Surface functionalization using catalyst-free azide-alkyne cycloaddition

Article abstract of DOI:10.1021/bc100306u

The utility of catalyst-free azide-alkyne [3 + 2] cycloaddition for the immobilization of a variety of molecules onto a solid surface and microbeads was demonstrated. In this process, the surfaces are derivatized with aza-dibenzocyclooctyne (ADIBO) for the immobilization of azide-tagged substrates via a copper-free click reaction. Alternatively, ADIBO-conjugated molecules are anchored to the azide-derivatized surface. Both immobilization techniques work well in aqueous solutions and show excellent kinetics under ambient conditions. We report an efficient synthesis of aza-dibenzocyclooctyne (ADIBO), thus far the most reactive cyclooctyne in cycloaddition to azides. We also describe convenient methods for the conjugation of ADIBO with a variety of molecules directly or via a PEG linker.

Full text of DOI:10.1021/bc100306u

2076
Bioconjugate Chem. 2010, 21, 2076–2085
Surface Functionalization Using Catalyst-Free Azide-Alkyne Cycloaddition
Alexander Kuzmin,† Andrei Poloukhtine,† Margreet A. Wolfert,‡ and Vladimir V. Popik*,†
Department of Chemistry and Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Road, Athens,
Georgia 30602, United States. Received July 6, 2010; Revised Manuscript Received September 29, 2010
The utility of catalyst-free azide-alkyne [3 + 2] cycloaddition for the immobilization of a variety of molecules
onto a solid surface and microbeads was demonstrated. In this process, the surfaces are derivatized with aza-
dibenzocyclooctyne (ADIBO) for the immobilization of azide-tagged substrates via a copper-free click reaction.
Alternatively, ADIBO-conjugated molecules are anchored to the azide-derivatized surface. Both immobilization
techniques work well in aqueous solutions and show excellent kinetics under ambient conditions. We report an
efficient synthesis of aza-dibenzocyclooctyne (ADIBO), thus far the most reactive cyclooctyne in cycloaddition
to azides. We also describe convenient methods for the conjugation of ADIBO with a variety of molecules directly
or via a PEG linker.
INTRODUCTION
Among reactive cyclooctyne derivatives, dibenzocyclooctynes
are synthetically more accessible (42). The major limitation of
these click reactants is the relatively low rate of azide cycload-
dition (ca. 0.05 M-1 s-1) (34-37). Substitution of one of the
saturated carbons in the cyclooctyne ring for a nitrogen atom
not only improves its reactivity, but also simplifies the
synthesis (28-33). To extend the arsenal of bioorthogonal
copper-free click reagents, we have developed an efficient
synthesis of aza-dibenzocyclooctyne-containing compounds for
azide-coupling reactions. In the present report, we describe a
novel approach to efficient surface-functionalization using a
catalyst-free azide click reaction. This method allows for the
site-specific covalent anchoring of proteins and other substrates
to various surfaces. The same metal-free click reaction is
employed for the PEGylation of nonfunctionalized areas of the
surface. Such treatment allows for a dramatic reduction or
complete elimination of nonspecific binding. The copper-free
click immobilization strategy discussed in the present report can
be applied to the preparation of various types of arrays, as well
as to the derivatization of microbeads and nanoparticles.
Surface immobilization of biomolecules is a very important
step in the manufacturing of biosensors, microbeads, biochips,
probe arrays, medical implants, and other devices (1-5). The
key requirements for this process are the preservation of
biochemical properties of immobilized substrates and robustness
of the linkage. Copper(I)-catalyzed Huisgen 1,3-dipolar cy-
cloaddition of azides to terminal acetylenes has emerged as one
of the most convenient methods for the functionalization of
various surfaces (3, 5-10). The triazol linker formed in the azide
“click” reaction has excellent chemical stability due to the
aromatic character of the heterocycle. Azide tags can be
incorporated into biomolecules using a variety of different
strategies, such as postsynthetic modification (11-13), in vitro
enzymatic transfer (14, 15), the use of covalent inhibitors (16),
and metabolic labeling by feeding cells a biosynthetic precursor
modified with azido functionality (17). While conventional
copper(I)-catalyzed click chemistry has become commonplace
in surface derivatization, as well as in polymer and materials
synthesis (6-10, 18-20), the use of metal catalyst often limits
the utility of the method. Copper ions are cytotoxic (21), can
cause degradation of DNA molecules (22, 23), and induce
protein denaturation (24). In addition, the use of catalysts
complicates kinetics of the immobilization process, requires
polar solvents, and can alter surface properties (25).
Conventional copper-catalyzed azide click coupling methods
employ terminal acetylenes, since internal alkynes react with
azides only at elevated temperatures. Cyclooctynes, on the other
hand, are known to form triazoles without a catalyst under
ambient conditions, albeit at a rather slow rate (26). The triple
bond incorporated into an eight-membered ring is apparently
already bent into a geometry resembling the transition state of the
cycloaddition reaction, thus reducing its activation barrier (27).
Recently developed cyclooctyne derivatives are substantially more
reactive toward azides and offer a convenient metal-free alternative
to the copper-catalyzed click reaction (28-33). Metal-free click
chemistry has been successfully employed for the modification of
luminescent quantum dots (25) and protein labeling and purification
(34-37), as well as for the introduction of fluorescent tags into
live cells (25, 38, 39) and organisms (40, 41).
EXPERIMENTAL PROCEDURES
Materials and Methods. Purification of products by column
chromatography was performed using 40-63 µm silica gel. All
NMR spectra were recorded on a 400 MHz instrument in CDCl3
and referenced to TMS unless otherwise noted. Images of
fluorophore-patterned slides were recorded, and fluorescence
intensity was quantified using a Typhoon 9400 variable mode
imager (GE Healthcare) at an excitation/emission setting ap-
propriate for fluorescein (488/520 nm) or Lissamine rhodamine
B (532/580 nm) fluorophores in PBS solution; images of
fluorescent microbeads were obtained using Zeiss Observer
AX10 inverted microscope with an X-cite series 120 fluorescent
light source and Chroma Technology filters. The relative
fluorescent intensity (spot/background) was quantified using
ImageJ program (NIH).
Tetrahydrofuran was distilled from a sodium/benzophenone
ketyl; ether and hexanes were distilled from sodium; N,N-
dimethylformamide (DMF) was dried by passing through an
alumina column. Biotin-dPEG3+4-azide and Biotin-PEG4-acid
were purchased from Quanta BioDesign; fluorescein SE, Oregon
Green SE, Lissamine rhodamine B sulfonyl chloride, and FITC-
Avidin were obtained from Invitrogen; (3-glycidyloxypropyl)-
* E-mail: vpopik@chem.uga.edu.
† Department of Chemistry.
‡ Complex Carbohydrate Research Center.
10.1021/bc100306u  2010 American Chemical Society
Published on Web 10/21/2010

Catalyst-Free Azide-Alkyne Cycloaddition
Bioconjugate Chem., Vol. 21, No. 11, 2010 2077
dimethoxymethylsilane was purchased from TCI America;
0.0067 M phosphate buffered saline (PBS) with pH 7.4 was
obtained from Thermo Scientific. Bovine serum albumin (BSA)
was obtained from Fisher BioReagents. Other reagents were
purchased from Aldrich or VWR and used as received unless
otherwise noted. Polished glass slides were obtained from VWR;
NanoLink streptavidin magnetic microspheres (1% aqueous
suspension) were purchased from SoluLink Biosciences. Aza-
dibenzocycloctyne 6 conjugates with fluorescein (ADIBO-fluor,
10), Oregon green (ADIBO-OG, 11), and Lissamine rhodamine
B (ADIBO-Rhodamine, 12) were prepared by treating 6 with
equimolar amounts of fluorescein SE, Oregon Green SE,
Lissamine rhodamine B sulfonyl chloride, respectively, in DMF
in the presence of DIEA (ethyldiisopropylamine). Oregon green
azide (13) and Lissamine rhodamine B azide (14) were prepared
by reacting equimolar amounts of Oregon green SE or Lissamine
rhodamine B sulfonyl chloride with 3-azidopropyl amine in
DMF in the presence of DIEA. Conjugates 10-14 were purified
by chromatography (CHCl3/MeOH/AcOH 100:5:0.5) and their
purity was confirmed by HPLC analysis. 1-Amino-11-azido-
3,6,9-trioxaundecane (AminoPEG4azide) (43) and 6-azidohexy-
lamine (44) were prepared according to literature procedures.
