Synthesis of crosslinked peptidoglycan fragments for investigation of their immunobiological functions

Article abstract of DOI:10.1016/j.tetlet.2009.03.081

The synthesis of crosslinked peptidoglycan (PGN) fragments from Streptococcus pneumoniae cell wall was achieved. The immunostimulatory activities, including the induction of IL-6 and the stimulation of the intracellular receptor Nod2, were also determined. The crosslinked PGN fragments were not major human Nod2 ligands.

Full text of DOI:10.1016/j.tetlet.2009.03.081

Tetrahedron Letters 50 (2009) 3631–3634
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis of crosslinked peptidoglycan fragments for investigation
of their immunobiological functions
Yukari Fujimoto a, Yasuko Konishi a, Osamu Kubo a, Mizuho Hasegawa b, Naohiro Inohara b, Koichi Fukase a,
*
a Department of Chemistry, Graduate School of Science, Osaka University Toyonaka, Osaka 560-0043, Japan
b Department of Pathology, The University of Michigan Medical School, Ann Arbor, MI 48109, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of crosslinked peptidoglycan (PGN) fragments from Streptococcus pneumoniae cell wall was
achieved. The immunostimulatory activities, including the induction of IL-6 and the stimulation of the
intracellular receptor Nod2, were also determined. The crosslinked PGN fragments were not major
human Nod2 ligands.
Received 15 January 2009
Revised 10 March 2009
Accepted 12 March 2009
Available online 16 March 2009
Ó 2009 Elsevier Ltd. All rights reserved.
The innate immunity is an evolutionary ancient defense system,
which is activated by microbial molecules with various sensor pro-
teins, called pathogen-recognizing receptors (PRRs).1 The bacterial
cell wall peptidoglycan (PGN) is one of the components, which
activates the immune system. PGN consists of polysaccharide
chains, which are crosslinked with peptides, to form a three-
dimensional mesh-like structure (Fig. 1). Polysaccharide is a
b(1?4) glycan composed of alternating N-acetylglucosamine (Glc-
NAc) and N-acetylmuramic acid (MurNAc), and the carboxyl group
of the MurNAc is linked to the peptide. At the branched position of
(GlcNAc)
HO
(MurNAc)
HO
HO
HO
O
O
O
O
O
O
O
O
O
O
HO
HO
O
NHAc
L-Ala-D-GluNH2
NHAc
NHAc
NHAc
CH3
L-Ala-D-GluNH
CH3
C
O
C
O
2
L-Lys-D-Ala
L-Lys D-Ala
D-Ala-(L-Ala-L-Ser)n
HO
HO
O
HO
O
O
O
O
O
O
the peptide, a diaminocarboxylic acid, such as L-Lys (in Gram-posi-
O
HO
HO
NHAc
L-Ala-D-GluNH2
NHAc
tive bacteria) or meso-diaminopimelic acid (meso-DAP, in Gram-
negative bacteria and some Gram-positive bacteria), is typically
present.
To investigate the recognition of PGN with PRRs and the func-
tion of PGN fragments, we have previously synthesized several
PGN fragments, including Lys-type,2,3 and DAP-type fragments
including tracheal cytotoxin (TCT).4 We and French group have
independently shown that the intracellular Nod1 and Nod2, which
are founding members of the Nod-like receptors (NLRs), are PGN
NHAc
CH3
C
O
L-Lys-D-Ala
(L-Ala-L-Ser)n
D-Ala-(L-Ala-L-Ser)n
n= 0, 1
Figure 1. Schematic structure of peptidoglycan (Lys-type) from Streptococcus
pneumoniae (n = 0: major structure). Segment enclosed by the dashed line is one
unit of the linked structure.
receptors; Nod1 recognizes
(iE-DAP)5 and the muropeptides containing iE-DAP,6 whereas
Nod2 recognizes N-acetylmuramyl- -alanyl- -isoglutamine (mura-
c-D-glutamyl diaminopimelic acid
it also plays important roles together with other immune receptors
(Fig. 2).10,11
Because natural PGN is a heterogeneous polymer often contam-
inated with other immunostimulating substances, detailed bio-
L
D
myl dipeptide: MDP) and muropeptides containing MDP.7,8,3 PGN
is also considered to be recognized by Toll-like receptor 2 (TLR2)
that is a member of membrane-bound Toll-like receptors (TLRs).
