Alkene-tetrazine ligation for imaging cellular DNA

Article abstract of DOI:10.1002/anie.201403580

5-Vinyl-2′-deoxyuridine (VdU) is the first reported metabolic probe for cellular DNA synthesis that can be visualized by using an inverse electron demand Diels-Alder reaction with a fluorescent tetrazine. VdU is incorporated by endogenous enzymes into the genomes of replicating cells, where it exhibits reduced genotoxicity compared to 5-ethynyl-2′-deoxyuridine (EdU). The VdU-tetrazine ligation reaction is rapid (k≈0.02 M^-1 s ^-1) and chemically orthogonal to the alkyne-azide "click" reaction of EdU-modified DNA. Alkene-tetrazine ligation reactions provide the first alternative to azide-alkyne click reactions for the bioorthogonal chemical labeling of nucleic acids in cells and facilitate time-resolved, multicolor labeling of DNA synthesis. Diels-Alder on DNA: 5-Vinyl-2′-deoxyuridine (VdU) is metabolically incorporated into cellular DNA where it can be visualized by using inverse electron demand Diels-Alder reactions with fluorescent tetrazines. VdU-tetrazine ligation reactions are rapid (k≈0.02 M^-1 s^-1) and chemically orthogonal to alkyne-azide "click" reactions, thereby enabling time-resolved, multicolor labeling of DNA synthesis in individual cells.

Full text of DOI:10.1002/anie.201403580

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DOI: 10.1002/anie.201403580
DNA Labeling Very Important Paper
Alkene–Tetrazine Ligation for Imaging Cellular DNA**
Ulrike Rieder and Nathan W. Luedtke*
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Abstract: 5-Vinyl-2’-deoxyuridine (VdU) is the first reported
metabolic probe for cellular DNA synthesis that can be
visualized by using an inverse electron demand Diels–Alder
reaction with a fluorescent tetrazine. VdU is incorporated by
endogenous enzymes into the genomes of replicating cells,
where it exhibits reduced genotoxicity compared to 5-ethynyl-
2’-deoxyuridine (EdU). The VdU–tetrazine ligation reaction is
rapid (k ꢀ 0.02mÀ1 sÀ1) and chemically orthogonal to the
alkyne–azide “click” reaction of EdU-modified DNA.
Alkene–tetrazine ligation reactions provide the first alternative
to azide–alkyne click reactions for the bioorthogonal chemical
labeling of nucleic acids in cells and facilitate time-resolved,
multicolor labeling of DNA synthesis.
alternatives to immunohistochemical staining.[3] In this
approach, a highly chemoselective reaction is used to ligate
an exogenous probe to a metabolically labeled biomolecule.[4]
For example, 5-ethynyl-2’-deoxyuridine (EdU, 2; Scheme 1a)
can be detected by using a CuI-mediated azide–alkyne “click”
cycloaddition (CuAAC) upon the addition of a fluorescent
azide.[5] Since small-molecule fluorescent probes are utilized,
EdU detection is more sensitive than the immunofluorescent
detection of BrdU, especially in deep tissues. EdU itself,
however, is highly toxic and its addition to living cells causes
DNA damage, cell cycle arrest, and apoptosis.[6] To minimize
these effects, “clickable” nucleosides that exhibit reduced
toxicity, including F-ara-EdU (3; Scheme 1a), have been
developed.[2,7] All such bioorthogonal chemical reporters of
DNA and RNA synthesis have hitherto utilized azide–alkyne
click reactions. The development of alternative bioorthogonal
chemical reactions for DNA imaging would be highly
desirable in multidimensional pulse-chase labeling of DNA
synthesis,[1,2] in experiments where CuAAC reactions are
utilized for tagging other cellular components,[3,4] and for
potentially minimizing the biological impact of the nucleoside
analogue itself.[6]
Inverse electron demand Diels–Alder (invDA) reactions
between electron-deficient tetrazines and electron-rich dieno-
philes are particularly attractive for bioorthogonal chemical
reporter strategies since invDA reactions are irreversible, do
not require a catalyst, and are compatible with cell media.[8]
To date, invDA reactions have been used for labeling
synthetic oligonucleotides in vitro, as well as cellular and
cell-surface proteins, by using strained dienophiles such as
norbornene,[8b–d,9] trans-cyclooctene,[9a,10] and cyclopropene.[11]
The addition of such large substituents to nucleosides is
known to inhibit their cellular metabolism.[12] We therefore
sought the smallest possible dienophile as a bioorthogonal
chemical reporter of cellular DNA synthesis. Since vinyl
aromatic compounds are known to react with tetrazines,[13] we
identified 5-vinyl-2’-deoxyuridine (VdU, 4; Scheme 1a) as
a potential metabolic label for DNA.