128.5, 128.2, 128.1, 128.0, 127.3, 127.2, 117.5, 114.6, 54.8,
39.6, 34.3, 28.2, 25.9, 24.3. HRMS (ESI+) m/z calcd for
C23H24F3N2O2 [M+H]+ 417.1790, found 417.1783.
N-(6-Trifluoroacetamidohexanoyl)-5,6-dihydro-11,12-didehy-
drodibenzo[b,f]azocine (5). Pyridine hydrobromide perbromide
(0.948 g, 2.97 mmol) was added to a solution of 3 (1.05 g,
2.70 mmol) in CH2Cl2 (4 mL) at rt, and the reaction mixture
was stirred overnight. The reaction mixture was diluted with
CH2Cl2 (20 mL), washed with 5% aqueous hydrochloric acid
(20 mL), dried over MgSO4, and solvent removed under
vacuum. The residue was passed through a short pad of silica
gel (CH2Cl2) to give 1.2 g of crude N-(trifluoroacetamidohex-
anoyl)-5,6,11,12-tetrahydro-11,12-dibromodibenzo[b,f]azo-
cine (4) as an oil. A solution of crude 4 (1.2 g, 2.08 mmol) in
THF (5 mL) was added to a solution of potassium t-butoxide
(0.584 g, 5.21 mmol) in THF (10 mL) at rt; the reaction mixture
was stirred for 1 h, diluted with ethyl acetate (20 mL), washed
with 5% aqueous hydrochloric acid and brine, then dried over
MgSO4, and the solvent was removed under reduced pressure.
The crude product was purified by chromatography (hexanes/
ethyl acetate, 2:1 to 1:1) to afford 0.76 g (1.83 mmol, 88%) of
5 as brown oil. 1H NMR: δ 7.68 (d, J ) 7.6 Hz, 1 H), 7.45-7.21
(m, 7 H), 6.79 (br s, 1 H), 5.16 (d, J ) 14.4 Hz, 1 H), 3.67 (d,
J ) 13.6 Hz, 1 H), 3.22-3.14 (m, 1 H), 3.11-3.02 (m, 1 H),
2.24-2.16 (m, 1 H), 1.41-1.22 (m, 4 H), 1.11-0.9 (m, 2 H).
MS: m/z 414 [M+]. Calcd for C23H21F3N2O2 414.
5,6-Dihydrodibenzo[b,f]azocine (2). A solution of dibenzo-
suberenone (25 g, 121 mmol) and hydroxylamine hydrochloride
(6.81 mL, 164 mmol) in pyridine (70 mL) was refluxed for 20 h.
The reaction mixture was concentrated and poured into 5%
aqueous hydrochloric acid (with crushed ice), stirred for 20 min,
filtered, and dried in the air to provide 28.1 g of crude
dibenzosuberenone oxime, as a white precipitate. Dibenzosuber-
enone oxime (16 g, 72.3 mmol) was added to 250 mL
polyphosphoric acid at 125 °C; the reaction mixture was stirred
for 60 min at this temperature, poured onto crushed ice (∼700
mL), stirred for another 30 min, and filtered. The filter cake
was washed with water and dried under vacuum to provide crude
dibenzo[b,f]azocin-6(5H)-one 1 (11.6 g, 52.4 mmol, 73%) as a
gray powder.
N-(6-Aminohexanoyl)-5,6-dihydro-11,12-didehydrodibenzo-
[b,f]azocine (ADIBO-C6-amine, 6). A solution of K2CO3 (2 g,
14.47 mmol) in 15 mL of water was added to a solution of
N-(6-trifluoroacetamido hexanoyl)-5,6-dihydro-11,12-didehy-
drodibenzo[b,f]azocine (5, 2.95 g, 7.12 mmol) in MeOH (30
mL) at rt and stirred overnight. Solvents were removed under
reduced pressure, the residue was redissolved in CH2Cl2/ethyl
acetate (1:4), then washed with brine and water. The organic
layer was dried over anhydrous Na2SO4 and concentrated in
vacuum. The crude product was purified by chromatography
(CH2Cl2/MeOH 10:1 to 10:4) to provide 1.31 g (4.11 mmol,
A suspension of 1 (7.4 g, 33.4 mmol) and lithium aluminum
hydride (2.494 mL, 66.9 mmol) in anhydrous ether (200 mL)
was refluxed for 15 h. The reaction mixture was quenched by
water and filtered, and the filter cake was washed with ether.
The filter cake was dispersed in ether (100 mL), stirred for 10
min, and filtered. The combined organic layers were dried over
MgSO4, solvent was removed under vacuum, and the product
was purified by chromatography (hexanes/ethyl acetate 2:1) to
provide 4.04 g (19.49 mmol, 58%) of 5,6-dihydrodibenzo[b,f]-
1
58%) of 6 as slightly yellow oil. H NMR: δ 7.71 (d, J ) 7.6
Hz, 1 H), 7.45-7.21 (m, 7 H), 5.18 (d, J ) 14.4 Hz, 1 H), 3.63
(d, J ) 13.6 Hz, 1 H), 3.32 (br, 2 H), 2.67 (t, J ) 5.6 Hz, 1 H),
2.52 (t, J ) 5.6 Hz, 1 H), 2.21-2.19 (m, 1 H), 1.95-1.90 (m,
1 H), 1.45-1.35 (m, 2 H) 1.28-1.20 (m, 2 H), 1.11-0.9 (m, 2
H). 13C NMR: δ 173.3, 151.8, 147.8, 132.3, 132.1, 128.9, 128.2,
128.0, 127.8, 127.5, 126.5, 122.1, 123.0, 114.8, 107.9, 55.8,
41.4, 34.7, 31.9, 26.0, 24.8. HRMS (ESI+) m/z calcd for
C21H23N2O [M+H]+ 319.1810, found 319.1799.
1
azocine (2). H NMR: δ 7.27-7.23 (m, 1 H), 7.2-7.1 (m, 3
H), 6.96-6.9 (m, 1 H), 6.9-6.8 (m, 1 H), 6.65-6.55 (m, 1 H),
6.54-6.48 (m, 1 H), 6.40 (d, J ) 8 Hz, 1 H), 6.38-6.29 (m, 1
H), 4.51 (d, J ) 6.8 Hz, 2 H), 4.2 (br s, 1 H). 13C NMR: δ
147.3, 139.3, 138.3, 134.9, 132.7, 130.3, 129.0, 128.1, 127.8,
127.6, 127.5, 121.8, 118.1, 117.9, 49.6. HRMS (ESI+) calcd
for C15H14N [M+H]+ 208.1126, found 208.1120.
