Genetically encoded cyclopropene directs rapid, photoclick-chemistry- mediated protein labeling in mammalian cells

Full text of DOI:10.1002/anie.201205352

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Chemie
DOI: 10.1002/anie.201205352
Bioorthogonal Chemistry
Genetically Encoded Cyclopropene Directs Rapid, Photoclick-
Chemistry-Mediated Protein Labeling in Mammalian Cells**
Zhipeng Yu, Yanchao Pan, Zhiyong Wang, Jiangyun Wang,* and Qing Lin*
Dedicated to Professor Andrew D. Hamilton on the occasion of his 60th birthday
The introduction of bioorthogonal organic reporters into
proteins site-selectively through genetic,[1] metabolic,[2] or
enzyme-catalyzed ligation methods,[3] in conjunction with
a growing repertoire of bioorthogonal reactions,[4] has allowed
for the visualization and regulation of proteins in their native
environment.[5] Numerous small organic functional groups,
such as ketones,[6] azides,[7] terminal alkynes,[8] and terminal
alkenes,[9] as well as larger reactive bioorthogonal groups,
such as cyclooctyne,[10] norbornene,[11] transcyclooctene,[10b,11b]
tetrazole,[12] and tetrazine[13] have been genetically encoded
for site-selective protein labeling in vivo. To track fast protein
dynamics in vivo, it is imperative that these genetically
encoded bioorthogonal reporters direct fast and selective
bioorthogonal labeling with the cognate biophysical probes,
preferably with spatiotemporal control.
In our continued effort to genetically encode substrates
that are suitable for photoclick chemistry,[14] we envisioned
that non-natural amino acids with strained alkenes attached
may show higher rate of cycloaddition without undergoing the
Michael addition side reactions with biological nucleophiles
typically associated with electron-deficient alkenes. While we
reported recently that norbornene exhibited robust reactivity
in the cycloaddition reaction with the macrocyclic tetra-
zoles,[15] norbornene is relatively bulky and may perturb the
structure of the encoded protein. Therefore, we tried to
genetically encode cyclopropene because of its small size and
inherently high ring strain (54.1 kcalmolÀ1 [16] versus 21.6 kcal
molÀ1 for norbornene[17]), much of which is released after the
cycloaddition reaction (ring strain of cyclopropane =
28.7 kcalmolÀ1).[18] Herein, we report the synthesis of
a stable cyclopropene amino acid, the characterization of its
reactivity in the photoinduced cycloaddition reaction with
two tetrazoles, its site-specific incorporation into proteins
both in E. coli and in mammalian cells, and its use in directing
bioorthogonal labeling of proteins both in vitro and in vivo.
To design a cyclopropene-containing amino acid suitable
for genetic incorporation, we decided to focus on pyrrolysyl/
tRNA synthetase (PylRS)/tRNACUA pair from Methanosar-
cina barkeri (Mb) because: 1) this pair is orthogonal to all
endogenous tRNAs and aminoacyl-tRNA synthetases in
E. coli and eukaryotic cells;[19] and 2) many non-natural
lysine-derived amino acids have been efficiently incorporated
into proteins based on this pair.[20] Preliminary studies showed
that 3,3-disubstituted cyclopropenes such as 1c exhibited
excellent chemical stability at room temperature. 1-Methyl-
cycloprop-2-enecarboxylic acid (1c) can be easily prepared
from the commercially available starting material ethyl-2-
methylacetoacetate, through a three-step procedure with an
overall yield of 21% (Scheme 1). The cyclopropene carbox-
ylic acid 1c was then coupled with the e-amino group of
Fmoc-lysine, which upon removal of the protecting group
[*] Y. Pan,[+] Prof. Dr. J. Wang
Laboratory of Noncoding RNA, Institute of Biophysics,
Chinese Academy of Sciences
Beijing 100101 (P.R. China)
E-mail: jwang@ibp.ac.cn
Dr. Z. Yu,[+] Dr. Z. Wang, Prof. Dr. Q. Lin
Department of Chemistry, State University of New York at Buffalo
Buffalo, NY 14260 (USA)
afforded
Ne-(1-methylcycloprop-2-enecarboxamido)lysine
(CpK, 1) in 74% yield over three steps. The crystal structure
of 1c was obtained, which showed a hydrogen-bonded dimer
of 1c (Scheme 1; see Supporting Information, Table S1 for
crystal data and structure refinement). As expected, a bond
E-mail: qinglin@buffalo.edu
[+] These two authors contributed equally to the work.
