Neoglycoconjugates based on dendrimer poly(aminoamides)

Article abstract of DOI:10.1023/A:1021293532046

Neoglycoconjugates containing 4, 8, 32, and 64 terminal residues of B-disaccharide (B_DI) or N-acetylneuraminic acid (Neu5Ac) attached to poly(aminoamide)-type dendrimers (PAMAMs) were synthesized. The ability of B_DI conjugates to bind natural xenoantibodies (anti-B_DI antibodies) and the ability of Neu5Ac conjugates to inhibit the hemagglutinin-mediated adhesion of influenza virus were studied. The biological activity of PAMAM conjugates turned out to be higher than that of free carbohydrate ligands, but less than that of multivalent glycoconjugates based on other types of synthetic polymeric carriers. A conformational analysis of PAMAM matrices and resulting conjugates was performed to determine the statistical distances between carbohydrate ligands. The computations revealed the tendency of the PAMAM chains toward compaction and formation of dense globules. The process results in a decrease in the distances between the carbohydrate ligands in the conjugates and, hence, could affect the ability of glycoconjugates to efficiently bind the polyvalent carbohydrate-recognizing proteins.

Full text of DOI:10.1023/A:1021293532046

Russian Journal of Bioorganic Chemistry, Vol. 28, No. 6, 2002, pp. 470–486. Translated from Bioorganicheskaya Khimiya, Vol. 28, No. 6, 2002, pp. 518–534.
Original Russian Text Copyright © 2002 by Tsvetkov, Cheshev, Tuzikov, Chinarev, Pazynina, Sablina, Gambaryan, Bovin, Rieben, Shashkov, Nifant’ev.
Neoglycoconjugates Based on Dendrimer Poly(aminoamides)
D. E. Tsvetkov*, P. E. Cheshev*, A. B. Tuzikov**, A. A. Chinarev**, G. V. Pazynina**,
M. A. Sablina**, A. S. Gambaryan***, N. V. Bovin**, R. Rieben****,
1
A. S. Shashkov*, and N. E. Nifant’ev*
*Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninskii pr. 47, Moscow, 119991 Russia
**Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,
ul. Miklukho-Maklaya 16/10, Moscow, 117997 Russia
***Chumakov Institute of Poliomyelitis and Viral Encephalitides, Russian Academy of Medical Sciences,
Moscow oblast, Russia
****Department of Cardiology, Bern University Hospital, Bern, Switzerland
Received October 4, 2001; in final form, December 25, 2001
Abstract—Neoglycoconjugates containing 4, 8, 32, and 64 terminal residues of B-disaccharide (BDI) or
N-acetylneuraminic acid (Neu5Ac) attached to poly(aminoamide)-type dendrimers (PAMAMs) were synthe-
sized. The ability of BDI conjugates to bind natural xenoantibodies (anti-BDI antibodies) and the ability of
Neu5Ac conjugates to inhibit the hemagglutinin-mediated adhesion of influenza virus were studied. The bio-
logical activity of PAMAM conjugates turned out to be higher than that of free carbohydrate ligands, but less
than that of multivalent glycoconjugates based on other types of synthetic polymeric carriers. A conformational
analysis of PAMAM matrices and resulting conjugates was performed to determine the statistical distances
between carbohydrate ligands. The computations revealed the tendency of the PAMAM chains toward compac-
tion and formation of dense globules. The process results in a decrease in the distances between the carbohy-
drate ligands in the conjugates and, hence, could affect the ability of glycoconjugates to efficiently bind the
polyvalent carbohydrate-recognizing proteins.
Key words: B-disaccharide, dendrimers, influenza virus, N-acetylneuraminic acid, neoglycoconjugates,
poly(aminoamide), xenoantigen
1
INTRODUCTION
acrylic acid or its derivatives, are used as synthetic car-
riers [2, 3].
