Relative performance of alkynes in copper-catalyzed azide-alkyne cycloaddition

Article abstract of DOI:10.1021/bc300672b

Copper-catalyzed azide-alkyne cycloaddition (CuAAC) has found numerous applications in a variety of fields. We report here only modest differences in the reactivity of various classes of terminal alkynes under typical bioconjugative and preparative organic conditions. Propargyl compounds represent an excellent combination of azide reactivity, ease of installation, and cost. Electronically activated propiolamides are slightly more reactive, at the expense of increased propensity for Michael addition. Certain alkynes, including tertiary propargyl carbamates, are not suitable for bioconjugation due to copper-induced fragmentation. A fluorogenic probe based on such reactivity is available in one step from rhodamine 110 and can be useful for optimization of CuAAC conditions.

Full text of DOI:10.1021/bc300672b

Article
pubs.acs.org/bc
Relative Performance of Alkynes in Copper-Catalyzed Azide−Alkyne
Cycloaddition
Alexander A. Kislukhin, Vu P. Hong, Kurt E. Breitenkamp, and M. G. Finn*
Department of Chemistry and The Skaggs Institute of Chemical Biology, The Scripps Research Institute, La Jolla, California, United
States
S
* Supporting Information
ABSTRACT: Copper-catalyzed azide−alkyne cycloaddition
(CuAAC) has found numerous applications in a variety of
fields. We report here only modest differences in the reactivity
of various classes of terminal alkynes under typical
bioconjugative and preparative organic conditions. Propargyl
compounds represent an excellent combination of azide
reactivity, ease of installation, and cost. Electronically activated
propiolamides are slightly more reactive, at the expense of
increased propensity for Michael addition. Certain alkynes,
including tertiary propargyl carbamates, are not suitable for
bioconjugation due to copper-induced fragmentation. A
fluorogenic probe based on such reactivity is available in one step from rhodamine 110 and can be useful for optimization of
CuAAC conditions.
ore than ten years have now passed since first reports of
copper-catalyzed azide−alkyne cycloaddition (CuAAC)
propiolates and can be rather difficult to functionalize.15 Most
importantly, these substrates, particularly in the terminal alkyne
form required for CuAAC, are too reactive as Michael
acceptors16 to be bioorthogonal. We regarded propiolamides
as having a good combination of synthetic accessibility,
electronic activation of the CuAAC process, and attenuated
Michael reactivity. The second-order rate constant of the
conjugate addition of a model cysteine-containing peptide to N-
phenylpropiolamide can be estimated as approximately 0.13
M−1 s−1 (pH 7.0).17 The reaction rates of maleimide18 and
oxanorbornadiene reagents19,20 with thiols are more than 500
times greater.
Standard unactivated alkynes, often derived from propargyl
building blocks, perform very well with good CuAAC catalysts
and are used in most applications of the reaction.
Propiolamides, among the first groups used for CuAAC,1
have been usually considered only when even greater reactivity
is required, such as when especially low concentrations of
reagents must be used, or when that particular structure type is
desired for other reasons. Thus, a search for optimal yields in
radiolabel synthesis among aromatic, aliphatic, propargylamido,
and propiolate alkyne possibilities found the last to be most
effective;21 and several demanding bioconjugative applications
involving propiolamides have been reported.22−25 A literature
search for carbamoyltriazoles, the product of CuAAC between
propiolamides and azides, revealed that most have been used in
larger screening efforts and without special consideration of
M
by Meldal1 and Fokin/Sharpless.2 The ability to reliably form a
stable linkage between these two functional groups in a
multitude of settings was immediately appreciated as a powerful
tool, placing CuAAC at the center of the click chemistry
arsenal.3
Azides and alkynes are rare in nature, making the CuAAC
process of great use as a bioorthogonal transformation4 for
making linkages to biological molecules,5 usually involving low
concentrations of reagents and mild conditions. The limited
compatibility of copper with living organisms has inspired the
development of metal-free azide−alkyne cycloaddition with
highly strained cyclooctynes.6,7 The discovery of other reactions
that fit these requirements, most notably tetrazine−olefin
cyclooaddition,8−10 quickly followed, and this field remains one
of rapid development.11
We have optimized the ligand-accelerated CuAAC bio-
conjugation reaction under practical conditions, ameliorating
some of the adverse effects of the ability of copper ions and
ascorbate to generate species harmful to biomolecules.12 Using
this procedure, we wanted to identify the substrates that enable
the most rapid triazole formation. We assumed from limited
anecdotal information that CuAAC reaction rates are more
sensitive to the nature of the alkyne component than the azide.
