Second-generation difluorinated cyclooctynes for copper-free click chemistry

Article abstract of DOI:10.1021/ja803086r

The 1,3-dipolar cycloaddition of azides and activated alkynes has been used for site-selective labeling of biomolecules in vitro and in vivo. While copper catalysis has been widely employed to activate terminal alkynes for [3 + 2] cycloaddition, this method, often termed click chemistry , is currently incompatible with living systems because of the toxicity of the metal. We recently reported a difluorinated cyclooctyne (DIFO) reagent that rapidly reacts with azides in living cells without the need for copper catalysis. Here we report a novel class of DIFO reagents for copper-free click chemistry that are considerably more synthetically tractable. The new analogues maintained the same elevated rates of [3 + 2] cycloaddition as the parent compound and were used for imaging glycans on live cells. These second-generation DIFO reagents should expand the use of copper-free click chemistry in the hands of biologists. ? 2008 American Chemical Society.

Full text of DOI:10.1021/ja803086r

Published on Web 08/05/2008
Second-Generation Difluorinated Cyclooctynes for
Copper-Free Click Chemistry
Julian A. Codelli,† Jeremy M. Baskin,† Nicholas J. Agard,† and
Carolyn R. Bertozzi*,†,‡,§,|
Departments of Chemistry and Molecular and Cell Biology and Howard Hughes Medical
Institute, UniVersity of California, Berkeley, California 94720, and The Molecular Foundry,
Lawrence Berkeley National Laboratory, Berkeley, California 94720
Received May 1, 2008; E-mail: crb@berkeley.edu
Abstract: The 1,3-dipolar cycloaddition of azides and activated alkynes has been used for site-selective labeling
of biomolecules in vitro and in vivo. While copper catalysis has been widely employed to activate terminal alkynes
for [3 + 2] cycloaddition, this method, often termed “click chemistry”, is currently incompatible with living systems
because of the toxicity of the metal. We recently reported a difluorinated cyclooctyne (DIFO) reagent that rapidly
reacts with azides in living cells without the need for copper catalysis. Here we report a novel class of DIFO
reagents for copper-free click chemistry that are considerably more synthetically tractable. The new analogues
maintained the same elevated rates of [3 + 2] cycloaddition as the parent compound and were used for imaging
glycans on live cells. These second-generation DIFO reagents should expand the use of copper-free click
chemistry in the hands of biologists.
to produce the 1,5-disubstituted isomer.6 These metal-catalyzed
Introduction
variants of the Huisgen [3 + 2] cycloaddition are now central
tools for combinatorial library synthesis,7 construction of
peptidomimetics and glycomimetics,8 supramolecular synthesis,9
and labeling of biomolecules in vitro or in fixed samples.10-14
However, the reliance of these chemistries on cytotoxic transi-
tion metals has largely precluded their use for applications in
vivo, despite parallel growth in the use of azides as chemical
reporters in biological systems.15
The term “click chemistry” describes a collection of organic
reactions that proceed rapidly and selectively under mild
conditions to covalently link molecular components.1 Among
the many click reactions described to date, the Huisgen 1,3-
dipolar cycloaddition of azides and alkynes2 has received the
most attention. The reaction is highly exergonic (∆G° ≈ -61
kcal/mol),3 the starting materials are easily prepared, the 1,2,3-
triazole products are exceptionally stable, and the reaction occurs
readily in both organic and aqueous solvents. However, elevated
temperatures or pressures are necessary to accelerate the reaction
when simple alkynes and azides are employed, since the
activation energy for the cycloaddition is high (∆Gq ≈ +26
kcal/mol).3
As an alternative approach to lower the activation barrier for
[3 + 2] cycloaddition, we have employed the intrinsic ring strain
of cyclooctynes, following the precedent of Wittig and Krebs.16,17
These highly strained alkynes (18 kcal/mol of ring strain18) react
selectively with azides to form regioisomeric mixtures of
In the past decade, several strategies have been pursued in
order to lower the activation barrier. Sharpless and co-workers4
and Meldal and co-workers5 first reported that Cu(I) catalysis
dramatically accelerates the reaction of terminal alkynes and
azides, regioselectively forming the 1,4-disubstituted triazoles.
More recently, ruthenium-based catalysts have been employed
(6) Zhang, L.; Chen, X.; Xue, P.; Sun, H. H. Y.; Williams, I. D.; Sharpless,
K. B.; Fokin, V. V.; Jia, G. J. Am. Chem. Soc. 2005, 127, 15998–
15999.
(7) Kolb, H. C.; Sharpless, K. B. Drug DiscoVery Today 2003, 8, 1128–
1137.
(8) Angell, Y. L.; Burgess, K. Chem. Soc. ReV. 2007, 36, 1674–1689.
(9) Lutz, J. F. Angew. Chem., Int. Ed. 2007, 46, 1018–1025.
(10) Wang, Q.; Chan, T. R.; Hilgraf, R.; Fokin, V. V.; Sharpless, K. B.;
Finn, M. G. J. Am. Chem. Soc. 2003, 125, 3192–3193.
(11) Speers, A. E.; Cravatt, B. F. Chem. Biol. 2004, 11, 535–546.
(12) Hsu, T.-L.; Hanson, S. R.; Kishikawa, K.; Wang, S.-K.; Sawa, M.;
Wong, C.-H. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 2614–2619.
(13) Salic, A.; Mitchison, T. J. Proc. Natl. Acad. Sci. U.S.A. 2008, 105,
2415–2420.
† Department of Chemistry, University of California.
‡ Department of Molecular and Cell Biology, University of California.
§ Howard Hughes Medical Institute, University of California.
| Lawrence Berkeley National Laboratory.
(1) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed.
2001, 40, 2004–2021.
(14) van Kasteren, S. I.; Kramer, H. B.; Jensen, H. H.; Campbell, S. J.;
Kirkpatrick, J.; Oldham, N. J.; Anthony, D. C.; Davis, B. G. Nature
2007, 446, 1105–1109.
(2) Huisgen, R. Angew. Chem. 1963, 75, 604–637.
(3) Himo, F.; Lovell, T.; Hilgraf, R.; Rostovtsev, V. V.; Noodleman, L.;
Sharpless, K. B.; Fokin, V. V. J. Am. Chem. Soc. 2005, 127, 210–
216.
(15) Prescher, J. A.; Bertozzi, C. R. Nat. Chem. Biol. 2005, 1, 13–21.
(16) Agard, N. J.; Prescher, J. A.; Bertozzi, C. R. J. Am. Chem. Soc. 2004,
126, 15046–15047.
(4) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew.
Chem., Int. Ed. 2002, 41, 2596–2599.
(17) Wittig, G.; Krebs, A. Chem. Ber. 1961, 94, 3260.
(18) Turner, R. B.; Jarrett, A. D.; Goebel, P.; Mallon, B. J. J. Am. Chem.
Soc. 1973, 95, 790–792.
(5) Tornoe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67,
3057–3064.
9
11486 J. AM. CHEM. SOC. 2008, 130, 11486–11493
10.1021/ja803086r CCC: $40.75
2008 American Chemical Society

Difluorinated Cyclooctynes for Cu-Free Click Chemistry
A R T I C L E S
without further purification, unless otherwise noted. All of the
reactions were performed in a N2 atmosphere, unless otherwise
noted. Liquid reagents were added with a syringe, unless otherwise
noted. THF and CH2Cl2 were dried in vacuo over alumina. Flash
chromatography was carried out with Merck 60 230-400 mesh
silica gel according to the procedure described by Still et al.24 When
necessary, deactivated silica gel was prepared by rinsing silica gel
thoroughly with a solution of 1% Et3N in the starting solvent
mixture used for flash chromatography. Reactions and chromatog-
raphy fractions were analyzed with Analtech 250 µm silica gel G
plates and visualized by staining with ceric ammonium molybdate,
anisaldehyde, vanillin, or 2,4-dinitrophenylhydrazine or by absor-
bance of UV light at 245 nm. Solvents were removed using a rotary
evaporator at reduced pressure (20 torr). Unless otherwise noted,
1H, 13C{1H}, 19F, and 31P{1H} NMR spectra were obtained with
300, 400, or 500 MHz Bruker spectrometers. Chemical shifts (δ)
are reported in parts per million referenced to the solvent peaks
for 1H and 13C, to CFCl3 for 19F, and to H3PO4 for 31P. Coupling
constants (J) are reported in hertz. Low-resolution and high-
resolution (HR) fast atom bombardment (FAB), electron impact
(EI), and electrospray ionization (ESI) mass spectra were obtained
at the UC Berkeley Mass Spectrometry Facility. Elemental analyses
were obtained at the UC Berkeley Micro-Mass Facility. Reversed-
phase HPLC was performed using a Rainin Dynamax SD-200
HPLC system with 210 nm detection on a Microsorb C18 analytical
or preparative column.