[**] We gratefully acknowledge the National Institutes of Health (GM
085092 to Q.L.), the Major State Basic Research Program of China
(2010CB912301 and 2009CB825505 to J.W.), National Science
Foundation of China (90913022 to J.W.), and CAS (KZCX2-YW-JC101
and KSCX2-EW-G-7 to J.W.) for financial support. We thank William
Brennessel at the University of Rochester for X-ray crystallography
and Reyna K. Lim in the Q.L. lab for plasmid preparation, Alan
Siegel at SUNY Buffalo Biological Sciences Imaging Facility
(supported by National Science Foundation Major Research
Instrumentation grant DBI-0923133) for assistance in microscopy.
CCDC 808108 (1c) contains the supplementary crystallographic
data for this paper. These data can be obtained free of charge from
The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.
uk/data_request/cif.
À
À
angle of only 508 at C1 C2 C1A provides very high strain in
the ring structure. Importantly, the carbonyl group in 1c is
essentially perpendicular to cyclopropene double bond,
preventing conjugation between these two p systems. Perhaps
as a result of this geometry, CpK was found to be very stable
around glutathione, an abundant biological nucleophile inside
cells; greater than 95% of CpK remained after incubation
with 10 mm glutathione (in reduced form) for more than 60 h
(Supporting Information, Figure S1).
Using ethyl-1-methylcycloprop-2-enecarboxylate (2) as
a model substrate, we examined the reactivity of cyclo-
propene with two representative tetrazoles: a 302 nm wave-
length photoreactive tetrazole 3[21] and a water-soluble
365 nm wavelength photoreactive tetrazole 4,[22] in acetoni-
Supporting information for this article (experimental details) is
available on the WWW under http://dx.doi.org/10.1002/anie.
201205352.
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Table 1: Kinetic characterization of ethyl-1-methylcycloprop-2-enecar-
boxylate (2) reactivity using photoclick chemistry in an acetonitrile/PBS
(1:1) mixture.[a]
Scheme 1. Synthesis of Ne-(1-methylcycloprop-2-enecarboxyamido)-
lysine (CpK, 1). Crystal structure of 1c (below). Tf=triflate; TBAB=
tetra-n-butyl ammonium bromide; TMS=trimethylsilyl; TPA=tri-
phenylacetato; NHS=N-hydroxysuccinimide; DCC=dicyclohexylcarbo-
diimide; Fmoc=fluorenylmethyloxycarbonyl.
Entry Alkene
Tetrazole Photoactivation
wavelength
Product k2 [mÀ1 sÀ1
]
1
2
3
4
302 nm
365 nm
5
6
58Æ16
4.6Æ1.3
3
4
3
4
302 nm
365 nm
7
8
46Æ8.6
9.2Æ0.7
trile/PBS buffer (1:1) mixture. We compared the reactivity of
this cyclopropene to those of acrylamide and norbornene
(Table 1 and Figure S2–S7). In the cycloaddition reactions
with tetrazole 3, cyclopropene 2 showed a k2 value of 58 Æ
16mÀ1 sÀ1, similar to acrylamide (entry 1 vs. 3) but significantly
faster than norbornene (entry 1 vs. 5). In the cycloaddition
reactions with tetrazole 4, however, cyclopropene 2 showed
more than a tenfold drop in k2 value (entry 1 vs. 2), which is
also 50% slower than that of acrylamide (entry 2 vs. 4) but
similar to norbornene (entry 2 vs. 6). The decrease in reaction
kinetics at the longer wavelength is likely due to the filtering
effect associated with the formation of fluorescent pyrazoline
adducts.[23] Compared to other bioorthogonal reactions
involving genetically encoded alkenes, the cycloaddition of
cyclopropene with tetrazole 3 is about 60 times faster than the
cycloaddition of O-allyltyrosine under the same conditions
(k2 = 0.95mÀ1 sÀ1),[21] at least six times faster than the ligation
between 5-norbornene-2-ol and a pyrimidine-substituted
tetrazine (k2 = 9mÀ1 sÀ1 in 95:5 H2O/MeOH),[11a] but three
orders of magnitude slower than tetrazine ligation with
transcyclooctene (k2 = 35000 Æ 3000mÀ1 s1 in biological buf-
fer).[11b]
5
6
3
4
302 nm
365 nm
9
10
32Æ12
5.8Æ1.4
[a] Reactions were set up by incubating 100 mm of tetrazole and 500 mm
of alkene in 0.5 mL PBS/MeCN (1:1). The mixtures were exposed to
a handheld UV lamp (302 nm: UVM-57, 0.16 A; 365 nm: UVGL-25,
0.16 A) for a specified time under ambient conditions. The product
mixtures were analyzed by reversed-phase HPLC and quantified by
comparing the integrated peaks to that of a standard. The kinetic
experiments were repeated three times to derive the average kinetic
constants along with the standard deviations. See Supporting Informa-
tion, Figures S2–S7 for details.