M
etabolic labeling and fluorescent detection of DNA are
essential techniques for deciphering the timing and location
of DNA synthesis in vivo.[1] The most commonly used label
for this purpose is 5-bromo-2’-deoxyuridine (BrdU, 1; Sche-
me 1a). BrdU is incorporated into the DNA of dividing cells
by endogenous enzymes and it can be detected with
fluorescent antibodies upon permeabilization of the cells
and denaturation of the DNA. However, the detection of
BrdU is limited by the poor tissue permeability of anti-
bodies.[2] Bioorthogonal chemical reactions provide attractive
To investigate the chemical reactivity of VdU in a model
invDA reaction, VdU (4) and tetrazine 5 (1.2 eq) were
incubated in a 2:1 mixture of 1,4-dioxane/water for 16 h at
room temperature (Scheme 1b). The reaction afforded dihy-
dropyridazine 6 (VdU-Tz) as a mixture of tautomeric and
diastereomeric isomers, as well as pyridazine 7 (VdU-Tz-ox),
with an 81% overall yield of isolated products (Scheme 1b,
and Scheme S3 and Figure S1 in the Supporting Information).
Similar product mixtures have been reported for the strained
alkenes that are widely used in cell-based conjugation
reactions.[8–11] It was unclear, however, whether a terminal
alkene such as VdU could exhibit sufficient reactivity towards
tetrazines for the effective intracellular labeling of DNA.[14,15]
To evaluate the reaction kinetics of the VdU–tetrazine
ligation as compared to styrene and 4-penteneamide, reac-
tions were conducted under pseudo-first-order conditions by
monitoring the consumption of tetrazine 5 upon the addition
of each dienophile (Figure 1 and Figure S2 in the Supporting
Information). 4-Penteneamide exhibited a relatively low rate
constant (k) of 0.42 ꢀ 10À2 mÀ1 sÀ1, while VdU and styrene
exhibited 5- to 10-fold higher rates of 2.1 ꢀ 10À2 mÀ1 sÀ1 and
Scheme 1. a) Thymidine analogues used for the metabolic labeling of
DNA and their respective detection methods (underlined). b) The
reaction between VdU (4) and 3,6-di-2-pyridyl-1,2,4,5-tetrazine 5 (py2-
Tz) in the presence of ambient oxygen. See the Supporting Information
for the synthesis and characterization of compounds 4, 6 and 7.
[*] Dr. U. Rieder, Prof. Dr. N. W. Luedtke
Department of Chemistry, University of Zurich
Winterthurerstrasse 190, 8057 Zurich (Switzerland)
E-mail: nathan.luedtke@chem.uzh.ch
Homepage: http://www.bioorganic-chemistry.com
[**] We gratefully acknowledge the Swiss National Science Foundation
(146754 to N.W.L.) and the University of Zꢀrich Forschungskredit
(FK-13-113 to U.R.) for financial support. We thank Dr. Kathrin Lang
and Prof. Dr. Jason Chin for providing the fluorescent tetrazine
“Tamra-Tz”. We thank Dr. Anne B. Neef, Dr. Kai J. Neelsen and
Therese Triemer for helpful discussions.
Supporting information for this article, including experimental
details, is available on the WWW under http://dx.doi.org/10.1002/
anie.201403580.