Annexin V-PE was purchased from BD Biosciences. Dulbecco’s
phosphate-buffered saline (PBS) and fetal bovine serum (FBS) were
purchased from HyClone Laboratory, and RPMI-1640 and F12
media were obtained from Invitrogen Life Technologies, Inc.
Fluorescein isothiocyanate (FITC)-labeled avidin was purchased
from Sigma-Aldrich. Flow cytometry analysis was performed on a
BD FACSCalibur flow cytometer using a 488 nm argon laser. At
least 2 × 104 cells were analyzed for each sample. Cell viability
was ascertained on the basis of forward scatter (to sort by size)
and side scatter (to sort by granularity). The average fluorescence
intensity was calculated from each of three replicate experiments
in order to obtain a representative value in arbitrary units. For all
of the flow cytometry experiments, data points were collected in
triplicate and represent at least two separate experiments. Fluores-
cence microscopy was performed on a Zeiss 200 M epifluorescence
microscope, and the images were deconvolved using the nearest-
neighbor algorithm of Slidebook 4.2 (Intelligent Imaging Innova-
tions, Inc.).
Figure 1. Panel of difluorinated cyclooctyne reagents (1-3) for copper-
free click chemistry.
triazoles at ambient temperatures and pressures without the need
for metal catalysis and with no apparent cytotoxicity (eq 1):
We further enhanced the cycloaddition rate by installing
propargylic fluorine atoms intended to lower the LUMO, thereby
increasing its interaction energy with the HOMO of the azide,19
an effect that was recently studied using density functional
theory calculations.20 The difluorinated cyclooctyne we termed
“DIFO” (compound 1 in Figure 1) reacted with azides on intact
proteins at a rate comparable to that of Cu-catalyzed click
chemistry and has since been employed for dynamic imaging
of cell-surface glycans in live cells19 and in developing zebrafish
embryos.21 Other groups have reported the use of strained
oxanorbornadiene22 and dibenzocyclooctyne23 reagents as sub-
strates for [3 + 2] cycloaddition with azides, suggesting a rich
future for Cu-free click chemistry.
Still, the canonical Cu-catalyzed reaction has the advantage
of synthetic convenience. Terminal alkynes can be installed in
biomolecules using simple building blocks, such as commercial
alkynoic acids. In contrast, the synthesis of DIFO comprised
12 steps and an overall yield of ∼1%.19 The final step of the
sequence, elimination of a vinyl triflate to form the cyclooctyne,
suffered from significant decomposition and low yield. Thus,
synthetically tractable cyclooctynes with the capabilities of
DIFO are needed in order to expand the use of this Cu-free
click reagent in biological settings.
Here we report the design, synthesis, and biological evaluation
of the second-generation DIFO reagents 2 and 3 (Figure 1),
which retain the difluorinated cyclooctyne core but possess a
C-C bond to a linker substituent at C4 rather than a C-O bond
at C6 as in 1 (Figure 1). This structural change dramatically
simplified the synthesis without impact on the kinetics or
bioorthogonality of [3 + 2] cycloaddition with azides. These
synthetically tractable second-generation DIFO reagents should
facilitate further applications of Cu-free click chemistry.
Synthesis of New Compounds. 2,2-Difluoro-1,3-cyclooctanedi-
one (4). A flame-dried round-bottom flask was charged with 1,3-
cyclooctanedione (5.10 g, 36.4 mmol) and MeCN (260 mL).
Cs2CO3 (24.3 g, 74.6 mmol) was added, and the reaction mixture
was stirred at room temperature (rt) for 30 min. The reaction mixture
was cooled to 0 °C, and Selectfluor (31.0 g, 87.4 mmol) was added,
after which the mixture was stirred for an additional 15 min. The
system was allowed to warm to rt, stirred for 1.5 h, concentrated
under reduced pressure, diluted with 1 M HCl (200 mL), and
extracted with diethyl ether (4 × 200 mL). The combined organic
layers were washed with brine (2 × 100 mL), dried over MgSO4,
and filtered through a glass frit. The solution was concentrated under
reduced pressure and purified by flash chromatography (9:1 hexanes/
EtOAc) to yield a white solid (4.70 g, 73%). Rf ) 0.40 (4:1 hexanes/
Experimental Section
General Materials and Methods. All of the chemical reagents
were analytical grade, obtained from commercial suppliers, and used
1
EtOAc); mp 42.7-43.7 °C. H NMR (500 MHz, CDCl3): δ 2.67
(19) 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.
(m, 4H), 1.81 (m, 4H), 1.63 (m, 2H). 13C NMR (125 MHz, CDCl3):
δ 197.9 (t, J ) 25.1 Hz), 109.5 (t, J ) 261.5 Hz), 38.7, 26.2, 24.7.
19F NMR (376 MHz, CDCl3): δ -118.35 (s). IR (thin film, cm-1):
3468, 2946, 2867, 1732. HRMS (EI+): calcd for C8H10O2F2,
176.0649; found, 176.0646.
(20) Ess, D. H.; Jones, G. O.; Houk, K. N. Org. Lett. 2008, 10, 1633–
1636.
(21) Laughlin, S. T.; Baskin, J. M.; Amacher, S. L.; Bertozzi, C. R. Science
2008, 320, 664–667.
Compound 6. To a flame-dried round-bottom flask were added
diketone 4 (1.57 g, 8.92 mmol), phosphonium bromide 5 (4.60
(22) van Berkel, S. S.; Dirks, A. 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, 1504–1508.
(23) Ning, X.; Guo, J.; Wolfert, M. A.; Boons, G.-J. Angew. Chem., Int.
Ed. 2008, 47, 2253–2255.
(24) Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43, 2923–
2925.
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A R T I C L E S
Codelli et al.
g, 9.37 mmol), and THF (200 mL). The system was cooled to 0
°C, and DBU (1.34 mL, 8.92 mmol) was added, after which the
reaction mixture was stirred for 20 min at 0 °C. After the reaction
mixture was warmed to rt, it was stirred for an additional 48 h;
the reaction was quenched with AcOH (1.5 mL), and the mixture
was diluted with MeOH (20 mL), concentrated under reduced
pressure, and purified by flash chromatography (0-5% EtOAc
in a 2:1 mixture of hexanes/toluene) to yield a white solid (2.63
g, 96%). Rf ) 0.55 (4:1 hexanes/EtOAc); mp 58.8-61.1 °C. 1H
NMR (400 MHz, CDCl3): δ 8.05 (d, 2H, J ) 8.4 Hz), 7.38 (d,
2H, J ) 8.2 Hz), 7.23 (s, 1H), 3.92 (s, 3H), 2.70 (t, 2H, J ) 6.6
Hz), 2.52 (app t, 2H, J ) 6.2 Hz), 1.86 (m, 2H), 1.53 (m, 4H).