Various carbohydrate chains bound to membrane
Glycoconjugates on the basis of linear polymeric
carriers are widely used in bioanalytical investigations,
because they allow the achievement of an effective
polyvalent binding with complementary proteins.
However, the relative positions of ligands, the number
and the kind of ligands that are recognized by proteins,
and the type of ligands that are situated inside the poly-
2
lipids and proteins are exposed on the surface of cells.
The glycocalix fragments participate in the processes
of biological recognition by interacting with cellular
and extracellular proteins and carbohydrates. Usually,
binding constants of the monovalent carbohydrate–pro-
tein interactions are low, and the processes they medi-
ate occur via multipoint binding, when several carbohy- meric globule and are inaccessible for interaction in the
conjugate are always unknown. In some cases, this
complicates both the correct interpretation of the exper-
imental results and the further structure optimization of
the glycoconjugates for the design of more active com-
pounds.
drate ligands interact with several protein receptors [1].
Therefore, the use of polyvalent glycoconjugates on the
basis of high-molecular carries rather than monovalent
glycoconjugates is often advantageous for studying the
processes that include the carbohydrate–protein inter-
actions. For the construction of polyvalent neoglyco-
conjugates, proteins or polysaccharides are usually
used as natural carriers; and linear polymers, e.g., poly-
These problems can largely be solved using den-
drimers as the carriers of carbohydrate ligands. The
dendrimers are the hyperbranched polymeric systems
obtained by a sequential elongation of the chains of the
branched central fragment with branched monomeric
units. The structural and chemical characteristics of
dendrimers, namely, topology, hydrophilic–hydropho-
bic balance, and functionality of terminal groups, can
be controlled and changed in the synthesis performed
by a stepwise reiterative sequence of reactions [4, 5].
1
The author may be contacted by phone, +7 (095) 135-8784, or e-
mail, nen@ioc.ac.ru.
2
Abbreviations: APT, Attached Proton Testing, NMR experiments
to examine the number of attached protons; B , B-disaccharide,
DI
Galα1–3Galβ; EDA, ethylenediamine; HA, hemagglutinin;
Neu5Ac, N-acetylneuraminic acid; PAA, poly(acrylamide); and
PAMAM, poly(aminoamide).
1068-1620/02/2806-0470$27.00 © 2002 MAIK “Nauka /Interperiodica”

NEOGLYCOCONJUGATES BASED ON DENDRIMER POLY(AMINOAMIDES)
471
The next
CH2CH2COOMe
CH2CH2COOMe
a)
CH2CH2C(O)NHCH2CH2NH2
CH2CH2C(O)NHCH2CH2NH2
I + II
I
II
N
generation
NH2
N
of the dendrimer
b)
III
IV) X–COOY –HOY
CH2CH2C(O)NHCH2CH2NH(CO)X
CH2CH2C(O)NHCH2CH2NH(CO)X
CH2CH2COOY
CH2CH2COOY
N
N
IV) NH2–X –HOY
O
CH2CH2CONHX
CH2CH2CONHX
Y = activating group –ë6H4–NO2-p or
ï = spacered carbohydrate ligand
N
O
N
Scheme 1. The synthetic strategy for PAMAM dendrimers and neoglycoconjugates on their basis: (I) chain branching by the
Michael addition of methyl acrylate; (II) chain elongation by aminolysis with EDA; (III) hydrolysis of terminal ester groups and
the activation of resulting carboxyl groups; and (IV) conjugation; (a, b) methods of conjugation with glycosides bearing (a) amino-
or (b) activated carboxyl group in the spacer.