amber codon at position four and a C-terminal His tag was
carried out in E. coli cells transformed with the CpKRS/
MbtRNACUA pair and grown in LB medium supplemented
with 1 mm CpK. The CpK-encoded mutant myoglobin
proteins (Myo-CpK) were obtained at a concentration of
3.0 mgLÀ1. Importantly, Myo-CpK was produced only when
CpK was added (Figure 1a), indicating that CpK incorpo-
ration is highly specific. ESI-TOF mass spectrometry showed
a mass of 18476.0 Da for Myo-CpK (Figure S8), matching the
calculated mass of 18476.3 Da. Subsequent tryptic digestion
and tandem mass spectrometric analysis confirmed the
presence of CpK at position four of the myoglobin (Fig-
ure 1b).
To assess whether CpK can serve as a bioorthogonal
reporter for protein labeling, we incubated Myo-CpK with
tetrazole 3 in PBS buffer and subjected the mixture to 302 nm
photoirradiation with a handheld UV lamp for a period of 1–
12 minutes. In-gel fluorescence analysis revealed the time-
dependent appearance of fluorescence at the Myo-CpK band,
with the highest intensity reached after 10 min (Figure 2a),
consistent with the formation of fluorescent pyrazoline
To evolve an orthogonal tRNA/aminoacyl-tRNA synthe-
tase pair that selectively charges CpK in response to a TAG
amber codon in proteins in E. coli, an MbPylRS library was
constructed in which five residues (L266, L270, Y271, L274,
and C313) were randomized through overlapping extension
PCR using synthetic oligonucleotide primers (Table S2).
After three rounds of positive and two rounds of negative
selections,
a CpK-specific aminoacyl-tRNA synthetase,
termed CpKRS, was identified. Sequencing of this clone
revealed the following five mutations: L266M, L270I, Y271L,
L274A, and C313I. To test whether the CpKRS/MbtRNACUA
pair allows for efficient and selective CpK incorporation in
E. coli, the expression of sperm whale myoglobin carrying an
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Figure 2. Selective labeling of CpK-encoded myoglobin using photo-
click chemistry. a) Coomassie blue stain (bottom) and inverted fluores-
cence image (top) of the protein gel after the reaction mixtures were
resolved by SDS-PAGE. For the photoclick reaction, a solution of Myo-
CpK (0.5 mgmLÀ1) and tetrazole 3 (100 mm) in PBS buffer was exposed
to 302 nm wavelength photoirradiation for a period of 1–12 min.
b) MS–MS spectrum of the N-terminal peptide fragment of the pyrazo-
line-modified myoglobin (Myo-Pyr) containing the pyrazoline-linked
lysine (K*).
