Novel mannosyl derivatives of peptidoglycan monomer: Synthesis and biological evaluation of immunomodulatory properties

Article abstract of DOI:10.1016/j.bmc.2009.06.016

The aim of this work was to prepare mannosyl derivatives of peptidoglycan monomer (PGM, β-d-GlcNAc-(1→4)-d-MurNAc-l-Ala-d-isoGln-mesoDAP(εNH₂)-d-Ala-d-Ala) in order to study the effects of mannosylation on adjuvant (immunostimulating) activity.

Full text of DOI:10.1016/j.bmc.2009.06.016

Bioorganic & Medicinal Chemistry 17 (2009) 6096–6105
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel mannosyl derivatives of peptidoglycan monomer: Synthesis
and biological evaluation of immunomodulatory properties
a
b
b
a,
b
*
´
´
´
Rosana Ribic , Lidija Habjanec , Marija Brgles , Srdanka Tomic , Jelka Tomašic
a Department of Chemistry, Faculty of Science, University of Zagreb, Horvatovac 102a, HR-10000 Zagreb, Croatia
b Institute of Immunology, Rockfellerova 2, HR-10000 Zagreb, Croatia
a r t i c l e i n f o
a b s t r a c t
Article history:
The aim of this work was to prepare mannosyl derivatives of peptidoglycan monomer (PGM, b-
(1?4)- -MurNAc- -Ala- -isoGln-mesoDAP( NH2)- -Ala- -Ala) in order to study the effects of mannosyla-
tion on adjuvant (immunostimulating) activity. Novel Man-OCH2CH(CH3)CO–PGM isomers were sub-
strates for N-acetylmuramyl- -alanine amidase, like the parent PGM molecule. Adjuvant activity of
Man-OCH2CH(CH3)CO–PGM was tested in the mouse model using ovalbumin as an antigen.
Ó 2009 Elsevier Ltd. All rights reserved.
D-GlcNAc-
Received 20 February 2009
Revised 29 May 2009
Accepted 5 June 2009
Available online 13 June 2009
D
L
D
e
D
D
L
Keywords:
Peptidoglycan monomer
Mannosides
Immunostimulating activity
N-Acetylmuramyl-L-alanine amidase
1. Introduction
pattern-recognition receptors (PRR) that recognize and bind
PAMPs. The mechanism of action of peptidoglycans leading to
immunostimulation in the host is not yet completely understood.
However, several reports have demonstrated that peptidoglycans
bind to Toll-like receptors12,13 on the cell surface and that smal-
ler peptidoglycan fragments bind intracellular NOD-recep-
tors.14,15 The recognition and binding of peptidoglycans leads to
the activation of genes coding for pro-inflammatory cytokines.
The interaction of PGM with the receptors has not been studied
extensively and only the involvement of CD14 in the activation
of human monocytes by PGM was reported.16
Mannose receptors (MR) present on the cell surface of
macrophages and dendritic cells are also considered to be
pattern-recognition receptors binding compounds comprising
mannose (Man), N-acetylglucosamine (GlcNAc) and fucose. These
receptors mediate endocytosis and phagocytosis but are also asso-
ciated with signal transduction leading to cytokine release and
production.17,18 They are considered to be a link between innate
and adaptive immunity. MR binds efficiently to GlcNAc, hence this
receptor is a potential candidate to interact with PGM.
Furthermore, it was demonstrated that mannosylation of
antigen leads to selective targeting and improved presentation in
antigen presenting cells and, eventually, alters the potential for
immunoregulation in vivo.19,20
This study was designed to investigate how mannosylation of
PGM might affect the immunostimulating activity in mice. Direct
linking of Man molecule to peptidoglycan fragments has not been
reported so far, although the conjugates of mannosylated proteins
with muramyl dipeptide (MDP) were described.21,22 Such
Peptidoglycans, essential and unique components of bacterial
cell walls, possess diverse biological activities that depend upon
the size and composition of peptidoglycan fragments. Both high
and low molecular mass peptidoglycan fragments, as well as syn-
thetic peptidoglycans, affect the immune system of mammalian
hosts.1,2 Immunostimulating activity of a smaller size peptidogly-
cans with well defined structures, that are not immunogenic and
are devoid of pyrogenicity and toxicity, has been studied exten-
sively, since such compounds could be considered for the use as
adjuvants for human and animal vaccines.3,4
Peptidoglycan monomer (PGM) studied in this work is a disac-
charide pentapeptide b-
D-GlcNAc-(1?4)-D-MurNAc-L-Ala-D-isoGln-
mesoDAP( NH2)- -Ala- -Ala (Fig. 1) originating from Brevibacterium
e
D
D
divaricatum. It was obtained after lysozyme hydrolysis of un-cross
linked peptidoglycan polymer isolated from the culture fluid of pen-
icillin treated bacteria.5 Its structure was confirmed and described in
several papers,6,7 as well as its immunostimulating activity.8,9 Two
lipophilic derivatives of PGM were prepared and characterized and
both derivatives exhibited immunostimulating properties in mice
comparable to the parent molecule.10,11
Peptidoglycans in general are highly conserved structures
unique to microorganisms that are not associated with human
cells. These unique molecules are called pathogen-associated
molecular patterns (PAMPs). Most body defense cells have
* Corresponding author. Tel.: +385 1 460 6411; fax: +385 1 460 6401.
E-mail address: stomic@chem.pmf.hr (S. Tomic´).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.06.016

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R. Ribic et al. / Bioorg. Med. Chem. 17 (2009) 6096–6105
carbodiimide (EDC ꢁ HCl). Water-soluble urea was removed by
OH
OH
extraction with water and upon column chromatography on sil-
O
HO
HO
O
O
ica gel in CHCl3/MeOH 9:1 5a or 5b were obtained. Their purity
O
OH,H
NHAc
H3C
was determined by thin layer chromatography (TLC) and the
compounds were identified by 1H NMR and 13C NMR spectrosco-
pies and elemental (C, H and N) analyses.
NHAc
O
O
CONH2
HN
N
O
CH3
O
H
N
H
N
H
CH3
COOH
CH3
N
2.1.4. Preparation of benzylated Man-OCH2CH(CH3)CO–PGM
H
O
derivatives (6
Benzylated Man-OCH2CH(CH3)CO–PGM derivatives 6
were prepared as pure anomers by condensation of unprotected
PGM 1 with an excess of N-hydroxysuccinimide esters 5 or 5b
a, 6b)
1: R = H
H
a
and 6b
α
: R =
α
2
-Man-OCH2CH(CH3)CO-
N
R
2
β
:
R = -Man-OCH2CH(CH3)CO-
β
CONH2
a
OH
in the presence of triethylamine in dimethylformamide (DMF).
The course of the reaction was monitored by TLC. No side-products
were detected, only traces of unreacted PGM. The reaction mixture
was then directly applied on a column of Sephadex LH-20 which
was eluted with the 20% solution of ethanol in water (Fig. 2). The
R =
OH
O
HO
HO
CH3
CO
O
Figure 1. Structures of the peptidoglycan monomer 1 and mannosyl derivatives 2
a
fractions containing 6a and 6b were pooled, concentrated in vacuo
and 2b.
and used in the following step without further purification
(Scheme 1). Compounds were identified by TLC and mass spec-
trometry which in both compounds revealed the molecular ion
[M+H]+ at m/z 1617.6.
conjugates exhibited enhanced immunostimulating and antitumor
properties in comparison to conjugates without Man.
Derivatives 6
a
and 6b were also prepared directly from carbox-
and
We now report the preparation of novel conjugates using
Man linked to a chiral spacer and coupled to the unprotected pep-
tidoglycan monomer through the free the amino group on diami-
nopimelic acid (Fig. 1). The effects of two newly formed
diastereoisomers on immune reaction in mice and the susceptibil-
ylic acids 4 and 4b. Unprotected PGM reacted with acids 4
a
a
4b (1.2 equiv) in the presence of N-hydroxybenzotriazole (HOBt)
(1.2 equiv), EDC ꢁ HCl (1.2 equiv) and triethylamine (1.2 equiv)
in DMF. Products 6a and 6b were identified by TLC. But, side-prod-
ucts and unreacted PGM were also detected and it was impossible
ity of these derivatives of PGM to the enzyme N-acetylmuramyl-L-
alanine amidase were investigated.
to isolate compounds 6 and 6b from this complex mixture.
