Synthesis and protective activity of β-glycosides of N-acetylmuramyl-L-alanyl-D-isoglutamine with alkylalicyclic and arylaliphatic aglycons

Article abstract of DOI:10.1007/s11171-005-0079-4

The following glycosides of N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) were synthesized: β-4-tert-butylcyclohexyl MDP, β-2-(adamant-1-yl) ethyl MDP, β-2,2-diphenylethyl MDP, and β-2-(p-biphenyl)ethyl MDP. The starting peracetylated β-N-acetylglucosaminides were prepared by the oxazoline method. They were converted into 4,6-O-isopropylidene-N-acetyl-D- muramic acids, which were coupled with L-Ala-D-Glu(NH₂)OBn. The target glycopeptides were obtained after their deprotection. The stimulation of the anti-infection resistance of mice against Staphylococcus aureus by the MDP glycosides was studied.

Full text of DOI:10.1007/s11171-005-0079-4

Russian Journal of Bioorganic Chemistry, Vol. 31, No. 6, 2005, pp. 576–582. Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 6, 2005, pp. 637–644.
Original Russian Text Copyright © 2005 by Zemlyakov, Tsikalova, Tsikalov, Chirva, Mulik, Kalyuzhin.
Synthesis and Protective Activity of b-Glycosides
of N-Acetylmuramyl-L-Alanyl-D-Isoglutamine
with Alkylalicyclic and Arylaliphatic Aglycons
1
A. E. Zemlyakov*, V. N. Tsikalova*, V. V. Tsikalov*, V. Ya. Chirva*,
E. L. Mulik**, and O. V. Kalyuzhin**
* Vernadsky Tauric National University, ul. Yaltinskaya 4, Simferopol, 95007 Ukraine
** Research Institute of Human Morphology, Russian Academy of Sciences, ul. Tsyurupy 3, Moscow, 117418 Russia
Received March 14, 2005; in final form, April 14, 2005
Abstract—The following glycosides of N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) were synthesized:
β-4-tert-butylcyclohexyl MDP, β-2-(adamant-1-yl)ethyl MDP, β-2,2-diphenylethyl MDP, and β-2-(p-biphe-
nyl)ethyl MDP. The starting peracetylated β-N-acetylglucosaminides were prepared by the oxazoline method.
They were converted into 4,6-O-isopropylidene-N-acetyl-D-muramic acids, which were coupled with L-Ala-
D-Glu(NH2)OBn. The target glycopeptides were obtained after their deprotection. The stimulation of the anti-
infection resistance of mice against Staphylococcus aureus by the MDP glycosides was studied.
Key words: glycopeptides, muramyl dipeptide, oxazoline synthesis of muramyl dipeptide glycosides, antibacte-
rial resistance
1
INTRODUCTION
the structure and the nature of aglycones of MDP gly-
cosides and their immunostimulating activity.
The glycosides of N-acetylmuramyl-L-alanyl-D-
isoglutamine (muramyl dipeptide) with various ë4–ë8-
aglycones have been shown to exhibit amphiphilic
properties and high immunostimulating activity in var-
ious tests in vitro and in vivo [1, 2]. In contrast, the gly- sic scheme. The oxazoline method [12] providing the
RESULTS AND DISCUSSION
The MDP glycosides were synthesized by the clas-
2
formation of strictly 1,2-trans-glycoside bond was cho-
sen for the preparation of starting peracetylated β-gly-
cosides of N-acetylglucosamine (IIa)–(IId). β-Substi-
tuted ethanols were glycosylated with excess oxazoline
(I) in dichloroethane at the temperature of approxi-
mately 90°ë in the presence of catalytic amounts of p-
toluenesulfonic acid. Glycosides (IIb)–(IId) were iso-
lated by crystallization or column chromatography in
68–81% yields.
The glycosylation of cis,trans-4-tert-butylcyclohex-
anol was carried out with a lack of oxazoline (I). Two
glycosylation products, the more mobile minor glyco-
side (IIe) and the main glycoside (IIa), were present in
the reaction mixture at the ratio of ~1 : 15 according to
TLC and were separated by column chromatography.
