Optimizing the selectivity of DIFO-based reagents for intracellular bioorthogonal applications

Article abstract of DOI:10.1016/j.carres.2013.05.014

One of the most commonly employed bioorthogonal reactions with azides is copper-catalyzed azide-alkyne [3+2] cycloaddition (CuAAC, a 'click' reaction). More recently, the strain-promoted azide-alkyne [3+2] cycloaddition (SPAAC, a copper-free 'click' reaction) was developed, in which an alkyne is sufficiently strained to promote rapid cycloaddition with an azide to form a stable triazole conjugate. In this report, we show that an internal alkyne in a strained ring system with two electron-withdrawing fluorine atoms adjacent to the carbon-carbon triple bond reacts to yield covalent adducts not only with azide moieties but also reacts with free sulfhydryl groups abundant in the cytosol. We have identified conditions that allow the enhanced reactivity to be tolerated when using such conformationally strained reagents to enhance reaction rates and selectivity for bioorthogonal applications such as O-GlcNAc detection. Published by Elsevier Ltd.

Full text of DOI:10.1016/j.carres.2013.05.014

Carbohydrate Research 377 (2013) 18–27
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Optimizing the selectivity of DIFO-based reagents for intracellular
bioorthogonal applications
a
b
c
d
d
d
Eun J. Kim , Dong W. Kang , Hans F. Leucke , Michelle R. Bond , Salil Ghosh , Dona C. Love ,
e
f
d,
⇑
Jong-Seog Ahn , Dae-Ook Kang , John A. Hanover
a
Department of Science Education-Chemistry Major, Daegu University, GyeongBuk 712-714, South Korea
b
c
d
e
f
Department of Pharmaceutical Science and Technology, Catholic University of Daegu, GyeongBuk 712-702, South Korea
Laboratory of Bioorganic Chemistry, NIDDK, National Institute of Health, Bethesda, MD 20892, USA
Laboratory of Cell Biochemistry and Biology, NIDDK, National Institute of Health, Bethesda, MD 20892, USA
Chemical Biology Research Center, Bio-Therapeutics Research Institutes, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Cheongwon 363-883, South Korea
Department of Biochemistry and Health Science, College of Natural Sciences, Changwon National University, Changwon 641-773, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
One of the most commonly employed bioorthogonal reactions with azides is copper-catalyzed azide–
alkyne [3+2] cycloaddition (CuAAC, a ‘click’ reaction). More recently, the strain-promoted azide–alkyne
Received 25 March 2013
Received in revised form 15 May 2013
Accepted 19 May 2013
[3+2] cycloaddition (SPAAC, a copper-free ‘click’ reaction) was developed, in which an alkyne is suffi-
ciently strained to promote rapid cycloaddition with an azide to form a stable triazole conjugate. In this
report, we show that an internal alkyne in a strained ring system with two electron-withdrawing fluorine
atoms adjacent to the carbon–carbon triple bond reacts to yield covalent adducts not only with azide
moieties but also reacts with free sulfhydryl groups abundant in the cytosol. We have identified condi-
tions that allow the enhanced reactivity to be tolerated when using such conformationally strained
reagents to enhance reaction rates and selectivity for bioorthogonal applications such as O-GlcNAc
detection.
Available online 28 May 2013
Keywords:
Copper-catalyzed azide–alkyne
cycloaddition
Strain-promoted azide–alkyne cycloaddition
O-GlcNAc
Glycoconjugates
Published by Elsevier Ltd.
1
. Introduction
modified-Staudinger ligation with a triaryl phosphine probe3,10,11
(
Fig. 1A) and the copper(I)-catalyzed cycloaddition with a terminal
1
2,13
14,15
Detection and isolation of metabolites and posttranslationally
alkyne probe, which is called CuAAC
(Fig. 1B).
a ‘click’ reaction
modified biomolecules such as glycoproteins and glycolipids can
be achieved by tagging target biomolecules with a bioorthogonal
functional group, a ‘chemical handle’, and using highly selective
chemical reactions (chemoselective reactions) between the chem-
ical handle and an appropriate reagent probe. The chemical handle
should be unreactive with native biological functional groups (i.e.,
bioorthogonal), and it should be incorporated into the target bio-
Compared to the Staudinger ligation, Cu(I)-catalyzed ‘click’
chemistry is faster and more sensitive, making this method attrac-
1
6–18
tive for bioconjugate chemistry.
However, applying CuAAC to
bioconjugate chemistry introduces several challenges, specifically
in biological settings. First, the more stable Cu(II) ion is initially
introduced as a Cu(II) salt and the Cu(I) catalyst is generated
in situ by reduction of Cu(II) using a reducing agent such as sodium
ascorbate. The Cu(I)-catalyzed ‘click’ reaction requires optimiza-
tion of several factors such as metal, ligand, and auxiliary reductant
concentrations. Furthermore, the copper ion(II) used in many
1
–6
molecules using either the cell’s biosynthetic machinery
or
7
added to macromolecules by the action of enzymes in vitro. Once
incorporated into bioconjugates, chemical handles can be cova-
lently ligated to either a visualization tag or an enrichment tag
through a chemoselective reaction.8 Among the chemical handles
that have been used, the azide is valuable as it is not typically
found in biological systems and does not interact with any biolog-
ical functionality. In addition, the azide has unique reactivity with
phosphine- and alkyne-based probes. The most commonly
employed reactions involving azido-functionality include the
,9
19
CuAAC is known to oxidize amino acid side chains and is attrib-
2
0,21
uted to DNA cleavage.
More recently, strain-promoted [3+2]
azide–alkyne cycloaddition (SPAAC), also known as copper-free
2
2–24
‘click’ chemistry was developed
in order to eliminate the use
of a cytotoxic copper catalyst. Copper-free ‘click’ reactions use a
cyclooctyne structure in which a triple bond in a ring is sufficiently
strained to promote rapid cycloaddition with an azide in order to
form a stable triazole conjugate (Fig. 1C). Unfortunately, the
chemical reaction of cyclooctyne and azide-functionality generally
⇑
Corresponding author. Tel.: +1 301 496 0943; fax: +1 301 496 9431.
E-mail address: jah@helix.nih.gov (J.A. Hanover).
0
008-6215/$ - see front matter Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.carres.2013.05.014

E. J. Kim et al. / Carbohydrate Research 377 (2013) 18–27
19
O
O
P
O
P
A
OCH3
N
H
OCH3
H2O
+
N
N
N
N
N
N
O
P
-N2
-CH3OH
Ph
Ph
Ph
Ph Ph
Ph
Cu(I)
N
N
B
C
C
CH
+
N
N
N
N
N
N
N
+
N
Figure 1. Chemoselective reactions with azide. Molecules containing azide react via the Staudinger ligation (A), Cu-catalyzed ‘click’ chemistry (B), or strain-promoted
cycloaddition (C) to produce ligated products.
proceeds at a slower rate than that of CuAAC.25 Therefore, several
groups have made considerable effort toward improving the
reaction rate of strain-promoted copper-free 1,3-dipolar cycloaddi-
tion between azide-moieties and cyclooctynes. These efforts have
lowers the LUMO energy and increases the HOMO energy thereby
narrowing the HOMO–LUMO gap and enhancing the reactivity.33
Herein, we report that difluorinated cyclooctyne core structures
conjugated with either a rhodamine-based fluorophore (TAMRA)
or a biotin tag (1 and 2, see Fig. 2) exhibit reactivity that causes
these reagents to form an adduct with not only azide-functionality
but also reactive sulfhydryls, which are abundant in many biolog-
ical systems. Since reactive sulfhydryls are maintained by a reduc-
ing intracellular environment this is particularly problematic when
examining intracellular glycosylation events. We describe a strat-
egy that greatly improves the specificity of the DIFO reactivity un-
der these conditions.
produced a number of strained cycloalkynes with enhanced reac-
tivity2
2,26–28
making SPAAC more practical and effective for appli-
26,29–32
cation in biological and physiological studies.
