Sulfated ligands for the copper(I)-catalyzed azide-alkyne cycloaddition

Article abstract of DOI:10.1002/asia.201100385

The copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), the prototypical reaction of click chemistry, is accelerated by tris(triazolylmethyl)amine-based ligands. Herein, we compare two new ligands in this family-3-[4-({bis[(1-tert-butyl-1H-1,2,3-triazol-4-yl)methyl]amino}methyl)- 1H-1,2,3-triazol-1-yl]propanol (BTTP) and the corresponding sulfated ligand 3-[4-({bis[(1-tert-butyl-1H-1,2,3-triazol-4-yl)methyl]amino}methyl)-1H-1,2, 3-triazol-1-yl]propyl hydrogen sulfate (BTTPS)-for three bioconjugation applications: 1)labeling of alkyne-tagged glycoproteins in crude cell lysates, 2)labeling of alkyne- or azide-tagged glycoproteins on the surface of live mammalian cells, and 3)labeling of azides in surface proteins of live Escherichia coli. Although BTTPS exhibits faster kinetics than BTTP in accelerating the CuAAC reaction in in vitro kinetic measurements, its labeling efficiency is slightly lower than BTTP in modifying biomolecules with a significant amount of negative charges due to electrostatic repulsion. Nevertheless, the negative charge conferred by the sulfate at physiological conditions significantly reduced the cellular internalization of the coordinated copper(I), thus making BTTPS-Cu^I a better choice for live-cell labeling.

Full text of DOI:10.1002/asia.201100385

FULL PAPERS
DOI: 10.1002/asia.201100385
Sulfated Ligands for the Copper(I)-Catalyzed Azide–Alkyne Cycloaddition
Wei Wang,[a] Senglian Hong,[b] Andrew Tran,[a] Hao Jiang,[a] Rebecca Triano,[a] Yi Liu,[c]
Xing Chen,[b] and Peng Wu*[a]
On the occasion of the 10th anniversary of click chemistry
Abstract:
The
copper(I)-catalyzed
fate (BTTPS)—for three bioconju-
gation applications: 1) labeling of
alkyne-tagged glycoproteins in crude
cell lysates, 2) labeling of alkyne- or
azide-tagged glycoproteins on the sur-
face of live mammalian cells, and 3) la-
beling of azides in surface proteins of
live Escherichia coli. Although BTTPS
exhibits faster kinetics than BTTP in
accelerating the CuAAC reaction in in
vitro kinetic measurements, its labeling
efficiency is slightly lower than BTTP
in modifying biomolecules with a sig-
nificant amount of negative charges
due to electrostatic repulsion. Never-
theless, the negative charge conferred
by the sulfate at physiological condi-
tions significantly reduced the cellular
internalization of the coordinated cop-
azide–alkyne cycloaddition (CuAAC),
the prototypical reaction of click
chemistry, is accelerated by tris(triazo-
lylmethyl)amine-based ligands. Herein,
we compare two new ligands in this
family—3-[4-({bis[(1-tert-butyl-1H-
1,2,3-triazol-4-yl)methyl]amino}meth-
yl)-1H-1,2,3-triazol-1-yl]propanol
(BTTP) and the corresponding sulfated
ligand 3-[4-({bis[(1-tert-butyl-1H-1,2,3-
triazol-4-yl)methyl]amino}methyl)-1H-
1,2,3-triazol-1-yl]propyl hydrogen sul-
Keywords: bioconjugation
· click
chemistry · copper · cycloaddition ·
glycoconjugates
per(I), thus making BTTPS–CuI
better choice for live-cell labeling.
