Sequential click - Photo-click cross-linker for catalyst-free ligation of azide-tagged substrates

Article abstract of DOI:10.1021/jo500143v

Heterobifunctional linker allows for selective catalyst-free ligation of two different azide-tagged substrates via strained-promoted azide-alkyne cycloaddition (SPAAC). The linker contains an azadibenzocyclooctyne (ADIBO) moiety on one end and a cyclopropenone-masked dibenzocyclooctyne (photo-DIBO) group on the other. The first azide-derivatized substrate reacts only at the ADIBO end of the linker as the photo-DIBO moiety is azide-inert. After the completion of the first SPAAC step, photo-DIBO is activated by brief exposure to 350 nm light from a fluorescent UV lamp. The unmasked DIBO group then reacts with the second azide-tagged substrate. Both click reactions are fast (k = 0.4 and 0.07 M^-1 s^-1, respectively) and produce quantitative yield of ligation in organic solvents or aqueous solutions. The utility of the new cross-linker has been demonstrated by conjugation of azide functionalized bovine serum albumin (azido-BSA) with azido-fluorescein and by the immobilization of the latter protein on azide-derivatized silica beads. The BSA-bead linker was designed to incorporate hydrolytically labile fragment, which permits release of protein under the action of dilute acid. UV activation of the second click reaction permits spatiotemporal control of the ligation process.

Full text of DOI:10.1021/jo500143v

Article
pubs.acs.org/joc
Sequential “Click” − “Photo-Click” Cross-Linker for Catalyst-Free
Ligation of Azide-Tagged Substrates
Selvanathan Arumugam and Vladimir V. Popik*
Department of Chemistry, University of Georgia, Athens, Georgia 30602, United States
S
* Supporting Information
ABSTRACT: Heterobifunctional linker allows for selective
catalyst-free ligation of two different azide-tagged substrates
via strained-promoted azide−alkyne cycloaddition (SPAAC).
The linker contains an azadibenzocyclooctyne (ADIBO) moiety
on one end and a cyclopropenone-masked dibenzocyclooctyne
(photo-DIBO) group on the other. The first azide-derivatized
substrate reacts only at the ADIBO end of the linker as the
photo-DIBO moiety is azide-inert. After the completion of the
first SPAAC step, photo-DIBO is activated by brief exposure to
350 nm light from a fluorescent UV lamp. The unmasked DIBO
group then reacts with the second azide-tagged substrate. Both
click reactions are fast (k = 0.4 and 0.07 M−1 s−1, respectively)
and produce quantitative yield of ligation in organic solvents or aqueous solutions. The utility of the new cross-linker has been
demonstrated by conjugation of azide functionalized bovine serum albumin (azido-BSA) with azido-fluorescein and by the
immobilization of the latter protein on azide-derivatized silica beads. The BSA−bead linker was designed to incorporate
hydrolytically labile fragment, which permits release of protein under the action of dilute acid. UV activation of the second click
reaction permits spatiotemporal control of the ligation process.
These strategies provide excellent selectivity but require
derivatization of substrates with different functionalities. To
avoid this complication, sequential click ligation may employ
reactions of the same type, most commonly azide−alkyne
cycloaddition. This approach relies on the differences in the
reactivity of alkyne moieties10c,13c,14c,18 or on the deprotection
of terminal acetylenes.10a,11c,19 The use of cytotoxic copper(I)
catalyst and/or deprotecting reagents, however, somewhat
reduces the utility of this method. In addition, terminal
acetylene were found to inhibit cysteine proteases by forming
thioether with the catalytically active thiol.20
Here, we report the development of the SPAAC-based
selective sequential click strategy, which permits cross-linking
of two different azide-tagged substrates without the use of
catalysts or activating reagents (Scheme 1). Dibenzocyclooc-
tynes are apparently too large to fit into the active site cavity
of a protease. Selection of the reactive moieties for the
construction of the sequential SPAAC cross-linking agent was
based on the orthogonality requirement. The first click
reaction should occur only at one end of the linker, while the
second ligation should not degrade connection to the first
substrate. We have chosen azadibenzocyclooctyne (ADIBO,
Scheme 1)21 as the first SPAAC moiety, as this cyclooctyne
combines high reactivity toward azides with excellent aqueous
stability and long shelf life.22 PhotoDIBO (Scheme 1), on the
INTRODUCTION
■
Structural modifications of biomolecules and polymers, as well
as derivatization of particles and surfaces, are commonly
achieved using ″click chemistry″.1 This term describes a set of
bimolecular reactions that permits the efficient formation of a
covalent link between two substrates (a.k.a. ligation in
biochemistry) or between a substrate and a surface. The
majority of ″click″ strategies are based on 1,3-dipolar or
Diels−Alder cycloadditions, imine formation, and addition to
carbon−carbon multiple bonds.2−4 Since azide and alkyne
moieties are very uncommon in natural products, i.e.,
″bioorthogonal″, the copper-catalyzed (CuAAC) or strain-
promoted azide−alkyne cycloadditions (SPAAC) have found
many applications in labeling biomolecules,5 protein mod-
ification,6 synthesis of bioconjugates,7 and developing biotech
tools.8 The chemical stability of the triazole linker made
azide−alkyne click reactions a popular tool in material
chemistry.1b,e,9
Chemoselective sequential click ligation brings the ability to
introduce multiple functionalities to biological molecules,10
drug delivery vehicles,11 polymer,12 or surfaces.13 Cross-
linking of biological molecules using sequential click chemistry
allows for the preparation of complex macromolecules.14
There are two different approaches to selective sequential
click ligations. One method relies on two (or more) mutually
orthogonal click reactions, e.g., CuAAC and Diels−
Alder,11a,12a−d SPAAC and SPANC,15,13a,b SPAAC and
hetero-Diels−Alder,16 as well as other combinations.11b,17
Received: January 31, 2014
Published: February 18, 2014
© 2014 American Chemical Society
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a
Scheme 1. Sequential Click−Photoclick Conjugation of Two Azides
a
(a) R1,R2 = n-Bu; (b) R1,R2 = Bn; (c) R1 = BSA, R2 = Fluorescein; (d) R1 = SiO2-beads, R2 = BSA.