Foremost
among the new generation of reagents are difluorinated cyclooc-
tyne (so-called DIFO)-based reagents.2
6,27
Ess et al. explained
33
the accelerated rate effect of introduction of an electron-withdraw-
ing group (fluorine) adjacent to the alkyne on the SPAAC reaction
by calculating the energy of the transition states of 1,3-dipolar cyc-
loadditions involving azide and different alkynes using density
functional theory (B3LYP). Calculations of the transition states of
different azide–alkyne combinations disclosed that difluoro substi-
tution lowers the activation energy by 2.0 kcal/mol compared to
that of non-fluorinated cyclooctyne, likely due to the increase in
the stabilizing interaction caused by fluorination. Consistent with
this result, calculations on the frontier orbitals of the cyclooctyne
and difluorinated cyclooctyne revealed that difluoro substitution
2. Results and discussion
2.1. Syntheses of DIFO-based reagents
DIFO-based reagents (TAMRA–DIFO 1 and Biotin–DIFO 2) used
in copper-free strain-promoted cycloadditions were prepared
2
7
according to previously published procedures
with minor
CH3
N
CH3
CH3
N
CH
3
+
O
S
+
O
N
O
N
H3C
O
CH3
H C
CH3
3
HN
NH
COO-
H
N
H
N
COO-
O
O
O
F
O
O F
O
Biotin-DIFO (2)
F
F
N
H
N
H
O
N
O
O
3
TAMRA-DIFO (1)
O
O
O
S
CH3
N
CH3
+
CH3
CH
N
3
O
N
3
H C
CH3
HN
NH
HN
NH
+
N
O
H3C
CH3
H
N
OH
O
O
NH2
COO-
S
O
COO-
5
O
6
O
N
O
O
H
4
H
O
7
N3
N
H
O
HN
NH
O
N
N(CH3)2
O
H
N
O
HN
NH
O
PPh2
OCH3
S
O
N
H
O
H
O
O C
O
N
9
S
O
N
4
H
O
10
8
O
N(CH3)2
Figure 2. Structures of DIFO-based reagents (1, 2), synthetic intermediates (3–6), terminal alkyne reagents (7, 8), triaryl phosphine reagent (9), and TAMRA–Mal (10).

2
0
E. J. Kim et al. / Carbohydrate Research 377 (2013) 18–27
modifications. Syntheses of these reagents are presented in detail
in the Section 3 and the structures of DIFO-based reagents and syn-
thetic intermediates are found in Figure 2. Briefly, commercially
available 5/6-carboxy-tetramethylrhodamine succinimidyl ester 3
was reacted with 3-azidopropylamine to produce mixed isomers
of azide-tagged tetramethylrhodamine. Azide-tagged tetra-
methyl-rhodamine single isomer 4 was obtained by flash column
chromatography from mixed isomers. Compound 4 was subjected
to hydrogenation and subsequent coupling reaction with 2-(2,2-di-
fluorocyclooct-3-yn-1-yl)acetic acid to produce DIFO–TAMRA 1 in
of each reaction were monitored by gel electrophoresis and the
immunoblot analysis. As necessary, blots were normalized to the
intensity of the Ponceau S stained gels quantified by Image J. As ex-
pected, copper-catalyzed ‘click’ reactions of azide-labeled C. ele-
gans lysate (Fig. 3A, lane 5) and HeLa cell lysate (Fig. 3B, lane 5)
with TAMRA–alkyne were specific for azide-moiety compared with
those reactions of unlabeled lysates (Fig. 3A, lane 2; Fig. 3B, lane 4).
However, strain-promoted copper-free ‘click’ reactions with TAM-
RA–DIFO did not show specificity for azide-functionality, since
treatment of unlabeled lysates with TAMRA–DIFO also generated
normalized fluorescent signals as strong as those generated from
the treatment of azide-labeled lysates (Fig. 3A, lane 3; Fig. 3B, lane
2). Previous results from Boons et al. indicated low background
interactions of cell lysates with cyclooctyne reagents and provided
a possible explanation about the source of background staining. By
careful experimentation, they excluded the possibility of unwanted
side reaction of the cyclooctyne reagents with proteins and sug-
gested the background was generated from potential noncovalent
interactions of the FITC-conjugated avidin agent used for capturing
biomolecules. Although dyes are known to interact with lysates in
a non-specific manner, the TAMRA dye itself is unlikely to have
generated the numerous non-specific bands detected in the sam-
9
6% yield. Biotin–DIFO 2 was prepared by direct amide coupling
of biotinyl-4,7,10-trioxatridecanediamine and 2-(2,2-di-
6
2
3
fluorocyclooct-3-yn-1-yl)acetic acid using a HATU coupling re-
agent in 35% yield.
2
.2. DIFO-based reagents are reactive toward both azide-labeled
and unlabeled proteins
Additional reactivity of DIFO-based reagents was observed in
unlabeled cell lysates (Fig. 3). In this study, azide groups were in-
stalled on proteins in two ways: in vitro enzymatic transfer and
metabolic labeling. In in vitro enzymatic transfer, proteins in Cae-
norhabditis elegans’ lysates were tagged with the azide-functional-
ity using a mutant Gal-T1 enzyme that is capable of transferring an
unnatural N-azidoacetylgalactosamine (GalNAz) sugar from UDP-
GalNAz onto proteins that have a terminal N-acetylglucosamine
ples lacking azide-functionality depicted in
Figure 3. As
demonstrated in Figure 3A (lane 2) and B (lane 4), although
TAMRA–Alkyne (7) possesses the same dye component as found
in TAMRA–DIFO, it did not give distinctive signals arising from
non-specific interactions with unlabeled lysates. Only one band
found in the lanes 1, 2, and 4 of Figure 3A most likely represents
a noncovalent interaction with C. elegans lysates. Likewise, treat-
ment of a different tag-containing DIFO reagent, Biotin–DIFO 2,
with azide-labeled and unlabeled cell lysates yielded the same
observation as obtained with TAMRA–DIFO (Fig. 4).