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minimal during this labeling period. In the case of EdU,
experimental results can be perturbed by DNA damage and
cell cycle arrest.[2,6] To assess the cytotoxicity of VdU as
compared to EdU, cell cultures were grown in the presence of
various nucleoside concentrations for 24–72 h, and total
metabolic activities were measured by using the Alamar
Blue assay. In all of the cell lines tested, EdU was 2- to 15-fold
more toxic than VdU (Figure S10 and Table S1 in the
Supporting Information). When HeLa cells were incubated
with the standard labeling concentration of EdU (10 mm) for
4–16 h, no changes in cellular morphology or total cellular
respiration were observed, but a dramatic accumulation of
tetraploid (4n) cells that stained positively for the phosphor-
ylation of histone H2AX was detected (Figure 3). This
Figure 1. Pseudo-first-order reaction rates (k’) versus alkene concentra-
tion for the consumption of tetrazine 5 (0.1 mm) in a 1:1 (v/v) mixture
of methanol and water at 258C. The slope of each linear regression
provides the rate constant k. R1 =2’-deoxyribose.
4.8 ꢀ 10À2 mÀ1 sÀ1, respectively (Figure 1). These values are
lower than those reported for angle-strained olefins,[16] but are
directly comparable to those of strain-promoted azide–alkyne
cycloaddition “SPAAC” reactions (k ꢀ 10À3–10À1mÀ1 sÀ1)
widely used for cellular labeling purposes.[17]
To evaluate the ability of endogenous enzymes to
incorporate VdU into DNA, HeLa cells were treated with
variable doses of VdU for 16 h before being fixed and stained
with a tetramethylrhodamine–tetrazine conjugate (Tamra-Tz;
Scheme S1 in the Supporting Information).[8c] Dose-depen-
dent nuclear staining was observed in cells treated with 1 to
100 mm of VdU (Figure 2 and Figure S3). Similar results were
Figure 3. Flow cytometry analysis of gH2AX versus DAPI staining of
HeLa cells treated with 30 mm VdU or 10 mm EdU for 16 h. These
concentrations were selected based upon the equivalent staining
frequencies and intensities observed for EdU and VdU (Figure 4b,c,
and Figure S15,16). gH2AX positive cells were detected by using
a phosphospecific antibody. a) The dot plots illustrate gH2AX abun-
dance versus the total cellular DNA content of each cell. The insets
display histograms of the DNA content (from the cell cycle phases
G0/1 to G2/M) versus cell count. The dashed lines show the thresholds
used for defining gH2AX positive/negative cells based on the control.
For a positive control, cells were treated for 1 h with camptothecin
(CPT, 0.5 mm), which causes DNA damage.[21] b) A graphical represen-
tation of results where n=5, *P<0.02, **P<0.002, ***P<0.0001,
ns=not significant, compared to the control. For 4 h time points and
U2OS cells see Figure S11.
Figure 2. Bioorthogonal invDA ligation of VdU in newly synthesized
DNA upon the addition of the fluorescent tetrazine Tamra-Tz. HeLa
cells were treated with 30 mm VdU for 16 h, followed by fixation, DNA
denaturation, and incubation with 5 mm Tamra-Tz for 4 h. Total cellular
DNA was stained with DAPI. The negative control (neg. ctrl.) samples
received identical treatment but were not incubated with VdU prior to
staining. Scale bars: 50 mm. DIC=differential interference contrast
image, merge=overlay of Tamra-Tz and DAPI channels.
gH2AX formation is associated with DNA strand break-
age.[18] Similar results were obtained with U2OS cells (Fig-
ure S11). By contrast, little or no gH2AX formation or G2/M
cell cycle arrest was observed in cells treated with 30 mm of
VdU (Figure 3 and Figure S11). While the reasons for these
differences are unclear, previous studies have established
a relationship between the chemical stability of modified
nucleotides and their ability to initiate the DNA damage
response.[19] EdU is known to initiate spontaneous DNA
strand breaks in the presence of nucleophilic amines.[20]
Interestingly, EdU (2) decomposed with a half-life of 8 h in
a 20% aqueous methyl amine solution at room temperature,
while VdU (4) exhibited no detectable decomposition even
after 48 h (Figure S12).
observed in U2OS, A549, Vero, and MRC-5 cells (Figures S4–
S7). The addition of 5 mm Tamra-Tz to VdU-treated cells
resulted in rapid intranuclear staining after only 30 min at
378C (Figure S8). To evaluate the selectivity of VdU for
incorporation into cellular DNA versus RNA, living HeLa
cells were treated with 30 mm of VdU in the presence of
aphidicolin, an inhibitor of DNA synthesis, for 16 h. Subse-
quent staining revealed no detectable VdU labeling (Fig-
ure S9). Together, these results demonstrate selective meta-
bolic incorporation of VdU into the DNA of replicating cells.
