A cascading reaction sequence involving ligand-directed azaelectrocyclization and autooxidation-induced fluorescence recovery enables visualization of target proteins on the surfaces of live cells

Article abstract of DOI:10.1039/c3ob42267d

A general probe designed to induce a cascading sequence of reactions on a target protein was efficiently synthesized. The cascading reaction sequence involved (i) ligand-directed azaelectrocyclization with lysine and (ii) the autooxidation-induced release of a fluorescence quencher from the labeled protein. The probe was linked to a cyclic RGDyK peptide to enable the selective visualization of integrin α_Vβ₃ on the surfaces of live cells.

Full text of DOI:10.1039/c3ob42267d

Organic &
Biomolecular Chemistry
View Article Online
View Journal | View Issue
PAPER
A cascading reaction sequence involving
ligand-directed azaelectrocyclization and
autooxidation-induced fluorescence recovery
enables visualization of target proteins on the
surfaces of live cells†
Cite this: Org. Biomol. Chem., 2014,
12, 1412
Katsunori Tanaka,*a Masataka Kitadani,b Ayumi Tsutsui,a Ambara R. Pradipta,a
Rie Imamaki,c Shinobu Kitazume,c Naoyuki Taniguchic and Koichi Fukase*b
A general probe designed to induce a cascading sequence of reactions on a target protein was efficiently
synthesized. The cascading reaction sequence involved (i) ligand-directed azaelectrocyclization with
lysine and (ii) the autooxidation-induced release of a fluorescence quencher from the labeled protein.
The probe was linked to a cyclic RGDyK peptide to enable the selective visualization of integrin αVβ3 on
the surfaces of live cells.
Received 14th November 2013,
Accepted 19th December 2013
DOI: 10.1039/c3ob42267d
www.rsc.org/obc
Introduction
A variety of novel chemical methods have been recently com-
bined with biological techniques to achieve protein labeling.
Successful examples include the Cu(I)-mediated Huisgen
[3 + 2] cycloaddition,1 Staudinger ligation,2 and the strain-pro-
moted Cu(I)-free Huisgen cycloadditon3 involving an azide that
has been genetically, metabolically, or enzymatically intro-
duced at a desired position within a protein or on a cell
surface. Variants of the Cu(I)-free reaction,4 e.g., the tetrazine/
norbornene D–A reaction4a–f and the base- or photo-induced
nitrile imine/norbornene cycloaddition reaction,4g–i have been
examined in an effort to improve the reactivity, and chemo-
orthogonal and bio-orthogonal profiles. Chemical methods in
which a protein-specific ligand is used to directly introduce a
probe molecule have been investigated by Hamachi and co-
workers.5 Their methods were successfully applied to the fluo-
rescence labeling of proteins in cells, tissues, and living mice.6
Scheme 1 Cascading reaction involving 6π-azaelectrocyclization,
autooxidation, and hydrolysis.
Independently of these efforts, we have been investigating a
lysine-based protocol for labeling peptides, proteins, and
living cell surfaces (Scheme 1).7,8 The labeling reaction
involves Schiff base formation and a subsequent rapid 6π-aza-
electrocyclization.9 The 1,2-dihydropyridine products are then
spontaneously oxidized in aqueous media, which accelerates
the hydrolysis of the ester and provides a zwitterion as the
amino modification product. This method was used to
efficiently and selectively introduce DOTA (1,4,7,10-tetraaza-
cyclodecane-1,4,7,10-tetraacetic acid) or fluorescent groups
(substituents at R1, see Scheme 1) onto the lysine residues
through a reaction with the unsaturated aldehyde probes 1 at
very low concentrations (∼10–8 M) within a short period of
time (10–30 min) at room temperature. This electrocyclization
protocol was used to enable the successful visualization
of receptor-mediated accumulation, circulatory residence, and
aBiofunctional Synthetic Chemistry Laboratory, RIKEN, 2-1 Hirosawa, Wako-shi,
Saitama 351-0198, Japan. Fax: +81-48-467-9405; Tel: +81-48-467-9379;
E-mail: kotzenori@riken.jp
bDepartment of Chemistry, Graduate School of Science, Osaka University, 1-1
Machikaneyama-cho, Toyonaka-shi, Osaka 560-0043, Japan
cDisease Glycomics Team, Systems Glycobiology Research Group, RIKEN-Max Planck
Joint Research Center for Systems Chemical Biology, RIKEN Global Research Cluster,
RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
†Electronic supplementary information (ESI) available: Experimental details,
characterization data and selected copies of NMR spectra. See DOI: 10.1039/
c3ob42267d
1412 | Org. Biomol. Chem., 2014, 12, 1412–1418
This journal is © The Royal Society of Chemistry 2014

View Article Online