(
O-GlcNAc). Alternatively, HeLa cell proteins were metabolically la-
beled by culturing the cells in a medium containing 100 M per-
acetylated N-azidoacetylglucosamine ((Ac) GlcNAz). In order to
l
4
examine reaction specificity, both azide-labeled and unlabeled cell
lysates were treated with either a DIFO-based probe (1) or terminal
alkyne (TAMRA–Alkyne 7) at room temperature for 20 min. Results
Figure 3. Ponceau S-stained nitrocellulose membrane (upper) and immunoblots (lower) of C. elegans (A) and HeLa cell (B) extracts treated with and without 1 or 7.
Ò
Immunoblots were probed with rabbit anti-TAMRA antibody followed by IRDye 800CW-conjugated goat anti-rabbit antibody. Quantitation of Ponceau S-stained gels was
performed using Image J and used to generate normalized values referred to in the text. In (A), Ponceau S staining of lanes 4–6 was 62% of the intensity of lanes 1–3 and was
used to normalize the fluorescence intensity values for comparison.

E. J. Kim et al. / Carbohydrate Research 377 (2013) 18–27
21
Figure 4. Ponceau S.-stained nitrocellulose membrane (left) and immunoblot (right) of HeLa cell extracts reacted with and without 2, 8, or 9. The immunoblot was probed
with IRDye800CW-Streptavidin.
It is important to note that copper-catalyzed ‘click’ reaction of
terminal alkyne and azide-containing molecules does not occur
in the commercial T-PER or M-PER lysis buffer (Thermo Scientific
Pierce). We speculate that the copper catalyst is no longer available
for alkyne–azide cycloaddition due to chelation of copper ions by
these commercial buffers.
6; Fig. 5B, lanes 4 and 5). In contrast, only azide-labeled a-crystal-
lin treated with either terminal alkyne reagent (7 or 8) or Biotin–
Phosphine (9) showed strong immunoreactivity, indicating the
high specificity of these reagents for azide-functionality (Fig. 5A,
lanes 2 and 3 for reaction with 7; Fig. 5B, lanes 2 and 3 for reaction
with 8; Fig. 5B, lanes 7 and 8 for reaction with 9).
As expected, copper-catalyzed ‘click’ reaction and Staudinger
ligation using biotinylated terminal alkyne (Biotin–Alkyne, 8) in
combination with Cu(I) or triaryl phosphine (Biotin–Phosphine,
According to a previous report from Agard et al., non-specific
covalent adduct formation between proteins lacking azido-moie-
ties and cyclooctyne-based probes can occur when the reaction
2
5
9), exhibited high specificity for azide-functionality. Immuno-reac-
temperature is elevated to boiling. Our experiments were per-
formed either at, or lower than, room temperature, ruling out ele-
vated temperature as the cause for the non-specific adduct
formation. Additionally, noncovalent interactions due to Biotin
and TAMRA do not appear to play a major role in adduct formation
since reactions of unlabeled proteins with the other Biotin- or
TAMRA–containing reagents (7, 8, and 9) gave little to no
reactivity.
tive bands were detected only in the azide-labeled lysates (Fig. 4,
lanes 5 and 6, respectively) and not in lanes containing unlabeled
lysates (lanes 4 and 6). In contrast, Biotin–DIFO (2) produced
numerous strong signals in both the absence and presence of
azide-labeled lysates (Fig. 4, lanes 2 and 3, respectively).
We used purified
the DIFO-based probes.
a
-crystallin to further test the specificity of
-crystallin is a protein modified by sev-
a
eral mapped O-GlcNAc posttranslational modifications and has a
mass range of 15–25 kDa. Azide-labeling of this protein was per-
formed via an in vitro enzymatic transfer strategy using UDP-Gal-
NAz and the Gal-Tl enzyme. Consistent with the observations in
Figure 4, treatment of unlabeled or azido-labeled a-crystallin with
either DIFO-based probes (1 or 2) produced strong immuno-reac-
2.3. Sulfhydryls are highly reactive toward DIFO-based reagents
A study by van Geel et al. reported the azide-independent reac-
tions of three strained cyclooctynes, dibenzocyclooctyne (DIBO),
azadibenzocyclooctyne (DIBAC), and bicyclo[6.1.0]nonyne (BCN)
and suggested that a free-thiol moiety could serve as a reactive
tive bands, indicating the formation of adducts between DIFO-
3
4
based reagents and
a
-crystallin, regardless of the presence of an
functional group for strained cyclooctynes. To determine if the
reactivity we observed was due to a free thiol, we used purified
azide-functional group, as shown in Figure 5 (Fig. 5A, lanes 5 and
Figure 5. Ponceau S.-stained nitrocellulose membranes (upper) and corresponding immunoblots (lower) of
crystallin is treated with TAMRA-tagged reagents (Cu /terminal alkyne 7 or DIFO-based reagent 1) (A) and with Biotin-tagged reagents (Cu /terminal alkyne 8, DIFO-based
reagent 2, or triaryl Phosphine reagent 9) (B). Immuoblots were analyzed using rabbit anti-TAMRA antibody followed by IRDye 800CW-conjugated goat anti-rabbit antibody
a-crystallin reacted with various azide-reactive reagents: a-
+
+
Ò
for A and IRDye800 CW-conjugated Streptavidin for B.

2
2
E. J. Kim et al. / Carbohydrate Research 377 (2013) 18–27
recombinant human nuclear pore protein Nup62, which is heavily
modified with O-GlcNAc and contains three cysteine residues with
at least one cysteine that is not part of a disulfide-bridge. We
confirmed the presence of a free thiol moiety in Nup62 using the
fluorophore-labeled sulfhydryl alkylating tetramethylrhodamine–
reactivity due to their surrounding context within a protein could
potentially add to DIFO-based probes. These results are different
3
4
from those obtained with other cyclooctynes. Previously, DIBO,
DIBAC, and BCN were reported to produce their adducts with a
nonpeptidyl thiol such as DTT, 2-mercaptoethanol, and glutathi-
3
4
5
-maleimide (TAMRA–Mal, 10; data not shown). Exposing
unlabeled Nup62 to Biotin–DIFO (2) produced a distinct signal
Fig. 6, lane 3). Prior treatment of Nup62 with TAMRA–Mal to block
one. It implies that DIFO-based probes are less prone to thiol-
yne addition than three other cyclooctynes.
(
Although the addition of thiols to alkynes was described as
3
5
free cysteine’s thiol(s) resulted in an almost complete abolishment
of the adduct formation between the DIFO-based probe and Nup62
early as the 1930s, photoinitiated radical addition of thiols to
various alkynes including cyclooctyne was only recently investi-
3
6–38
(Fig. 6, lane 2).
gated.
Thiol-yne coupling is generally known to proceed by a
Likewise, prior treatment of
itol (DTT) to generate free cysteines and blocking the moieties
using another sulfhydryl-alkylating reagent, 2-iodoacetamide
IAM), followed by its exposure to DIFO-based probes completely
abolished the adduct formation (Fig. 7A, lanes 2 and 3 for the reac-
tion with TAMRA–DIFO, 1; lanes 5 and 6 for the reaction with Bio-
tin–DIFO, 2).