Metabolic labeling experiments can last anywhere from
minutes to weeks prior to fixation and analysis.[1,2] The
biological impact of the metabolic label should thus be
Pulse-chase labeling experiments, in which multiple
metabolic probes are introduced into DNA over time, are
used in a wide variety of important biological investigations,
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such as characterizing the timing of DNA replication, visual-
izing embryogenesis, and in stem cell research.[1,2] We there-
fore examined the possibility of using VdU–tetrazine ligation
in combination with EdU–azide cycloaddition for introducing
orthogonal chemical labels into cellular DNA. One potential
pitfall to this approach is that alkynes can cross-react with
tetrazines.[13,16] The reactivity of tetrazine 5 towards EdU was
therefore evaluated in vitro (Figure 4a). The addition of
excess VdU (4) caused rapid consumption of tetrazine 5, but
no reaction was observed with EdU (2). This finding was
confirmed by NMR experiments conducted over 48 h (Fig-
ure S13). These results demonstrate the feasibility of orthog-
onal DNA labeling with both VdU and EdU.
To further evaluate the relative efficiencies of nucleoside
incorporation and detection, optimized concentrations of
VdU (30 mm) and EdU (10 mm) were prepared as a mixture
and co-administered to cells for 8 h. Following staining,
extensive colocalization of VdU (red) and EdU (green) with
approximately equal staining intensities were observed,
thereby giving a yellow color in the merged images (Fig-
ure 4c). Taken together, these results indicate that the
methods used for the chemical detection of VdU and EdU
are both efficient and mutually orthogonal in cellular DNA.
The orthogonal chemical labeling of VdU and EdU could,
in principal, be expanded to a third level of spatial and
temporal resolution if the immunohistochemical staining of
BrdU was included. Previous studies, however, have demon-
strated that BrdU and EdU are often incompatible because
EdU pulses can inhibit the subsequent incorporation of
BrdU[2] and most anti-BrdU antibodies ( ꢀ 90%) exhibit
cross-reactivity with EdU.[22] We therefore selected F-ara-
EdU as a relatively nontoxic EdU analogue (Figure S11C,D)
that is compatible with subsequent BrdU incorporation and
detection.[2] To investigate the compatibility of VdU with
BrdU and F-ara-EdU, HeLa cells were sequentially treated
with BrdU (30 mm), VdU (30 mm), and F-ara-EdU (10 mm).
Subsequent staining revealed well-resolved, non-overlapping
fluorescent signals (Figure 5 and Figure S17). The spatial and
Figure 4. a) The consumption of tetrazine 5 (0.1 mm) according to
normalized absorbance changes at 530 nm in the presence of 5 mm of
EdU or VdU in a 1:1 (v/v) mixture of methanol and water at 258C.
b) Confocal fluorescence microscopy images of cells treated sequen-
tially with 10 mm EdU (4 h) and 30 mm VdU (4 h), followed by fixation,
DNA denaturation, and staining with Tamra-Tz (red) and AF-azide
(green). The overlay of the two color channels reveals both distinct
and dual labeling, indicated by circles and squares, respectively.
c) Cells were treated simultaneously with a mixture of VdU (30 mm)
and EdU (10 mm) for 8 h and stained as in (b). Scale bars: 50 mm;
merge=overlay of AF-azide, Tamra-Tz, and DAPI channels. See Fig-
ures S15,16 for additional control experiments and quantitative image
analyses.
Figure 5. Visualizing the progression of the S-phase in a single cell by
using BrdU (30 mm, 1st pulse, green), VdU (30 mm, 2nd pulse, red), and
F-ara-EdU (10 mm, 3rd pulse, blue). HeLa cells were incubated for 2 h
45 min with each nucleoside and washed for 15 min with fresh media
in between nucleoside treatments. After fixation and DNA denatura-
tion, the cells were stained with Tamra-Tz (red), BrdU antibody
(green), and AF-azide (blue). Scale bars: 5 mm. See Figure S17 for
additional examples.