a
2.1.5. Preparation of Man-OCH2CH(CH3)CO–PGM derivatives
(2 , 2b)
Deprotected Man-OCH2CH(CH3)CO–PGM derivatives 2
were prepared by subjecting benzylated PGM conjugates 6
2. Results
a
a
and 2b
2.1. Chemistry
a and 6b
to hydrogen atmosphere in 50% ethanol and in the presence of cat-
alytic quantities of palladium on charcoal. The catalyst was filtered
off and the residues purified by column chromatographies on Bio-
gel P-2 in water (Fig. 3). Fractions containing the desired products
2.1.1. Preparation of methyl esters of R-(ꢀ)-(2,3,4,6-tetra-O-
benzyl- -mannopyranosyloxy)-2-methylpropionic acid (3 , 3b)
Commercially available methyl (R)-3-hydroxy-2-methylpropio-
nate was O-mannosylated using benzyl protected mannosyl tri-
chloroacetimidate23 as a good glycosyl donor. Glycosylation was
promoted by the catalytic amount of trimethylsilyl trifluorometh-
D
a
2a
and 2b were concentrated, passed through the 0.45
lm mem-
brane and lyophilized to give pure 2 in 87% yield and pure 2b in
a
84% yield. The structures of both compounds were confirmed by
1H NMR and 13C NMR spectroscopies, mass spectrometry and total
acid hydrolysis. Full assignment of 1H-signals was not possible be-
cause of severe overlaps but partial assignments were possible by
anesulfonate (TMSOTf) and gave the anomeric mixture of 3
a, 3b
(a/b ratio was found to be approximately 3:1). Pure anomers 3a
and 3b were relatively easily separated from anomeric mixture
by column chromatography on silica gel in diethyl ether/petroleum
ether 1:1. Compounds were identified by TLC, 1H NMR and 13C
NMR spectroscopies and elemental (C, H and N) analyses.
comparison with 1H NMR spectra of PGM.11 Mass spectra for 2
a
and 2b revealed the molecular ions [M+H]+ at m/z 1257.5 and
m/z 1257.4, respectively.
2.1.2. Preparation of R-(ꢀ)-(2,3,4,6-tetra-O-benzyl-
ranosyloxy)-2-methylpropionic acids (4 , 4b)
The hydrolysis of the methyl ester of each anomer 3
D
-mannopy-
a
a
and 3b
5
4
3
2
230 nm
254 nm
was performed by saponification using 1 M sodium hydroxide in
dioxane. Mixtures were acidified, extracted with diethyl ether
and the residues were submitted to column chromatography on
silica gel in CHCl3/MeOH 9:1. Pure anomers of benzylated O-man-
I
nosyl carboxylic acids 4
a
and 4b were identified by TLC, 1H NMR
and 13C NMR spectroscopies and elemental (C, H and N) analyses.
1
0
2.1.3. Preparation of N-hydroxysuccinimide esters of
anomers of R-(ꢀ)-3-(2,3,4,6-tetra-O-benzyl- -mannopyranosy-
loxy)-2-methylpropionic acids (5 , 5b)
Activation of the carboxyl groups of
- and b-anomers of R-(ꢀ)-
(2,3,4,6-tetra-O-benzyl-mannopyranosyloxy)-2-methylpropionic
and 4b was achieved by condensation of pure anomers of
acids 4 and 4b with an excess of N-hydroxysuccinimide (HOSu)
in the presence of 1-ethyl-3-(3-dimethylaminopropyl)
a- and b-
D
a
105
125
145
Fraction No.
165
a
4
a
Figure 2. Separation of the benzyl protected Man-OCH2CH(CH3)CO–PGM 6
a
and 6b
a or 6b
a
on a Sephadex LH-20 column (90 ꢁ 2.5 cm) in 20% EtOH. Peak I contains 6
with traces of HOSu.

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R. Ribic et al. / Bioorg. Med. Chem. 17 (2009) 6096–6105
OR1
OR
OR
O
OR
OR
OR1
O
O
R1O
R1O
RO
RO
RO
RO
b
c
CH3
O
CH3
O
CH3
O
+
H
N
PGM-NH2
O
O
PGM
O
O
COOR2
N
3α,3β
R
1 = Bn, R2 = CH3
5α 5β
R1 = Bn
1
6α 6β R1 = Bn
2α, 2β
O
a
d
4α,
4β
R1 = H
R1 = Bn, R2 = H
Scheme 1. Synthesis of mannosyl derivatives of PGM 2
EtOH.
a
and 2b. Reagents: (a) 1 M NaOH, dioxan; (b) EDC ꢁ HCl, HOSu, dry DCM; (c) Et3N, dry DMF; (d) H2, 10% Pd/C, 50%
more, three distinct peaks were obtained by RP-HPLC separation
4
(Fig. 4), denoting the
anhydro-form in the PGM part of the molecules.24 The retention
time for the compound was 15.25 min (31.8%), for the
b-anomer, 16.41 min (61.8%) for the -anomer and 19.01 min
(3.5%) for the anhydro-PGM, respectively. Retention times for
compound 2b were 15.47 min (26.4%) and 16.60 min (59.2%) for
a- and b-anomeric configuration and the
I
3
2
1
0
2a
a
II
b- and
a-anomers and 19.17 min (3.2%) for the anhydro-PGM,
respectively.
In order to unequivocally determine the site of binding of the
a
- and b-
to PGM, products 2
sis with N-acetylmuramyl-
D
-mannopyranosyloxy-2-methylpropionic acid residue
and 2b were submitted to enzymatic hydroly-
-alanine amidase as described bellow.
a
40
60
80
L
Fraction No.
Figure 3. Purification of Man-OCH2CH(CH3)CO–PGM 2
column (90 ꢁ 2.5 cm) in water. Peak I; glycoconjugate 2
a
and 2b on Biogel P-2
2.1.6. Hydrolysis of Man-OCH2CH(CH3)CO–PGM derivatives (2a,
a
or 2b, peak II: HOSu.
2b) with N-acetylmuramyl-L-alanine amidase
It was previously reported that PGM25, as well as its (adamant-
1-yl)-CH2CO–PGM10 and Boc-Tyr derivatives26 can be enzymati-
In order to unequivocally determine the structure of products
2a
and 2b total acid hydrolysis of both compounds with 6 M
cally hydrolyzed with N-acetylmuramyl-L-alanine amidase from
hydrochloric acid was performed at 100 °C, for 16 h, followed by
TLC to reveal the expected amino acid composition (Ala, Glu and
diaminopimelic acid (DAP)). Acid hydrolysis in 2 M hydrochloric
acid at 100 °C, for 6 h, followed by TLC revealed the expected
monosaccharide composition (Man, GlcNH2 and MurNH2). Further-
human serum as a catalyst. In all the cases reported regiospecific
hydrolysis of the lactylamide bond connecting the disaccharide
and pentapeptide or modified pentapeptide moieties occured.
Thus, both 2a and 2b were treated with partially purified amidase
which was isolated from human serum according to the
0.24
100.00
100.00
a
b
0.16
0.22
0.20
0.14
80.00
80.00
0.18
0.12
0.16
0.10
0.14
60.00
60.00
0.12
0.08
A
A
0.10
40.00
40.00
0.06
0.08
0.06
0.04
20.00
20.00
0.04
0.02
0.02
0.00
12.00
0.00
12.00
0.00
0.00
14.00
16.00
18.00
20.00
14.00
16.00
18.00
20.00
t / min
t / min
Figure 4. Chromatogram of (a)
a-Man-OCH2CH(CH3)CO–PGM 2a; peak 1, b-anomer; peak 2, a-anomer; peak 3, anhydro-component; (b) b-Man-OCH2CH(CH3)CO–PGM 2b; 1,
b-anomer; peak 2, -anomer; peak 3, anhydro-component.
a

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R. Ribic et al. / Bioorg. Med. Chem. 17 (2009) 6096–6105
O
CONH2
H2N
OH
N
H
O
CH3
O
H
N
H
CH3
N
COOH
CH3
N
H
OH
OH
OH
O
O
HO
HO
CH3
O
H
N
HO
HO
O
O
NHAc
H3C
O
O
OH,H
NHAc
O
CONH2
O
O
CONH2
8α, 8β
HN
N
H
O
CH3
O
N-acetylmuramyl-
-alanine amidase
H
N
H
N
OH
CH3
O
COOH
CH3
+
L
N
H
OH
O
O
OH
O
HO
HO
OH
O
CH3
H
N
HO
O
HO
O
OH,H
NHAc
COOH
O
CONH2
NHAc
H3C
2α, 2β
7
Scheme 2. Products of the enzymatic hydrolysis of Man-OCH2CH(CH3)CO–PGM 2a and 2b, respectively, with N-acetylmuramyl-L-alanine amidase.
3
I
determined in the sera by ELISA after the second booster
(Fig. 6a). PGM tested in parallel induced significantly higher levels
of anti-OVA IgG in comparison to values induced by OVA alone.
2.5
2
Man-OCH2CH(CH3)CO–PGM 2a and the mixture of 2a and 2b
had no effect on anti-OVA IgG level in comparison to the group
treated only with OVA. Man-OCH2CH(CH3)CO–PGM 2ß caused
the decrease in antibody level that was significantly lower than
in groups treated only with OVA, or with OVA and PGM. Therefore,
it could be concluded that the novel compounds have no adjuvant
activity. However, the modulation of immune response was ob-
served following analyses of IgG subtypes, IgG1 and IgG2a, as indi-
cators of Th2 or Th1 immune response. The effects on IgG1
induction were basically the same as those observed for total spe-
cific IgG and in the levels of IgG2a there were no significant differ-
ences, although PGM-treated group had higher values.