Additionally, prior treatment of unlabeled C. elegans lysate with
DTT/IAM resulted in a marked reduction of non-specific reactions
arising from the formation of adducts between unlabeled lysate
and DIFO-based probes (Fig. 7B, lane 2 and lane 3 for the reaction
with TAMRA–DIFO and lane 5 and lane 6 for the reaction with Bio-
tin–DIFO). In the C. elegans negative control, three strong bands ap-
pear representing non-specific binding between IR-dye conjugated
to Streptavidin and C. elegans proteins supporting that these three
bands are not due to covalent labeling reactions of the reagent. Un-
like the pure proteins, DTT/IAM-treatment of unlabeled C. elegans
extract did not completely remove the background signals shown
in the lane 3 of Figure 7B. Since azide-lacking C. elegans lysates
treated with TAMRA–Alkyne reagent (7) produced the same back-
ground signal (Fig. 3A, lane 2) as negative controls of C. elegans
a
-crystallin with 1,4-dithio-d-thre-
radical mechanism as shown in Scheme 1. Briefly, a thiyl radical
generated under the photoinduced reaction condition adds to a tri-
ple bond to give a vinyl sulfide radical which abstracts hydrogen
from the thiol to afford the thiyl radical and a regioisomeric mix-
ture of vinyl sulfide. Subsequently, the regenerated thiyl radical
adds to the double bond of the vinyl sulfide eventually yielding a
dithioether through a dithioether radical. The second addition of
thiol to the vinyl sulfide is known to depend on the initial yne
(
3
7
structure. In addition to the general photoinitiated thiol-yne
reaction, spontaneous thiol addition to cyclooctyne under the
‘dark’ reaction condition (absence of light or a photoinitiator)
3
7
was also reported by Fairbanks et al. Although, the precise mech-
anism of the ‘dark’ reaction of thiols with cyclooctyne has not been
elucidated, this reaction is thought to proceed through a radical
mechanism probably initiated by some reactive oxygen species
introduced by the diffusion of molecular oxygen into the reaction
3
7
mixture. This may be of particular importance in the cytoplasm
of the cell and cell extracts where free radical generation is thought
to occur under tightly controlled conditions.
2.4. Free thiol-blocking is necessary prior to reaction with DIFO-
based probes
(
Fig. 3A, lane 1 and lane 4), we can exclude the possibility that
higher background signals shown in DTT/IAM-treated unlabeled
C. elegans exposed to TAMRA–DIFO are simply due to non-covalent
TAMRA–dye interaction with unlabeled proteins. Faint signals
were also seen in the unlabeled lysate that was sequentially re-
acted with DTT/IAM and Biotin–DIFO.
Exhaustive alkylation of all sulfhydryl groups of unlabeled
crystallin was conducted by protein treatment with DTT to first
break all disulfide bonds followed by subsequent treatment with
a-
IAM. Treated
a-crystallin was then completely inactive toward
It is interesting that not all sulfhydryl-containing molecules are
reactive toward DIFO-based reagents: when sulfhydryl-containing
DIFO-based probes as demonstrated before (Fig. 8A and B, lane
2). However, azide-labeling of DTT/IAM and Gal-T/UDP-GalNAz-
small molecules such as DTT,
L
-cysteine, and glutathione were re-
treated a-crystallin followed by the reaction with DIFO-based re-
acted with DIFO-based reagents at the same molar ratio at room
temperature for 1 h, corresponding adduct products were not de-
tected at all. However, when 2-mercaptoethanol was reacted with
Biotin–DIFO, a small amount of the adduct product was observed
by MS at m/z 709.3 and the percentage of adduct product increased
as the concentration of 2-mercaptoethanol was increased (data not
shown). Thus, we speculate that sulfhydryls with increased
agents restored a strong signal which represents the adduct forma-
tion between the azide-labeled protein and the DIFO-based probe
(Fig. 8A, lane 3 and B, lane 3). In the parallel experiment, copper-
catalyzed ‘click’ reactions using TAMRA–Alkyne (7) and Biotin–Al-
kyne (8) and Staudinger ligation reaction using Biotin–Phosphine
(9) were carried out as positive controls for the chemoselective
cycloaddition of the azide-labeled
B, lane 5 and lane 7).
a-crystallin (Fig. 8A, lane5 and
Treatment of GalNAz-labeled HeLa cell extract with DTT/IAM
followed by exposure to the Biotin–DIFO (2) generated many sig-
nals as shown in Fig. 9 (lane 4). The reaction of GalNAz-labeled
HeLa cell extracts with Biotin–Alkyne 8 in combination with
Cu(I) (Fig. 9, lane 9) and the reaction with Biotin–Phosphine 9 were
performed (Fig. 9, lane 7) as positive controls. The two background
signals depicted in the immunoblot in Fig. 9 (lanes 1, 5, and 8) are
due to the non-specific interaction with IR-dye conjugated Strepta-
vidin. Although DTT/IAM-treatment affected the blocking of reac-
tive functionalities toward DIFO-based probes, there are still
DIFO-reactive species remaining (Fig. 9, lane 3). It is currently un-
clear whether these remaining weak signals are caused from failing
to complete sulfhydryls’ alkylation or from the existence of
another functional group that can also react with DIFO-based
probes. However, based on the reaction results obtained with the
Figure 6. Ponceau S.-stained nitrocellulose membranes (left) and corresponding
immunoblots (right) of Nup62: lane 1 is Nup62 as a negative control, lane 2 is
Nup62 sequentially treated with TAMRA–Mal (10) and then with Biotin–DIFO (2),
and lane 3 is Nup62 treated with only DIFO–Biotin. Western blots were analyzed
using IRDye800 CW-conjugated Streptavidin.
pure proteins such as Nup62 and
a-crystallin where DTT/IAM-
treatment of the proteins completely blocked the non-specific,

E. J. Kim et al. / Carbohydrate Research 377 (2013) 18–27
23
Figure 7. Ponceau S.-stained nitrocellulose membrane (upper in A and left in B) and immunoblots (bottom in A and right in B) of
a
-crystallin (A) and C. elegans lysate (B)
treated with DTT/IAM, followed by reactions with and without either DIFO–TAMRA (1) or DIFO–Biotin (2). Each immunoblot was cut into two pieces and probed with rabbit
anti-TAMRA antibody and IRDye800CW-conjugated goat anti-rabbit antibody (a) or with IRDye800CW-Streptavidin (b). Three strong signals in the negative control of C.
elegans (Fig. 6B, lane 4) represent non-specific interactions with IRDye800CW-Streptavidin.
R3 SH
R2
hν
H
R3
S
R3
S
R3 SH R3
S
R3 S
H
R3
S
R3
S
R1
+
+
R1
R1
R1
R1
R2
R2
R2
R2
R3
S
R3
R3
S
R3 SH
R3
S
S
R3
R3
S
S
S
R3
H
R3
S
H
+
R1
H
R1
H
R2
R1
R2
R2
Scheme 1. Possible photoinduced thiol-yne mechanism.37
Figure 8. Ponceau S-stained nitrocellulose membranes (upper) and immunoblots (lower) of sequentially reacted a-crystallin: a-crystallin is first subjected to reduction of
+
disulfide bond and then alkylation of all sulfhydryls generated. Next, the protein was subjected to GalNAz labeling followed by reaction with TAMRA-tagged reagents (Cu /
+
terminal alkyne 7 or TAMRA–DIFO 1) (A) or with Biotin-tagged reagents (Cu /terminal alkyne 8 or Biotin–DIFO 2, or Biotin–Phosphine 9) (B). Immunoblots were analyzed
using rabbit anti-TAMRA antibody and IRDye800CW-conjugated goat anti-rabbit antibody for A and IRDye800CW-Streptavidin for B.
covalent additions with DIFO-based probes, it is more likely that
the background observed in Figure 9, lane 3 is from the incomplete
alkylation of free thiols.
