Synthesis of a fluorogenic cyclooctyne activated by Cu-free click chemistry

Article abstract of DOI:10.1021/ol2025026

Cyclooctyne-based probes that become fluorescent upon reaction with azides are important targets for real-time imaging of azide-labeled biomolecules. The concise synthesis of a coumarin-conjugated cyclooctyne, coumBARAC, that undergoes a 10-fold enhanceme

Full text of DOI:10.1021/ol2025026

ORGANIC
LETTERS
2011
Vol. 13, No. 22
5937–5939
Synthesis of a Fluorogenic Cyclooctyne
Activated by Cu-Free Click Chemistry
John C. Jewett and Carolyn R. Bertozzi*
Departments of Chemistry and Molecular and Cell Biology and Howard Hughes Medical
Institute, University of California, Berkeley, California 94720, United States
crb@berkeley.edu
Received September 14, 2011
ABSTRACT
Cyclooctyne-based probes that become fluorescent upon reaction with azides are important targets for real-time imaging of azide-labeled
biomolecules. The concise synthesis of a coumarin-conjugated cyclooctyne, coumBARAC, that undergoes a 10-fold enhancement in
fluorescence quantum yield upon triazole formation with organic azides is reported. The design principles embodied in coumBARAC
establish a platform for generating fluorogenic cyclooctynes suited for biological imaging.
Molecules that become fluorescent upon chemical sti-
mulus, be it change in pH, metal chelation, or cleavage/
formation of covalent bonds, are highly desirable for
sensitive real-time imaging applications.1 Our laboratory
has had a longstanding interest in the ability to image
bioorthogonal chemical reporters introduced metaboli-
cally into biological systems.2 In particular, we have
employed the azide as a reporter of cellular glycan bio-
synthesis. Azidosugars delivered metabolically to cell-sur-
face glycans can be visualized by covalent reaction with
triarylphosphines (i.e., Staudinger ligation3) or cyclooctynes
(i.e., Cu-free click chemistry4) conjugated to imaging probes.
We have reported several strategies whereby triarylphosphine
reagents used in the Staudinger ligation can be rendered fluo-
rogenic,5 that is, where quantum yield increases upon reac-
tion. These fluorogenic phosphines were highly effective pro-
bes for cell-based imaging studies but had limited utility for in
vivo imaging experiments because of slow reaction kinetics,
an inherent liability of the Staudinger ligation reaction.2
Cyclooctynes appear to be more promising reagents for
in vivo imaging applications. Optimization efforts have led
to cyclooctynes that are ∼500-fold more reactive with
azides (via 1,3-dipolar cycloaddition) than triarylpho-
sphines.6 Other important properties such as solubility7
and stability8 have been modulated as well, with an eye for
developing analogues that are well-suited for use in ani-
mals. However, fluorogenic capabilities, which can be
critical for high-sensitivity biological imaging, have not
yet been bestowed upon cyclooctynes.
The notion that an azideÀalkyne cycloaddition reaction
can alter a substrate’s photophysical properties is certainly
well-precedented. Several azide- or terminal alkyne-func-
tionalizeddyes have beenreportedtoundergofluorescence
enhancement upon triazole formation, typically under
(6) (a) 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, 16793–16797. (b) Ning, X.; Guo, J.;
Wolfert, M. A.; Boons, G.-J. Angew. Chem., Int. Ed. 2008, 47, 2253–
2255. (c) Debets, M. F.; van Berkel, S. S.; Schoffelen, S.; Rutjes, F. P. J.
T.; van Hest, J. C. M.; van Delft, F. L. Chem. Commun. 2010, 46, 97–99.
(d) Jewett, J. C.; Sletten, E. M.; Bertozzi, C. R. J. Am. Chem. Soc. 2010,
132, 3688–3690. (e) Dommerholt, J.; Schmidt, S.; Temming, S.;
Hendriks, L. J. A.; Rutjes, F. P. J. T.; van Hest, J. C. M.; Lefeber, D. J.;
Friedl, P.; van Delft, F. L. Angew. Chem., Int. Ed. 2010, 49, 9422–9425.
