Cell surface biotinylation by azaelectrocyclization: Easy-handling and versatile approach for living cell labeling

Article abstract of DOI:10.1016/j.bmc.2011.12.043

Versatile method for living cell labeling has been established. Cell surfaces are initially biotinylated by azaelectrocyclization, and then treated with the fluorescence-labeled avidin or the anti-biotin antibody.

Full text of DOI:10.1016/j.bmc.2011.12.043

Bioorganic & Medicinal Chemistry 20 (2012) 1865–1868
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Cell surface biotinylation by azaelectrocyclization: Easy-handling
and versatile approach for living cell labeling
Katsunori Tanaka a, , Satomi Yokoi b, Koji Morimoto c, Takayuki Iwata a, Yuka Nakamoto a,
⇑
Kaori Nakayama b, Koichi Koyama b, Takeshi Fujiwara c, Koichi Fukase a,
⇑
a Department of Chemistry, Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka-shi, Osaka 560-0043, Japan
b Kishida Chemical Co., Ltd, 14-10 Technopark, Sanda-shi, Hyogo 669-1339, Japan
c Division of Biochemistry, Department of Molecular Biology and Biochemistry, Graduate School of Medicine/Faculty of Medicine, Osaka University, 1-3 Yamada-oka, Suita-shi,
Osaka 565-0871, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 30 December 2011
Versatile method for living cell labeling has been established. Cell surfaces are initially biotinylated by
azaelectrocyclization, and then treated with the fluorescence-labeled avidin or the anti-biotin antibody.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Biotin
Electrocyclic reactions
Fluorescence
Living cell
Lysine
1. Introduction
Although our reaction is non-bioorthogonal to the primary amino
groups on cell surfaces, under such mild reaction conditions the
Covalent chemical labeling of living cells has garnered significant
attention in the fields related to molecular imaging,1 and has broad
and general applicability to both in vitro and in vivo studies of living
cells.2 Undesirable modification of key functions on cell surfaces
should be avoided in order to retain the cell’s native functions.
Therefore, bioorthogonal approaches, which can be combined with
biological techniques, have actively been investigated.3 Successful
examples include Bertozzi’s Staudinger ligation4–6 and strain-accel-
erated Huisgen 1,3-cycloaddtion reaction.7–10 These methods have
been applied to labeling of cells expressing oligosaccharides with
azido-containing sugar residues on the cell surfaces through biosyn-
thetic pathways,8 which subsequently reacted with the methoxy-
carbonyl phenyldiphenylphosphine or cyclooctyne derivatives.
Variety of the reactive cyclooctyne derivatives have continuously
been developed in pursuit of the efficient bioconjugation on the liv-
ing cell surfaces by this strategy.10b
most exposed and densely expressed amines that is, lysines of
membrane proteins and/or ethanol amine derivatives at cell sur-
faces react rapidly and selectively; hence minimizing the indis-
criminative amino modification as well as the significant probe
internalization, which cause the interferences with their native
functions. Pyridine derivatives as the electrocyclization products,
which retain the cationic charges as those of the inherent lysines,
might also contribute to the retention of the cell functions. In vivo
imaging of the Cy5 (cyanine fluorescence dye)-labeled lympho-
cytes was also achieved as an application of the present protocol;
the trafficking of the cells into the immune-related organs was
clearly visualized with markedly high imaging contrasts.13,14
In widely applying our electrocyclization chemistry to the cell
surface labeling by various fluorescence dyes and/or the other small
molecules,14 however, a ‘generality problem’, in a sense of the con-
venient use of our chemistry in both organic chemistry and biology
laboratories, still remains unsolved. Namely, the unsaturated alde-
hydes 1a are to be synthesized for each different fluorescence probe.
Although 1a with TAMRA or Cy5 dyes could readily be prepared,
some fluorescence structures, such as Alexa FluorÒ series and fluo-
resceins, were also foundto decompose duringthe oxidation process
from the precursor alcohol to the probes 1a. Because the structures
of most of the fluorescence dyes are not even disclosed, the stability
during the preparation of the probes cannot be predicted. In order to
generalize the cell-surface labeling, herein we reporta two-step pro-
tocol by combining our electrocyclization chemistry with the biotin/
On the other hand, we recently developed an amine-based
labeling through rapid 6p
-azaelectrocyclization.11,12 We have used
this method to efficiently and conveniently introduce the fluores-
cent groups to the amino groups on the cell surfaces13 via a reac-
tion with unsaturated aldehyde probes (such as probe 1a in
Scheme 1a) at very low concentrations (can be decreased down
to 10À8 M) within a short time (10 min) at room temperature.
⇑
Corresponding authors.
E-mail address: koichi@chem.sci.osaka-u.ac.jp (K. Fukase).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.12.043