However, our previous studies using the synthetic PGN partial
structures indicated that TLR2 does not sense muropeptides,2,3
although it is still debated.9 PGN is also a target of other types of
proteins, which include peptidoglycan recognition proteins
(PGRPs) and lectins. PGRP is a major protein family responsible
for the defense against microorganisms in insects, and in animals
HO
HO
HO
HO
O
O
HO
O
O
OH
O
HO
O
OH
NHAc
-Ala-
R
NHAc
CH3
NHAc
peptide
R
CH3
CO
L
D
-isoGln
CO
peptide:
L
L
L
-Ala-
D
-isoGln
DS-2P
MDP
:
:
-Ala-
-Ala-
D
D
-isoGln-
-isoGln-
L
L
-Lys
DS-3P
-Lys-D-Ala
DS-4P
* Corresponding author. Tel.: +81 6 6850 5388; fax: +81 6 6850 5419.
E-mail address: koichi@chem.sci.osaka-u.ac.jp (K. Fukase).
Figure 2. Structures of MDP, DS-2P, DS-3P, and DS-4P.
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.03.081

3632
Y. Fujimoto et al. / Tetrahedron Letters 50 (2009) 3631–3634
b), c), d)
89%
functional studies of PGN have been investigated using synthetic
specimens. However, the biological activity of the crosslinked frag-
ments has not been well elucidated, due to the synthetic difficul-
ties. Previously, Boons and Mariuzza’s group has reported the
synthesis of a crosslinked PGN structure from Staphylococcus aur-
eus by solid-phase method for analysis of PGRPs recognition, the
structure only contained a monosaccharide moiety.12 Herein, we
report the successful synthesis of crosslinked PGN structures with
a repeating disaccharide (GlcNAc-MurNAc) unit using a solution-
phase synthesis. Fragments with monosaccharides and/or shorter
peptide chains were also synthesized. The synthesized structures
are the major partial structures of Streptococcus pneumoniae PGN,
a)
Fmoc-
L
-Lys(Boc)-OBn
Fmoc-Lys(Boc)-OH
quant.
13
12
f)
Boc-
L
-Lys(R)-
D
-Ala
b), c), e)
Boc-
D
-Ala
Fmoc-
L-Lys-OBn
Fmoc-
L-Lys-OBn
14
R = Z 89% 10
Ac 91% 11
g)
Boc-
L
D
-Lys(R)- -Ala
D
Boc-
L-Lys(R)-
D
-Ala
Fmoc-
L
-Ala-
-isoGln-L-Lys-OBn
H-
L
-Lys-OBn
R = Z quant. 15
Ac quant. 16
R = Z 79% 17
Ac 78% 18
which has the simplest peptide chain containing
L-alanine, D-iso-
glutamine, -lysine, and
L
D
-alanine.13 The established synthetic ap-
Boc-
L-Ala-
D
-isoGln-
L
-Lys(R)-
-isoGln-
D
-Ala
h)
proach should be applicable to the synthesis of crosslinked PGN
structures with longer glycan chains. We also evaluated their
immunostimulatory activities and compared these activities to
the previously synthesized PGN fragments with shorter peptide
structures.3
Scheme 1 shows the synthetic strategy for the linked com-
pounds. Initially, we prepared the peptide parts 7 and 8 by the con-
densation of 9 and 10/11; and then peptides 70 and 80 were
connected to the monosaccharide 5 or disaccharide 6, to obtain
crosslinked PGN partial structures 1–4.