13C NMR (125 MHz, CDCl3): δ 202.1 (t, J ) 28.9 Hz), 166.8,
140.0, 134.6 (t, J ) 19.6 Hz), 131.2 (t, J ) 10.3 Hz), 130.0,
129.7, 129.0, 115.2 (t, J ) 253.4 Hz), 52.4, 37.5, 27.3, 26.0,
25.7, 25.3 (t, J ) 2.5 Hz). 19F NMR (376 MHz, CDCl3): δ
-111.09 (s). HRMS (FAB): calcd for C17H18O3F2 [M + H]+,
309.1302; found, 309.1302.
Compound 7. To a round-bottom flask were added olefin 6 (2.63
g, 8.53 mmol) and MeOH (100 mL). The system was flushed with
N2, and a catalytic amount of Pd/C was added. The system was
again flushed with N2 followed by H2, and the reaction mixture
was stirred under an H2 atmosphere (using a balloon) for 24 h.
The system was then flushed thoroughly with N2, after which the
reaction mixture was diluted with CH2Cl2 (100 mL), filtered through
Celite, and concentrated under reduced pressure. The crude product
was purified by flash chromatography (0-2% EtOAc in a 2:1
mixture of hexanes/toluene) to yield a white solid (2.47 g, 93%).
Rf ) 0.60 (4:1 hexanes/EtOAc); mp 70.1-71.4 °C. 1H NMR (400
MHz, CDCl3): δ 7.99 (dm, 2H, J ) 8.3 Hz), 7.27 (d, 2H, J ) 8.14
Hz), 3.92 (s, 3H), 3.31 (dd, 1H, J ) 13.6, 2.9 Hz), 2.82-2.73 (m,
1H), 2.66-2.48 (m, 2H), 2.45-2.28 (m, 1H), 2.17-2.02 (m, 1H),
1.97-1.84 (m, 1H), 1.66-1.42 (m, 4H), 1.41-1.29 (m, 1H),
1.29-1.11 (m, 1H). 13C NMR (125 MHz, CDCl3): δ 205.7 (dd, J
) 30.4, 25.1 Hz), 167.1, 144.7, 130.0, 129.4, 128.6, 119.4 (dd, J
) 258.0, 250.7 Hz), 52.2, 46.4 (t, J ) 21.6), 39.1, 33.8 (t, J ) 4.8
Hz), 27.2, 24.3 (d, J ) 6.9 Hz), 24.1 (d, J ) 3.4 Hz), 22.9. 19F
NMR (376 MHz, CDCl3): δ -102.72 (d, 1F, J ) 245.5 Hz),
-122.67 (d, 1F, J ) 251.7 Hz). HRMS (FAB): calcd for
C17H20O3F2 [M + H]+, 311.1459; found, 311.1467.
dropwise to a solution of diisopropylamine (3.59 mL, 25.4 mmol)
in THF (93.8 mL) while stirring at -78 °C. A portion of the LDA
solution (37.4 mL, 7.44 mmol) was added dropwise to the first
mixture over 1 h using a syringe pump at -20 °C. The reaction
mixture was then brought to rt over 20 min, and the reaction was
quenched with MeOH (10 mL); the mixture was then concentrated
under reduced pressure and purified by flash chromatography
(0-3% EtOAc in 2:1 hexanes/toluene) to yield a white solid (1.58
1
g, 87%). Rf ) 0.50 (1:2 hexanes/toluene); mp 78.5-82.7 °C. H
NMR (400 MHz, CDCl3): δ 7.99 (d, 2H, J ) 8.1 Hz), 7.27 (d, 2H,
J ) 7.9 Hz), 3.92 (s, 3H), 3.16 (app d, 1H, J ) 11.2 Hz), 2.60-2.43
(m, 2H), 2.41-2.24 (m, 2H), 2.10-1.88 (m, 2H), 1.87-1.68 (m,
2H), 1.62-1.44 (m, 1H), 1.21-1.08 (m, 1H). 13C NMR (125 MHz,
CDCl3): δ 167.2, 145.5, 130.0, 129.4, 128.4, 119.5 (t, J ) 238.6
Hz), 109.9 (t, J ) 11.1 Hz), 85.1 (dd, J ) 47.2, 41.6 Hz), 58.2 (t,
J ) 24.3 Hz), 52.2, 34.5 (d, J ) 4.7 Hz), 32.6, 30.8 (d, J ) 4.4
Hz), 28.0, 20.4. 19F NMR (376 MHz, CDCl3): δ -94.32 (d, 1F, J
) 260.2 Hz), -101.36 (dm, 1F, J ) 259.8 Hz). HRMS (FAB):
calcd for C17H18O2F2 [M + H]+, 293.1353; found, 293.1357.
Compound 2. To a round-bottom flask fitted with a reflux
condenser were added cyclooctyne methyl ester 9 (1.58 g, 5.42
mmol), LiOH (2.59 g, 108 mmol), water (6 mL), and dioxane (24
mL) under an air atmosphere. The reaction mixture was heated to
55 °C and stirred for 3 h. The mixture was then cooled to rt and
diluted with 1 M HCl until the pH of the solution was <2. The
solution was extracted with CH2Cl2 (4 × 50 mL). The combined
organic layers were then washed (1:1 1 M HCl/brine, 50 mL), dried
over MgSO4, filtered through a glass frit, and concentrated under
reduced pressure. The crude product was then purified by flash
chromatography (9:1 to 3:1 hexanes/EtOAc with 1-2% AcOH) to
yield a white solid (1.08 g, 72%). Rf ) 0.45 (4:1 hexanes/EtOAc
with 1% AcOH); mp 181.0-182.0 (dec). 1H NMR (400 MHz,
CD3CN): δ 10.10-8.80 (br s, 1H), 7.94 (d, 2H, J ) 8.2 Hz), 7.35
(d, 2H, J ) 8.1 Hz), 3.10 (d, 1H, J ) 10.9 Hz), 2.70-2.50 (m,
2H), 2.43-2.24 (m, 2H), 2.04-1.84 (m, 2H) 1.83-1.67 (m, 2H),
1.55-1.41 (m, 1H), 1.22-1.11 (m, 1H). 13C NMR (125 MHz,
acetone-d6): δ 167.6, 146.2, 130.8, 130.2, 129.6, 120.2 (dd, J )
239.1, 235.4 Hz), 111.6 (app t, J ) 11.3 Hz), 85.5 (dd, J ) 46.7,
41.9 Hz), 58.6 (t, J ) 24.2 Hz), 34.9 (d, J ) 4.9 Hz), 33.3 (d, J )
2.1 Hz), 31.6 (d, J ) 4.7 Hz), 28.4, 20.5. 19F NMR (376 MHz,
CD3CN): δ -93.81 (d, 1F, J ) 258.7 Hz), -101.32 (ddt, 1F, J )
258.8, 20.3, 7.0 Hz). HRMS (FAB): calcd for C16H16O2F2 [M +
H]+, 279.1197; found, 279.1190.
Compound 10. To a flame-dried round-bottom flask were added
DCC (3.66 g, 17.7 mmol), DMAP (98.6 mg, 0.807 mmol),
3-methyl-3-oxetanemethanol (1.59 mL, 16.1 mmol), and CH2Cl2
(20 mL). The mixture was cooled to 0 °C with stirring. A solution
of iodoacetic acid (3.00 g, 16.1 mmol) in CH2Cl2 (30 mL) was
then added via syringe. The reaction mixture was stirred for 1 h at
0 °C and allowed to warm to rt over an additional 1.5 h; the reaction
was quenched with acetic acid (1 mL), and the mixture was stirred
for an additional 30 min. The mixture was then diluted with CH2Cl2
and filtered through Celite. The filtrate (∼300 mL total) was then
washed with water (200 mL), saturated NaHCO3 (2 × 200 mL),
and brine (200 mL). The organic layer was then dried over MgSO4,
filtered through a glass frit, and concentrated under reduced
pressure. The crude product was diluted with CH2Cl2 and again
filtered through Celite to remove any residual dicyclohexylurea that
had precipitated. The filtrate was again concentrated to yield a
yellow oil. This material was then transferred to a new round-bottom
flask and dissolved in THF (100 mL). Triphenylphosphine (4.65
g, 17.7 mmol) was added, and the reaction mixture was stirred under
N2 at rt for 40 h and then diluted with diethyl ether (100 mL) and
filtered through a glass frit to isolate the precipitated product.
