Synthesis and immunomodulating activity of new glycopeptides of glycyrrhizic acid containing residues of L-glutamic acid

Article abstract of DOI:10.1134/S1068162006060136

New glycopeptides of glycyrrhizic acid (GA) containing Glu residues and their α-methyl esters, γ-methyl esters, and α,γ-dimethyl esters were synthesized using N,N′-dicyclohexylcarbodiimide in the presence of N-hydroxybenzotriazole or N-hydroxysuccinimide.

Full text of DOI:10.1134/S1068162006060136

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2006, Vol. 32, No. 6, pp. 595–601. © Pleiades Publishing, Inc., 2006.
Original Russian Text © R.M. Kondratenko, L.A. Baltina, Jr., L.A. Baltina, N.Zh. Baschenko, G.A. Tolstikov, 2006, published in Bioorganicheskaya Khimiya, 2006, Vol. 32, No. 6,
pp. 660–666.
Synthesis and Immunomodulating Activity of New Glycopeptides
of Glycyrrhizic Acid Containing Residues of L-Glutamic Acid
a
b
b 1
b
R. M. Kondratenko , L. A. Baltina, Jr. , L. A. Baltina , N. Zh. Baschenko ,
c
and G. A. Tolstikov
a
Bashkortostan State Medical University, Ufa, Bashkortostan, Russia
b
Institute of Organic Chemistry, Bashkir Research Center, pr. Oktyabrya 71, Ufa, 450054 Bashkortostan, Russia
c
Vorozhtsov Institute of Organic Chemistry, Siberian Division, Russian Academy of Sciences, Novosibirsk, Russia
Received December 26, 2005; in final form, April 6, 2006
Abstract—New glycopeptides of glycyrrhizic acid (GA) containing Glu residues and their α-methyl esters,
γ-methyl esters, and α,γ-dimethyl esters were synthesized using N,N'-dicyclohexylcarbodiimide in the presence
of N-hydroxybenzotriazole or N-hydroxysuccinimide. Formation of amide bonds was observed on all the three
COOH groups of GA, or selectively on the COOH groups of the GA carbohydrate part in dependence on the
ratio of reagents and the reaction conditions. The GA glycopeptide with three residues of Glu(OH)-OMe at a
dose of 2 mg/kg stimulated the production of antibody-forming cells in mouse spleen in comparison with the
control. The GA glycopeptide containing Glu residues only in the GA carbohydrate part turned out to be an
immunosuppressor. The glycopeptide of the 30-methyl ester of GA with residues of free Glu in its carbohydrate
part increased the hemagglutinine titer at oral doses of 2 and 10 mg/kg. All the studied compounds had practi-
cally no effect on the delayed-type hypersensitivity in mice.
DOI: 10.1134/S1068162006060136
Key words: glutamic acid, glycopeptides, glycyrrhizic acid, immunomodulating activity
INTRODUCTION
Hydrogenolysis of (II) in 75% acetic acid in the
presence of 10% Pd/C gave glycopeptide (III) contain-
ing three residues of α-methyl ester of glutamic acid.
The crude product was purified by column chromatog-
raphy on silica gel and was finally obtained in 62%
yield.
Derivatives of glycyrrhizic acid, a triterpene glyco-
side from basic saponin of liquorice (Glycyrrhiza glabra L.
and Uralian Glycyrrhiza uralensis Fisher) containing
residues of amino acids or peptides are promising
immunomodulators with the properties interesting for
2
medicine [1–5].
Glycopeptides (IV), (VI), (VII), (VIII), and (IX)
containing the residues of amino acid esters only in the
GA carbohydrate part were predominantly formed after
activation of the GA carboxyl groups by DCC/HONSu
in THF or dioxane at 0–+5°C and 1 : 4–5 : 2.5–3.0 GA–
HONSu–DCC molar ratio [6, 7]. The compounds con-
taining free C30-COOH group in the triterpene part of
the molecule were separated to homogeneous com-
In this paper, we describe synthesis of new GA gly-
copeptides (II)–(IX) containing the residues of
glutamic acid or its esters.