To test the chemical orthogonality of VdU and EdU in the
context of cellular DNA, HeLa cells were independently
treated with EdU or VdU before being fixed and stained with
Tamra-Tz followed by azide-modified AlexaFluor 488 (AF-
azide) under CuAAC conditions. The ligation reactions did
not show any cross-reactivity since Tamra-Tz did not label
EdU-treated cells and VdU-treated cells were not stained by
AF-azide (Figure S14). To evaluate the orthogonality of VdU
and EdU in individual cells containing one or both labels, cells
were treated sequentially with a pulse of EdU for 4 h followed
by a VdU “chase” for an additional 4 h. The cells were then
fixed and stained with Tamra-Tz and AF-azide. The resulting
staining revealed cells containing only one of the two labels
(circles, Figure 4b), as well as cells with well-resolved signals
from both labels (squares, Figure 4b). Quantitative image
analyses indicated that VdU and EdU were incorporated and
detected in the cells with similar efficiencies (Figures S15,16).
temporal distribution of the labels were characteristic of
S-phase progression,[23] where the first label stained euchro-
matin in the early S-phase as numerous small replication foci
throughout the nucleus, the second label was observed at
a fewer number of larger foci located around the nucleoli, and
the final label gave staining in distinct globular regions at the
nuclear envelope and perinucleolar heterochromatin corre-
sponding to the late S-phase. The order of nucleoside addition
did not influence these patterns, thereby demonstrating that
none of the three nucleosides interfered with metabolism or
the detection of the other two (Figure S17).
In conclusion, VdU is selectively incorporated into
cellular DNA, where it can be detected by using alkene–
tetrazine ligation. This is the first example of an inverse
electron demand Diels–Alder reaction on cellular nucleic
acids. The reaction rate between VdU (4) and dipyridyl
tetrazine 5 is similar to those for strain-promoted azide–
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alkyne cycloaddition “SPAAC” reactions,[17d–f] and the addi-
tion of Tamra-Tz to VdU-treated cells results in rapid
intranuclear staining apparent after only 30 min. These results
demonstrate that highly reactive strained alkenes are not
a prerequisite for effective intracellular labeling through
tetrazine ligation.[9–11,14] The biological properties of VdU are
highly desirable since it causes relatively little gH2AX
formation or cell cycle arrest when it is applied at optimal
labeling concentrations. These results open the door to
alternative bioorthogonal chemical reactions for the modifi-
cation of cellular nucleic acids, for example, tetrazole photo-
ligation[24] or palladium-catalyzed Heck reactions.[25]
[10] a) 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 – 10320; b) J. Schoch, M. Staudt, A. Samanta, M. Wiessler,
A. Jꢁschke, Bioconjugate Chem. 2012, 23, 1382 – 1386; c) N. K.
Devaraj, R. Upadhyay, J. B. Haun, S. A. Hilderbrand, R.
Weissleder, Angew. Chem. 2009, 121, 7147 – 7150; Angew.
Chem. Int. Ed. 2009, 48, 7013 – 7016.
ˇ
ˇ
˙
[11] a) C. M. Cole, J. Yang, J. Seckute, N. K. Devaraj, ChemBioChem
2013, 14, 205 – 208; b) D. M. Patterson, L. A. Nazarova, B. Xie,
D. N. Kamber, J. A. Prescher, J. Am. Chem. Soc. 2012, 134,
18638 – 18643.
[12] A. Verri, F. Focher, R. J. Duncombe, I. Basnak, R. T. Walker,
P. L. Coe, E. de Clercq, G. Andrei, R. Snoeck, J. Balzarini, S.
Spadari, Biochem. J. 2000, 351 Pt 2, 319 – 326.
[13] J. Sauer, D. K. Heldmann, J. Hetzenegger, J. Krauthan, H.
Sichert, J. Schuster, Eur. J. Org. Chem. 1998, 2885 – 2896.
[14] During the course of our investigations, Wittmann and cow-
orkers reported that aliphatic terminal alkenes containing 4-
penteneamide (Figure 1) could be used for cell surface imaging
of glycans when incubated with highly concentrated solutions of
tetrazine probes (1 mm) for 6–12 h.[15] It was unclear, however, if
the probe concentrations typically used for intracellular labeling
purposes (1–10 mm) would exhibit sufficiently rapid reactions
with aromatic terminal alkenes for selective intracellular stain-
ing.