Residual solvent was removed under reduced pressure to yield a
pale-yellow solid (7.93 g, 92% over two steps). Mp 152.8-154.6
°C. 1H NMR (400 MHz, CDCl3): δ 7.93-7.84 (m, 6H), 7.84-7.78
(m, 3H), 7.73-7.65 (m, 6H), 5.51 (d, 2H, J ) 13.5 Hz) 4.26 (s,
4H), 4.15 (s, 2H), 1.22 (s, 3H). 13C NMR (125 MHz, CDCl3): δ
Compound 8. To a flame-dried round-bottom flask was added
THF (175 mL) followed by KHMDS (14.81 mL of a 0.5 M solution
in toluene, 7.40 mmol). The reaction mixture was cooled to -78
°C with stirring, and ketone 7 (2.19 g, 7.05 mmol) in THF (70
mL) was added dropwise over 20 min. The reaction mixture was
stirred for an additional 40 min, and then a solution of Tf2NPh
(2.65 g, 7.40 mmol) in THF (70 mL) was added via syringe. The
system was warmed to rt with stirring over 21 h, and the reaction
was then quenched with MeOH (10 mL). The reaction mixture was
concentrated under reduced pressure and purified by flash chro-
matography (0-5% EtOAc in 4:1 hexanes/toluene with 1% Et3N)
to yield a pale-yellow oil (2.74 g, 88%). Rf ) 0.60 (4:1 hexanes/
1
EtOAc). H NMR (400 MHz, CD3CN): δ 7.93 (d, 2H, J ) 8.2
Hz), 7.36 (d, 2H, J ) 8.2 Hz), 6.25 (t, 1H, J ) 9.4 Hz), 3.85 (s,
3H), 3.24 (dd, 1H, J ) 13.5, 3.8 Hz), 2.88-2.70 (m, 1H), 2.61
(dd, 1H, J ) 13.5, 10.2 Hz), 2.56-2.42 (m, 1H), 2.41-2.30 (m,
1H), 1.68-1.52 (m, 3H), 1.51-1.44 (m, 2H), 1.43-1.34 (m, 1H).
13C NMR (125 MHz, CD3CN): δ 167.6, 145.8, 143.6 (t, J ) 29.4
Hz), 130.5, 130.4, 129.5, 129.1 (t, J ) 4.0 Hz), 120.5 (dd, J )
246.2, 243.1 Hz), 119.5 (q, J ) 319.1 Hz), 52.7, 46.8 (app t, J )
22.0 Hz), 35.2 (app d, J ) 5.4 Hz), 27.0, 26.2, 23.0, 21.8. 19F
NMR (376 MHz, CD3CN): δ -74.45 (s, 3F), -93.93 (d, 1F, J )
272.2 Hz), -105.18 (d, 1F, J ) 266.9 Hz). HRMS (FAB): calcd
for C18H19O5F5S [M + H]+, 443.0952; found, 443.0960.
Compound 9. To a flame-dried round-bottom flask was added
vinyl triflate 8 (2.74 g, 6.20 mmol) in THF (160 mL). The mixture
was cooled to -20 °C with stirring. In a separate flame-dried round-
bottom flask, a 0.199 M solution of LDA was made by adding
n-butyllithium (8.46 mL of a 2.5 M solution in hexanes, 21.2 mmol)
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11488 J. AM. CHEM. SOC. VOL. 130, NO. 34, 2008

Difluorinated Cyclooctynes for Cu-Free Click Chemistry
A R T I C L E S
164.3 (d, J ) 3.5 Hz), 135.4 (d, J ) 3.1 Hz), 133.9 (d, J ) 10.8
Hz), 130.4 (d, J ) 13.2 Hz), 117.4 (d, J ) 89.2 Hz), 79.1, 71.1,
38.8, 33.6 (d, J ) 56.6 Hz), 21.0. 31P NMR (162 MHz, CDCl3): δ
20.41 (s). HRMS (ESI+): calcd for C25H26O3P, 405.1620; found,
405.1631.
NMR (400 MHz, CDCl3): δ 6.05 (t, 1H, J ) 9.6 Hz), 3.89 (s, 6H),
2.74-2.56 (m, 1H), 2.51-2.31 (m, 2H), 2.20 (dd, 1H, J ) 14.5,
1.7 Hz), 2.00-1.87 (m, 1H), 1.72-1.49 (m, 6H), 0.80 (s, 3H). 13
C
NMR (125 MHz, CDCl3) δ 143.2 (t, J ) 30.2 Hz), 126.9, 119.2
(app t, J ) 246.5 Hz), 118.6 (q, J ) 320.0 Hz), 108.9, 72.8, 40.9
(app t, J ) 22.3 Hz), 35.1, 30.5, 27.2, 26.6, 22.6, 21.8, 14.6. 19F
NMR (376 MHz, CDCl3): δ -74.52 (s, 3F), -93.80 (d, 1F, J )
269.8 Hz), -104.75 (dm, 1F, J ) 278.0 Hz). HRMS (FAB): calcd
for C16H21O6F5S [M + H]+, 437.1057; found, 437.1050.
Compound 11. To a flame-dried round-bottom flask were added
phosphonium iodide 10 (5.66 g, 10.6 mmol), CH2Cl2 (75 mL), and
DBU (1.51 mL, 10.1 mmol), and the reaction mixture was stirred
for 20 min. In a separate flame-dried round-bottom flask, difluo-
roketone 4 (1.78 g, 10.1 mmol) was dissolved in CH2Cl2 (125 mL),
and this solution was then added to the phosphonium iodide
solution. The reaction mixture was allowed to stir for 48 h,
concentrated under reduced pressure, and purified by flash chro-
matography (10:1 to 4:1 hexanes/EtOAc), yielding a white solid
(2.87 g, 94%). Rf ) 0.50 (2:1 hexanes/EtOAc); mp 44.2-46.0 °C.
1H NMR (500 MHz, CDCl3): δ 6.49 (s, 1H), 4.51 (d, 2H, J ) 6.0
Hz), 4.40 (d, 2H, J ) 6.0 Hz), 4.25 (s, 2H), 2.81 (t, 2H, J ) 6.6
Hz), 2.66 (t, 2H, J ) 6.7 Hz), 1.82 (m, 2H), 1.72 (m, 2H), 1.52
(m, 2H), 1.35 (s, 3H). 13C NMR (125 MHz, CDCl3): δ 200.2 (t, J
) 28.1 Hz), 165.1, 150.4 (t, J ) 20.0 Hz), 121.1 (t, J ) 9.6 Hz),
114.2 (t, J ) 254.6 Hz), 79.7, 69.3, 39.2, 37.5, 26.7, 26.6, 26.0,
25.5, 21.3. 19F NMR (376 MHz, CD3CN): δ -113.03 (s). HRMS
(FAB): calcd for C15H20O4F2 [M + H]+, 303.1408; found,
303.1404.