1866
K. Tanaka et al. / Bioorg. Med. Chem. 20 (2012) 1865–1868
the general and versatile approach to the living cell surface labeling
and/or engineering.
(a) Previous work
H2N
CO2Et
N
(flu = TAMRA or Cy5)
CO2Et
CHO
O
O
H
N
flu HN
flu
N
N
H
H
2. Results and discussion
2.1. Probe preparation
cells
1a
Azaelectrocyclization
10 min, 24-37 o
C
~10-8 M of probe conc
Although we have previously reported in situ generation of the
biotin-containing unsaturated adehyde14 through the Staudinger
ligation using the 2-methoxycarbonylphenyldiphenylphosphine
reagent,5d the facile air oxidation of the phosphine sometimes
caused the reproducibility problems during the cell labeling
experiments. We therefore used in this experiment the probe
1b, where the biotin is covalently introduced to the unsaturated
aldehyde in advance through the amide bond. Oxidation of the
alcohol 2b, which is now commercialized15 as the precursor of
(b) This work
H2N
CO2Et
CO2Et
1)
biotin
O
H
N
O
N
CHO
N
H
biotin
HN
cells
1b
N
H
Azaelectrocyclization
CO2Et
O
N
flu
2)
flu
1b, was achieved by the improved procedures using a few
lg-
biotin
HN
N
H
scale in Eppendorf-tube (Scheme 2). Thus, the alcohol 2b was
treated with IBX-resin (2-iodoxybenzoic acid) in small amount
of DMSO for 30 min.13 After the resin was filtered off, the result-
ing aldehyde probe 1b was directly used by diluting with PBS
buffer solution (final PBS solution containing 0.01% of DMSO).
Alternatively, the alcohol 2b could be oxidized by Dess–Martin
periodinane in DMSO (see details in Section 4); the reaction solu-
tion was diluted by PBS buffer and directly treated with the living
cells. Any significant effects on cell viability and/or functions
could not be observed by the latter procedure (vide infra). For
both cases, the sulfur atom in a half amount of 2b was oxidized
to the sulfoxide as judged by MALDI-TOF-MS analysis; although
the oxidized derivatives of biotin are known to show the 10-fold
reduced interaction with the avidins,16 they still retained very
small Kd value of 10À14 M, and therefore used for cell labeling
without separating the two compounds. In addition to the bio-
tin-aldehyde probe 1b, the TAMRA (carboxytetramethylrhod-
amine)-fluorescence probe 1a,13 as well as biotin 3, hydrazone
linked 4, and PEG-amine linked 5, were used as the positive
and negative controls for the labeling experiments (Scheme 2).
Labeled mAb, avidin
Scheme 1. Schematic presentation of cell surface labeling by azaelectrocyclization.
(a) Previous work. (b) This work.
avidin and/or biotin/anti-biotin monoclonal antibody (mAb) inter-
actions, as shown in Scheme 1b. Thus, the cell surfaces are initially
biotinylated by azaelectrocyclization using the probe 1b, and then
treated with the fluorescence-labeled avidin or the anti-biotin anti-
body. Considering the commercial availability of variously labeled
avidins and mAbs, and the numerous reports on the biotin-based
techniques in biology-directed research, the present method offers
O
CO2Et
HN NH
H
H
O
H
N
H
N
O
O
N
H
OH
O
O
S
O
O
2b
in Eppendorf tube (10 µg)
Method (a)
IBX-resin / DMSO (2 µL)
then, filtration and PBS dilution
2.2. Labeling experiments
Labeling experiments were performed using MDCK (Madin-
Darby canine kidney) and HeLa (cervical cancer cell line) cells
(see details in Section 4): We initially treated the cells (2 Â 105/
Method (b)
Dess-Martin periodinane / DMSO (2 µL)
then, PBS dilution
700
lL), which were cultured on glass cover slips coated with
-lysine, with biotin-probe 1b (1.0 Â 10À6 M in PBS) for
O
poly-
L
CO2Et
HN NH
5 min at 37 °C, 5% CO2 (Scheme 1b). After the cells were washed
with culture medium twice in order to remove the excess alde-
hyde probe, they were subsequently treated with Cy3-labeled
anti-biotin mAb (excitation: 547 nm, emission: 563 nm) or
Alexa594-labeled streptavidin (590 nm/617 nm) in medium (dilu-
tion of 1:100 and 1:200, respectively) for 30 min, under the iden-
tical conditions performed for the biotin-labeling conditions. As
seen in Figure 1, any significant differences in morphology were
not found between the labeled cells (Fig. 1a and b) and the con-
trol cells (Fig. 1c and d). Cell division could also be observed in
some labeled cells; thus any notable effects on cell viability could
not be observed during a two-step labeling procedure, but the
prolonged subjection of cells to the PBS solution of 1b, that is,
more than 20 min, caused round-shaped outward appearance.
The fluorescence labeled cells were also treated with Hoe-
chst33342 as a blue dye to visualize the nuclei and fixed with
formaldehyde. All the control experiments using 1a (TAMRA)
and 3–5 were also performed according to the procedures de-
scribed above, and analyzed by confocal microscopy (Fig. 1).
H
H
O
H
N
H
N
O
O
N
H
H
O
O
O
R
O
O
1b: R = S, S(O) (1 : 1)
PBS buffer solution (10-6M~10-7M)
positive control
CO2Et
Me2N
O
O
H
N
N
H
H
O
N
H
O
O
CO2
1a (TAMRA)
Me2N
negative control
O
O
O
HN NH
HN NH
HN NH
H
H
H
H
H
H
H
N
OH
NHNH2
O
NH2
O
O
S
S
S
O
O
O
3
4
5
Scheme 2. Oxidation of biotin-alcohol to 1b and the other biotin derivatives as
control for labeling experiments.