Organic & Biomolecular Chemistry
Paper
was loaded via an ester linkage. The structural arrangement
shown in 3 achieved efficient quenching of the coumarin fluo-
rescence by DABCYL so that the probe 3 itself was non-fluo-
rescent. Incubation of albumin with the probe 3 selectively
labeled the lysine moieties proximal to the ligand-binding site,
and the subsequent autooxidation and hydrolysis reactions
released the DABCYL quencher to selectively recover the cou-
marin fluorescence within the albumin. Although probe 3
blocked the protein-binding site after labeling (this technique
could be characterized as invasive protein labeling), this strat-
egy enabled the sensitive detection of the target albumin, even
in a mixture of several proteins and peptides, by achieving a
high fluorescence contrast.
We envisioned that the simultaneous introduction of bio-
active peptide- or glycan-based ligands bound to a fluorescence
quencher and to probe 3 through an ester linkage could
permit the “switched on” labeling and imaging of receptors in
live systems, e.g., on the surfaces of live cells or in live
animals. Here, we report a general route to the preparation of
fluorescence “switch on” probes that can conveniently bind to
a variety of amine-containing ligands during the last stage of
synthesis through a combination of azaelectrocyclization and
the strain-promoted click reactions. As a model case, the cyclic
RGDyK peptide integrin αVβ3 ligand was loaded onto a new
“switched on” probe, and the integrin αVβ3 was used to label
live RAW264.7 cells (mouse leukemic monocyte macrophage
cells). The cells were then selectively visualized through a cas-
cading sequence of ligand-directed azaelectrocyclization and
autooxidation-induced fluorescence recovery.
Scheme 2 Two previously reported ligand-directed strategies for
protein labeling. (a) Lysine-selective and noninvasive fluorescence label-
ing. (b) Lysine-selective and invasive “switched on” labeling.
trafficking of glycoproteins and/or lymphocytes via PET and
non-invasive fluorescence imaging.7,8
The site-selective and non-destructive modification of
target proteins has been achieved by directing reactive groups
to a specific site using a small molecule ligand of the protein
(Scheme 2a).10 After the selective azaelectrocyclization of the
probe 2 with the target lysine in human serum albumin (HSA),
the ligand connected to the ester linkage was then spon-
taneously cleaved from the conjugated albumin. This ligand-
directed approach may be applied to the selective and noninva-
sive labeling of target proteins in cell lysates, on cell surfaces,
or even in living animals. The selective labeling of a target
protein in a mixture of biomolecules, e.g., using fluorescence
reporter groups, usually yields a poor fluorescence contrast
between the labeled protein and the unreacted probes. The
development of labeling probes that are fluorescently silent by
default (“caged” fluorescence) but are “switched on” when
they selectively react with the target proteins would go a long
way toward removing the unlabeled background fluorescence
signal.6b,11
We therefore incorporated a fluorescence quenching system
into an unsaturated aldehyde probe 3 (Scheme 2b),12 which
could be unlocked only after an azaelectrocyclization-induced
cascading reaction had proceeded in the presence of the target
lysine. The coumarin moiety, which functioned as both a
ligand of serum albumin and as a “caged” fluorescence repor-
ter group, was introduced at the left terminus of the probe 3,
and DABCYL (4-[4-(dimethylamino)phenylazo]benzoic acid)
Results and discussion
The application of the fluorescence “switched on” probes to
imaging of various proteins in biological systems required that
the synthetic route be general and practical, and that a variety
of protein-selective ligands, i.e., peptides, could be installed
during the last stages of synthesis. The introduction of
complex and often hydrophilic molecules, e.g., peptides
or glycans, to the relatively unstable (E)-3-alkoxycarbonyl-
5-phenyl-2,4-dienal (see structure 3 in Scheme 2) through an
appropriate ester linkage similar to that used to link the cou-
marin fluorophore and DABCYL quencher was not trivial. The
conventional thiol-based or photo-induced ligation conditions
led to the decomposition, polymerization, or isomerization of
the (E)-carbonyl-2,4-dienal system. The dienals themselves
were not appropriate substrates for the amide coupling reac-
tion due to a rapid azaelectrocyclization reaction that occurred
upon exposure to the free amino groups in the linker and/or
the dienal molecules.
