and will be useful for chemical genetic strategies.
C
dx.doi.org/10.1021/ja401989p | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
Lin, Q. J. Am. Chem. Soc. 2008, 130, 9654. (d) van Berkel, S. S.; Dirks,
A. T. J.; Debets, M. F.; van Delft, F. L.; Cornelissen, J. J. L. M.; Nolte,
R. J. M.; Rutjes, F. P. J. T. ChemBioChem 2007, 8, 1504.
In conclusion, we have developed a new bioorthogonal
ligation that is enabled by click hetero-Diels−Alder cyclo-
addition of o-quinolinone quinone methide (oQQM) and vinyl
thioether (VT). We have also demonstrated that this ligation is
efficient and robust under physiological conditions and is
compatible with the widely used strain-promoted azide−alkyne
cycloaddition. Furthermore, we have successfully utilized this
bioorthogonal ligation for site-specific labeling of proteins as
well as imaging of a taxol derivative inside live cells. Hence, we
can expect that the oQQM−VT ligation will become a
promising addition to the bioorthogonal chemistry compen-
dium and facilitate studies of complex biological processes.
(8) (a) de Arauljo, A. D.; Palomo, J. M.; Cramer, J.; Kohn, M.;
́
̈
Schroder, H.; Wacker, R.; Niemeyer, C.; Alexandrov, K.; Waldmann,
̈
H. Angew. Chem., Int. Ed. 2006, 45, 296. (b) de Arauljo, A. D.; Palomo,
́
J. M.; Cramer, J.; Seitz, O.; Alexandrov, K.; Waldmann, H. Chem.
Eur. J. 2006, 12, 6095.
(9) (a) Taylor, M. T.; Blackman, M. L.; Dmitrenko, O.; Fox, J. M. J.
Am. Chem. Soc. 2011, 133, 9646. (b) Li, Z.; Cai, H.; Hassink, M.;
Blackman, M. L.; Brown, R. C. D.; Conti, P. S.; Fox, J. M. Chem.
Commun. 2010, 46, 8043. (c) Blackman, M. L.; Royzen, M.; Fox, J. M.
J. Am. Chem. Soc. 2008, 130, 13518.
(10) (a) Karver, M. R.; Weissleder, R.; Hilderbrand, S. A. Angew.
Chem., Int. Ed. 2012, 51, 920. (b) Devaraj, N. K.; Weissleder, R.;
Hilderbrand, S. A. Bioconjugate Chem. 2008, 19, 2297.
(11) Patterson, D. M.; Nazarova, L. A.; Xie, B.; Kamber, D. N.;
Prescher, J. A. J. Am. Chem. Soc. 2012, 134, 18638.
(12) Sletten, E. M.; Bertozzi, C. R. J. Am. Chem. Soc. 2011, 133,
17570.
ASSOCIATED CONTENT
* Supporting Information
Experimental procedures and characterization data for all new
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
■
S
(13) Agarwal, P.; van der Weijden, J.; Sletten, E. M.; Rabuka, D.;
Bertozzi, C. R. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 46.
(14) For recent reviews of oQMs, see: (a) Willis, N. J.; Bray, C. D.
Chem.Eur. J. 2012, 18, 9160. (b) Ferreira, S. B.; Silva, F. C.; Pinto,
A. C.; Gonzaga, D. T. G.; Ferreira, V. F. J. Heterocycl. Chem. 2009, 46,
1080. (c) Van De Water, R. W.; Pettus, T. R. R. Tetrahedron 2002, 58,
5367.
(15) For selected recent works, see: (a) Liao, D.; Li, H.; Lei, X. Org.
Lett. 2012, 14, 18. (b) Green, J. C.; Jiminez-Alonso, S.; Brown, E. R.;
Pettus, T. R. R. Org. Lett. 2011, 13, 5500. (c) Bender, C. F.;
Yoshimoto, F. K.; Paradise, C. L.; De Brabander, J. K. J. Am. Chem. Soc.
2009, 131, 11350. (d) Lumb, J. P.; Choong, K. C.; Trauner, D. J. Am.
Chem. Soc. 2008, 130, 9230.
(16) (a) Arumugam, S.; Popik, V. V. J. Am. Chem. Soc. 2012, 134,
8408. (b) Arumugam, S.; Popik, V. V. J. Am. Chem. Soc. 2011, 133,
15730. (c) Arumugam, S.; Popik, V. V. J. Am. Chem. Soc. 2011, 133,
5573. (d) Arumugam, S.; Popik, V. V. J. Am. Chem. Soc. 2009, 131,
11892.