a
Introduction
brought new technique advances in numerous biological
fields.[6,7] For example, CuAAC has offered simpler and
highly specific methods for bioorthogonal conjugation in the
covalent labeling of biomolecules.[8–11] It also allows straight-
forward derivatization of natural products to generate new
antibiotic activities.[12,13] However, the current copper(I) cat-
alyst formulation has two major problems: toxicity, which
hinders its use in living systems,[14,15] and slow kinetics,[16]
which hampers its use in quantitative functionalization of
biomolecules of limited quantities.[17]
The past 30 years have witnessed a growing synergy be-
tween advanced organic chemistry and biomedical re-
search.[1] Featuring exquisite selectivity and bioorthogonali-
ty, the copper(I)-catalyzed azide–alkyne cycloaddition
(CuAAC),[2–4] the prototypical reaction of click chemistry,[5]
is one of the prominent chemical transformations that have
[a] Dr. W. Wang, A. Tran, Dr. H. Jiang, R. Triano, Prof. P. Wu
Department of Biochemistry
Our initial screening of a small library consisting of 14
water-soluble tris(triazolylmethyl)amine-based ligands led to
the discovery of a potent ligand—2-[4-({bis[(1-tert-butyl-1H-
1,2,3-triazol-4-yl)methyl]amino}methyl)-1H-1,2,3-triazol-1-
yl]ethyl hydrogen sulfate (BTTES)—that dramatically accel-
erated the rate of the azide–alkyne cycloaddition by coordi-
nating with the in situ generated copper(I), and also ren-
dered the CuAAC nontoxic.[18] The new catalyst formulation
Albert Einstein College of Medicine
Yeshiva University
1300 Morris Park Ave, Bronx, NY 10461 (USA)
E-mail: peng.wu@einstein.yu.edu
[b] S. Hong, Prof. X. Chen
Beijing National Laboratory for Molecular Sciences
and Department of Chemical Biology
College of Chemistry and Molecular Engineering
Peking University
Beijing 100871 (P.R. China)
is BTTES–CuSO4: for live-cell labeling, [ligand]/ACTHUNRGTNEUNG[CuSO4]=
[c] Dr. Y. Liu
6:1, [CuSO4]=50–75 mm and 2.5 mm sodium ascorbate to
reduce copper(II) to copper(I) in situ (Scheme 1). Although
this catalytic system holds great promise for biocompatible
applications, further improvement of its activity and lower-
ing of the copper loading are desirable for broader biomedi-
cal applications. Building upon this discovery, we performed
The Molecular Foundry
Lawrence Berkeley National Laboratory
One Cyclotron Road, MS 67R6110
Berkeley, CA 94720 (USA)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/asia.201100385.
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Chem. Asian J. 2011, 6, 2796 – 2802

BTTES showed comparable
rate acceleration and TBTA ex-
hibited the lowest activity.
BTTPS–CuI gave the largest
slope of the reaction curve,
yielding more than 50% cyclo-
addition product within the first
30 min when a 75 mm solution
of CuSO4 was used and the
ligand/CuSO4 ratio was 6:1.
Under the same conditions,
quantitative conversion was re-
alized with increasing concen-
tration of propargyl alcohol
(300 mm). By contrast, the
TBTA-mediated reaction was
significantly slower, thereby
yielding less than 20% cycload-
dition product (Figure 1b).
Labeling of Glycoproteins
in Crude Cell Lysates
Scheme 1. CuAAC is accelerated by the copper(I)-stabilizing ligands. TBTA=tris[(1-benzyl-1H-1,2,3-triazol-4-
yl)methyl]amine, BTTPS=3-[4-({bis[(1-tert-butyl-1H-1,2,3-triazol-4-yl)methyl]amino}methyl)-1H-1,2,3-triazol-
1-yl]propyl hydrogen sulfate, BTTP=3-[4-({bis[(1-tert-butyl-1H-1,2,3-triazol-4-yl)methyl]amino}methyl)-1H-
1,2,3-triazol-1-yl]propanol.
To compare the efficacy of
BTTPS- and BTTP-mediated
CuAAC in labeling glycopro-
teins, we sought to investigate
bioconjugation
of
affinity
a structure–activity relationship study, in which we identified
a new ligand (BTTPS) that shows better kinetics in acceler-
ating the CuAAC.
probes to alkyne-tagged sialylated glycoproteins in crude
cell lysates, which is the first step in enriching these proteins
for glycoproteomic analysis. We cultured Jurkat cells, a
human T lymphocyte cell line, in medium supplemented
Herein, we compare the activity of BTTPS and the corre-
sponding unsulfated ligand (BTTP) in a set of bioconju-
gation studies: 1) labeling of alkyne-tagged glycoproteins in
crude cell lysates, 2) labeling of azide- and alkyne-tagged
glycoproteins on the surface of live mammalian cells, and
3) labeling of azide-bearing proteins on the surface of live
Escherichia coli. We discovered that both ligands were
highly efficient for all these bioconjugation applications with
biocompatibility of the catalyst further improved by ligand
sulfation.