We eventually hit upon the idea that the unsaturated alde-
hydes with an azide functionality, such as 6 (Scheme 3), could
be coupled to the acetylene-functionalized ligands 5 via a
strain-promoted click reaction3 during the last stage of syn-
thesis. Previous investigations13 indicated that the strain-pro-
moted click reaction was chemoselective toward the azides
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem., 2014, 12, 1412–1418 | 1413

View Article Online
Paper
Organic & Biomolecular Chemistry
and strained acetylenes and did not destroy the dienal system.
In this work, dibenzocyclooctyne (DIBO), developed by Boons
and co-workers,3e was used as the strained acetylene because
this acetylene displays a relatively high reactivity and is readily
prepared from a simple starting material.
We also envisioned that various bioactive ligands, e.g., pep-
tides, could be readily functionalized with the strained acety-
lene, DIBO, by submitting the DIBO aldehyde 4 to a rapid
azaelectrocyclization reaction with lysine amino groups
(Scheme 3). The last two steps of the probe synthesis, i.e.,
peptide functionalization with DIBO (from 4 to 5) and coup-
ling to the azide–aldehyde (5 + 6), and the subsequent ligand-
directed “switched on” labeling of the target proteins, could
all be handled in a one-pot process without isolating the
unstable and complex intermediates. The synthetic route
shown in Scheme 3 is applicable to a variety of peptides and
other amine-containing ligands, once the DIBO-aldehyde 4
and azide–aldehyde 6 have been prepared as the two key inter-
mediates of the probe synthesis.
Throughout this study, we focused on the use of an RGD
peptide as an αVβ3-integrin ligand. αVβ3-Integrin is a cell
adhesion molecule and is highly expressed on endothelial and
tumor cells. The cyclic RGDyK peptide (see the structure
shown in Scheme 5) is used as a strong integrin agonist in
breast, brain, and lung cancer models.7b,14 This peptide pre-
sents an attractive model ligand for the selective imaging of
cell surface integrin molecules via the fluorescence “switched
on” strategy.
Scheme 3 One-pot sequence involving ligand functionalization with
DIBO (1st step), loading onto a fluorescently “caged” probe (2nd step),
and ligand-directed azaelectrocyclization and oxidation-induced fluo-
rescence recovery (3rd step).
The synthesis of both intermediates 4 and 6 was carried out
from the Boc-protected amines
8 and 11, which were
previously synthesized through a key Stille coupling step
(Scheme 4).7a,10 Thus, the Boc and THP protecting groups of 8
Scheme 4 Synthesis of the DIBO- and azide-containing unsaturated ester aldehydes. HATU: O-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluro-
nium hexafluorophosphate; TBAF: tetra-n-butylammonium fluoride.
1414 | Org. Biomol. Chem., 2014, 12, 1412–1418
This journal is © The Royal Society of Chemistry 2014

View Article Online
Organic & Biomolecular Chemistry
Paper
were simultaneously removed by treatment with 6 M HCl in
methanol. The resulting amino group was selectively acylated
by DIBO 9 activated by the p-nitrophenylcarbonate to yield the
alcohol 10 in 43% yield over two steps. Oxidation with the
Dess Martin periodinane in CH2Cl2 for 20 min gave one of the
key intermediates, the DIBO-aldehyde 4.