The e,e- and a,e-configurations of the disubstituted
cyclohexane cycle were proposed for compounds (IIa)
and (IIe), respectively, on the basis of a greater reactiv-
ity of trans-4-tert-butylcyclohexanol with the equato-
rial disposition of hydroxyl group and a higher stability
of the corresponding glycoside derivative.
cosides with predominating lipophylic properties have
not been studied methodically. However, some experi-
ments [3, 4] indicated that β-hexadecyl MDP [5], β-
(2,3-didodecyloxypropyl) MDP [6], and β-cholesteryl
MDP [7] activate immune reactions more effectively
than MDP itself. A keen interest in lipophilic MDP
derivatives is also associated with the fact that ε-octa-
decanoyl MDP-L-lysine (Romurtid) [8] and phosphati-
dylethanolamide of MDP-L-alanine [9] are allowed for
the therapeutic use. Moreover, a number of compounds,
such as cholesteryl ester of MDP-L-alanine [10] and 6-
O-(2-tetradecylhexadecanoyl)-MDP [11] are on the fin-
ishing stages of clinical trials.
We synthesized β-4-tert-butylcyclohexyl, β-2-(ada-
mant-1-yl)ethyl, β-2,2-diphenylethyl, and β-2-(p-
biphenyl) glycosides of MDP (VIIa)–(VIId) contain-
ing alicyclic, cycloalkylaliphatic, and aromatic alco-
hols as aglycones in order to search for highly affective
immunomodulators and to find a relationship between
1
Corresponding author; phone: +38 (0652) 23-3885; fax: +38
The structures of glycosides (IIa)–(IIe) were con-
(0652) 23-2310; e-mail: alex_z@crimea.edu
1
2
firmed by their H NMR spectra in which the reso-
Abbreviations: MDP, N-acetylmuramyl-L-alanyl-D-isoglutamine
(muramyl dipeptide); TosOH, p-toluenesulfonic acid.
nances of all protons were completely assigned (see
1068-1620/05/3106-0576 © 2005 Pleiades Publishing, Inc.

SYNTHESIS AND PROTECTIVE ACTIVITY
577
Table 1). The protons of cyclohexane ring in the agly- blets at 7.52 and 7.56 ppm of p-phenylene groups of the
cone of (IIa) resonate in the region of 0.88–1.74 ppm. biphenyl derivative (IId).
The exception is the multiplet of methine proton at δ
The ethyl part of aglycones of glycosides (IIb) and
3.43 ppm attached to the Cl linked with oxygen atom.
(IId) is represented in the spectra as two doublets of
The intensive singlet of methyl groups of tert-butyl rad-
triplets of nonequivalent H1' protons at 3.53–3.72 ppm
ical resonates at 0.77 ppm (cf. Table 1). The protons of
and 3.93–4.18 ppm areas and as a doublet of triplets of
its isomer (IIe) have similar values of chemical shifts
the β-methylene group at 1.37 and 2.93 ppm. The pro-
and the spin-coupling constants. The main differences
tons of methylene group of (IIc) resonate as two dou-
blets of doublets at 4.29 and 4.42 ppm. The protons of
are downfield shifts of the resonances of H2 in the gly-
coside residue and the resonance of methine H1' of
methine group are characterized by the triplet at
aglycone. This fact confirms the isomeric structures of
4.02 ppm.
glycosides (IIa) and (IIe).
The adamantyl protons of glycoside (IIb) resonate
The β-configuration of D-glucosamines (IIa)–(IId)
in a high field (1.48 to 1.92 ppm). The aromatic protons is confirmed by the presence of doublet of the anomeric
are exhibited in the spectra as two multiplets at 7.21 and proton in the area of 4.67–4.85 ppm with the spin-cou-
7.27 ppm of two phenyl groups for (IIc) or a broad mul- pling constant of 8–8.5 Hz characteristic of 1,2-trans-
tiplet of phenyl group at 7.27–7.46 ppm and two dou- glycoside bond.
OAc
OR'
O
O
AcO
AcO
R'O
R'O
OR
N
NHAc
O
Me
(I)
(II‡–e) R' = Ac
(III‡–d) R' = H
Me
Me
OH
O
O
O
HO
H
O
R'O
O
OR
OR
NHAc
NHAc
Me
CO-L-Ala-D-Glu(NH2)OR'
(IV‡–e) R' = H
(VI‡–d) R' = Bzl (VII‡–g) R' = H
(V‡–e) R' = CH(Me)COOH
2'
Me
1'
Me
‡: R =
e: R =
b: R =
c: R =
d: R =
g: R =
2'
1'
2'
Me
1'
1'
Me
Me
Me
Me
Me
Me
Me
Me
Me
1'
f: R =
Me
Me
Peracetates (IIa)–(IId) were deacetylated by the bromopropionic acid. The reaction was carried out
Zemplen reaction. The 4,6-diol grouping of the result- under the conditions described in [13] that favored the
ing triols (IIIa)–(IIId) was blocked by the action of nucleophylic substitution with the inversion of config-
2,2-dimethoxypropane. The free hydroxyl group at C3 uration of the chiral center in the propionic acid deriv-
remained free in derivatives (IVa)–(IVd) was treated ative. As a result, the protected N-acetyl-D-muramic
with sodium hydride in dioxane and alkylated with L-2- acids (Va)–(Vd) were synthesized. They were con-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 6 2005

578
ZEMLYAKOV et al.