Compound 12. To a round-bottom flask were added compound
11 (804 mg, 2.66 mmol) and MeOH (30 mL). The system was
flushed with N2, and a catalytic amount of Pd/C was added. The
system was then flushed again with N2 followed by H2, and the
reaction mixture was stirred under an H2 atmosphere for 24 h. The
system was flushed thoroughly with N2, and the reaction mixture
was diluted with CH2Cl2 (30 mL) and filtered through Celite to
remove the catalyst. The filtrate was then concentrated under
reduced pressure. The crude material was dissolved in CH2Cl2 (17
mL) and transferred via syringe to a new flame-dried round-bottom
flask, which was under a N2 atmosphere and contained activated 4
Å molecular sieves. The mixture was then cooled to 0 °C with
stirring. In a separate flame-dried conical flask, a 0.20 M solution
of BF3 ·OEt2 (100 µL, 0.780 mmol) in CH2Cl2 (3.9 mL) was
prepared, and a portion of this solution (1.0 mL, 0.20 mmol) was
added to the reaction mixture via syringe. The reaction mixture
was warmed to rt and stirred for an additional 20 h before the
reaction was quenched with Et3N (0.5 mL). The reaction mixture
was then concentrated under reduced pressure and purified by flash
chromatography (20:1 hexanes/EtOAc with 1% Et3N over deacti-
vated silica gel) to yield a white solid (731 mg, 90% over two
steps). Rf ) 0.70 (2:1 hexanes/EtOAc); mp 80.3-83.3 °C. 1H NMR
(500 MHz, CDCl3): δ 3.89 (s, 6H), 2.65 (m, 1H), 2.62-2.46 (m,
2H), 2.21 (d, 1H, J ) 14.6 Hz), 2.10-1.94 (m, 2H), 1.90 (br s,
1H), 1.60 (m, 2H), 1.56-1.42 (m, 2H), 1.36 (m, 2H), 0.80 (s, 3H).
13C NMR (125 MHz, CDCl3): δ 205.8 (dd, J ) 29.7, 25.7 Hz),
119.3 (dd, J ) 257.6, 250.2 Hz), 109.0, 72.8, 39.2 (t, J ) 21.2
Hz), 38.9, 34.6 (t, J ) 4.7 Hz), 30.5, 27.0, 26.3 (d, J ) 7.2 Hz),
24.8 (d, J ) 2.7 Hz), 23.3, 14.7. 19F NMR (376 MHz, CD3CN): δ
-103.10 (d, 1F, J ) 246.4 Hz), -124.20 (dm, 1F, J ) 249.0 Hz).
HRMS (FAB): calcd for C15H22O4F2 [M + H]+, 305.1564; found,
305.1558.
Compound 14. In a round-bottom flask, vinyl triflate 13 (407
mg, 0.932 mmol) was dissolved in toluene (20 mL) and
concentrated under reduced pressure to remove trace moisture.
This procedure was repeated four times. The material was then
dissolved in THF (20 mL), and the solution was cooled to -20
°C with stirring. In a separate flame-dried round-bottom flask, a
0.20 M solution of LDA was made by adding n-butyllithium
(1.12 mL of a 2.5 M solution in hexanes, 2.80 mmol) dropwise
to a solution of diisopropylamine (475 µL, 3.36 mmol) in THF
(12.4 mL) at -78 °C. A portion of the LDA solution (5.6 mL,
1.12 mmol) was added dropwise via syringe pump to the first
mixture over 1 h. The reaction mixture was then brought to rt
over 30 min, and the reaction was quenched with deactivated
silica gel; the mixture was then concentrated under reduced
pressure and purified by flash chromatography (20:1 to 15:1
hexanes/EtOAc with 1% Et3N over deactivated silica gel) to yield
a white solid (159 mg, 59%). Rf ) 0.73 (2:1 hexanes/EtOAc);
mp 80.1-86.0 °C. 1H NMR (500 MHz, CDCl3): δ 3.90 (s, 6H),
2.54 (app dq, 1H, J ) 23.5, 9.1 Hz), 2.40-2.25 (m, 2H),
2.25-2.11 (m, 2H), 2.10-2.02 (m, 2H), 1.81-1.71 (m, 1H),
1.65 (dd, 1H, J ) 14.6, 10.3 Hz), 1.50 (app quint, 1H, J ) 7.5
Hz), 1.33 (m, 1H), 0.81 (s, 3H). 13C NMR (125 MHz, CDCl3):
δ 120.0 (t, J ) 238.3 Hz), 109.8 (t, J ) 11.2 Hz), 85.4 (dd,
47.1, 41.8 Hz), 72.8, 51.9 (t, J ) 23.8 Hz), 35.2 (dd, J ) 4.5,
1.6 Hz), 32.8 (d, J ) 4.8 Hz), 32.7 (d, J ) 2.1 Hz), 30.5, 29.9,
28.2, 20.5, 14.8. 19F NMR (376 MHz, CD3CN): δ -94.82 (d,
1F, J ) 258.7 Hz), -101.81 (ddt, 1F, J ) 259.3, 24.1, 7.2 Hz).
HRMS (FAB): calcd for C15H20O3F2 [M + H]+, 287.1459; found,
287.1461.
Compound 3. To a scintillation vial under an air atmosphere
were added cyclooctyne orthoester 14 (90.4 mg, 0.316 mmol),
MeOH (4.5 mL), water (450 µL), and PPTS (159 mg, 0.632
mmol). The reaction mixture was stirred at rt for 24 h, after
which the reaction was quenched with saturated NaHCO3 (2 mL)
and the mixture concentrated under reduced pressure. The crude
product was diluted with brine (8 mL) and extracted with EtOAc
(3 × 10 mL). The combined organic layers were washed with
brine (10 mL) and a HCl/brine solution (1:1 1 M HCl/brine, 2
× 10 mL), dried over MgSO4, and filtered through a glass frit.
The filtrate was concentrated under reduced pressure to yield a
white solid (106.8 mg). A portion of this material (57.8 mg)
was transferred to a round-bottom flask, where it was dissolved
in dioxane (1 mL) and water (200 µL). To this was added LiOH
(91 mg, 3.8 mmol), and the reaction mixture was stirred at rt
for 3 h under an air atmosphere, after which the reaction was
quenched with 1 M HCl (5 mL). The reaction mixture was further
diluted with brine (3 mL) and extracted with EtOAc (4 × 10
mL). The combined organic layers were washed (1:1 1 M HCl/
brine, 10 mL), dried over MgSO4, and filtered through a glass
frit. The filtrate was concentrated and purified by flash chroma-
tography (20:1 hexanes/EtOAc with 2% AcOH) to give a white
solid (33 mg, 96% over two steps). Rf ) 0.66 (1:1 hexanes/
EtOAc with 1% AcOH); mp 87.4-88.9 °C. 1H NMR (400 MHz,
CDCl3): δ 11.90-10.60 (br s, 1H), 2.84-2.69 (m, 2H),
2.46-2.28 (m, 3H), 2.21-2.05 (m, 2H), 1.90-1.74 (m, 2H),
1.67 (m, 1H), 1.41 (m, 1H). 13C NMR (125 MHz, CDCl3): δ
177.8, 119.1 (t, J ) 238.6 Hz), 110.7 (t, J ) 11.2 Hz), 84.7
(dd, J ) 47.0, 41.6 Hz), 52.6 (t, J ) 24.4 Hz), 33.7 (app d, J )
4.4 Hz), 32.9 (d, J ) 4.6 Hz), 32.7 (d, J ) 2.0 Hz), 27.9, 20.5.
19F NMR (376 MHz, CDCl3) δ -94.64 (d, 1F, J ) 260.0 Hz),
Compound 13. To a flame-dried round-bottom flask was added
THF (30 mL) followed by KHMDS (2.66 mL of a 0.5 M solution
in toluene, 1.33 mmol). The reaction mixture was cooled to -78
°C with stirring, and ketone 12 (354 mg, 1.16 mmol) in THF (15
mL) was added dropwise via syringe over 15 min. The reaction
mixture was stirred for an additional 3 h, and then a solution of
Tf2NPh (457 mg, 1.28 mmol) in THF (15 mL) was added via
syringe. The system was allowed to slowly warm to rt with stirring
over 19 h. The reaction was then quenched with deactivated silica
gel, and the mixture was concentrated under reduced pressure and
purified by flash chromatography (20:1 to 15:1 hexanes/EtOAc with
1% Et3N over deactivated silica gel) to yield a white solid (407
mg, 80%). Rf ) 0.72 (2:1 hexanes/EtOAc); mp 81.2-83.3 °C. 1H
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A R T I C L E S
Codelli et al.
-100.82 (ddt, 1F, J ) 260.2, 21.1, 6.8 Hz). HRMS (ESI-): calcd
for C10H11O2F2 [M]-, 201.0722; found, 201.0729.