The two Boc protecting groups in 11 were simultaneously
removed by treatment with 20% TFA in CH2Cl2 to give the
unstable diamine. The more reactive aliphatic amine at the C3
ester group was selectively reacted with the in situ-generated
pentafluorophenyl ester of the DABCYL-azide acid 12 to
provide 13 in a 52% yield in two steps. The remaining anilino
nitrogen group in 13 was acylated with 7-diethylaminocou-
marin-3-carboxylic acid in the presence of HATU and triethyl-
amine in DMF to provide the coupling product 14 in a 79%
yield. TBDPS deprotection was then achieved in a 47% yield by
reaction with TBAF buffered with acetic acid in THF. Finally,
the allylic alcohol was oxidized by the Dess Martin periodinane
to afford the azide–aldehyde 6 as the second intermediate. The
relatively unstable aldehyde 6, the structure of which was con-
1
firmed by the presence of the characteristic H NMR and MS
signatures, was then immediately used in a strain-releasing
click reaction with the acetylene-functionalized cRGDyK
ligands to enable the labeling experiments (vide infra).
Prior to performing the one-pot synthesis, “click” ligand
loading, and cell surface “switched on” imaging studies, the
efficiency of the cascade process involving the azaelectrocycli-
zation and autooxidation-induced fluorescence recovery steps
was determined using the azide–aldehyde 6 (Fig. 1). As
designed, the fluorescence spectroscopic analysis of 6 (concen-
tration of 6: 1.0 × 10–6 M, 10% MeOH in PBS) detected a fluo-
rescence signal intensity that was 10% of the fluorescence
intensity due to the 7-diethylaminocoumarin carboxylic acid
moiety at 480 nm (excitation at 420 nm) as a result of efficient
quenching. The quenching resulted from fluorescence reson-
ance energy transfer (FRET) between the two proximal dyes in
6 (Fig. 1a). Incubation at 25 °C of probe 6 with the n-octyl-
amine (used as a model compound for lysine) resulted in a
rapid two-fold increase in the coumarin fluorescence over
60 min (Fig. 1b). No significant increase in fluorescence was
observed in the absence of the amine (Fig. 1c), indicating that
the DABCYL quencher had been released from the coumarin-
modified octylamine through the azaelectrocyclization-
initiated autooxidation and hydrolysis reactions. The for-
mation of the zwitterion product was confirmed by MALDI-
TOF-MS analysis (m/z = 570.3 theoretical; 570.3 for C34H40N3O5
(M + H)+); thus, the amino group could be sensitively detected
using the new “switched on” probe. Because the 1,2-dihydro-
pyridine could be rapidly oxidized and hydrolyzed during
ionization by FAB, CI, ESI, and MALDI, an MS-based time-
course analysis of the 1,2-dihydropyridine formation, oxi-
dation, and hydrolysis processes was not possible, as reported
previously.12
Fig. 1 Monitoring the azaelectrocyclization-induced fluorescence
recovery (10% MeOH in PBS, rt). (a) Comparison of the fluorescence
intensities of the azide–aldehyde 6 and the 7-diethylaminocoumarin
carboxylic acid. Time-course fluorescence spectra of 6 (b) in the pres-
ence of n-octylamine, and (c) in the absence of n-octylamine.
strained acetylene, (2) “click” ligand loading of the probe 6,
and (3) selective fluorescence “switched on” imaging of the
integrins expressed on the cell surfaces (Scheme 5). The lysine
amino group in the cyclic RGDyK peptide initially reacted with
an equimolar amount of the DIBO-aldehyde 4 at room temp-
erature. The peptide ligand was functionalized with the
strained acetylene in quantitative yield over 30 min, as
judging from the TLC and MS analysis. The reaction mixture
was then directly treated with the azide–aldehyde 6 at 55 °C for
5 min, providing the desired RGDyK-loaded aldehyde 7 in
approximately 50% yield based on the direct MS analysis of
the reaction mixtures. 7 was found to decompose under
either prolonged reaction times (in an attempt to improve the
The promising fluorescence results led to an examination
of the one-pot sequence of ligand-directed “switched on” label-
ing reactions, including (1) peptide modification by the
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem., 2014, 12, 1412–1418 | 1415

View Article Online
Paper
Organic & Biomolecular Chemistry
RAW264.7 cells, which express high levels of αVβ3-integrin on
the cell surfaces, and Hep3B cells, which express lower levels
of αVβ3-integrin. The cells were cultured on glass coverslips
(2 × 105/50 μL) to facilitate imaging. Control experiments, in
which the cells were treated with the azide–aldehyde 6 without
the cyclic RGDyK ligand, were performed under identical
conditions. After treatment with the probes, the live cells
were immediately analyzed (within 1 min)15 by confocal
microscopy.
