Synthesis and protective anti-infective action of anomeric lipophilic glycosides of N-acetylmuramyl-L-alanyl-D-isoglutamine

Article abstract of DOI:10.1134/S1068162006040091

Anomeric pairs of α-and β-dodecyl, α-and β-(1-pentylhexyl), and α-and β-cyclododecyl glycosides of N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) were synthesized. The starting β-D-glucosaminides were obtained by the oxazoline method, and the corresponding α-isomers, by the mercuric iodide-catalyzed glycosylation of alcohols with α-glucosaminyl chloride peracetate in nitromethane at ～90°C. No reliable differences between the stimulation of mouse resistance to the infection with Staphylococcus aureus (doses of 2, 20, and 200 μg/mouse) and Escherichia coli (doses of 0.05, 1, and 20 μg/mouse) with the MDP α-and β-glycosides were found. Pleiades Publishing, Inc., 2006.

Full text of DOI:10.1134/S1068162006040091

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2006, Vol. 32, No. 4, pp. 382–388. © Pleiades Publishing, Inc., 2006.
Original Russian Text © A.E. Zemlyakov, V.N. Tsikalova, V.V. Tsikalov, V.Ya. Chirva, E.L. Mulik, O.V. Kalyuzhin, 2006, published in Bioorganicheskaya Khimiya, 2006, Vol. 32,
No. 4, pp. 424–431.
Synthesis and Protective Anti-Infective Action
of Anomeric Lipophilic Glycosides
of N-Acetylmuramyl-L-Alanyl-D-Isoglutamine
A. E. Zemlyakova, 1, V. N. Tsikalovaa, V. V. Tsikalova, V. Ya. Chirvaa,
E. L. Mulikb, and O. V. Kalyuzhinb
a Vernadsky Tauric National University, pr. akademika Vernadskogo 4, Simferopol, Crimea, 95007 Ukraine
b Research Institute of Human Morphology, Russian Academy of Medical Sciences, ul. Tsuryupy 3, Moscow, 117418 Russia
Received June 20, 2005; in final form, September 3, 2005
Abstract—Anomeric pairs of α- and β-dodecyl, α- and β-(1-pentylhexyl), and α- and β-cyclododecyl glyco-
sides of N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP) were synthesized. The starting β-D-glucosaminides
were obtained by the oxazoline method, and the corresponding α-isomers, by the mercuric iodide–catalyzed
glycosylation of alcohols with α-glucosaminyl chloride peracetate in nitromethane at ~90°C. No reliable dif-
ferences between the stimulation of mouse resistance to the infection with Staphylococcus aureus (doses of 2,
20, and 200 µg/mouse) and Escherichia coli (doses of 0.05, 1, and 20 µg/mouse) with the MDP α- and β-gly-
cosides were found.
DOI: 10.1134/S1068162006040091
Key words: anti-infective resistance, glycopeptides, muramyldipeptide, muramyldipeptide glycosides
INTRODUCTION
ric secondary alcohols and cycloaliphatic alcohols as
aglycones, in order to determine how the configuration
of anomeric center and the structure and nature of agly-
cones of MDP glycosides affect their immunostimulat-
ing activity. On the one hand, these aglycons have sim-
ilar lipophilicities (ClogP*);3 they are 4.95, 4.20, and
4.52 for the corresponding alcohols. On the other hand,
the conformational mobilities of hydrocarbon chains
and the effective surface in this series of aglycons
decrease, which undoubtedly affects the interaction
with biomembranes. In particular, a relatively rigid
double-stranded structure could form due to hydropho-
bic interactions for the aglycones of glycopeptides
(IXc)–(IXd) in aqueous solutions.