Fmoc-
L-Ala-
D
L
-Lys-OBn
R = Z 80%
Ac 92%
7
8
Scheme 2. Preparation of peptides 7 and 8. (a) BnBr, Cs2CO3, DMF, overnight; (b)
TFA; (c) HClꢀEt2O; (d) Boc-
Lys(R), WSCD.HCl, Et3N, HOBt, DMF, overnight; (f) 20% piperidine, DMF; (g) Fmoc-
Ala- -Ala-
-isoGln, WSCD.HCl, HOBt, Et3N, DMF/DMSO (2:1), overnight; (h) Boc-
D
-Ala, WSCD.HCl, HOBt, Et3N, DMF, overnight; (e) Boc-
L
-
-
-
L
D
L
D
isoGln, HATU, DMF/DMSO (1:1), overnight. WSCD = 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride, HOBt = 1-Hydroxy-1H-benzotriazole, HATU = 2-
(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate
methanaminium.
Branched peptides 7 and 8 were prepared as shown in Scheme
2. We initially attempted to introduce two molecules of dipeptide
peptides were solubilized with DMF/DMSO to carry out the
reactions.
Boc-
Lys-
dipeptide 9 to compounds 10 or 11 in a stepwise manner. Because
of the relative reactivities of lysines, Fmoc protected -amino
group of lysine was first reacted. Starting from protected Lys 12,
-Ala and -Lys residues were introduced to the side chain of 12
L-Ala-D-isoGln 9 to the two lysines of the branched L-Lys-[e-(L-
D-Ala)], but were unsuccessful. Hence, we introduced the
Glycan parts 5 and 6 were prepared from 19 and 20, which were
derived from D
-glucosamine as described (Scheme 3).2–4 The Troc
a
group of 19 was replaced to acetyl group, and then the ethyl ester
at the lactate was cleaved to give monosaccharide unit 5. To pre-
pare disaccharide unit 6, selective cleavage of the benzylidene ace-
tal of compound 19 yielded 21.14 Synthesis of disaccharide 22 with
a b(1?4) linkage was achieved by means of neighboring group
participation of the N-Troc (Troc = 2,2,2-trichloroethoxycarbonyl)
group and the appropriate reactivity of N-Troc-glucosaminyl tri-
chloroacetimidate of 20 and 21. The Troc group of 21 was then con-
verted to an acetyl group to obtain 22, and the ethyl ester was
cleaved with LiOH for the further coupling reaction with the pep-
tides, to give 6.
D
L
to give 10 and 11. In the preparation, Boc group was cleaved with
trifluoroacetic acid (TFA), and the obtained compound was precip-
itated with Et2O/HCl as a HCl salt, and then coupled with another
amino acid. The Fmoc group of 10 or 11 was then cleaved to give
15 or 16. Dipeptides Fmoc-L-Ala-D-isoGln and Boc-L-Ala-D-isoGln
9 were then introduced to afford 7 and 8, respectively. After depro-
