A combined 6π-azaelectrocyclization/staudinger approach to protein and cell engineering: Noninvasive tumor targeting by N-glycan-engineered lymphocytes

Article abstract of DOI:10.1080/07328303.2010.483042

Combined azaelectrocyclization and Staudinger ligation allowed proteins and living cells to be modified by small molecules (i.e., biotin or N-glycans). Chemically engineered lymphocytes modified by complex-type N-glycan targeted DLD-1 tissues implanted in nude mice at the whole-body level. Copyright Taylor & Francis Group, LLC.

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A Combined 6π-Azaelectrocyclization/
Staudinger Approach to Protein and
Cell Engineering: Noninvasive Tumor
Targeting by N-Glycan-Engineered
Lymphocytes
Katsunori Tanaka a , Kaori Minami a , Tsuyoshi Tahara b , Eric R.
O. Siwu a , Koichi Koyama c , Satoshi Nozaki b , Hirotaka Onoe b ,
Yasuyoshi Watanabe b & Koichi Fukase a
a Department of Chemistry, Graduate School of Science , Osaka
University , Machikaneyama 1-1, Toyonaka, Osaka, 560-0043, Japan
b RIKEN Center for Molecular Imaging Science 6-7-3 Minatojima-
minamimachi , Chuo-ku, Kobe-shi, Hyogo, 650-0047, Japan
c Kishida Chemical Co. Ltd , 14-10 Technopark, Sanda-shi, Hyogo,
669-1339, Japan
Published online: 02 Jun 2010.
To cite this article: Katsunori Tanaka , Kaori Minami , Tsuyoshi Tahara , Eric R. O. Siwu , Koichi
Koyama , Satoshi Nozaki , Hirotaka Onoe , Yasuyoshi Watanabe & Koichi Fukase (2010) A Combined
6π-Azaelectrocyclization/Staudinger Approach to Protein and Cell Engineering: Noninvasive Tumor
Targeting by N-Glycan-Engineered Lymphocytes, Journal of Carbohydrate Chemistry, 29:3, 118-132,
DOI: 10.1080/07328303.2010.483042
To link to this article: http://dx.doi.org/10.1080/07328303.2010.483042
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Journal of Carbohydrate Chemistry, 29:118–132, 2010
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C
Copyright Taylor & Francis Group, LLC
ISSN: 0732-8303 print / 1532-2327 online
DOI: 10.1080/07328303.2010.483042
A Combined
6π-Azaelectrocyclization/
Staudinger Approach to
Protein and Cell Engineering:
Noninvasive Tumor Targeting
by N-Glycan-Engineered
Lymphocytes
Katsunori Tanaka,1 Kaori Minami,1 Tsuyoshi Tahara,2
Eric R. O. Siwu,1 Koichi Koyama,3 Satoshi Nozaki,2
Hirotaka Onoe,2 Yasuyoshi Watanabe,2 and Koichi Fukase1
1Department of Chemistry, Graduate School of Science, Osaka University,
Machikaneyama 1-1, Toyonaka, Osaka 560-0043, Japan
2RIKEN Center for Molecular Imaging Science 6-7-3 Minatojima-minamimachi,
Chuo-ku, Kobe-shi, Hyogo 650-0047, Japan
3Kishida Chemical Co., Ltd 14-10 Technopark, Sanda-shi, Hyogo 669-1339, Japan
Graphical Abstract
Combined azaelectrocyclization and Staudinger ligation allowed proteins and living
cells to be modified by small molecules (i.e., biotin or N-glycans). Chemically engineered
lymphocytes modified by complex-type N-glycan targeted DLD-1 tissues implanted in
nude mice at the whole-body level.
Received January 21, 2010; accepted March 31, 2010.
Address correspondence to Katsunori Tanaka and Koichi Fukase, Department
of Chemistry, Graduate School of Science, Osaka University, Machikaneyama 1-
1, Toyonaka, Osaka 560-0043, Japan. E-mail: ktzenori@chem.sci.osaka-u.ac.jp and
koichi@chem.sci.osaka-u.ac.jp
118