The green fluorescence derived from the coumarin fluoro-
phore was clearly observed on the RAW264.7 cells treated with
probe 7 (Fig. 2a, middle panel), whereas no significant fluo-
rescence was detected in the control cells treated with the
azide–aldehyde 6 (Fig. 2a, left panel). Fluorescence intensity
(averaged at each cell) was 5.7 times higher for the cells
treated by the probe 7, than those treated by the control 6. The
fluorescence signals on the Hep3B cells treated with probe 6 or
7 were indistinguishable (Fig. 2b). These results indicated that
the selective “switched on” labeling of the cell surface αVβ3-
integrin was efficiently achieved through the strong RGD–
integrin interaction. The results were further supported by
treating the RAW264.7 cells with probe 7 in the presence of an
excess of the acetylated RGDyK peptide (1 × 10–4 M). The fluo-
rescence intensity was significantly reduced by the presence of
the competitive inhibitor (Fig. 2a, right panel). Despite
repeated trials, the fluorescently labeled αVβ3-integrin could
not be detected by SDS-PAGE analysis of the 7-treated
RAW264.7 cell lysate, presumably due to the presence of small
amounts of the αVβ3-integrin and/or instabilities in the 7-integ-
rin conjugates during the lysis procedure. The data shown
in Fig. 2 suggested that the selective fluorescence recovery
on the RAW264.7 cells was derived from the integrin-selective
“switched on” labeling procedure.
Scheme 5 One-pot DIBO functionalization of the cyclic RGDyK ligand
via (1) azaelectrocyclization and (2) loading onto a “switched on” probe
by means of a strain-promoted click reaction.
conversion to 7) or HPLC purification. The decomposition
reaction resulted from the instability of the densely function-
alized dienal moiety in 7. Nevertheless, the cell surface reac-
tion of the azide–aldehyde 6 was much slower than the
reaction involving the integrin ligand in 7 (vide infra), and the
mixture could be used directly in labeling experiments.
Thus, the one-pot solution of the probe 7, as prepared in
Scheme 5, was diluted to a concentration of 1 × 10–5 M in PBS
(containing 0.05% DMSO) and was applied directly to
The cell morphologies were not observed to vary during the
labeling process. Thus, the “switched on” imaging of live cell
surfaces under mild conditions, i.e., an exposure time of less
than 1 min at 37 °C, did not detectably affect the cell viability.
Fig. 2 Optical microscopy images of RAW264.7 and Hep3B cells treated with the fluorescence “switched on” probes 6 and 7. The bars indicate
20 μm. (a) RAW264.7 cells treated with the probes 6 (left panel), 7 (middle panel), and 6 in the presence of the NAc-cyclic RGDyK peptide (right
panel). (b) Hep3B cells treated with 6 (left panel) and 7 (middle panel).
1416 | Org. Biomol. Chem., 2014, 12, 1412–1418
This journal is © The Royal Society of Chemistry 2014

View Article Online
Organic & Biomolecular Chemistry
Paper
Fig. 2a suggested that not all fluorescent probes appeared to
be present on the cell surfaces. The azaelectrocyclization reac-
tion with the unsaturated aldehydes proceeded smoothly at
the lysine amino groups of the αVβ3-integrin on the cell
surface, and it is possible that the labeled integrin was rapidly
endocytosed within the experimental timeframe, as has been
observed previously.8,13
J. W. Chin, J. C. Anderson and P. G. Schultz, J. Am.
Chem. Soc., 2003, 125, 11782–11783; (e) A. E. Speers and
B. F. Cravatt, Chem. Biol., 2004, 11, 535–546; (f) J. Gierlich,
G. A. Burley, P. M. E. Gramlich, D. M. Hammond and
T. Carell, Org. Lett., 2006, 8, 3639–3642; (g) P. V. Chang,
X. Chen, C. Smyrniotis, A. Xenakis, T. Hu, C. R. Bertozzi
and P. Wu, Angew. Chem., Int. Ed., 2009, 48, 4030–4033.