Our in vitro experimental results also show that not only azide-moi-
eties but also reactive sulfhydryl groups in protein lysates can rap-
idly react with highly reactive strained alkynes such as DIFO-based
reagents to give adduct products. The observation that two
DIFO-based reagents examined here do not form detectable adducts
with nonpeptidyl thiols suggests they are less prone to thiol-yne
addition than other cyclooctynes such as DIBO, DIBAC, and BCN. In
More recently, thiol-yne reactions using more biologically-rele-
34,39
vant biomolecules were explored by Conte et al.
reports
Three previous
examining thiol-yne reactions, expressed concern
about orthogonal reactions with the reactive cyclooctyne probes.
3
4,37,39

2
4
E. J. Kim et al. / Carbohydrate Research 377 (2013) 18–27
Figure 9. Ponceau S-stained nitrocellulose membrane (left) and immunoblot (right) of unlabeled HeLa cell extracts sequentially treated with or without DTT/IAM, GalNAz
labeling reagents, and azide-reactive reagent 2, 8, or 9. Immunoblot was analyzed using IRDye800CW-Streptavidin.
addition, in order to use DIFO-based reagents to tag azide-labeled
biomolecules in biological contexts, the bioconjugate thiol moieties
can be blocked first to diminish the potential unwanted side-reac-
tion with DIFO-based reagents. Although this step is probably less
important for cell surface applications where free thiols are less
USA), DTT, b-mercaptoethanol, and
ceived. UDP-GalNAz, Gal-T1 enzyme, and
tained in the Click-iT™ O-GlcNAc Enzymatic Labeling Kit
(Invitrogen, Carlsbad, CA, USA). TAMRA–Alkyne and Biotin–
Alkyne with copper(II)-salt and reducing agent are contained in
the Click-iT™ Protein Analysis Detection Kits (Invitrogen).
L
-cysteine were used as re-
a
-crystallin are con-
6
likely to exist, this thiol-blocking step is essential when applying
DIFO-based labeling to intracellular glycans.
Ò
DMEM, FBS, PBS, NuPAGE pre-cast gels, 4Â NuPAGE LDS sample
buffer, and rabbit anti-TAMRA antibody were purchased from
Invitrogen (Carlsbad, CA, USA). Complete, mini, EDTA-free prote-
ase inhibitor cocktail tablets were purchased from Roche Applied
Science (Indianapolis, IN, USA). M-PER and T-PER lysis buffers,
and BCA protein assay reagent were purchased from Thermo Sci-
2
.5. Conclusion
Difluorinated cyclooctynes (DIFO) conjugated with either a rho-
damine-based fluorophore (TAMRA) or a biotin tag are useful dipo-
larphiles developed for rapid Strain-Promoted Azide Alkyne
Cycloaddition (SPAAC) protein labeling and have been widely ap-
plied in several outstanding studies. Nevertheless, the bioorthogo-
nality of these probes is not absolute and minimizing non-specific
labeling (background labeling) is still a major concern in these
chemical biology approaches. Previous reports illustrate the poten-
tial non-specific labeling with strained cyclooctynes such as DIBO,
DIBAC, and BCN.3 Here, we extend these studies suggesting that
two widely used DIFO-based probes also show the same concern
for bioorthogonality. In this study, we demonstrated that reactive
thiols present in biomolecules rapidly add to DIFO-based probes
to form the covalent adducts and thus potentially limit the bioor-
thogonality of the strain-promoted copper-free azide–alkyne cyc-
loadditions. However, we demonstrate that thorough blocking of
the free-thiols using exhaustive alkylation prior to copper-free
Ò
entific Pierce (Rockford, IL, USA). IRDye 800CW-conjugated goat
anti-rabbit antibody was purchased from LI-COR Biosciences
(Lincoln, NE, USA). Recombinant human nuclear pore glycopro-
tein, p62 (Nup62) was purchased from Bioclone (San Diego, CA,
USA). Synthetic intermediates, and target compounds (TAMRA–
DIFO and Biotin–DIFO) were confirmed by high-resolution ESI
MS analysis. High-resolution mass measurements were per-
formed on a Micromass/Waters LCT Premier Electrospray Time
of Flight (TOF) mass spectrometer coupled with a Waters HPLC
system. The immunoblots were imaged according to the manu-
facturer’s instructions using the Odyssey Infrared Imaging Sys-
tem (LI-COR Biosciences).
4
3.2. Syntheses of DIFO-based reagents
‘
click’ reactions restores the bioorthogonal nature of these reac-
Difluorinated cyclooctyne (DIFO) core structure [2-(2,2-di-
fluoro-cyclooct-3-yn-1-yl)acetic acid] was prepared according to
the procedure reported by Codelli et al.
tions and this may be particularly important when examining
intracellular glycans such as the widespread O-GlcNAc
modification.
2
7
3
.2.1. 5-(3-Azidopropylcarbamoyl)-2-(6-dimethylamino-3-di-
methylimino-3H-xanthen-9-yl) benzoate (4)
A solution of 2-(6-dimethylamino-3-dimethyliminio-3H-xan-
then-9-yl)-5(and 6)-[(2,5-dioxopyrrolidin-1-yloxy)carbonyl]ben-
3
3
. Experimental
.1. Material
zoate
10.1 mg, 0.0998 mmol) in methanol (1 mL) was treated with 3-
azidopropylamine (11.6 mg, 0.116 mmol) and then stirred for
6 h at ambient temperature. Methanol was removed by evapora-
tion and the residue was separated by flash column chromatogra-
phy eluting with CH Cl :MeOH = 10:1–4:1. The product fractions
were collected, concentrated, and lyophilized to afford the
3 (9.8 mg, 0.019 mmol, Invitrogen) and triethylamine
(
All chemicals were purchased from Sigma–Aldrich (St. Louis,
MO, USA) unless otherwise indicated. All solutions were pre-
pared using ultrapure deionized water. 5-(and 6)-carboxytetram-
ethylrhodamine, succinimidyl ester (3, Invitrogen, Carlsbad, CA,
USA), 3-azidopropylamine,
ediamine, 2-iodoacetamide (Pierce Biotechnology, Rockford, IL,
1
D
-Biotin, 4,7,10-trioxa-1,13-tridecan-
2
2

E. J. Kim et al. / Carbohydrate Research 377 (2013) 18–27
25
compound 4 (5.7 mg, 60%). HRMS (ESI): [M+H]+ calculated for
HEPES buffer was subject to N-azidoacetylgalactosamine (Gal-
C
28
H
29
N
6
O
4
: m/z = 513.2250; found m/z = 513.2230.