1
2
*
Table 1. H NMR spectra of β-glycosides (IIa)–(IIe) in C HCl3
Chemical shifts, ppm (spin-coupling constants, Hz)
Group or atom
(IIa)
(IIb)
4.67d (8)
(IIc)
(IId)
(IIe)
H1 (J1,2
H2 (J2,3
H3 (J3,4
H4 (J4,5
)
)
)
)
4.85d (8.5)
4.69d (8.5)
4.67d (8.5)
3.86ddd (10)
5.26dd (9.5)
5.08dd (9.5)
4.79d (8.5)
3.54ddd (10)
5.37dd (9.5)
4.98dd (9.5)
3.64ddd (2.5; 5)
3.80ddd (10.5)
5.32dd (9.5)
5.07dd (9.5)
3.74ddd (10.5)
5.25dd (9.5)
5.05dd (9.5)
3.79ddd (10.5)
5.37dd (9.5)
5.08dd (9.5)
3.68ddd (2.5; 5)
H5 (J5,6a; J5,6b
)
3.70ddd (2.5; 4.5) 3.68ddd (2.5; 4.5) 3.69ddd (2.5; 4.5)
H6a,b (J6a,6b
)
4.04dd,
4.20dd (12.5)
4.13dd,
4.28dd (12.5)
4.12dd,
4.26dd (12.5)
4.14dd,
4.27dd (12.5)
4.12dd,
4.26dd (12.5)
NAc, OAc
1.88s, 1.95s,
1.96s, 2.02s
1.95s, 2.02s,
2.03s, 2.09s
1.66s, 1.99s,
2.01s, 2.08s
1.83s, 2.02s (6 H),
2.09s
1.94s, 2.03s,
2.04s, 2.09s
NH (J2,NH
)
5.43d (8)
5.48d (8.5)
5.07d (8.5)
5.35d (8.5)
5.54d (8.5)
R
H1'
3.43m
–
3.53dt, 3.93dt
1.37dt
4.29dd, 4.42dd
4.02t
3.72dt, 4.18dt
2.93dt
3.97m
–
H2'
Alk/Ar
0.77s (9 H),
0.88–1.74m
1.48m, 1.65m,
1,92m
7.21m, 7.27m
7.27–7.46m,
7.52d, 7.56d
0.83s (9 H),
0.86–1.52m
* Working frequency was 300 MHz [400 MHz for (IIe)].
Abbreviations: dd, doublet of doublets; ddd, doublet of doublet of doublets; dt, doublet of triplets.
densed with the benzyl ester of L-alanyl-D-iso-
glutamine through the formation of the corresponding
N-succinimide esters. The acetal protection was
removed from the resulting glycopeptides by acidic
hydrolysis.
A low solubility of the lipoglycopeptides in water
and their ability for aggregation have a considerable
influence on the results of studies of their immunostim-
ulating activity using in vitro tests. Therefore, the in
vivo studies of the action of lipophylic muramyl dipep-
tides on the immune system is more correct. For exam-
ple, the test of stimulation of the antibacterial resistance
of mice after an intraperitoneal introduction of
S. aureus has been recommended [15].
1H NMR spectra were recorded for (VIa)–(VId),
and the resonances corresponding to aglycone, carbo-
hydrate fragment, and lactylpeptide component were
interpreted. These are similar to those for MDP itself
(see Table 2). The concluding catalytic hydrogenolysis
of the benzyl esters in the isoglutamine residue of gly-
copeptides (VIa)–(VId) resulted in the target MDP β-
glycosides (VIIa)–(VIId).
All the studied glycosides of muramyl dipeptide
exhibit the protective activity (Fig. 1). The opposite
effects of two MDP arylethyl glycosides (in the active
doses) on the stimulation of the antiinfection resistance
should be noted. The activity of β-2-(p-biphenyl)ethyl-
200 µg per mouse 20 µg per mouse 2 µg per mouse
100
80
60
40
20
0
Control (VII‡) (VIIb) (VIIc) (VIId) (VIIf) (VIIg)
Fig. 1. Protective effect of MDP glycosides (VIIa)–(VIIg) at doses of 2, 20, and 200 µg per mouse after an intraperitoneal injection
6
of mice with S. aureus (10 cells per mouse).