RESULTS AND DISCUSSION
Glycopeptide (II) was prepared in 72% yield by
coupling of L-glutamic acid α-methyl, γ-benzyl ester pounds (TLC) by the column chromatography on silica
hydrochloride (4 mmol) with tris-N-hydroxybenzotria- gel in 50–55% yields. The selective formation of
zole ester of GA, formed by activation of the GA (I) CONH bonds from the carboxyl groups of the GA car-
carboxyl groups by DCC in the presence of HOBt at
1 : 4 : 3 GA–HOBt–DCC molar ratio, in THF at 0–
+5°ë in the presence of triethylamine (7 mmol) at 20–
22°C.
bohydrate part was confirmed by preservation of the
value of chemical shift of the resonance from C30 agly-
cone atom in the 13C NMR spectra of these compounds
(the same chemical shift (180 ppm) was observed for
the GA molecule) [8, 9]. GA derivative (V) with two
Glu(OH)-OMe residues was prepared by the selective
hydrogenolytic deprotection of γ-COOH in (IV) in
75% acetic acid in the presence of 10% Pd/C and by the
subsequent hydrolysis of glycopeptide (VI) with TFA.
1
Corresponding author; phone: (3472) 35-5288;
e-mail: baltina@anrb.ru.
2
Abbreviations: AFC, antibody-forming cells; DTH, delayed-type
hypersensitivity; CC, column chromatography; GA, glycyrrhizic
acid; and ShE, sheep erythrocytes.
595

596
KONDRATENKO et al.
30
29
(I) R' = R = OH
Me
COR'
20
(II) R' = R = Glu(OBzl)-OMe
19
21
22
H
12
(III) R' = R = Glu(OH)-OMe
O
18
13
14
17
16
Me 11Me
(IV) R' = OH; R = Glu(OBzl)-OMe
(V) R' = OH; R = Glu(OH)-OMe
Me
15
1
9
10
5
8
2
3
6' OR
t
Me
C
(VI) R' = OH; R = Glu(OBu )-OMe
7
27
4'
4
6
5'
O
O
HO
HO
(VII) R' = OH; R = Glu(OMe)-OMe
(VIII) R' = OH; R = Glu(OMe)-OH
(IX) R' = OH; R = Glu(OBu )-OH
(X) R' = OH; R = Glu(OH)-OH
(XI) R' = OMe; R = Glu(OBzl)-OBzl
(XII) R' = OMe; R = Glu(OH)-OH
2'
Me
Me
1'
3'
23
24
t
6''
O
COR
4''
5''
O
HO
HO
2''
1''
3''
OH
The removal of tert-Bu group by the treatment of mediated immunity, although (III) slightly inhibited
(IX) with TFA gave glycopeptide (X) with free carbox- the DTH reaction.
yls of the L-Glu residues. Glycopeptide (XII) was syn-
thesized by DCC/HOBt coupling of C30-methyl ester
of GA [5] with L-glutamic acid dibenzyl ester hydro-
chloride, followed by hydrogenolysis and column chro-
matography on silica gel.
The immunotropic activity of glycopeptide (XII)
was studied at two doses: 10 mg/kg administration for
7 days and 2 mg/kg for 14 days, as earlier described [7].
The glycopeptide was orally introduced. The response
of immune system was evaluated according to the value
of log2 titers of hemagglutinine antibodies (Table 4).
Compound (XII) exerted a statistically reliable
increase in the hemagglutinine level after 7 days (see
Table 4). In the case of 14-days introduction, the agglu-
tinin titer twofold increased in control. The titer of
experimental group somewhat exceeded that in control
and was reliably higher than that obtained after the
7-day introduction of (XII) despite the use of its lower
dose. This GA derivative had no influence on the cell-
mediated immunity (Table 5).
The structures of all the synthesized compounds
were confirmed by spectral methods (IR, UV, 1H NMR,
and 13C NMR spectroscopy) and elemental analysis
(Tables 1, 2). The assignment of C8 and C14 reso-
nances atoms in the 13C NMR spectra of the new GA
glycopeptides was based on a high-resolution 13C NMR
spectra of GA and its esters [9].
The residues of Glu and its esters were identified in
the 13C NMR spectra by the additional resonances of
the carbon atoms in low field (172–176 ppm) (COOH,
COOR). Methoxycarbonyl groups and α-carbon atoms
of Glu formed a group of resonances in the area of
52−53 ppm.
The effects of glycopeptides (III) and (X) on the
production of AFC to ShE in sterile mice were studied.
The examined substances were tested by a single intra-
peritoneal injection to mice at a dose of 2.0 mg/kg one
day after their immunization. The AFC number was
determined for the whole spleen calculated per 106
splenocytes. Isotonic solution was injected to control
animals.