2 (a) E. Saxon and C. R. Bertozzi, Science, 2000, 287, 2007–
2010; (b) K. L. Kiick, E. Saxon, D. A. Tirrell and
C. R. Bertozzi, Proc. Natl. Acad. Sci. U. S. A., 2002, 99, 19–24;
(c) D. J. Vocadlo, H. C. Hang, E.-J. Kim, J. A. Hanover and
C. R. Bertozzi, Proc. Natl. Acad. Sci. U. S. A., 2003, 100,
9116–9121; (d) P. V. Chang, J. A. Prescher, M. J. Hangauer
and C. R. Bertozzi, J. Am. Chem. Soc., 2007, 129, 8400–8401;
(e) M. J. Hangauer and C. R. Bertozzi, Angew. Chem., Int.
Ed., 2008, 47, 2394–2397.
3 (a) N. J. Agard, J. A. Prescher and C. R. Bertozzi, J. Am.
Chem. Soc., 2004, 126, 15046–15047; (b) M. Fernández-
Suárez, H. Baruah, L. Martínez-Hernández, K. T. Xie,
J. M. Baskin, C. R. Bertozzi and A. Y. Ting, Nat. Biotechnol.,
2007, 25, 1483–1487; (c) J. M. Baskin, J. A. Prescher,
S. T. Laughlin, N. J. Agard, P. V. Chang, I. A. Miller, A. Lo,
J. A. Codelli and C. R. Bertozzi, Proc. Natl. Acad. Sci. U. S. A.,
2007, 104, 16793–16797; (d) S. T. Laughlin, J. M. Baskin,
S. L. Amacher and C. R. Bertozzi, Science, 2008, 320, 664–
667; (e) X. Ning, J. Guo, M. A. Wolfert and G.-J. Boons,
Angew. Chem., Int. Ed., 2008, 47, 2253–2255;
(f) B. C. Sanders, F. Friscourt, P. A. Ledin, N. E. Mbua,
S. Arumugam, J. Guo, T. J. Boltje, V. V. Popik and
G.-J. Boons, J. Am. Chem. Soc., 2011, 133, 949–957;
(g) J. Z. Yao, C. Uttamapinant, A. Poloukhtine,
J. M. Baskin, J. A. Codelli, E. M. Sletten, C. R. Bertozzi,
V. V. Popik and A. Y. Ting, J. Am. Chem. Soc., 2012, 134,
3720–3728.
Conclusions
In conclusion, we established a method for preparing a
general “switched on” labeling probe for the detection of
target proteins using a fluorescence quenching system. A
variety of amine-containing high-affinity ligands of target pro-
teins, e.g., peptides or oligosaccharides, may be readily loaded
onto the fluorescently “caged” probe 6 via azaelectrocyclization
and a strain-releasing click reaction. The method was demon-
strated using the cyclic RGDyK peptide as an integrin αVβ3
ligand loaded onto the “switched on” probe. The cell surface
αVβ3-integrins were selectively imaged after carrying out a cas-
cading sequence of ligand-directed azaelectrocyclization and
autooxidation-induced fluorescence recovery reactions in the
presence of the cells. Although many fluorogenic protocols
have been combined with biological techniques in the past,
purely chemical methods that employ novel reactivity strategies
are quite rare.5,6 The results described here are applicable to
the efficient imaging of target proteins in cell lysates or on live
cell surfaces without the need for isolation and/or washing
procedures. Direct labeling in living animals could enable
in vivo molecular imaging immediately following injection
of the probe. These studies are currently in progress in our
laboratory.