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SYNTHESIS AND PROTECTIVE ACTIVITY
579
1
Table 2. H NMR spectra of (VIa)–(VIe) measured in DMSO-d6; working frequency of 300 MHz
Chemical shifts, ppm (spin-coupling constants, Hz)
Group or atom
(VIa)
(VIb)
(VIc)
(VId)
β-MDP [14]
–
C1-OR
0.82s, 0.94m,
1.16m, 1.72m
1.18t, 1.45m,
1.62m, 1.89m
3.96t, 7.25m
2.82t, 7.30–7.47m,
7.55d, 7.63d
H1 (J1,2
HNAc
NHAc
C4-OH
C6-OH
)
4.40d (8)
1.75s
4.24d (8.5)
1.74s
4.39d (8.5)
1.57s
4.34d (8.5)
1.73s
4.41d
1.77s
7.93d
6.60d
4.24t
1.23d
1.24d
7.51d
7.74d
7.80d
7.69d
7.76d
5.22d
5.24d
5.27d
5.25d
4.53bt*
1.24d
4.59bt
4.61t
4.59bt
CH3CHCO
Ala: CH3
NH
1.23d
1.21d
1.24d
1.24d
1.23d
1.22d
1.24d
7.38d
7.38d
7.39d
7.39d
iGln: CO2CH2Ph
γ-CH2
5.08s, 7.36m
2.36t
5.08s, 7.36m
2.36t
5.08s, 7.36m
2.35t
5.08s, 7.36m
2.36t
–
2.20t
β-CH2
CONH2
1.77m, 1.97m
7.10s, 7.32s
8.10d
1.77m, 2.02m
7.11s, 7.31s
8.11d
1.75m, 2.02m
7.12s, 7.18s
8.11d
1.78m, 2.01m
7.09s, 7.30s
8.09d
1.69m, 1.94m
7.11s, 7.33s
8.10d
NH
* Abbreviation: bt, broadened triplet.
MDP (VIId) decreased with a decrease in the immuno- Mercury 400 (400 MHz) with Me4Si used as an internal
modulator dose, whereas that of β-2,2-diphenylethyl-
MDP (VIIc) dramatically increased. Previously, we
synthesized [16] β-n-(1,1,3,3-tetramethylbutyl)phenyl
MDP (VIIg), a glycopeptide with alkylphenyl aglycone
also containing 14 carbon atoms; in general, it proved
to be more active than glycosides (VIIc) and (VIId).
β-2-(Adamant-1-yl)ethyl MDP (VIIb) demon-
strated a moderate protective effect, whereas β-4-tert-
butylcyclohexyl MDP (VIIa) exhibited the lowest
immunostimulating activity among all the compounds.
Its protective effect was significantly lower than that of
the corresponding aromatic analogue, β-4-tert-
butylphenyl MDP (VIIf) [16].
A tendency to a growing protective effect with an
increase in the aglycone lipophylicity was observed in
the group of MDP glycosides at the lowest examined
dose for which the dose-dependent effects are possible
to the least degree (Fig. 2). The same biological activity
was observed for three compounds with similar agly-
cone lipophylicity: β-4-tert-butylcyclohexyl MDP
(VIIa), β-2-(p-biphenyl)ethyl MDP (VIId), and β-4-
tert-butylphenyl MDP., β-2,2-Diphenylethyl MDP
(VIIc) obviously falls out of this series, because it
exhibits the maximal effect at this dose.
standard. Chemical shifts (δ scale) and spin–spin cou-
pling constants (J, Hz) are given. The techniques of
double homonuclear resonance, the spectra of previ-
ously prepared compounds [2 7], and literature data
[14] were used for the assignment of resonances.
TLC was carried out on Sorbfil-AFB-UF plates
(Sorbpolimer, Russia). Substances were detected in
UV-light (254 nm) and by the action of 5% solution of
sulfuric acid in ethanol with the subsequent heating to
CLogP
6
5
(VIIa)
4
3
2
1
(VIIb)
(VIIc)
(VIId)
(VIIf)
(VIIg)
0
20
40
60
80
100
Survival, %
EXPERIMENTAL
Fig. 2. The dependence of protective effect of MDP glyco-
sides on the aglycone lipophylicity after the intraperitoneal
injection to mice of S. aureus at the dose of 2 µg/mouse.
CLogP is a calculated value of the logarithm of the distribu-
tion coefficient for the n-octanol–water system.
Melting points were determined on a PTP device.
Optical rotations were measured on a Polamat-A pola-
rimeter at 20–25°C at λ 546 nm. 1H NMR spectra were
recorded on a Varian VXR-300 (300 MHz) and Varian
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 6 2005

580
ZEMLYAKOV et al.