Thus, the effect of GA glycopeptides containing Glu
residues and their methyl esters on humoral immunity
depends on the structure of molecules and the way of
their introduction.
EXPERIMENTAL
Melting points were determined on a Boetius micro-
plate. IR spectra were recorded on a Specord M80 in a
paste with Vaseline oil. UV spectra were recorded on a
Specord M400 in methanol. The optical rotation was
measured on a Perkin-Elmer 214 MC polarimeter in a
1
tube of 1 dm in length. The H NMR and 13C NMR
Glycopeptide (III) stimulated the AFC production
in comparison with control (Table 3). Glycopeptide (X)
containing Glu residues only in the carbohydrate part
proved to be an immunosuppressor (the relative AFC
number was three times lower in the mice spleen in
comparison with control).
spectra were recorded on a Bruker AM-300 spectrome-
ter (300 and 75.5 MHz) in ëD3éD in δ-scale with tet-
ramethylsilane as an internal standard; chemical shifts
are given in ppm and spin-coupling constants, in Hz.
TLC was carried out on Silufol plates (Czech
The effect of GA derivatives (III) and (X) on the Republic) or Sorbfil plates (ZAO Sorbpolimer) in the
functional capacity of T-effector cells was studied in the chromatographic system 45 : 10 : 1 chloroform–meth-
DTH model. The intensity of DTH was determined as a anol–water. The substances were detected by the treat-
difference in the masses of the control and experimental ment with 20% solution of phosphotungstic acid in eth-
legs of mice and expressed in percents. The examined anol with the subsequent heating at 110–120°ë for
compounds exerted practically no effect on the cell- 2−3 min.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 6 2006

SYNTHESIS AND IMMUNOMODULATING ACTIVITY
597
Table 1. Parameters of the 13C NMR spectra of GA glycopeptides (aglycone and carbohydrate parts) (CD3OD, δ, ppm, 25°C,
75.5 MHz)
Number of
carbon atom
(III)*
(IV)
(V)
(VII)
(VIII)
(X)
(XII)
1
2
39.42
26.50
89.13
39.64
55.22
18.70
32.81
45.37
61.82
36.89
200.00
128.51
171.48
43.18
27.47
27.68
31.88
47.36
41.81
43.66
31.43
37.62
28.45
16.46
17.50
19.38
23.31
29.36
29.87
175.88
40.38
27.67
90.60
40.70
56.55
18.51
33.90
46.78
63.18
38.13
202.34
129.03
171.45
44.59
27.75
28.22
33.01
–
40.33
27.62
90.59
40.67
56.39
18.45
33.83
46.68
63.06
38.01
202.42
129.10
172.47
44.54
27.87
28.42
32.87
–
40.38
27.62
90.71
40.68
56.51
18.45
33.82
46.76
63.16
38.08
202.48
129.24
171.86
44.89
27.88
28.21
32.97
–
40.16
27.47
90.96
40.50
56.34
18.29
33.68
46.72
63.04
37.90
202.20
128.69
172.41
44.54
27.47
28.22
32.88
–
40.33
27.35
90.90
40.78
56.79
18.42
33.82
46.79
63.18
38.13
202.58
128.97
171.74
44.64
27.61
28.13
33.10
–
40.57
27.49
90.45
40.75
56.45
18.38
33.60
46.76
63.14
38.06
202.50
129.03
171.19
44.57
27.64
28.22
32.90
–
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
1'
42.52
44.92
32.21
38.50
28.54
17.02
17.22
19.45
23.91
28.87
29.23
180.31
42.48
44.80
32.01
38.69
28.63
17.09
17.31
19.38
23.86
29.18
29.32
178.97
42.46
45.08
32.14
39.02
28.48
17.05
17.30
19.37
23.93
28.85
29.21
180.35
42.24
44.90
31.89
38.88
28.22
16.99
17.23
19.25
23.80
28.77
29.17
180.18
42.47
44.94
32.04
39.05
28.80
16.54
17.06
19.35
23.68
28.80
29.23
180.39
42.65
44.64
32.06
38.79
28.22
16.98
17.12
19.36
23.76
29.26
29.43
177.72
51.50
105.62
84.08
77.13
72.84
77.54
168.33
106.29
74.64
76.28
73.01
77.37
170.72
104.00
80.80
76.02
72.90
76.25
170.22
104.46
75.09
74.07
72.98
77.57
170.67
105.05
82.15
76.59
73.31
77.63
171.57
105.32
76.03
76.09
73.88
77.32
171.89
105.03
82.24
76.20
73.22
77.62
171.37
105.11
75.92
75.92
73.42
77.09
171.57
105.02
81.72
76.25
73.29
77.66
171.61
105.08
75.94
76.18
73.51
77.18
171.50
105.00
83.07
76.98
72.98
77.49
171.46
105.58
75.79
76.03
73.42
77.18
171.46
105.28
84.17
77.32
72.99
77.70
169.84
106.17
73.57
75.82
72.47
77.38
170.08
2'
3'
4'
5'
6'
1''
2''
3''
4''
5''
6''
* CDCl + C D N.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 6 2006
3
5 5

598
KONDRATENKO et al.