Comparison of the immunostimulating action of
anomeric pairs of amphiphilic N-acetylmuramyl-L-ala-
nyl-D-isoglutamine (muramyldipeptide) glycosides,2
namely, α- and β-butyl- [1], α- and β-heptyl-, and
α-and β-cyclohexyl muramyldipeptides [2, 3], showed
that α-glycosides both in vitro and in vivo are less
active than the corresponding muramyldipeptide β-gly-
cosides and even MDP itself. The β-anomers of MDP
glycosides exhibit a more intensive adjuvant action
than the corresponding α-glycosides also in the case of
methyl [4] and benzyl [5] glycosides of MDP. The
effect of glycoside center configuration on the biologi-
cal activity was not studied for the predominantly lipo-
philic MDP glycosides, although it is known that
changes in lipophilicity can cardinally alter the mecha-
nism of glycopeptide penetration through the macro-
phage membranes. In particular, the processes of
lipoglycopeptide incorporation into lipid membrane
and exit from it change.
The
starting
peracetylated
β-N-acetylglu-
cosaminides (IIIa), (IIIc), and (IIIe) were synthesized
by the glycosylation of alcohols with an excess oxazo-
line (I) [6] in dichloroethane at ~90°ë in the presence
of catalytic amounts of TosOH. The corresponding
α-anomers (IIIb), (IIId), and (IIIf) were synthesized
by the reaction of the alcohols with α-glucosaminyl
chloride (II) in nitromethane at ~90°ë in the presence
of mercuric iodide [7].
RESULTS AND DISCUSSION
We synthesized α- and β-dodecyl-, α- and β-(1-pentyl-
hexyl)-, and α- and β-cyclododecyl glycosides of MDP
(IXa)–(IXf) from aliphatic linear primary and symmet-
The structures of glycosides (IIIa)–(IIIf) were con-
1
firmed by H NMR spectra, in which all protons were
completely assigned (see Table 1). Methylene protons
1
in the aglycones of (IIIa)–(IIIf) resonate in the region
Corresponding author: phone: (065) 223-3885; fax: (065) 223-
2310; e-mail: alex_z@crimea.edu
Abbreviations: HONSu, N-hydroxysuccinimide; MDP, muramyl-
dipeptide, N-acetylmuramyl-L-alanyl-D-isoglutamine.
2
3
The calculated value of the logarithm of the n-octanol/water dis-
tribution coefficient.
382

SYNTHESIS AND PROTECTIVE ANTI-INFECTIVE ACTION
383
OAc
N
AcO
AcO
O
a
Me
O
OAc
OH
Me
O
O
AcO
AcO
HO
HO
O
O
O
c
d
e
Me
(I)
HO
R
R
R
O
AcNH
AcNH
R'
(III)
AcNH
R'
(IV)
R'
OAc
b
(V)
AcO
AcO
O
a. R =
AcNH
Cl
(II)
R' = H
b. R = H
R' =
O
Me
OH
Me
HO
O
O
O
O
O
R
g
c. R = O
H
Me
O
R
H
AcNH
R'
R' = H
AcNH
Me
COR''
R'
L-Ala-D-Glu(NH2)OR'' d. R = H
(VI) R'' = OH
(VII) R'' = L-Ala-D-Glu(NH2)OBzl
(VIII) R'' = Bzl
(IX) R'' = H
f
h
R' =
O
O
e. R =
R' = H
f. R = H
a. ROH, TsOH; b. ROH, HgI2; c. MeOH, MeONa; d. Me2C(OMe)2,
TsOH; e. NaH, (S)-MeCHBrCOOH, 65–95°C; f. HOSu, DCC,
L-Ala-D-Glu(NH2)OBzl; g. AcOH, H2O, 100°C; h. H2, Pd/C
R' =
O
of 1.19–1.63 ppm. High-field signals of the protons of (δ 4.25–4.27 ppm as compared with 3.64–3.80-ppm
shift for the corresponding β-anomers) and the amide
proton at δ 5.56–5.59 vs. 5.45–5.50 ppm, respectively.
terminal methyl groups (δ from 0.80 to 0.88 ppm) are
represented by a triplet for each of (IIIa) and (IIIb) and
by two triplets for glycosides (IIIc) and (IIId). The sig-
nals of the methyne proton coupled with oxygen atom
at C1 are shifted to a low field: δ 3.48 and 3.52 ppm for
(IIIc) and (IIId) and 3.71 ppm for (IIIe) and (IIIf).