K. Tanaka et al. / Bioorg. Med. Chem. 20 (2012) 1865–1868
1867
Figure 1. Detection of biotin-labeled HeLa cells by azaelectrocyclization; bars: 20 lm. (a, b) Cells treated by the method using 1b to label the cell surface. (c, d) Cells treated
with either PBS (c) or biotin (d) instead of method using 1b. For each condition, cells were further labeled with either Cy3-conjugated anti-biotin mouse monoclonal antibody
(a, c, d) or Alexa594-conjugated streptavidin (SA) (b, c, d). Nuclei and chromosomes were labeled with Hoechst33342 (Blue). (e) Cells treated by the method using 1a to label
the cell surface with TAMRA (red). (a–d) Left panels: merge of Cy3 or Alexa594 and Hoechst33342, middle panels: Cy3 or Alexa594, right panels: merge of phase contrast and
Cy3 or Alexa594. (e) Left panel: TAMRA, right panel: merge of phase contrast and TAMRA.
2.3. Confocal microscopic analysis
individually heterogeneous in functional and physical properties
and those of amino-riched domain could preferentially be labeled,
of which investigation is currently in progress.
Red fluorescence derived from the Cy3 and Alexa594 dyes was
observed on both HeLa and MDCK cells reacted with the aldehyde
1b (Data shown only for HeLa cells in Figure 1a and b, and see
Supplementary data for MDCK cells), whereas no fluorescence
was observed in cells treated with PBS (without 1b) and fluores-
cence-labeled anti-biotin mAb or streptavidin (Fig. 1c). Further-
more, any fluorescence could not be observed by using the biotin
derivatives 3–5, instead of 1b, which lack the unsaturated alde-
hyde structures necessary for azaelectrocyclization (Fig. 1d and
Supplementary data). Detailed 2D and 3D microscopic analysis of
the labeled cells by the biotin/avidin and the biotin/antibody con-
jugates, clearly showed the fluorescence localization on the cell
membrane. These results clearly show that biotin-probe 1b rapidly
reacts with the amino groups of the lysines and/or other amino-
containing cell membrane constitutes, such as phosphatidyl etha-
nolamines, to covalently anchor the biotin molecule through
azaelectrocyclization,13 which the antibody or the streptavidin
interacted with.
It should be noted from Figure 1a and b that not all cells were
equally labeled, therefore showed selective cell surface labeling.
Namely, rather than the cells with cell–cell contact showing colo-
nization, the cells that do not contact with neighboring cells are
preferentially labeled possibly by more exposed cell surface to
the probes. Such labeling properties were same as those performed
by directly reacted with TAMRA-fluorescence probe 1a (Fig. 1e).
Based on previous results on the reactivity of 1a and other unsat-
urated aldehyde probes,12,13 azaelectrocyclization selectively pro-
ceeds at the most exposed surface regions to the probes and/or
at the high protein assembly (high concentration of Lys) on the cell
surface. Taking these into consideration, the biotin-labeling might
also proceed most probably at the accessible cell surfaces rather
than those inside the aggregates under the mild labeling condi-
tions, that is, at 37 °C for 5 min. Alternatively, the cell lines are still
3. Summary
In summary, we established labeling of whole cells through
chemical biotinylation followed by the anti-biotin mAb or avidin
treatments. With our hands, the labeling by biotin-probe 1b was
still effective at a 10À7 M concentration (Supplementary data), pro-
viding one of the highly diluted chemical methods realized so far
through a covalent bond formation. Because variously labeled
and functionalized biotin antibodies and avidins are commercially
available, and easily prepared if necessary, the present two-step
protocol offers versatile and convenient chemical approaches to la-
bel and engineer the living cells. Based on the results described
herein, it may further be possible to label living cells with the me-
tal-incorporated DOTA,12 which would expand the method to the
radiotracer-based targeting or imaging of whole cells for PET,
MRI,17 or other radiopharmaceutical purposes. Research in this
direction is currently under way in our laboratory.
4. Experimental
4.1. Preparation of biotin probe 1b
Dess–Martin periodinane (22
l
g, 52 nmol) in 2
lL of DMSO was
added at room temperature to allylic alcohol 2b (10
lg, 13 nmol) in
an Eppendorf tube. After the reaction mixture was shaken at room
temperature for 10 min, the resulting DMSO solution of 1b was di-
luted by adding the 198
l
L of PBS (ca. 6 Â 10À5 M). Various con-
centrations of solution of probe 1a were then prepared by
further dilution by PBS; the 1.0 Â 10À6 M solution of 1b contains
0.01% DMSO, by this procedure.