with
peracetylated
N-(4-pentynoyl)mannosamine
(Ac4ManNAl) to introduce terminal alkynes onto the cell-
surface sialylated glycoconjugates.[20] After three days, we
lysed the cells and reacted the cell lysates with either
biotin–azide or FLAG–azide by the CuAAC. In these label-
ing reactions, 100 mm of biotin-azide or FLAG–azide was
used as the coupling partner, and the ratio of azide, ligand,
CuSO4, and sodium ascorbate was held at 1:5:2.5:25–a label-
ing condition optimized in our lab. After reaction for one
hour, we probed the modified cell lysates with anti-biotin or
anti-FLAG Western blots. Robust labeling was observed in
both cases for lysates isolated from cells treated with
Ac4ManNAl, whereas no signals were detectable for lysates
obtained from cells cultured in the absence of the sugar
(Figure 2). Consistent with the kinetic measurements, stron-
ger signals were detected for BTTPS–CuI-treated lysates
than the BTTP–CuI-treated counterparts when biotin was
used as the probe (Figure 2a). Interestingly, when FLAG
was used as the probe, the BTTP–CuI catalyst afforded
stronger signals (Figure 2b). At neutral pH, the FLAG pep-
tide has a large amount of negative charges, which may in-
terfere with the approach of BTTPS functionalized with a
negatively charged arm, thus lowering the labeling efficien-
cy.
Kinetic Evaluation of the Ligand-Accelerated
CuAAC
The relative reactivity of the copper(I) catalysts in the form
of TBTA–CuI, BTTES–CuI, BTTP–CuI, and BTTPS–CuI
complexes was determined in a fluorogenic assay by treating
propargyl alcohol (50 mm) with 3-azido-7-hydroxycoumarin
(100 mm) (Figure 1a).[19] In all studies, CuSO4·5H2O was
used as the copper source to form complexes with the li-
gands. The active catalysts were generated in situ by using a
2.5 mm solution of sodium ascorbate. BTTPS showed the
highest activity of the four tris(triazolylmethyl)amine-based
ligands evaluated in accelerating the CuAAC; BTTP and
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Labeling of Live Mammalian Cells
In our ligand design, sulfation of BTTP is used to generate a
negatively charged ligand, BTTPS, to minimize the cellular
internalization of the coordinated copper. To evaluate if sul-
fation confers the ligand with the desired property, we com-
pared BTTPS–CuI and BTTP–CuI in live-cell labeling ex-
periments. We metabolically labeled Jurkat cells with
Ac4ManNAl and reacted the treated cells with biotin–azide
(50 mm) in the presence of BTTPS–CuI or BTTP–CuI for 5–
15 min at room temperature (catalyst formulation: [ligand]/ACHTUNG-
TERNN[UG CuSO4]=6:1, [CuSO4]=75 mm). After the reaction was
quenched with bathocuprioine disulfate (BCS), the biotiny-
lated cells were incubated with Alexa Fluor 488-streptavi-
din. The cells were then stained with 7-aminoactinomycin D
(7-AAD), which is a fluorescent molecule with strong affini-
ty for double-stranded DNA, and analyzed by flow cytome-
try. 7-AAD does not pass through intact membrane, but it
readily enters damaged cells with compromised membranes.
Therefore, healthy and damaged cells can be easily distin-
guished. As shown in Figure 3a, cell-associated Alexa Fluor
488 fluorescence was detected for both BTTPS–CuI or
BTTP–CuI treated cells, and the fluorescence increased
along with increasing reaction time. Although both catalytic
systems showed comparable labeling efficiency, BTTPS–CuI
was significantly better in protecting cells from the cop-
per(I)-associated toxicity, especially for labeling with ex-
tended reaction time (15 min). More than 60% of cells were
still undamaged after treatment with BTTPS–CuI (7-AAD
negative), whereas only 26% of cells treated with BTTP–
CuI remained normal (Figure 3b). Furthermore, we noticed
that the labeling temperature also played a significant role
in modulating the copper-associated toxicity. When both
Jurkat cells and the labeling reagents were precooled to 48C
before triggering the CuAAC, more than 85% of cells re-
mained viable after a BTTPS–CuI-mediated reaction for
15 min with high labeling efficiency achieved (mean fluores-
cence intensity 2200, for reactions performed at 48C to
room temperature, versus 3679, for reactions performed at
room temperature).