It has long been known that alkynes can be activated toward
uncatalyzed [3 + 2] cycloaddition by conjugating the alkyne
unit with electron-withdrawing ester groups due to a lowering
of the LUMO energy of the alkyne.13,14 Thus, it is reasonable to
test the idea that propiolate derivatives, ethynyl ketones, and
ethynyl aldehydes could be more reactive in a copper-catalyzed
AAC as well. However, acetylenic ketones, aldehydes, and alkyl
Received: December 19, 2012
Revised: March 13, 2013
© XXXX American Chemical Society
A
dx.doi.org/10.1021/bc300672b | Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
Article
Figure 1. Performance of various alkyne substrates in the ligand-accelerated (LA) CuAAC process under bioconjugation conditions. (Top) Reaction
scheme and structures of alkynes used. (Bottom) Time required to reach 50% and 90% of maximum fluorescence under various conditions.
Figure 2. Competition experiment under typical organic conditions.
their potentially high reactivity. Often, these triazoles were
made by aminolysis of corresponding methyl triazole-4-
carboxylate, as exemplified by a synthesis of the anticonvulsant
drug rufinamide.26 To date, no quantitative comparison of
ligand-accelerated triazole-forming reactivity (LACuAAC) has
been reported for candidate alkyne types including propiola-
mides and propargylic compounds, using any of the more active
catalyst formulations.
Toward this end, we tested the performance of a panel of
alkynes in a standard kinetic assay using the fluorogenic
azidocoumarin of Wang and co-workers.27 We included several
types of alkynes that are typically attached to molecules of
B
dx.doi.org/10.1021/bc300672b | Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
Article
Figure 3. (A) CuAAC reaction of propiolamide PyrPRA with azidohomoalanine. (B) Emission spectra (excitation at 348 nm) before (PyrPRA) and
after (PyrTRZ) CuAAC reaction with AHA. (C) Time course of polyvalent Qβ nanoparticle-azide labeling with PyrPRA (excitation at 348 nm,
emission at 394 and 488 nm).
interest, including secondary propiolamide A, tertiary propio-
lamide E, propargyl derivatives B−D and F−J, longer-chain
alkyne L, and aromatic alkynes K and M (Figure 1). In this
survey we excluded poorly soluble and potentially volatile
unfunctionalized alkynes.
fluorophores, and thus may prove useful for development of
fluorogenic substrates for LACuAAC. For example, we
observed that N-(pyren-1-ylmethyl)propiolamide (PyrPRA)
exhibited an 8-fold increase in fluorescence intensity at 394 nm
upon reaction with a model azide γ-L-azidohomoalanine
(AHA), suggesting that PyrPRA may be quenched by a
photoinduced electron-transfer mechanism (Figure 3A,B).
Well-structured emission bands at 370−430 nm are typically
associated with monomeric excited pyrene species (Figure 3B).
Interestingly, upon reaction of PyrPRA with a polyvalent azide
(Qβ virus-like particles bearing several hundred azide groups on
the 28-nm-diameter surface31), the intensity of monomer
emission at 394 nm grew slightly, but excimer fluorescence at
488 nm increased 3-fold (Figure 3C). This suggests that
excimers are formed from nearby pyrene units on the
nanoparticle scaffold. Further development of these results
requires a more polar derivative, as precipitation of PyrPRA
was observed even when significant amounts of DMSO
cosolvent were used. Other fluorogenic alkynes have been
reported based on coumarin,27,32 naphthalimide,33 benzothia-
zole,34,35 benzoxadiazole,36 and cyclooctyne37 scaffolds.