N-(6-(Dibenzo[b,f]azocin-5(6H)-yl)-6-oxohexyl)trifluoroac-
etamide (3). 6-(Trifluoroacetamido)hexanoyl chloride (45) (0.984
g, 4.52 mmol) was added to a solution of 2 (0.75 g, 3.62 mmol)
and pyridine (0.859 g, 10.86 mmol) in CH2Cl2 (ca. 10 mL) at
rt and stirred for 30 min. The reaction mixture was diluted with
CH2Cl2 (ca. 20 mL), washed with water (2 × 30 mL), and dried
over anhydrous MgSO4, and the solvent was removed under
reduced pressure. The residue was purified by chromatography
(hexanes/ethyl acetate 2:3) to provide 1.064 g (2.56 mmol, 71%)
of 3 as yellowish oil. 1H NMR: δ 7.32-7.22 (m, 4 H),
7.19-7.11 (m, 4 H), 6.77 (d, J ) 13.2 Hz, 1 H), 6.58 (d, J )
13.2 Hz, 1 H), 5.46 (d, J ) 14.8 Hz, 1 H), 4.20 (d, J ) 14.8
Hz, 1 H), 3.29-3.15 (m, 2 H), 2.09-2.02 (m, 1 H), 1.93-1.85
(m, 1 H), 1.51-1.32 (m, 4 H), 1.25-1.04 (m, 2 H). 13C NMR:
δ 172.7, 157.5, 141.1, 136.1, 135.8, 134.8, 132.4, 131.8, 130.5,
N-(3-Aminopropionyl)-5,6-dihydro-11,12-didehydrodiben-
zo[b,f]azocine (ADIBO-C3-amine, 7). 7 was prepared following
the same protocol as the preparation of 6. 1H NMR (500 MHz):
δ 7.68 (d, J ) 7.5 Hz, 1 H), 7.45-7.33 (m, 5 H), 7.29 (t, J )
7.5 Hz, 1 H), 7.25 (t, J ) 7 Hz, 1 H), 5.15 (d, J ) 14 Hz, 1 H),
3.16 (d, J ) 14 Hz, 1 H), 2.82-2.67 (m, 2H), 2.45-2.35 (m,
1 H), 2.01-1.92 (m, 1 H), 1.6-1.4 (br s, 2 H). 13C NMR: δ
172.14, 151.48, 148.01, 132.12, 129.08, 128.29, 128.21, 127.99,
127.63, 127.01, 125.43, 122.85, 122.57, 114.97, 107.66, 55.25,
38.25, 38.15. HRMS (ESI+) calcd for C18H17N2O [M+H]+
277.1341, found 277.1339.
Aza-dibenzocyclooctyne-Biotin Conjugate (ADIBO-biotin,
8). HBTU (1.916 g, 5.05 mmol) was added to a solution of
biotin-PEG4-acid (1.9 g, 3.87 mmol) and DIEA (0.647 g, 5.61
mmol) in CH2Cl2 (15 mL) at rt and stirred for 15 min. A solution
of 7 (1.238 g, 4.48 mmol) in CH2Cl2 (2 mL) was added
dropwise, the reaction mixture was stirred for 3 h and
concentrated under reduced pressure. The product was purified
by chromatography (CH2Cl2 to CH2Cl2/MeOH 20:1 to 100:15)
to provide 2.44 g (3.26 mmol, 87%) of 8 as colorless semisolid.
1H NMR (500 MHz, CDCl3): δ 7.66 (d, J ) 7 Hz, 1 H),

2078 Bioconjugate Chem., Vol. 21, No. 11, 2010
Kuzmin et al.
7.42-7.3 (m, 7 H), 7.28-7.25 (m, 1 H), 7.05-6.99 (m, 1 H),
6.79-6.75 (m, 1 H), 6.61 (br s, 1 H), 5.13 (d, J ) 14 Hz, 1
H), 4.5-4.45 (m, 1 H), 4.32-4.25 (m, 1 H), 3.68 (d, J ) 14
Hz, 1 H), 3.65-3.45 (m, 17 H), 3.44-3.36 (m, 2 H), 3.32-3.22
(m, 2 H), 3.15-3.07 (m, 2 H), 2.9-2.82 (m, 1 H), 2.75-2.65
(m, 1 H), 2.55-2.46 (m, 1 H), 2.33 (q, J ) 6 Hz, 2 H), 2.2 (t,
J ) 7.5 Hz, 2 H), 2.0-1.92 (m, 1 H), 1.75-1.6 (m, 4 H),
1.45-1.35 (m, 5 H). 13C NMR: δ 173.51, 171.99, 171.13,
164.01, 151.1, 148.09, 132.13, 129.14, 128.68, 128.21, 127.83,
127.19, 125.58, 123.07, 122.45, 114.72, 107.86, 70.45, 70.43,
70.39, 70.3, 70.17, 70.03, 69.98, 97.17, 61.83, 60.26, 55.69,
55.52, 53.67, 42.0, 40.52, 39.14, 36.79, 35.89, 35.26, 34.71,
28.29, 28.11, 25.63, 18.59, 17.44, 11.88. HRMS (ESI+) m/z
calcd for C39H53N5O8S [M+H]+ 750.3537, found 750.3542.
Aza-dibenzocyclooctyne-PEG4-Amine (ADIBO-PEG4-amine,
9). EDC (0.75 g, 4.68 mmol) was added to a solution of Boc-
NH-(CH2CH2O)4-CH2CH2CO2H (46) (1.57 g, 4.32 mmol) in
CH2Cl2 (15 mL) and DIEA (0.7 g, 5.4 mmol) at rt and stirred
for 15 min. A solution of ADIBO-amine 7 (1 g, 3.6 mmol) in
CH2Cl2 (1 mL) was added to the reaction mixture and stirred
for 4 h, at which time the solvent was removed under reduced
pressure and the crude product purified by chromatography
(ethyl acetate/hexanes 1:1 to 9:1) to provide 1.8 g (2.8 mmol,
80%) of crude N-Boc-protected ADIBO-PEG4-amine (9-Boc)
as yellow oil.
Slides were sonicated two times in methanol for 15 min,
thoroughly rinsed with acetone, and dried under a stream of
nitrogen.
Preparation of Aza-dibenzocyclooctyne (ADIBO, 6)-
Coated Glass Slides. Freshly prepared epoxy-coated glass slides
were placed in a solution of aza-dibenzocyclooctyne amine 6
(75 mg) and DIEA (1 mL) in DMF (100 mL) and incubated
overnight at rt. Slides were then rinsed with acetone, sonicated
for 15 min in methanol, rinsed with acetone, and dried under a
stream of nitrogen.
Preparation of Azide-Coated Glass Slides. Freshly prepared
epoxy-coated glass slides were immersed in a solution of
6-azidohexylamine (1 mL) and DIEA (1 mL) in DMF (100 mL)
overnight at rt. The slides were then sonicated for 15 min in
methanol, rinsed with acetone, and dried under a stream of
nitrogen.
Patterned Derivatization of Glass Slides with Aza-diben-
zocyclooctyne 6. One microliter drops of a 10 mM PBS solution
of dibenzocyclooctyne 6 were spotted using a micropipet on a
freshly prepared epoxy-coated slide, followed by incubation in
a humidity chamber containing PBS buffer for 12 h at rt. The
slide was washed with copious amounts of acetone, then water,
and sonicated in DMF for 30 min.
Patterned Immobilization of Fluorescent Dyes on ADIBO-
Coated Slides. Solutions of Oregon green azide (13, 0.1 mM
and 1 mM in PBS) or Lissamine rhodamine B azide (14, 0.1
mM) were spotted using a pipet (2 µL) on a freshly prepared
dibenzocyclooctyne plate and incubated in a humidity chamber
for various periods of time (vide infra). Slides were rinsed with
acetone, then sonicated for 15 min in methanol, rinsed with
acetone, and dried under a stream of nitrogen.