[15] A. Niederwieser, A. K. Spꢁte, L. D. Nguyen, C. Jꢂngst, W.
Reutter, V. Wittmann, Angew. Chem. 2013, 125, 4359 – 4363;
Angew. Chem. Int. Ed. 2013, 52, 4265 – 4268.
[16] F. Thalhammer, U. Wallfahrer, J. Sauer, Tetrahedron Lett. 1990,
31, 6851 – 6854.
[17] a) J. M. Baskin, J. A. Prescher, S. T. Laughlin, N. J. Agard, P. V.
Chang, I. A. Miller, A. Lo, J. A. Codelli, C. R. Bertozzi, Proc.
Natl. Acad. Sci. USA 2007, 104, 16793 – 16797; b) X. Ning, J.
Guo, M. A. Wolfert, G. J. Boons, Angew. Chem. 2008, 120, 2285 –
2287; Angew. Chem. Int. Ed. 2008, 47, 2253 – 2255; c) J.
Dommerholt, S. Schmidt, R. Temming, L. J. A. Hendriks,
F. P. J. T. Rutjes, J. C. M. van Hest, D. J. Lefeber, P. Friedl, F. L.
van Delft, Angew. Chem. 2010, 122, 9612 – 9615; Angew. Chem.
Int. Ed. 2010, 49, 9422 – 9425; d) M. F. Debets, S. S. van Berkel, J.
Dommerholt, A. T. Dirks, F. P. Rutjes, F. L. van Delft, Acc.
Chem. Res. 2011, 44, 805 – 815; e) N. E. Mbua, J. Guo, M. A.
Wolfert, R. Steet, G. J. Boons, ChemBioChem 2011, 12, 1912 –
1921; f) J. Tummatorn, P. Batsomboon, R. J. Clark, I. V. Alabu-
gin, G. B. Dudley, J. Org. Chem. 2012, 77, 2093 – 2097.
[18] a) O. Fernandez-Capetillo, A. Lee, M. Nussenzweig, A. Nus-
senzweig, DNA Repair 2004, 3, 959 – 967; b) J. Lukas, C. Lukas, J.
Bartek, Nat. Cell Biol. 2011, 13, 1161 – 1169.
[19] X. J. Liu, B. Nowak, Y. Q. Wang, W. Plunkett, Chin. J. Cancer
2012, 31, 373 – 380.
[20] a) P. Ding, D. Wunnicke, H. J. Steinhoff, F. Seela, Chem. Eur. J.
2010, 16, 14385 – 14396; b) S. Ikeda, K. Tainaka, K. Matsumoto,
Y. Shinohara, K. L. Ode, E. A. Susaki, H. R. Ueda, PLoS One
2014, 9, e92369.
[21] T. Furuta, H. Takemura, Z.-Y. Liao, G. J. Aune, C. Redon, O. A.
Sedelnikova, D. R. Pilch, E. P. Rogakou, A. Celeste, H. T. Chen,
A. Nussenzweig, M. I. Aladjem, W. M. Bonner, Y. Pommier, J.
Biol. Chem. 2003, 278, 20303 – 20312.
[22] R. Liboska, A. Ligasovꢃ, D. Strunin, I. Rosenberg, K. Koberna,
PLoS One 2012, 7, e51679.
[23] V. O. Chagin, J. H. Stear, M. C. Cardoso, Cold Spring Harbor
Perspect. Biol. 2010, 2, a000737.
[24] R. K. V. Lim, Q. Lin, Acc. Chem. Res. 2011, 44, 828 – 839.
[25] M. E. Ourailidou, J.-Y. van der Meer, B.-J. Baas, M. Jeronimus-
Stratingh, A. L. Gottumukkala, G. J. Poelarends, A. J. Minnaard,
F. J. Dekker, ChemBioChem 2014, 15, 209 – 212.
Received: March 21, 2014
Published online: June 30, 2014
Keywords: bioorthogonal reporters · Diels–Alder cycloaddition ·
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fluorescent probes · metabolic labeling · nucleosides
[1] a) D. E. Wagner, I. E. Wang, P. W. Reddien, Science 2011, 332,
811 – 816; b) J. J. Collins III, B. Wang, B. G. Lambrus, M. E.