Table 2. Chemical shifts of C atom signals of the amino acid moieties in the 13C NMR spectra of GA glycopeptides (CD3OD,
δ, ppm, 25°C, 75.5 MHz)
Amino acid fragments in the com-
C1
C2
C3
C4
C5
C6
pounds
6
4
51.63
51.52
51.16
172.72
172.61
171.74
52.47
52.31
52.02
27.62
27.45
26.57
31.25
31.11
30.76
174.86
174.73
173.47
3
2
1
5
HOOCCH2CH2CHCOOCH3
(III)
NH
3
2
1
5
6
4
52.43
52.37
173.73
173.08
53.13
52.86
27.84
27.63
31.25
31.11
174.95
174.52
HOOCCH2CH2CHCOOCH3
(V)
NH
6
2
5
4
3
1
52.99
52.82
173.32
173.19
53.11
53.02
27.28
26.30
31.29
30.79
176.02
174.77
CH3OOCCH2CH2CHCOOCH3
(VII)*
NH
6
2
1
5
4
3
173.42
172.97
52.32
52.50
53.26
53.10
27.23
26.89
31.31
31.15
176.77
175.25
CH3OOCCH2CH2CHCOOH
(VIII)
NH
4
3
2
5
1
173.16
172.90
51.85
51.70
27.07
26.63
37.59
36.22
174.98
174.76
HOOCCH2CH2CHCOOH
(X)
NH
5
4
3
2
1
172.59
172.30
52.70
52.50
26.21
26.44
33.88
33.73
174.97
174.42
HOOCCH2CH2CHCOOH
(XII)
NH
* C7 (52.59; 52.40).
Triethylamine and N-ethylmorpholine were kept for ing until the formation of a solution and for 6 h at 20–
one day over KOH and distilled. THF and dioxane were 22°C. The solvent and thionyl chloride excess were
purified by the procedure [10].
removed in a vacuum. The syrup-like residue was tritu-
rated with ether. The obtained powder was filtered and
two times recrystallized from a mixture of methanol–
diethyl ether to get the title compound; yield 5.6 g
(92%); mp 187–189°ë. [α]2D0 +21.9° (Ò 0.05, ethanol);
IR spectrum (ν, cm–1); 3150 (NH), 1730 (COOR),
1680 (C=O), 1610 (C6H5), 1580 (NH+3 ), 1510 (C6H5).
GA was prepared by the procedure [8] and had the
purity of approximately 95%. DCC (Aldrich),
L-glutamic acid γ-tert-butyl ester, γ-methyl ester, and
γ-benzyl ester (Reanal, Hungary) were used in this
study. 30-Monomethyl ester of GA and L-glutamic acid
dibenzyl ester hydrochloride were prepared according
to the procedures [5] and [11], respectively.
Found, %: C 54.28, H 6.74, N 4.76. Calc. for
C13H18O4NCl, %: C 54.17, H 6.29, N 4.79.
L-Glutamic acid a-methyl, g-benzyl diester
hydrochloride. A freshly distilled thionyl chloride
(10 ml) was added to a solution of L-glutamic acid
γ-benzyl ester (5 g) in anhydrous methanol (300 ml) at
0 to +5°ë. The reaction mixture was stirred under cool-
L-Glutamic acid a-methyl, g-tert-butyl diester. A
solution of diazomethane in ether was added to a solu-
tion of γ-tert-butyl ester of L-glutamic acid (5.0 g) in
methanol (100 ml) at 0 to +5°C to a stable yellow color.