1.5
1
II
0.5
0
50
70
90
Fraction No.
Figure 5. Separation of products of the enzymatic hydrolysis of Man-
OCH2CH(CH3)CO–PGM 2
, 2b on Sephadex G-25 fine column (90 ꢁ 2.5 cm) in
water. Peak I, disaccharide 7; peak II, Man-OCH2CH(CH3)CO–pentapeptide 8 or 8b.
a
a
When IgG1/IgG2a ratios were calculated (Table 1) it was evi-
dent that the group treated with OVA alone had the lowest ratio,
and all groups treated with PGM or mannosylated derivatives
had higher values, indicating the switch toward Th2 type of re-
sponse. The highest value, significantly different from the control
group treated with OVA, was obtained for group treated with
previously reported procedure in TRIS–HCl buffer, pH 7.9.25 The
reaction was monitored by thin layer chromatography. The
products of hydrolysis (Scheme 2) were isolated by column chro-
matography on Sephadex G-25 fine in water (Fig. 5). Eluted first
in both cases (hydrolysis of 2
followed by the - or b-anomer of Man-OCH2CH(CH3)CO–penta-
peptide, 8 and 8b, respectively. These results proved that enzy-
matic hydrolysis of 2 and 2b occurs at the same site as in the
a and 2b) was the disaccharide 7,
a
Man-OCH2CH(CH3)CO–PGM 2a.
a
a
previously reported cases leading to the regiospecific cleavage
3. Discussion
of the lactylamide bond.
The structure of the obtained disaccharide from both hydrolysis
reactions co-migrated on TLC with the authentic sample of GlcNAc-
MurNAc formed by the hydrolysis of PGM catalyzed by N-acety-
Natural peptidoglycan molecules exhibit various biological
activities, and the most often investigated were their immunomod-
ulating characteristics.2,27–29 Smaller synthetic molecules, such as
muramyl dipeptides and tripeptides were investigated as well.
Up to now a number of structure–activity studies were reported.
These investigations include synthetic modifications in the first
step, possibilities of which are numerous since they can be affected
in both, the saccharidic and peptide parts of the molecule. Syn-
thetic modifications described so far were mostly carried out on
smaller, synthetic molecules since the protection and deprotection
steps needed in the synthesis of multifunctional compounds get
more complicated in more complex compounds such as the bacte-
rial PGM monomer which is a disaccharide pentapeptide. Thus,
several synthetically modified MDP, syntheses of which were pat-
ented, have already been included in clinical trials.30 Furthermore,
formulations in liposomes of these compounds were found to be of
lmuramyl-
by 1H NMR and 13C NMR spectroscopies.
The structures of - and b-Man-OCH2CH(CH3)CO–pentapep-
tides 8
L
-alanine amidase.6 Its structure was further determined
a
a
and 8b obtained by enzymatic hydrolysis were confirmed
by 1H NMR and 13C NMR spectroscopies and mass spectroscopy
which in both cases revealed the molecular ion [M+H]+ at m/z
779.7.
2.2. Testing of adjuvant activity
Adjuvant activity of Man-OCH2CH(CH3)CO–PGM 2a and 2b was
estimated by their immunostimulatory effect on secondary hu-
moral immune response to ovalbumin in mice. Anti-OVA IgG was

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R. Ribic et al. / Bioorg. Med. Chem. 17 (2009) 6096–6105
a
b
c
600
500
400
*
40000
2500
2000
1500
1000
500
*
35000
30000
*
25000
*
300
200
100
0
20000
15000
10000
*
*
*
5000
0
0
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
experimental groups
experimental groups
experimental groups
Figure 6. The effect of PGM 1 and its mannosyl derivates 2
in NIH/OlaHsd mice immunized with OVA as an antigen. Sera were analyzed after the second booster. Experimental group: 1—OVA alone, 2—1, 3—2
a
and 2b on production of anti-OVA IgG (a) and its subtypes, anti-OVA IgG1 (b) and anti-OVA IgG2a (c) respectively,
, 4—2 b, 5—mixture of 2
a
a
and 2b. d denotes group mean value, s denotes each serum separately. The difference between two experimental groups was statistically evaluated by Mann–Whitney U-
nonparametric test (Statistica 6.0 for Windows, StatSoft Inc.) with probability value less then 0.05 (p < 0.05) considered as significant. *p < 0.05 in comparison to antigen
alone-treated group or in comparison to the group connected with dashed line.
to our long-standing interest in monosaccharide conjugates,32,33
Table 1
their synthesis34,35 and enzymatic transformations36–40 modifica-
The ratio of anti-OVA IgG1 and anti-OVA IgG2a (IgG1/IgG2a) antibodies developed in
mice after immunisation and two boosters with PGM 1 and its mannosyl derivates 2
and 2b
a
tion of an unprotected, complex and immunomodulating molecule
such as PGM was a special challenge. Substitution with saccharides
on the other hand may contribute to the recognition of the biolog-
ically active molecule by lectins, cell proteins which specifically
and reversibly bind sugar containing structures.41 Therefore, we
chose Man, as one of monosaccharides that specific lectins recog-
nize, to be incorporated as a substituent in the PGM structure.
PGM was connected to Man through an O-glycosidically bonded
linker ((R)-2-methylpropionate) (Fig. 1). We decided to use a chiral
linker of R-configuration to study the regioselectivity of N-acety-
Experimental groups
Average of log10 IgG1/IgG2a SD
1. OVA
2. OVA + 1
3. OVA + 2
4. OVA + 2b
2.2 0.3
2.6 0.7
3.0 0.6*
2.5 0.5
2.5 0.6
a
5. OVA + mixture of 2a and 2b
Log10 of IgG1/IgG2a value was calculated for each mouse serum; results for each
experimental group (n = 5) are reported as average standard deviation (SD).
*
p < 0.05 in comparison to OVA-treated group.
lmuramyl-L-alanine amidase since the absolute configuration of
the linker was the same as in the lactyl part on MurNAc. Further-
more, spacer arms are useful for possible increased accessibility
of the ligand to its receptor, since we expected that Man might di-
rect the novel mannosylated PGM to MR.
interest. Therefore, modifications with lipophilic substituents have
been the mainstream of research since lipophilic moieties in parent
compounds makes them better constituents for incorporation in
liposomes. Several successful attempts to synthetically modify
PGM monomer as a parent compound were made in spite of its
complex structure which contains several groups prone to chemi-
cal modifications such as hydroxyls in the disaccharide and the free
carboxyl and amino groups in the pentapeptide part of the mole-
cule. However, the preferential conformation of PGM in aqueous
and dimethylsulfoxide solutions was determined, and it was
shown that the amino group of meso-diaminopimelic acid
(meso-DAP) is exposed and therefore readily available for some
reactions without the interference of other reactive groups.11 In
our previous paper on the synthesis of Boc-Tyr-PGM31 and
(adamant-1-yl)-CH2CO–PGM10 it was demonstrated that the PGM
monomer can be modified in its pentapeptide part without any
protection when reagents such as N-hydroxysuccinimide esters
and anhydrides are used. Electrophilic carbonyl on anhydrides
and N-hydroxysuccinimide esters react much faster with the free
amino group of meso-DAP than with free hydroxyls in the carbohy-
drate moiety due to the greater nucleophilicity of the amino group
when compared to hydroxyls.
The desired mannosyl derivatives of PGM (2
thesized by a sequence of steps (Scheme 1), the first of which was
the preparation of benzylated mannosyl esters 3 and 3b (in /b
a and 2b) were syn-
a
a
ratio of approximately 3:1) containing a chiral linker of R-
configuration. Saponification of chromatographically separated
anomers led to acids 4
ing them to N-hydroxysuccinimide esters 5
of the amide bond with the free amino group on PGM was per-
formed in the next step to give 6 and 6b followed by the removal
of the benzyl protection of hydroxyls on a Man moiety. This final
a
and 4b which were activated by transform-
a
and 5b. The formation
a
step led to the formation of the desired final diastereoisomers 2
a
and 2b.
It was also shown that the method involving isolation of acti-
vated N-hydroxysuccinimide esters followed by the formation of
the amide bond with the free amino group on PGM is much better
than the strategy of in situ activation of the carboxyl group by cou-
pling reagents EDC ꢁ HCl/HOBt.
The two diastereoisomers 2a and 2b are the first PGM deriva-
tives comprising carbohydrate substituents. They are also the first
mannose substituted peptidoglycan fragments of lower molecular
mass synthesized so far. To the best of our knowledge, Man was
not linked to either peptide or saccharide part of peptidoglycans.