(7) Sletten, E. M.; Bertozzi, C. R. Org. Lett. 2008, 10, 3097–3099.
(1) For a recent review on fluorogenic probes designed for protein
labeling, see: (a) Sadhu, K. K.; Mizukami, S.; Hori, Y.; Kikuchi, K.
ChemBioChem 2011, 12, 1299–1308. For a nice example of a pH sensitive
probe, see: (b) Hilderbrand, S. A.; Kelly, K. A.; Niedre, M.; Weissleder,
R. Bioconjugate Chem. 2008, 19, 1635–1639.
(2) Sletten, E. M.; Bertozzi, C. R. Acc. Chem. Res. 2011, 44, 666–676.
(3) Saxon, E.; Bertozzi, C. R. Science 2000, 287, 2007–2010.
(4) Agard, N. J.; Prescher, J. A.; Bertozzi, C. R. J. Am. Chem. Soc.
2004, 126, 15046–15047.
(5) (a) Lemieux, G. A.; de Graffenried, C. L.; Bertozzi, C. R. J. Am.
Chem. Soc. 2003, 125, 4708–4709. (b) Hangauer, M. J.; Bertozzi, C. R.
Angew. Chem., Int. Ed. 2008, 47, 2394–2397.
€
(8) Stockmann, H.; Neves, A. A.; Stairs, S.; Ireland-Zecchini, H.;
Brindle, K. M.; Leeper, F. J. Chem. Sci. 2011, 2, 932–936.
r
10.1021/ol2025026
2011 American Chemical Society
Published on Web 10/26/2011

conditions of copper catalysis.9 Recently, Boons and co-
workers reported a cyclooctyne-based probe whose fluor-
escence was quenched upon formation of the triazole,10
but to date, a fluorogenic cyclooctyne remains elusive,
perhaps reflecting the difficulty inherent to synthesizing
cyclooctynes that are electronically coupled to a fluorophore.
Scheme 1. Synthesis of CoumBARAC
color can be tuned.12 These considerations motivated the
design of synthetic target 3 (Figure 1B) bearing a 4-phe-
nylcoumarin moiety,13,14 which we call coumBARAC.
In a retrosynthetic sense, we decided to install the cou-
marin’s lactone moiety as late in the synthesis as possible to
take maximum advantage of the modular scaffolds that we
were fusing. The late-stage synthetic steps would parallel
our previous synthesis of BARAC, and as such, coumBAR-
AC (3) was envisioned to derive from the fused indole
coumarin 4 (Figure 1B). This coumarin could in turn come
from a Pechmann condensation between a 5-methoxyindole,
5, and a β-ketoester. As before, the indole would arise from a
Fischer indole synthesis between 4-methoxyphenylhydrazine
(6) and 1-indanone (7). In theory, the benzylic silylation
could be accomplished at various stages of the synthesis.
The synthesis of coumBARAC began with a Fischer
indole synthesis to make known tetracyclic 5-methoxyin-
dole 8 (Scheme 1).15 All attempts to silylate the N-methy-
lated indole proved unsuccessful. This was a surprising
result that we attribute to the reported capricious nature of
N-alkyl-5-methoxyindoles.16 We circumvented this pro-
blem by double deprotonation of the free NH-indole to
allow for benzylic silylation. By quenching the reaction
with methyliodide we alkylated the deprotonated nitrogen
atom to give 9 directly. The use of a tert-butyldimethylsilyl
Figure 1. (A) Fluorogenic BARAC becomes fluorescent upon
reaction with an azide to form a triazole. (B) Retrosynthesis of a
coumarin-fused BARAC.