Chemical Engineering by Oligosaccharides
119
Keywords Cancer; Chemical Engineering; N-Glycan; Lymphocytes; Noninvasive
Imaging; 6π-Azaelectrocyclization
Chemical methods for labeling or modification of proteins and living cells
with functional molecules, such as oligosaccharides or biofunctional peptides
(termed “bioconjugation”), have garnered significant attention, especially in
the fields of molecular imaging and bioengineering. These methods offer the
advantages of easy operation[1] and general applicability.[2] The chemical mod-
ification of biomolecules or living cells should be performed at low concentra-
tions under mild conditions, with the goal of preserving the native bioactivities
of the biomolecules in addition to attaining new functions from the conjugated
molecules. Recently, a variety of new methods, which can also be combined
with biological techniques, have been actively investigated,[3] for example, the
Cu(I)-mediated Huisgen 1,3-dipolar cycloaddition reaction (Sharpless/Meldal
click reaction) with the azide moiety genetically introduced at desired posi-
tions within proteins.[4] Cell-friendly versions of the Cu(I)-free click reaction
using strained acetylenes,[5–7] as well as the Staudinger reaction,[8,9] were de-
veloped by Bertozzi and coworkers. These reactions were successfully used
for fluorescence imaging on living cell surfaces[10] and in living animals.[7c]
The use of transition metal catalysts in the conjugation of biomolecules has
also been reported. Davis and coworkers introduced mono- and disaccharides
onto proteins by employing Ru-catalyzed cross-metathesis[11] and Pd-catalyzed
Suzuki-Miyaura coupling.[12] Chemistry-based unique bio-orthogonal ap-
proaches have been extensively investigated by Hamachi and coworkers.[13]
Hamachi’s group developed a single molecule containing the fluorescent la-
bels, the anchoring functional groups, and the protein ligands to selec-
tively label regions near ligand-binding sites. After cleavage of the ligands
from labeled proteins, proteins with recovered activity were used as sensi-
tive reporter molecules for fluorescent imaging (e.g., for visualization of gly-
colysis rates inside cells). Hamachi’s group also developed many efficient
variants to this strategy, which involves post affinity labeling modification
(P-ALM).
We recently developed a primary amine-based labeling of peptides,
proteins (antibodies),[14,15] and even living cells,[16] which is based on a
rapid 6π-azaelectrocyclization (Sch. 1).[17] This method was used to effi-
ciently and selectively introduce both fluorescent groups and DOTA (1,4,7,10-
tetraazacyclodecane-1,4,7,10-tetraacetic acid), a metal-chelating agent, to
amino groups. Amines in target proteins react with unsaturated aldehyde
probes, such as probe 1a (Sch. 1), at low concentrations (∼10−8 M) within a
short time (10 to 30 minute) at room temperature. Our method could be ap-
plied to visualization of the in vivo dynamics of glycoproteins[14] and cellular
trafficking of lymphocytes[16] by means of noninvasive PET and fluorescence

K. Tanaka et al.
120
Scheme 1: Two-step procedure for protein engineering through azaelectrocyclization-
Staudinger ligation. (Figure available in color online.)
imaging. Our new method precisely controls the introduction of DOTA or flu-
orescence labels onto amines, such as lysines or ethanolamines, in target pro-
teins or cells by adjusting the probe concentration so that the activity of the
biomolecules can be retained.

Chemical Engineering by Oligosaccharides
121
Based on these initial results, we speculated that our azaelectrocyclization
chemistry could also be used to covalently modify proteins or cells with func-
tional molecules other than labels under mild physiological conditions. Herein,
we report a combined azaelectrocyclization/Staudinger method useful as a new
bioconjugation procedure to install biotin or complex-type N-glycans on pro-
teins or living cells. Remarkable effects of chemical engineering with N-glycan
were discovered during whole-body imaging of lymphocyte trafficking in living
animals.
PROTEIN ENGINEERING
Electrocyclization probes such as aldehydes 2–4 (Sch. 1) are difficult to pre-
pare, with the synthetic route involving oxidation of an allylic alcohol to the
conjugated aldehyde as the last step.[14a] According to our preliminary trials,
biotin or oligosaccharide moieties with numerous hydroxyl groups were in-
compatible with various oxidation conditions, and the reactions only provided
decomposition products. To overcome this challenge, we developed a two-step
engineering procedure shown in Scheme 1. Proteins are first tagged with an
azide residue through an azaelectrocyclization. The cyclized products are then
treated with phosphine reagents to complete the chemical bioconjugation via
Bertozzi’s Staudinger ligation.[8,9]
Human serum albumin (HSA)[15] was first investigated as a model protein
(MW = 66,000 and containing 59 Lys). HAS was incubated with azide probe
1a[14a,16] in 0.1 M phosphate buffer at 25◦C for 30 min (HSA: 1.6 × 10−5 M,
probe 1a: 3.2 × 10−3 M). We “tagged” several azide groups onto HSA (vide in-
fra) using the electrocyclization procedure performed at high concentrations to
maximize the number of the azide functions on the protein template. Because
the reactivity of the Staudinger ligation was previously shown to be relatively
low, increasing the number of azide groups on the protein was crucial for the
success of the two-step procedure. The excess of probe 1a was removed by size-
partitioning gel filtration, using the Microcon (Millipore, filter: MW = 10,000)
under centrifugal conditions.[14a,16] Based on the MALDI-TOF-MS spectrum
of the azide-tagged HSA, approximately 12 molecules of the probe were con-
jugated to each molecule of HSA (Fig. 1b). The azide-tagged HSA was then
treated with Bertozzi’s 2-methoxycarbonylphenyldiphenylphosphine reagents
2 and 3,[8d] derived from the complex-type N-glycan and biotin (Sch. 1),[8] re-
spectively, at 25◦C for 3.5 h (N3-modified HSA: 1.6 × 10−5 M, phosphines 2
and 3: 2.0 × 10−3 M) to complete the protein engineering. The success of the
bioconjugation was analyzed by using MALDI-TOF-MS after centrifugal sepa-
ration with the Microcon. Although the broadening signals showed a mixture
of reacted and unreacted N3-HSAs from the Staudinger ligation, 1 molecule
of N-glycan and 4 molecules of biotin were introduced onto HSA under these
conditions on average, as shown in Figure 1c and d.

K. Tanaka et al.
122
Figure 1: MALDI-TOF-MS spectra of azide-, biotin-, and N-glycan-engineered HSA: (a) Intact
HSA (MW = 66,000); (b) Electrocyclization-modified HSA with an average of 12 molecules of
azide; (c) Staudinger-modified HSA with an average of 1 molecule of N-glycan; (d)
Staudinger-modified HSA with an average of 4 molecules of biotin. (Figure available in color
online.)
CELL ENGINEERING
The engineering procedure was next applied to living cells, specifically to
rat C6 glioma cells.[18] Unfortunately, application of the conditions shown
in Scheme 1 led to the death of the cells, presumably because of exposure
of the living cells to the harsh chemical reagents (aldehydes, phosphines, or
DMSO solvent, see Experimental section) for a prolonged time (total reaction
time over 4h). However, the cell engineering was successfully realized when
the procedure was performed in the alternative reaction order, that is, by
performing the Staudinger ligation of the probe and phosphine reagents first
followed by azaelectrocyclization with the cells (Sch. 2). The azide-containing
aldehyde 1a was initially treated with phosphines 3 and 4 in PBS buffer at
24◦C (reaction conc. for both 1a and phosphines: 1.2 × 10−3 M). The N-glycan
structures of 2 and 4 are slightly different in terms of the sialic acid linkages to
the galactose residues. The glycan in 4 has two NeuNAcα(2,6)Gal nonreducing
ends, whereas that in 2 has both NeuNAcα(2,6)Gal and NeuNAcα(2,3)Gal
moieties (Sch. 1).
Based on the MALDI-TOF-MS spectra, the Staudinger ligation proceeded
about 50% within 20 min, accompanied by the CHO-reduced byproduct (alco-
hol derivative of 1a). A reaction concentration greater than 10−3 M and a pro-
longed reaction time led to the oxidation of the phosphine and the reduction

Chemical Engineering by Oligosaccharides
123
Scheme 2: One-pot procedure for cell engineering. (Figure available in color online.)
of the aldehyde in 1a. Unpurified intermediates 1b (biotin) and 1c (N-glycan)
were subsequently reacted with the C6 glioma cell in a one-pot procedure
at 37◦C for 10 to 30 min (engineering concentration: 2.0 × 10−5 M for 1b
and c). The modified cell surfaces were evaluated by treatment with TRITC
(rhodamine)-labeled avidin and TRITC-labeled SNA (Sambucus nigra, Elder-
berry, Neuα(2,6)Gal-specific lectin)[19] (Fig. 2).
Although TRITC-derived red fluorescence was observed for the 1b (biotin)-
labeled cell (Fig. 2a), only a negligible level of fluorescence was detected for the
control cell, which was treated with phosphine reagent 3 instead of 1b (Fig.
2b). The covalent modification of the cells with biotin was achieved through a
combined sequence of azaelectrocyclization/Staudinger ligation. Similarly, con-
focal microscopy detected red fluorescence on the 1c (N-glycan)-labeled C6 cell
upon treatment with TRITC-labeled SNA (Fig. 2c). Red fluorescence was also
observed for the intact cell (Fig. 2d) because the C6 glioma cell itself had na-
tive N- and/or O-glycans with a nonreducing end Neuα(2,6)Gal motif on the
cell surface. The SNA lectin was able to interact with these native glycans.
However, the 1c-modified glial cell showed higher fluorescence intensity than
that of the intact cell, thereby confirming the modification by N-glycan.
NONINVASIVE IMAGING OF N-GLYCAN-ENGINEERED LYMPHOCYTES
IN A CANCER MOUSE MODEL
Finally, we investigated how N-glycan engineering affects the trafficking prop-
erties of lymphocytes in living animals. Lymphocytes (1 mL, 1.0 × 105 cells)
directly extracted from the abdominal cavity of nude mice were fluorescently
labeled by azaelectrocyclization (Cy5-fluorescence),[16,20] and the labeled lym-
phocytes (100 µL/mouse, 104 cells) were administrated to the tail vein of a

K. Tanaka et al.
124
Figure 2: Confocal microscopy of biotin- and N-glycan-modified C6 glioma cells (DAPI-,
TRITC, and PH detections). (a) 1b-Modified cell on TRITC-avidin treatments; (b) control cell
treated with biotin-containing phosphine 3 on TRITC-avidin treatments; (c) 1c-modified cell
on SNA lectin (Neuα(2,6)Gal-specific) treatments; (d) intact cell on SNA lectin treatments.
PH: phase contrast. (Figure available in color online.)
mouse that had a DLD-1 human colon carcinoma implanted to the dorsal divi-
sion (Experimental Section). The dynamic fluorescence images were recorded
over a week by using eXplore Optix, GE Healthcare, Bioscience (Fig. 3).[21] The
lymphocytes gradually accumulated over 6 h, mainly in the spleen and intesti-
nal lymph nodes, and the fluorescence intensity in the spleen was found to
decrease over time (Fig. 3a). These trafficking properties were similar to our
earlier findings in the normal nude mice,[16] although the accumulation to the
spleen was slightly slower in the cancer model. However, no fluorescence was
detected in the tumor tissues over the course of a week. On the other hand, we
made a remarkable observation when the lymphocytes were simultaneously
labeled by the Cy5-fluorophore and engineered by N-glycan 1c under the con-
ditions shown in Scheme 2. The fluorescence intensity of the engineered cells
remained strong in the spleen after 48 h (Fig. 3b), and the cells gradually be-
gan to accumulate into DLD-1 (TM). The fluorescence intensity in the tumor
regions became even stronger over a week.

Chemical Engineering by Oligosaccharides
125
Figure 3: Fluorescence imaging of lymphocytes in mice. Labeled and/or engineered cells
were administrated intravenously (n = 3, 100 µL/mouse, 104 cells) and the whole body was
scanned from the back side by eXplore Optix, GE Healthcare, Bioscience (excitation at
646 nm, emission 663 nm), 1 h, 2 h, 4 h, 6 h, 48 h, and 1 week after injection. Data were
normalized. SP: spleen; LN: lymph node of epidermal intestinal tract; TM: DLD-1 human colon
carcinoma. (a) Cy5-labeled cells injected to nude mice that were implanted with DLD-1 at
the dorsal division; (b) both Cy5-labeled and N-glycan-engineered lymphocytes in the
tumor model. (Figure available in color online.)
In order to explain the imaging results in Figure 3, further investigations
are needed to examine the mechanisms at the molecular level. Nevertheless,
since neither the native lymphocytes nor the complex-type N-glycan[22] traffics
to the cancer regions on their own, the engineering of the lymphocyte surfaces
is key to the tumor targeting. The low efficiency of lymphocyte-based cancer
immunotherapy, such as using NK, LAK, CTL, and TIL as the immune effec-
tor cells, is well documented, and in order to circumvent the problems, (1) coin-
jection with cytokines, costimulatory molecules or cancer antigens, and more

K. Tanaka et al.
126
recently, (2) engineering of the lymphocytes by the chimeric antigen receptors
have widely been applied.[23]
The reason for N-glycan-enriched lymphocyte trafficking to tumor is un-
clear. There are two possible explanations: (1) the N-glycan-enriched lym-
phocytes might interact with the tumor via additional interaction with
lectins on cancer cells or (2) the interaction of Siglec (Sialic acid-binding,
immunoglobulin-like lectin) and internal sialoglycans on the lympocytes,
which stimulates the immunosuppressive signals through the ITIM (im-
munoreceptor tyrosine-based inhibitory motif) molecules,[24] could be inter-
rupted by the N-glycan 1c externally introduced by chemical engineering. This
eventually might activate the lymphocytes to target the tumor. These prelim-
inary results indicate the potential for the electrocyclization-based engineer-
ing to be applied to whole cell-based targeting to specific cancers or immune-
related organs, and therefore highlight the present glycan-engineering
protocol.
In summary, a combined electrocyclization/Staudinger protocol enables
functional groups, such as biotin and complex-type N-glycans 2 and 4, to be
loaded onto proteins and living cells. The present method was applied to mod-
ification of lymphocytes with N-glycan and the artificial lymphocytes targeted
a tumor in living animals due to the synergistic effects of both functions of
lymphocytes and oligosaccharides. The current method is not yet generally
applicable to most systems because of the low reactivity of the Staudinger
ligation and the facile oxidation of phosphine reagents under physiological
conditions. Nevertheless, combining our electrocyclization with other conjuga-
tion reactions, such as the Cu(I)-mediated Huisgen 1,3-dipolar cycloaddition or
strain-releasing reactions, and/or generating the aldehyde function at the last
stage of the probe synthesis (i.e., enzymic oxidation or protecting groups con-
version under the mild conditions), may significantly broaden the scope of this
approach to chemical engineering. The development of more efficient engineer-
ing probes and the biological evaluation of the N-glycan-engineered proteins
and living cells are now in progress in our laboratory.
EXPERIMENTAL SECTION
Ethyl (E,E)-4-Hydroxy-2-{4-[2-(7-azidoheptanamide)
acetamide]styryl}but-2-enoate (alcohol precursor of 1a)
To a solution of ethyl (E,E)-[2-(N-tert-butoxycarbonyl-2-aminoacetamide)
styryl-4-(tetrahydro-2H-pyran-2-yloxy)]but-2-enoate (40.0 mg, 81.9 µmol) in
MeOH (1.25 mL) was added 6N aqueous HCl (1.25 mL) dropwise at 0◦C.
After the reaction mixture was stirred at rt for 1 h, the mixture was extracted
with hexane. The aqueous layer was neutralized with 1N aqueous NaOH and

Chemical Engineering by Oligosaccharides
127
desalted through a column filled with HP-20 to afford ethyl (E,E)-4-hydroxy-
2-[4-(2-aminoacetamide)styryl]but-2-enoate as a yellow solid, which was used
without further purification.
To a solution of the crude aminoalcohol obtained above (20.1 mg,
66.1 µmol) in DMF (1 mL) was added the succinimidyl ester of 7-
azidoheptanoic acid (17.7 mg, 66.1 µmol) at rt. After the mixture was stirred
at rt for 2 h, the solution was concentrated in vacuo. The residue was directly
purified by using preparative chromatography on silica gel (CH3Cl: CH3OH =
30:1) to give the title compound (8.1 mg, 22% for two steps) as a white solid:
1
mp 105◦C; IR (neat, cm−1) 3350, 1748, 1707; H NMR (500 MHz, CD3OD) δ
7.55 (2H, d, J = 8.7 Hz), 7.43 (2H, d, J = 8.9 Hz), 6.86 (1H, d, J = 16.3 Hz),
6.77 (1H, d, J = 16.3 Hz), 6.76 (1H, t, J = 6.0 Hz), 4.51 (1H, br, s), 4.46 (2H,
d, J = 6.1 Hz), 4.26 (2H, q, J = 7.2 Hz), 3.99 (2H, s), 3.28 (2H, t, J = 6.9 Hz),
2.30 (2H, t, J = 7.5 Hz), 1.69–1.63 (2H, m), 1.62–1.57 (2H, m), 1.42–1.39 (4H,
m), 1.32 (3H, t, J = 7.2 Hz); 13C NMR (100 MHz, CD3OD) δ 176.75, 169.66,
168.40, 142.57, 139.61, 135.43, 134.31, 131.58, 128.21, 121.15, 120.88, 62.10,
59.99, 52.38, 44.03, 36.69, 29.75 (x2), 27.51, 26.63, 14.53; ESI HRMS m/z calcd
for C23H31N5O5Na (M+Na)+ 480.2223, found 480.2204.
General Procedure to Chemically Engineer Cell Surfaces by
Modification with Biotin and N-Glycans
IBX-resin (10 mg, 8.0 µmol) was added at rt to a solution of the alcohol
precursor of 1a (200 µg, 440 nmol) in DMF (25 µL) and CH2Cl2 (25 µL) in an
Eppendorf tube. After the reaction mixture was shaken at rt for 30 min, the
IBX-resin was removed by using a centrifugal filtration tube (Cosmospin Fil-
ter H, 0.45 µm, Nacalai Tesque), and the solvents were removed by centrifugal
concentration at rt. The aldehyde probe 1a remaining in the Eppendorf tube
was dissolved in the appropriate volume of the PBS containing 20% DMSO,
and the solution concentration of probe 1a was adjusted to 3.14 × 10−3 M. To
the resultant solution (140 µL, 3.14 × 10−3 M) was added a PBS solution of
the biotin/phosphine reagent 3[8d] (220 µL, 2.0 × 10−3 M, cont. 20% of DMSO)
(reaction concentrations: 1.22 × 10−3 M for both 1a and 3). The resulting so-
lution was kept at rt for 20 min (model Staudinger product with the stable
alcohol precursor of 1a; MALDI-TOF HRMS m/z calcd for C61H78N7NaO13PS
(M+Na)+ 1202.5, found 1202.9). The mixture was then treated with the sus-
pended C6 glioma cells (cell number: 1.0 × 105/mL, final labeling concentra-
tions of the probe: 2.0 × 10−5 M, cont. 0.2% of DMSO, 1.0 mL). After maintain-
ing the mixture at 37◦C for 10 min under a carbon dioxide atmosphere, the
resulting cells were washed three times with the culture medium to remove
excess probe.

K. Tanaka et al.
128
Rat Glioma Cell Culture and Cell Engineering Procedure
Rat glioma C6 cells were grown in Dulbecco’s modified Eagle’s medium
supplemented with 10% fetal calf serum (FCS) and antibiotics. The cells
(1 × 105) were trypsinized and washed with PBS three times to exclude any
inhibitors for engineering probes, such as amines. The washing buffer was re-
placed with new buffer containing probe (2.0 × 10−5 M). After incubation at
37◦C, the cells were washed with cultured medium to stop the reaction. Then
engineered cells were seeded into culture dishes to estimate the reaction effi-
ciency by viewing the cell surface by microscopy.
Preparation of Lymphocytes, Cancer Model, and in vivo
Fluorescence Imaging
A wild-type mouse (BALB/cAJcl, CLEA Japan, Inc.) was euthanized by us-
ing excess anesthetic (Isoflurane). After creating an incision into the abdomen,
about 1 mL of blood was collected from the aorta descendens. Heparin sodium
(100 µL, 100 units/mL) was added to the collected blood and the blood was
stored at rt for 2 h, whereupon the lymphocytes were extracted. Mouse pe-
ripheral blood diluted with medium was added onto the lymphosepar II layer,
and the mixture was centrifuged. The lymphocyte fraction in the middle layer
(suspended cell culture) was collected, medium was added, and the mixture
was centrifuged again. The pellet was then suspended in cell culture medium
and the number of cells was counted. By repeating this procedure, a total of
3.6 × 106 cells were collected from 3.5 mL of the blood sample. After adjusting
the cell number using culture medium, the cells were immobilized on antibody-
coated plates and cultured at 37◦C under a 5% carbon dioxide atmosphere. The
cultured lymphocytes were stored for 2 h at rt in a physiological salt solution
before performing the labeling and engineering procedures described above. A
total of 1 × 105 cells/1 mL PBS buffer of the lymphocytes were labeled with a
Cy5-probe16 (incubation concentration: 2.0 × 10−5 M), and these lymphocytes
were engineered with the N-glycan probe 1c at an incubation concentration of
5.1 × 10−5 M.
Eight-week-old mice (BALB/cAJcl–nu/nu, CLEA Japan, Inc.) were used for
the in vivo fluorescence imaging study. The cancer model was prepared by sub-
cutaneously injecting DLD-1 (107 cells/100 µL) to the dorsal division of the
mouse (BALB/cAJcl–nu/nu, CLEA Japan, Inc.). A tumor was grown up to a
proper size for imaging purposes over a 2- to 3-week period from the injec-
tion. Therefore, 10-week-old mice (2 weeks from the injection of DLD-1) were
used for imaging. Fluorescence-labeled or N-glycan-engineered lymphocytes
(1 × 104 cells/100 µL of a physiological salt solution) were injected from the
caudal vein without anesthesia, and a whole-body scan was performed by us-
ing eXplore Optix, GE Healthcare, Bioscience, over 1 week after injection.

Chemical Engineering by Oligosaccharides
129
The in vivo fluorescence image was taken under inhalation anesthesia with
Isoflurane, with the concentration of Isoflurane kept at 4% from 15 min to 1 h
after the injection and then at 1.5% to 2% during the rest of the measurements.
ACKNOWLEDGMENTS
We thank Hiroaki Asai, Dr. Michio Sasaoka, Kazuhiro Fukae, and Azusa
Hashimoto, Otsuka Chemical Co., Ltd., for supplying N-glycans. This work
was supported in part by Grants-in-Aid for Scientific Research No. 19681024
and 19651095 from the Japan Society for the Promotion of Science, Collabo-
rative Development of Innovative Seeds from Japan Science and Technology
Agency (JST), New Energy and Industrial Technology Development Organi-
zation (NEDO, project ID: 07A01014a), research grants from Yamada Science
Foundation and the Molecular Imaging Research Program, and Grants-in-Aid
for Scientific Research from the Ministry of Education, Culture, Sports, Science
and Technology (MEXT) of Japan.
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