Tharp, H. Iyer, P. A. Newmark, Nature 2013, 494, 476 – 479.
[2] A. B. Neef, N. W. Luedtke, Proc. Natl. Acad. Sci. USA 2011, 108,
20404 – 20409.
[3] a) E. Saxon, C. R. Bertozzi, Science 2000, 287, 2007 – 2010; b) Q.
Wang, T. R. Chan, R. Hilgraf, V. V. Fokin, K. B. Sharpless, M. G.
Finn, J. Am. Chem. Soc. 2003, 125, 3192 – 3193; c) A. J. Link,
D. A. Tirrell, J. Am. Chem. Soc. 2003, 125, 11164 – 11165;
d) M. D. Best, Biochemistry 2009, 48, 6571 – 6584; e) C. P.
Ramil, Q. Lin, Chem. Commun. 2013, 49, 11007 – 11022.
[4] E. M. Sletten, C. R. Bertozzi, Angew. Chem. 2009, 121, 7108 –
7133; Angew. Chem. Int. Ed. 2009, 48, 6974 – 6998.
[5] a) A. Salic, T. J. Mitchison, Proc. Natl. Acad. Sci. USA 2008, 105,
2415 – 2420; b) J. Gierlich, G. A. Burley, P. M. Gramlich, D. M.
Hammond, T. Carell, Org. Lett. 2006, 8, 3639 – 3642.
[6] a) P. V. Danenberg, R. S. Bhatt, N. G. Kundu, K. Danenberg, C.
Heidelberger, J. Med. Chem. 1981, 24, 1537 – 1540; b) F.
Kohlmeier, A. Maya-Mendoza, D. Jackson, Chromosome Res.
2013, 21, 87 – 100; c) H. Zhao, H. D. Halicka, J. Li, E. Biela, K.
Berniak, J. Dobrucki, Z. Darzynkiewicz, Cytometry Part A 2013,
83, 979 – 988.
[7] a) D. Qu, G. Wang, Z. Wang, L. Zhou, W. Chi, S. Cong, X. Ren, P.
Liang, B. Zhang, Anal. Biochem. 2011, 417, 112 – 121; b) L.
Guan, G. W. van der Heijden, A. Bortvin, M. M. Greenberg,
ChemBioChem 2011, 12, 2184 – 2190; c) A. B. Neef, F. Samain,
N. W. Luedtke, ChemBioChem 2012, 13, 1750 – 1753; d) I. H.
Wang, M. Suomalainen, V. Andriasyan, S. Kilcher, J. Mercer, A.
Neef, N. W. Luedtke, U. F. Greber, Cell Host Microbe 2013, 14,
468 – 480; e) A. B. Neef, N. W. Luedtke, ChemBioChem 2014, 15,
789 – 793.
[8] a) M. L. Blackman, M. Royzen, J. M. Fox, J. Am. Chem. Soc.
2008, 130, 13518 – 13519; b) N. K. Devaraj, R. Weissleder, S. A.
Hilderbrand, Bioconjugate Chem. 2008, 19, 2297 – 2299; c) K.
Lang, L. Davis, J. Torres-Kolbus, C. Chou, A. Deiters, J. W. Chin,
Nat. Chem. 2012, 4, 298 – 304; d) E. Kaya, M. Vrabel, C. Deiml,
S. Prill, V. S. Fluxa, T. Carell, Angew. Chem. 2012, 124, 4542 –
4545; Angew. Chem. Int. Ed. 2012, 51, 4466 – 4469.
[9] a) T. Plass, S. Milles, C. Koehler, J. Szymanski, R. Mueller, M.
Wiessler, C. Schultz, E. A. Lemke, Angew. Chem. 2012, 124,
4242 – 4246; Angew. Chem. Int. Ed. 2012, 51, 4166 – 4170; b) J.
Schoch, M. Wiessler, A. Jꢁschke, J. Am. Chem. Soc. 2010, 132,
8846 – 8847.
9172
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