Here we describe the synthesis and photophysical char-
acterization of a fluorogenic cyclooctyne reagent compris-
ing previously reported biarylazacyclooctynone (named
BARAC)5 fused to a coumarin dye (1, Figure 1A). The
parent compound BARAC reacts more rapidly with azides
than any other reported cyclooctyne. We postulated that
its modular synthesis using mild reactions would enable
the incorporation of a fluorogenic moiety. Coumarin was
an obvious first choice based on precedent from Zhou and
Fahrni, who demonstrated that functionalization of the
coumarin scaffold with a terminal alkyne can suppress fluo-
rescence relative to the triazole-functionalized congener.9b
Their observation is consistent with the general consensus
that an electron-donating functionality at the position
meta to the oxygen atom in the lactone ring augments
coumarin’s fluorescence quantum yield and that triazoles
are electron-rich.11 Additionally, coumarins are highly
modular fluorophores and by simply changing the electro-
nics of the functionality appended to the unsaturated
lactone (R1 and R2, 1, Figure 1A) their brightness and
€
(12) Schiedel, M.-S.; Briehn, C. A.; Bauerle, P. J. Organomet. Chem.
2002, 653, 200–208.
(13) We found that the phenyl ring at the coumarin 4-position
facilitated the synthesis because of its lack of acidic protons.
(14) The 4-phenylcoumarin moiety has shown success in other
fluorogenic applications, albeit with a different turn-on mechanism.
See: Do, J. H.; Kim, H. N.; Yoon, J.; Kim, J. S.; Kim, H.-J. Org. Lett.
2010, 12, 932–934.
(9) For a recent review of fluorogenic click reactions, see: (a) Le
Droumaguet, C.; Wang, C.; Wang, Q. Chem. Soc. Rev. 2010, 39, 1233–
1239. Also see:(b) Zhou, Z.; Fahrni, C. J. J. Am. Chem. Soc. 2004, 126,
8862–8863.
(10) Mbua, N. E.; Guo, J.; Wolfert, M. A.; Steet, R.; Boons, G.-J.
ChemBioChem 2011, 12, 1912–1921.
(11) Kleinpeter, E.; Wilde, H.; Hauptmann, S. Magn. Reson. Chem.
(15) Brown, D. W.; Graupner, P. R.; Sainsbury, M.; Shertzer, H. G.
Tetrahedron 1991, 47, 4383–4408.
1986, 24, 53–54.
(16) Sundberg, R. J.; Parton, R. L. J. Org. Chem. 1976, 41, 163–165.
5938
Org. Lett., Vol. 13, No. 22, 2011

group was advantageous for stability during subsequent
reactions and purification schemes.
After trying many traditional procedures for functiona-
lization at the 6-position of both the 5-methoxy and
5-hydroxyindoles, we found that most conditions would
leave the starting material untouched, or at times remove
the silyl group. FriedelÀCrafts acylation with acid chlor-
ides under forcing conditions was possible, but only with
manydeleterioussidereactions. Withthe5-hydroxyindole,
we observed products arising from reaction at the undesir-
able C4 position. Ultimately, we observed that methox-
yindole 9 would condense with ethyl benzoyl acetate in the
presence of triflic acid in dichloromethane. These nontra-
ditional conditions were expected to furnish an R,β un-
saturated ester with the C5-methoxy group remaining
intact,17 but at a slow rate, they provided the highly
fluorescent desired coumarin, 10, with few side products.
Oxidative cleavage of the indole C2ÀC3 bond in 10 with
m-CPBA afforded keto-amide 11, which was only weakly
fluorescent using standard hand-held UV-lamps set at
365 nm. This observation provided a qualitative indica-
tion that electronic perturbations at this position of the
substituted coumarin would affect fluorescent output.
Keto-amide 11 was taken on to silylenol triflate 12
using KHMDS and triflic anhydride. Consistent with a
previous account of strained alkyne formation from TBS-
enoltriflates,18 12 was impervious to cesium fluoride and
required TBAF to afford coumBARAC (3).