Over the years of active use of CuAAC, a few reports on the
failure of certain substrates to convert cleanly and quantitatively
to the desired triazoles have emerged. One of the best known
cases is the formation of bis-triazoles by oxidative coupling of
triazolylcopper intermediate, an occasional outcome under
bioconjugative conditions that use low concentration of
reagents.38 Other complications arise from azide-independent
reactivity of copper acetylides. For example, 4-pentynoic acid
and its derivatives such as propargyl glycine may give enol
lactone side products using either CuBr or Cu(II)/ascorbate as
a source of Cu(I).39 Predictably, this occurs for free acids but
not for 4-pentynoic esters and amides. Using accelerating ligand
THPTA, this pathway appears to be suppressed to a significant
extent, as evidenced by the observed CuAAC reactivity of 4-
pentynoic acid L.
Propiolamides were initially confirmed as good substrates for
CuAAC using accelerating ligand THPTA (Figure 1).28
Tertiary propiolamide E reached >90% conversion within 1 h
even with 5 μM Cu catalyst, a lower concentration than we
typically recommend.12,29,30 All alkyne substrates were quite
reactive in the presence of 100 μM Cu+, being completely
consumed in less than 30 min. Under demanding conditions
(10 μM Cu+) the differences were more distinct. Due to
varying quantum yields, the comparison between kinetic traces
of the reactions of coumarin azide with alkynes A−M is not
straightforward. Therefore, we compare the relative rates by
reporting the times required to reach 50% and 90% of the
maximum fluorescence in each case (Figure 1). Propiolamide A
was the fastest, followed not by its analogue E, but by propargyl
ethers B−D. N-propargylamide F, propargylamines G−I, and
propargyl alcohol J all reacted reasonably well, while aromatic
and aliphatic alkynes K−M were slower. The kinetic traces
from which these data were extracted are shown in the
Supporting Information.
Competition experiments performed under typical organic
LACuAAC conditions (using ligand BimPy2 instead of
THPTA)30 revealed that propiolamide E was more reactive
than the aromatic and propargylic alkynes shown in Figure 2. In
neither case (aqueous bioconjugation-like conditions, Figure 1,
and organic synthesis-like conditions, Figure 2) were the
observed differences in reactivity between alkynes great enough
to warrant a firm recommendation to use one type of substrate
over another. The relatively close reactivities of a variety of
easily installed terminal alkyne motifs serves to illustrate the
robustness of these LACuAAC protocols.
As evident from Figures 1 and 2, electronic activation of the
alkyne does not bring about as great a rate increase in
LACuAAC as it does in the uncatalyzed reaction (methyl
propiolate reacts with phenyl azide 32 times faster than
phenylacetylene).13 However, the electron-poor propiolamides
are capable of altering the spectral properties of attached
Propargylic esters, carbamates, and carbonates readily lose
their leaving groups upon formation of the corresponding Cu-
acetylide to form copper-stabilized propargyl cations, which can
be readily trapped by nucleophiles present in the reaction
mixture. If the reaction is performed in water, propargylic
C
dx.doi.org/10.1021/bc300672b | Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
Article
Figure 4. (Left) Cu(I)-induced cleavage of alkynyl carbamate releases fluorescent Rhodamine 110. (Right) Kinetic traces of Ynoc2Rho cleavage in
the presence of indicated amount of catalyst (5:1 THPTA:Cu+) at 37 °C. In the absence of THPTA, the reaction is more than 100 times slower
(Supporting Information).
alcohols are formed.40,41 This pathway is not a major one for
primary propargyl substrates (Figure 2, entry 1), but becomes
dominant if the propargyl cation is stabilized further by methyl
groups. We applied this reaction to the tertiary propargyl
carbamate (Ynoc group) to create a copper-sensitive
fluorogenic probe, as shown in Figure 4. Under typical aqueous
CuAAC conditions, both carbamate groups of the readily
available rhodamine alkynyl carbamate Ynoc2Rho were cleaved.