33.5 (d, J ) 4.5 Hz), 30.6, 30.0, 29.7, 29.0, 27.1, 20.8, 14.6.
19F NMR (376 MHz, CD3OD): δ -95.57 (d, 1F, J ) 259.0 Hz),
-102.08 (ddt, 1F, J ) 259.4, 20.9, 6.8 Hz). HRMS (FAB): calcd
for C30H48N4O6F2S [M + H]+, 631.3341; found, 631.3324.
Kinetic Evaluation of [3 + 2] Cycloaddition of 2 and 3
with Benzyl Azide. Cyclooctyne 2 or 3 was mixed at a 1:1 molar
ratio (∼15 mM) with benzyl azide in either (a) CD3CN or (b) a
7:3 mixture of CD3CN and 25 mM potassium phosphate in D2O
Compound 15b. To a flame-dried round-bottom flask were
added cyclooctyne 2 (10.5 mg, 0.0377 mmol), CH2Cl2 (0.5 mL),
and diisopropylethylamine (16.4 µL, 0.0943 mmol), and the
mixture was cooled to 0 °C with stirring. Pentafluorophenyl
trifluoroacetate (7.1 µL, 0.042 mmol) was then added, and after
10 min, the system was warmed to rt. The reaction mixture was
stirred an additional 1.5 h, concentrated, filtered through a plug
of silica gel eluting with hexanes, and then concentrated to yield
a white solid. This material was transferred to a new round-
bottom flask and dissolved in anhydrous DMF (0.5 mL).
Biotinylated amine 1625 (12.0 mg, 0.0268 mmol) was then added,
followed by diisopropylethylamine (7.0 µL, 0.040 mmol). The
reaction mixture was stirred overnight, concentrated, and purified
twice by flash chromatography (1% Et3N in CH2Cl2, then 5%
MeOH in CH2Cl2) to yield a clear oil (9.7 mg, 36% over two
steps). Rf ) 0.65 (9:1 CH2Cl2/MeOH). 1H NMR (400 MHz,
CD3OD): δ 8.41 (m, 1H), 7.93 (m, 1H), 7.77 (d, 2H, J ) 8.2
Hz), 7.32 (d, 2H, J ) 8.2 Hz), 4.48 (dd, 1H, J ) 7.8, 4.7 Hz),
4.29 (dd, 1H, J ) 7.9, 4.5 Hz), 3.68-3.53 (m, 10H), 3.48 (q,
4H, J ) 6.0 Hz), 3.28-3.16 (m, 4H), 3.10 (d, 1H, J ) 10.8
Hz), 2.92 (dd, 1H, J ) 12.7, 5.0 Hz), 2.70 (d, 1H, J ) 12.7
Hz), 2.63-2.47 (m, 2H), 2.44-2.27 (m, 2H), 2.19 (t, 2H, J )
7.4 Hz), 2.08-1.99 (m, 1H), 1.98-1.79 (m, 4H), 1.79-1.69 (m,
4H), 1.69-1.55 (m, 3H), 1.51 (dd, 1H, J ) 15.9, 8.1 Hz),
1.47-1.37 (m, 2H), 1.31 (t, 1H, J ) 7.3 Hz), 1.23-1.12 (m,
1H). 13C NMR (125 MHz, CD3OD): δ 176.1, 170.1, 166.3,
145.2, 134.0, 130.5, 128.7, 121.0 (dd, J ) 238.9, 236.0 Hz),
111.2 (t, J ) 11.2 Hz), 86.0 (dd, J ) 46.8, 41.8 Hz), 71.7, 71.7,
71.4, 71.4, 70.4, 70.1, 63.5, 61.8, 59.5 (t, J ) 24.3 Hz), 57.2,
41.2, 38.9, 38.0, 37.0, 35.2 (d, J ) 4.0 Hz), 33.8 (d, J ) 1.5
Hz), 32.0 (d, J ) 4.6 Hz), 30.6 (d, J ) 3.8 Hz), 30.0, 29.7,
29.1, 27.0, 20.8, 9.4. 19F NMR (376 MHz, CD3OD): δ -95.40
(d, 1H, J ) 259.9 Hz), -102.76 (ddt, 1H, J ) 259.9, 20.1, 6.8
Hz). HRMS (FAB): calcd for C36H52N4O6F2S [M + Li]+,
713.3736; found, 713.3736.
Compound 15c. To a flame-dried round-bottom flask were
added cyclooctyne 3 (26.3 mg, 0.130 mmol), CH2Cl2 (2.0 mL),
and diisopropylethylamine (57.0 µL, 0.325 mmol), and the
mixture was cooled to 0 °C with stirring. Pentafluorophenyl
trifluoroacetate (25.0 µL, 0.143 mmol) was added, and after 10
min, the system was warmed to rt. The reaction mixture was
stirred for an additional 1 h, concentrated, and filtered through
a plug of silica gel using 1% EtOAc in hexanes as the eluent to
yield a white solid (41.4 mg). A portion of this material (6.5
mg, 0.018 mmol) was then transferred to a flame-dried conical
flask and dissolved in anhydrous DMF (0.5 mL). Biotinylated
amine 1625 (7.9 mg, 0.018 mmol) was added, followed by
diisopropylethylamine (5.0 µL, 0.027 mmol). The reaction
mixture was stirred for 4 h, concentrated, and purified twice by
flash chromatography (0-10% MeOH in CH2Cl2) to yield a clear
oil. Rf ) 0.37 (9:1 CH2Cl2/MeOH). The oil was further purified
by reversed-phase HPLC (gradient of 5-70% CH3CN in H2O
over 40 min, eluting at ∼27 min) to yield a clear oil (6.4 mg,
50% over two steps). 1H NMR (400 MHz, CD3OD): δ 4.49 (dd,
1H, J ) 7.9, 4.3 Hz), 4.30 (dd, 1H, J ) 7.9, 4.5 Hz), 3.68-3.55
(m, 8H), 3.52 (dt, 4H, J ) 6.2, 1.4 Hz), 3.27 (m, 4H), 3.21 (m,
1H), 2.93 (dd, 1H, J ) 12.8, 5.0 Hz), 2.82-2.66 (m, 2H), 2.49
(dd, 1H, J ) 14.7, 3.9 Hz), 2.46-2.28 (m, 2H), 2.20 (t, 2H, J
) 7.4 Hz), 2.18-2.00 (m, 3H), 1.85-1.70 (m, 7H), 1.70-1.52
(m, 4H), 1.50-1.33 (m, 3H). 13C NMR (125 MHz, CD3OD): δ
176.1, 173.8, 166.3, 120.9 (app t, J ) 237.4 Hz), 111.8 (app t,
J ) 11.0 Hz), 85.7 (dd, J ) 46.6, 42.0 Hz), 71.7, 71.7, 71.4,
71.4, 70.1, 70.0, 63.5, 61.8, 57.2, 54.6 (t, J ) 24.3 Hz), 41.2,
38.1, 38.0, 37.0, 36.3 (d, J ) 4.0 Hz), 33.9 (d, J ) 1.9 Hz),
1
(pH 7), and the reaction was monitored by H NMR. The kinetic
data were derived by following the change in integration of
resonances corresponding to the benzylic protons in benzyl azide
(δ ∼4.4) compared to those of the benzylic protons in the triazole
products (δ ∼5.5-5.7). The second-order rate constants for the
reaction were determined by plotting 1/[azide] versus time and
subsequently using analysis by linear regression. The second-order
rate constant k (M-1 -1
s ) corresponds to the determined slope.
Cell Culture. Jurkat or Chinese hamster ovary (CHO) cells were
maintained in a 5% CO2, water-saturated atmosphere and grown
in RPMI-1640 (Jurkat) or F12 (CHO) media supplemented with
10% FBS, penicillin (100 units/mL), and streptomycin (0.1 mg/
mL). Cell densities were maintained between 1 × 105 and 1.6 ×
106 cells/mL.