Nonequivalent protons of the α-methylene group of the
aglycone of glycosides (IIIa) and (IIIb) are repre-
sented by two doublets of triplets with δ 3.44–3.46 and
3.62–3.86 ppm.
The scheme of further synthesis (see the scheme)
involved the successive deacetylation of peracetates
(IIIa)–(IIIf) by Zemplen and isopropylidenation of tri-
ols (IVa)–(IVf) with 2,2-dimethoxypropane. Free
hydroxyl groups at C3 in (Va)–(Vf) were treated with
sodium hydride in dioxane and alkylated with L-2-bro-
mopropionic acid as described in [8] to give N-acetyl-
D-muramic acid derivatives (VIa)–(VIf). The muramic
acids were activated with HONSu and DCC and then
coupled with L-alanyl-D-isoglutamine benzyl ester.
The isopropylidene protection was removed from gly-
copeptides (VIIa)–(VIIf) by acidic hydrolysis.
The β-configuration of D-glucosaminides (IIIa),
(IIIc), and (IIIe) follows from the doublet of anomeric
proton at 4.68–4.77 ppm with a spin coupling constant
of 8.5 Hz characteristic of 1,2-trans-glycoside bond.
The signals of anomeric protons in the case of α-D-glu-
cosaminides (IIIb), (IIId), and (IIIf) are shifted to low
field (δ 4.75–4.87 ppm) and have spin coupling con-
stants of 3.5 Hz characteristic of 1,2-cis-glycosides. In
addition, the spectra of the latter compounds exhibit a
The benzyl groups were removed from the iso-
glutamine residues of glycopeptides (VIIIa)–(VIIIf)
by catalytic hydrogenolysis to give the target MDP gly-
cosides (IXa)−(IXf). The structures of (VIIIa)–(VIIIf)
1
and (IXa)–(IXf) were confirmed by their H NMR
typical downfield shift of the proton signal at C2 spectra, in which the signals of protons belonging to the
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 4 2006

384
ZEMLYAKOV et al.
1
Table 1. H NMR spectra of glycosides (IIIa)–(IIIf) in C2HCl3
Chemical shift, ppm (spin coupling constant, Hz)
Group or atom
(IIIa)
(IIIb)
(IIIc)
(IIId)
(IIIe)
(IIIf)
H1 (J1, 2
H2 (J2, 3
H3 (J3, 4
H4 (J4, 5
)
4.69 d (8.5)
4.75 d (3.5)
4.68 d (8.5)
4.87 d (3.5)
4.77 d (8.5)
3.71 m (10.5)
5.37 dd (9.5)
5.02 dd (9.5)
4.87 d (3.5)
)
3.80 ddd (10.5) 4.27 ddd (10)
3.64 ddd (10.5) 4.25 ddd (10)
4.25 ddd (10.5)
5.13 dd (9.5)
5.03 dd (9.5)
)
5.32 dd (9.5)
5.07 dd (9.5)
5.15 dd (9.5)
5.05 dd (9.5)
5.29 dd (9.5)
4.96 dd (9.5)
5.13 dd (9.5)
5.03 dd (9.5)
)
H5 (J5, 6a; J5, 6b) 3.70 ddd (2; 5) 3.87 ddd (2; 4.5) 3.61 ddd (2.5; 5) 3.95 ddd (2; 4.5) 3.71 ddd (2; 5) 3.98 ddd (2; 5)
H6a, b (J6a, 6b
)
4.13 dd, 4.27 dd 4.02 dd, 4.17 dd 4.05 dd, 4.14 dd 3.99 dd, 4.16 dd 4.12 dd, 4.22 dd 4.00 dd, 4.17 dd
(12.5)
(12.5)
(12)
(12.5)
(12.5)
(12.5)
NAc, OAc
1.95 s, 2.02 s,
2.03 s, 2.09 s
1.88 s, 1.95 s,
1.96 s, 2.03 s
1.86 s, 1.96 s
(6H), 2.00 s
1.86 s, 1.95 s,
1.96 s, 2.02 s
1.94 s, 2.03 s
(6H), 2.08 s
1.87 s, 1.95 s,
1.96 s, 2.02 s
NH (J2, NH
C1-OCH
CH3CH2
(CH2)n
)
5.50 d (8.5)
5.59 d (9)
5.45 d (8.5)
5.59 d (9)
5.50 d (8)
3.71 m
5.56 d (9.5)
3.71 m
3.46 dt, 3.86 dt 3.44 dt, 3.62 dt 3.48 quintet
0.88 t 0.81 t 0.80 t, 0.82 t
3.52 quinte
0.82 t, 0.83 t
1.25 m, 1.56 m 1.20 m, 1.54 m 1.19 m, 1.36 m, 1.20 m, 1.38 m, 1.32 m, 1.44 m, 1.27 m, 1.48 m,
1.46 m 1.46 m 1.63 m 1.61 m
main moieties of the molecules: aglycone, glycoside derivatives and then a lethal dose of Gram-negative
residue, and lactyldipeptide component were assigned bacteria are administered to the animal body, the tested
(see Table 2). The signals of protons of benzyl protect- compounds begin to act, depending on dose, in syner-
ing groups were absent from the spectra of deblocked gism/antagonism with various bacterial products and
glycopeptides (IXa)–(IXf).