Figure 1. Incorporation of CpK (1) site-selectively into sperm whale
myoglobin: a) Coomassie-blue-stained gel of the wildtype and mutant
myoglobin expressed in the absence or presence of 1 mm CpK. A
concentration of 3.0 mgLÀ1 was obtained for Myo-CpK. b) MS–MS
spectrum of the N-terminal peptide fragment containing CpK (K*). The
CpK mass of 207.48 Da was determined from the b3 and b4 ions
(mass=b4Àb3), which closely matches the calculated mass of
208.13 Da.
To examine whether CpK can direct bioorthogonal label-
ing in mammalian cells, we co-transfected human embryonic
kidney (HEK) 293 cells with the pCMV-CpKRS plasmid, in
which the transcription of CpKRS is under the control of
a CMV promoter and the transcription of MbtRNACUA is
under the control of a human U6 promoter,[20c] together with
the pSwan-EGFP37TAG reporter.[20d] The cells were allowed
to grow in the presence 4 mm CpK for 36 h, treated with 40 mm
tetrazole 4 for 1.5 h followed by brief photoirradiation at
365 nm for two minutes, and finally imaged using a fluores-
cence microscope. Using the EGFP channel (lex = 488 nm,
lem = 499-578 nm), green fluorescent cells were detected only
in plates where CpK was included in the culture medium
(Figure 3b,e vs. Figure 3h), indicating that the CpKRS/
MbtRNACUA pair supports site-specific incorporation of
CpK into EGFP37TAG protein in HEK293 cells. Using the
pyrazoline channel (lex = 405 nm, lem = 410-498 nm), only the
tetrazole 4-treated cells expressing CpK-encoded EGFP
showed cyan fluorescence (Figure 3a vs. Figure 3d,g; see
Figure S14 for the fluorescence spectrum of the pyrazoline
adduct). Notably, fluorescence images were acquired using
two separate channels, with a single laser source exciting one
wavelength a time to avoid any possible fluorescence leakage
to the unintended channel. As seen in the overlaid images, the
adduct (Figure 2a).[24] As a control, photoirradiation of
a mixture of wild-type myoglobin and tetrazole 3 did not
yield fluorescent bands on the SDS-PAGE gel (Figure S9),
confirming that labeling of Myo-CpK was mediated by the
cyclopropene moiety. Furthermore, ESI-TOF mass spectro-
metric analysis of the product mixture revealed greater than
85% conversion with a new mass peak at 18701.87 Da,
matching the calculated mass of the pyrazoline-labeled
myoglobin, 18701.56 (Figure S10). Tryptic digestion and
tandem mass spectrometry confirmed the presence of the
pyrazoline-modified lysine at position four by the appearance
of expected masses of the three fragment ions b92+, b162+, and
3+
y14
(Figure 2b). For comparison, the same photoclick
reaction with a mutant myoglobin encoding the recently
reported norbornene-modified lysine (NorK)[11a] at position
four[25] showed a time-dependent, weakly fluorescent cyclo-
adduct formation (Figure S12), affording the pyrazoline
cycloadduct in 60% yield, based on mass spectrometric
analysis, after ten minutes of photoirradiation (Figure S13).
The slightly higher reactivity of CpK relative to NorK in the
context of intact proteins is consistent with the kinetic data
described in Table 1.
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Received: July 7, 2012
Revised: August 21, 2012
Published online: && &&, &&&&
Keywords: bioorthogonal · click chemistry · cyclopropenes ·
.
proteins · tetrazoles
[1] C. C. Liu, P. G. Schultz, Annu. Rev. Biochem. 2010, 79, 413.
[2] a) E. Saxon, C. R. Bertozzi, Science 2000, 287, 2007; b) D. C.
Dieterich, J. J. Hodas, G. Gouzer, I. Y. Shadrin, J. T. Ngo, A.
Triller, D. A. Tirrell, E. M. Schuman, Nat. Neurosci. 2010, 13,
897.
[3] M. Fernꢀndez-Suꢀrez, H. Baruah, L. Martꢁnez-Hernꢀndez, K. T.
Xie, J. M. Baskin, C. R. Bertozzi, A. Y. Ting, Nat. Biotechnol.
2007, 25, 1483.
[4] a) E. M. Sletten, C. R. Bertozzi, Angew. Chem. 2009, 121, 7108;
Angew. Chem. Int. Ed. 2009, 48, 6974; b) R. K. Lim, Q. Lin,
Chem. Commun. 2010, 46, 1589.
[5] E. M. Sletten, C. R. Bertozzi, Acc. Chem. Res. 2011, 44, 666.
[6] L. Wang, Z. Zhang, A. Brock, P. G. Schultz, Proc. Natl. Acad.
Sci. USA 2003, 100, 56.
[7] J. W. Chin, S. W. Santoro, A. B. Martin, D. S. King, L. Wang, P. G.
Schultz, J. Am. Chem. Soc. 2002, 124, 9026.
Figure 3. Confocal micrographs of human embryonic kidney 293 cells
transfected with the plasmids expressing CpKRS, MbtRNACUA, and
EGFP37TAG, and grown in the presence of 4 mm CpK (a–f) or the
absence of CpK (g–i). The cells in (a–c) and (h–j) were treated with
40 mm tetrazole 4 for 1.5 h before 365 nm wavelength photoirradiation
with a handheld UV lamp for 2 min. White arrowheads indicate cells
positive for both pyrazoline and EGFP fluorescence. Scale
bar=200 mm.