Patterned Immobilization of Avidin on ADIBO-Coated
Slides. One microliter drops of biotin-dPEG3+4-azide PBS
solutions of different concentrations (10 mM, 1 mM, and 0.1
mM) were spotted on a ADIBO-coated glass slide. Slides were
incubated in a humidity chamber for 1 h at rt, rinsed with
copious amounts of acetone, then water, and sonicated in DMF
for 30 min. Slides were then immersed in a blocking solution
containing 1% aminoPEG4azide in DMF and incubated over-
night at rt. The slides were then rinsed with acetone, sonicated
in DMF for 30 min, and rinsed with distilled water, followed
by immersion into a solution of avidin-FITC (50 µL of 2 mg/
mL in 10 mL of PBS) at 2 °C for 15 min. The slides were
sonicated in PBS containing 0.1% Tween 20 for 30 min, washed
with distilled water, incubated in PBS containing 0.1% of BSA
for 12 h at 2 °C, sonicated again in PBS (1% of Tween 20) for
30 min, and rinsed with distilled water.
A solution of TFA (0.48 g, 4.2 mmol) in THF (15 mL) was
added to a solution of 9-Boc (1.8 g, 2.8 mmol) in THF (30
mL) at rt. The reaction mixture was stirred overnight, and the
solvent was removed under reduced pressure. The residue was
purified by chromatography (CH2Cl2/MeOH 10:1 to 10:4) to
provide 1.15 g (2.2 mmol, 79%) of ADIBO-PEG4-amine (9) as
1
slightly yellow oil. H NMR (500 MHz): δ 7.67 (d, J ) 7.5
Hz, 1 H), 7.43-7.34 (m, 5 H), 7.31 (t, J ) 7.5 Hz, 1 H),
7.29-7.24 (m, 1 H), 6.95-6.88 (m, 1 H), 5.13 (d, J ) 14 Hz,
1 H), 4.45-4.2 (br s, 2 H), 3.7-3.5 (m, 22 H), 3.37-3.2 (m,
3 H), 2.68 (t, J ) 5 Hz, 2 H), 2.57-2.42 (m, 1 H), 2.4-2.32
(m, 2 H), 2.02-1.92 (1 H). 13C NMR (100 MHz, CDCl3):
172.13, 171.21, 151.20, 148.16, 129.3, 129.2, 128.78, 128.44,
128.31, 127.9, 127.29, 127.25, 125.68, 123.16, 122.55, 114.81,
107.92, 70.46, 70.39, 70.35, 70.32, 70.30, 70.26, 70.1, 55.62.
48.86, 36.69, 35.41, 34.67. HRMS (ESI+) m/z calcd for
C29H38N3O6 [M+H] 524.2761, found 524.2756. (Note: Integra-
1
tion of H spectra of compounds containing PEG commonly
overestimates the number of protons due to the presence of
tightly bound water and/or hydroxylic solvents.)
Cleaning and Activation of Glass Slide Surfaces. All glass
slides were sonicated for 30 min in methanol, rinsed with
acetone, and dried in an oven at 145 °C for 1 h. Piranha solution
(90 mL) was prepared by the addition of H2O2 (25 mL of 35%
v/v) in one portion to 65 mL of conc H2SO4 in a 100 mL Pyrex
beaker, which was kept in a water bath (Caution: Piranha
solution reacts violently with organic compounds and should
be handled with extreme care. Wear thick plastic gloves, lab
coat, and safety glasses. Any piranha solutions should be handled
in a fume hood at all times). The solution was carefully stirred
with a glass rod, followed by inserting glass slides into the
solution. After 1 h, slides were removed from the solution and
rinsed with copious amounts of distilled water, then acetone,
and dried in an oven for 20 min at 145 °C. Glass slides prepared
in this fashion were submitted to derivatization procedures
immediately.
Patterned Immobilization of Fluorescent Probes on Azide-
Coated Slides. One microliter drops of 1 mM of ADIBO-fluor
(10) solution in PBS were spotted on an azide plate, followed
by incubation in a humidity chamber for 12 h at rt. Slides were
rinsed with acetone, then sonicated for 15 min in methanol,
rinsed with acetone, and dried under a stream of nitrogen.
For two-color derivatization, azide-coated slides patterned
with ADIBO-fluor (10) spots as described above were immersed
in a solution of ADIBO-Rhodamine (12, 1 mM in PBS) and
incubated for 3 h at rt. Slides were rinsed with acetone, then
sonicated for 15 min in methanol, rinsed with acetone, and dried
under a stream of nitrogen.
Patterned Immobilization of Avidin on Azide-Coated
Slides. One microliter drops of ADIBO-biotin (8) solutions of
different concentrations (10 mM, 1 mM, 0.1 mM, and 0.01 mM
in PBS) were spotted on an azide-coated glass slide. Slides were
incubated in a humidity chamber for 1 h at rt, rinsed with
copious amounts of acetone, then water, and sonicated in DMF
for 30 min. Slides were then immersed in a blocking solution
containing 0.1% ADIBO-PEG4-amine (9) in DMF and incu-
Preparation of Epoxy-Derivatized Glass Slides. Freshly
activated glass slides were immersed in a solution of freshly
distilled (3-glycidyloxypropyl)dimethoxy-methylsilane (1% v/v)
and DIEA (1% v/v) in dry toluene (100 mL) at 25 °C for 16 h.

Catalyst-Free Azide-Alkyne Cycloaddition
Bioconjugate Chem., Vol. 21, No. 11, 2010 2079
Scheme 1. Synthesis of ADIBO-C6-Amine (6)a
a Reagents and conditions: (a) NH2OH.HCl, pyridine, 60%; (b) PPA, 125 °C, 73%; (c) LiAlH4, ether, 58%; (d) pyridine, CH2Cl2, 71%; (e)
pyridinium tribromide, 78%; (f) t-BuOK, THF, 88%; (g) K2CO3, aq MeOH, 58%.
Scheme 2. Synthesis of ADIBO-Biotin (8) and ADIBO-PEG4-Amine (9)a
a Reagents and conditions: (a) HBTU, DIEA, CH2Cl2, 89%; (b) Boc-NH-(CH2CH2O)4-CH2CH2CO2H, EDC, DIEA, CH2Cl2, 80%; (c) TFA,
THF, 79%.
bated overnight at rt. The slides were rinsed with acetone,
sonicated in DMF for 30 min, and rinsed with distilled water,
followed by immersion into a solution of avidin-FITC (50 µL
of 2 mg/mL in 10 mL PBS) at 2 °C for 15 min. The slides
were sonicated in a PBS solution containing 0.1% Tween 20
for 30 min, washed with distilled water, incubated in PBS
containing 0.1% BSA for 12 h at 2 °C, sonicated again in PBS
(1% Tween 20) for 30 min, and rinsed with distilled water.
Preparation of ADIBO-Coated Magnetic Beads. A solution
of ADIBO-biotin conjugate (8) (25 µL, 10 mM in PBS) was
added to a suspension of streptavidin-coated magnetic beads
(25 µL of 10 mg/mL) in 450 µL PBS. The resulting mixture
was stirred by shaking for 2 h at rt. The reaction mixture was
centrifuged at 11 000 rpm for 2 min, the supernatant liquid was
decanted, and the pellet resuspended in PBS (450 µL). The
washing step was repeated two times.