In glycoconjugates based on dendrimers (named herein (IIb)–(Vb) were obtained starting from 1,6-diamino-
glycodendrimers for short), the residues of carbohy- hexane (I) by the modified reiterative method [17],
drate ligands are attached only to the functional groups which involved (i) the step of chain branching by the
disposed on the surface of the carrier molecule. As a treatment with methyl acrylate and (ii) the step of chain
result, a carbohydrate environment is formed around elongation using EDA (Scheme 2); in both cases the
the dendrimer carrier, which makes the structure of gly- excess of the reagents were used for the reactions to be
codendrimers to be similar to that of the natural glyco- completed and the possible side processes to be dimin-
conjugates. Some samples of the syntheses of glyco- ished.
dendrimers were previously reported [6–12].
The necessity of excesses of the reagents used was
Among known dendrimer carriers suitable for the
conjugation with carbohydrate ligands, PAMAM den-
drimers are of especial interest [13]. These compounds
have a low toxicity [14] and are readily soluble in
organic and aqueous media. Furthermore, we assumed
that the PAMAM dendrimers can be easily prepared via
sequential reactions of chain (I) branching and (II)
elongation and then conjugated with carbohydrate moi-
eties (steps III + IV, Scheme 1).
previously shown [13]. However, we did not observe
any decrease in the yield and deterioration in the purity
of the resulting PAMAMs when using only 1.4–5 equiv
excess of the reagent per reacting group, which was
essentially less than 30 eqiuv suggested previously
[13]. The formation of crosslinked dendrimer mole-
cules was observed upon aminolysis of (Va) under the
EDA deficiency as a result of interaction between the
terminal amino groups and the unreacted ester groups
of the products of the incomplete condensation
(Scheme 2). According to gel chromatography data, the
average molecular mass of (VIb) was approximately
400 kDa.
In this paper, we report the synthesis, the structure,
and the investigation of some biological activities of
two series of glycodendrimers bearing the residues of
either BDI or Neu5Acα. The glycoconjugates of the first
series were synthesized as the blockers of natural anti-
BDI immunoglobulins causing the rejection of trans-
planted organs upon pig-to-human xenotransplantation
[15]. The glycoconjugates of the second series were
designed as inhibitors of adhesion of the influenza virus
[16].
Completeness of the elongation of dendrimer chains
1
was monitored by H NMR spectroscopy by the inte-
gration of the signals of the fragments a + e and d in the
spectra of (IIb)–(Vb) (Table 1). At the step of branch-
ing, the completeness of the reaction was checked by
the integration of the signals at ~3.6 ppm that corre-
spond to the CH3OC(O) groups and those at ~3.2 ppm
1
that correspond to the C(O)NHCH2 groups in the H
RESULTS AND DISCUSSION
NMR spectra of aminoesters (IIa)–(Va). The signals
were assigned using the data on homonuclear and het-
eronuclear correlation spectroscopy (COSY 1H–1H and
1H–13C) and APT experiments.
Synthesis of PAMAM Carriers and Glycodendrimers
PAMAM dendrimers (IIb)–(Vb) and (VIIb) with 4,
8, 16, 32, and 64 terminal amino groups and multivalent
product (VIb) that were built on the basis of the linked