[8] a) A. Deiters, T. A. Cropp, M. Mukherji, J. W. Chin, J. C.
Anderson, P. G. Schultz, J. Am. Chem. Soc. 2003, 125, 11782;
b) D. P. Nguyen, H. Lusic, H. Neumann, P. B. Kapadnis, A.
Deiters, J. W. Chin, J. Am. Chem. Soc. 2009, 131, 8720; c) T.
Fekner, X. Li, M. M. Lee, M. K. Chan, Angew. Chem. 2009, 121,
1661; Angew. Chem. Int. Ed. 2009, 48, 1633.
[9] a) Z. Zhang, L. Wang, A. Brock, P. G. Schultz, Angew. Chem.
2002, 114, 2964; Angew. Chem. Int. Ed. 2002, 41, 2840; b) W.
Song, Y. Wang, J. Qu, Q. Lin, J. Am. Chem. Soc. 2008, 130, 9654;
c) H. W. Ai, W. Shen, E. Brustad, P. G. Schultz, Angew. Chem.
2010, 122, 947; Angew. Chem. Int. Ed. 2010, 49, 935.
[10] a) T. Plass, S. Milles, C. Koehler, C. Schultz, E. A. Lemke,
Angew. Chem. 2011, 123, 3964; Angew. Chem. Int. Ed. 2011, 50,
3878; b) K. Lang, L. Davis, S. Wallace, M. Mahesh, D. J. Cox,
M. L. Blackman, J. M. Fox, J. W. Chin, J. Am. Chem. Soc. 2012,
134, 10317.
cyan fluorescent cells coincided with cells that showed high
green fluorescence (indicated by white arrows in Figure 3c),
suggesting that the labeling reaction was indeed directed by
the cyclopropene moiety. However, not all green fluorescent
cells were labelled, indicating some variability in the pene-
tration of the tetrazole into the highly confluent HEK293
cells. Notably, repeated attempts to incorporate NorK in
HEK293 cells using wild-type MbPylRS and identical trans-
fection conditions were not successful (Figure S15,S16),
precluding the comparison of the reactivity of these two
strained alkenes in photoclick chemistry in vivo.
In conclusion, we have demonstrated the genetic incor-
poration of a cyclopropene-containing amino acid, CpK, site-
specifically into target proteins, and the use of CpK as
a bioorthogonal reporter for directing rapid (approximately
two minutes) fluorescent labeling of the target protein in
mammalian cells. Compared to other genetically encoded,
bioorthogonal labeling reactions reported recently,[10–13] the
main advantage of the cyclopropene-directed photoclick
chemistry lies in its potential for spatiotemporally controlled
protein labeling in mammalian cells, which requires the
development of highly reactive laser-activatable tetrazole
reagents using either a single-photon (for example 405 nm) or
two-photon laser source; work in this area is currently in
progress. Because of its small size, a cyclopropene moiety
such as 1c can also be readily incorporated into small-
molecule substrates and inhibitors for the study of pro-
teomes[26] and lipids.[27]
[11] a) K. Lang, L. Davis, J. Torres-Kolbus, C. Chou, A. Deiters, J. W.
Chin, Nat. Chem. 2012, 4, 298; b) T. Plass, S. Milles, C. Koehler, J.
´
Szymanski, R. Mueller, M. Wießler, C. Schultz, E. A. Lemke,
Angew. Chem. 2012, 124, 4242; Angew. Chem. Int. Ed. 2012, 51,
4166; c) E. Kaya, M. Vrabel, C. Deiml, S. Prill, V. S. Fluxa, T.
Carell, Angew. Chem. 2012, 124, 4542; Angew. Chem. Int. Ed.
2012, 51, 4466.
[12] J. Wang, W. Zhang, W. Song, Y. Wang, Z. Yu, J. Li, M. Wu, L.
Wang, J. Zang, Q. Lin, J. Am. Chem. Soc. 2010, 132, 14812.
[13] J. L. Seitchik, J. C. Peeler, M. T. Taylor, M. L. Blackman, T. W.
Rhoads, R. B. Cooley, C. Refakis, J. M. Fox, R. A. Mehl, J. Am.
Chem. Soc. 2012, 134, 2898.
[14] a) Y. Wang, Q. Lin, Org. Lett. 2009, 11, 3570; b) R. K. Lim, Q.