components, e.g., lipopolysaccharide. We plan further
studies to unambiguously find a relationship between
the configuration of the anomeric center of lipoglyco-
peptides and their biological action.
It is correct to assess the action of lipophilic
muramyldipeptides on the immune system in vivo
rather than using in vitro experiments. We used the test
involving the stimulation of antibacterial resistance of
mice after the intraperitoneal injection of our prepara-
tions [9]. This permits one to ignore the problem of sol-
ubility of lipoglycopeptides in aqueous solutions and
possible aggregation. The study of stimulation of the
mouse resistance to the infection with Staphylococcus
aureus (Fig. 1a) showed that there is no reliable differ-
ence in the immunostimulating action between α- and
β-dodecyl-MDPs (IXa) and (IXb) and between α- and
β-cyclododecyl-MDPs (IXe) and (IXf). Moreover,
unlike the MDP glycosides with alkylalicyclic, alkyl-
aryl, cycloalkyl, and arylethyl aglycones described in
the earlier communication [10], all of these glycopep-
tides demonstrated 100% protective effect. Presum-
ably, the influence of the aglycone lipophilicity on the
protective action dominates over the effect of configu-
ration of its glycoside center.
EXPERIMENTAL
Melting points were measured on a PTP device, and
optical rotation was registered at 20–25°C on a Pola-
mat-A polarimeter at λ 546 nm. 1H NMR spectra were
recorded on a Varian VXR-300 spectrometer
(300 MHz) with Me4Si as internal standard. Chemical
shifts in δ-scale (ppm) and spin coupling constants (J,
Hz) are presented. Signals were assigned using the
methods of double homonuclear resonance, the spectra
of compounds obtained previously (including those
reported in [3, 7, 10]), and the published data [11].
TLC was carried out on Sorbfil-AFV-UV plates
(Sorbpolimer, Russia). Substances were detected using
a 5% sulfuric acid solution in ethanol followed by heat-
The protective effect of glycosides (IXa)–(IXf) ing at 200–300°ë. The following solvent systems were
against the infection of mice with E. coli was studied used: (A) 10 : 1 benzene–isopropanol, (B) 19 : 1 diethyl
using lower doses and a greater dilution step (Fig. 1b). ether–isopropanol, (C) 10 : 1 chloroform–isopropanol,
However, the testing did not reveal any distinct influ- (D) 5 : 1 chloroform–isopropanol, and (E) 3 : 1 chloro-
ence of either the configuration of the glycoside center form–isopropanol. Column chromatography was car-
or the administration of lipophilic aglycones on the pro- ried out on silica gel 60 (63–200 µm; Merck, Germany)
tective anti-infectious action of lipophilic MDP glyco- columns [(A) 1.8 × 12 cm and (B) 1.2 × 12 cm]. The
sides. Presumably, this is related to the fact that, under data of elemental analysis of key compounds corre-
the conditions of the in vivo model when the MDP spond to the calculated values.