4 (a) K. Lang, L. Davis, J. Torres-Kolbus, C. Chou, A. Deiters
and J. W. Chin, Nat. Chem., 2012, 4, 298–304; (b) E. Kaya,
M. Vrabel, C. Deiml, S. Prill, V. S. Fluxa and T. Carell,
Angew. Chem., Int. Ed., 2012, 51, 4466–4469;
(c) L. I. Willems, N. Li, B. I. Florea, M. Ruben, G. A. van der
Marel and H. S. Overkleeft, Angew. Chem., Int. Ed., 2012, 51,
4431–4434; (d) H.-S. Han, N. K. Devaraj, J. Lee,
S. A. Hilderbrand, R. Weissleder and M. G. Bawendi, J. Am.
Chem. Soc., 2010, 132, 7838–7839; (e) A. Niederwieser,
A.-K. Späte, L. D. Nguyen, C. Jüngst, W. Reutter and
V. Wittmann, Angew. Chem., Int. Ed., 2013, 52, 4265–4268;
(f) R. Rossin, P. R. Verkerk, S. M. van den Bosch,
R. C. M. Vulders, I. Verel, J. Lub and M. S. Robillard, Angew.
Chem., Int. Ed., 2010, 49, 3375–3378; (g) W. Song, Y. Wang,
J. Qu, M. M. Madden and Q. Lin, Angew. Chem., Int. Ed.,
2008, 47, 2832–2835; (h) W. Song, Y. Wang, J. Qu and
Q. Lin, J. Am. Chem. Soc., 2008, 130, 9654–9655; (i) J. Wang,
W. Zhang, W. Song, Y. Wang, Z. Yu, J. Li, M. Wu, L. Wang,
J. Zang and Q. Lin, J. Am. Chem. Soc., 2010, 132, 14812–
14818; representative review on Cu(I)-free reactions, see:
( j) J. C. Jewett and C. R. Bertozzi, Chem. Soc. Rev., 2010, 39,
1272–1279.
Acknowledgements
This work was supported in part by Grants-in-Aid for Scientific
Research from the Japan Society for the Promotion of Science,
23681047, 23241074, and 25560410, by the New Energy and
Industrial Technology Development Organization (NEDO), by a
research grant from the Mizutani Foundation for Glycoscience,
and by a MEXT Grant-in-Aid for Scientific Research on the
Innovative Areas “Organic Synthesis Based on Reaction Inte-
gration, Development of New Methods and Creation of New
Substances” and “Chemical Biology of Natural Products:
Target ID and Regulation of Bioactivity”.
Notes and references
1 (a) Q. Wang, T. R. Chan, R. Hilgraf, V. V. Fokin,
K. B. Sharpless and M. G. Finn, J. Am. Chem. Soc., 2003,
125, 3192–3193; (b) A. E. Speers, G. C. Adam and
B. F. Cravatt, J. Am. Chem. Soc., 2003, 125, 4686–4687;
(c) A. J. Link and D. A. Tirrell, J. Am. Chem. Soc., 2003, 125,
11164–11165; (d) A. Deiters, T. A. Cropp, M. Mukherji,
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem., 2014, 12, 1412–1418 | 1417

View Article Online
Paper
Organic & Biomolecular Chemistry
5 (a) I. Hamachi, T. Nagase and S. Shinkai, J. Am. Chem. Soc.,
2000, 122, 12065–12066; (b) T. Nagase, S. Shinkai and
I. Hamachi, Chem. Commun., 2001, 229–230; (c) T. Nagase,
E. Nakata, S. Shinkai and I. Hamachi, Chem.–Eur. J., 2003,
9, 3660–3669; (d) E. Nakata, Y. Koshi, E. Koga, Y. Katayama
and I. Hamachi, J. Am. Chem. Soc., 2005, 127, 13253–13261;
(e) Y. Takaoka, H. Tsutsumi, N. Kasagi, E. Nakata and
I. Hamachi, J. Am. Chem. Soc., 2006, 128, 3273–3280;
(f) A. Ojida, H. Tsutsumi, N. Kasagi and I. Hamachi, Tetra-
hedron Lett., 2005, 46, 3301–3305; (g) Y. Koshi, E. Nakata,
M. Miyagawa, S. Tsukiji, T. Ogawa and I. Hamachi, J. Am.
Chem. Soc., 2008, 130, 245–251.