molecules of dendrimer (Vb) were used as the carriers fragments of the PAMAM matrix depend on pH of
for obtaining the target glycoconjugates. Compounds solution, and the maximum variations in chemical
The chemical shifts of the protons of all structural
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 28 No. 6 2002

R
R
R
R
R
O
N
N
N
O
N
N
N
H
R
R
R
R
R
N
H
NH2
N
R
N
H2N
N
O
NH
O
R
R
O
H
N
O
R
N
N
N
N
R
N
N
H
N
H
H
O
R
H
1
(iii)
(IIa) (82%)
O
O
R
N
2
R
O
(i)
O
N
N
N
N
(ii)
N
N
H
N
N
H
R
O
O
R
H O
N
H
N
R
N
R
R
N
O
N
H
N
H
[(CH2)3NHR]2
(IIb) (67%)
H
N
O
R
H
N
2
O
(iii)
(Ic) (76%)
(i)
(IIIa) (62%)
N
N
H
H
N
N
O
R
(ii)
O
N
(IIc) (51%)
N
N
H
O
R
(iv)
R
(IIIc) (35%)
(IIIb) (74%)
H
O
N
N
(i)
(IVa) (64%)
R
(iii)
N
(ii)
H
H
N
O
N
R
(IIIc) (86%)
(IVb) (73%)
R
N
(v)
(i)
(iii)
R
R
2
(Va) (58%)
(IVd) (67%) (IVc) (77%)
(ii)
(vi)
(Vb) (73%)
(iii)
(v)
(Vd) (46%) (Vc) (66%)
(v)
a: R = MeOOCCH2CH2–
(VId) (40%)
b: R = NH2CH2CH2NHC(O)CH2CH2–
OH
OH
O
O
O
OH
O
H
OH
O
c: R =
d: R =
HO
H
N
CH2
O
HO
O
N
N
H
O
(VIb) (8%)
O
HO
HO
OH
COOH
O
O
H
HO
AcNH
HN
O
N
CH2
H
N
H
N
HO
O
Scheme 2. Synthesis of PAMAM glycodendrimers. (i)) Chain branching: 1.4–4 equiv of methyl acrylate, MeOH, 20°C, 18 h. (ii) Chain elongation: ~5 equiv of EDA, MeOH,
20°C, 48 h. Conjugation: (iii) (VIII), DMF, 20°C, 18 h; (iv) (IX), DMF, 18 h, 20°C, 1 N NaOH; (v) (XI), DMF, 20°C, 18 h, 1 N NaOH; (vi) 0.83 equiv of EDA, MeOH, 20°C.
The syntheses of (VIII), (IX), and (XI) are given in Scheme 4.

NEOGLYCOCONJUGATES BASED ON DENDRIMER POLY(AMINOAMIDES)
473
Table 1. Chemical shifts (δ, ppm) of resonances in the 1H and 13C NMR spectra of PAMAMs (IIb)–(Vb) (D2O)
Core
c
d
e
f
O
CH2CH2CNHCH2CH2NH2
1
2
3
a
b
CH2CH2CH2N
CNHCH2CH2N
O
pH 1
pH 10
Fragment
1H
13C
1H
13C
core: 1 + 2
1.25–1.35; 1.60–1.70
2.65–2.85
23.8; 26.3
52.6
1.35–1.45; 1.55–1.65
2.45–2.60
26.4; 27.6
53.3–54.0
49.8–50.2
33.1–33.7
41.5–42.0
51.5–53.0
38.0–39.5
40.2–40.8
core: 3
a
b
c
d
e
f
3.53–3.57
34.5–35.5
52.0–53.0
49.8–52.0
29.4–29.9
39.3–40.2
37.2–38.3
3.30–3.43
3.23–3.32
2.60–2.80
3.40–3.52
2.65–3.00
2.65–2.85
2.45–2.60
3.03–3.10
3.30–3.43
3.40–3.52
2.65–3.00
shifts are observed for the groups attached to the salt- drimer (IIIb) can be reached after 18-h reaction at 20°ë
forming nitrogen atoms (Table 1). The line broadening in the presence of 20% (mol) excess of the protected
and decreased intensity of some signals correspond to glycoside (IX). The completeness of the conjugation
the data reported previously for various classes of den- was verified by the disappearance of free amino groups
drimers [18–20].
in the reaction products, which were detected by the
ninhydrin test. After the deprotection with 1 N NaOH
in aqueous methanol (Scheme 2, step iv), (IIIÒ) was
isolated by gel chromatography in 35% yield.
Conjugation of the carbohydrate ligands with
PAMAM carriers could be performed by two methods.
The first of them is based on the use of dendrimer matri-
ces with terminal carboxyl groups (in the form of active
esters) and a glycoside containing an NH2 group in the
spacer arm (Scheme 1, ‡). According to the second
approach (Scheme 1, b), the amino terminated PAM-
AMs (Scheme 2, series b, and Scheme 3) are coupled
with carbohydrate ligands bearing an activated car-
boxyl group in the spacer moiety (see Scheme 4 for the
syntheses of ligands).