The resulting release of Rhodamine 110 induced an 1800-fold
increase of fluorescence within 30 min at 50 μM Cu+ only in
the presence of THPTA (5:1 L:Cu+).
The similarity of the response of Ynoc cleavage and CuAAC
reactions to the nature of the catalyst is striking even though
the former uses no azide and does not generate triazole.
Nonetheless, just like the CuAAC reaction, the Ynoc2Rho
probe did not react in the presence of 1 mM of other soft ions,
including Hg2+ and Ag+,42 and responded very poorly to Cu+
without an accelerating ligand (Supporting Information). Both
Ynoc2Rho cleavage and CuAAC were most efficient in the
presence of THPTA (5:1 L:Cu+). In both cases, BimPy2, a
strong copper chelator, worked well only at ligand:Cu+ ratios
less than or equal to 1:1.29 Ynoc2Rho could therefore be a
useful tool for ligand screening for CuAAC and related alkyne-
dependent processes. In addition, Ynoc should be a useful
protecting group for amines, since it is removed under very
mild aqueous conditions with catalytic copper(I), in contrast to
the strongly acidic conditions required for cleavage of the
related tert-butoxycarbonyl (Boc) group.
conclusion that moderate changes in the electronic nature of
the alkyne do not have a dramatic effect on CuAAC rate when
good accelerating ligands are used. The results, recommenda-
tions, and potential pitfalls are summarized below.
1. Most alkyne substrates behave well under optimized
CuAAC conditions, as seems to be the case from the
wide use of the process. Substrates derived from simple
alkyne building blocks, such as propargyl ethers, N-
propargylamides, and propiolamides, are recommended
over more expensive and slower aryl- and alkylacetylenes.
2. Propiolamides are very good CuAAC substrates,
particularly when Michael reactivity with biological
nucleophiles is not a concern. The electron-poor nature
of propiolamides may serve well for the development of
turn-on fluorogenic substrates for CuAAC, as illustrated
by the dequenching of pyrene-propiolamide (PyrPRA)
substrate upon reaction with an azide.
3. Tertiary propargyl esters and carbamates should not be
used in CuAAC process. Mild and catalytic copper-
induced cleavage of Ynoc carbamate may be useful when
traditional protecting groups for an amino group cannot
be used due to senstitivity of the substrtate to the
cleavage protocols.
4. Future ligand screens may be facilitated by the
introduction of Ynoc2Rho, a fluorogenic probe contain-
ing cleavable alkynyl carbamate groups that responds to
Cu(I) when an appropriate accelerating ligand is present.
We did not explore the reactivity of azides, and until recently
there were no reports of significant effects of azide structure on
the reaction rate,43 excluding such activated species as carbonyl-
and sulfonylazides. However, Ting et al. have recently described
the use of 2-picolyl azides for bioconjugation.44 The chelating
effect allows the reaction to proceed at low micromolar
concentration of reactants without accelerating ligands, but
with added benefits from their use.
ASSOCIATED CONTENT
■
S
* Supporting Information
Improved synthetic procedure for the THPTA ligand, synthesis
of alkyne substrates, conditions for “bioconjugative” and
“organic” CuAAC (Tables S1 and S2), kinetic traces for the
data in Figure 1 (Figure S2), fluorescent response of Ynoc2Rho
to other metals (Figure S3). The material is available free of
charge via the Internet at http://pubs.acs.org.
Here we compared the performance of common non-
Michael-reactive alkyne building blocks, resulting in the
D
dx.doi.org/10.1021/bc300672b | Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
Article
(18) Tournier, E. J. M., Wallach, J., and Blond, P. (1998)
Sulfosuccinimidyl 4-(N-maleimidomethyl)-1-cyclohexane carboxylate
as a bifunctional immobilization agent. Optimization of the coupling
conditions. Anal. Chim. Acta 361, 33−44.