In the present work we have shown that the free amino group
on meso-DAP could be substituted with completely different,
hydrophilic substituents such as monosaccharides, as well. Due

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R. Ribic et al. / Bioorg. Med. Chem. 17 (2009) 6096–6105
In one report several carbohydrate analogues of MDP were pre-
pared and their immunoadjuvant activity tested; -gluco, -manno
and -galacto-analogues showed strong activity comparable to
toward Th2. Although these results are not sufficient to document
fully the effects on immune response, they indicate that the prop-
erties of novel Man-OCH2CH(CH3)CO–PGM isomers merit further
research. Several aspects should be covered: (a) the study of con-
formation of both isomers in order to investigate the differences
in these diastereoisomers, but also the differences in conformation
of novel derivatives and more lipophilic Boc-Tyr-PGM and (ada-
mant-1-yl)-CH2CO–PGM described earlier, (b) direct investigation
of possible binding to MR on antigen presenting cells and (c) a de-
tailed study of effects on cellular immunity, that is, cytokine profile
induced by novel derivatives in comparison to PGM. Each of these
aspects requires a separate, comprehensive study. It could be ex-
pected, that the data on conformation and biological activity of
Man-OCH2CH(CH3)CO–PGM isomers might contribute to the
knowledge of molecular requirements of sugar binding to cell sur-
face receptors and to structure–activity relationship.
D
D
D
that of MDP, whereas numerous other positional, configurational
and optical analogues had no activity.42 It should be noted that
in the described D-manno analogue Man was not linked to MDP,
but GlcNAc moiety was substituted by the mannopyranose ring.
Several reports concern the linking of Man to polymers comprising
covalently linked MDP. MDP bound to poly-L-lysine substituted
with Man21 and MDP bound to serum albumin substituted with
Man22,43 were more efficient in activation of macrophages than
MDP alone or conjugated to protein without mannose. In another
report, the preparation of macromolecular conjugate comprising
Man, D-glucose analogue of MDP and carboxymethyl dextran was
described and the immunological activities investigated.44 The
mannosylated conjugate had increased activity in comparison to
the conjugate without Man.
The properties and biological activity of our mannosylated PGM
5. Experimental
2a
and 2b were studied in two models. One aspect was the study of
susceptibility to hydrolysis with N-acetylmuramyl- -alanine ami-
L
5.1. Materials and methods
dase (Scheme 2), since both lipophilic derivatives of PGM described
earlier10 were good substrates for this enzyme. We demonstrated
that Man did not affect the susceptibility of lactylamide bond to
Peptidoglycan monomer was prepared in PLIVA, Chemical and
Pharmaceutical Works (Zagreb, Croatia), according to the previ-
ously described procedure.5 Bovine serum albumin (BSA), Tween
20, monoclonal anti-chicken egg albumin (clone OVA-14, mouse
IgG1 isotype), avidin-peroxidase and o-phenylenediamine dihy-
drochloride (OPD) were from Sigma (USA). Ovalbumin (OVA) was
purchased from Serva, Germany. Horseradish peroxidase conju-
gated goat anti-mouse IgG (HRP-anti-mouse IgG) was from Bio-
Rad Laboratories, USA. Biotin-conjugated rat anti-mouse IgG1
and anti-mouse IgG2a monoclonal antibodies and streptavidin-
peroxidase were purchased from PharMingen, Becton Dickinson
(USA). Chemicals for buffers and solutions were from Kemika,
Croatia, unless stated otherwise.
hydrolysis with N-acetylmuramyl-L-alanine amidase in both iso-
mers. Further, hydrolysis was regiospecific; only lactylamide bond
was cleaved, despite the structural similarity with the amide bond
connecting amino group of meso-DAP and the chiral linker.
The second aspect was testing of immunomodulating properties
in vivo in a well defined mouse model. In this first and preliminary
testing, the levels of specific anti-OVA IgG were determined. Anti-
OVA IgG level reflects the properties of a potential adjuvant to in-
crease the humoral immune response. We found that mannosyl
derivatives of PGM 2a and 2ß did not stimulate the immune re-
sponse specific for OVA in comparison to PGM 1. Moreover, 2ß
even exhibited immunosuppressive properties. Analysis of IgG1
Starting compound, 2,3,4,6-tetra-O-benzyl-a-D-mannopyrano-
and IgG2a, respectively, indicated that 2
a further improved the
syl trichloroacetimidate, was prepared as previously described.23
Most of the chemical reagents used in syntheses were obtained
from Fluka and Aldrich Corp. All organic solvents were purified
using standard procedures.
switch toward Th2 direction in comparison to PGM 1.
The results clearly showed that mannosylation changed the bio-
logical activity of PGM. The possible reasons could be:
Column chromatography (solvents and proportions are given in
the text) was performed on Merck Silica Gel 60 (size 70–230 mesh
ASTM), Sephadex LH-20 (Pharmacia, Sweden) and Biogel P-2
(Bio-rad Laboratories, USA). For TLC monitoring Fluka Silica Gel
(60 F 254) plates (0.25 mm) were used. Visualization was effected
by use of UV light, iodine and by charring with H2SO4 and
ninhydrin.
1. The drastic change in the tertiary structure and conformation
caused by the hydrophilic substituent—the molecule with dif-
ferent conformation might hinder the binding to receptors rele-
vant for PGM. In any case, both diastereoisomers irrespective of
changes in conformation were still good substrates for the
enzyme N-acetylmuramyl-L-alanine amidase, so probably only
the peptide portion distant from lactylamide bond was changed.
2. Binding to MR—this could result in induction of different cyto-
kine program than binding to Toll or Nod receptors. In several
reports it was suggested that binding to MR was associated
with decreased immune response.45,46 We have no evidence
Optical rotations were measured at room temperature using the
Schmidt + Haensch Polartronic NH8. NMR spectra were recorded
using Bruker Avance (300 MHz) spectrometer. C, H and N analyses
were provided by the Analytical Services Laboratory of Ruder Boš-
´
kovic Institute, Zagreb. Mass spectra were recorded with Waters
so far, that PGM 1 or the novel mannosylated PGM 2a, 2ß bind
MS—Quattro micro instrument. Absorbance was measured on a
Perkin–Elmer Lambda 3 UV–vis spectrometer.
Chromatographic separations were carried using Waters HPLC
System: Waters 600 System Controller, Waters 600 Pump, Waters
In-Line Degasser AF, 2996 photodiode array detection (DAD) sys-
tem, Empower Software (Milford, MA, USA). A Lichrosorb RP-18
to MR, or that such receptors are in any way implicated in the
mechanism of action of these compounds, and further research
on this topic is required.
4. Conclusion
column, 244 mm ꢁ 4 mm, 5
samples were applied using a Rheodyne 7725i injector with an
effective loop volume of 20 L. Analyses were performed at a flow
rate of 1.0 mL/min at room temperature and the eluate was mon-
itored at 214 nm. The gradient solvent system used was made of
acetonitrile containing 0.035% TFA and water containing 0.05%
TFA. The percentage of acetonitrile at 0, 20 and 21 min was 3, 17
and 3, respectively and a running time was 25 min.
lm (Merck, Germany) was used. The
The data obtained in this study indicate that mannosylation of
PGM alters its effects on the immune system. The assay of total
specific IgG showed that the immunostimulating activity of two
novel Man-OCH2CH(CH3)CO–PGM isomers was decreased in com-
parison to the parent molecule. The assay of IgG subtypes also
showed that the isomers switched the type of immune reaction
l

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R. Ribic et al. / Bioorg. Med. Chem. 17 (2009) 6096–6105
6102
5.2. Mannosylation of methyl (R)-3-hydroxy-2-methylpropionate
5.3.1. (R)-(2,3,4,6-Tetra-O -benzyl-
methylpropionic acid (4
Rf = 0.61 (CHCl3/MeOH 9:1); [
a
-
D
-mannopyranosyloxy)-2-
a)
Methyl (R)-3-hydroxy-2-methylpropionate (193.4
1.2 equiv) was suspended in dry DCM (5 mL) at 0 °C under N2 and
TMSOTf was added (7.9 L, 0.04 mmol, 0.03 equiv). 2,3,4,6-Tetra-
O-benzyl- -mannopyranosyl trichloroacetimidate (1000 mg,
lL, 1.75 mmol,
a]
D +13.1 (c 0.21, CHCl3); 1H NMR
(CDCl3): d 7.36–7.13 (m, 20H, H–Ar), 4.85 (d, 1H, J1,2 = 1.77 Hz, H-
1), 4.84 (d, 1H, Jgem = 10.69 Hz, CH2Ph), 4.74–4.51 (m, 6H, 3CH2Ph),
4.49 (d, 1H, Jgem = 10.80 Hz, CH2Ph), 3.96 (app t, 1H, J = 9.29 Hz,
J = 9.36 Hz, H-4), 3.84 (dd, 1H, J 2,3 = 3.00 Hz, J3,4 = 9.21 Hz, H-3),
3.77–3.69 (m, 5H, OCH2, H-2, H-6a, H-6b), 3.60–3.55 (m, 1H, H-
5), 2.76–2.64 (m, 1H, CH), 1.13 (d, 3H, J = 7.12 Hz, CH3). 13C NMR
(CDCl3): d 179.51 (C@O), 138.39, 138.33, 138.26, 138.26 (4C–Ar),
128.31 127.46 (CH–Ar), 98.41 (C1), 79.93, 74.81, 74.81, 72.04
(C2–C5), 75.06, 73.33, 72.63, 72.22 (4CH2Ph), 69.17 (C6), 69.17
(OCH2), 39.83 (CH), 13.85 (CH3). Anal. Calcd for C38H42O8: C,
72.82; H, 6.75. Found: C, 72.75; H, 6.66.
l
a-D
1.46 mmol) suspended in dry DCM (10 mL) was then added drop-
wise within 15 min after which the reaction mixture was stirred
for the additional 2 h and monitored by TLC (diethyl ether/petro-
leum ether 1:1). Reaction mixture was than diluted with DCM
(20 mL) and extracted with 1 M NaHCO3 (20 mL). Organic layer
was washed with water (20 mL) and dried over Na2SO4. After filtra-
tion, the organic layer was concentrated in vacuo and the residue
was purified by flash chromatography on silica gel (diethyl ether/
petroleum ether 1:1). The product was isolated as anomeric pale yel-
low oil mixture (3
73.10; H, 6.92. Found: C, 72.91; H, 6.84.