Figure 1. Comparison of CuAAC kinetics in the presence of various ac-
celerating ligands. a) A fluorogenic assay for the qualitative measurement
of CuAAC kinetics. b) Conversion–time profiles of CuAAC in the pres-
ence of various ligands. Reaction conditions: propargyl alcohol (50 mm),
3-azido-7-hydroxycoumarin (100 mm), CuSO4 (75 mm), 0.1m potassium
phosphate buffer (pH 7.0)/dimethylsulfoxide (DMSO) 95:5, sodium as-
corbate (2.5 mm), room temperature ([ligand]/ACTHNURTGNENG[U CuSO4]=6:1). The reac-
tion yields were calculated against 100% conversion curve, which was ob-
tained under the same reaction conditions using BTTPS with increasing
concentration of propargyl alcohol (300 mm). Error bars represent the
standard deviation of three replicate experiments.
Our previous studies showed that significant labeling of
Ac4ManNAl-treated Jurkat cells was achieved with BTTES–
CuI-mediated click chemistry when 50–75 mm CuSO4 was
used as the copper source. With the observation that BTTPS
confers CuAAC with faster kinetics than BTTES, we were
eager to test if efficient cell labeling could be achieved with
BTTPS when using a lower copper loading. To this end, we
treated alkyne-bearing Jurkat cells with biotin–azide (50 mm)
in the presence of BTTPS–CuI for one minute at room tem-
Figure 2. Comparison of the efficiency of the BTTPS–CuI- and BTTP–
CuI-mediated azide–alkyne cycloaddition in labeling glycoproteins in
crude cell lysates. Cell lysates prepared from Ac4ManNAl-treated or un-
treated Jurkat cells were reacted with a) biotin–azide (100 mm) or
b) FLAG–azide (100 mm) in the presence of sodium ascorbate (2.5 mm),
and CuSO4 (250 mm) premixed with ligands BTTPS or BTTP (500 mm).
Reactions were allowed to proceed for 1 h at room temperature, and
were analyzed by Western blots using a horseradish peroxidase (HRP)-
conjugated anti-biotin antibody (a, top panel) or an HRP-conjugated
anti-FLAG antibody (b, top panel). Anti-tubulin Western blots were per-
form to confirm equal protein loadings (bottom panel).
perature (catalyst formulation: [ligand]/ACTHNUGTRENUNG[CuSO4]=6:1,
[CuSO4]=20–75 mm). After the reaction was quenched with
BCS, the biotinylated cells were incubated with Alexa Fluor
488-streptavidin, stained with 7-AAD, and analyzed by flow
cytometry. As shown in Figure 3c, significant labeling was
realized with as little as 30 mm copper loading.
The BTTPS–CuI catalyst is equally active in detecting
cell-surface azides in Jurkat cells metabolically treated with
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Copper(I)-Catalyzed Cycloaddition
Figure 3. Comparison of the efficiency of the BTTPS–CuI- and BTTP–CuI-mediated azide–alkyne cycloaddition in labeling sialylated glycoconjugates in
live cells. Jurkat cells were cultured in the presence or absence of Ac4ManNAl for 3 days. Cells were then treated with biotin azide (50 mm) in the pres-
ence of sodium ascorbate (2.5 mm), and CuSO4 premixed with ligands BTTPS or BTTP ([ligand]/ACHTNUGTRENUNG[CuSO4]=6:1). Reactions were quenched with BCS,
stained with Alexa Fluor 488-streptavidin, 7-AAD, and analyzed by flow cytometry. a) Mean fluorescence intensity (MFI) of cells treated with the
BTTPS–CuI or BTTP–CuI catalyst ([CuSO4]=75 mm) in the course of 5–15 min reactions. b) Percentage of viable cells without cell-membrane damage
after the click reactions with the BTTPS–CuI or BTTP–CuI catalyst ([CuSO4]=75 mm) in the course of 5–15 min reactions. c) MFI of cells treated with
the BTTPS–CuI catalyst ([CuSO4]=20–75 mm) in a reaction for 1 min. d) Percentage of viable cells without cell-membrane damage after the click reac-
tions with the BTTPS–CuI catalyst ([Cu]=20–75 mm) in a reaction for 1 min.
peracetylated N-azidoacetylmannosamine (Ac4ManNAz).[21]
To evaluate if the faster kinetic behavior of BTTPS–CuI rel-
ative to BTTES–CuI could be transferred in vivo, we com-
pared these two catalysts directly in biotinylation of azido
sialic acid (SiaNAz)-bearing Jurkat cells. Under exactly the
same labeling conditions, about 15% higher labeling effi-
ciency was achieved when using the BTTPS–CuI catalyst
(Figure 4).