tection of 10 and 11, the peptides were insoluble in DMF; thus, the
Peptides 7/8 and glycans 5/6 were then linked to give PGN frag-
ments 1–4 (Scheme 4). We initially attempted to introduce two
+
Boc L-Lys(R)-D-ALa
L-Lys
Boc L-Ala-D-isoGln
9
Fmoc
OBn
Ph
O
O
O
O
Ph
O
R=Z: 10
R=Ac: 11
Ph
O
O
O
a), b)
O
O
O
O
TrocNH
OAllyl
AcNH
CH
CO2Et
AcNH
R CH
OAllyl
CH3
CH
CO2H
OAllyl
CH3
c)
CH3
CO2H
19
R' L-Ala-D-isoGln L-Lys(R)-D-ALa
5
5
OBn
L-Lys
Fmoc L-Ala-D-isoGln
or
BnO
+
Ph
O
O
O
BnO
O
HO
O
O
O
Ph
BnO
O
R'=Boc
R'=Boc
7
8
R=Z:
H
O
+
CCl3
NH
O
O
TrocNH
R=Ac:
H
O
TrocNH
CH
CO2Et
BnO
OAllyl
CH3
AcNH
OAllyl
20
NHAc
CH3R
6
21
CO2H
BnO
Ph
O
O
O
d)
O
O
BnO
O
TrocNH
HO
O
TrocNH
CH
CO2Et
HO
HO
OAllyl
O
CH3
O
22
O
HO
n
AcNH
OR'
NHAc
CH3R
L-Ala-D-isoGln L-Lys(R)-D-ALa
BnO
Ph
O
C
O
O
O
O
b)
e)
O
BnO
O
6
n= 0 (H), R=H, R"= propyl:
n= 0 (H), R=Ac, R"= propyl:
HO
1
2
3
AcNH
HO
HO
AcNH
O
O
CH
OAllyl
O
CH3
23
CO2Et
n= 1,
n= 1,
R=H, R"= propyl:
O
HO
n
AcNH
R=Ac, R"= propyl: 4
NHAc
OR'
CH3R
L-Ala-D-isoGln L-Lys
C
O
Scheme 3. Synthesis of glycans 5 and 6. (a) Zn–Cu, AcOH then Ac2O, Py., 69%; (b)
LiOH, THF/1,4-dioxane/H2O = 4/2/1; (c) Me3NꢀBH3, BF3ꢀEt2O, CH3CN; (d) TMSOTf
(0.1 equiv), CH2Cl2, ꢁ15 °C, 10 min, 88%; (e) Zn/Cu, THF, AcOH/Ac2O = 1/1/1, 46%.
TMSOTf = trimethylsilyl trifluoromethanesulfonate.
Scheme 1. Synthetic strategy for the crosslinked peptidoglycan partial structures.

Y. Fujimoto et al. / Tetrahedron Letters 50 (2009) 3631–3634
3633
BnO
O
O
Ph
O
Ph
O
O
O
O
O
O
O
OAllyl
NHAc
-Ala-
Fmoc-
OAllyl
NHAc
-Ala-
Fmoc-
BnO
Boc-
L
-Ala-D
-isoGln-
L
-Lys(R)-
D
-Ala
a), b)
c)
5 or 6,
NHAc
Fmoc-
L
-Ala-
D
-isoGln-L-Lys-OBn
CH3
L
D
-isoGln-
-Ala-
(from 6)
L
-Lys(R)- -Ala
D
CO
CH3
L
D
-isoGln-
L
-Lys(R)- -Ala
D
CO
or
L
D
-isoGln-L-Lys-OBn
R = Ac: 7
L-Ala-
D
-isoGln-L-Lys-OBn
Z:
8
R = Ac: 24 (96%)
R = Ac: 26 (75%)
Z : 27 (82%)
(from 5)
Z : 25 (92%)
HO
HO
BnO
O
HO
HO
BnO
O
O
5 (for 28 and 29) or 6 (for 30 and 31)
d), e)
or
O
O
O
OAllyl
OAllyl
c)
NHAc
NHAc
CH3
NHAc
CH3
L-Ala-
D
-isoGln- -Ala
L
-Lys(R)-
D
CO
L-Ala-
D
-isoGln-
L
-Lys(R)- -Ala
D
CO
H- -Ala-D-isoGln-L-Lys-OBn
L
H- -Ala-
L
D
-isoGln-L-Lys-OBn
R = Ac: 30 (from 26)
Z : 31 (from 27)
R = Ac: 28 (from 24)
Z : 29 (from 25)
HO
BnO
HO
HO
BnO
O
O
O
HO
O
O
OAllyl
O
OAllyl
NHAc
NHAc
CH3
NHAc
CH3
L
-Ala-
D
-isoGln-
O
L
-Lys(R)-
D-Ala
CO
Ph
L-Ala-
D
-isoGln-
L-Lys(R)-D-Ala
CO
1 (from 32)
2 (from 33)
3 (from 34)
f)
or
O
BnO
Ph
O
quant.