(19) Kislukhin, A. A., Higginson, C. J., Hong, V. P., and Finn, M. G.
(2012) Degradable conjugates from oxanobornadiene reagents. J. Am.
Chem. Soc. 134, 6491−6497.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: mgfinn@gatech.edu.
Notes
The authors declare no competing financial interest.
(20) Hong, V., Kislukhin, A., and Finn, M. G. (2009) Thiol-selective
fluorogenic probes for labeling and release. J. Am. Chem. Soc. 131,
9986−9994.
ACKNOWLEDGMENTS
■
This work was supported by the NIH (F31CA165653 to A. A.
K.), NSF (CH-1011796), and an Eli Lilly Fellowship to A. A. K.
(21) Glaser, M., and Arstad, E. (2007) ″Click labeling″ with 2-
[18F]fluoroethylazide for positron emission tomography. Bioconjugate
Chem. 18, 989−993.
(22) Yang, Y., and Kluger, R. (2010) Efficient CuAAC click
formation of functional hemoglobin bis-tetramers. Chem. Commun.
46, 7557−7559.
(23) Billing, J. F., and Nilsson, U. J. (2005) C2-Symmetric
macrocyclic carbohydrate/amino acid hybrids through copper(I)-
catalyzed formation of 1,2,3-triazoles. J. Org. Chem. 70, 4847−4850.
(24) Wang, J., Li, H., Zou, G., and Wang, L.-X. (2007) Novel
template-assembled oligosaccharide clusters as epitope mimics for
HIV-neutralizing antibody 2G12. Design, synthesis, and antibody
binding study. Org. Biomol. Chem. 5, 1529−1540.
(25) Srinivasachari, S., Fichter, K. M., and Reineke, T. M. (2008)
Polycationic β-cyclodextrin “click clusters”: monodisperse and versatile
scaffolds for nucleic acid delivery. J. Am. Chem. Soc. 130, 4618−4627.
(26) Mudd, W. H., and Stevens, E. P. (2010) An efficient synthesis of
rufinamide, an antiepileptic drug. Tetrahedron Lett. 51, 3229−3231.
(27) Sivakumar, K., Xie, F., Cash, B. M., Long, S., Barnhill, H. N., and
Wang, Q. (2004) A Fluorogenic 1,3-dipolar cycloaddition reaction of
3-azidocoumarins and acetylenes. Org. Lett. 6, 4603−4606.
(28) We also report in Supporting Information an improved
synthesis of THPTA suitable for large scale production. The procedure
uses (Ph3P)2CuOAc catalyst in organic solvent [ Shao, C., Cheng, G.,
Su, D., Xu, J., Wang, X., and Hu, Y. (2010) Copper(I) acetate: a
structurally simple but highly efficient dinuclear catalyst for copper-
catalyzed azide-alkyne cycloaddition. Adv. Synth. Catal. 352, 1587−
1592. Gonda, Z., and Novak, Z. (2010) Highly active copper catalysts
for azide-alkyne cycloaddition. Dalton Trans. 39, 726−729 ], and
provides material without the bis(triazole) contaminant that can
inhibit the CuAAC reaction..
REFERENCES
■
(1) Tornøe, C. W., Christensen, C., and Meldal, M. (2002)
Peptidotriazoles on solid phase: [1,2,3]-triazoles by regiospecific
copper(I)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to
azides. J. Org. Chem. 67, 3057−3062.
(2) Rostovtsev, V. V., Green, L. G., Fokin, V. V., and Sharpless, K. B.
(2002) A stepwise Huisgen cycloaddition process: copper(I)-catalyzed
regioselective ligation of azides and terminal alkynes. Angew. Chem.,
Int. Ed. 41, 2596−2599.
(3) Kolb, H. C., Finn, M. G., and Sharpless, K. B. (2001) Click
chemistry: diverse chemical function from a few good reactions.