NAz)-labeling using a Click-iT™ O-GlcNAc Enzymatic Labeling
Kit. Briefly, 500
buffer containing 0.1% SDS was placed into a 1.5 mL microcentri-
fuge tube and 122.5 L of DPEC-treated water, 200 L of labeling
buffer, 27.5 L of 100 mM MnCl , 25 L of 0.5 mM of UDP-GalNAz,
and 20 L of Gal-T1 were added. The reaction mixture was incu-
bated at 4 °C overnight. Finally, the buffer was exchanged into a
buffer appropriate for the following chemoselective reaction using
a 10 K-cutoff Amicon Ultra-0.5 Centrifugal filter device. Metabolic
azide-labeling of HeLa cells was achieved by culturing the cells in
a 10 mL DMEM supplemented with 10% FBS in the presence of
lg of C. elegans proteins in 100 lL of 20 mM HEPES
3
.2.2. 5-{3-[2-(2,2-Difluorocyclooct-3-yn-1-yl)acetamido]pro-
pylcarbamoyl}-2-(6-dimethyl-amino-3-dimethyliminio-3H-
xanthen-9-yl)benzoate (1)
l
l
l
2
l
To a solution of 4 (2.0 mg, 0.0039 mmol) in ethanol (4 mL) was
added 5% Pd on carbon (2 mg) and hydrogenated for 24 h. Reaction
progress was monitored by MS. When the azide compound 4 dis-
appeared and amine compound existed alone, the catalyst was re-
moved by filtration. The filtrate was concentrated and lyophilized.
The amine compound was dissolved in DMF (1 mL) and 2-(2,2-di-
fluorocyclooct-3-yn-1-yl)acetic acid (1.5 mg, 0.0074 mmol), HATU
l
100
l
M of peracetylated N-azidoacetylglucosamine (Ac
4
GlcNAz)
(
0
3.2 mg, 0.0082 mmol), and diisopropyl-ethylamine (5.0 mg,
.039 mmol) were added and stirred for 2 h at ambient tempera-
in a humidified 5% CO
2
incubator for 2 days. Cultured cells were
washed with PBS at pH 7.2, transferred into a 15 mL-felcon tube
and then centrifuged at 2500Âg for 10 min at 4 °C. The pellets were
lysed in 0.6 mL of M-PER protein extraction buffer containing an
EDTA-free protease inhibitor cocktail, according to the manufac-
turer’s protocol. The supernatant was obtained after centrifugation
at 20,000Âg for 15 min at 4 °C and stored in aliquots at À80 °C un-
til use. GalNAz-labeling of unlabeled HeLa cell extracts was per-
formed using a Click-iT™ O-GlcNAc Enzymatic Labeling Kit as
described above.
ture. The reaction mixture was lyophilized to remove DMF. The
residue was separated by flash column chromatography eluting
with CH
lected, concentrated, and lyophilized to afford the titled compound
1
2 2
Cl :MeOH = 10:1–4:1. The product fractions were col-
+
(2.5 mg, 96%). HRMS (ESI): [M+H] calculated for C38
41 4 5 2
H N O F :
m/z = 671.3045; found m/z = 671.3054.
3
.2.3. N-(3-{2-[2-(3-Aminopropoxy)ethoxy]ethoxy}propyl)-5-
(4S)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentana-
mide (6)
To a solution of biotin 5 (109.5 mg, 0.444 mmol) in 1,4-dioxane
3 mL) were added 4,7,10-trioxa-1,13-tridecanediamine (102 mg,
[
3.3.2. Preparation of azide-labeled
GalNAz-labeling of the pure proteins such as
Nup62 was achieved by in vitro enzymatic labeling strategy using
a Click-iT™ O-GlcNAc Enzymatic Labeling Kit. Briefly, 50 g of
either -crystallin or Nup62 was reconstituted in 10 L of 20 mM
HEPES buffer containing 0.1% SDS. Sequentially, 8.2 L of DPEC-
treated water, 16 L of labeling buffer, 2.2 L of 100 mM MnCl
L of 0.5 mM of UDP-GalNAz, and 1.6 L of Gal-T1 were added
a-crystallin and Nup62
a-crystallin and
(
0
0
.449 mmol), EDC (132 mg, 0.689 mmol), HOBT (95 mg,
.703 mmol), and triethylamine (145 mg, 1.43 mmol). The reaction
l
a
l
l
mixture was stirred for 16 h at ambient temperature and then 1,4-
dioxane was removed. The residue was separated by flash column
chromatography eluting with CH
l
l
2
,
Cl
2 2
:MeOH:AcOH = 100:10:2–
2
l
l
3
0:10:2. The product fractions were collected, and concentrated to
and the reaction mixture was incubated at 4 °C overnight. Then,
reaction buffer was exchanged into an appropriate buffer for cop-
per-catalyzed or copper-free ‘click’ reaction or Staudinger ligation
using a 10 K-cutoff Amicon Ultra-0.5 Centrifugal filter device.
+
afford the titled compound 6 (56.1 mg, 28%). HRMS (ESI): [M+H]
calculated for C20 S: m/z = 447.2641; found m/z = 447.2659.
39 4 5
H N O
3
.2.4. N-[1-(2,2-Difluorocyclooct-3-yn-1-yl)-2-oxo-7,10,13-
trioxa-3-azahexadecan-16-yl]-5-[(4S)-2-oxohexahydro-1H-
thieno[3,4-d]imidazol-4-yl]pentanamide (2)
3.3.3. Reactions of azide-labeled and unlabeled proteins with
various azide-specific reagents
To a solution of 6 (4.4 mg, 0.00985 mmol) in DMF (0.5 mL) was
added 2-(2,2-difluorocyclooct-3-yn-1-yl)acetic acid (1.8 mg,
Before carrying out the reaction of cell lysates or pure proteins
with various azide-specific reagents, buffer-exchange into an
appropriate buffer was performed. For copper-catalyzed or cop-
per-free ‘click’ reactions, 50 mM Tris–HCl, pH 7.4 buffer containing
0.1% SDS was used as reaction buffer. For Staudinger ligation reac-
tion, PBS was used.
0
.00890 mmol), HATU (4.7 mg, 0.0120 mmol), and diisopropyleth-
ylamine (2.0 mg, 0.0155 mmol). The reaction mixture was stirred
for 16 h at room temperature and then lyophilized to remove
DMF. The residue was separated by flash column chromatography
2 2
eluting with CH Cl :MeOH = 20:1–5:1. The product fractions were
collected, and concentrated to afford the titled compound 2
3.3.4. Reactions of azide-labeled and unlabeled proteins with
DIFO-based probes (copper-free ‘click’ reaction)
+
(
2.2 mg, 35%). HRMS (ESI): [M+H] calculated for C30
49 4 6 2
H N O F S:
m/z = 631.3341; found m/z = 631.3359.