O
O
O
O
O
O
OAllyl
O
OAllyl
NHAc
-Ala- -isoGln-
BnO
4 (from 35)
NHAc
-Ala- -isoGln-L-Lys-OBn
NHAc
L
D
CH3
CO
L
D
L
-Lys-OBn
CH3
CO
R = Ac: 32 (42%) (from 28)
Z : 33 (14%) (from 29)
R = Ac: 34 (42%) (from 30)
Z : 35 (26%) (from 31)
Scheme 4. Synthesis of crosslinked PGN fragments 1–4. (a) TFA; (b) HClꢀEt2O; (c) HATU, Et3N, DMF/DMSO = 1:1; (d) 10% TFA/CH2Cl2; (e) 20% piperidine/DMSO; (f) H2,
Pd(OH)2, AcOH.
glycan chains to the two
tivity of amino groups was insufficient to obtain the desired com-
pounds. Hence, the N-Boc-protection of one of the -Ala residues
was cleaved and glycan moiety 5 or 6 was subsequently introduced
using HATU and Et3N in DMF/DMSO (1:1) to give 24–27. We then
L
-Ala of the linked structure, but the reac-
and benzylidene groups of 32–35 were removed by catalytic
hydrogenation with Pd(OH)2 and H2 in acetic acid to give 1–4,
respectively. Moreover, we synthesized the PGN fragments with
pentapeptides 42–45, which contain the branched part of the pep-
L
tides, L-Ala-D-isoGln-L-Lys-D-Ala-(e)-L-Lys, using a method similar
tried the deprotection of the N-Fmoc group of another
L
-Ala in or-
to that used to prepare compounds 1–415 (Scheme 5).
der to introduce a second glycan to 24–27. However, Fmoc depro-
tection was difficult due to the very low solubility of compounds
24–27. Hence, we cleaved the benzylidene acetal at the 4- and 6-
positions of glucosamine with 10% trifluoroacetic acid (TFA) before
Fmoc cleavage. Another unit of glycan 5 or 6 was introduced in a
manner similar to that using HATU. All benzyl, benzyloxycarbonyl
The biological activities of synthetic PGN fragments, that is,
linked structures 3 (DS2-link(Ac)) and 4 (DS2-link)), and the mono-
saccharides (42 (MS-B5P(Ac)) and 43 (MS-B5P)) and disaccharides
(44 (DS-B5P(Ac)) and 45 (DS-B5P)) with branched pentapeptide,
were evaluated along with the previously synthesized monosac-
charides and disaccharides with a series of the peptides (MDP,
DS-2P, DS-3P, and DS-4P). The ability of synthesized PGN frag-
ments to induce cytokine (IL-6) secretion from human peripheral
blood mononuclear cells (PMNs) was determined (Fig. 3).3 The
samples were incubated with human blood mononuclear cells
(PMNs) for 12 h, and the cytokine production was determined by
ELISA. The PGN fragments, that is, MDP, DS-2P, DS-3P, and DS-4P
showed potent IL-6 inducing activity. The potency was decreased
in ascending order of peptide chain length. The compounds having
c)
a), b)
5 or 6,
Boc-L-Ala-
D
-isoGln-
L
-Lys(R)-
D
-Ala
Z-L-Lys-OBn
R = Ac: 36
Z: 37
O
Ph
Ph
O
O
O
OAllyl
NHAc
CH3
L-Ala-
D
-isoGln-
L
-Lys(R)-
D
-Ala
-Lys-OBn
CO
Z-
L
R = Ac: 38 (61%)
Z : 39 (63%)
or
8
d)
0.01 ug/mL
0.1 ug/mL
1 ug/mL
7
quant.