We next evaluated the use of BTTP- and BTTPS-mediat-
ed CuAAC for direct fluorescence imaging of glycans on
live-cell surfaces. HeLa cells, which are a human epithelial
carcinoma cell line, were incubated with 50 mm Ac4ManNAz
to metabolically incorporate the corresponding SiaNAz into
Figure 4. Comparison of the efficiency of BTTPS–CuI and BTTES–CuI in
labeling azido sialic acids in live cells. Jurkat cells were cultured in the
presence or absence of Ac4ManNAz for 3 days. Cells were then treated
with biotin alkyne (50 mm) in the presence of sodium ascorbate (2.5 mm),
cell-surface glycoconjugates. The resulting HeLa cells with
azides on their cell surface were treated with Alexa Fluor
and CuSO4·5H2O premixed with ligands BTTPS or BTTES ([CuSO4]=
75 mm, [ligand]/[CuSO4]=6:1). After 3 min, the reactions were quenched
488-alkyne (50 mm) in the presence of BTTP–CuI or
AHCTUNGTRENNUNG
BTTPS–CuI ([ligand]/
ACHTNUTRGNEUG[N CuSO4]=6:1, [CuSO4]=50 mm). After
with BCS, stained with Alexa Fluor 488-streptavidin, 7-AAD, and ana-
lyzed by flow cytometry.
five minutes, the reaction was quenched with BCS. As
shown by confocal fluorescence microscopy, robust Alexa
Fluor 488 fluorescence was detected in the cell membrane
(Figure 5). Under exactly the same imaging conditions, com-
parable labeling efficiency was achieved by using BTTPS–
CuI and BTTP–CuI. No clear cytotoxicity was observed after
the copper(I) treatment, as confirmed by trypan blue stain
(data not shown).
Labeling of Azide-Bearing Surface Proteins in E.
Coli
The development of genetically encoded unnatural amino
acid technology based on amber nonsense codons[22] and the
complementary metabolic approach[23] allows the incorpora-
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P. Wu et al.
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Figure 5. Fluorescent imaging of SiaNAz-containing glycans on cell surfa-
ces by using biocompatible CuAAC. HeLa cells were incubated with (a–
h) or without (i–l) 50 mm Ac4ManNAz for 3 days. The cells were subse-
quently labeled with Alexa Fluor 488-alkyne by using BTTP–CuI and
Figure 6. Labeling E. coli by using CuAAC. E. coli bearing OmpC were
cultured in the presence or absence of AHA until an optical density
(O.D.) of 1 was reached, and then treated with biotin–azide (50 mm) in
the presence of sodium ascorbate (2.5 mm), and CuSO4 (75 mm) premixed
with ligands BTTPS or BTTP at room temperature. Reactions were
quenched by BCS after 10 min, and probed with Alexa Fluor 488-strepta-
vidin and analyzed by flow cytometry. MFI of E. coli reacted with
BTTPS–CuI-catalyzed AAC for 5 min with 50 mm CuSO4, [ligand]/
ACTHNUTRGNE[UNG Cu-
SO4]=1:6, sodium ascorbate 2.5 mm (a–d) ligand: BTTP, e–l) ligand:
BTTPS). The cell nuclei were stained with Hoechst 33342 prior to mi-
croscopy analysis. First column: bright field; second column: Hoechst
33342 channel; third column: Alexa Fluor 488 channel; and fourth
column: overlay. Scale bars: 20 mm.
a) BTTPS–CuI or BTTP–CuI ([ligand]/
ACHTUNGRTEN[NUNG CuSO4]=6:1 complex) and
b) BTTPS–CuI or BTTP–CuI ([ligand]/
AHCTUNGTRENNUNG
responding E. coli growth curves after the click reactions are shown in c)
and d), respectively.