To 10
porated azides)
containing 0.1% SDS was added 0.2
or Biotin–DIFO). Reaction mixtures were agitated at room temper-
l
g of GalNAz-labeled and unlabeled (without any incor-
-crystallin in 39.8 L of 50 mM Tris–HCl buffer
L of 10 mM TAMRA–DIFO
a
l
l
3
3
.3. Biological assays
.3.1. Preparation of azide-labeled C. elegans and HeLa cell
(
ature for 20 min. In the parallel experiments, same reaction mix-
tures were carried out in the dark at room temperature for
20 min. The reactions were quenched by removing remaining
small molecules including excess DIFO-TAMRA reagent using a
10 K-cutoff Amicon Ultra-0.5 Centrifugal filter device. Therefore,
the whole reaction mixture was transferred into a 10 K-cutoff Ami-
con filter device and centrifuged at 14,000Âg at 4 °C for 15 min.
lysates
2
0–50 lL of worms was lysed in 100 lL T-PER lysis buffer with
protease inhibitors (complete, mini, EDTA-free protease inhibitor
cocktail and PMSF). Worm lysate was sonicated in an ice bath in
Misonic sonicator for 10 min followed by centrifugation at
2
0,000Âg for 10 min at 4 °C. Supernatant was transferred into a
fresh tube and protein concentration was determined by BCA assay
and samples were stored at À80 °C until use. 500 g of wild-type C.
elegans proteins prepared in T-PER lysis buffer was buffer-ex-
changed into 100 L of 20 mM HEPES buffer containing 0.1% SDS
using a 10 K-cutoff Amicon Ultra-0.5 Centrifugal filter device
EMD Millipore, Billerica, MA, USA) according to the manufac-
turer’s protocol. Then, 500 g of C. elegans proteins in the 20 mM
Washes and buffer exchange were performed by placing 250 lL
l
of 50 mM Tris–HCl buffer containing 0.1% SDS into the centrifuged
filter device and by re-centrifuging at 14,000Âg at 4 °C for 30 min.
This buffer exchange process was repeated three times. In order to
l
recover the residues remaining in the filter device, 30
Tris–HCl buffer containing 0.1% SDS was added into the filter to
give a total volume of 30 L and the filter device was placed upside
lL of 50 mM
(
l
l

2
6
E. J. Kim et al. / Carbohydrate Research 377 (2013) 18–27
down in a clean microcentrifuge tube and was centrifuged at
000Âg at 4 °C for 2 min. The reaction results were evaluated by
gel electrophoresis followed by Western blot analysis. Samples
were prepared for gel electrophoresis by adding 5 L of distilled
L of 4Â NuPAGE LDS sample buffer to give a total
L. After incubation at 80 °C for 5 min, 20 L of each
fer containing 0.1% SDS, using a 10 K-cutoff Amicon Ultra-0.5 Cen-
trifugal filter device. The reaction results were determined by gel
electrophoresis followed by Western blot analysis.
1
l
Ò
water and 15
volume of 60
l
l
3
.3.6. Reactions of azide-labeled and unlabeled proteins with
l
Biotin–Phosphine (Staudinger ligation)
reaction sample was loaded onto a SDS–PAGE gel (10% or 4–12%
Staudinger ligation reactions of azide-labeled and unlabeled a-
Ò
Ò
NuPAGE Bis–Tris gel) and run with 1Â NuPAGE MOPS SDS Run-
ning Buffer for 50 min at 200 V. Then, the proteins were electro-
phoretically transferred onto the nitrocellulose membrane for
Western blot analysis. The membrane was probed either with rab-
bit anti-TAMRA antibody at 1:1000 dilution, followed by IR-
crystallin were performed using Biotin–Phosphine (9). To 2
.1% SDS, 50 mM Tris–HCl buffer containing either 10 g of Gal-
NAz-labeled or unlabeled -crystallin was added 36 L of PBS buf-
fer. Subsequently, 2 L of 10 mM Biotin–Phosphine (9) was added
lL of
0
l
a
l
l
and the reaction mixture was agitated at 37 °C for 90 min. Reaction
was quenched by removing excess Biotin–Phosphine reagent by
performing buffer-exchange into a total 40 lL volume of 50 mM
Tris–HCl buffer containing 0.1% SDS, using a 10 K-cutoff Amicon
Ultra-0.5 Centrifugal filter device. The reaction results were deter-
mined by gel electrophoresis followed by Western blot analysis.
Staudinger ligation reactions of azide-labeled and unlabeled HeLa
Ò
Dye 800CW-conjugated goat anti-rabbit secondary antibody at
1
:10,000 dilution for the evaluation of reactions with TAMRA-
Ò
tagged reagent, or with IRDye 800CW-Streptavidin at 1:5000 dilu-
tion for the evaluation of reactions with Biotin-tagged reagent. The
blot was imaged according to the manufacturer’s instructions by
using the Odyssey Infrared Imaging System.
To 150
HeLa cell lysates) in 30
.1% SDS was added 1 L of 10 mM TAMRA–DIFO (or Biotin–DIFO).
lg of GalNAz-labeled and unlabeled C. elegans lysates (or
cell lysates were performed using Biotin–Phosphine (9). To 20
of 0.1% SDS, 50 mM Tris–HCl buffer containing either 100 g of
GalNAz-labeled or unlabeled HeLa cell lysate were added 52
of PBS and 8 L of 10 mM Biotin–Phospine (9). The reaction mix-
ture was agitated at 37 °C for 90 min. Then, buffer-exchange into
a total 60 L volume of 50 mM Tris–HCl buffer containing 0.1%
lL
l
L of 50 mM Tris–HCl buffer containing
l
0
l
l
L
Reaction mixtures were agitated at room temperature for 20 min.
In the parallel experiments, same reaction mixtures were carried
out in the dark at room temperature for 20 min. Then, reaction buf-
l
l
fer was exchanged into 80
.1% SDS to remove unreacted TAMRA–DIFO (or Biotin–DIFO),
using a 10 K-cutoff Amicon Ultra-0.5 Centrifugal filter device. For
gel electrophoresis and subsequent Western blot analysis, 10
lL of 50 mM Tris–HCl buffer containing
SDS was performed to remove the excess, unreacted reagent using
a 10 K-cutoff Amicon Ultra-0.5 Centrifugal filter device. The reac-
tion results were determined by gel electrophoresis followed by
Western blot analysis.
0
l
L
Ò
of distilled water and 30
l
L of 4Â NuPAGE LDS sample buffer
were added to each reacted sample. Protocols for gel electrophore-
3
.3.7. Treatment of unlabeled Nup62 with TAMRA–Mal and
reaction of prior TAMRA–Mal-treated Nup62
g of Nup62 in 49 L of PBS was treated with and without
of 10 mM tetramethylrhodamine–5-maleimide (TAMRA–
sis and Western blot analysis were identical as described above.