Cell-Surface Labeling of Azide-Bearing Glycans with
Biotinylated Conjugates. Jurkat cells were incubated for 3 days
in media containing 25 µM Ac4ManNAz or no sugar. The cells
were distributed into a 96-well V-bottom tissue culture plate and
washed three times by sequential concentration by centrifugation
(2500g, 3 min, 4 °C) and resuspension in 200 µL of labeling buffer
[PBS (pH 7.4) containing 1% FBS]. The cells were then incubated
at rt with 100 µL of 0-10 µM 15a-c in labeling buffer for 0-60
min, with dilutions made from a 2.5 mM stock solution in 7:3 PBS/
DMF. After incubation, cells were washed three times and
resuspended in 100 µL of FITC-labeled avidin (1:200 dilution in
labeling buffer of a 1 mg/mL stock solution). After a 15 min
incubation in the dark at 4 °C, the cells were washed once and
then incubated with FITC-labeled avidin for an additional 15 min
at 4 °C. The cells were washed three times and then diluted to a
volume of 400 µL for flow cytometry analysis. Annexin V-PE
staining was performed according to instructions from the manu-
facturer immediately prior to flow cytometry analysis.
Live-Cell Imaging of Azide-Labeled Glycans. CHO cells were
incubated for 3 days in media containing 25 µM Ac4ManNAz or
no sugar in an eight-well LabTek II chambered coverglass (Nunc).
The cells were washed three times by sequential gentle aspiration
of the media and addition of 500 µL of media. The cells were then
treated with 100 µL of a 10 µM solution of 15b or 15c (1:250
dilution in media from a 2.5 mM stock solution in 7:3 PBS/DMF)
for 60 min at rt. The cells were washed three times, stained with
FITC-labeled avidin (100 µL of a 1:200 dilution in media from a
1 mg/mL stock solution) for 10 min at rt, washed three times, treated
with Hoechst 33342 dye (100 µL of a 1:1000 dilution in media
from a 1 mg/mL DMSO stock solution) for 2 min at rt to stain the
nuclei, washed twice, and imaged by epifluorescence microscopy.
Results and Discussion
Synthesis of Second-Generation DIFO Reagent 2. The syn-
thesis of cyclooctyne 2 (Scheme 1) began with 1,3-cyclooc-
tanedione, which was prepared in ∼67% yield as previously
described.26 Difluorination of 1,3-dicarbonyl compounds with
Selectfluor has been reported to occur sluggishly under neutral
conditions27 and more rapidly using enamine intermediates28
or microwave-assisted strategies.29 We found that simply
(26) Pirrung, M. C.; Webster, N. J. G. J. Org. Chem. 1987, 52, 3603–
3613.
(27) Banks, R. E.; Lawrence, N. J.; Popplewell, A. L. J. Chem. Soc., Chem.
Commun. 1994, 343–344.
(25) Wilbur, D. S.; Hamlin, D. K.; Vessella, R. L.; Stray, J. E.; Buhler,
K. R.; Stayton, P. S.; Klumb, L. A.; Pathare, P. M.; Weerawarna,
S. A. Bioconjugate Chem. 1996, 7, 689–702.
(28) Peng, W.; Shreeve, J. M. J. Org. Chem. 2005, 70, 5760–5763.
(29) Xiao, J.-C.; Shreeve, J. M. J. Fluorine Chem. 2005, 126, 475–478.
9
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Difluorinated Cyclooctynes for Cu-Free Click Chemistry
A R T I C L E S
Scheme 1. Synthesis of Second-Generation DIFO Reagent 2
Scheme 2. Synthesis of Second-Generation DIFO Reagent 3
treating the diketone with Selectfluor and Cs2CO3 at 0 °C
produced 2,2-difluoro-1,3-cyclooctanedione (4) in 73% yield.
In order to install a linker with a protected carboxylic acid, we
performed a Wittig reaction using phosphonium salt 5 and DBU.
Fortuitously, we observed exclusive formation of monosubsti-
tuted product 6 even when excess amounts of 5 and base were
used, thus enabling efficient desymmetrization of the diketone.
The observed chemoselectivity is likely due to the high
electrophilicity of the first ketone equivalent compounded with
the low nucleophilicity of the stabilized ylide. Hydrogenation
of the olefin to saturated compound 7 and subsequent conversion
of the ketone to a vinyl triflate (8) proceeded in good yield (82%
over two steps). Cyclooctyne 9 was formed by LDA-mediated
elimination, and the methyl ester was saponified to yield DIFO
reagent 2 in a total of six steps in 36% overall yield from 1,3-
cyclooctanedione. This yield is ∼25-fold higher than that for
the previously reported synthesis of 119 and was achieved in
half as many steps.
which bears an oxetane ester, an OBO ester precursor.30 Again,
we observed selective monosubstitution to yield olefin 11 in
85% yield. Hydrogenation of the olefin and conversion of the
oxetane ester to the orthoester using BF3 ·OEt231 yielded ketone
12 in 90% yield. Formation of vinyl triflate 13 and elimination
to form cyclooctyne 14 proceeded in 80% and 59% yields,
respectively. Two-step deprotection of the OBO ester to
carboxylic acid 3 was accomplished by acidic deprotection using
PPTS to form a simple ester and subsequent saponification using
LiOH (86% yield).32 The synthesis of 3 was accomplished with
an overall yield of 28% from 1,3-cyclooctanedione.
Kinetic Evaluation of [3 + 2] Cycloaddition of 2 and 3
1
with Benzyl Azide. With these reagents in hand, we used H
NMR to measure the kinetics of the copper-free click reaction
with the model compound benzyl azide. For the reactions with
2 and 3 performed in CD3CN, we obtained second-order rate
constants of (4.2 ( 0.1) × 10-2 and (5.2 ( 0.2) × 10-2 M-1
s-1, respectively; these values are comparable to that for the
reaction of 1 with benzyl azide under identical conditions (7.6
Synthesis of Second-Generation DIFO Reagent 3. A lesson
from previous studies is that the hydrophobicity of DIFO
reagents can contribute to nonspecific protein and cell binding.
Thus, we also synthesized a second-generation DIFO analogue
lacking the nonessential phenyl moiety in the linker. The target,
compound 3 (Figure 1), possessed a carboxymethyl group to
which probes were later attached. A key protecting-group
strategy masked the carboxymethyl group as an oxabicycloortho
(OBO) ester in order to avoid acidic protons that would later
interfere with vinyl triflate formation and elimination.
× 10-2 M-1 -1
s
).19 To evaluate the kinetics of the [3 + 2]
cycloaddition in an aqueous environment, we performed reac-
tions of 2 and 3 with benzyl azide in a 7:3 mixture of CD3CN
and 25 mM potassium phosphate in D2O (pH 7) and obtained
k values of (9.0 ( 0.3) × 10-2 and (8.6 ( 0.9) × 10-2 M-1
(30) Corey, E. J.; Raju, N. Tetrahedron Lett. 1983, 24, 5571–5574.
(31) Blaskovich, M. A.; Lajoie, G. A. J. Am. Chem. Soc. 1993, 115, 5021–
5030.
(32) Martynow, J. G.; Jozwik, J.; Szelejewski, W.; Achmatowicz, O.;
Kutner, A.; Wisniewski, K.; Winiarski, J.; Zegrocka-Stendel, O.;
Golebiewski, P. Eur. J. Org. Chem. 2007, 689–703.