The reaction mixture was kept for 6 h at 20–22°ë, and
several drops of glacial acetic acid were added. The
reaction mixture was evaporated in a vacuum at 30–
35°ë to dryness. The residue was dissolved in ethyl
acetate (50 ml) and washed with 1 N NaHCO3 (3 ×
50 ml) and water. The organic phase was dried with
MgSO4 and evaporated. The residue was recrystallized
from an ethyl acetate–hexane mixture; yield 91%;
Table 3. Effect of GA glycopeptides (III) and (X) on the
AFC production and DTH reaction in mice
AFC number in AFC number
DTH (%) of
Com-
pound
the whole
spleen
per 106 spleno- edema of the
cytes
mouse paw
(III)
(X)
17393 3423
3640 605
2299 448*
443 109*
1392 301
10.4 2.0
17.0 4.4
16.7 4.0
mp 135−136°ë; [α]2D0 +17° (c 0.03, methanol) (lit.
mp 135–136.5°ë and [α]2D0 +21.7° (c 2%, methanol)
Control 15039 2635
* Reliable in comparison with control (p < 0.05).
[11]).
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 6 2006

SYNTHESIS AND IMMUNOMODULATING ACTIVITY
599
Table 4. Effect of GA glycopeptide (XII) on the hemagglu-
3-é-{2-é-[N-(b-D-Glucopyranosyluronoyl)-L-
glutamic acid a-methyl ester]-N-(b-D-glucopyrano-
syluronoyl)-L-glutamic acid a-methyl ester]-(3b,
20b)-11-oxo-30-norolean-12-en-30-oyl-(L-glutamic
acid a-methyl ester) (III). 1. HOBt (0.54 g, 4 mmol)
and DCC (0.70 g, 3.2 mmol) were added to a solution
of GA (0.82 g, 1 mmol) in dioxane (20 ml) at 0 to +5°C.
The reaction mixture was stirred for 1 h at 0 to +5°C
and for 6 h at 20–22°C. The precipitate of N,N'-dicyclo-
hexylurea was filtered off. The filtrate was cooled on ice
bath, and hydrochloride of L-Glu(OBzl)-OMe (1.10 g,
4 mmol) and triethylamine (1.0 ml, 7.2 mmol) were
added. The reaction mixture was kept under occasional
stirring at 20–22°C for 24 h, diluted with cool water,
and acidified to pH 4 with citric acid. The precipitated
glycopeptide (II) was filtered, washed with water, and
dried. Recrystallization from an acetone–hexane mix-
ture gave 1.10 g (72%) of product; Rf 0.73; mp
tinin titer of the sensitized mice
After 7 days
(10 mg/kg)
After 14 days
(2 mg/kg)
Compound
(XII)
Control
1.0 0.2*
0.4 0.2
1.5 0.1*
0.8 0.2
* Reliable in comparison with the control (p < 0.05).
were added to a solution of GA (0.82 g, 1 mmol) in
THF (20 ml) at 0 to +5°C. The reaction mixture was
stirred for 2 h at 0 to +5°C and kept overnight in a
fridge. The precipitated N,N'-dicyclohexylurea was fil-
tered off. Hydrochloride of L-Glu(OBzl)-OMe (0.82 g,
3 mmol) and triethylamine (0.7 ml, 5 mmol) were
added to the filtrate at 0 to +5°C. The reaction mixture
was stirred under cooling for 1 h and at 20–22°C for
20 h, diluted with cool water, and acidified with citric
acid to pH 4. The precipitate was filtered, washed with
water, and dried. The product was fractionated on a sil-
145−148°ë; IR spectrum, (ν, cm–1): 3600–3200 (OH,
NH), 1740 (COOR), 1650 (C11=O), 1550 (CONH),
1500 (Ph); UV spectrum, [λmax, nm, ( (logε )]: 249
(4.20). Found, %: N 2.79. Calc. for C81H107O25N3, %:
N 2.76.