tion of functional groups beyond those found in the 20 can-
onical amino acids in proteins of live cells. As demonstrated
elegantly by the Tirrell group, azide- or alkyne-bearing
groups can be introduced onto the surface of E. coli and be
further derivatized by the CuAAC.[24,25] However, bacteria
subjected to the overexpression of outer-membrane protein
C (OmpC) containing azidohomoalanine (AHA) and then
treated with canonical CuAAC reagents (i.e., CuSO4,
TBTA,[26] tris(2-carboxyethyl)phosphine) were unable to
divide after being transferred to rich medium.[24,25] To evalu-
ate if the BTTP–CuI or BTTPS–CuI catalyst could improve
the viability of the treated bacteria, we reacted E. coli cells
pulsive interactions with the negatively charged BTTPS,
thus lowering the labeling efficiency of the corresponding
BTTPS–CuI complex.[27]
To evaluate if the copper catalysts caused any long-term
perturbations to the treated bacteria, we cultured the
copper-treated and untreated E. coli in nutrient-rich
medium and followed their growth by O.D. reading. As
shown in Figure 6c and d, the bacteria bearing AHA
showed about a four-hour delay in their cell proliferation
compared with the bacteria growing with all 20 natural
amino acids through this series of experiments. Noteworthy,
copper treatment barely had any influence on the bacteria
growing with 20 natural amino acids—the copper(I)-treated
and untreated bacteria showed similar proliferation trends.
However, after bacteria bearing AHA were subjected to
CuAAC, a one-hour delay in cell proliferation was ob-
served. Nevertheless, both the copper(I)-treated and un-
treated bacteria reached same cell density 15 hours after
being transferred to the rich medium, thus suggesting only
minor toxicity was imparted to the labeled E. coli.
expressing OmpC with biotin–alkyne. Here, two [ligand]/ACTHNUTRGNEUNG[-
CuSO4] ratios, 6:1 and 4:1, were used. After reaction for ten
minutes, the bacteria were stained with Alexa Fluor 488-
streptavidin and subjected to flow cytometry analysis. As ex-
pected, bacteria expressing OmpC and induced in the pres-
ence of 20 natural amino acids had the same mean fluores-
cence as the unlabeled cells. In contrast, bacteria induced in
the presence of AHA showed a 2.2-fold increase in the fluo-
rescence of Alexa Fluor 488 over the background when the
6:1 ligand–CuI complex was used (Figure 6a). When the [li-
gand]/ACHTUNGTRENNUNG[CuSO4] ratio decreased to 4:1, more than 30-fold
fluorescence over the background was observed (Figure 6b).
Notably, with the canonical CuAAC conditions, the compa-
rable labeling level was only achieved after reaction for
16 hours.[25] In our E. Coli labeling experiments, BTTP–CuI
gave a 10% stronger signal than BTTPS–CuI (Figure 6b). In
phosphate buffered saline (PBS; pH 7.4), the surface of E.
coli is known to be negatively charged, which may have re-
Conclusion
Our systematic investigation of BTTPS–CuI and BTTP–CuI
in this series of labeling studies showed that both catalysts
were highly efficient for bioconjugation. Consistent with the
criteria of our ligand design, the negatively charged sulfate
minimized the membrane permeability of the coordinated
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Copper(I)-Catalyzed Cycloaddition
prepared sodium ascorbate. Ligands used included BTTP and BTTPS.
The samples were lightly vortexed and allowed to react for 1 h (258C,
800 rpm in eppendorf Theromomixer R). Reacted samples (8.3 mg each)
were heated in SDS-loading buffer (40% glycerol, 200 mM Tris-HCl pH
6.8, 8% SDS, 0.4% bromophenol blue) at 958C for 5 min and resolved
on 4–20% Criterion XT Precast Gels. The samples were transferred to
nitrocellulose and incubated for 1 h at room temperature in blocking
buffer (5% non-fat milk in TBST (tris-buffered saline with 0.1% Tween-
20, pH 7.5)). The blocked membrane was incubated for 1 h at room tem-
perature with an HRP anti-FLAG antibody (1:3000 dilution) or an HRP
anti-biotin antibody (1:100000 dilution) in blocking buffer, washed three
times with TBST. The membranes were developed by using SuperSignal
West Pico chemiluminescent substrate and imaged on film. To confirm
equal protein loading, the primary antibody-treated membranes were
stripped (http://www.abcam.com/ps/pdf/protocols/Stripping%20for%20re-
probing.pdf). The membranes were then incubated for 1 h at room tem-
perature in blocking buffer and then incubated with monoclonal anti-tu-
bulin clone AA13 (1:1000 dilution) for 1 h at room temperature. After
being washed three times with TBST, the membranes were incubated
with goat anti-mouse HRP (1:60000 dilution) for 1 h at room tempera-
ture, then washed three times with TBST. The membranes were incubat-
ed with SuperSignal West Pico chemiluminescent substrate and imaged
on film.
copper(I) at physiological conditions and significantly re-
tained cell viability post-copper(I) treatment. Therefore, the
BTTPS–CuI catalyst is the clear choice for live-cell surface-
labeling experiments. Importantly, a ligand/CuSO4 ratio
greater than one is critical to reduce copper(I)-associated
toxicity, a phenomenon discovered by us and the Finn group
previously.[18,28] However, the negative charge conferred by
the sulfate functionality may impose electrostatic repulsion
with probes or biomolecules bearing significant negative
charges, thus reducing the catalytic efficiency of the corre-
sponding BTTPS–CuI complex. For these reasons, BTTP–
CuI may be a better choice for in vitro labeling of negatively
charged biomolecules.
Noteworthy, this study has identified highly reliable cop-
per(I) catalyst formulations that can be easily scaled up and
adopted for various bioconjugation applications. The new
catalysts reported herein also solved longstanding problems
of canonical CuAAC, including toxicity and poor labeling
efficiency at low-substrate concentration, thus opening new
possibilities for bioorthogonal click chemistry-based molecu-
lar imaging and proteomic analysis.
HeLa Cell Labeling by CuAAC Chemistry and Imaging with Confocal
Microscopy
HeLa cells were cultured in Dulbeccoꢂs modified eagle medium
(DMEM) supplemented with or without Ac4ManNAz (50 mm) on Lab-
Tek Chambered cover glass for 3 days. The cells were washed three times
with PBS (100 mL) and treated with Alexa Flour 488-alkyne (50 mm) in a
100 mL reaction vessel containing premixed ligand—Cu complex
Experimental Section
Metabolic Labeling of Jurkat Cell and Detection by CuAAC Click
Chemistry and Flow Cytometry Analysis
([CuSO4]=50 mm, [ligand]/ACHTUNTRGNE[NUG CuSO4]=6:1) and 2.5 mm freshly prepared
sodium ascorbate for 5 min at room temperature. The reaction was
quenched with 1 mm BCS. The cells were washed three times with PBS
and stained with Hoechst 33342 at 48C for 5 min. A laser scanning confo-
cal microscope (Nikon, A1R-si) 60ꢁ was used for imaging Alexa Flour
488 on the HeLa cell surfaces.
Jurkat cells were cultured for 3 days in untreated RPMI 1640 medium or
medium containing 50 mm Ac4ManNAl (or Ac4ManNAz). The cells then
were distributed into a 96-well round-bottomed tissue culture plate (0.4–
0.5 million cells per well), pelleted (300ꢁg, 3 min), and washed twice
with labeling buffer (200 mL, PBS, pH 7.4, containing 1% fetal bovine
serum (FBS)). Cells were then resuspended in labeling buffer (92 mL),
followed by addition of 100 mm biotin–alkyne (or biotin–azide to react
Metabolic Labeling of E. Coli and Detection with CuAAC Chemistry and
Flow Cytometry Analysis
with SiaNAl), BTTP or BTTPS–Cu complex ([ligand]/ACTHNUGTRENUNG[CuSO4]=6:1) and
2.5 mm sodium ascorbate in labeling buffer at room temperature. The re-
actions were quenched by adding bathocuproine disulfonate (BCS)
copper chelator (2 mL, 50 mm). Then the cells were pelleted, washed
three times with labeling buffer, and resuspended in the same buffer con-
taining 1 mgmLÀ1 streptavidin-Alexa Fluor 488 and incubated in the dark
at 48C for 30 min. Following incubation, cells were washed three times
with labeling buffer and resuspended in FACS buffer (400 mL; Hankꢂs
Balanced Salt Solution, pH 7.4, 1% FBS, 2 mgmLÀ1 7-AAD, 0.2% NaN3)
for flow cytometry analysis. Flow cytometry experiments were performed
on a Becton Dickinson FACScan flow cytometer using a 488 nm argon
laser. At least 15000 cells were recorded for each sample. Flow cytome-
try data were analyzed by using FlowJo software. MFI was calculated for
live cells.