Scheme 2. Preparation of ADIBO−PhotoDIBO Cross-Linker (1)
other hand, does not react with azides in the dark and
possesses excellent thermal stability.23 Exposure of photo-
DIBO moiety to a low intensity 350 nm light results in the
efficient decarbonylation and the formation of azide-reactive
dibenzocyclooctyne (DIBO).23,24 ADIBO, DIBO, and corre-
sponding triazole adducts have virtually no absorbance at 350
nm, and, therefore, are stable under photodecarbonylation
conditions.23
observed pseudo-first-order rate constants on azide concen-
tration was linear and produced second-order rate constant k=
0.406 0.001 M−1 s−1. This value is consistent with literature
data for ADIBO.21,22 After the completion of the first click
reaction, the cyclopropenone protection of the triple bond in
2 was removed by 2 min irradiation of the reaction mixture
with 350 nm fluorescent lamps (Scheme 1). The conversion
of photo-DIBO was followed by the disappearance of the
characteristic cyclopropenone bands at 331 and 347 nm and
the formation of the DIBO band at 319 nm (Figure 1a). The
disappearance of the latter band due to the addition of the
second molecule of benzyl azide to 3 also showed clean first-
order kinetics (Figure 1b). The second-order rate of this
reaction (k = 0.072 0.004 M−1 s−1) is similar to the values
reported for DIBO.22,23a
HPLC analysis of the “click” − photoactivation − second
“click” sequence indicates clean and quantitative conversion at
every step (Figure 2). Thus, 50 μM methanol solution of 1
(Figure 2a) was treated with an equimolar amount of butyl
acid and incubated for 48 h under ambient conditions to
ensure complete conversion. The HPLC trace (Figure 2b)
and ESI-HRMS of the resulting product (MH+, Calc. for
C50H54N5O7 836.4018, found 836.4016) confirmed the
quantitative formation of 2a (Note: Apparently, head-to-tail
and head-to-head isomers have very similar retention times on
C-18 column). Photodecarbonylation step produces 3a (MH+,
Calc. for C49H54N5O6 808.4069, found 808.4067) with no
detectable amounts of side products (Figure 2c). The addition
of the second equivalent of butyl azide cleanly gives the final
RESULTS AND DISCUSSION
■
The heterobifunctional ADIBO-photoDIBO cross-linker (1)
was prepared by the DCC/DMAP-assisted coupling of
ADIBO-acid (5)25 with of pehtamethylenehydroxy-derivatized
photoDIBO (6, Scheme 2).
The efficiency and selectivity of cross-linker 1 were
evaluated using its reaction with benzyl azide. The accurate
rate measurements of the first and second click reaction were
conducted by UV spectroscopy at 25
0.1 °C in methanol
under pseudo-first-order conditions. The UV spectrum of
ADIBO-photoDIBO (1) contains characteristic features of
both ADIBO (a band at 292 nm) and photoDIBO (intense
bands at 331 and 347 nm) chromophores (Figure 1a, black
trace). All stages of the conversion of 1 can be, therefore,
conveniently monitored by UV spectroscopy. The addition of
the excess of benzyl azide (0.1−20 mM) to the methanol
solution of 1 leads to the rapid disappearance of the band at
292 nm, while photoDIBO bands remain unchanged (Figure
1a, red trace). The decay ADIBO band followed the single
exponential equation well (Figure 1b). The dependence of the
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Figure 1. (a) UV spectra of 50 μM methanol solutions of ADIBO-photoDIBO (1, black line); product of the first click reaction 2b (red line);
product of photoactivation 3b (blue line); product of the second click reaction 4b (purple line). (b) Kinetics traces of the first (black circles) and
the second (blue hexagons) click reactions in the presence of 10 mM of benzyl azide.
adduct 4a after 48 h incubation (Figure 2d; MH+, Calc. for
Scheme 3. Conjugation of Azido-Derivatized Bovine Serum
Albumin (BSA) with Azido-Fluorescein (FL) Using
ADIBO-PhotoDIBO Cross-Linker
C53H63N8O6 907.4865, found 907.4867).
Figure 2. HPLC traces of the sequential click ligation (Scheme 1):
(a) starting linker 1; (b) reaction mixture after the reaction of 1 with
equimolar amount of butyl azide; (c) product of photodecarbony-
lation step (3a); (d) product (4a) of the second click reaction with
equimolar amount of azide.
(Scheme 3). The resulting Fluorescein-derivatized BSA (4c)
clearly shows characteristic absorbance and emission of the
Fluorescein chromophore (Figure 3).
The protein content of the solution of 4c was determined
using Coomassie brilliant blue dye assay, while the emission
intensity of 4c and the absorbance in the fluorescein region
were compared to the fluorescence and absorbance of an
aqueous solution of an authentic fluorescein sample of the
same concentration (Figure 3). These experiments confirm
high cross-linking efficiency (84−92%) of the ADIBO-
photoDIBO (1). The high yield of azido-BSA (7) labeling
was somewhat surprising result since it is commonly accepted
that commercial BSA samples contain 0.6−0.8 equiv of free
thiols. The rest of Cys34 moieties are believed to form a
disulfide with cysteine or glutathione.26 We believe that free
thiol content of the BSA is actually higher than reported
previously, since Ellman’s test, which uniformly used in these
measurements, often underestimates SH contents in pro-
As an illustrative example, we have employed ADIBO-
photoDIBO cross-linker (1) for the selective conjugation of
azido-derivatized BSA (7) with (3-azidopropylcarbamoyl)-
fluorescein (azido-Fl, 8, mixture of 5- and 6-isomers, Scheme
3). After each click reaction the functionalized protein was
isolated and characterized by MALDI. The azido-derivatized
BSA (7) was prepared by treating the native BSA with 1-
azido-3-iodopropane. Protein 7 was incubated with an excess
of the cross-linker 1 in aqueous solution overnight and
resulting BSA derivative 2c (Scheme 3) was isolated by gel
filtration. An aqueous solution of 2c was irradiated for 2 min
using 350 nm fluorescent lamps and treated with 50-fold
excess of azidoFL 8 overnight and purified by gel filtration
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Scheme 4. Photo-Diels-Alder Click Derivatization of Silica Microbeads with Azide Moiety
Silica beads bearing azido functionality (11) were first
derivatized with linker 1 (click 1) by overnight incubation
with ADIBO-photoDIBO (1) in DMF (Scheme 5). The
resulting microbeads were added to 10 μM PBS solution of
azido-BSA (7) and irradiated with 350 nm light for 2 min
(Scheme 5). The reaction mixture was incubated under
ambient conditions for 16 h and the beads were separated and
washed. The Bradford assay of the combined supernatants
from the reaction mixture after click reaction 2 showed that
there was virtually no protein present in the solution. This
observation illustrates the high efficiency of azido-BSA (7)
immobilization using linker 1.
As BSA was immobilized to silica beads via an acid-sensitive
2-alkoxybenzochroman fragment,30 the protein can be cleaved
from the solid support at low pH (Scheme 6). To validate the
release of the protein, silica microbeads bearing immobilized
BSA were incubated in 0.1 M perchloric acid overnight. The
protein released from the beads was isolated from the
supernatant via spin filtration and reconstituted to the original
volume in PBS. Total protein concentration in the resulting
solution was determined to be 9.12 μM by Bradford assay,
which corresponds to the ca. 91% recovery. The BSA protein
is stable in an aqueous 0.1 M perchloric acid solution, at least
for the time frame (16 h) used for the release of protein from
silica microbeads.
Figure 3. Absorption (dash−dotted line) and emission spectra (solid
line) of 11 μM PBS solution of BSA−fluorescein conjugate (10) and
emission spectrum of a 11 μM PBS solution of fluorescein (dotted
line).
teins,27 especially in proteins containing acidic Cys
residues.28,29
To test the suitability of ADIBO-photoDIBO (1) for
protein immobilization, we have employed this cross-linking
agent for the attachment of azido-BSA (7) to azide-
functionalized silica microbeads. First, a photo-Diels−Alder
click reaction30 between 8-(2-(2-(2-aminoethoxy)ethoxy)-
ethoxy)-3-(hydroxymethyl)naphthalen-2-ol (9, NH2-TEG-
NQMP)17d and (2-(2-azidoethoxy)ethoxy)ethylene produced
heterobifunctional linker 10 (Scheme 4). It is important to
note that 2-alkoxybenzochroman 10 is stable under neutral
conditions, but becomes hydrolytically labile at pH < 3.30
Linker 10 was then EDC-coupled to commercial carboxylate-
functionalized silica microbeads to yield target azide-
derivatized microbeads bearing acid-labile linker (11, Scheme
4).
CONCLUSIONS
■
The heterobifunctional ADIBO-photoDIBO linker (1) allows
for the efficient ligation of azide-tagged substrates, or for the
immobilization of azide-functionalized molecules on an azide-
coated surfaces. Since the covalent conjugation is achieved via
strained-promoted azide−alkyne cycloaddition (SPAAC) and
photochemical activation, this method does not require any
catalysts or other reagents. This sequential click strategy is
applicable to both homogeneous and heterogeneous cross-
linking. The use of phototrigger in the sequential click system
enables the spatiotemporal control to the cross-linking
chemistry. Additionally, the incorporation of a hydrolytically
Scheme 5. Immobilization of Azido-BSA on Azide-Functionalized Silica Microbeads
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Scheme 6. Release of BSA from Silica Microbeads
ca. 1 μmol) in 3 mL of 0.1 N phosphate buffer (pH = 8) containing
0.5 mL of acetonitrile and gently shaken for 12 h at r.t. The aqueous
layer was washed with ethyl acetate and was freeze-dried to produce
azido-derivatized BSA (80 mg, contains some phosphate salt).
Ellman’s test performed after completion of the reaction gave
negative results. Azido-derivatized BSA (7) was further purified by
spin filtration. MALDI TOF: 66546.
0.3 mg of linker 1 (400 μmol) was added to the solution of azido-
BSA 7 (13.2 mg, ca. 0.2 μmol) in 2 mL of PBS and the mixture was
incubated for 16 h at r.t. on a mechanical shaker. The excess of 1
was removed by filtration through a Dextran desalting column. The
product 2c was characterized by MALDI-TOF: 67283.
labile fragment in protein−surface linker, allows for the release
of a protein under the action of dilute acid.
EXPERIMENTAL SECTION
■
General Information. All organic solvents were dried and freshly
distilled before use. Flash chromatography was performed using 40−
63 μm silica gel. All NMR spectra were recorded on 400 MHz
instruments in CDCl3 and referenced to TMS unless otherwise
noted. Solutions for HPLC and UV−vis analysis were prepared using
HPLC grade solvents. HPLC analysis was conducted using analytical
C-18 column and a diode array detector. Photochemical decarbon-
ylation of photoDIBO moiety was conducted by the irradiation of
the reaction mixture for 1−2 min in a photochemical reactor
equipped with four fluorescent UV lamps (4 W, 350 nm).
A solution of BSA 2c (13.2 mg, ca. 0.2 μmol) in 2 mL of PBS was
irradiated for 2 min and a solution of azido-Fl 8 (0.5 mg ca 1.1 μM)
in 2 μL of DMF was added. The reaction mixture was incubated 16
h at r.t. on a mechanical shaker. The unreacted azido-fluorescein was
removed by filtration through a Dextran desalting column.
Fluorescein−BSA conjugate 4c was characterized by MALDI-TOF:
67713. Total protein content in the solution of 4c was determined
by Bradford assay using Brilliant Blue G-250 dye. The Bradford assay
standard curve was obtained from a series of standard BSA solution
in the concentration range 1−15 μM and the unknown protein
concentration was determined from the quadratic fit of the standard
data. The concentration of protein in fluorescein−BSA conjugate 4c
was found to be 11.13 μM. The emission (519 nm) and absorbance
(494 nm) of this solution were compared to corresponding spectral
features of 11 μM solution of fluorescein, confirming 84−92% of
BSA derivatization.
Preparation of Azide-Derivatized Silica Beads. (2-Azidoethoxy)-
ethyl vinyl ether (96 mg, 0.6 mmol) was added to a solution of 8-(2-
(2-(2-aminoethoxy)ethoxy)ethoxy)-3-(hydroxymethyl) naphthalen-2-
ol (9, NH2-TEG-NQMP, 70 mg, 0.22 mmol) in 20 mL of aqueous
acetonitrile (1:1). The reaction mixture was irradiated for 1 h. The
solvent and excess of (2-azidoethoxy)ethyl vinyl ether was removed
under reduced pressure. The crude amine 11 was taken into the next
step without further purification. EDC.HCl (66 mg, 0.3 mmol) and a
catalytic amount of DMAP were added to a dispersion of carboxy-
functionalized silica microspheres (2 g) in 20 mL of dry DMF,
followed by addition of amine 11. The mixture was stirred for 48 h
at r.t. and the solvent was removed by centrifugation. The azido
derivatized silica beads (12) were thoroughly washed with DMF trice
and dried under nitrogen.
Materials. Bovine serum albumin (BSA) was purchased from
Boehringer Mannheim. ADIBO-carboxylic acid (5),25 cycloprope-
none-masked DIBO-OH (6),23a 8-(2-(2-(2-aminoethoxy)ethoxy)-
ethoxy)-3-(hydroxymethyl)naphthalen-2-ol (9),17d (2-azidoethoxy)-
ethyl vinyl ether,17d 5- and 6-(3-azidopropylcarbamoyl)fluorescein
(azido-Fl, 8, mixture of isomers)23b were prepared following
previously reported procedures. Carboxy-functionalized silica micro-
spheres (0.01 mmol of acid/g, 1 μm) was purchased from
Polysciences Inc.
ADIBO-PhotoDIBO Cross-Linker (1). N,N′-Dicyclohexylcarbodii-
mide (62 mg, 0.3 mmol) and a catalytic amount of DMAP were
added to a solution of ADIBO-carboxylic acid (5) (150 mg, 0.38
mmol) in 8 mL of dry DMF, followed by a dropwise addition of a
solution of alcohol 6 (151 mg, 0.42 mmol) in 2 mL of DMF. The
mixture was stirred for 12 h at r.t., the solvent was removed in a
vacuum, the residue was dissolved in DCM, washed with NaHCO3
solution, brine, dried over anhydrous magnesium sulfate, and
concentrated under reduced pressure. The residue was purified by
column chromatography (10% MeOH in DCM) to yield 225 mg
1
(80%) of ADIBO-photoDIBO (1). H NMR: 7.93 (dd, J = 8.9, 6.6
Hz, 2H), 7.66 (dd, J = 7.4, 1.5 Hz, 1H), 7.43−7.21 (m, 7H), 6.83−
6.93 (m, 4H), 6.06 (t, J = 6.6 Hz, 1H), 5.12 (d, J = 13.9 Hz, 1H),
4.07 (t, J = 6.4 Hz, 2H), 4.02 (t, J = 6.4 Hz, 2H), 3.87 (s, 3H), 3.59
(d, J = 13.9 Hz, 1H), 3.29−3.39 (m, 3H), 3.16−3.27 (m, 1H), 2.55−
2.65 (m, 2H), 2.30−2.50 (m, 1H), 2.28 (t, J = 7.5 Hz, 2H), 2.04 (t,
J = 7.5 13 Hz, 2H), 1.76−1.99 (m, 3H), 1.72−1.38 (m, 6H). 13C
NMR: 173.6, 172.7, 172.3, 162.9, 162.5, 154.2, 151.5, 148.5, 148.3,
148.3, 142.8, 142.6, 136.3, 136.3, 132.6, 129.5, 129.1, 128.9, 128.8,
128.3, 127.7, 126.1, 123.4, 123.0, 116.8, 116.7, 116.3, 115.3, 112.8,
112.3, 108.2, 68.6, 64.8, 63.3, 55.99, 55.98, 35.9, 35.7, 35.2, 34.0,
29.5, 29.1, 26.2, 21.3. FW calc [(C46H44N2O7)H+]: 737.3221; ESI-
HRMS: 737.3217.
Immobilization of Azido-BSA 7 on Azide-Coated Silica Microbe-
ads (12). A solution of ADIBO-photoDIBO 1 (1.5 mg, 2 μM) in 10
μL of methanol was added to a suspension of the azide-derivatized
silica beads 11 (100 mg) in DMF (10 mL) and incubated overnight.
The beads were filtered, washed, and allowed to dry. Excess of silica
beads (50 mg, 0.5 μmol in ADIBO-photo DIBO) were added to 3.5
mL of 10 μM PBS solution azido-BSA 7 (2.33 mg, 0.035 μmol) and
Sequential Labeling of BSA with Azido-Fl. 3-Azidoprop-1-yl
iodide (2.1 mg, 10 μmol) was added to a solution of BSA (66 mg,
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irradiated under vigorous stirring with 350 nm light for 2 min
(Scheme 5). The reaction mixture was incubated under ambient
conditions for 16 h and the beads were separated by centrifugation
and washed.
Release of BSA from Solid Support. 50 mg of silica microbeads
with immobilized BSA were incubated in 1 mL of 0.1 M perchloric
acid overnight under mild shaking. The beads were separated and
washed with 1 mL PBS buffer 5 times. The protein was isolated from
combined supernatants (6 mL) by spin-filtration and reconstituted in
3.5 mL of PBS. The resulting solution was analyzed by the Bradford
Assay.
Sharpless, K. B.; Finn, M. G. J. Am. Chem. Soc. 2003, 125, 3192.
(c) Strable, E.; Prasuhn, D. E., Jr.; Udit, A. K.; Brown, S.; Link, A. J.;
Ngo, J. T.; Lander, G.; Quispe, J.; Potter, C. S.; Carragher, B.;
Tirrell, D. A.; Finn, M. G. Bioconjugate Chem. 2008, 19, 866.
(8) (a) Panda, S.; Sato, T. K.; Hampton, G. M.; Hogenesch, J. B.
Trends Cell Biol. 2003, 13, 151. (b) MacBeath, G.; Schreiber, S. L.
Science 2000, 289, 1760. (c) Delehanty, J. B.; Ligler, F. S. Anal.
Chem. 2002, 74, 5681. (d) Chen, C. S.; Alonso, J. L.; Otsuni, E.;
Whiteside, G. M.; Ingber, D. E. Biochem. Biophys. Res. Commun.
2003, 307, 355. (e) Chiellini, F.; Bizzarri, R.; Ober, C. K.;
Schmaljohann, D.; Yu, T.; Solaro, R.; Chiellini, E. Macromol.
Rapid. Commum. 2001, 22, 1284. (f) Wu, H.; Ge, J.;
Uttamchandani, M.; Yao, S. Q. Chem. Commun. 2011, 47, 5664.
(9) (a) Moses, J. E.; Moorhouse, A. D. Chem. Soc. Rev. 2007, 36,
1242. (b) Nandivada, H.; Jiang, X.; Lahann, J. Adv. Mater. 2007, 19,
2197.
ASSOCIATED CONTENT
* Supporting Information
■
S
NMR spectra of newly synthesized compounds. This material
is available free of charge via the Internet at http://pubs.acs.
org.
(10) (a) Gramlich, P. M. E.; Warncke, S.; Gierlich, J.; Carell, T.
Angew. Chem., Int. Ed. 2008, 47, 3442. (b) Yi, L.; Sun, H.; Itzen, A.;
Triola, G.; Waldmann, H.; Goody, R. S.; Wu, Y.-W. Angew. Chem.,
Int. Ed. 2011, 50, 8287. (c) Beal, D. M.; Albrow, V. E.; Burslem, G.;
Hitchen, L.; Fernandes, C.; Lapthorn, C.; Roberts, L. R.; Selby, M.
D.; Jones, L. H. Org. Biomol. Chem. 2012, 10 (3), 548.
AUTHOR INFORMATION
Corresponding Author
*E-mail: vpopik@uga.edu.
■
Notes
(11) (a) Chan, D. P. Y.; Owen, S. C.; Shoichet, M. S. Bioconjugate
Chem. 2013, 24, 105. (b) Goswami, L. N.; Houston, Z. H.; Sarma, S.
J.; Jalisatgi, S. S.; Hawthorne, M. F. Org. Biomol. Chem. 2013, 11,
1116. (c) Valverde, I. E.; Lecaille, F.; Lalmanach, G.; Aucagne, V.;
Delmas, A. F. Angew. Chem., Int. Ed. 2012, 51, 718.
(12) (a) Durmaz, H.; Sanyal, A.; Hizal, G.; Tunca, U. Polym. Chem.
2012, 3, 825. (b) Glassner, M.; Oehlenschlaeger, K. K.; Gruendling,
T.; Barner-Kowollik, C. Macromolecules 2011, 44, 4681. (c) Durmaz,
H.; Dag, A.; Altintas, O.; Erdogan, T.; Hizal, G.; Tunca, U.
Macromolecules 2007, 40, 191. (d) Dag, A.; Sahin, H.; Durmaz, H.;
Hizal, G.; Tunca, U. J. Polym. Sci., Part A: Polym. Chem. 2011, 49,
886. (e) Galibert, M.; Dumy, P.; Boturyn, D. Angew. Chem., Int. Ed.
2009, 48, 2576. (f) Kempe, K.; Hoogenboom, R.; Jaeger, M.;
Schubert, U. S. Macromolecules 2011, 44, 6424. (g) Ehlers, I.; Maity,
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors thank NSF (CHE-0842590) and NIH/NCI
(R01CA157766) for the support of this project.
■
REFERENCES
■
(1) Some recent reviews: (a) Takaoka, Y.; Ojida, A.; Hamachi, I.
Angew. Chem., Int. Ed. 2013, 52, 4088. (b) Wong, C. H.;
Zimmerman, S. C. Chem. Commun. 2013, 49, 1679. (c) Adzima, B.
J.; Bowman, C. N. AIChE J. 2012, 58, 2952. (d) El-Sagheer, A. H.;
Brown, T. Acc. Chem. Res. 2012, 45, 1258. (e) Gunay, K. A.; Theato,
P.; Klok, H. A. J. Polym. Sci., Part A 2013, 51, 1.
́
P.; Aube, J.; Konig, B. Eur. J. Org. Chem. 2011, 2474.
̈
(2) (a) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int.
Ed. 2001, 40, 2004. (b) Tornøe, C. W.; Christensen, C.; Meldal, M.