(17) For details, see the Supporting Information.
(18) Højfeldt, J. W.; Blakskjær, P.; Gothelf, K. V. J. Org. Chem. 2006,
71, 9556.
AUTHOR INFORMATION
Corresponding Author
leixiaoguang@nibs.ac.cn
■
Author Contributions
∥Q.L. and T.D. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Mr. Daohong Liao and Ms. Xiaoying Yang for their
assistance with this research, Ms. Mingyan Zhao and Ms. Wei
Li (NIBS) for NMR and HPLC-MS analysis, and Dr. Jiang
Zhou (Peking University) for HRMS analysis. Financial
support from the National High Technology Project 973
(2012CB837400), the Program for New Century Excellent
Talents in University (NCET-10-0614), and the NNSFC
(21072150 and 21222209) is gratefully acknowledged.
(19) Lin, F. L.; Hoyt, H. M.; Van Halbeek, H.; Bergman, R. G.;
Bertozzi, C. R. J. Am. Chem. Soc. 2005, 127, 2686.
(20) Rokita, S. E. In Quinone Methides; Rokita, S. E., Ed.; Wiley:
Hoboken, NJ, 2009; p 297.
(21) For an excellent review of chemical genetics, see: Walsh, D. P.;
Chang, Y. T. Chem. Rev. 2006, 106, 247.
(22) Devaraj, N. K.; Hilderbrand, S.; Upadhyay, R.; Mazitschek, R.;
Weissleder, R. Angew. Chem., Int. Ed. 2010, 49, 2869.
(23) Guy, R. K.; Scott, Z. A.; Sloboda, R. D.; Nicolaou, K. C. Chem.
Biol. 1996, 3, 1022.
REFERENCES
■
(1) For excellent reviews, see: (a) Bertozzi, C. R. Acc. Chem. Res.
2011, 44, 651. (b) Jewett, J. C.; Bertozzi, C. R. Chem. Soc. Rev. 2010,
39, 1272. (c) Sletten, E. M.; Bertozzi, C. R. Angew. Chem., Int. Ed.
2009, 48, 6974. (d) Prescher, J. A.; Bertozzi, C. R. Nat. Chem. Biol.
2005, 1, 13.
(2) For an excellent review of click chemistry, see: Kolb, H. C.; Finn,
M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001, 40, 2004.
(3) Rideout, D. Science 1986, 233, 561.
(4) Saxon, E.; Bertozzi, C. R. Science 2000, 287, 2007.
(5) (a) Soriano del Amo, D.; Wang, W.; Hao, J.; Besanseney, C.; Yan,
A. C.; Levy, M.; Liu, Y.; Marlow, F. L.; Wu, P. J. Am. Chem. Soc. 2010,
132, 16893. (b) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.;
Sharpless, K. B. Angew. Chem., Int. Ed. 2002, 41, 2596.
(6) (a) Friscourt, F.; Fahrni, C. J.; Boons, G. J. J. Am. Chem. Soc.
2012, 134, 18809. (b) Jewett, J. C.; Sletten, E. M.; Bertozzi, C. R. J.
Am. Chem. Soc. 2010, 132, 3688. (c) Codelli, J. A.; Baskin, J. M.;
Agard, N. J.; Bertozzi, C. R. J. Am. Chem. Soc. 2008, 130, 11486.
(d) Ning, X. H.; Guo, J.; Wolfert, M. A.; Boons, G. J. Angew. Chem.,
Int. Ed. 2008, 47, 2253. (e) Baskin, J. M.; Prescher, J. A.; Laughlin, S.
T.; Agard, N. J.; Chang, P. V.; Miller, I. A.; Lo, A.; Codelli, J. A.;
Bertozzi, C. R. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 16793. (f) Agard,
N. J.; Prescher, J. A.; Bertozzi, C. R. J. Am. Chem. Soc. 2004, 126,
15046.
(7) (a) Song, W.; Wang, Y.; Qu, J.; Madden, M. M.; Lin, Q. Angew.
Chem., Int. Ed. 2008, 47, 2832. (b) Wang, Y.; Hu, W. J.; Song, W.; Lim,
R. K. V.; Lin, Q. Org. Lett. 2008, 10, 3725. (c) Song, W.; Wang, Y.;
D
dx.doi.org/10.1021/ja401989p | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX