Self and nonself recognition with bacterial and animal glycans, surveys by synthetic chemistry

Article abstract of DOI:10.1016/S0076-6879(10)78016-2

In this chapter, we describe synthetic studies on partial structures of lipopolysaccharide (LPS) and peptidoglycan (PGN), which work as tags for nonself recognition in innate immune system. Our previous studies demonstrated that lipid A is the endotoxic p

Full text of DOI:10.1016/S0076-6879(10)78016-2

C H A P T E R S I X T E E N
Self and Nonself Recognition with
Bacterial and Animal Glycans,
Surveys by Synthetic Chemistry
Yukari Fujimoto, Katsunori Tanaka, Atsushi Shimoyama, and
Koichi Fukase
Contents
324
1. Overview
2. Bacterial Glycoconjugates for Nonself
Recognition—Lipopolysaccharide (LPS)
3. Synthesis of H. pylori Kdo–Lipid A Backbone
4. Glycosylation with Kdo Donor 11
325
327
327
5. Cytokine (IL-6) Induction in Human Peripheral Whole-Blood
Cell Cultures
328
329
6. Bacterial Glycoconjugates for Nonself
Recognition—Peptidoglycan (PGN)
7. Synthesis of Disaccharide Moiety 15 in Tracheal Cytotoxin with
b-Selective Glycosylation
331
332
333
333
334
335
337
8. Synthesis of Tracheal Cytotoxin 20 and Its Fragment 21
9. Immunostimulatory Activities of DAP Containing PGN Fragments
10. Visualizing the In Vivo Dynamics of Animal N-Glycans
11. PET Imaging of Glycoproteins
12. Method for 68Ga-DOTA Labeling and MicroPET Imaging in Rabbit
13. PET Imaging of Glycoclusters
14. Method for Preparation of N-Glycan Clusters and PET Imaging
in Mouse
Acknowledgments
338
339
340
References
Abstract
In this chapter, we describe synthetic studies on partial structures of lipopoly-
saccharide (LPS) and peptidoglycan (PGN), which work as tags for nonself
recognition in innate immune system. Our previous studies demonstrated that
Department of Chemistry, Graduate School of Science, Osaka University, Machikaneyama, Toyonaka,
Osaka, Japan
#
Methods in Enzymology, Volume 478
ISSN 0076-6879, DOI: 10.1016/S0076-6879(10)78016-2
2010 Elsevier Inc.
All rights reserved.
323

324
Yukari Fujimoto et al.
lipid A is the endotoxic principle of LPS. The synthetic homogeneous prepara-
tions have enabled not only precise structure–activity relationships, but also
recognition mechanisms of LPS with innate immune receptor complexes,
including the TLR4/MD-2 complex, to be studied. Synthetic studies of lipid A
and Kdo–lipid A from parasitic Helicobacter pylori revealed their low inflamma-
tory activities, suggesting the molecular evolution to escape from the host
immune system. A synthetic study of the partial structures of PGN has also
contributed to the understanding of the innate immune mechanism. The
biological activities of the synthetic fragments have revealed that the intracel-
lular receptor Nod2 recognizes partial structures containing the muramyl dipep-
tide (MDP) moiety. The PGN of Gram-negative bacteria and some Gram-positive
bacteria contain meso-diaminopimelic acid (meso-DAP), and recent studies
have revealed that the intracellular receptor Nod1 recognizes DAP-containing
peptides. We have synthesized DAP-containing PGN fragments, including the
first chemical synthesis of tracheal cytotoxin (TCT). The ability of these frag-
ments to stimulate human Nod1 as well as differences in Nod1 recognition for
various synthesized ligand structures was elucidated. Cell-surface glycans such
as N-glycans and O-glycans on glycoproteins and glycoconjugates work as
signaling molecules for self-recognition and control immune system. Our new
strategy using glycan-imaging in whole-body system is expected to unveil the
dynamics of glycans in the body. Positron emission tomography (PET) is a
noninvasive method that visualizes the locations and levels of radiotracer
accumulation. We developed the facile labeling of peptides and proteins for
PET imaging. The labeled glycoproteins and glycoclusters were then subjected
to PET imaging in order to examine their in vivo dynamics, visualizing the
differences in the circulatory residence of glycoproteins and glycoclusters in
the presence or absence of sialic acid residues.
1. Overview
Self and nonself recognition is a fundamental function to maintain the
life of a multicellular organism with preventing the invasion of microorgan-
isms, components from other species/individuals, or problematic materials.
In order to distinguish the difference between self and nonself, glycan
structures or glycoconjugates on the cell surface are often used as the signal
motifs. One of significant roles in nonself recognition is to recognize
microorganism including bacteria, which has recently been exhibited to
have close relationships with the innate immunity activation. On the other
hand, the cell/cell and cell/protein interactions mediated by the N-glycans,
for example, stimulating the immunosuppressive signals through Siglec
families, constitute the significant roles in self-recognition process. In this
chapter, we show key chemical synthesis methods to build a compound
library of bacterial glycoconjugates, and the immunostimulating functions.

Self and Non-Self Recognition with Glycans
325
In addition, the methods for visualizing the in vivo dynamics of N-glycans
and glycoproteins, by means of the noninvasive molecular imaging as the
new tool for investigating the oligosaccharides functions, will be described.
2. Bacterial Glycoconjugates for Nonself
Recognition—Lipopolysaccharide (LPS)
LPS of Gram-negative bacteria is one of major signaling motifs for the
recognition of bacteria invading to the host organism. The compound is
also known as endotoxin due to its potent immunostimulation and the
toxicity. LPS is recognized with Toll-like receptor 4 (TLR4)/MD-2
complex on cell surface to produce mediators, for example, cytokines,
prostagrandins, the platelet activating factor, oxygen-free radicals, and
NO, which all activate and modulate the immune system (Kusumoto and
Fukase, 2006; Raetz and Whitfield, 2002). LPS consists of a glycolipid
component named lipid A, which is covalently connected to the polysac-
charide part. Lipid As from various bacteria have common structural
features, that is, GlcNAcb(1-6)GlcNAc possessing phosphono groups at
the reducing end and 4-positon of the nonreducing glucosamine, and
long-chain acyl groups at 2, 20, 3, and 30 positions. The total synthesis of
Escherichia coli lipid A 1 (synthetic 1 is termed 506) confirmed that lipid A is
the chemical entity responsible for the innate immunostimulatory activity of
LPS (Fig. 16.1) (Kusumoto and Fukase, 2006). Various lipid A structures are
known such as compound 2, 3, 4, and 5 (Fig. 16.1) (Kusumoto et al., 1999,
2009; Takada and Kotani, 1992), and we have synthesized various natural or
designed lipid A structures to investigate the structure–activity relationships
and analyze the action mechanisms (Fujimoto et al., 2005).
One of the characteristic groups of lipid As are the compounds from
parasitic bacteria such as Helicobacter pylori and Porphyromonas gingivalis.
H. pylori is one of Gram-negative bacteria and an etiological agent of
gastroduodenal diseases, including chronic gastritis, gastroduodenal ulcers,
and gastric cancer. H. pylori LPS shows a lower endotoxic activity compared
to other enterobacterial preparations such as E. coli LPS, but it is considered
to have relationships to the chronic inflammation. H. pylori LPS has charac-
teristic lipid A structures; namely it has fewer but longer acyl groups, and
does not have the 40-phosphate group, and the glycosyl phosphate often has
an ethanolamine group (8b; Fig. 16.1). H. pylori LPS has one Kdo as a
linkage between lipid A and the polysaccharide part. We have synthesized
triacyl type H. pylori lipid A with or without ethanolamine (8a, 8b)
(Fujimoto et al., 2007; Sakai et al., 2000), and also H. pylori lipid A
connecting to one Kdo residue (9) (Fujimoto et al., 2007), in order to
elucidate their biological activities.

326
Yukari Fujimoto et al.
OH
OH
OH
O
O
O
O
O
O
O
O
O
O
O
O
O
O
(HO) P
2
O
O
(HO) P
(HO) P
2
O
O
HO
O
HO
O
HO
2
O
O
O
O
P(OH)
NH
NH
NH
NH
O
O
NH
NH
O
O
O
O
O
OH
P(OH)
O
OH
O
O
P(OH)
O
O
O
O
2
2
2
O
O
O
OH
OH
O
O
O
OH
O
O
OH
OH
O
O
O
(C14)
(C14)
(C14)
(C14)
(C14)
(C14)
(C14)
(C14)
(C12)
(C14)
(C12)
(C14)
(C14)
(C16)
(C14)
(C14)
(C14)
2
3
1
Escherichia coli
Escherichia coli lipid A
strongly immunostimulative
Salmonella typhimurium lipid A
biosynthetic precursor lipid IVa
antagonistic
immunostimulative
OMe
OH
O
O
O
O
O
O
O
O
O
O
(HO) P
(HO) P
O
HO
O
HO
2
2
O
O
O
O
NH
NH
NH
NH
O
O
OH
P(OH)
2
O
O
P(OH)
O
O
O
2
OMe
OH
O
O
O
O
O
(C10)
(C10)
(C10)
(C10)
(C14)
(C14)
(C18)
O
(C14)
4 : RSLA
(C14)
O
5 : E5564
Antagonistic
Antagonistic
OH
OH
O
O
O
O
O
O
NH
(HO) P O
(HO) P O
HO
HO
O
2
2
O
O
O
O
P(OH)
NH
O
NH
O
NH
O
O
O
CO H
2
O
O
2
O
O
OH
O
OH
OH
OH
O
O
O
O
O
O
O
O
(C10)
(C10)
(C10)
(C10)
(C10)
(C12)
(C10)
(C10)
(C10)
(C12)
6 : Ru. gelatinosus lipid A
(C12)
(C12)
7 : Ru. gelatinosus CM-analog
antagonistic (Limulus activity ++)
immunostimulative (Limulus activity +)
OH
Kdo
OH
HO
HO
O
OH
O
CO H
2
O
O
HO
HO
HO
O
R2O
O
HO
O
P
O
O
HN
HN
OH
OR
HO
HO
O
O
1
O
O
P
HO
HN
HN
OH
OH
O
O
OH
O
O
O
O
OH
O
Helicobacter pylori
lipid A
and
Kdo-lipidA
1
1
2
R
= –H, R = –H: 8a
antagonistic
R
= –CH CH –NH ,
2
2
2
(C18)
(C18)
2
(C18)
R
= –H: 8b
9
immunostimulative
(C18)
Antagonistic
(C18)
(C18)
Figure 16.1 Structures of various lipid A and LPS partial structures.

Self and Non-Self Recognition with Glycans
327
OBn
O
BnO
TBSO
5
OBn
O
R O
BnO
TBSO
O
CO Bn
2
TBSO
BnO
R O
BnO
O
O
4
3
R O
O
+
O
NH
CO Bn
2
TBSO
NH
OAllyl
BnO
O
BnO
O
O
O
1
2
R
3
4
F
R
R O
R O
11
NH
NH
OAllyl
3
4
5
O
O
O
10 (R = Bn, R = Bn, R = TES)
1
2
R
12
R
BnO
1
R = CH (CH )
3
2 14
H. pylori Kdo-lipid A 9
O
2
R = CH (CH )
3
2 16
CH (CH )
3
2 14
Figure 16.2 Synthesis of Helicobacter pylori Kdo–lipid A backbone.
3. Synthesis of H. pylori Kdo–Lipid A Backbone
For the synthesis of Kdo–lipid A, the lipid A backbone (10) was first
prepared, and connected with Kdo (11) with an a-selective glycosylation
(Fig. 16.2) (Fujimoto et al., 2007; Yoshizaki et al., 2001). The molecular
sieve 5A (MS5A) was found to be more effective for this reaction than
MS4A, presumably because MS5A contains calcium, which traps fluoride
anions and promotes glycosylation. The stereochemistry at the anomeric
position was determined with the chemical shift of the protons at the
1
30-position from H NMR in comparison with the previously reported
data (Fujimoto et al., 2007; Yoshizaki et al., 2001).
4. Glycosylation with Kdo Donor 11
1. To a mixture of 10 (48.8 mg, 0.0275 mmol), Kdo-fluoride 11 (52.7 mg,
˚
0.0713mmol)andMS5AindryCHCl3 (2mL)isaddedBF3ꢀOEt2 (0.052ml,
0.288 mmol) at –20 ꢁC and the mixture is stirred at –20 ꢁC for 1.5 h.
2. After addition of saturated NaHCO3, the mixture is extracted with
CHCl3. The organic layer is washed with saturated NaHCO3 and
brine, dried over anhydrous Na2SO4, filtered, and concentrated under
reduced pressure.
3. The residue is purified by column chromatography (silica gel 50 g,
CHCl3/acetone ¼ 30/1) to give 12 as a white solid (55.3 mg, 85%).
Rf (CHCl3/acetone ¼ 30/1) ¼ 0.25; ESI-MS (positive) m/z 2402.51
1
[(MþNa)þ]; H NMR (600 MHz, CDCl3) d ¼ 7.35–7.20 (m, 40H,
PhCH2 8ꢂ), 6.24 (d, J ¼ 9.0 Hz, 1H, NH), 5.96 (d, J ¼ 7.2 Hz, 1H,
NH0), 5.68 (m, 1H, OCH2CH¼CH2), 5.17 (d, J ¼ 10.8 Hz, 1H,

328
Yukari Fujimoto et al.
PhCH2OCO), 5.15–5.13 (m, 1H, OCH2CH¼CH2), 5.12–5.07 (m, 1H,
OCH2CH¼CH2), 5.04 (d, J ¼ 10.8 Hz, 1H, PhCH2OCO), 5.01
(d, J ¼ 6.0 Hz, 1H, b-CH of acyloxyacyl), 4.97 (d, J ¼ 7.8 Hz, 1H, H10),
4.77 (d, J ¼ 10.8 Hz, 1H, PhCH2), 4.74–4.72 (m, 1H, PhCH2), 4.70 (d,
J ¼ 3.7 Hz, 1H, H1), 4.69–4.67 (m, 1H, PhCH2), 4.63 (d, J ¼ 10.8 Hz, 1H,
PhCH2), 4.61 (d, J ¼ 10.8 Hz, 1H, PhCH2), 4.57 (d, J ¼ 11.4 Hz, 1H,
PhCH2), 4.55 (d, J ¼ 11.4 Hz, 2H, PhCH2), 4.53 (d, J ¼ 12.0 Hz,
1H, PhCH2), 4.52 (d, J ¼ 10.8 Hz, 1H, PhCH2), 4.50 (d, J ¼ 11.4 Hz,
1H, PhCH2), 4.48 (d, J ¼ 10.8 Hz, 1H, PhCH2), 4.46 (d, J ¼ 11.4 Hz, 1H,
PhCH2), 4.39 (d, J ¼ 12.0 Hz, 1H, PhCH2), 4.31–4.28 (m, 1H, H2), 4.24
(t, J ¼ 8.5 Hz, 1H, H30), 4.13–4.07 (m, 4H, H400, H500, H600, OCH2-
CH¼CH2), 3.99–3.94 (m, 2H, H4, H700), 3.89–3.87 (m, 2H, H60, H800),
3.83 (d, J ¼ 9.0 Hz, 1H, H5), 3.80–3.77 (m, 1H, b-CH of acyl), 3.73–3.63
(m, 6H, H3, H6 2ꢂ, H60, H800, OCH2-CH¼CH2), 3.59–3.56 (m, 1H, H50),
3.39 (t, J ¼ 8.5 Hz, 1H, H40), 3.25 (q, J ¼ 9.0 Hz, 1H, H20), 2.34 (dd,
J ¼ 15.6, 7.2 Hz, 2H, a-CH2 of acyloxyacyl, a-CH2 of acyl), 2.25 (dd,
J ¼ 15.0, 7.8 Hz, 1H, a-CH2 of acyl), 2.18 (dd, J ¼ 15.6, 4.8 Hz, 1H,
a-CH2 of acyloxyacyl), 2.13–2.07 (m, 3H, OCOCH2 of acyloxyacyl 2ꢂ,
H300), 1.96 (dd, J ¼ 12.0, 3.6 Hz, 1H, H300), 1.58–1.53 (m, 1H, g-CH2 of
acyl), 1.48–1.45 (m, 2H, g-CH2 of acyl, OCOCH2CH2 of acyloxyacyl), 1.38
(q, J ¼ 6.6 Hz, 1H, g-CH2 of acyloxyacyl), 1.31–1.04 (m, 82 H, CH2 of acyl
82ꢂ), 0.92–0.82 (m, 27H, CH3 of acyl 9ꢂ, t-BuSi 2ꢂ), 0.11–0.01 (m, 12H,
CH3Si 12ꢂ).
For obtaining the final compound, the anomeric position of trisaccha-
ride was changed to the phosphate, and all the protecting groups were
cleaved by hydrogenation (Fujimoto et al., 2007).
5. Cytokine (IL-6) Induction in Human Peripheral
Whole-Blood Cell Cultures
1. The synthetic samples (lipid A 8a and Kdo–lipid A 9 with noted concen-
trations in 25 mL of saline) and heparinized human peripheral whole-blood
(HWBC) (25 mL) collected from an adult volunteer in RPM1 1640
medium (75 mL; Flow Laboratories, Irvine, Scotland, UK) are incubated
in triplicate in a 96-well plastic plate at 37 ꢁC in 5% CO2. An LPS specimen
prepared by Westphal method from E. coli O111:B4 (Sigma Chemicals
Co.) is used as a positive control at the concentration of 0.5 ng/mL.
2. After 24 h of the incubation, the plate is centrifuged at 300ꢂg for 2 min
and cytokines in the supernatant are assayed (Suda et al., 1995).
3. The levels of IL-6 induced by stimulating HWBC cultures with test
samples are measured by means of an enzyme-linked immunosorbent assay
(ELISA)usingHumanIL-6ELISAkit(eBioscience,SanDiego,CA,USA).

Self and Non-Self Recognition with Glycans
329
1.4
1.2
50ng/ml
500ng/ml
1
5000ng/ml
0.8
0.6
0.4
0.2
0
9
9
8a
8a
+ LPS (E.coli)
LPS (E.coli)
+ LPS (E.coli)
LPS (E.coli)
Figure 16.3 IL-6 induction and inhibition by Helicobacter pylori Kdo–lipid A 9 and lipid
A 8a with heparinized human peripheral whole-blood (HWBC). E. coli (O111:B4) LPS
is used as a positive control at the concentration of 0.5 ng/mL.
Data represent averages of three repeated assays with standard deviations
from individual experiments.
H. pylori Kdo–lipid A 9 and lipid A 8a inhibited IL-6 induction by E. coli
LPS. Kdo–lipid A 9 showed more potent antagonistic activity than 8a
(Fig. 16.3) (Fujimoto et al., 2007). On the other hand, Lipid A 8b having
ethanolamine induced low levels of cytokines such as IL-18 and TNF-a via
TLR4/MD-2 complex (Ogawa et al., 2003). These results demonstrated that
the number of anionic charges influences the biological activity of lipid A and
LPS. Similar charge effects to biological activities were also observed in our
studiesofRu.gelatinosuslipidAandlipidAanalogscontainingacidicaminoacid
residues(Fujimotoetal.,2005).Immunostimulatingorantagonisticactivitycan
be controlled by the anionic charges in these analogs and H. pylori lipid A. The
present study also explains why some strains of H. pylori LPS show weak
immunostimulating activity, while others show antagonistic activity. Weak
immunostimulating activity may be correlated with the ability of H. pylori to
induce chronic inflammation, whereas the antagonistic activity should be
importantforsuppressingtheinnateimmuneresponseandsurvivalasaparasite.
6. Bacterial Glycoconjugates for Nonself
Recognition—Peptidoglycan (PGN)
PGN is a component of bacterial cell walls, and has conserved struc-
tural characteristics, which makes PGN fragment structures to be good
motifs for nonself recognition. PGN has polysaccharide chains linked to

330
Yukari Fujimoto et al.
MurNAc
HO
GlcNAc
HO
HO
HO
O
O
O
O
O
O
HO
O
O
O
O
HO
O
NHAc
NHAc
NHAc
NHAc
D
D
C
CH3
L-Ala-D-Glu-(NH )
CH3
L-Ala-D-Glu-(NH2)n
C
2 n
g
g
O
O
AA-D-Ala-(peptide)
AA-D-Ala-(peptide)
D-Ala
HO
L
HO
HO
O
O
O
O
H2N CH CO2H
O
O
O
O
CH2
CH2
CH2
HO
HO
NHAc
NHAc
NHAc
CH3
D
L-Ala-D-Glu-(NH2)n
C
g
O
H2N CH CO2H
D
AA-D-Ala-(peptide)
meso-Diaminopimelic acid
(meso-DAP)
D-Ala
Lys-type peptidoglycan of Staphylococcus aureus: n = 1, AA: L-Lys, peptide: (L-Gly)5
DAP-type peptidoglycan of Escherichia coli: n = 0 or 1, AA: meso-DAP, peptide: D-Ala
Figure 16.4 Schematic structures of bacterial cell wall peptidoglycan; Lys-type pepti-
doglycan of Staphylococcus aureus, and DAP-type peptidoglycan of Escherichia coli.
peptides, and the polysaccharide is composed of alternating b(1!4) linked
N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc)
glycan. At the branched position of the peptide, there is a diaminocar-
boxylic acid such as L-Lys (in many of Gram-positive bacteria) or meso-
diaminopimelic acid (meso-DAP, in Gram-negative bacteria and some
Gram-positive bacteria) as shown in Fig. 16.4. The structure of the dipep-
tide (L-Ala-D-Glu) linked MurNAc (MDP) is highly conserved in bacterial
species (Schleifer and Kandler, 1972).
PGN has been known as a potent immunostimulator and an immune
adjuvant, but the mechanism of the immune stimulation had not been
unveiled until the recent findings of the receptors. In 2003, two groups
independently found that the intracellular protein Nod1, which is the
founding member of the NLR protein family, is a receptor of PGN
fragments (Chamaillard et al., 2003; Girardin et al., 2003a). Philpott and
coworkers reported that Nod1 senses DAP containing muropeptides, such
as GlcNAc-MurNAc-L-Ala-g-D-Glu-meso-DAP (Girardin et al., 2003a),
whereas Inohara and coworkers found a DAP-containing smaller peptide,
iE-DAP (g-D-glutamyl diaminopimelic acid), activates Nod1 using PGN
synthetic peptide fragments (Chamaillard et al., 2003). It was also shown

Self and Non-Self Recognition with Glycans
331
HO
HO
HO
HO
HO
HO
HO
HO
O
O
HO
O
O
O
HO
HO
HO
O
O
O
OH
AcNH
L-Ala-D-isoGln
OH
O
O
OR
O
O
m
AcNH
AcNH
AcNH
AcNH
CH CHCO
CH CHCO peptide
3
OR'
3
CH CHCO peptide
NHAc
CH
3
MDP
L-Ala-D-isoGln L-Lys(R)-D-ALa
C
3
O
HO
Peptides: L-Ala-D-isoGln
O
HO
O
HO
HO
O
HO
O
HO
HO
HO
L-Ala-D-isoGln-L-Lys
L-Ala-D-isoGln-L-Lys(Ac)
HO
O
O
HO
O
O
O
O
AcNH
O
O
HO
AcNH
peptide
OR
O
AcNH
L-Ala-D-isoGln-L-Lys-D-Ala
m
AcNH
OR'
L-Ala-D-isoGln L-Lys
CH CHCO
AcNH
CH CHCO peptide
NHAc
L-Ala-D-isoGln-L-Lys(Ac)-D-Ala
L-Ala-D-isoGln-L-Lys-D-Ala-D-Ala
L-Ala-D-isoGln-L-Lys(Ac)-D-Ala-D-Ala
3
CH
C
O
3
3
HO
HO
HO
O
HO
O
HO
O
O
O
HO
O
HO
O
O
Linked structures
AcNH
O
HO
O
HO
O
O
AcNH
O
HO
O
AcNH
HO
O
HO
O
O
O
AcNH
AcNH
CH CHCO-L-Ala-D-isoGln
3
HO
AcNH
O
OR
AcNH
CH CHCO-L-Ala-D-isoGln
AcNH
3
CH CHCO-L-Ala-D-isoGln
3
CH CHCO-L-Ala-D-isoGln
3
HO
HO
HO
O
HO
O
O
H
C
L-Ala-D-Glu
O
3
Repeating unit of DAP-type PGN
OH
O
n
O
HN CH CO
R
Tracheal cytotoxin (TCT)
(n = 1, R = D-Ala): DS(anh)-4P
(n = 1, R = D-Ala): DS-4P
(n = 1, R = OH) : DS-3P
R
O
NHAc
NHAc
L-Ala-D-Glu
DAP
CH
O
(CH )
2 3
N
CH CO
DAP
H
C
H
H
HO
HO
HO
3
HNCHCO
O
2
(n = 1, R = OH) : DS(anh)-3P
2
DAP
O
DAP
(CH
)
(n = 0, R = D-Ala): MS-4P
O
NHAc
DAP
2 3
NCH CO
(n = 0, R = D-Ala) : MS(anh)-4P
(n = 0, R = OH) : MS(anh)-3P
DAP
DAP
DAP
DAP
H
H
2
DAP
(n = 0, R = OH) : MS-3P
n
AcNH
2
Figure 16.5 Chemically synthesized PGN fragment library (Inamura et al., 2001,
2006; Kawasaki et al., 2008; Kusumoto et al., 2009).
that the MDP is a ligand of Nod2, which is another NLR family of
intracellular proteins (Girardin et al., 2003b; Inohara et al., 2003).
We constructed the PGN fragments library (Fig. 16.5), which included
Lys-type linear and linked fragments, and also DAP-type fragments such as a
repeating unit of the PGN fragment (GlcNac(b1-4)MurNAc-L-Ala-g-D-
Glu-meso-DAP-D-Ala), tracheal cytotoxin (TCT; GlcNac(b1-4)MurNAc
(anh)-L-Ala-g-D-Glu-meso-DAP-D-Ala), and their fragments (Kawasaki
et al., 2008). The synthesis of TCT is shown in Fig. 16.6, and the one of
the key reactions is the b-selective glycosylation as used to prepare 15 from
glycosyl donor 13 and glycosyl acceptor 14.
7. Synthesis of Disaccharide Moiety 15 in
Tracheal Cytotoxin with b-Selective
Glycosylation
1. TMSOTf (5 ml, 0.030 mmol) is added to the solution of glucosamine
imidate 13 (200 mg, 0.296 mmol), 1ꢁ4 (193.6 mg, 0.443 mmol), and
30 min under Ar atmosphere.
˚
MS4A in dry CH2Cl2 (3 mL) at ꢃ17 C, and the mixture is stirred for
2. The reaction is quenched with saturated aqueous NaHCO3.
3. The organic layer is washed with brine, dried over Na2SO4 and con-
centrated in vacuo.
4. The residue is purified by silica-gel flash column chromatography
(toluene/AcOEt ¼ 7/1) to give 15 (182 mg, 65%).

332
Yukari Fujimoto et al.
(R)
CH CH COOEt
(R)
O
Ph
3
NH
CCl
O
O
O
O
CH CH COOEt
O
Zn-Cu
AcOH/THF/Ac2O
= 1/1/1
3
BnO
14
O
O
O
O
3
NHTroc
O
Ph
O
O
81%
O
BnO
NHTroc
TMSOTf, MS4A, CH Cl ,
2
2
OH
NHTroc
–15°C, 65%
NHTroc
15
13
(R)
L-Ala-D-Glu-OBn
L (S)
CH CH CO
3
Tripeptide 16
LiOH
THF/1, 4-dioxane/
H2O = 4/2/1
O
O
O
1
or
HN CH COR
tetrapeptide 17
O
Ph
(CH )
2 3
O
O
Quant.
ZHN CH COOBn
WSCD HCl, HOBt,
BnO
O
NHAc
D (R)
Et N, DMF
3
NHAc
1
18 92%: R = D-Ala-OBn
19 85%: R = OBn
HCl H-L-Ala-D-Glu-OBn
1
L
CH COOBn
H N
2
(R)
(CH )
2 3
L-Ala-D-Glu-OH
CH CH CO
3
Pd(OH) ,
L (S)
O
O
O
ZHN CH COOBn
2
2
D
H
(20kg/cm )
HN CH COR
2
2
16
HO
HO
HO
(CH )
2 3
HCl H-L-Ala-D-Glu-OBn
THF
O
L
H N CH COOH
O
HN CH CO D-Ala-OBn
2
NHAc
D (R)
(CH )
NHAc
2 3
2
ZHN CH COOBn
20 Quant. (from 18), R = D-Ala; Tracheal cytotoxin (TCT)
2
D
21 Quant. (from 19), R = H
17
Figure 16.6 Synthesis of Tracheal cytotoxin (TCT) and its fragments. Abbreviations:
Bn, benzyl; Tf, trifluoromethanesulfonyl; Troc, 2,2,2-trichloroethoxycarbonyl; TMS,
trimethylsilyl; WSCDꢀHCl, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydro-
chloride; HOBt, 1-hydroxybenzotriazole; Z, benzyloxycarbonyl.
1
ESI-TOF-MS (positive) m/z 949.17 [MþH]þ; H NMR (500 MHz,
CDCl3) d ¼ 7.52–7.18 (10H, m, ArH 2ꢂ), 5.60 (1H, s, Ph-CH¼O2), 5.33
(1H, s, Hanh-1), 5.03 (1H, d, J ¼ 7.7 Hz, NH), 4.91 (1H, d, J ¼ 12 Hz,
-O-CH2-Ph), 4.82 (1H, d, J ¼ 8 Hz, H-1), 4.76 (2H, dd, J ¼ 12 Hz,
20 Hz, -CH2-CCl3), 4.7 (1H, d, J ¼ 12 Hz, -O-CH2-Ph), 4.60 (1H, d,
J ¼ 12 Hz, NH), 4.49 (1H, d, J ¼ 5.7 Hz, Hanh-5), 4.33 (1H, dd,
J ¼ 5 Hz, 10 Hz, H-6), 4.22 (1H, q, J ¼ 3.6 Hz, Lac-aH), 4.20–4.14
(3H, m, -CH2CH3, Hanh-60), 3.95 (1H, d, J ¼ 9.8 Hz, Hanh-2), 3.86–
3.76 (3H, m, H-3, H-6, H-4), 3.74–3.72 (2H, m, Hanh-60, Hanh-4), 3.58
(1H, brs, Hanh-3), 3.52–3.42 (2H, m, H-2, H-5). Found: C, 47.02; H, 4.42;
N, 2.96%. Calcd for C37H42Cl6N2O14: C, 46.71; H, 4.45; N, 2.94%.
8. Synthesis of Tracheal Cytotoxin 20
and Its Fragment 21
After preparation of the disaccharide 15, 2,2,2-trichloroethoxycarbonyl
(Troc) groups were changed to acetyl groups via cleavage of Troc group
using Zn–Cu in the presence of acetic anhydride. The ethyl ester was cleaved

Self and Non-Self Recognition with Glycans
333
with LiOH, and then the liberated carboxyl group was connected to the
DAP-containing peptides 16 or 17 by using 1-ethyl-3-(3-dimethylamino-
propyl)carbodiimide (WSCD; water-soluble carbodiimide), 1-hydroxyben-
zotriazole (HOBt), and triethylamine (Et3N) in DMF. After the coupling, all
benzyl and benzyloxycarbonyl groups of 18 and 19 were removed by catalytic
hydrogenation with Pd(OH)2 and H2 to give 20 (TCT) and 21 (Kawasaki
et al., 2008).
9. Immunostimulatory Activities of DAP
Containing PGN Fragments
The human Nod1 stimulating activity of the DAP-type synthetic
PGN fragments have been evaluated by HEK293T bioassay expressed
human Nod1 as previously described (Chamaillard et al., 2003). In these
compounds, 20 (TCT) shows only very weak human-Nod1 stimulatory
activity, whereas 21 (DS(anh)-3PDAP) shows approximately 10-fold higher
activity than that of known ligand A-iE-DAP (Kawasaki et al., 2008). These
results are consistent with a report using TCT from a natural source
(Magalhaes et al., 2005), and demonstrate that a free carboxyl group at
the 2-position of DAP is favorable for the human Nod1 recognition.
In case of human Nod1, TCT is a only weak stimulant, but it plays a
fundamental role in innate immune systems of other species such as
Drosophila with the activation of PGRP-LC (Kaneko et al., 2004; Stenbak
et al., 2004). It has also been reported that recognition of DAP-type PGN
by PGRP-LE in Drosophila is crucial for the induction of autophagy, which
prevents intracellular growth of Listeria monocytogenes and promotes host
survival after an infection (Yano et al., 2008).
The structurally defined synthesized PGN fragments have been funda-
mental to understand the activation mechanism of innate immune system.
10. Visualizing the In Vivo Dynamics of
Animal N-Glycans
Among the various types of oligosaccharide structures, asparagine-linked
oligosaccharides (N-glycans) are the most prominent in terms of diversity and
complexity. In particular, N-glycans containing sialic acid residues are involved
in a variety of important physiological events, including cell–cell recognition,
adhesion, signal transduction, and quality control (Kamerling et al., 2007).
Moreover, it has long been known that the sialic acids in N-glycans on soluble
proteins or peptides enhance circulatory residence (Morell et al., 1968), that is,
N-glycan-engineered erythropoietin (EPO) (Elliott et al., 2003) or insulin

334
Yukari Fujimoto et al.
(Sato et al., 2004) exhibits a remarkably higher stability in serum, which effects
the prolonged bioactivity. Antibody-dependent cellular cytotoxicity (ADCC)
and/or complement-dependent cytotoxicity (CDC) has also been proposedto
be modulated by the sialic acids of N-glycans in immunoglobulin (IgG)
through Siglec interactions by glycosylating or removing the sialic acids
(Kaneko et al., 2006). However, these important findings and previous efforts
in investigating N-glycan functions have been mostly based on in vitro experi-
ments using isolated lectins, cultured cells, and dissected tissues. Recently,
interest has shifted to the dynamics of these glycoproteins and/or glycans
in vivo, that is, how the function and/or interaction of the individual N-glycan
works synergistically through dynamic processes in the body to eventually
exhibit biological phenomena. Molecular imaging (Tanaka and Fukase, 2008)
is the most promising tool to visualize the ‘‘on-time’’ N-glycan dynamics
in vivo. Although fluorescence imaging is the method of choice due to the
convenient experimental and detection procedures at the small animal levels,
magnetic resonance(MR), andmore preferably, the positron emissiontomog-
raphy (PET) imaging, which have technologically improved sensitivity and
resolution, are well suited for diagnostic applications. Nevertheless, molecular
imaging ofglycanshasnotbeenthoroughlyexamined, exceptforthe18F-FDG
tracer (technically a monosaccharide) and the very limited examples of lipo-
some-conjugated oligosaccharides (Chen et al., 2008; Hirai et al., 2007). This is
due to the lack of the efficient labeling methods of glycoproteins, and the
bioactivity of the oligosaccharides is affected by the multivalency and/or
heterogeneous environment, that is, on cell surfaces that are composed of
oligosaccharide clusters along with other biomolecules. A single molecule of
the N-glycan, either obtained from a natural or synthetic source, is readily
excreted from the body (Vyas et al., 2001). Thus, efficiently labeling
and mimicking such a N-glycan-involved bioenvironment, for example, by
conjugating the N-glycans, to the liposomes, or to the clusters, may provide
information on the ‘‘in vivo dynamics’’ of N-glycans. Below we discuss
the methods for microPET imaging of (1) glycoproteins and (2) dendrimer-
type glycoclusters by making much use of the multivalency effects of 16
molecules of N-glycans.
11. PET Imaging of Glycoproteins
In order to investigate the effects of N-glycans, especially the sialic acid
residue at the nonreducing end of the glycans, on the metabolic stability
of the proteins, a microPET of glycoproteins, orosomucoid, and asialoor-
osomucoid could be investigated (Tanaka et al., 2008). Based on the
recently developed ‘‘azaelectrocyclization protocol’’ (Fig. 16.7A), glyco-
proteins available in only small amounts (62 mg of orosomucoid and

Self and Non-Self Recognition with Glycans
335
A
HO C
CO H
2
2
N
N
N
N
N-glycan chain
CO Et
2
NH
NH
O
2
2
H
N
CHO
HO C
2
N
H
NH
O
2 Molecules of DOTA
3 Molecules of DOTA
N
Orosomucoid
2
+
22 (STELLA kit)
NH
2
1) 24°C, 30min
NH
0.1M phosphate buffer (pH 7.4)
2
NH
68
2
2) GaCl
Asialoorosomucoid
3
62mg, 4.5×10–6M
HO
:
C
C
CO
H
2
OH
OH
2
2
OH
OH
O
HO
N
N
CO
O
2
68 3+
:
:
Ga
NH
O
O
O
O
N
N
HN
AcHN
OH
HO
HO
O
Sialic acid
Galactose
EtO
C
B
2
0–10min
Liver
20–30min
60–80min
180–240min
Lung
Liver
Gall bladder
Spleen
Kidney
Figure 16.7 (A) Labeling of glycoproteins by DOTA by STELLAþ kit (B) Dynamic
microPET images of [68Ga]DOTA-glycoproteins in rabbits. Time course of accumula-
tion of [68Ga]DOTA-orosomucoid (upper) and [68Ga]DOTA-asialoorosomucoid
(lower) in some peripheral organs (axial views). These PET images were fused to
anatomical images obtained by using CT.
asialoorosomucoid) were labeled with the incorporation of ꢄ2–3 units of
DOTA (1,4,7,10-tetraazacyclodecane-1,4,7,10-tetraacetic acid) bþy incu-
bating the respective protein with aldehyde probe 22 (STELLA , a kit
available from Kishida, Co., Ltd.) for 30 min (Tanaka et al., 2010). The
DOTA-labeled glycoproteins were subsequently radiometallated with 68Ga
and their in vivo kinetics were analyzed in rabbit by means of microPET.
12. Method for 68Ga-DOTA Labeling and
MicroPET Imaging in Rabbit
1. PBS solutions of orosomucoid and asialoorosomucoid (62 mg, 1.4 nmol,
295 mL, pH ¼ 7.4) are reacted with a DMF solution of probe
22 (STELLAþ kit, 14 nmol, 1.5 mL) at room temperature for 30 min

336
Yukari Fujimoto et al.
(reaction concentrations: 4.5 ꢂ 10ꢃ6
4.5 ꢂ 10ꢃ5 M for 22).
M
for orosomucoid,
2. DOTA-labeled proteins are purified by centrifugal filtration using
MicroconÒ (Millipore, 30,000 cut).
3. DOTA-proteins prepared above (10 mg) are incubated with 68GaCl3
solution (pH ¼ 7.0, 1.68 mCi, 500 mL) obtained from 68Ge/68Ga
radionuclide generator, at 40 ꢁC for 10 min.
4. A solution of DOTA (1.0 mmol, 10 mM in H2O) is added in order
to chelate and excrete the excess 68Ga from the body during the PET
study.
5. 68Ga-DOTA-glycoproteins at a dose of 15.8–16.1 MBq in 2.2 mL
are injected via an ear vein of female Japanese white rabbits weighing
2.1–2.2 kg at 13 weeks of age (Japan SLC, Inc., Hamamatsu, Japan) under
a general anesthesia with ketamine (60 mg/kg, KetalarÒ, Sankyo, Tokyo,
Japan) and xylazine (6 mg/kg, SelactarÒ, Bayer Yakuhin, Tokyo, Japan).
6. During the imaging experiments, the rabbits are sedated continuously
with intravenously administered a mixture of ketamine (60 mg/kg/h)
and xylazine (6 mg/kg/hr).
7. PET images are obtained by using a small animal PET scanner, the
microPET P4 system (Siemens Medical Solutions Inc., Knoxville, TN,
USA), and the emission data is collected for 240 min postinjection as 12
frames (6 ꢂ 400 s, 3 ꢂ 1000 s, 2 ꢂ 1600 s, and 1 ꢂ 3400 s), and is
acquired with an energy window of 400–650 keV and a coincidence
timing window of 6 ns.
8. The images are reconstructed from 120 to 240 min after injection
of 68Ga-DOTA-orosomucoid or asialoorosomucoid by an ordered
subset expectation maximization (OSEM) algorithm with attenuation
correction using CT data or no scatter correction, and are
smoothed by using a Gaussian kernel with an FWHM of 3 mm in the
all directions.
9. Quantitative analysis is performed using ASIPro VM version 6.3.3.0
software (Siemens Medical Solutions, Inc., Knoxville, TN, USA).
Regions of interest (ROIs) are placed on the tissue region.
MicroPET images of 68Ga-DOTA-orosomucoid and asialoorosomu-
coid in Fig. 16.7B detected the asialo-glycoprotein being cleared through-
kidney faster than orosomucoid through the well-known asialoglycoprotein
receptor (Morell et al., 1968), thus achieving the visualization of sialic
acid-dependent circulatory residence of glycoproteins. PET images also
detected another clearance pathway of asialo-glycoprotein through the
gallbladder, that is, intestinal excretion pathway, as well the accumulation
to the lung and spleen. These promising PET images of glycoproteins
suggest future uses for the glycoproteins in pharmacological and/or clinical
applications.

Self and Non-Self Recognition with Glycans
337
13. PET Imaging of Glycoclusters
In order to image the ‘‘in vivo dynamics’’ of glycans, it is necessary to
mimic the ‘‘glycan-cluster environment,’’ as well as to make advantage of
the glycan multivalency effects for the stronger interaction with lectins. The
polylysine-based dendrimer-type glycoclusters 23a-c with 16 molecules of
glycans (Fig. 16.8) were developed as the excellent templates for investigat-
ing the N-glycan dynamics in vivo (Tanaka et al., unpublished results); thus,
the dendrimer core 23 with the N-benzyl histidine and the terminal
acetylene embodied in the propargyl glycine residue (Fig. 16.8A) could be
smoothly reacted with the 16 molecules of the azide-containing N-glycans
with large and complex structures, that is, many hydroxyls and molecular
weight of ca. 1500, based on the ‘‘self-activating’’ Huisgen 1,3-dipolar
im
N
-Bn-His
A
1)
Acetylene
N
3
20 eq
CuSO4 (16 eq), Na ascorbate (48 eq),
DIPEA (16 eq), DMF, Ar, 40min
then, DOTA (32 eq), 40min
O
OEt
2)
O
Y
N
H
N
H
O
H
23
23a
H O, rt, 30min
2
SO
H
+
2
(STELLA kit)
O
Y =
O
O
N
a
NeuNAca2,6Galb1,4GlcNAcb1,2Mana1,6
NeuNAca2,6Galb1,4GlcNAcb1,2Mana1,3
N
N
O
Manb1,4GlcNAcb1,4GlcNAcbAsp-linker
68
N
HO
O
Ga
N
N
B
O
30min
1h
2h
4h
O
L
GB
68
Ga-DOTA
O
O
Cy5
O
H
K
B
B
NeuNAca2,3Galb1,4GlcNAcb1,2Mana1,6
NeuNAca2,3Galb1,4GlcNAcb1,2Mana1,3
Galb1,4GlcNAcb1,2Mana1,6
Galb1,4GlcNAcb1,2Mana1,3
Manb1,4GlcNAcb1,4GlcNAcbAsp-linker
Manb1,4GlcNAcb1,4GlcNAcbAsp-linker
b
c
30min
H
1h
2h
4h
30min
H
1h
2h
4h
L
L
K
B
K
B
B
Figure 16.8 (A) Preparation of glycoclusters. (B) Dynamic PET imaging of glycoclus-
ters 23a-c in normal BALB/c nude mice. 68Ga-DOTA-Labeled glycoclusters (10 MBq)
were administered from the tail vein of the mice (n ¼ 3, 500 pmol, 100 mL/mouse) and
the whole body was scanned by a small animal PET scanner, microPET Focus 220
(Siemens Medical Solutions, Inc., Knoxville, TN, USA), over 0–4 h after injection;
H, heart; K, kidney; L, liver; B, urinary bladder; GB, gallbladder. (a) glycocluster 23a,
(b) glycocluster 23b, (c) glycocluster 23c.

338
Yukari Fujimoto et al.
cycloaddition.’’ The clusters were designed to have a terminal lysine
e-amino group so that they could be efficiently labeled by 68Ga-DOTA as
the PET radiolabel, and if required, fluorescentþgroups, in the presence of
numerous hydroxyls by labeling kit ‘‘STELLA ’’ under mild conditions
(Tanaka et al., unpublished results).
14. Method for Preparation of N-Glycan
Clusters and PET Imaging in Mouse
1. Acetylene-containing polylysine 23 (16-mer, 158 mg, 2.0 ꢂ 10ꢃꢃ45 mmol)
is reacted with azide-containing N-glycans (1.0 mg, 4.0 ꢂ 10 mmol)
in DMF (50 mL) and H2O (50 mL) at room temperature in the presence of
CuSO4 (64 mg, 3.2 ꢂ 10ꢃ4 mmol), sodium L-ascorbate (238 mg,
1.2 ꢂ 10ꢃ3 mmol), and diisopropylethylamine (74 nL).
2. Excess copper ion is removed by chelating with DOTA (647 mg,
1.56 ꢂ 10ꢃ3 mmol) for 40 min at room temperature, and low-molecular
weight compounds are filtered off using the centrifugal filtration by
MicroconÒ (10,000 cut, Millipore).
3. Lyophilization of the aqueous solution and the purification by reverse-
phase HPLC provide the desired glycoclusters.
4. Labeling by DOTA and 68Ga, and MicroPET imaging were performed
as described above, except using BALB/c mice for imaging (see Fig. 16.8
Caption).
Figure 16.8B shows the microPET in mice of the N-glycoclusters
(Y ¼ 68Ga-DOTA) with the glycan structure of bis-Neua(2-6)Gal glycan
23a, asialo-glycan 23b, and bis-Neua(2-3)Gal glycan 23c. 68Ga-radio-
activity derived from 23a was retained after 4 h in the liver (Fig. 16.8B
(a)), and was excreted slowly from the kidney/urinary bladder and from
the gallbladder (intestinal excretion pathway). On the other hand, asialo-
glycan cluster 23b rapidly cleared through the kidney to the bladder
(Fig. 16.8B(b)), although some accumulation was observed in the liver
because the asialoglycoproten receptors are highly expressed in this organ
(Morell et al., 1968). The results are consistent with the PET analyses of
glycoproteins discussed above (Tanaka et al., 2008), where the asialo-
congener is more rapidly excreted than orosomucoid through the kidney.
However, the a-linking to the 3-OH of galactose in glycocluster 23c,
which also contains sialic acid, was readily excreted through the kidney/
urinary bladder as shown in Fig. 16.8B(c). These PET results on the 16-
mer glycoclusters 23a-c suggest that the specific sialoside linkage to
galactose, that is, Neua(2-6)Gal linkage, in N-glycan structures plays an
important role in the circulatory residence of N-glycans, which in turn

Self and Non-Self Recognition with Glycans
339
results in uptake of 23a in the liver. In addition, this specific sialoside
linkage markedly differentiates the excretion mechanism from those of the
asialo- and Neua(2-3)Gal cases, which proceed via a biofiltration pathway
through the kidney.
The notable difference in the serum stability due to the sialoside bond
linkages to the galactose, that is, the a(2-6)- versus a(2-3)-linkages, is an
intriguing observation. These dynamic PET images suggest a new recep-
tor-mediated excretion mechanism for Neua(2-3)Gal-containing glycans.
Namely, Neua(2-3)Gal-cluster 23c, which usually cannot be found in
serum, is probably recognized as an invader and smoothly excreted by
the vascular endothelial cells, erythrocytes, leucocytes, and via the phago-
cytosis by a macrophage; the smaller sized degradation products may be
filtered in the kidney. Alternatively, the ‘‘excretion-escaping’’ mechanism
by stimulating the immunosuppressive signals through the ITIM (immu-
noreceptor tyrosine-based inhibitory motif) molecules via Siglec families
(Varki and Angata, 2006), may account for the higher stability of Neua(2-
6)Gal-glycan. It is reported that the Neua(2-6)Gal-containing BSA
reduces but does not prevent binding to the asialoglycoprotein receptor,
while the Neua(2-3)Gal-congener abolishes the binding (Park et al.,
2005). Therefore, the prolonged half-life coupled to uptake by the asia-
loglycoprotein receptor account for the high accumulation of 23a in the
liver (Fig. 16.8B(a)). Biantennary Neua(2-6)Gal-chains especially reduce
binding in comparison with tri- and tetraantennary glycans (Lee et al.,
1983), nevertheless, the combination of high valency and long circulatory
half life (reduced clearance) likely leads to the uptake of 23a by hepato-
cytes via the Gal/GalNAc lectin receptor. Note that the slow clearance of
bis-Neua(2-6)Gal cluster 23a through the gallbladder may be due to the
‘‘polar transport mechanism’’ (Nakagawa et al., 2006), which ‘‘tags’’ the
fucose to specific N-glycans in the liver. Elucidation of a detailed mecha-
nism will be the subject of future investigations.
ACKNOWLEDGMENTS
This work was supported in part by Grants-in Aid for Scientific research (No. 19310144,
19681024, 19651095, and 20241053) from Japan Society for the Promotion of Science, by
grants from the Institute for Fermentation, Osaka (IFO), Collaborative Development of
Innovative Seeds from Japan Science and Technology Agency ( JST), New Energy 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 Education, Culture,
Sports, Science and Technology (MEXT) of Japan.

340
Yukari Fujimoto et al.
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