J. Org. Chem. 2002, 67, 3057.
(3) (a) Nebhani, L.; Barner-Kowollik, C. Adv. Mater. 2009, 21,
3442. (b) Wong, L. S.; Khan, F.; Micklefield, J. Chem. Rev. 2009,
109, 4025. Ratner, D. B. in Biomaterials science: an introduction to
materials in medicine; Hoffman, A. S., Schoen, F. J., Lemons, J. E.,
Eds.; Academic Press: San Diego, 2004.
(13) (a) Wendeln, C.; Singh, I.; Rinnen, S.; Schulz, C.; Arlinghaus,
H. F.; Burley, G. A.; Ravoo, B. J. Chem. Sci. 2012, 3, 2479. (b) Sun,
X. L.; Stabler, C. L.; Cazalis, C. S.; Chaikof, E. L. Bioconjugate Chem.
2005, 17, 52. (c) Deng, X.; Friedmann, C.; Lahann, J. Angew. Chem.,
Int. Ed. 2011, 50, 6522. (d) Broyer, R. M.; Schopf, E.; Kolodziej, C.
M.; Chen, Y.; Maynard, H. D. Dual Soft Matter 2011, 7, 9972. (e) Li,
Y.; Niehaus, J. C.; Chen, Y.; Fuchs, H.; Studer, A.; Galla, H. J.; Chi,
L. Soft Matter 2011, 7, 861.
(4) (a) Iha, R. K.; Wooley, K. L.; Nystrom, A. M.; Burke, D. J.;
̈
(14) (a) Ichikawa, S.; Ueno, H.; Sunadome, T.; Sato, K.; Matsuda,
A. Org. Lett. 2013, 15, 694. (b) Kii, I.; Shiraishi, A.; Hiramatsu, T.;
Matsushita, T.; Uekusa, H.; Yoshida, S.; Yamamoto, M.; Kudo, A.;
Hagiwara, M.; Hosoya, T. Org. Biomol. Chem. 2010, 8, 4051.
(c) Yuan, Z.; Kuang, G. C.; Clark, R. J.; Zhu, L. Org. Lett. 2012, 14,
2590. (d) Xiong, H.; Seela, F. J. Org. Chem. 2011, 76, 5584.
(e) Pujari, S. S.; Xiong, H.; Seela, F. J. Org. Chem. 2010, 75, 8693.
(15) Sanders, B. C.; Friscourt, F.; Ledin, P. A.; Mbua, N. E.;
Arumugam, S.; Guo, J.; Boltje, T.; Popik, V. V.; Boons, G.-J. J. Am.
Chem. Soc. 2011, 133, 949.
Kade, M. J.; Hawker, C. J. Chem. Rev. 2009, 109, 5620.
(b) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B.
Angew. Chem., Int. Ed. 2002, 41, 2596. (c) Hein, J. E.; Fokin, V. V.
Chem. Soc. Rev. 2010, 39, 1302. (d) Mamidyala, S. K.; Finn, M. G.
Chem. Soc. Rev. 2010, 39, 1252. (e) Im, S. G.; Bong, K. W.; Kim, B.-
S.; Baxamusa, S. H.; Hammond, P. T.; Doyle, P. S.; Gleason, K. K. J.
Am. Chem. Soc. 2008, 130, 14424.
(5) (a) Agard, N. J.; Baskin, J. M.; Prescher, J. A.; Lo, A.; Bertozzi,
C. R. ACS Chem. Biol. 2006, 1, 644−648. (b) Link, A. J.; Vink, M. K.
S.; Agard, N. J.; Prescher, J. A.; Bertozzi, C. R.; Tirrell, D. A. Proc.
Natl. Acad. Sci. U.S.A. 2006, 103, 10180. (c) Tanrikulu, I. C.;
Schmitt, E.; Mechulam, Y.; Goddard, W. A., III; Tirrell, D. A. Proc.
(16) Neves, A. A.; Stockmann, H.; Wainman, Y. W.; Kuo, J. C-H.;
̈
Fawcett, S.; Leeper, F. J.; Brindle, K. M. Bioconjugate Chem. 2013,
24, 934.
Natl. Acad. Sci. U.S.A. 2009, 106, 15285. (d) Fernan
Baruah, H.; Martínez-Hernandez, L.; Xie, K. T.; Baskin, J. M.;
́ ́
dez-Suarez, M.;
(17) (a) Lee, L. A.; Wang, Q. Angew. Chem., Int. Ed. 2012, 51,
4004. (b) Meyer, A.; Spinelli, N.; Dumy, P.; Vasseur, J. J.; Morvan,
F.; Defrancq, E. J. Org. Chem. 2010, 75, 3927. (c) Arumugam, S.;
Orski, S.; Locklin, J.; Popik, V. V. J. Am. Chem. Soc. 2012, 134, 179.