Preparation of Azide-Coated Magnetic Beads. A solution
of Biotin-dPEG3+4-azide (25 µL, 10 mM in PBS) was added
to a suspension of streptavidin-coated magnetic beads (25 µL
of 10 mg/mL) in 450 µL PBS. The resulting mixture was stirred
by shaking for 2 h at rt. The reaction mixture was centrifuged
at 11 000 rpm for 2 min, the supernatant liquid was decanted,
and the pellet resuspended in PBS (450 µL, pH 7.4). The
washing step was repeated twice.
containing 0.1% Tween 20, centrifuged, washed with PBS,
centrifuged, and resuspended in 450 µL PBS for fluorescent
microscopy imaging.
Fluorescent Labeling of Azide-Coated Magnetic Beads.
A solution of ADIBO-fluor (10, 25 µL of 10 mM in PBS) was
added to a suspension of azide-coated magnetic beads (25 µL
of 10 mg/mL) in 450 µL PBS and incubated for 3 h at rt. Beads
were centrifuged at 11 000 rpm for 1 min, the supernatant liquid
was decanted, and the pellet was resuspended in 450 µL PBS
containing 0.1% of Tween 20, centrifuged, washed with PBS,
centrifuged, and resuspended in 450 µL PBS for fluorescent
microscopy imaging.
RESULTS AND DISCUSSION
Synthesis of Aza-dibenzocyclooctyne-Amine Conjugate
(ADIBO-amine, 6). The efficient preparation of ADIBO-amine
developed in our lab is outlined in Scheme 1. It starts with the
polyphosphoric acid-catalyzed Beckman rearrangement of diben-
zosuberenone oxime, which is readily prepared by treating
commercially available dibenzosuberenone with hydroxylamine.
The lactam 1 produced in this reaction is then reduced with
lithium aluminum hydride to give dihydrodibenzo[b,f]azocine
(2). The secondary amino group in 2 is converted to amide 3
by reacting with 1.25 equiv of 6-(trifluoroacetamido)hexanoyl
chloride in the presence of pyridine. The olefin in 3 is smoothly
converted into an acetylene moiety via a bromination-dehydro-
bromination procedure to give aza-dibenzocyclooctyne 5 in
excellent yield (88%). Saponification of the trifluoroacetamide
moiety with potassium carbonate in aqueous methanol gives
Fluorescent Labeling of ADIBO-Coated Magnetic Beads.
A solution of Oregon Green-azide (13) (10 mM in PBS) was
added to a suspension of ADIBO-coated magnetic beads (25
µL of 10 mg/mL) and incubated for 3 h at rt. Beads were
centrifuged at 11 000 rpm for 1 min, the supernatant liquid was
decanted, and the pellet was resuspended in 450 µL PBS

2080 Bioconjugate Chem., Vol. 21, No. 11, 2010
Kuzmin et al.
Figure 1. Schematic representation of ADIBO derivatization of an epoxy-coated slide followed by copper-free click immobilization of Oregon
green dye. Inserts show fluorescent images ADIBO slides patterned with (A) Oregon green azide (13) and (B) Lissamine rhodamine B azide (14).
target ADIBO-C6-amine (6). ADIBO-amine with a shorter
aminoakyl side chain, ADIBO-C3-amine (7), was prepared
following the same synthetic sequence by replacing 6-(trifluo-
roacetamido)hexanoyl chloride in step “d” with 3-(trifluoroac-
etamido)propionyl chloride.
Aza-dibenzocyclooctyne-Biotin Conjugate (ADIBO-bio-
tin, 8). 8 was prepared by HBTU (1-[bis(dimethylamino)meth-
ylene]-1H-benzotriazolium-3-oxide hexafluorophosphate)-pro-
moted coupling of ADIBO-C3-amine (7) with biotin-PEG4-acid
(Scheme 2). EDC-induced coupling of compound 7 with N-Boc-
15-amino-4,7,10,13-tetraoxapentadecanoic acid, followed by
trifluoroacetic acid-catalyzed removal of N-Boc protection gave
Aza-dibenzocyclooctyne-PEG4-amine (ADIBO-PEG4-amine, 9,
Scheme 2).
Figure 2. Integral fluorescent intensity of Oregon green azide (13) spots
on ADIBO-derivatized slide versus reaction time.
ADIBO and Azide Functionalization of Glass Slides.
Derivatization experiments were performed on glass surfaces
due to the ready availability, low cost, high mechanical stability,
low intrinsic fluorescence, and easy surface modification
techniques of the glass substrate. Freshly prepared epoxide-
coated slides were incubated in a DMF solution of ADIBO-
amine 6 or 6-azidohexylamine in the presence of Hu¨nig’s base
(N-ethyl-N,N-diisopropylamine) overnight and washed with
acetone, then methanol (Figures 1 and 5).
Oregon Green azide (13) was used as a model compound to
assess the efficiency and kinetics of the metal-free azide click
immobilization on ADIBO-coated slides. To ensure that a
fluorescent dye is immobilized on the slides only by the click
reaction and not due to physical absorption or other chemical
reactions, we spotted the epoxy-coated plate with ADIBO-amine
(6). After overnight incubation and washing, these plates were
immersed in a PBS solution of Oregon green azide (13, 0.01
mM) for 100 min and washed with acetone, then sonicated for
15 min in methanol, rinsed with acetone, and dried under a
stream of nitrogen. The fluorescence image of the resulting slide
demonstrates that dye 13 specifically binds to the ADIBO-
derivatized surface and not to the rest of the slide (Figure 1,
insert A). While the value of relative fluorescence intensity (spot
versus background) depends on the starting epoxy plate (we
have also tested VWR Microarray Epoxy 2 Slides and Corning
Epoxide Slides), Oregon green spots always showed bright
fluorescence with 2000-6000 contrast ratio. In the following
fluorescent dye patterning experiments, 2 µL drops of 0.1 mM
or 0.01 mM PBS solutions of azide-dye conjugate were applied
onto ADIBO-derivatized slides. Thus, 0.1 mM solution of
Lissamine rhodamine B azide (14) was spotted on the ADIBO-
Figure 3. Integral fluorescent intensity of Oregon green azide (13) spots
on ADIBO-derivatized slide versus concentration (10 µM, 50 µM, 0.1
mM, 1 mM, and 5 mM).
slide, incubated for 1 h, and thoroughly washed. The fluores-
cence image also has a good contrast ratio (Figure 1, insert B).
Kinetics of Metal-Free Click Immobilization. To evaluate
the kinetics of the ADIBO-azide reaction on the surface, we
spotted 2 µL drops of a 0.1 mM Oregon green azide (13) PBS
solution onto ADIBO-derivatized slides. The first spot was
allowed to react for 284 min; subsequent drops were applied at
different times, with the last drop applied just 5 min before
washing. The slides were stored in a humidity chamber during
this procedure. The immobilization reaction shows excellent
kinetics. Within 5 min, the relative fluorescent intensity reaches

Catalyst-Free Azide-Alkyne Cycloaddition
Bioconjugate Chem., Vol. 21, No. 11, 2010 2081
Figure 4. Schematic representation of patterned biotinylation of ADIBO-coated slide followed by selective immobilization of avidin-FITC. The
insert shows the fluorescent image of ADIBO-slide spotted with 1 µL of the following solutions: lanes 1-3, 10 mM, 1 mM, and 0.1 mM PBS
solutions of Biotin-dPEG3 + 4-azide; lane 4, 1 mM biotin-PEG4-Ct CH; lane 5, 0.1 mM of Oregon green azide (13). The slide was then treated
with aminoPEG4azide and developed with avidin-FITC solution.
Figure 5. Schematic representation of azide derivatization of an epoxy-coated slide followed by copper-free click immobilization of ADIBO-fluor
(10). Inserts show fluorescent images of azide slides patterned with (A) ADIBO-fluor (10) and (B) ADIBO-Rhodamine (12).