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K. Tanaka et al. / Bioorg. Med. Chem. 20 (2012) 1865–1868
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buffer solution (pH 8.4) and grown overnight at 37 °C, 5% CO2. Cells
were washed twice with PBS (pH 7.2–7.4) to exclude residual label-
ing inhibitors. Washing buffer was replaced with either biotin-con-
jugated cell labeling probe or biotin derivative diluted in PBS at
1 Â 10À6–1 Â 10À7 M concentration. After incubation for 5 min at
37 °C, 5% CO2, cells were washed twice with cultured medium to
stop the labeling reaction. Biotinylated cells were further incubated
with Cy3-conjugated anti-biotin mouse monoclonal antibody (Jack-
son ImmunoResearch Inc.) or Alexa FluorÒ 594-conjugated strepta-
vidin (Invitrogen) in culture medium for 30 min at 37 °C, 5% CO2.
Nuclei and chromosomes were labeled with 2 lg/mL Hoechst33342
(Invitrogen) in addition to the secondary antibody. Labeled cells
were fixed with 2% paraformaldehyde and 4% sucrose in PBS for
20 min at room temperature. They were subsequently washed with
PBS and mounted with Prolong Gold (Invitrogen). Images were cap-
tured by confocal laser microscopy Fluoview FV1000 system (Olym-
pus) using 20Â objective lens 4Â zoom.
Acknowledgments
9. (a) Baskin, J. M.; Prescher, J. A.; Laughlin, S. T.; Agard, N. J.; Chang, P. V.; Miller, I.
A.; Lo, A.; Codelli, J. A.; Bertozzi, C. R. Proc. Natl. Acad. Sci. U.S.A. 2007, 104,
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2008, 130, 11486; (c) Laughlin, S. T.; Baskin, J. M.; Amacher, S. L.; Bertozzi, C. R.
Science 2008, 320, 664.
This work was supported in part by Grants-in-Aid for Scientific
Research Nos. 19681024 and 19651095 from the Japan Society for
the Promotion of Science, Collaborative Development of Innovative
Seeds from Japan Science and Technology Agency (JST), New En-
ergy and Industrial Technology Development Organization (NEDO,
project ID: 07A01014a), Research Grants from Yamada Science
Foundation as well as Molecular Imaging Research Program,
Grants-in-Aid for Scientific Research from the Ministry of Educa-
tion, Culture, Sports, Science and Technology (MEXT) of Japan.
10. (a) Ning, X.; Guo, J.; Wolfert, M. A.; Boons, G.-J. Angew. Chem., Int. Ed. 2008, 47,
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Supplementary data
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Supplementary data associated with this article can be found, in
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