Pure anomers 3 and 3b were separated from anomeric mixture
using column chromatography on silica gel (diethyl ether/petro-
leum ether 1:1) and /b ratio was approximately 3:1.
a
, 3b) (767 mg, 82%; Anal. Calcd for C39H44O8: C,
5.3.2. (R)-(2,3,4,6-Tetra-O -benzyl-b-
methylpropionic acid (4b)
D-mannopyranosyloxy)-2-
a
Rf = 0.50 (CHCl3/MeOH 9:1); [
a
]
D ꢀ61.2 (c 0.44, CHCl3); 1H NMR
(CDCl3): d 7.57–7.28 (m, 20 H, H–Ar), 5.06 (d, 1H, Jgem = 12.34 Hz,
CH2Ph), 5.01 (d, 1H, Jgem = 10.96 Hz, CH2Ph), 4.94 (d, 1H,
Jgem = 12.59 Hz, CH2Ph), 4.64 (d, 1H, Jgem = 10.71 Hz, CH2Ph), 4.77–
4.56 (m, 4H, 2CH2Ph), 4.53 (s, 1H, H-1), 4.29–4.24 (m, 1H, H-4),
4.03–3.94 (m, 2H, H-2, H-3), 3.91–3.86 (m, 2H, OCH2), 3.76–3.71
(m, 1H, H-6a), 3.64–3.55 (m, 2H, H-5, H-6b), 2.98–2.91 (m, 1H,
CH), 1.37 (d, J = 7.06 Hz, 3H, CH3). 13C NMR (CDCl3): d 177.43
(C@O), 138.75, 138.39, 138.39, 138.19 (4C–Ar), 128.37–127.32
(CH–Ar), 101.74 (C1), 82.20, 75.99, 74.91, 73.46 (C2–C5), 75.04,
73.95, 73.86, 71.63 (4CH2Ph), 71.22 (C6), 69.77 (OCH2), 39.88
(CH), 13.71 (CH3). Anal. Calcd for C38H42O8: C, 72.82; H, 6.75.
Found: C, 72.67; H, 6.62.
a
5.2.1. Methyl (R)-(2,3,4,6-tetra-O-benzyl-
loxy)-2-methylpropionate (3
Rf = 0.38 (diethyl ether/petroleum ether 1:1); [a]D +17.3 (c 0.62,
a-D-mannopyranosy-
a)
CHCl3); 1H NMR (CDCl3): d 7.37–7.16 (m, 20 H, H–Ar), 4.86 (d, 1H,
Jgem = 10.38 Hz, CH2Ph), 4.85 (s, 1 H, H-1), 4.74–4.53 (m, 6H,
3CH2Ph), 4.50 (d, 1H, Jgem = 10.79 Hz, CH2Ph), 3.97 (app t, 1H,
J = 9.38 Hz, J = 9.41 Hz, H-4), 3.84 (dd, 1H, J2,3 = 2.96 Hz,
J3,4 = 9.33 Hz, H-3), 3.77 (dd, 1H, J5,6a = 5.50 Hz, J6a,6b = 11.14 Hz,
H-6a), 3.73–3.68 (m, 4H, OCH2, H-2, H-6b), 3.64 (s, H, OCH3),
3.59–3.56 (m, 1H, H-5), 2.72–2.66 (m, 1H, CH), 1.12 (d,
J = 7.15 Hz, 3H, CH3). 13C NMR (CDCl3): d 174.85 (C@O), 138.52,
138.52, 138.47, 138.39 (4C–Ar), 128.40–127.31 (CH–Ar), 98.36
(C1), 80.08, 74.92, 74.84, 72.16 (C2–C5), 75.03, 73.36, 72.61,
72.22 (4CH2Ph), 69.35 (C6), 69.33 (OCH2), 51.63 (OCH3), 39.96
(CH), 13.97 (CH3).
5.4. General procedure for synthesis of N-hydroxysuccinimide
esters
To solution of pure anomer of each carboxylic acid 4
a or 4b
(150 mg, 0.24 mmol) in dry DCM (5 mL) at 0 °C EDC ꢁ HCl
(68.9 mg, 0.36 mmol, 1.5 equiv) and HOSu (41.7 mg, 0.36 mmol,
1.5 equiv) were added. The mixture was stirred for 1 h at 0 °C
and than overnight at room temperature. The solution was then di-
luted with DCM (10 mL) and extracted twice with water (10 mL).
Organic layer was dried over Na2SO4 and after filtration, evapo-
rated. The residue was purified by column chromatography on sil-
5.2.2. Methyl (R)-(2,3,4,6-tetra-O -benzyl-b-
loxy)-2-methylpropionate (3b)
D
-mannopyranosy-
D ꢀ39.3 (c 0.51,
Rf = 0.28 (diethyl ether/petroleum ether 1:1); [a]
CHCl3); 1H NMR (CDCl3): d 7.45–7.18 (m, 20 H, H–Ar), 4.95 (d, 1H,
Jgem = 12.49 Hz, CH2Ph), 4.90 (d, 1H, Jgem = 10.74 Hz, CH2Ph), 4.81
(d, 1H, Jgem = 12.43 Hz, CH2Ph), 4.53 (d, 1H, Jgem = 10.65 Hz, CH2Ph),
4.65–4.42 (m, 4H, 2CH2Ph), 4.39 (s, 1H, H-1), 4.16–4.14 (m, 1H, H-
4), 3.88–3.79 (m, 2H, H-2, H-3), 3.76–3.71 (m, 2H, OCH2), 3.66 (s, 3
H, OCH3), 3.59 3.56 (m, 1H, H-6a), 3.50–3.48 (m, 1H, H-6b), 3.45–
3.43 (m, 1H, H-5), 2.84–2.80 (m, 1H, CH), 1.24 (d, 3H, J = 6.95 Hz,
CH3). 13C NMR (CDCl3): d 174.72 (C@O), 138.77, 138.51, 138.38,
138.19 (4C–Ar), 128.40–127.31 (CH–Ar), 101.85 (C1), 82.27,
76.09, 74.89, 73.62 (C2–C5), 75.13, 73.70, 73.47, 71.44 (4CH2Ph),
71.37 (C6), 69.62 (OCH2), 51.70 (OCH3), 40.09 (CH), 13.93 (CH3).
ica gel (CHCl3/MeOH 9:1) to afford product 5a or 5b (172.6 mg or
173.7 mg, 78%) as pale yellow oil.
5.4.1. (R)-3-(2,3,4,6-Tetra-O -benzyl-
2-methylpropionic acid N-hydroxysuccinimide ester (5
Rf = 0.85 (CHCl3/MeOH 9:1); [
+8.5 (c 0.47, CHCl3); 1H NMR
a-D-mannopyranosyloxy)-
a
)
a]
D
(CDCl3): d 7.38–7.15 (m, 20H, H–Ar), 4.90 (d, 1H, J1,2 = 1.51 Hz, H-
1), 4.87 (d, 1H, Jgem = 10.90 Hz, CH2Ph), 4.78–4.60 (m, 5H, CH2Ph),
4.54 (d, 1H, Jgem = 12.42 Hz, CH2Ph), 4.49 (d, 1H, Jgem = 10.40 Hz,
CH2Ph), 4.00–3.60 (m, 8H, OCH2, H-2, H-3, H-4, H-5, H-6a, H-6b),
3.08–2.97 (m, 1H, CH), 2.78 (s, 4H, 2CH2), 1.26 (d, 3H, J = 3.52 Hz,
CH3). 13C NMR (CDCl3): d 169.98, 168.86 (C@O), 138.6, 138.52,
138.34 (4C–Ar), 128.24–127.41 (CH–Ar), 98.82 (C1), 79.93, 74.81,
74.66, 72.22 (C2–C5), 74.96, 73.33, 72.77, 71.92 (4 CH2Ph), 69.28
(C6), 68.93 (OCH2), 38.18 (CH), 25.55 (CH2), 13.65 (CH3). Anal.