BnO
O
6
10 ug/mL
O
O
O
O
NHAc
O
OAllyl
NHAc
-Ala-
BnO
5
4
3
2
1
0
CH3
CO
L
D
-isoGln-
L
-Lys(R)-
D
-Ala
-Lys-OBn
Z-L
R = Ac: 40 (51%)
Z : 41 (63%)
HO
HO
HO
HO
O
O
O
n=0 (H),
O
OPr
R = Ac: 42
H : 43
n
DS-2PDS-3P DS-4P 44
45
42
43
3
4
MDP control
NHAc
NHAc
Ligand (µg/mL)
n=1,
CH3
R = Ac: 44
H : 45
L-Ala-
D
-isoGln-
L
-Lys(R)-
D
-Ala
CO
L
-Lys
Figure 3. IL-6 induction of the PGN fragments; DS-2P, DS-3P, DS-4P, DS-B5P(Ac) 44,
DS-B5P 45, MS-B5P(Ac) 42, MS-B5P 43, (DS)2-Link(Ac) 3, (DS)2-Link 4, and MDP.
Samples were incubated with human peripheral blood mononuclear cells (PMNs)5
for 12 h, and cytokine production was determined by ELISA.
Scheme 5. Synthesis of PGN fragments 42–45. (a) TFA; (b) HClꢀEt2O; (c) HATU,
Et3N, DMF/DMSO (1:1); (d) H2, Pd(OH)2, AcOH.

3634
Y. Fujimoto et al. / Tetrahedron Letters 50 (2009) 3631–3634
branched pentapeptide (MS-B5P(Ac), MS-B5P, DS-B5P(Ac), and DS-
B5P) scarcely showed the activity. As for the linked compounds,
(DS)2-Link(Ac) 3 showed low activation, whereas (DS)2-Link 4
showed slightly higher activity. Similar tendency was observed in
IL-8 induction (see Supplementary data).
The human Nod2 stimulating activity of each synthetic PGN
fragment was then evaluated by the HEK293T bioassay as previ-
ously described16 (Fig. 4). MDP and DS-2P exhibited similar Nod2
stimulatory activities, while DS-3P showed a 10-fold lower activity
than MDP. Notably, DS-4P showed only very weak Nod2 stimula-
tory activity, and DS-B5P, MS-B5P, (DS)2-Link, and (DS)2-Link(Ac)
degradation of bacteria in the phago-lysosome proved to be impor-
tant for activation of the macrophage through Nod2.17
In the host defence system against bacterial infection, along
with the NLRs and TLRs, some other recognizing proteins are also
known; one of major families is peptidoglycan recognition protein
(PGRP) family. PGRP seems to recognize a relatively longer glycan
with a peptide moiety, but the recognizing structures and its func-
tions are only partially known. The compounds synthesized in the
present research would also be useful to investigate these struc-
tural bases.18,19
In conclusion, we successfully synthesized glycan linked PGN
fragments, and observed their biological activities. The established
synthesis in this Letter offers possibilities to construct longer PGN
structures, which are useful for the investigation in innate immu-
nostimulation of PGN. Based on these PGN fragments library,
exploring of other biological activities, and determination of the
detailed recognition structures of other types of PGN recognition
molecules including PGRPs and PGN recognizing lectins would be
possible.
did not show the Nod2 stimulatory activities less than 1 lg/ml.
These results clearly indicated that crosslinked PGN fragments
with peptides are not major Nod2 ligands. These results along with
previous studies3 suggest that Nod2 should recognize PGN frag-
ments containing MDP, which are digested by enzymes from both
bacteria and host animals. In fact, our recent studies revealed that
many bacteria excrete soluble Nod1 and Nod2 ligands, which
should be PGN fragments produced by bacterial enzymes.16 These
PGN fragments might be transported into the cells through phago-
cytosis, molecular adhesion, or specific transport system.