A single colony of M15MA[pQE-60/OmpC] was grown in M9 minimal
medium supplemented with all 20 natural amino acids and with carbeni-
cillin (100 mgLÀ1) and kanamycin (35 mgLÀ1) until O.D.600 =1. The bacte-
ria were pelleted (2500ꢁg, 10 min), resuspended in M9 medium (50 mL;
supplemented with 19 amino acids), and agitated at 378C for 10 min. The
cells were pelleted, resuspended in the same volume M9 medium (sup-
plemented with19 amino acids), and divided into two equal portions (
ꢀ25 mL each): methionine (40 mgLÀ1) was added to one and AHA
(40 mgLÀ1) was added to the other. Isopropyl b-d-1-thiogalactopyrano-
side (IPTG; 1 mm) was added to each culture and then shaken at 378C
for 3 h. The E. Coli cultures (ꢀ25 mL each) were centrifuged at 2500ꢁg
for 5 min and washed once with PBS (12.5 mL). The cells were centri-
fuged again and resuspended in PBS (2.5 mL). In a 96-well round-bot-
tomed tissue culture plate, biotin–alkyne (100 mm) and premixed ligand–
Metabolic Labeling of Jurkat Cell Glycoproteins and Detection by
CuAAC Click Chemistry and Western Blot
Cu complex ([CuSO4]=75 mm, [ligand]/ACHTNURGTNEUNG[CuSO4]=4:1 or 6:1) were added
to each well containing an aliquot of these bacteria (200 mL). After
10 min, BCS was added (1 mm) to the bacteria to quench the reaction.
Bacteria were then washed three times with PBS (200 mL), then were re-
suspended in PBS (200 mL), and divided into two portions. Portion one
was diluted with PBS to 1 mL, from which 10 mL was taken and diluted
with M9 medium containing all 20 amino acids (190 mL; 40 mgLÀ1 each)
as well as carbenicillin (100 mgLÀ1) and kanamycin (35 mgLÀ1), and
shaken at 378C. O.D.600 was measured every 15 min for an 18 h period by
using a Synergy Hybrid plate reader. Portion two was incubated with
Alexa Fluor 488-streptavidin (final concentration 1 mgmLÀ1) at 48C for
25 min. Bacteria were then washed three times with PBS (200 mL), resus-
pended in PBS (200 mL), and analyzed by flow cytometry. Flow cytome-
try experiments were performed on an Eclipese iCyt flow cytometer
Jurkat cells were incubated for 3 days in RPMI medium or RPMI
medium containing 50 mm Ac4ManNAl. The cells were washed with PBS,
harvested by centrifugation (300g, 3 min), and homogenized in lysis
buffer (100 mm sodium phosphate, 150 mm NaCl, 1% NP-40, pH 7.4)
containing protease inhibitors (Roche complete tablets, ethylenediamine-
tetraacetic acid (EDTA) free) by 10 freeze–thaw cycles. Insoluble debris
was removed by centrifugation (10000g, 10 min) and the soluble protein
concentration was determined by using the DC protein assay kit. To
label the sialylated glycoproteins, protein was resuspended at a concen-
tration of 0.69 mgmLÀ1 and treated with 100 mm FLAG–azide or 100 mm
biotin–azide in a 100 mL reaction vessel containing premixed ligand–Cu
complex ([CuSO4]=250 mm, [ligand]/
ACHTUNGRTEN[NUNG CuSO4]=2:1) and 2.5 mm freshly
Chem. Asian J. 2011, 6, 2796 – 2802
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemasianj.org
2801

P. Wu et al.
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Acknowledgements
This work was partially supported by the National Institutes of Health
grants GM080585 and GM093282 (P.W.). Part of the ligand synthesis was
performed as a User Project at the Molecular Foundry, Lawrence Berke-
ley National Laboratory, which was supported by the Office of Science,
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Received: April 15, 2011
Published online: September 8, 2011
2802
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Chem. Asian J. 2011, 6, 2796 – 2802