(d) Arumugam, S.; Popik, V. V. J. Am. Chem. Soc. 2011, 133, 15730.
(18) Elamari, H.; Meganem, F.; Herscovici, J.; Girard, C.
Tetrahedron Lett. 2011, 52, 658.
(19) (a) Valverde, I. E.; Delmas, A. F.; Aucagne, V. Tetrahedron
2009, 65, 7597. (b) Kele, P.; Mezo, G.; Achatz, D.; olfbeis, O.
Angew. Chem., Int. Ed. 2009, 48, 344.
́
Bertozzi, C. R.; Ting, A. Y. Nat. Biotechnol. 2007, 25, 1483. (e) Neef,
A. B.; Schultz, C. Angew. Chem., Int. Ed. 2009, 48, 1498.
(6) (a) Nessen, M. A.; Kramer, G.; Back, J. W.; Baskin, J. M.;
Smeenk, L. E. J.; de Koning, L. J.; van Maarseveen, J. H.; de Jong, L.;
Bertozzi, C. R.; Hiemstra, H.; de Koster, C. G. J. Proteome Res. 2009,
8, 3702. (b) Kele, P.; Mezo, G.; Achats, F.; Wolfbeis, O. S. Angew.
̈
Chem., Int. Ed. 2009, 48, 344−347.
(7) (a) Punna, S.; Kaltgrad, E.; Finn, M. G. Bioconjugate Chem.
2005, 16, 1536. (b) Wang, Q.; Chan, T. R.; Hilgraf, R.; Fokin, V V.;
2707
dx.doi.org/10.1021/jo500143v | J. Org. Chem. 2014, 79, 2702−2708

The Journal of Organic Chemistry
Article
(20) (a) Arkona, C.; Rademann, J. Angew. Chem., Int. Ed. 2013, 52,
8210. (b) Ekkebus, R.; van Kasteren, S. I.; Kulathu, Y.; Scholten, A.;
Berlin, I.; Geurink, P. P.; de Jong, A.; Goerdayal, S.; Neefjes, J.;
Heck, A. J. R.; Komander, D.; Ovaa, H. J. Am. Chem. Soc. 2013, 135,
2867. (c) Sommer, S.; Weikart, N. D.; Linne, U.; Mootz, H. D.
Bioorg. Med. Chem. 2013, 21, 2511.
(21) (a) Debets, M. F.; van Berkel, S. S.; Schoffelen, S.; Rutjes, F.
P. J. T.; van Hest, J. C. M.; van Delft, F. L. Chem. Commun. 2010,
46, 97. (b) Kuzmin, A.; Poloukhtine, A.; Wolfert, M.; Popik, V. V.
Bioconjugate Chem. 2010, 21, 2076.
(22) Orski, S.; Sheppard, G. R.; Arumugam, S.; Popik, V. V.;
Locklin, J. Langmuir 2012, 28, 14693.
(23) (a) Poloukhtine, A. A.; Mbua, N. E.; Wolfert, M. A.; Boons,
G.-J.; Popik, V. V. J. Am. Chem. Soc. 2009, 131, 15769. (b) Orski, S.
V.; Poloukhtine, A. A.; Arumugam, S.; Mao, L.; Popik, V. V.; Locklin,
J. J. Am. Chem. Soc. 2010, 132, 11024.
(24) Ning, X.; Guo, J.; Wolfert, M. A; Boons, G.-J. Angew. Chem.,
Int. Ed. 2008, 47, 2253.
(25) Cheng, Z.; Elias, D. R.; Kamat, N. P.; Johnston, E. D.;
Poloukhtine, A.; Popik, V.; Hammer, D. A.; Tsourkas, A. Bioconjugate
Chem. 2011, 22, 2021.
(26) (a) Janatova, J.; Fuller, J. K.; Hunter, M. J. J. Biol. Chem. 1968,
243, 3612. (b) Chen, S.-L.; Kim, K.-H. Arc. Biochem. Biophys. 1985,
239, 163. (c) Yasuhara, T.; Nokihara, K. Anal. Chem. 1998, 70, 3505.
(27) (a) Riener, C. K.; Kada, G.; Gruber, H. J. Anal. Bioanal. Chem.
2002, 373, 266. (b) Faulstich, H.; Tews, P.; Heintz, D. Anal.
Biochem. 1993, 206, 357. (c) Wright, S. K.; Viola, R. E. Anal.
Biochem. 1998, 265, 8.
(28) Lewis, S. D.; Misra, D. C.; Shafer, J. A. Biochemistry 1980, 19,
6129.
(29) K. Brocklehurst, K.; Little, G. Biochem. J. 1973, 133, 67.
(30) Arumugam, S.; Popik, V. V. J. Am. Chem. Soc. 2011, 133, 5573.
2708
dx.doi.org/10.1021/jo500143v | J. Org. Chem. 2014, 79, 2702−2708