As shown in Scheme 2, 2,2-difluoro-1,3-cyclooctanedione (4)
was reacted under basic conditions with phosphonium salt 10,
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Scheme 3. Synthesis of Biotinylated Derivatives of Difluorinated Cyclooctynes (15a-c)
s-1, respectively. All three reagents are considerably more
reactive than cyclooctynes lacking the difluoromethylene
group.33
We then compared the efficacy of cell-surface azide labeling
of 15b and 15c to the first-generation reagent 15a in a 60 min
reaction at 10 µM (Figure 2D). The observed labeling with 15b
and 15c was approximately half of that with 15a, consistent
with their relative rate constants. These labeling efficiencies are
all significantly higher than those previously reported for mono-
Additionally, we investigated the stabilities of compounds 2
and 3 with respect to biologically relevant nucleophiles. We
dissolved each cyclooctyne (20 mM) in a 7:3 mixture of CD3CN
and 25 mM potassium phosphate in D2O (pH 7) and then added
either 2-mercaptoethanol or 2-aminoethanol (20 mM). We
1
observed no reaction after 24 h, as monitored by H and 19F
NMR, suggesting that compounds 2 and 3 are stable to water,
thiols, amines, and alcohols at physiological pH. Finally,
compounds 2 and 3 were found to be stable for many months
when stored at -20 °C.
Copper-Free Click Labeling of Cell-Surface Azido Glycans
Using Biotinylated Conjugates. An important application of
copper-free click chemistry is the nontoxic and rapid detec-
tion of azides within living systems, either for molecular
imaging or for subsequent affinity capture. DIFO reagent 1
and its derivatives (e.g., the biotinylated derivative 15a19)
have proven useful for bioorthogonal labeling of cell-surface
glycans bearing azides, and we set out to evaluate the second-
generation DIFO reagents in this context. We first derivatized
carboxylic acids 2 and 3 as the biotinylated reagents 15b
and 15c, respectively, via formation of the corresponding
activated pentafluorophenyl esters and subsequent conjugation
to an amine-derivatized biotin reagent (Scheme 3).
Jurkat cells were incubated with 25 µM peracetylated
N-azidoacetylmannosamine (Ac4ManNAz) for 3 days, result-
ing in the metabolic labeling of cell-surface glycans with
azido sialic acid (SiaNAz) residues (Figure 2A).34 The cells
were washed and labeled with biotinylated reagents 15b and
15c either at various concentrations (0-10 µM) for 60 min
(Figure 2B) or for various reaction times (0-60 min) at 10
µM (Figure 2C). In all cases, the cells were subsequently
stained with FITC-labeled avidin and analyzed by flow
cytometry, as described previously.35 Both of the second-
generation DIFO reagents displayed concentration- and time-
dependent reaction profiles with cell-surface-associated azides.
We were pleased to observe that both reagents displayed very
little background fluorescence labeling, though the more
hydrophobic 15b resulted in slightly higher background
fluorescence than 15c.
Figure 2. Labeling of cell-surface glycans with azido sugars and DIFO
reagents. (A) Bioorthogonal chemical reporter strategy for two-step detection
of glycans. Cells were first incubated with Ac4ManNAz, which is metaboli-
cally converted to cell-surface SiaNAz residues, and subsequently reacted
with difluorinated cyclooctyne probes for visualization. (B-E) Jurkat cells
were metabolically labeled with 25 µM Ac4ManNAz (+Az) or no sugar
(-Az) for 3 days. The cells were labeled with 15b or 15c either (B) for 60
min at 0, 1, 2, 5, or 10 µM or (C) for 0, 15, 25, 35, or 65 min at 10 µM.
The cells were then stained with FITC-labeled avidin and analyzed by flow
cytometry. (D-E) The cells were labeled with 15a-c or no reagent (no
rgt) for 60 min at 10 µM and then sequentially stained with FITC-labeled
avidin and Annexin V-PE, followed by flow cytometry analysis. Gray bars
denote no sugar and black bars 25 µM Ac4ManNAz. The error bars indicate
the standard deviation of three replicate samples. Shown in (D) is the mean
fluorescence intensity (MFI) in arbitrary units (au). Shown in (E) is the
percentage of cells in each sample belonging to the population that stains
highly with Annexin V-PE.
(33) Agard, N. J.; Baskin, J. M.; Prescher, J. A.; Lo, A.; Bertozzi, C. R.
ACS Chem. Biol. 2006, 1, 644–648.
(34) Saxon, E.; Luchansky, S. J.; Hang, H. C.; Yu, C.; Lee, S. C.; Bertozzi,
C. R. J. Am. Chem. Soc. 2002, 124, 14893–14902.
(35) Laughlin, S. T.; Agard, N. J.; Baskin, J. M.; Carrico, I. S.; Chang,
P. V.; Ganguli, A. S.; Hangauer, M. J.; Lo, A.; Prescher, J. A.;
Bertozzi, C. R. Methods Enzymol. 2006, 415, 230–250.
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Difluorinated Cyclooctynes for Cu-Free Click Chemistry
A R T I C L E S
ground fluorescence in the negative control for both 15b and
15c (Figure 3), indicating that these second-generation DIFO
reagents can be utilized for live-cell imaging applications.
Conclusion
We have developed synthetically tractable second-generation
difluorinated cyclooctyne reagents for copper-free click chem-
istry. The synthesis of these reagents involves a novel method
for one-step difluorination of 1,3-diketones, a selective Wittig
reaction to install the linkers, and the use of an orthoester as a
cyclooctyne-compatible protecting group. Furthermore, these
reagents can be prepared efficiently, with overall yields that are
more than 20-fold higher than that of the first-generation reagent.
Critically, we determined that these novel DIFO reagents are
nontoxic to cells and can selectively tag azide-labeled biomol-
ecules in living systems with efficiencies approaching that of
the parent compound. We envision that these reagents will
enable widespread use of copper-free click chemistry in the
context of in vivo imaging and ex vivo labeling of biomolecules
and in the generation of novel biocompatible materials.36
Figure 3. Live-cell imaging of cell-surface glycans using second-generation
difluorinated cyclooctynes. CHO cells were metabolically labeled with (A,
B) 25 µM Ac4ManNAz or (C, D) no sugar for 3 days. The cells were labeled
for 60 min with 10 µM (A, C) 15b or (B, D) 15c, washed, stained with
FITC-labeled avidin and Hoechst 33342, and imaged by epifluorescence
microscopy. Images were deconvolved using the nearest-neighbor algorithm
of Slidebook 4.2 and are shown as maximum intensity z-projection
fluorescence images over 7.5 µm. Green, FITC; blue, Hoechst 33342. Scale
bar: 10 µm.
or nonfluorinated cyclooctyne reagents or triarylphosphines
capable of Staudinger ligation.33
Acknowledgment. This work was supported by a grant to
C.R.B. from the National Institutes of Health (GM058867). J.A.C.
was supported by an undergraduate scholarship from the Amgen
Foundation, and J.M.B. was supported by National Science
Foundation and National Defense Science and Engineering pre-
doctoral fellowships. We thank Ellen Sletten, Pamela Chang, and
Scott Laughlin for helpful discussions and Phung Gip and Kapil
Amarnath for technical assistance.
Finally, we tested the toxicity of probes 15a-c by incubation
of cells labeled as above (60 min, 10 µM) with phycoerythrin-
conjugated Annexin V, a marker of apoptosis. As shown in
Figure 2E, none of the reagents caused a significant change in
the percentage of Annexin V-positive cells, indicating that DIFO
reagents 15a-c are not toxic to Jurkat cells.
Live-Cell Imaging of Membrane-Associated Glycans Using
Second-Generation DIFO Reagents. Finally, we applied these
novel cyclooctynes to image cell-surface glycans in live CHO
cells. The cells were incubated for 3 days with 25 µM
Ac4ManNAz or with no sugar as a negative control and labeled
with 10 µM 15b or 15c for 60 min. Subsequently, the cells
were treated with FITC-labeled avidin and Hoechst 33342, a
live-cell nuclear stain, and imaged by epifluorescence micros-
copy. We observed clear azide-dependent labeling at the cell
surface in the Ac4ManNAz-treated cells and minimal back-
Supporting Information Available: 1H and 13C NMR spectra
for all of the new compounds, kinetic data, and flow cytometry
plots. This material is available free of charge via the Internet
at http://pubs.acs.org.
JA803086R
(36) Johnson, J. J.; Baskin, J. M.; Bertozzi, C. R.; Koberstein, J. T.; Turro,
N. J Chem. Commun. 2008, 3064–3066.
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