K. Fukase, J. Carbohydr. Chem., 2010, 29, 118–132;
(c) K. Tanaka, S. Yokoi, K. Morimoto, T. Iwata,
Y. Nakamoto, K. Nakayama, K. Koyama, T. Fujiwara and
K. Fukase, Bioorg. Med. Chem., 2012, 20, 1865–1868;
(d) K. Tanaka, K. Moriwaki, S. Yokoi, K. Koyama,
E. Miyoshi and K. Fukase, Bioorg. Med. Chem., 2013, 21,
1074–1077.
9 (a) K. Tanaka, M. Kamatani, H. Mori, S. Fujii, K. Ikeda,
M. Hisada, Y. Itagaki and S. Katsumura, Tetrahedron, 1999,
55, 1657–1686; (b) K. Tanaka, H. Mori, M. Yamamoto and
S. Katsumura, J. Org. Chem., 2001, 66, 3099–3110;
(c) K. Tanaka and S. Katsumura, J. Am. Chem. Soc., 2002,
124, 9660–9661; (d) K. Tanaka, T. Kobayashi, H. Mori and
S. Katsumura, J. Org. Chem., 2004, 69, 5906–5925.
6 (a) S. Tsukiji, M. Miyagawa, Y. Takaoka, T. Tamura and
I. Hamachi, Nat. Chem. Biol., 2009, 5, 341–343;
(b) S. Tsukiji, H. Wang, M. Miyagawa, T. Tamura, 10 K. Tanaka, Y. Fujii and K. Fukase, ChemBioChem, 2008, 9,
Y. Takaoka and I. Hamachi, J. Am. Chem. Soc., 2009, 131,
9046–9054.
7 (a) K. Tanaka, T. Masuyama, K. Hasegawa, T. Tahara,
2392–2397.
11 S. Mizukami, S. Watanabe, Y. Hori and K. Kikuchi, J. Am.
Chem. Soc., 2009, 131, 5016–5017.
H. Mizuma, Y. Wada, Y. Watanabe and K. Fukase, Angew. 12 K. Tanaka, M. Kitadani and K. Fukase, Org. Biomol. Chem.,
Chem., Int. Ed., 2008, 47, 102–105; (b) K. Tanaka and 2011, 9, 5346–5349.
K. Fukase, Org. Biomol. Chem., 2008, 6, 815–828; 13 K. Tanaka, Y. Nakamoto, E. R. O. Siwu, A. R. Pradipta,
(c) K. Tanaka, E. R. O. Siwu, K. Minami, K. Hasegawa,
S. Nozaki, Y. Kanayama, K. Koyama, W. C. Chen,
J. C. Paulson, Y. Watanabe and K. Fukase, Angew. Chem.,
K. Morimoto, T. Fujiwara, S. Yoshida, T. Hosoya,
Y. Tamura, G. Hirai, M. Sodeoka and K. Fukase, Org.
Biomol. Chem., 2013, 11, 7326–7333.
Int. Ed., 2010, 49, 8195–8200; (d) K. Tanaka, K. Fukase and 14 X. Chen, R. Park, M. Tohme, A. H. Shahinian,
S. Katsumura, Chem. Rec., 2010, 10, 119–139; (e) K. Tanaka,
K. Fukase and S. Katsumura, Synlett, 2011, 2115–2139.
J. R. Bading and P. S. Conti, Bioconjugate Chem., 2004,
15, 41–49.
8 Discussions of live cell labeling and modification using 15 Due to the non-specific reaction on the cell surface, i.e.,
small molecules via a 6π-azaelectrocyclization reaction are
provided in: (a) K. Tanaka, K. Minami, T. Tahara, Y. Fujii,
E. R. O. Siwu, S. Nozaki, H. Onoe, S. Yokoi, K. Koyama,
Y. Watanabe and K. Fukase, ChemMedChem, 2010, 5, 841–
845; (b) K. Tanaka, K. Minami, T. Tahara, E. R. O. Siwu,
K. Koyama, S. Nozaki, H. Onoe, Y. Watanabe and
cRGDyK ligand-independent reaction, the microscopic ana-
lysis should be performed as soon as possible after the
probe treatment. When the microscopic analysis was per-
formed after 10 min, the significant fluorescence contrasts
could not be detected between the cells treated by 7 and
the control 6.
1418 | Org. Biomol. Chem., 2014, 12, 1412–1418
This journal is © The Royal Society of Chemistry 2014