Calcd for C42H45NO10: C, 69.69; H, 6.27; N, 1.94. Found: C, 69.93;
H, 6.17; N, 2.00.
5.3. General procedure for methyl ester hydrolysis
To a solution of pure anomer of each ester derivative 3
a or 3b
(200 mg, 0.31 mmol) in dioxan (3 mL) 10 equiv of 1 M NaOH
(3.13 mL) was added and the reaction mixture was stirred for
24 h at room temperature and monitored by TLC (diethyl ether/
petroleum ether 1:1). Reaction mixtures were then neutralized
with 0.5 M HCl. Mixtures were extracted with diethyl ether
(10 mL) and organic layers dried over Na2SO4. After filtration, the
organic layers were concentrated in vacuo and the residues puri-
fied by column chromatography on silica gel (CHCl3/MeOH 9:1).
5.4.2. (R)-3-(2,3,4,6-Tetra-O-benzyl-b-D-mannopyranosyloxy)-2-
methylpropionic acid N-hydroxysuccinimide ester (5b)
Rf = 0.83 (CHCl3/MeOH 9:1); [
a
]
D ꢀ77.8 (c 0.27, CHCl3); 1H NMR
(CDCl3): d 7.46–7.15 (m, 20H, H–Ar), 4.96 (d, 1H, Jgem = 12.56 Hz,
CH2Ph), 4.89 (d, 1H, Jgem = 10.81 Hz, CH2Ph), 4.79 (d, 1H,
Jgem = 12.53 Hz, CH2Ph), 4.65–4.40 (m, 5H, CH2Ph), 4.44 (s, 1H,
Product 4a (182.8 mg, 94%) or 4b (162.3 mg, 83%) were afforded
as pale yellow oil.

´
6103
R. Ribic et al. / Bioorg. Med. Chem. 17 (2009) 6096–6105
H-1), 4.21 (dd, 1H, J6a,6b = 10.04 Hz, J6a,5 = 4.06 Hz, H-6a), 4.05 (d,
1H, J2,3 = 2.84 Hz, H-2), 3.90–3.71 (m, 4H, OCH2, H-4, H-6b), 3.55
(dd, 1H, J2,3 = 2.94 Hz, J3,4 = 9.37 Hz, H-3), 3.49–3.44 (m, 1H, H-5),
3.15–3.05 (m, 1H, CH), 2.80 (s, 4H, 2CH2), 1.40 (d, 3H, J = 7.05 Hz,
CH3). 13C NMR (CDCl3): d 169.31, 168.87 (C@O), 138.96, 138.37,
138.35, 138.25 (4C–Ar), 128.27–127.19 (CH–Ar), 102.23 (C1),
82.12, 75.91, 74.67, 73.78 (C2–C5), 75.07, 74.07, 73.43, 71.11
(4CH2Ph), 70.81 (C6), 68.59 (OCH2), 38.12 (CH), 25.56 (2CH2),
13.52 (CH3). Anal. Calcd for C42H45NO10: C, 69.69; H, 6.27; N,
1.94. Found: C, 69.71; H, 6.13; N, 2.15.
DAP, CH2-
c
; isoGln), 27.07 (CH2-b; isoGln), 22.07, 22.03, (2CH3;
a-MurNAc, b-GlcNAc), 21.50 (CH2-
c
; DAP), 18.13 (CH3; Lac),
17.39, 16.65, 16.63 (3CH3; 3Ala), 13.19 (CH3). ESI-MS: calcd for
C50H84N10O27: 1256.6; found [M+H]+ at m/z 1257.5.
5.6.2. N-Acetyl-b-
nyl- -iso-glutaminyl-{N -[(R)-3-(2,3,4,6-tetra-O-benzyl-b-
nopyranosyloxy)-2-methylpropanoyl]}-meso-2,6-diaminopimel-
yl- -alanyl- -alanine (2b)
Rf = 0.52 (2-propanol/ethanol/ammonia/H2O 5:3:2:2); 1H NMR
(D2O): d 5.22 (d, <1H, J = 3.19 Hz, H-1; -MurNAc), 4.82 (d, 1H,
D
-glucosaminyl-(1?4)-N-acetylmuramyl-
L
-ala-
e
D
D-man-
D
D
a
5.5. General procedure for preparation of benzylated Man-OCH2
CH(CH3)CO–PGM
J1,2 = 0.59 Hz, H-1; b-Man), 4.61 (d, 1H, J = 7.73 Hz, H-1; b-GlcNAc),
4.60–4.50 (m, 1H), 4.42–4.21 (m, 5H), 4.08 (q, 1H, J = 7.22 Hz, CH-
a; Lac), 3.95–3.29 (m, 21 H), 2.85–2.77 (m, 1H, CH), 2.40–2.35 (m,
To a solution of N-hydroxysuccinimide esters 5
0.06 mmol, 1.2 equiv) in dry DMF (2 mL), peptidoglycan monomer
(50 mg, 0.05 mmol) was added, followed by triethylamine
(13.9 L, 1 mmol, 2 equiv). The reaction was carried out at 0 °C
for 1 h and then at room temperature over 48 h. The reaction
was monitored by TLC in solvent system 2-propanol/ammonia
7:3. After 48 h the reaction mixture was directly applied on a
Sephadex LH-20 column (90 ꢁ 2.5 cm) and eluted with 20% EtOH
in water. Absorbance of fractions (3 mL) was measured at
230 nm and 254 nm. Those corresponding to benzyl protected
a
or 5b (43.4 mg,
2H), 2.18–1.46 (m, 10H), 2.03 (s, 3H, CH3; MurNAc), 1.94 (s, 3H,
CH3; GlcNAc), 1.42 (d, 3H, J = 7.22 Hz, CH3; Ala), 1.36 (pt, 3H,
J = 7.42 Hz, J = 7.34 Hz, CH3; Lac, CH3; Ala), 1.31 (d, 3H,
J = 7.27 Hz, CH3; Ala), 1.06 (d, 3H, J = 6.91 Hz, CH3). 13C NMR
(D2O, dioxane): d 179.71 (COOH), 177.83, 176.66, 175.78, 175.57,
174.97, 174.88, 174.57, 174.03, 173.64, 173.38 (5CONH, 2CONH2,
2CH3CO, lactyl CO), 100.36 (C1; b-GlcNAc), 99.59 (C1; b-Man),
1
l
90.17 (C1;
70.99, 70.38, 70.20, 66.79 (C2–C5;
C3, C4, C5; -MurNAc, CH- ; Lac), 71.10 (C6; b-Man), 61.01,
61.01, 59.69 (C6; -MurNAc, C6; b-GlcNAc, OCH2), 55.96, (CH-
DAP), 53.80, 53.58, 53.22, 52.70, 50.97, 49.90, 49.35 (3CH-
3Ala, CH- ; isoGln, CH- ; DAP, C2;
40.28 (CH), 31.45, 30.42, 30.31 (CH2-d; DAP, CH2-b; DAP, CH2-
isoGln), 27.06 (CH2-b; isoGln), 22.06, 22.03 (2CH3; -MurNAc,
b-GlcNAc), 21.49 (CH2- ; DAP), 18.12 (CH3; Lac), 17.36, 16.64,
a
-MurNAc), 77.43, 76.17, 76.04, 75.14, 73.49, 72.80,
a-Man, C3, C4, C5; b-GlcNAc,
a
a
Man-OCH2CH(CH3)CO–PGM 6
a
and 6b were pooled, evaporated
a
e
a
;
;
and used in the following step. Rf values (2-propanol/ammonia
7:3) of 6
a
and 6b were 0.40 and 0.37, respectively. ESI-MS: calcd
a
a
a-MurNAc, C2; b-GlcNAc),
for C78H108N10O27: 1616.7; found [M+H]+ at m/z 1617.6.
c;
a
5.6. General procedure for preparation of Man-OCH2CH-
(CH3)CO–PGM
c
16.61 (3CH3, 3Ala), 12.90 (CH3). ESI-MS: calcd for C50H84N10O27
:
1256.6; found [M+H]+ at m/z 1257.4.
To a solution of PGM conjugate 6
a
or 6b in 50% EtOH in water
Total acid hydrolysis of products 2a and 2b in 6 M hydrochloric
(20 mL) 40 mg of 10% Pd/C was added. The mixture was subjected
to hydrogen atmosphere under 2.5 bars at room temperature and
stirred for 24 h. The catalyst was filtrated and the filtrate concen-
trated in vacuo. The residue was purified by column chromatogra-
phy on Biogel P-2 in water. Absorbance of fractions (3 mL) was
measured at 230 nm and those containing Man-OCH2CH(CH3)-
acid at 100 °C, for 16 h, followed by TLC in solvent system n-buta-
nol/acetic acid/H2O 6:1.5:2.5 revealed the expected amino acid
composition (Ala, Glu and DAP), and acid hydrolysis in 2 M HCl
at 100 °C for 6 h, followed by TLC in solvent system acetonitrile/
H2O 5:1 revealed the expected monosaccharide composition
(Man, GlcNH2 and MurNH2).