Lin, Acc. Chem. Res. 2011, 44, 828.
[15] Z. Yu, R. K. Lim, Q. Lin, Chem. Eur. J. 2010, 16, 13325.
[16] R. D. Bach, O. Dmitrenko, J. Am. Chem. Soc. 2004, 126, 4444.
[17] P. v. R. Schleyer, J. E. William, K. R. Blanchard, J. Am. Chem.
Soc. 1970, 92, 2377.
[18] M. S. Gordon, J. Am. Chem. Soc. 1980, 102, 7419.
[19] a) S. K. Blight, R. C. Larue, A. Mahapatra, D. G. Longstaff, E.
Chang, G. Zhao, P. T. Kang, K. B. Green-Church, M. K. Chan,
J. A. Krzycki, Nature 2004, 431, 333; b) A. Ambrogelly, S.
Gundllapalli, S. Herring, C. Polycarpo, C. Frauer, D. Sçll, Proc.
Natl. Acad. Sci. USA 2007, 104, 3141.
[20] a) H. Neumann, S. Y. Peak-Chew, J. W. Chin, Nat. Chem. Biol.
2008, 4, 232; b) T. Mukai, T. Kobayashi, N. Hino, T. Yanagisawa,
K. Sakamoto, S. Yokoyama, Biochem. Biophys. Res. Commun.
4
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2008, 371, 818; c) X. Li, T. Fekner, J. J. Ottesen, M. K. Chan,
Angew. Chem. 2009, 121, 9348; Angew. Chem. Int. Ed. 2009, 48,
9184; d) P. R. Chen, D. Groff, J. Guo, W. Ou, S. Cellitti, B. H.
Geierstanger, P. G. Schultz, Angew. Chem. 2009, 121, 4112;
Angew. Chem. Int. Ed. 2009, 48, 4052; e) J. K. Takimoto, N.
Dellas, J. P. Noel, L. Wang, ACS Chem. Biol. 2011, 6, 733; f) Y.-S.
Wang, X. Fang, A. L. Wallace, B. Wu, W. R. Liu, J. Am. Chem.
Soc. 2012, 134, 2950.
[24] W. Song, Y. Wang, J. Qu, M. M. Madden, Q. Lin, Angew. Chem.
2008, 120, 2874; Angew. Chem. Int. Ed. 2008, 47, 2832.
[25] The NorK-encoded myoglobin mutant was expressed using
wildtype pylRS and a pBAD-pylT-Myo4TAG reporter in E. coli
in the presence of 1 mm NorK. A yield of the isolated product of
1.0 mgLÀ1 was obtained for the NorK-containing myoglobin (see
Figure S11 for characterization data).
[26] B. R. Martin, C. Wang, A. Adibekian, S. E. Tully, B. F. Cravatt,
Nat. Methods 2011, 9, 84.
[27] During the preparation of this manuscript, a report on the use of
1-methylcyclopropene as a lipid reporter appeared: J. Yang, J.
[21] Y. Wang, W. Song, W. J. Hu, Q. Lin, Angew. Chem. 2009, 121,
5434; Angew. Chem. Int. Ed. 2009, 48, 5330.
[22] Z. Yu, L. Y. Ho, Z. Wang, Q. Lin, Bioorg. Med. Chem. Lett. 2011,
21, 5033.
[23] Y. Wang, W. J. Hu, W. Song, R. K. Lim, Q. Lin, Org. Lett. 2008,
10, 3725.
ˇ
˙
Seckute, C. M. Cole, N. K. Devaraj, Angew. Chem. 2012, 124,
7594; Angew. Chem. Int. Ed. 2012, 51, 7476.
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Communications
Bioorthogonal Chemistry
Z. Yu, Y. Pan, Z. Wang, J. Wang,*
Q. Lin*
&&&& — &&&&
Genetically Encoded Cyclopropene
Directs Rapid, Photoclick-Chemistry-
Mediated Protein Labeling in Mammalian
Cells
We just click: Genetic incorporation of
a cyclopropene amino acid CpK (see
scheme) site-specifically into proteins in
E. coli and mammalian cells was achieved
using an orthogonal aminoacyl-tRNA
synthetase/tRNACUA pair (CpKRS/
MbtRNACUA). Cyclopropene exhibited fast
reaction kinetics in the photoclick reac-
tion and allowed rapid (ca. 2 min) label-
ing of proteins.
6
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