200–300°ë. The following chromatographic systems the (IIa)–(IIe) mixture by a repeated column chroma-
were used: (A) 15 : 1 chloroform–ethanol, (B) 5 : 1 tography with a benzene–ethanol gradient elution (75 :
chloroform–ethanol, (C) 10 : 1 benzene–ethanol, and 1 to 60 : 1); mp 159–162°ë, [α]546 –27° (Ò 1.0, chloro-
form); 1H NMR: Table 1.
(D) 3 : 1 : 1 N-butanol–acetic acid–water. Column chro-
matography was carried out on silica gel (Merck, 230–
400 mesh). The data of elemental analysis of key com-
pounds corresponded to the calculated values.
4-tert-Butylcyclohexanol and 2-(adamant-1-yl)eth-
anol were from Acros (Belgium). 2,2-Diphenylethanol
and 2-(p-biphenyl)ethanol were prepared by LiAlH4
reduction of diphenylacetic acid and p-biphenylacetic
acid (Acros), respectively. The constants of these alco-
hols corresponded to literature data.
2-(Adamant-1-yl)ethyl 2-acetamido-3,4,6-tri-O-
acetyl-2-deoxy-b-D-glucopyranoside (IIb) was pre-
pared from 2-(adamantyl-1)ethanol (0.66 g,
3.65 mmol) and oxazoline (I) (1.8 g, 5.47 mmol) and
isolated by column chromatography with a gradient
elution (from benzene to 20 : 1 benzene–ethanol mix-
ture); yield 1.5 g (81%). The analytical sample was
crystallized from ether; mp 123–125°ë, [α]546 –17° (Ò
1.0, chloroform); 1H NMR: Table 1.
The biological activity was studied by the procedure
[15]. White outbred mice (body mass 12–14 g, Kryu-
kovo Department of the Central Breeding Nursery of
Experimental Animals, Russia) at the age of 20–
25 days (groups of five animals) were used in experi-
ments. The examined preparations were intraperito-
neally introduced in 0.9% NaCl solution (final volume
was 0.5 ml) at doses of 200, 20, and 2 µg per mouse.
Mice of control group were intraperitoneally injected
with 0.9% NaCl (0.5 ml). The animals were infected
with a S. aureus culture (Wood 46 strain) after 24 h and
observed for six days. The efficiency of preparations
was estimated according to a number of animals sur-
vived. The number of microbial cells necessary for the
infection (109 cells) was determined in preliminary
experiments as a minimum dose causing 100% death of
animals at the intraperitoneal injection within the first
three days.
Glycosylation by the oxazoline method. An alco-
hol (at the alcohol–oxazoline molar ratio of 2 : 3) and
anhydrous TosOH (20–30 mg) were added to a solution
of 2-methyl-(3,4,6-tri-O-acetyl-1,2-dideoxy-α-D-glu-
copyranosido)-[2,1-d]-2-oxazoline (I) [17] in anhy-
drous dichloroethane [10 ml per g of (I)]. On the other
hand, a solution of oxazoline (I) in anhydrous dichloro-
ethane (the alcohol–glycosyl donor ratio of 3 : 2) was
added in portions to the 4-tert-butylcyclohexanol when
synthesizing (IIa). The reaction mixture was heated at
85–90°C (bath temperature) until the complete decom-
position of the oxazoline (TLC monitoring in systems
A and C), neutralized with pyridine (50 µl), and evapo-
rated. The residue was purified by column chromatog-
raphy or by crystallization.
2,2-Diphenylethyl
2-acetamido-3,4,6-tri-O-
acetyl-2-deoxy-b-D-glucopyranoside (IIc) was pre-
pared from 2,2-diphenylethanol (0.79 g, 4.0 mmol) and
oxazoline (I) (2.0 g, 6.08 mmol). The residue of (IIc)
was crystallized from ether and recrystallized from iso-
propyl alcohol; yield 1.43 g (68%); mp 176–178°C;
[α]546 –42° (Ò 1.0, chloroform); 1H NMR: Table 1.
2-p-Biphenylethyl
2-acetamido-3,4,6-tri-O-
acetyl-2-deoxy-b-D-glucopyranoside (IId) was pre-
pared from 2-p-biphenylethanol (0.67 g, 3.36 mmol)
and oxazoline (I) (1.66 g, 5.05 mmol). The residue of
(IId) was crystallized from ether and recrystallized
from isopropyl alcohol; yield 1.2 g (68%); mp 172–
1
174°C; [α]546 –15° (Ò 1.0, chloroform); H NMR:
Table 1.