Angew. Chem., Int. Ed. 40, 2004−2021.
(4) Sletten, E. M., and Bertozzi, C. R. (2009) Bioorthogonal
chemistry: fishing for selectivity in a sea of functionality. Angew. Chem.,
Int. Ed. 48, 6974−6998.
(5) Wang, Q., Chan, T. R., Hilgraf, R., Fokin, V. V., Sharpless, K. B.,
and Finn, M. G. (2003) Bioconjugation by copper(I)-catalyzed azide-
alkyne [3 + 2] cycloaddition. J. Am. Chem. Soc. 125, 3192−3193.
(6) Agard, N. J., Prescher, J. A., and Bertozzi, C. R. (2004) A strain-
promoted [3 + 2] azide-alkyne cycloaddition for covalent modification
of biomolecules in living systems. J. Am. Chem. Soc. 126, 15046−
15047.
(7) Laughlin, S. T., Baskin, J. M., Amacher, S. L., and Bertozzi, C. R.
(2008) In vivo imaging of membrane-associated glycans in developing
zebrafish. Science 320, 664−667.
(8) Blackman, M. L., Royzen, M., and Fox, J. M. (2008) Tetrazine
ligation: fast bioconjugation based on inverse-electron-demand Diels−
Alder reactivity. J. Am. Chem. Soc. 130, 13518−13519.
(29) Rodionov, V. O., Presolski, S., Díaz, D. D., Fokin, V. V., and
Finn, M. G. (2007) Ligand-accelerated Cu-catalyzed azide-alkyne
cycloaddition: a mechanistic report. J. Am. Chem. Soc. 129, 12705−
12712.
(30) Presolski, S. I., Hong, V., Cho, S.-H., and Finn, M. G. (2010)
Tailored ligand acceleration of the Cu-catalyzed azide-alkyne cyclo-
addition reaction: practical and mechanistic implications. J. Am. Chem.
Soc. 132, 14570−14576.
(9) Devaraj, N. K., Weissleder, R., and Hildebrand, S. A. (2008)
Tetrazine-based cycloadditions: application to pretargeted live cell
labeling. Bioconjugate Chem. 19, 2297−2299.
(10) Devaraj, N. K., and Weissleder, R. (2011) Biomedical
applications of tetrazine cycloadditions. Acc. Chem. Res. 44, 816−827.
(11) Debets, M. F., Van Berkel, S. S., Dommerholt, J., Dirks, A. J.,
Rutjes, F. P. J. T., and Van Delft, F. L. (2011) Bioconjugation with
strained alkenes and alkynes. Acc. Chem. Res. 44, 805−815.
(12) Hong, V., Presolski, S. I., Ma, C., and Finn, M. G. (2009)
Analysis and optimization of copper-catalyzed azide-alkyne cyclo-
addition for bioconjugation. Angew. Chem., Int. Ed. 48, 9879−9883.
(13) Huisgen, R. (1962) Kinetics and mechanism of 1,3-dipolar
cycloaddition. Angew. Chem., Int. Ed. Engl. 2, 633−645.
(14) Huisgen, R. (1984) 1,3-Dipolar cycloaddition - introduction,
survey, mechanism. In 1,3-Dipolar Cycloaddition Chemistry (Padwa, A.,
Ed.) pp 1−176, Vol. 1, Wiley, New York.
(31) Sen Gupta, S., Kuzelka, J., Singh, P., Lewis, W. G., Manchester,
M., and Finn, M. G. (2005) Accelerated bioorthogonal conjugation: a
practical method for the ligation of diverse functional molecules to a
polyvalent virus scaffold. Bioconjugate Chem 16, 1572−1579.
(32) Zhou, Z., and Fahrni, C. J. (2004) A fluorogenic probe for the
copper(I)-catalyzed azide-alkyne [3 + 2] cycloaddition: modulation of
3
the fluorescence emission via (n,π*)-1(π,π*) inversion. J. Am. Chem.