Figure 2. CoumBARAC is fluorogenic. (A) CoumBARAC
(blue) reacts with 2-azidoethanol to form a mixture of triazole
products (one shown in red). (B) Absorbance and emission
spectra of 22 μM solutions coumBARAC (blue, λex = 348
nm) and triazoles (red, λex = 305 nm) in a solution of 1%
DMSO in pH 7.4 phosphate buffer solution.
We were encouraged by the observation that coumBAR-
AC (3) is only weakly fluorescent (Figure 2B). However,
conversion of 3 to triazole 14 and its regioisomer (Figure 2A)
by treatment with 2-azidoethanol19 was accompanied by a
strong increase in fluorescence with a large Stokes shift into
a standard range for coumarin emission (λem = 438 nm,
Figure 2B). To quantitate the magnitude of this “turn-on”
effect, we measured the fluorescence quantum yields of
coumBARAC 3 (Φf ∼ 0.003) and its triazole products 14
(Φf ∼ 0.04).20 The observed ∼10-fold increase exhibited by
the triazoles versus coumBARAC is consistent with the
magnitude of turn-on observed by Zhou and Fahrni.9b
These results establish a platform for the design of
fluorogenic cyclooctynes, but improvements are war-
ranted for applications in biological imaging. First, the
calculated fluorescence quantum yield for triazoles 14
is quite low relative to standard fluorophores used in
microscopy.21 We speculate that the low quantum yield
reflects the inability of the aryl-triazole bond to freely
rotate and optimize orbital overlap with the coumarin
moiety. In addition, the optimal excitation wavelength
for the triazoles is relatively high energy (∼300 nm), and
their excitation profile was sharp and only slightly over-
lapping with excitation lasers commonly used for imaging
biological samples.22 Notably, the modular synthesis we
report here should be useful for further functionalization
of the coumarin scaffold toward achieving more conveni-
ent excitation (and emission) wavelengths. More broadly,
this work provides a starting point en route to a new class
of fluorogenic cyclooctynes for copper-free click chemistry.
Acknowledgment. We thank E. Sletten (UC Berkeley),
K. Beatty (UC Berkeley), K. Dehnert (UC Berkeley), B.
Beahm (UC Berkeley), and H. Aaron (UC Berkeley,
imaging facility) for fruitful discussions and A. Lambert
(UC Berkeley, C. Chang Laboratory) for technical assis-
tance with fluorimetric measurements. J.C.J was sup-
ported by a postdoctoral fellowship from the American
Cancer Society. This work was supported by a grant to C.
R.B. from the National Institutes of Health (GM58867).
(17) Plazuk, D.; Zakrzewski, J. J. Org. Chem. 2002, 67, 8672–8674.
~
ꢀ
ꢀ
(18) Pena, D.; Cobas, A.; Perez, D.; Guitian, E. Synthesis 2002, 10,
1454–1458.
(19) The rate of reaction of coumBARAC with benzyl azide was
comparable to that previously reported for the parent compound
BARAC (ref 6d). In work not yet published, we found that the addition
of electron-withdrawing or -donating substituents to BARAC’s aryl
rings has only a minor effect on reactivity.
(20) We considered the possibility that the two regioisomeric tria-
zoles formed by reaction of coumBARAC with 2-azidoethanol have
different photophysical properties. To address this, we separated the
∼1:1 mixture by column chromatography and analyzed their spectral
properties independently. The triazoles had identical excitation/emis-
sion profiles, making the selective formation of one over the other
irrelevant in the context of improving fluorescent properties.
Supporting Information Available. Detailed experimen-
tal details pertaining to the synthesis and characterization
of compounds 8À12 and 3. This material is available free
of charge via the Internet at http://pubs.acs.org.
(21) Thequantumyieldwasmeasuredusingquininesulfateasastandard.
(22) The DAPI filter set is commonly the highest energy light used to
excite biological samples, and the excitation is somewhat narrowly
centered at 350 nm.
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