ica gel column eluted with CHCl –MeOH–H O mix-
3
2
tures at the following volume ratios: 200 : 10 : 1, 100 :
10 : 1, and 50 : 10 : 1. The fractions homogenous
according to TLC were combined and evaporated.Yield
2. The protected glycopeptide (II) (0.90 g,
0.6 mmol) was subjected to hydrogenolysis in 75%
acetic acid (50 ml) in the presence of 10% Pd/C for
24 h. The catalyst was filtered off; the filtrate was evap-
orated; and the residue was fractionated on a silica gel
column eluted with a CHCl3–åÂéç–ç2é mixture at
the following volume ratios: 100 : 10 : 1, 50 : 10 : 1, and
25 : 10 : 1. The homogenous product (III) was eluted
with a 50 : 10 : 1 CHCl3–CH3OH–H2O mixture; yield
of (IV) 0.69 g (53%); [α]2D0 +60° (Ò 0.02, methanol); IR
spectrum (ν, cm–1): 3600–3200 (OH, NH), 1740
(COOR), 1650 (C11=O), 1550 (CONH), 1500 (Ph);
1
UV spectrum, [λmax, nm, (logε )]: 248 (4.2); H NMR
(ëD3OD, δ, ppm): 0.74, 1.04, 1.04, 1.10, 1.12, 1.16,
1.44 (21 ç, 7ëH3), 3.64, 3.74 (6 H, both s, 2éëç3),
5.70 (1 H, s, C12-H). Found, %: N 2.19. Calc. for
C70H94O22N2, %: N 2.12. The following impurities were
isolated and identified by TLC by a comparison with
the known samples: (II) (20%) and the starting GA
(approximately 10%).
0.46 g (62%); Rf 0.5; [α]2D0 +35° (Ò 0.02, methanol); IR
spectrum, (ν, cm–1): 3600–3200 (OH, NH), 1740
(COOMe), 1660 (C11=O), 1540 (CONH); UV spec-
trum, [λmax, nm, (logε )]: 249 (4.02); 1H NMR (ëD3OD,
δ, ppm): 0.74, 1.06, 1.08, 1.15, 1.18, 1.44 (21 ç, 7
ëç3), 3.74, 3.64 (6 H, both s, éëç3), 3.30 (3 H, s,
éëç3), 4.56, 4.72 (2 H, H1', H1''), 5.56 (1 H, s, 12-H),
2. Glycopeptide (VI) was prepared a described for
(IV) from GA (0.82 g, 1 mmol), HONSu (0.60 g,
5.2 mmol), DCC (0.65 g, 3 mmol), L-Glu(OÇut)-OMe
hydrochloride (0.52 g, 3 mmol), and N-ethylmorpho-
line (0.6 ml, 5 mmol) in THF (20 ml). The crude prod-
uct (0.90 g) was purified by precipitation from metha-
nol with ether; yield 0.68 g (55%); IR spectrum (ν,
cm−1): 3600–3200 (OH, NH), 1740 (COOR), 1670
(C11=O), 1540 (CONH). Found, %: N 2.35. Calc. for
7.90 (1 H, broadened s, NH); 13ë NMR spectrum is
given in Tables 1, 2. Found, %: N 3.53. Calc. for
C60H89O25N3, %: N 3.36.
3-é-{2-é-[N-(b-D-Glucopyranosyluronoyl)-L-
glutamic acid a-methyl ester]-N-(b-D-glucopyrano-
syluronoyl)-L-glutamic acid a-methyl ester}-
(3b,20b)-11-oxo-30-norolean-12-en-30-acid (V). 1.
HONSu (0.60 g, 5.2 mmol) and DCC (0.65 g, 3 mmol) C62H96O22N2, %: N 2.29.
Table 5. Effect of GA glycopeptide (XII) on the DTH reaction in mice sensitized for 7 and 14 days
Seven days (10 mg/kg)
Forteen days (2 mg/kg)
Compound
difference in the masses of increase in the paw vol- difference in the masses of increase in the paw vol-
paws, mg
ume, %
paws, mg
ume, %
(XII)
Control
8.40 1.05
8.14 0.45
7.56 1.05
7.59 0.73
9.60 1.21
8.14 0.45
8.24 1.00
7.59 0.73
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 6 2006

600
KONDRATENKO et al.
3a. The protected glycopeptide (IV) (0.65 g,
2. Glycopeptide (IX) (0.56 g, 0.5 mmol) was treated
0.5 mmol) was hydrogenolyzed in 75% acetic acid with TFA (10 ml) as described for (V) and (X) was puri-
(30 ml) in the presence of 10% Pd/C for 24 h. The cat- fied by column chromatography to homogenous state
alyst was filtered off, and the filtrate was evaporated. according to TLC; yield of 0.36 g (63%); X). Rf 0.4.