Deacetylation by the Zemplen reaction. A 0.1 N
solution of sodium methylate (0.01–0.05 equiv) in
methanol was added to a solution of one of acetates
(IIa)–(IId) in anhydrous methanol (10 ml/g). The reac-
tion mixture was kept for 12–24 h, and the precipitated
crystals were filtered and washed with methanol. The
filtrate was neutralized with the KU-2 (H+-form) cation
exchanger; the resin was filtered off and washed with
methanol; and the filtrate was evaporated.
4-tert-Butylcyclohexyl 2-acetamido-2-deoxy-b-
D-glucopyranoside (IIIa) was prepared from acetate
(IIa) (0.7 g, 1.44 mmol); yield 0.5 g (98%); mp 177–
179°C; [α]546 –25° (Ò 1.0, ethanol).
2-(Adamant-1-yl)ethyl 2-acetamido-2-deoxy-b-
D-glucopyranoside (IIIb) was prepared from (IIb)
(1.45 g, 2.85 mmol); yield 1.0 g (93%); mp 157–180°C
(with decomposition); [α]546 –25° (Ò 1.0, ethanol).
4-tert-Butylcyclohexyl 2-acetamido-3,4,6-tri-O-
acetyl-2-deoxy-b-D-glucopyranoside trans- and cis-
isomers (IIa) and (IIe) were synthesized from
cis,trans-4-tert-butylcyclohexanol (0.86 g, 5.62 mmol)
and oxazoline (I) (1.23 g, 3.75 mmol). Pure glycoside
(IIa) in yield of 1.0 g (37%) and a fraction containing a
mixture of glycosides (IIa) and (IIe) were isolated by a
column chromatography with gradient elution from
pure benzene to 30 : 1 benzene–ethanol mixture. Ana-
lytical sample of (IIa) was crystallized from ether; mp
164–166°C, [α]546 –8° (Ò 1.0, chloroform); for 1H NMR,
2,2-Diphenylethyl
2-acetamido-2-deoxy-b-D-
glucopyranoside (IIIc) was prepared from acetate
(IIc) (1.4 g, 2.66 mmol); yield 1.0 g (95%); mp 174–
175°C (with decomposition); [α]546 –10° (Ò 1.0, etha-
nol).
2-p-Biphenylethyl
2-acetamido-2-deoxy-b-D-
glucopyranoside (IIId) was prepared from (IId)
(1.1 g, 2.09 mmol); yield 0.7 g (84%); mp 210–214°C
(with decomposition); [α]546 –21° (Ò 1.0, ethanol).
Preparation of isopropylidene derivatives. 2,2-
see Table 1. Glycoside (IIe) (50 mg) was isolated from Dimethoxypropane (3 equiv) and anhydrous TosOH
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 6 2005

SYNTHESIS AND PROTECTIVE ACTIVITY
581
(10 mg) were added to a solution of one of triols (IIIa)– copyranoside (Vb) was prepared by alkylation of ace-
(IIId) in anhydrous THF (50 ml/g). The reaction mix- tal (IVb) (0.58 g, 1.38 mmol) with (S)-2-brompropi-
ture was heated at 60°C for 1 h with the TLC monitor- onic acid; yield 0.6 g (90%).
ing in systems A and C, neutralized with pyridine, and
2,2-Diphenylethyl 2-acetamido-2-deoxy-4,6-O-
evaporated. The residue was dissolved in chloroform
isopropylidene-3-O-(D-1-carboxyethyl)-b-D-glu-
(50 ml), washed with water (3 × 25 ml), dried over
copyranoside (Vc) was prepared by alkylation of ace-
tal (IVc) (0.59 g, 1.35 mmol) with (S)-2-bromopropi-
onic acid; yield 0.57 g (84%).
2-p-Biphenylethyl 2-acetamido-2-deoxy-4,6-O-
isopropylidene-3-O-(D-1-carboxyethyl)-b-D-gluco-
pyranoside (Vd) was prepared by the alkylation of
acetal (IVd) (0.5 g, 1.14 mmol) with (S)-2-bromopro-
pionic acid; yield 0.51 g (88%).
Preparation of glycopeptides and hydrolysis of
isopropylidene groups. HOSu (1.1 equiv) and DCC
(1.1 equiv) were added to a solution of one of muramic
acids (Va)–(Vd) in anhydrous THF (10 ml/g) under
stirring. The reaction mixture was monitored by TLC in
systems A and B. After 3–5 h, the precipitated dicyclo-
hexylurea was filtered off and washed with the solvent.
L-Alanyl-D-isoglutamine benzyl ester trifluoroacetate
(1 equiv) [18] and triethylamine (to pH 8) were added
to the filtrate. After the reaction was completed (TLC
monitoring in systems A and B), the solvent was evap-
orated.