44-80% of the maximum value, and saturation of fluorescence
is achieved at ca. 100 min at 0.1 mM of azide (Figure 2). As a
result, incubation with the coupling reagent for 100 min was
selected as a standard procedure for subsequent experiments.
To optimize the concentration of the substrate for the metal-
free click immobilization, we spotted an ADIBO slide with PBS
solutions of various concentration of Oregon green azide (13):
10 µM, 50 µM, 0.1 mM, 1 mM, and 5 mM. After incubation
for 100 min and washing, the image of the slide was recorded
and the integral fluorescent intensity analyzed (Figure 3). The
saturation of Oregon green fluorescence is achieved at ap-
proximately 0.1 mM concentration of the azide 13.
(47) is widely used in bioconjugation and surface immobilization
applications (1-5, 10, 48-53). Therefore, we decided to test
the efficiency of surface biotinylation using a copper-free azide
click reaction. As shown in Figure 4, 1 µL drops of three
different concentrations (10 mM, 1 mM, and 0.1 mM) of biotin-
dPEG3+4-azide solutions in PBS were applied onto an ADIBO-
functionalized glass slide. For a comparison, 1 µL drops of a
0.1 mM PBS solution of Oregon green azide (13) were also
spotted on the slide. To test for the possibility of nonspecific
absorption of biotin conjugates on the ADIBO slides, a 1 mM
PBS solution of biotin-PEG4-Ct CH was also spotted on the
slide. After an hour-long incubation and washing, slides were
immersed in a 1% DMF solution of aminoPEG4azide and
incubated overnight. Our initial experiment showed that diben-
zocyclooctynes have significant affinity toward proteins. Ex-
Biotinylation of ADIBO-Slides and Immobilization of
Avidin. The exceptional selectivity and high binding constant
between avidin and biotin (dissociation constant ) 10-15 M)

2082 Bioconjugate Chem., Vol. 21, No. 11, 2010
Kuzmin et al.
After immobilization, slides are often subjected to vigorous
washing procedures, including the use of detergents and
sonication in aqueous solutions and organic solvents. To test
the stability of the azide surface to the washing procedures
and to explore the feasibility of multisubstrate surface
derivatization using copper-free click chemistry, we studied
the sequential immobilization of two fluorescent dyes. The
azide-coated glass slide was initially spotted with 1 µL drops
of a 1 mM PBS solution of ADIBO-fluor (10) and incubated
in a humidity chamber for 12 h at rt. The slide was rinsed
with acetone, sonicated in DMF for 30 min, and rinsed with
distilled water, then immersed in a 0.1 mM solution of
ADIBO-Rhodamine (12) for another 12 h, then thoroughly
washed. The fluorescent image recorded with a green
(495-520 nm) filter clearly shows a pattern of fluorescein-
immobilized spots (Figure 6A). The bright background of
Lissamine rhodamine B fluorescence and dark spots, where
azide groups were consumed in the first step, are visible on
the image recorded with a red (532-580 nm) filter (Figure
6B). Figure 6C shows merged images A and B, which
demonstrates that reactivity of an azide-derivatized surface
is not significantly affected by washing procedures.
Figure 6. Two-color derivatization of azide slides. The slide was spotted
with ADIBO-fluor (10), washed, and immersed in a solution of ADIBO-
Rhodamine (12). (A) Fluorescent image recorded using 495/520 nm
filter; (B) image recorded using 532/580 nm filter; (C) images A and
B merged.
posure to the aminoPEG4azide solution converts unreacted aza-
dibenzocyclooctyne fragments into triazole-PEG conjugates.
Patterned biotinylated slides were developed with an avidin-
FITC PBS solution for 15 min at 2 °C and washed thoroughly.
The fluorescent image of this slide shows selective immobiliza-
tion of the protein in the biotinylated areas, while nonspecific
binding of avidin was not observed (Figure 4). Incubation of
the avidin-FITC-patterned slides in a PBS solution containing
BSA does not reduce the fluorescence, confirming the im-
mobilization of avidin via specific biotin-avidin interactions.
Patterning of Fluorescent Dyes on Azide-Coated Slides.
To assess the efficiency of a reverse copper-free click surface
derivatization, we prepared azide-coated glass slides (vide
supra). Such a surface provides a convenient platform for
immobilization using both conventional (i.e., copper-catalyzed)
and catalyst-free azide click reactions. One microliter drops of
a 1 mM PBS solutions of aza-dibenzocyclooctyne 6 conjugates
with fluorescein (ADIBO-fluor, 10) or with Lissamine rhodamine
B (ADIBO-Rhodamine, 12) were allowed to react for 12 h, then
washed thoroughly. Fluorescent images of the resulting slides
with fluorescein (Figure 5, insert A) and Lissamine rhodamine
B (Figure 5, insert B) illustrate the efficiency of substrate
immobilization on the azide-derivatized surface using metal-
free click chemistry.
Patterned Biotinylation of Azide-Slides and Selective
Immobilization of Avidin. To further demonstrate the ver-
satility of the copper-free click reaction for protein im-
mobilization, we have conducted patterned biotinylation of
azide-derivatized slides, followed by the immobilization of
avidin. Azide-functionalized glass slides were spotted with
1 µL drops of four different concentrations (10 mM, 1 mM,
0.1 mM, and 0.01 mM) of a PBS solution of ADIBO-biotin
(8). Slides were incubated in a humidity chamber for 30 min,
2.5 h, and 3.5 h and washed with copious amounts of acetone,
then water, and sonicated in DMF. To reduce nonspecific
binding of the protein, slides were immersed in a blocking
solution containing 0.1% ADIBO-PEG4-amine (9) in DMF
and kept overnight. The slides were then washed, incubated
Figure 7. Schematic representation of patterned biotinylation of azide-coated slides followed by selective immobilization of avidin-FITC. The
insert shows the fluorescent image of azide-derivatized slide spotted with 1 µL of ADIBO-biotin (8) solutions of the following concentrations: (1)
10 mM; (2) 1 mM; (3) 0.1 mM; (4) 0.01 mM. The first slide was incubated for 30 min, the second for 2.5 h, and the third for 3.5 h. Slides were
then immersed in a solution of ADIBO-PEG4-amine and developed with a solution of avidin-FITC.

Catalyst-Free Azide-Alkyne Cycloaddition
Bioconjugate Chem., Vol. 21, No. 11, 2010 2083
Figure 8. Schematic representation of derivatization of streptavidin-coated magnetic beads with azide and aza-dibenzocyclooctyne moieties, followed
by the metal-free azide click coupling to fluorescent dyes.
Figure 9. Fluorescent confocal microscope images of PBS suspensions of streptavidin-coated magnetic beads: (A) treated with ADIBO-biotin (8)
followed by reaction with Oregon green azide (13); (B) treated with biotin-PEG4-Ct CH and then with Oregon green azide (13); (C) incubated with
ADIBO-fluor only (10); (D) treated with biotin-dPEG3 + 4-azide and then with incubated ADIBO-fluor (10).
in a solution of avidin-FITC at 2 °C for 15 min, and washed
again. As in the case of patterned biotinylation of ADIBO
slides, fluorescent images show selective immobilization of
the fluorescently labeled protein in biotinylated areas. No
significant nonspecific binding of avidin to the slides was
observed (Figure 7). Fluorescent intensity of the spots
produced at various concentrations of ADIBO-biotin and
incubation time illustrate the efficiency of the reaction. Even
at 10 µM concentration of ADIBO-biotin (8), a 30 min
incubation produces a brightly fluorescent spot after avidin-
FTIC development.
dibenzocyclooctyne functionalization of streptavidin beads was
achieved by reacting the former with ADIBO-biotin (8, Figure
8).