Acknowledgments
DS-4P, (DS)2-Link, and (DS)2-LinkAc showed weak but signifi-
cant activities in IL-6 and IL-8 induction from human blood mono-
nuclear cells, suggesting the posibility of enzyme digestion of the
PGN fragments in mononuclear cells during the incubation period,
or facilitated up-take of these molecules in mononuclear cells. The
former mechanism was also reported recently; the intracellular
This work was supported in part by Grants-in Aid for Scientific
research (Nos. 17310128, 17510178, and 19310144) from the Ja-
pan Society for the Promotion of Science as well as grants from
Suntory Institute for Bioorganic Research (SUNBOR Grant), the
Houansha Foundation, and the Institute for Fermentation, Osaka
(IFO).
25
A
MDP
Supplementary data
DS-2P
DS-3P
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2009.03.081.
20
DS-4P
(DS)2-Link-Ac
(DS)2-Link
15
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Bertin, J.; Sansonetti, P. J.; Philpott, D. J. Science 2003, 300, 1584–1587.
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Inamura, S.; Kusumoto, S.; Hashimoto, M.; Foster, S. J.; Moran, A. P.; Fernandez-
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9. Zahringer, U.; Lindner, B.; Inamura, S.; Heine, H.; Alexander, C. Immunobiology
2008, 213, 205–224. and references cited therein.
0
0.1
1
10
100
1000
10000
60
B
MS-B5P
MS-B5P(Ac)
DS-B5P
50
40
30
20
10
0
DS-B5P(Ac)
MDP
10. Royet, J.; Dziarski, R. Nat. Rev. Microbiol. 2007, 5, 264–277.
11. Guan, R.; Mariuzza, R. A. Trends Microbiol. 2007, 15, 127–134.
12. Swaminathan, C. P.; Brown, P. H.; Roychowdhury, A.; Wang, Q.; Guan, R.;
Silverman, N.; Goldman, W. E.; Boons, G. J.; Mariuzza, R. A. Proc. Natl. Acad. Sci.
U.S.A. 2006, 103, 684–689.
13. Garcia-Bustos, J. F.; Chait, B. T.; Tomasz, A. J. Biol. Chem. 1987, 262, 15400–
15405.
14. Oikawa, M.; Liu, W. C.; Nakai, Y.; Koshida, S.; Fukase, K.; Kusumoto, S. Synlett
1996, 1179–1180.
15. Compound 3: ESI-MS (positive) m/z = 1849.76 [M+H]+; 1H NMR (500 MHz,
CD3OD): d = 4.44–4.15 (m, 9H), 3.86–3.30 (m, 11H), 3.08–3.20 (m, 4H), 2.06–
2.40 (m, 8H), 1.91 (s, 6H, NHC(O)CH3), 1.85 (s, 6H, NHC(O)CH3), 1.83 (m, 3H,
1
10
100
1000
Ligand (ng/ml)
Lys-e-NHC(O)CH3), 1.42–1.50 (m, 4H), 1.26–1.34 (m, 22H), 0.85–0.80 (m, 6H,
propyl CH3).
Figure 4. Stimulation of Nod2 by the PGN fragments: (A) MDP, DS-2P, DS-3P, DS-
4P, (DS)2-Link 4, and (DS)2-LinkAc 3. (B) MS-B5P 43, MS-B5P(Ac) 42, DS-B5P 45, and
DS-B5P(Ac) 44. HEK293T cells were transfected with human-Nod2, and the
indicated amount of each compound was added to the cells. Ability of each
16. Hasegawa, M.; Yang, K.; Hashimoto, M.; Park, J. H.; Kim, Y. G.; Fujimoto, Y.;
Nunez, G.; Fukase, K.; Inohara, N. J. Biol. Chem. 2006, 281, 29054–29063.
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18. Chaput, C.; Boneca, I. G. Microbes Infect. 2007, 9, 637–647.
19. Dziarski, R.; Gupta, D. Genome Biol. 2006, 7, 232.
compound to activate NF-j
B was determined by luciferase reporter assay.16