1
l
l
3
.3.5. Reactions of azide-labeled and unlabeled proteins with
1 lL
terminal alkyne and copper catalyst (copper-catalyzed ‘click’
reaction)
Mal) solution at room temperature in the dark for 1 h. The reaction
was quenched by removing excess, unreacted TAMRA–Mal reagent
Reactions of azide-labeled and unlabeled
a-crystallin with
by performing buffer-exchange into a total volume of 49
0 mM Tris–HCl buffer containing 0.1% SDS, using a 10 K-cutoff
Amicon Ultra-0.5 Centrifugal filter device. Then, resulting Nup62
was reacted with 1 L of 10 mM DIFO–biotin solution at room tem-
lL of
TAMRA–Alkyne (7) (or Biotin–Alkyne, 8) were carried out using a
Click-iT™ Protein Analysis Detection Kit according to the manufac-
5
turer’s protocol. Briefly, to 10
-crystallin in 2 L of 50 mM Tris–HCl buffer containing 0.1% SDS
were added 10 L of distilled water and 20
L of 2 Â Click-iT™
reaction buffer containing TAMRA–Alkyne (or Biotin–Alkyne). Sub-
sequently, 2 L of 40 mM CuSO solution and 2 L of Click-iT™
reaction buffer additive 1 were added. After 2–3 min, 4 L of
lg of GalNAz-labeled and unlabeled
l
a
l
perature for 30 min. Reaction results were determined by gel elec-
trophoresis and Western blot analysis as described before.
l
l
l
4
l
l
3.3.8. Treatment of GalNAz-labeled or unlabeled
Nup62) with DTT and IAM
a-crystallin (or
Click-iT™ reaction buffer additive 2 was added and the reaction
tube was covered with foil to minimize light exposure and agitated
at room temperature for 20 min. Reaction was quenched by
removing excess DIFO-based reagent by performing buffer-ex-
To 20
reconstituted in 99
added 1 L of 1 M DTT. The reaction was heated at 80 °C on a heat-
lg of either GalNAz-labeled or unlabeled
a-crystallin
l
L of 0.1% SDS, 50 mM Tris–HCl buffer was
l
change into a total volume of 40
l
L of 50 mM Tris–HCl buffer con-
ing block for 15 min and then cooled at room temperature for
15 min. Immediately before use, sulfhydryl alkylating agent was
prepared by dissolving 14.4 mg of iodoacetamide in 0.1% SDS,
50 mM Tris–HCl, pH 7.5, to give a total volume of 1.4 mL to make
taining 0.1% SDS, using a 10 K-cutoff Amicon Ultra-0.5 Centrifugal
filter device. The reaction results were determined by gel electro-
phoresis followed by Western blot analysis. Reactions of azide-la-
beled and unlabeled C. elegans lysates (or HeLa cell lysates) with
TAMRA–Alkyne (7) and Biotin–Alkyne (8) were carried out using
a Click-iT™ Protein Analysis Detection Kit, according to the manu-
55.6 mM iodoacetamide solution. Then, 100
lL of the 55.6 mM
iodoacetamide (IAM) was added to the DTT-treated
a-crystallin
and the reaction mixture was protected from light and agitated
at room temperature for 40 min. The reaction was quenched by
removing excess, unreacted IAM reagent by performing buffer-ex-
facturer’s protocol. Briefly, 150
beled C. elegans lysates (or HeLa cell lysates) in 30
Tris–HCl buffer containing 0.1% SDS were added 50
Click-iT™ reaction buffer containing TAMRA–Alkyne reagent (or
Biotin–Alkyne). Subsequently, 5 L of 40 mM CuSO solution, and
L of Click-iT™ reaction buffer additive 1 were added. After 2–
l
g of GalNAz-labeled and unla-
L of 50 mM
L of 2Â
l
l
change into a total volume of 40
taining 0.1% SDS, using a 10 K-cutoff Amicon Ultra-0.5 Centrifugal
filter device. GalNAz-labeling of prior DTT/IAM-treated -crystallin
lL of 50 mM Tris–HCl buffer con-
l
4
a
5
3
l
(or Nup62) was carried out using a Click-iT™ O-GlcNAc Enzymatic
Labeling Kit as described previously. GalNAz-labeled or unlabeled
min, 10 lL of Click-iT™ reaction buffer additive 2 was added.
The reaction tube was covered with foil to minimize light exposure
and agitated at room temperature for 20 min. Reaction was
quenched by removing excess DIFO-based reagent by performing
a-crystallin (or Nup62) prior treated with DTT/IAM was then re-
acted with various azide-reactive reagents as described above.
Reaction results were determined by gel electrophoresis and Wes-
tern blot analysis as described before.
buffer-exchange into a total 80 lL volume of 50 mM Tris–HCl buf-

E. J. Kim et al. / Carbohydrate Research 377 (2013) 18–27
27
3
.3.9. Treatment of unlabeled C. elegans lysate (or HeLa cell
lysate) with DTT and IAM
To 200 g of either GalNAz-labeled or unlabeled C. elegans ly-
sate (or HeLa cell lysate) constituted in a 198 L volume of 0.1%
SDS, 50 mM Tris–HCl buffer was added 2 L of 1 M DTT. Reaction
5. Rabuka, D.; Hubbard, S. C.; Laughlin, S. T.; Argade, S. P.; Bertozzi, C. R. J. Am.
Chem. Soc. 2006, 128, 12078–12079.
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Sampathkumar, S. G.; Jones, M. B.; Yarema, K. J. Nat. Protoc. 2006, 1, 1840–1851.
Wang, Z.; Udeshi, N. D.; O’Malley, M.; Shabanowitz, J.; Hunt, D. F.; Hart, G. W.
Mol. Cell. Proteomics 2010, 9, 153–160.
l
l
8.
Laughlin, S. T.; Agard, N. J.; Baskin, J. M.; Carrico, I. S.; Chang, P. V.; Ganguli, A.
S.; Hangauer, M. J.; Lo, A.; Prescher, J. A.; Bertozzi, C. R. Methods Enzymol. 2006,
l
was heated at 80 °C on a heating block for 20 min and then cooled
at room temperature for 15 min. Immediately before use, sulfhy-
dryl alkylating agent was prepared by dissolving 14.4 mg of iodo-
acetamide in 0.1% SDS, 50 mM Tris–HCl, pH 7.5, to give a total
volume of 1.4 mL to make 55.6 mM iodoacetamide solution.
4
15, 230–250.
9. Blackman, M. L.; Royzen, M.; Fox, J. M. J. Am. Chem. Soc. 2008, 2, 2930–2944.
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0. Prescher, J. A.; Dube, D. H.; Bertozzi, C. R. Nature 2004, 130, 13518–13519.
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002, 41, 2596–2599.
2
2
00 lL of the 55.6 mM iodoacetamide (IAM) was added to the
1
1
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DTT-treated lysate and the reaction mixture was protected from
light and agitated for 40 min at room temperature. Reaction was
quenched by removing excess, unreacted IAM reagent by perform-
1
1
1
ing buffer-exchange into a total volume of 80
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2
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2
2
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.3.10. Evaluation of adduct formation between Biotin–DIFO
and sulfhydryl-containing small molecules
To 298 L of PBS, were added 1 L of 10 mM Biotin–DIFO (2)
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-cysteine, b-mercaptoethanol, or glutathione. Reaction mixture
1
1486–11493.
l
l
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l
L
was stirred at room temperature for 1 h and analyzed by ESI Mass
spectrometric analysis.
Acknowledgements
This work was supported by the NIDDK intramural funds (NIH)
and the National Research Foundation of Korea (2011-0027257).
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