[α]2D0 +49° (Ò 0.02, methanol); IR spectrum (ν, cm–1):
3600–3200 (OH, NH), 1710 (COOH), 1660 (C11=O),
1530 (CONH); 13C NMR spectrum is given in Tables 1, 2.
Found, %: N 2.62. Calc. for C52H76O22N2, %: N 2.59.
The residue was fractionated on a silica gel column
eluted with CHCl –MeOH–H O mixtures at the fol-
3
2
lowing volume ratios: 100 : 10 : 1, 50 : 10 : 1, and 25 :
10 : 1. The homogenous (TLC) product (V) was eluted
with 50 : 10 : 1 CHCl –MeOH–H O mixture; yield
3
2
Compound (XII) was prepared as described for (II)
from C30-methyl ester of GA (1 mmol), HOBt
(3 mmol), DCC (2.5 mmol), and L-Glu(OBzl)-OBzl
hydrochloride in dioxane (20 ml) in the presence of tri-
ethylamine (5 mmol). The crude product (0.78 g) was
hydrogenolyzed in 75% acetic acid (30 ml) in the pres-
ence of Pd/C for 12 h. The solvent was evaporated. The
residue was twice purified on a column as described
above for (VI); yield of (XII) was 0.50 g (37%); Rf 0.42.
0.45 g (73%); Rf 0.45. [α]2D0 +47° (Ò 0.02, methanol);
IR spectrum (ν, cm–1): 3600–3200 (OH, NH), 1740
(COOMe), 1660 (C11=O), 1540 (CONH); UV spec-
trum, [λmax, nm, (logε )]: 249 (4.0); 13ë NMR spectrum
is given in Tables 1, 2. Found, %: N 2.55. Calc. for
C54H80O22N2, %: N 2.52.
3b. The protected glycopeptide (VI) (0.60 g,
0.5 mmol) was dissolved in TFA (5 ml), kept for 30 min
at 20–22°C, and evaporated to dryness with anhydrous
benzene (3 × 10 ml). The residue was purified on a col-
[α]2D0 +38° (Ò 0.028, methanol); IR spectrum (ν, cm–1):
3600–3200 (OH, NH), 1705 (COOH), 1650 (C11=O),
umn as described above; yield of (V) was 0.38 g (69%); 1530 (CONH); UV spectrum, [λmax, nm, (logε )]: 248
[α]2D0 +45° (Ò 0.02, methanol). This sample was identi-
cal to that described above.
13
(3.85); C NMR spectrum is given in Tables 1, 2.
Found, %: N 3.07. Calc. for C53H78O22N2, %: N 2.56.
Effects of the GA glycopeptides on immune sys-
tem of white outbred mice was evaluated according to
a change in the humoral immune response (production
of AFC) by the method of Jerne and Nordin [12] mod-
ified by Cunningham [13] and according to a change in
Compound (VII) was prepared as described for (V)
from GA (1 mmol), HONSu (5 mmol), DCC
(2.5 mmol),
L-Glu(OMe)-OMe
hydrochloride
(2.5 mmol), and triethylamine (5 mmol) in THF (20 ml)
yield of 0.62 g (55%); Rf 0.5. [α]2D0 +43 2° (Ò 0.03, the cell-mediated immunity on the DTH model with the
methanol); IR spectrum (ν, cm–1): 3600–3200 (OH,
use of ShE as test antigens. The examined substances
were intraperitoneally injected to the animals one time
NH), 1740 (COOMe), 1660 (C11=O), 1550 (CONH);
one day after the immunization at the dose of 2 mg/kg.
UV spectrum, [λmax, nm, (logε )]: 249 (4.0); 13C NMR
The number of AFC was determined on the seventh day
spectrum is given in Tables 1 and 2. Found, %: N 2.55.
visually in the whole spleen according to the number of
Calc. for C H84O22N2, %: N 2.46.
hemolysis zones and calculated per 106 splenocytes.
56
The influence of glycopeptide (XII) on the primary
immune response was evaluated according to the level
of hemagglutinines in the mice blood by the method
[14]. An examined compound was introduced to the
animals per os beginning from the first day of sensitiza-
tion of ShE at doses of 10 mg/kg (the introduction for
7 days) and 2 mg/kg (the introduction for 14 days). The
isotonic solution was introduced to control animals. On
the 7th and 14th days of the sensitization, the resolving
dose of ShE at a concentration of 108 was subplantary
injected into the right paw of the mice of each group
(n = 7). Another paw remained intact. One day later, the
animals were decapitated, their blood was taken, and
hemagglutinines were determined in the blood serum.