Soc. 126, 8862−8863.
(33) Sawa, M., Hsu, T.-., Itoh, T., Sugiyama, M., Hanson, S. R., Vogt,
P. K., and Wong, C.-H. (2006) Glycoproteomic probes for fluorescent
imaging of fucosylated glycans in vivo. Proc. Natl. Acad. Sci. U.S.A. 103,
12371−12376.
(15) Nagel, M., and Hansen, H.-J. (2000) Synthesis of
polyalkylphenyl prop-2-ynoates and their flash vacuum pyrolysis to
polyalkylcyclohepta[b]furan-2(2H)-ones. Helv. Chim. Acta 83, 1022−
1048.
(34) Qi, J. J., Han, M. S., Chang, Y. C., and Tung, C. H. (2011)
Developing visible fluorogenic ’click-on’ dyes for cellular imaging.
Bioconjugate Chem. 22, 1758−1762.
(35) Qi, J. J., and Tung, C. H. (2011) Development of benzothiazole
’click-on’ fluorogenic dyes. Bioorg. Med. Chem. Lett. 21, 320−323.
(36) Key, J. A., and Cairo, C. W. (2011) Identification of fluorogenic
and quenched benzoxadiazole reactive chromophores. Dyes Pigm. 88,
95−102.
(16) Bohme, A., Thaens, D., Schramm, F., Paschke, A., and
̈
Schuurmann, G. (2010) Thiol reactivity and its impact on the ciliate
̈
̈
toxicity of alpha,beta-unsaturated aldehydes, ketones, and esters. Chem.
Res. Toxicol. 23, 1905−1912.
(17) Shiu, H.-Y., Chan, T.-C., Ho, C.-M., Liu, Y., Wong, M.-K., and
Che, C.-M. (2009) Electron-deficient alkynes as cleavable reagents for
the modification of cysteine-containing peptides in aqueous medium.
Chem.Eur. J. 15, 3839−3850.
E
dx.doi.org/10.1021/bc300672b | Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
Article
(37) Friscourt, F., Fahrni, C. J., and Boons, G.-J. (2012) A
fluorogenic probe for the catalyst-free detection of azide-tagged
molecules. J. Am. Chem. Soc. 134, 18809−18815.
(38) Kwon, M., Jang, Y., Yoon, S., Yang, D., and Jeon, H. B. (2012)
Unusual Cu(I)-catalyzed 1,3-dipolar cycloaddition of acetylenic
amides: formation of bistriazole. Tetrahedron Lett. 53, 1606−1609.
(39) Mindt, T. L., and Schibli, R. (2007) Cu(I)-catalyzed
intramolecular cyclization of alkynoic acids in aqueous media: a
″click side reaction″. J. Org. Chem. 72, 10247−10250.
(40) Bertrand, P., and Gesson, J. P. (2007) Click chemistry with O-
dimethylpropargylcarbamate for preparation of pH-sensitive functional
groups. J. Org. Chem. 72, 3596−3599.
(41) Ljungdahl, N., and Kann, N. (2009) Transition-metal-catalyzed
propargylic substitution. Angew. Chem., Int. Ed. 48, 642−644.
(42) McNulty, J., and Keskar, K. (2012) Discovery of a robust and
efficient homogeneous silver(I) catalyst for the cycloaddition of azides
onto terminal alkyne. Eur. J. Org. Chem., 5462−5470.
(43) See reference 28 for a study of reactivity vs electronic effects in
aromatic azides.
(44) Uttamapinant, C., Tangpeerachaikul, A., Grecian, S., Clarke, S.,
Singh, U., Slade, P., Gee, K. R., and Ting, A. Y. (2012) Fast, cell-
compatible click chemistry with copper-chelating azides for bio-
molecular labeling. Angew. Chem., Int. Ed. 51, 5852−5856.
F
dx.doi.org/10.1021/bc300672b | Bioconjugate Chem. XXXX, XXX, XXX−XXX