The ADIBO-functionalized streptavidin beads were suspended
in a PBS solution of Oregon green azide (13) and incubated
for 3 h at rt. Beads were washed and resuspended in PBS for
imaging via fluorescent confocal microscopy. Figure 9A shows
the bright fluorescence of these Oregon green-labeled beads.
The starting (unmodified) streptavidin magnetic beads treated
with 13 under the same conditions show no detectable emission.
As an additional control experiment, we have derivatized the
beads with terminal acetylene groups by treating streptavidin
beads with biotin-PEG4-Ct CH. Incubation of the resulting
particles in an Oregon green azide (13) solution, followed by
thorough washing, did not induce detectable fluorescence in the
beads (Figure 9B). Azide-derivatized microbeads were labeled
with fluorescein by reacting them with an ADIBO-fluorescein
Catalyst-Free Derivatization of Microbeads. To demon-
strate the utility of the copper-free azide click reaction for the
modification of microparticle surfaces, we have explored the
application of this reaction to the fluorescent labeling of
streptavidin-coated magnetic beads. The surface of the beads
was derivatized with azide groups by treating a suspension of
the beads with biotin-dPEG3+4-azide (Figure 8). The aza-

2084 Bioconjugate Chem., Vol. 21, No. 11, 2010
Kuzmin et al.
(12) Weisbrod, S. H., and Marx, A. (2008) Novel strategies for
the site-specific covalent labelling of nucleic acids. Chem.
Commun. 5675–5685.
conjugate (10, Figure 9C). Magnetic streptavidin beads directly
treated with ADIBO-fluor (10) showed no fluorescence (Figure
9D).
(13) Sletten, E., and Bertozzi, C. (2009) Bioorthogonal chemistry:
fishing for selectivity in a sea of functionality. Angew. Chem.,
Int. Ed. 48, 6974–6998.
CONCLUSIONS
We have described an efficient synthesis of aza-dibenzocy-
clooctynes (ADIBO) starting from inexpensive precursors. This
method allows for preparation of ADIBO derivatives on a large
scale. The utility and excellent kinetics of catalyst-free ADIBO-
azide cycloaddition for the patterned derivatization of glass
slides and streptavidin beads has been demonstrated. The same
metal-free click reaction was employed for the PEGylation of
unfunctionalized areas of the surface. Such treatment allowed
for dramatic reduction or complete elimination of nonspecific
binding of proteins to the surface. The strategy described in
this report provides a convenient tool for the site-specific
covalent immobilization of various biomolecules. These pro-
cedures can be especially useful in cases where the presence of
copper ions has to be avoided.
(14) Fernandez-Suarez, M., Baruah, H., Martinez-Hernandez, L.,
Xie, K. T., Baskin, J. M., Bertozzi, C. R., and Ting, A. Y. (2007)
Redirecting lipoic acid ligase for cell surface protein labeling
with small-molecule probes. Nat. Biotechnol. 25, 1483–1487.
(15) Ochiai, H., Huang, W., and Wang, L.-X. (2008) Expeditious
chemoenzymatic synthesis of homogeneous N-glycoproteins
carrying defined oligosaccharide ligands. J. Am. Chem. Soc. 130,
13790–13803.
(16) Speers, A. E., Adam, G. C., and Cravatt, B. F. (2003) Activity-
based protein profiling in vivo using a copper(I)-catalyzed azide-
alkyne [3 + 2] cycloaddition. J. Am. Chem. Soc. 125, 4686–
4687.
(17) Baskin, J. M., and Bertozzi, C. R. (2007) Bioorthogonal click
chemistry: covalent labeling in living systems. QSAR Comb. Sci.
26, 1211–1219.
(18) Rostovtsev, V. V., Green, L. G., Fokin, V. V., and Sharpless,
K. B. (2002) A stepwise Huisgen cycloaddition process: cop-
per(I)-catalyzed regioselective “ligation” of azides and terminal
alkynes. Angew. Chem., Int. Ed. 41, 2596–2599.
(19) Hein, J. E., and Fokin, V. V. (2010) Copper-catalyzed azide-
alkyne cycloaddition (CuAAC) and beyond: new reactivity of
copper(I) acetylides. Chem. Soc. ReV. 39, 1302–1315.
(20) Mamidyala, S. K., and Finn, M. G. (2010) In situ click
chemistry: probing the binding landscapes of biological mol-
ecules. Chem. Soc. ReV. 39, 1252–1261.
ACKNOWLEDGMENT
Authors thank Ms. Sara Orski and Dr. Jason Locklin for the
useful discsussions and assistance in surface characterization;
Georgia Cancer Coalition for the support of this project.
Supporting Information Available: NMR spectra of newly
synthesized compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
(21) Gaetke, L. M., and Chow, C. K. (2003) Copper toxicity,
oxidative stress, and antioxidant nutrients. Toxicology 189, 147–
163.
(22) Gierlich, J., Burley, G. A., Gramlich, P. M. E., Hammond,
D. M., and Carell, T. (2006) Click chemistry as a reliable method
for the high-density postsynthetic functionalization of alkyne-
modified DNA. Org. Lett. 8, 3639–3642.
(23) Burrows, C. J., and Muller, J. G. (1998) Oxidative nucleobase
modifications leading to strand scission. Chem. ReV. 98, 1109–
1152.
(24) 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.
LITERATURE CITED
(1) Nebhani, L., and Barner-Kowollik, C. (2009) Orthogonal
transformations on solid substrates: efficient avenues to surface
modification. AdV. Mater. 21, 3442–3468.
(2) Wong, L. S., Khan, F., and Micklefield, J. (2009) Selective
covalent protein immobilization: strategies and applications.
Chem. ReV. 109, 4025–4053.
(3) Iha, R. K., Wooley, K. L., Nystro¨m, A. M., Burke, D. J., Kade,
M. J., and Hawker, C. J. (2009) Applications of orthogonal
“click” chemistries in the synthesis of functional soft materials.
Chem. ReV. 109, 5620–5686.
(4) Ratner, B. D., Hoffman, A. S., Schoen, F. J., and Lemons, J. E.
(2004) Biomaterials science: an introduction to materials in
medicine, Academic Press, San Diego, CA.
(5) Im, S. G., Bong, K. W., Kim, B.-S., Baxamusa, S. H.,
Hammond, P. T., Doyle, P. S., and Gleason, K. K. (2008)
Patterning nanodomains with orthogonal functionalities: solvent-
less synthesis of self-sorting surfaces. J. Am. Chem. Soc. 130,
14424–14425.
(6) Fleischmann, S., Hinrichs, K., Oertel, U., Reichelt, S., Eichhorn,
K.-J., and Voit, B. (2008) Modification of polymer surfaces by
click chemistry. Macromol. Rapid Commun. 29, 1177–1185.
(7) Fournier, D., Hoogenboom, R., and Schubert, U. S. (2007)
Clicking polymers: a straightforward approach to novel macro-
molecular architectures. Chem. ReV. Soc. 36, 1369–1380.
(8) Michel, O., and Ravoo, B. J. (2008) Carbohydrate microarrays
by microcontact “click” chemistry. Langmuir 24, 12116–12118.
(9) Ku, S.-Y., Wong, K.-T., and Bard, A. J. (2008) Surface
patterning with fluorescent molecules using click chemistry
directed by scanning electrochemical microscopy. J. Am. Chem.
Soc. 130, 2392–2393.
(25) Bernardin, A., Cazet, A. l., Guyon, L., Delannoy, P., Vinet,
´
F., Bonnaff, D., and Texier, I. (2010) Copper-free click chemistry
for highly luminescent quantum dot conjugates: application to
in vivo metabolic imaging. Bioconjugate Chem 21, 583–588.