The alkylidene derivatives were dissolved in 70%
acetic acid (10 ml/g) under heating on a boiling water
bath and kept for 5–15 min at this temperature with the
TLC monitoring in systems A and B. The solution was
evaporated to dryness and coevaporated with toluene.
The residue was purified by the column chromatogra-
phy.
Na2SO4, and evaporated to dryness.
4-tert-Butylcyclohexyl 2-acetamido-2-deoxy-4,6-
O-isopropylidene-b-D-glucopyranoside (IVa) was
prepared from triol (IIIa) (0.50 g, 1.40 mmol). The
glass-like target (IVa) was isolated by column chroma-
tography with a gradient elution (chloroform to 20 : 1
chloroform–ethanol mixture); yield 0.5 g (91%); [α]546
–77° (Ò 1.0, chloroform).
2-Adamant-1-yl)ethyl 2-acetamido-2-deoxy-4,6-
O-isopropylidene-b-D-glucopyranoside (IVb) was
prepared from triol (IIIb) (0.6 g, 1.58 mmol) and puri-
fied by a column chromatography with a gradient elu-
tion (from chloroform to 20 : 1 chloroform–ethanol); a
glass-like product; yield 0.6 g (91%); [α]546 –56° (Ò
0.67, chloroform).
2,2-Diphenylethyl 2-acetamido-2-deoxy-4,6-O-
isopropylidene-b-D-glucopyranoside (IVc) was pre-
pared from triol (IIIc) (0.6 g, 1.51 mmol) and purified
by column chromatography (a gradient elution from
chloroform to 20 : 1 chloroform–ethanol); amorphous;
yield 0.6 g (91%); [α]546 –56° (Ò 1.0, chloroform).
2-p-Biphenylethyl 2-acetamido-2-deoxy-4,6-O-
isopropylidene-b-D-glucopyranoside (IVd) was pre-
pared from triol (IIId) (0.5 g, 1.26 mmol) and purified
by column chromatography with a gradient elution
(from chloroform to 20 : 1 chloroform–ethanol); amor-
phous; yield 0.5 g (91%); [α]546 –48° (Ò 1.0, chloro-
form).
Preparation of muramic acids. Sodium hydride
(4 equiv) was added in portions to a solution of one of
acetals (IVa)–(IVd) in anhydrous dioxane (20 ml/g)
under stirring. The reaction mixture was heated at
95°C, kept at this temperature for 1 h, and cooled to
65°C. (S)-2-Bromopropionic acid (1.5 equiv) was
added, and the mixture was kept at 65°C for 1 h. The
reaction mixture was cooled. The excess sodium
hydride was quenched with ethanol, and the mixture
was concentrated and poured into cool water. The solu-
tion was acidified with 1 N HCl to pH 2–3, and
muramic acid was extracted with chloroform. The
extract was dried with Na2SO4 and evaporated. The
acids were used for coupling with the dipeptide without
an additional purification. The following compounds
were synthesized by this procedure:
4-tert-Butylcyclohexyl 2-acetamido-2-deoxy-4,6-
O-isopropylidene-3-O-(D-1-carboxyethyl)-b-D-glu-
copyranoside (Va) was prepared by the alkylation of
acetal (IVa) (500 mg, 1.26 mmol) with (S)-2-bro-
mopropionic acid; yield 510 mg (86%).
2-(Adamant-1-yl)ethyl 2-acetamido-2-deoxy-4,6- 1.12 mmol) and the subsequent hydrolysis and column
O-isopropylidene-3-O-(D-1-carboxyethyl)-b-D-glu- chromatography with a gradient elution (from chloro-
O-[(4-tert-Butylcyclohexyl
2-acetamido-2,3-
dideoxy-b-D-glucopyranos-3-yl] D-lactoyl-L-ala-
nyl-D-isoglutamine benzyl ester (VIa) was prepared
by coupling of the dipeptide with muramic acid (Va)
(0.51 g, 1.09 mmol), the subsequent hydrolysis, and
purification by column chromatography with a gradient
elution (from chloroform to 10 : 1 chloroform–etha-
nol); yield 310 mg (40%); amorphous powder; [α]546
−2° (Ò 1.0, ethanol); 1H NMR: Table 2.
O-[2-Adamant-1-ylethyl
2-acetamido-2,3-
dideoxy-b-D-glucopyranos-3-yl]-D-lactoyl-L-alanyl-
D-isoglutamine benzyl ester (VIb) was prepared by
coupling of the dipeptide with muramic acid (Vb)
(0.6 g, 1.22 mmol) and the subsequent hydrolysis. The
purification by the column chromatography with a gra-
dient elution (from chloroform to 10 : 1 chloroform–
ethanol) yielded 360 mg (40%) of (VIb) as amorphous
powder; [α]546 +12° (Ò 0.67, ethanol); 1H NMR:
Table 2.