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SYNTHESIS AND PROTECTIVE ANTI-INFECTIVE ACTION
385
200 µg/mouse
20 µg/mouse
2 µg/mouse
(a)
100
80
60
40
20
0
Control MDP
(a)
(b)
(e)
(f)
(b)
20 µg/mouse
1 µg/mouse
0.05 µg/mouse
60
40
20
0
Control MDP
(a)
(b)
(c)
(d)
(e)
(f)
9
Fig. 1. Effect of MDP glycosides on the protection against intraperitoneal infection of mice with (a) St. aureus (10 cells/mouse)
7
and (b) E. coli (2 × 10 cells/mouse). Doses are given in the figures.
Dodecanol-1 (Novocherkassk, Russia), undecanol-6 (1.87 g, 5.87 mmol) [6] in dry dichloroethane (15 ml).
(Lancaster), and cyclododecanol (Acros) were used.
The reaction was carried out at 85–90°C (bath temper-
ature) until the complete decomposition of oxazoline
under the monitoring with TLC in systems A and B.
The reaction mixture was neutralized by pyridine
(50 µl), and dichloroethane (15 ml) was added.
The organic layer was washed with water (10 ml), and
the extract was dried with anhydrous Na2SO4 and evap-
orated. The separation of the residue by column chro-
matography (column A, stepwise gradient of the eluent
benzene–isopropanol: 100 : 1, 75 : 1, and 50 : 1; volume
of a step 100 ml) and subsequent crystallization of the
product from diethyl ether yielded 1.60 g (82%) of gly-
coside (IIIa); mp 103–105°ë, [α]546 –15° (c 1.0; chlo-
The biological activity was examined as described
in [9]. White nonbred mice of 12–14-g body mass
(Central Nursery of Experimental Animals, branch
Kryukovo, Russia) of 20–25-day-old (five animals in
each group) were used.
A test preparation dissolved in 0.9% NaCl was
injected intraperitoneally (final volume of 0.5 ml) at
doses of 200, 20, and 2 µg per mouse. Mice of the con-
trol group were injected intraperitoneally with 0.5 ml of
0.9% NaCl. After 24 h, the animals were infected either
with a culture of S. aureus (Wood 46 strain) or an 18-h
culture of E. coli (strain 264). The culture was adminis-
tered in 0.2% agar to enhance the virulent properties of
the microorganisms. The animals were under observa-
tion for 6 days. The efficiency of the preparation was
estimated from the number of survived animals (in per-
cent of animals taken into experiment).
The amounts of microbial bodies necessary for
infection (109 for St. aureus and 2 × 107 for E. coli)
were determined in preliminary experiments; they were
the minimal doses inducing, upon intraperitoneal injec-
tion, 100% death of animals for the first three days.
Dodecyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-
b-D-glucopyranoside (IIIa). Dodecanol-1 (0.70 g,
3.79 mmol) and anhydrous TosOH (25 mg) were added
to a solution of 2-methyl-(3,4,6-tri-O-acetyl-1,2-
dideoxy-α-D-glucopyrano)-[2,1-d]-2-oxazoline
1
roform); for H NMR, see Table 1. Similarly, the fol-
lowing compounds were synthesized: (1-pentylhexyl)
2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopy-
ranoside (IIIc), yield 0.61 g (48%); mp 178–182°ë,
[α]546 –10° (c 1.0; chloroform); for 1H NMR, see Table 1;
and cyclododecyl 2-acetamido-3,4,6-tri-O-acetyl-2-
deoxy-β-D-glucopyranoside (IIIe): yield 0.70 g (45%);
mp 160–162°ë, [α]546 –21° (c 1.0; chloroform); for 1H
NMR, see Table 1.
Dodecyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-
a-D-glucopyranoside (IIIb). Mercuric iodide (2.88 g,
6.34 mmol) and dodecanol-1 (1.0 g, 5.37 mmol) were
added to a solution of 2-acetamido-3,4,6-tri-O-acetyl-
2-deoxy-α-D-glucopyranosyl chloride (II) [12] (2.0 g,
(I) 5.47 mmol) in dry nitromethane (30 ml). The reaction
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SYNTHESIS AND PROTECTIVE ANTI-INFECTIVE ACTION
387
mixture was stirred in the presence of molecular sieves (88%); [α]546 +96° (c 1.0, chloroform); (1-pentylhexyl)
3 Å at 80–90°ë until the glycosyl donor disappeared 2-acetamido-2-deoxy-4,6-O-isopropylidene-β-D-glu-
(monitoring by TLC in systems Ä and B) and then for copyranoside (Vc); yield 0.55 g (86%); [α]546 –62°
additional 30 min (total time 1.5 h). Molecular sieves (Ò 0.67, chloroform); (1-pentylhexyl) 2-acetamido-2-
and the catalyst were filtered off, and the filtrate was deoxy-4,6-O-isopropylidene-α-D-glucopyranoside (Vd);
evaporated. The residue was dissolved in chloroform 0.5 g (81%); [α]546 +93° (Ò 0.67, chloroform);
(50 ml) and washed with 10% potassium iodide solu- cyclododecyl 2-acetamido-2-deoxy-4,6-O-isopropyl-
tion (20 ml) and water. The organic layer was dried with idene-β-D-glucopyranoside (Ve); yield 0.36 g (67%);
anhydrous Na2SO4 and evaporated. The residue was [α]546 –85° (c 1.0, chloroform); and cyclododecyl
purified by chromatography on column A as described 2-acetamido-2-deoxy-4,6-O-isopropylidene-α-D-glucopy-
for (IIIa) to give 1.2 g (43%) of glycoside (IIIb); an ranoside (Vf); yield 0.45 g (85%); [α]546 +110° (Ò 1.0,
amorphous powder; [α]546 +138° (c 1.0; chloroform); chloroform).
1
for H NMR, see Table 1. Similarly, the following
Benzyl ester of O-(dodecyl 2-acetamido-2,3-
compounds were synthesized: (1-pentylhexyl) 2-acet-
dideoxy-b-D-glucopyranoside-3-yl)-D-lactoyl-L-alan-
amido-3,4,6-tri-O-acetyl-2-deoxy-α-D-glucopyrano-
side (IIId); yield 0.79 g (38%); mp 63°ë, [α]546 +94°
yl-D-isoglutamine (VIIIa). Sodium hydride (4 equiv)
was portionwise added to a stirred suspension of (Va)
(0.26 g, 0.61 mmol) in dry dioxane (10 ml). The reac-
tion mixture was heated to 95°ë and kept at this tem-
perature for 1 h. After cooling to 65°ë, (S)-2-bro-
mopropionic acid (0.075 ml, 0.9 mmol) was added, and
the mixture was kept at 65°C for additional 3 h. After
1
(c 1.0, chloroform); for H NMR, see Table 1, and
cyclododecyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-
α-D-glucopyranoside (IIIf); yield 0.95 g (34%); mp
1
108–110°ë, [α]546 +118° (c 0.67, chloroform); for H
NMR, see Table 1.