(26) Wittig, G., and Krebs, A. (1961) On the existence of low-
membered cycloalkynes. Chem. Ber. 94, 3260–3275.
(27) Ess, D. H., Jones, G. O., and Houk, K. N. (2008) Transition
states of strain-promoted metal-free click chemistry: 1,3-dipolar
cycloadditions of phenyl azide and cyclooctynes. Org. Lett. 10,
1633–1636.
(28) Baskin, J. M., Prescher, J. A., Laughlin, S. T., Agard, N. J.,
Chang, P. V., Miller, I. A., Lo, A., Codelli, J. A., and Bertozzi,
C. R. (2007) Copper-free click chemistry for dynamic in vivo
imaging. Proc. Natl. Acad. Sci. U.S.A. 104, 16793–16797.
(29) Sletten, E. M., and Bertozzi, C. R. (2008) A hydrophilic
azacyclooctyne for Cu-free click chemistry. Org. Lett. 10, 3097–
3099.
(30) Codelli, J. A., Baskin, J. M., Agard, N. J., and Bertozzi, C. R.
(2008) Second-generation difluorinated cyclooctynes for copper-
free click chemistry. J. Am. Chem. Soc. 130, 11486–11493.
(31) Jewett, J. C., Sletten, E. M., and Bertozzi, C. R. (2010) Rapid
Cu-free click chemistry with readily synthesized biarylazacy-
clooctynones. J. Am. Chem. Soc. 132, 3688–3690.
(10) Sun, X.-L., Stabler, C. L., Cazalis, C. S., and Chaikof, E. L.
(2006) Carbohydrate and protein immobilization onto solid
surfaces by sequential diels-alder and azide-alkyne cycloaddi-
tions. Bioconjugate Chem. 17, 52–57.
(11) Gramlich, P. M. E., Wirges, C. T., Manetto, A., and Carell,
T. (2008) Postsynthetic DNA modification through the copper-
catalyzed azide-alkyne cycloaddition reaction. Angew. Chem.,
Int. Ed. 47, 8350–8358.
(32) Ning, X., Guo, J., Wolfert, M. A., and Boons, G.-J. (2008)
Visualizing metabolically labeled glycoconjugates of living cells

Catalyst-Free Azide-Alkyne Cycloaddition
Bioconjugate Chem., Vol. 21, No. 11, 2010 2085
by copper-free and fast huisgen cycloadditions. Angew. Chem.,
Int. Ed. 47, 2253–2255.
(33) Debets, M. F., van Berkel, S. S., Schoffelen, S., Rutjes,
F. P. J. T., van Hest, J. C. M., and van Delft, F. L. (2010) Aza-
dibenzocyclooctynes for fast and efficient enzyme PEGylation
via copper-free (3 + 2) cycloaddition. Chem. Commun 46, 97–
99.
(42) Seitz, G., Pohl, L., and Pohlke, R. (1969) 5,6-Didehydro-
11,12-dihydrodibenzo[a,e]-cyclooctene. Angew. Chem., Int. Ed.
8, 447–448.
(43) Schwabacher, A. W., Lane, J. W., Schiesher, M. W., Leigh,
K. M., and Johnson, C. W. (1998) Desymmetrization reactions:
efficient preparation of unsymmetrically substituted linker mol-
ecules. J. Org. Chem. 63, 1727–1729.
(44) Lee, J. W., Jun, S. I., and Kim, K. (2001) An efficient and
practical method for the synthesis of mono-N-protected a,w-
diaminoalkanes. Tetrahedron Lett. 42, 2709–2711.
(45) Clark, J. S., Hodgson, P. B., Goldsmith, M. D., and Street,
L. J. (2001) Rearrangement of ammonium ylides produced by
intramolecular reaction of catalytically generated metal car-
benoids. Part 1. Synthesis of cyclic amines. J. Chem. Soc. Perkin
Trans. 1, 3312–3324.
(46) Pessi, A., Bianchi, E., and Ingallinella, P. (2009) Preparation
of cholesterol derivatives of peptidyl inhibitors of viral fusion.
PCT Int. Appl. 39.
(47) Green, N. M. (1990) Avidin and streptavidin. Methods
Enzymol. 184, 51–67.
(48) Jacobs, A. T., and Marnett, L. J. (2010) Systems analysis of
protein modification and cellular responses induced by electro-
phile stress. Acc. Chem. Res. 43, 673–683.
(49) Wilchek, M., and Bayer, E. A. (1990) Introduction to avidin-
biotin technology. Methods Enzymol. 184, 5–13.
(50) Mader, H. S., Link, M., Achatz, D. E., Uhlmann, K., Li, X.,
and Wolfbeis, O. S. (2010) Surface-modified upconverting
microparticles and nanoparticles for use in click chemistries.
Chem.sEur. J. 16, 5416–5424.
(51) Bo¨ttcher, T., and Sieber, S. A. (2010) Showdomycin as a
versatile chemical tool for the detection of pathogenesis-
associated enzymes in bacteria. J. Am. Chem. Soc. 132, 6964–
6972.
(52) Landi, F., Johansson, C. M., Campopiano, D. J., and Hulme,
A. N. (2010) Synthesis and application of a new cleavable linker
for “click”-based affinity chromatography. Org. Biomol. Chem.
8, 56–59.
(34) Link, A. J., Vink, M. K. S., Agard, N. J., Prescher, J. A.,
Bertozzi, C. R., and Tirrell, D. A. (2006) Discovery of aminoacyl-
tRNA synthetase activity through cell-surface display of non-
canonical amino acids. Proc. Natl. Acad. Sci. U.S.A. 103, 10180–
10185.
(35) Fernandez-Suarez, M., Baruah, H., Martinez-Hernandez, L.,
Xie, K. T., Baskin, J. M., Bertozzi, C. R., and Ting, A. Y.
(2007) Redirecting lipoic acid ligase for cell surface protein
labeling with small-molecule probes. Nat. Biotechnol. 25, 1483–
1487.
(36) Zou, Y., and Yin, J. (2008) Cu-free cycloaddition for
identifying catalytic active adenylation domains of nonribosomal
peptide synthetases by phage display. Bioorg. Med. Chem. Lett.
18, 5664–5667.
(37) Nessen, M. A., Kramer, G., Back, J., Baskin, J. M., Smeenk,
L. E. J., de Koning, L. J., van Maarseveen, J. H., de Jong, L.,
Bertozzi, C. R., Hiemstra, H., and de Koster, C. G. (2009)
Selective enrichment of azide-containing peptides from complex
mixtures. J. Proteome Res. 8, 3702–3711.
(38) Wilson, J. T., Krishnamurthy, V. R., Cui, W., Qu, Z., and
Chaikof, E. L. (2010) Noncovalent cell surface engineering
with cationic graft copolymers. J. Am. Chem. Soc. 132, 2845–
2845.
(39) Poloukhtine, A. A., Mbua, N. E., Wolfert, M. A., Boons, G.-
J., and Popik, V. V. (2009) Selective labeling of living cells by
a photo-triggered click reaction. J. Am. Chem. Soc. 131, 15769–
15776.
(40) 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.
(41) Chang, P. V., Prescher, J. A., Sletten, E. M., Baskin, J. M.,
Miller, I. A., Agard, N. J., Lo, A., and Bertozzi, C. R. (2010
Copper-free click chemistry in living animals. Proc. Nat. Acad.
Sci. U.S.A. 107, 1821–1826.
(53) Zhang, J., Wang, X., Wu, D., Liu, L., and Zhao, H. (2009)
Bioconjugated Janus particles prepared by in situ click chemistry.
Chem. Mater. 21, 4012–4018.
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