CO–PGM 2
0.45 m membrane and lyophilized, to give the pure 2
87%) or 2b (53 mg, 84%).
a
or 2b was combined, concentrated, passed through
For testing of biological activity, special batches of endotoxin-
l
a
(55 mg,
free 2a and 2b were prepared by passing the aqueous solution of
2a
and 2b through Detoxy gel column (Pierce, The Netherlands)
followed by lyophilization.
5.6.1. N-Acetyl-b-
nyl- -iso-glutaminyl-{N -[(R)-3-(2,3,4,6-tetra-O-benzyl-
nopyranosyloxy)-2-methylpropanoyl]}-meso-2,6-diaminopimelyl
-alanyl- -alanine (2
Rf = 0.55 (2-propanol/ethanol/ammonia/H2O 5:3:2:2); 1H NMR
(D2O): d 5.22 (d, <1H, J1,2 = 3.41 Hz, H-1; -MurNAc), 4.77 (d, 1H,
J1,2 = 1.73 Hz, H-1; -Man), 4.62 (d, 1H, J1,2 = 8.34 Hz, H-1; b-Glc-
NAc), 4.56–4.51 (m, 1H), 4.36–4.23 (m, 5H), 4.09 (q, 1H,
J = 7.20 Hz, CH- ; Lac), 3.94–3.39 (m, 21H), 2.81–2.75 (m, 1H,
D
-glucosaminyl-(1?4)-N-acetylmuramyl-
L
-ala-
e
D
a-D-man-
5.7. General procedure for hydrolysis of Man-OCH2CH(CH3)CO–
PGM with N-acetylmuramyl-L-alanine amidase
-D
D
a)
A solution of Man-OCH2CH(CH3)CO–PGM 2
0.04 mmol) in TRIS–HCl buffer (0.05 M, pH 7.9) containing
0.02 M MgCl2 was incubated with N-acetylmuramyl- -alanine
amidase from human serum25 (500
L, 0.816 i.u./mL) at 37 °C,
a or 2b (50 mg,
a
a
L
l
a
for 24 h. The reaction was monitored by TLC in solvent system
2-propanol/ammonia 7:3. The reaction mixture was directly ap-
plied to a Sephadex G-25 fine column (90 ꢁ 2.5 cm) in water.
Absorbance of fractions (3 mL) was measured at 230 nm and
CH), 2.40–2.37 (m, 2H), 2.23–1.45 (m, 10H), 2.03 (s, 3H, CH3; Mur-
NAc), 1.95 (s, 3H, CH3; GlcNAc), 1.42 (d, 3H, J = 7.23 Hz, CH3; Ala),
1.38 (d, 3H, J = 6.75 Hz, CH3; Lac), 1.35 (d, 3H, J = 7.15 Hz, CH3; Ala),
1.31 (d, 3H, J = 7.25 Hz, CH3; Ala), 1.07 (d, 3H, J = 7.01 Hz; CH3). 13
C
those containing Man-OCH2CH(CH3)CO–pentapeptide 8
a or 8b
NMR (D2O, dioxane): d 179.66 (COOH), 177.75, 176.51, 175.76,
175.58, 174.97, 174.85, 174.54, 174.09, 173.60, 173.34 (5CONH,
2CONH2, 2CH3CO, lactyl CO), 100.36 (C1; b-GlcNAc), 100.12 (C1;
and disaccharide 7 were pooled and evaporated in vacuo. Ob-
tained products were: Man-OCH2CH(CH3)CO–pentapeptide
8
a
(28.5 mg, 91%) or 8b (28.3 mg, 91%) and disaccharide
7
a
-Man), 90.18 (C1;
72.74, 71.00, 70.45, 70.20, 69.86, 66.66 (C2–C5;
C5; b-GlcNAc, C3, C4, C5; -MurNAc, CH- ; Lac), 69.49 (C6;
Man), 61.06, 61.05, 59.69 (C6; -MurNAc, C6; b-GlcNAc, OCH2),
55.96 (CH- ; DAP), 53.77, 53.59, 53.12, 52.70, 50.96, 49.89, 49.34
(3CH- ; 3Ala, CH- ; isoGln, CH- ; DAP, C2; -MurNAc, C2;
b-GlcNAc), 40.52 (CH), 31.46, 30.45, 30.36 (CH2-d; DAP, CH2-b;
a
-MurNAc), 77.42, 76.19, 76.05, 75.37, 73.50,
(20.7 mg, from hydrolysis of compound 2
lysis of compound 2b).
a; 20.9 mg, from hydro-
a-Man, C3, C4,
a
a
a-
a
5.7.1. N-Acetyl-b-D-glucosaminyl-(1?4)-N-acetylmuramyl
e
acid (7)
a
a
a
a
Rf = 0.40 (2-propanol/ethanol/ammonia/H2O 5:3:2:2); 1H NMR
(DMSO): d 10.43 (br s, 1H, COOH), 7.82 (d, 1H, J = 8.30 Hz, NH),

´
R. Ribic et al. / Bioorg. Med. Chem. 17 (2009) 6096–6105
6104
6.25 (s, 1H, NH), 5.34 (d, 1H, J = 2.78 Hz, H-1;
a-MurNAc), 4.36 (d,
5.8. Experiments in vivo
1H, J1,2 = 7.66 Hz, H-1; b-GlcNAc), 4.32–4.24 (m, 1H, H-5; MurNAc),
4.03 (q, 1H, J = 7.12 Hz, CH- ; Lac), 3.76–3.36 (m, 11H), 1.80 (s, 3H,
CH3; MurNAc), 1.78 (s, 3H, CH3; GlcNAc), 1.21 (d, 3H, J = 6.77 Hz,
CH3; Lac). 13C NMR (CD3OD): d 181.94 (COOH), 173.78, 173.66
(2CH3CO), 101.65 (C1; GlcNAc), 91.38 (C1; MurNAc), 79.89,
78.73, 77.62, 75.97, 75.39, 72.97 (C3, C4, C5; GlcNAc, C3, C4, C5;
a
Experiments in vivo were performed on NIH/OlaHsd (H-2q)
inbred mice strains. All mice used were females from 2 to
2.5 months old. During the experimental period animals were
housed in the animal facility of the Institute of Immunology. Com-
mercial food and water were provided ad libitum. All animal work
was performed according to Croatian Law on Animal Welfare
(1999).
MurNAc, CH-a; Lac), 63.49 (C6; MurNAc), 61.26 (C6; GlcNAc),
58.54 (C2; GlcNAc), 56.72 (C2; MurNAc), 23.05, 22.79 (2CH3; Mur-
NAc, GlcNAc), 19.80 (CH3; Lac).
Experimental groups of five mice were immunized and boosted
two times subcutaneously (s.c.) into the tail base at 21-days inter-
vals. Mice were anaesthetised prior to blood collection on 7th day
after each booster. Sera were prepared, decomplemented at 56 °C
for 30 min and stored at ꢀ20 °C until tested.
e
5.7.2.
zyl-
6-diaminopimelyl-
L
-Alanyl-
D
-iso-glutaminyl-{N -[(R)-3-(2,3,4,6-tetra-O-ben-
a-
D-mannopyranosyloxy)-2-methylpropanoyl]}-meso-2,
D
-alanyl- -alanine (8
D
a)
Rf = 0.29 (2-propanol/ethanol/ammonia/H2O 5:3:2:2);
[
a
]
The dose of antigen OVA was 10
l
g per mouse. The dose of PGM
D
+23.5 (c 0.17, DMSO/H2O 1:1); 1H NMR (DMSO-d6): d 8.73 (br s,
1H, NH), 8.26 (d, 1H, J = 7.31 Hz, NH), 7.98 (d, 1H, J = 7.82 Hz,
NH), 7.95 (d, 1H, J = 8.25 Hz, NH), 7.62 (d, 1H, J = 6.31 Hz, NH),
7.47 (s, 1H, NH), 7.25 (s, 1H, NH), 7.07 (s, 1H, NH), 6.98 (s, 1H,
and its derivates was 200
lg per mouse. The injection volume in all
experimental groups was 0.1 mL per mouse.