Dodecyl 2-acetamido-2-deoxy-b-D-glucopyrano- cooling, the sodium hydride excess was decomposed
side (IVa). A 0.1 N sodium methylate solution (1 ml) in by ethanol, and the mixture was concentrated and
methanol was added to a solution of (IIIa) (1.55 g, poured into cold water (30 ml). The solution was acid-
3.01 mmol) in dry methanol (50 ml), and the mixture ified with 1 N HCl to pH 3–4, and muramic acid (VIa)
was kept for 16 h. The precipitate was filtered off and was extracted with chloroform (3 × 20 ml). The extract
washed with cold methanol. The mother liquor was was dried with anhydrous Na2SO4 and evaporated. The
neutralized by cation exchanger KU-2 (ç+); the resin product (VIa) (230 mg, 75%) was dissolved without
was washed with methanol; and the filtrate was evapo- additional purification in dry THF (10 ml), and HONSu
rated. Total yield of (IVa) was 1.1 g (94%); mp 165– (58 mg, 0.50 mmol) and DCC (105 mg, 0.50 mmol)
167°ë, [α]546 –29° (c 1.0; ethanol). Similarly, the fol- were added under stirring. After 5 h, the dicyclohexyl-
lowing compounds were synthesized: dodecyl 2-acet- urea residue was filtered off and washed with the sol-
amido-2-deoxy-α-D-glucopyranoside (IVb); yield vent. L-Alanyl-D-isoglutamine benzyl ester trifluoro-
0.75 g (95%); mp 144–145°ë, [α]546 +117° (c 1.0; ethan- acetate [13] [obtained by the treatment of the corre-
ol); (1-pentylhexyl) 2-acetamido-2-deoxy-β-D-glu- sponding Boc-derivative (205 mg, 0.50 mmol) with
copyranoside (IVc); yield 0.6 g (95%); mp 178–182°ë, TFA followed by evaporation to dryness] and triethyl-
[α]546 –21° (c 1.0; ethanol); (1-pentylhexyl) 2-acet- amine were added to the filtrate to pH 8. After the reac-
amido-α-D-glucopyranoside (IVd); yield 0.58 g tion was over (monitored by TLC in system C), the
(98%); mp 163–168°ë, [α]546 +160° (c 1.0; ethanol); reaction mixture was evaporated. The residue was dis-
cyclododecyl 2-acetamido-2-deoxy-β-D-glucopyrano- solved in chloroform (50 ml), and the solution was
side (IVe); yield 0.5 g (96%); mp 204–207°ë, [α]546 washed with 1 N HCl, a saturated NaHCO3 solution,
−15° (c 1.0, ethanol); and cyclododecyl 2-acetamido-2- and water (15 ml each). The organic layer was dried
deoxy-α-D-glucopyranoside (IVf); yield 0.5 g (89%); with anhydrous Na2SO4 and evaporated. The resulting
mp 207–212°ë; [α]546 +158° (c 1.0, ethanol).
glycopeptide (VIIa) was dissolved in 70% acetic acid
(10 ml) upon heating on a boiling water bath and kept
at this temperature for 15 min (monitoring by TLC in
systems C and D). The solution was evaporated to dry-
ness, and the residue was coevaporated with toluene.
The residue was purified by chromatography on col-
umn B (stepwise gradient of chloroform–isopropanol
50 : 1, 25 : 1, 10 : 1, and 5 : 1; volume of a step 75 ml)
and crystallized by adding ether. Yield of glycopeptide
(VIIIa) 160 mg [35% from (Va)]; mp 174–180°ë
Dodecyl 2-acetamido-2-deoxy-4,6-O-isopropyl-
idene-b-D-glucopyranoside (Va). 2,2-Dimethoxypro-
pane (1.0 ml) and TosOH (10 mg) were added to a sus-
pension of (IVa) (0.5 g, 1.29 mmol) in dry THF (20 ml)
under stirring and heating at 50–55°ë. After 1 h (mon-
itoring by TLC in system A), the reaction mixture was
cooled, neutralized with pyridine, and evaporated. The
residue was purified by column chromatography (col-
umn B, stepwise gradient of isopropanol in benzene 1 :
50, 1 : 25, and 1 : 10 (a step volume of 75 ml). Crystal-
lization of the product from ether yielded 0.32 g (58%)
of acetal (Va) as a glassy solid; [α]546 –71° (c 1.0, chlo-
1
(dec.); [α]546 –22° (Ò 0.67, THF); for H NMR, see
Table 2.
Similarly, the following compounds were obtained:
roform). Similarly, the following compounds were benzyl ester of O-(dodecyl 2-acetamido-2,3-dideoxy-
obtained: dodecyl 2-acetamido-2-deoxy-4,6-O-isopro- α-D-glucopyranosid-3-yl)-D-lactoyl-L-alanyl-D-iso-
pylidene-α-D-glucopyranoside (Vb); yield 0.5 g glutamine (VIIIb); yield 400 mg (44%); mp 178–
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 4 2006

388
ZEMLYAKOV et al.