The reaction was evaluated according the value of
log2 antibody titers.
Compound (VIII) was prepared as described for
(V) from GA (1 mmol), HONSu (3 mmol), DCC
(2.5 mmol), and L-Glu(OMe)-OH (3 mmol) in dioxane
(20 ml) in the presence of triethylamine (5 mmol) in
yield of 0.54 g (49%). Glycopeptide (VIII) was twice
purified by the column chromatography on silica gel
and was homogeneous by TLC; Rf 0.42. [α]2D0 +62°
(Ò 0.04, methanol); IR spectrum (ν, cm–1): 3600–3200
(OH, NH), 1740 (COOMe), 1660 (C11=O), 1530
(CONH); UV spectrum, [λmax, nm, (logε )]: 248 (3.9);
13C NMR spectrum is given in Tables 1, 2. Found, %:
N 2.60. Calc. for C54H80O22N2, %: N 2.52.
Compound (X). 1. Compound (IX) was synthe-
sized as described for (V) from GA (1 mmol), HONSu
(4 mmol), DCC (2.5 mmol), L-Glu(OBut)-Oç
(3 mmol), and triethylamine (5 mmol) in THF (20 ml)
in yield of 0.60 g (51.5%); IR spectrum (ν, cm–1): 3600–
3200 (OH, NH), 1740 (COOBut), 1660 (C11=O), 1540
Effect of compounds (III), (X), and (XII) on the
cell-mediated immunity was studied in the DTH reac-
tion to ShE. The mice were sensitized by the intrave-
nous injection of ShE at the stimulating dose (2 × 106)
and by the subplantary injection at the resolving dose
(2 × 108) of ShE [15]. One day after the last injection,
(CONH); UV spectrum, [λmax, nm, (logε )]: 249 (3.9).
Found, %: N 2.42. Calc. for C60H82O22N, %: N 2.37.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 6 2006

SYNTHESIS AND IMMUNOMODULATING ACTIVITY
601
6. Kondratenko, R.M., Baltina, L.A., Vasil’eva, E.V., Bal-
tina, L.A. (Jr.), Ismagilova, A.F., Nasyrov, Kh.M.,
Baschenko, N.Zh., Kireeva, R.M., Fridman, S.M., and
Tolstikov, G.A., Bioorg. Khim., 2004, vol. 30, pp. 61–67;
Rus. J. Bioorg. Chem., 2004, vol. 30, pp. 53–59.
the intensity of DTH reaction was determined accord-
ing to an increase in the mass and % volume of the
mouse paw. The experimental results are given in
Tables 3–5.
7. Kondratenko, R.M., Baltina, L.A., Vasil’eva, E.V., Nasy-
rov, Kh.M., Kireeva, R.M., Baschenko, N.Zh., Frid-
man, S.M., Baltina, L.A. (Jr.), and Tolstikov, G.A.,
Bioorg. Khim., 2004, vol. 30, pp. 168–173; Rus. J.
Bioorg. Chem., 2004, vol. 30, pp. 148-153.
ACKNOWLEDGMENTS
This study was supported by the Russian Founda-
tion for Basic Research, BNTS, project no. 03-03-
20004; by the grants of the President of the Russian
Federation for support of young Russian scientists and
leading scientific schools, project no. NSh
1488.2003.3; by the grant of the Scientific School of
Academician G.A. Tolstikov, project no. RI-
112/001/375; and by the grant of Russian Science,
project no. 2005-RI-12.0/004/088 (State contract no.
02.434.11.7060).
8. Kondratenko, R.M., Baltina, L.A., Mustafina, S.R.,
Makarova, N.V., Nasyrov, Kh.M., and Tolstikov, G.A.,
Khim.-Farm. Zh., 2001, vol. 35, pp. 39–42.
9. Baltina, L.A., Kunert, O., Fatykhov, A.A., Kon-
dratenko, R.M., Spirikhin, L.V., Baltina, L.A., Galin, F.Z.,
Tolstikov, G.A., and Khaslinger, E., Khim. Prir. Soedin.,
2005, no. 4, pp. 347–350.
10. Gordon, A.J. and Ford, R.A., The Chemist’s Companion:
A Handbook of Practical Data, Techniques, and Refer-
ences, New York: Wiley, 1972.
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