5.9. Enzyme immunoassays for qualitative and quantitative
determination of immunoglobulin G specific for OVA (anti-OVA
IgG) in mice sera
NH), 4.56 (s, 1H, H-1), 4.24 (q, 1H, J = 7.30 Hz, CH-
(t, 1H, J = 7.34 Hz, CH- ; DAP), 4.17–4.12 (m, 2H, 2CH-
isoGln), 3.91–3.86 (m, 1H, CH- ; DAP), 3.72 (q, 1H, J = 6.63 Hz,
CH- ; Ala), 3.65 (pd, 1H, J = 10.28 Hz, H-6a), 3.55 (br s, 1H, H-
a
; Ala), 4.19
a
a
; Ala,
e
a
Enzyme immunoassays (ELISA) were performed on flat-bot-
tomed high binding microtitar plates (Costar, USA) according to
previously described procedure.47 Briefly, plates were coated with
15 lg/mL OVA solution in carbonate buffer, pH 9.6, and left over-
night at room temperature (rt). Non-specific antibody binding
2), 3.49–3.42 (m, 4H, OCH2, H-3, H-6b), 3.36 (app t, 1H,
J = 9.77 Hz, J = 9.15 Hz, H-4), 3.32–3.28 (m, 1H, H-5), 2.68–2.62
(m, 1H, CH), 2.20–2.12 (m, 3H, CH2;
c,
c0-isoGln, CH; b-isoGln),
2.02–1.96 (m, 1H, CH; b0-isoGln), 1.77–1.71 (m, 2H, 2CH; d-DAP,
b-DAP), 1.67–1.57 (m, 2H, 2CH; d0-DAP, b0-DAP), 1.49–1.44 (m,
was blocked by incubation with 0.5% (w/v) BSA in PBS-T (0.05%
2H, CH2;
c
,
c0-DAP), 1.30 (d, 3H, J = 6.84 Hz, CH3, Ala), 1.20 (app
(v/v) Tween 20 in PBS) buffer (200
lL/well) for 2 h at 37 °C. Mice
t, 6H, J = 6.57 Hz, J = 6.65 Hz, 2CH3; 2Ala), 0.97 (d, 3H,
J = 6.85 Hz, CH3).13C NMR (DMSO-d6): d 173.42, 173.30, 172.72,
171.53, 171.08 (5CONH, 2CONH2, COOH), 99. 84 (C1), 73.63,
70.74, 69.91, 66.95 (C2–C5), 68.80 (C6), 61.15 (OCH2), 52.39
sera to be tested (100 L/well) and standard preparation were
added in five binary dilutions in duplicates and incubated over-
l
night at rt. Plates were washed and 4000 ꢁ diluted HRP-anti-
mouse IgG was added (100 lL/well) and incubated for 2 h at
(CH-
48.29, 48.00 (CH-
(CH2- ; isoGln, CH2-b; DAP), 27.38 (CH2-b; isoGln), 21.48 (CH2-
e
; DAP), 51.95 (CH-
a
; DAP), 51.89 (CH-
a
; isoGln), 49.14,
37 °C. After washing, the OPD solution (0.6 mg/mL in citrate–phos-
phate buffer pH 5.0) was added (100 L/well) and incubated for
30 min at rt in the dark. The enzymatic reaction was stopped with
1 M H2SO4 (50 L/well) and absorbance at 492 nm (A492) was
measured using a microplate reader (Thermo Scientific Multiscan
Spectrum, Finland). All washings (three times after each step) were
done with PBS-T buffer in microplate washer (Multiwash
Labsystems, Finland). The relative quantities of anti-OVA IgG were
determined by parallel line assay comparing each serum to mono-
clonal anti-chicken egg albumin, declared as standard preparation,
to which 20,000 arbitrary units per milliliters (AU/mL) were
voluntarily assigned.9
a; 3Ala), 39.58 (CH), 31.34 (CH2-d; DAP), 31.10
l
c
c
; DAP), 18.19, 17.83, 17.51 (CH3, 3Ala), 14.23 (CH3). ESI-MS:
l
calcd for C31H54N8O15: 778.4; found [M+H]+ at m/z 779.7.
e
5.7.3.
L
-Alanyl-
D
-iso-glutaminyl-{N -[(R)-3-(2,3,4,6-tetra-O-ben-
zyl-b-
D-mannopyranosyloxy)-2-methylpropanoyl]}-meso-2,
6-diaminopimelyl-
D
-alanyl- -alanine (8b)
D
Rf = 0.26 (2-propanol/ethanol/ammonia/H2O 5:3:2:2);
[a]
D
+11.8 (c 0.17, DMSO/H2O 1:1); 1H NMR (DMSO-d6): d 8.71 (br s,
1H, NH), 8.24 (d, 1H, J = 7.26 Hz, NH), 7.99 (d, 1H, J = 7.75 Hz,
NH), 7.96 (d, 1H, J = 8.10 Hz, NH), 7.63 (d, 1H, J = 6.53 Hz, NH),
7.49 (s, 1H, NH), 7.25 (s, 1H, NH), 7.07 (s, 1H, NH), 6.98 (s, 1H,
5.10. Enzyme immunoassays for qualitative and quantitative
determination of immunoglobulin G subtypes specific for OVA,
anti-OVA IgG1 and anti-OVA IgG2a respectively, in mice sera
NH), 4.55 (s, 1H, H-1), 4.25–4.11 (m, 4H, 4CH-
isoGln), 3.89–3.87 (m, 1H, CH- ; DAP), 3.74 (q, 1H, J = 6.66 Hz,
CH- ; Ala), 3.64 (dd, 1H, J6a,6b = 11.24 Hz, J6a,5 = 1.12 Hz, H-6a),
a; 2Ala, DAP,
e
a
3.55 (d, 1H, J = 1.52 Hz, H-2), 3.48–3.41 (m, 4H, OCH2, H-3,
H-6b), 3.36 (app t, 1H, J = 9.54 Hz, J = 9.14 Hz, H-4), 3.32–3.28
(m, 1H, H-5), 2.68–2.62 (m, 1H, CH), 2.22–2.12 (m, 3H, CH2;
ELISAs for determination of anti-OVA IgG1 and anti-OVA IgG2a
were performed as follows: after sera incubation on microplates
coated with OVA, biotinylated rat anti-mouse IgG1 at 0.05
or biotinylated rat anti-mouse IgG2a at 0.5 g/mL was added,
respectively, 100 L to each well and incubated for 2 h at 37 °C.
lg/mL
c,
c0-isoGln, CH; b-isoGln), 2.02–1.96 (m, 1H, CH; b0-isoGln),
l
1.77–1.71 (m, 2H, 2CH; d-DAP, b-DAP), 1.67–1.57 (m, 2H, 2CH;
l
d0-DAP, b0-DAP), 1.52–1.44 (m, 2H, CH2; c0-DAP), 1.30 (d, 3H,
c,
After washing, streptavidin-peroxidase (100,000ꢁ) was added for
J = 6.90 Hz, CH3, Ala), 1.20 (app t, 6H, J = 6.15 Hz, J = 6.62 Hz,
2CH3; 2Ala), 0.97 (d, 3H, J = 6.86 Hz, CH3). 13C NMR (DMSO-d6):
d 173.69, 173.52, 173.44, 173.37, 172.90, 171.63, 171.29, 170.93
(5CONH, 2CONH2, COOH), 99.91 (C1), 73.87, 70.82, 70.05, 66.89
determination of IgG1, while avidin-peroxidase (50,000ꢁ) was
used for IgG2a determination, (100 lL/well) and incubated for an-
other 2 h at 37 °C. Plates were washed and the substrate solution of
100 L per well was added and incubated for 30 min at rt in the
dark. The enzymatic reaction was stopped with 1 M H2SO4
(50 L/well) and A492 was measured using a microplate reader.
l
(C2–C5), 68.83 (C6), 61.18 (OCH2), 52.43 (CH-
(CH- ; DAP), 52.00 (CH- ; isoGln), 49.02, 48.89, 48.18 (CH-
3Ala), 39.64 (CH), 31.55 (CH2-d; DAP), 31.34 (CH2-b; DAP), 31.18
(CH2- isoGln), 27.41 (CH2-b; isoGln), 21.72 (CH2- DAP),
e; DAP), 52.19
a
a
a;
l
The relative quantities of antibody subtypes were determined
by parallel line assay using appropriate standard preparations. A
monoclonal anti-OVA IgG1 was a standard for relative quantifica-
tion of anti-OVA IgG1 to which was assigned 400,000 AU/mL, while
c;
c;
18.34, 18.28, 17.82 (CH3, 3Ala), 14.57 (CH3). ESI-MS: calcd for
C31H54N8O15: 778.4; found [M+H]+ at m/z 779.7.

´
6105
R. Ribic et al. / Bioorg. Med. Chem. 17 (2009) 6096–6105
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20. Nadesalingam, J.; Dodds, A. W.; Reid, K. B.; Palaniyar, N. J. Immunology 2005,
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was used as a standard for relative quantification of IgG2a specific
antibodies with voluntarily assigned 20,000 AU/mL.
21. Derrien, D.; Midoux, P.; Petit, C.; Nègre, E.; Mayer, R.; Monsigny, M.; Roche, A.
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Acknowledgment
We wish to thank the Ministry of Science, Education and Sports
of the Republic of Croatia for support of this work (Projects 119-
1191344-3121 and 021-0212432-2431).
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