183°ë, [α]546 +77° (Ò 1.0, ethanol); for 1H NMR, see copyranosid-3-yl)-D-lactoyl-L-alanyl-D-isoglutamine
Table 2; benzyl ester of O-[(1-pentylhexyl) 2-acet- (IXf); yield 200 mg (91%); amorphous powder, [α]546
amido-2,3-dideoxy-β-D-glucopyranosid-3-yl]-D-lac- +93° (Ò 1.0, ethanol).
toyl-L-alanyl-D-isoglutamine (VIIIc); yield 340 mg
(35%); mp 210–213°ë, [α]546 –12° (Ò 0.67, ethanol);
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1H NMR: Table 2; benzyl ester of O-[(1-pentylhexyl)
2-acetamido-2,3-dideoxy-α-D-glucopyranosid-3-yl)-D-
lactoyl-L-alanyl-D-isoglutamine (VIIId); yield 360 mg
(32%); mp 186°ë, [α]546 +19° (Ò 0.33, ethanol–chlo-
1. Krivorutchenko,Yu.L., Andronovskaja, I.B., Hinkula, J.,
Krivoshein, Yu.S., Ljungdahl-Stahle, E., Pertel, S.S.,
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roform 2 : 1); H NMR: Table 2; benzyl ester of
2. Karaulov, A.V., Kalyuzhin, O.V., and Zemlyakov, A.E.,
O-(cyclododecyl 2-acetamido-2,3-dideoxy-β-D-glu-
copyranosid-3-yl)-D-lactoyl-L-alanyl-D-isoglutamine
(VIIIe); 180 mg (37%); mp 149–155°ë (dec.); [α]546
+6° (Ò 067, ethanol); 1H NMR: Table 2; benzyl ester of
O-(cyclododecyl 2-acetamido-2,3-dideoxy-α-D-glu-
copyranosid-3-yl)-D-lactoyl-L-alanyl-D-isoglutamine
(VIIIf); yield 260 mg (34%); mp 209–215°ë (dec.);
[α]546 +83° (Ò 1.0, ethanol); 1H NMR: Table 2.
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O-(Dodecyl 2-acetamido-2,3-dideoxy-b-D-glu-
copyranosid-3-yl)-D-lactoyl-L-alanyl-D-isoglutamine
(IXa). Benzyl ester (VIIIa) (165 mg, 0.22 mmol) was
dissolved in 9 : 1 THF–water mixture (15 ml) and sub-
jected to hydrogenolysis over 10% Pd/C (50 mg) at
room temperature for 4 h (monitoring by TLC in sys-
tem E). The catalyst was filtered off and washed with
5 ml of a mixture of solvents, and the filtrate was evap-
orated to dryness. The residue was triturated under a
layer of ether to get 120 mg (83%) of amorphous gly-
copeptide (IXa); [α]546 +4° (Ò 0.67, ethanol).
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Similarly, the following compounds were obtained:
O-(dodecyl 2-acetamido-2,3-dideoxy-α-D-glucopyra-
nosid-3-yl)-D-lactoyl-L-alanyl-D-isoglutamine (IXb);
yield 180 mg (86%); amorphous powder, [α]546 +85°
(Ò 1.0, ethanol); O-[(1-pentylhexyl) 2-acetamido-2,3-
dideoxy-β-D-glucopyranosid-3-yl]-D-lactoyl-L-alanyl-
D-isoglutamine (IXc); yield 250 mg (96%); mp 174–
175°ë, [α]546 +2° (Ò 1.0, ethanol); O-[(1-pentylhexyl)
2-acetamido-2,3-dideoxy-α-D-glucopyranosid-3-yl)-
D-lactoyl-L-alanyl-D-isoglutamine (IXd); yield 150 mg
(78%); mp 185–187°ë, [α]546 +90° (Ò 0.67, ethanol);
O-(cyclododecyl 2-acetamido-2,3-dideoxy-β-D-glu-
copyranosid-3-yl)-D-lactoyl-L-alanyl-D-isoglutamine
(IXe); yield 70 mg (98%); amorphous powder, [α]546
+3° (Ò 1.0, ethanol–chloroform 2 : 1); and
O-(cyclododecyl 2-acetamido-2,3-dideoxy-α-D-glu-
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