O-[2,2-Diphenylethyl 2-acetamido-2,3-dideoxy-
b-D-glucopyranos-3-yl]-D-lactoyl-L-alanyl-D-isoglu-
tamine benzyl ester (VIc) was prepared by coupling of
the dipeptide with muramic acid (Vc) (570 mg,
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 6 2005

582
ZEMLYAKOV et al.
form to 10 : 1 chloroform–ethanol); yield 385 mg
REFERENCES
(45%); amorphous powder; [α]546 +3° (c 0.67, ethanol);
1. Karaulov, A.V., Kalyuzhin, O.V., and Zemlyakov, A.E.,
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2. Zemlyakov, A.E., Tsikalov, V.V., Kalyuzhin, O.V.,
Kur’yanov, V.O., and Chirva, V.Ya., Bioorg. Khim.,
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O-[2-p-Biphenylethyl 2-acetamido-2,3-dideoxy-
b-D-glucopyranos-3-yl]-D-lactoyl-L-alanyl-D-isoglu-
tamine benzyl ester (VId) was prepared by coupling
of the dipeptide with muramic acid (Vd) (500 mg,
0.98 mmol), the subsequent hydrolysis, and column
chromatography with a gradient elution (from chloro-
form to 10 : 1 chloroform–ethanol); yield 305 mg
(41%); mp 172–175°C (with decomposition); [α]546
+9° (Ò 0.67, ethanol); 1H NMR: Table 2.
5. Zemlyakov, A.E. and Chirva, V.Ya., Khim. Prir. Soedin.,
1987, no. 5, pp. 714–718.
Hydrogenolysis of the benzyl protective groups.
Benzyl esters (VIa)–(VId) were dissolved in a 9 : 1
THF–water mixture (50 ml/g) and hydrogenolyzed
over 10% Pd/C an the ratio of 1 : 5 (to substance) at
room temperature for 3–6 h. The reaction was moni-
tored by TLC in system D. When the reaction com-
pleted, the catalyst was filtered off and washed with sol-
vent. The filtrate was evaporated.
6. Kur’yanov, V.O., Zemlyakov, A.E., and Chirva, V.Ya.,
Bioorg. Khim., 1994, vol. 20, pp. 439–447.
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2-acetamido-2,3-
19825k.
dideoxy-b-D-glucopyranos-3-yl)-D-lactoyl-L-alanyl-
D-isoglutamine (VIIa) was prepared as amorphous
powder from benzyl ester (VIa) (290 mg, 0.40 mmol);
yield 240 mg (96%); [α]546 +2° (Ò 0.83, ethanol).
11. Kusumoto, S., Inage, M., Shiba, T., Azuma, I., and
Yamamura, Y., Tetrahedron Lett., 1978, no. 49,
pp. 4899–4902.
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O-[2-(Adamant-1-yl)ethyl
2-acetamido-2,3-
13. Flowers, H.M. and Jeanloz, R.W., J. Org. Chem., 1963,
dideoxy-b-D-glucopyranos-3-yl]-D-lactoyl-L-alanyl-
D-isoglutamine (VIIb) was prepared as amorphous
powder from benzyl ester (VIb) (260 mg, 0.35 mmol);
yield 220 mg (96%); [α]546 +2° (Ò 0.83, ethanol).
vol. 28, pp. 2983–2986.
14. Fermand-Jian, S., Perly, B., Level, M., and Lefran-
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O-(2,2-Diphenylethyl 2-acetamido-2,3-dideoxy-
b-D-glucopyranos-3-yl]-D-lactoyl-L-alanyl-D-isoglu-
tamine (VIIc) was prepared as amorphous powder
from benzyl ester (VIc) (260 mg, 0.34 mmol); yield
220 mg (96%); [α]546 +4° (Ò 1.0, ethanol).
O-(2-p-Biphenylethyl 2-acetamido-2,3-dideoxy-
b-D-glucopyranos-3-yl)-D-lactoyl-L-alanyl-D-isoglu-
tamine (VIId) was prepared as amorphous powder
from benzyl ester (VId) (280 mg, 0.37 mmol); yield
240 mg (97%); [α]546 +6° (Ò 1.0, ethanol).
17. Lemieux, R.U. and Driguez, H., J. Am. Chem. Soc.,
1975, vol. 52, pp. 4063–4068.
18. Rostovtseva, L.I., Andronova, T.M., Mal’kova, V.P.,
Sorokina, I.B., and Ivanov, V.T., Bioorg. Khim., 1981,
vol. 7, pp. 1843–1858.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 31 No. 6 2005