To examine feasibility of the first approach, we tried
to synthesize the active ester from the model compound
(IIa). The hydrolysis of ester (IIa) with 1 N NaOH in
aqueous methanol gave the corresponding tetracar-
bonic acid in 93% yield, but we failed to esterify it
quantitatively with both trifluoroacetoxysuccinimide
and p-nitrophenyl trifluoroacetate in the presence of
pyridine.
Conjugation of NH2-terminated PAMAM matrices
with the carbohydrate ligands bearing the activated car-
boxyl residue in the aglycone according to the method
(b) (Scheme 1) allowed us to obtain glycodendrimers in
high yields. PAMAM matrices were conjugated with
BDI glycoside (VIII) (or its per-O-acetylated analogue
(IX)) or with the glycoside of per-é-acetylated
Neu5Acα methyl ester ((XI) synthesized according to
Scheme 4.
The conjugation of nonprotected glycoside (VIII)
instead of its acetylated analogue (IX) allowed us to
exclude the deprotection step and to increase the yield
of conjugate (IIIÒ) up to 74%. Therefore, in subsequent
experiments, we used glycoside (VIII) for the conjuga-
tion with NH2-terminated PAMAMs (IIb)–(VIIb) and
1,6-diaminohexane (I) (Schemes 2, 3). The yields of
(IÒ)–(VIIÒ) were 51–86% after chromatographic puri-
fication.
The derivative (XI) of Neu5Acα bearing the acti-
vated carboxyl moiety in the spacer was conjugated
with NH2-terminated PAMAMs (IVb) and (Vb) and
dendritic polymer (VIb) under similar conditions. The
yields of glycodendrimers (IVd)–(VId) were 40–67%.
The glycodendrimers we obtained were character-
ized by 1H and 13C NMR spectroscopy. The NMR spec-
tra contained all sets of the signals corresponding to the
PAMAM matrix, the spacer group, and also the BDI or
Neu5ÄÒα ligands (Tables 2, 3). When obtaining BDI
conjugates, the completeness of conjugation was con-
firmed by comparing the integral intensities for the sig-
nals corresponding to the protons of fragment d of the
matrix and the protons corresponding to fragments h, g,
and j of the spacer group of the BDI-ligand (see scheme
The conditions of the conjugation reaction of in Table 2). In a similar analysis of the spectra of glyco-
PAMAM matrices with carbohydrate ligands were opti- dendrimers (IVd)–(VId), the intensities of the proton
mized using octaamine (IIIb). We found that the signals of the fragment d of the carrier and the protons
exhaustive substitution of all amino groups in den- corresponding to fragments h and g of the spacer group
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 28 No. 6 2002

474
TSVETKOV et al.
R
R
R
N
N
R
R
NH
O
HN
O
N
R
O
N
R
N
O
O
N
R
H
HN
N
H
R
R
H
N
O
N
N
N
N
O
H
R
N
O
HN
N
O
N
R
NH
O
O
H
O
N
R
N
N
N
N
H
H
R
N
N
N
O
H
R
N
H
(iii)
N
R
R
(VIIc) (76%)
O
R
O
O
O
N
N
H
N
H
N
N
H
O
N
N
R
R
O
N
N
HN
H
O
H
N
O
N
R
H
N
N
N
H
N
O
R
O
O
N
N
O HN
R
O
N
N
N
H
HN
O
R
N
R
R
O
N
R
HN
HN
R
N
R
R
N
R
R
2
(VIIb)
Scheme 3. Synthesis of neoglycoconjugate (VIIc) from 64-dentate PAMAM (VIIb) and ligand (VIII). Designation of substituent
R for series (b) and (c), the reaction conditions, and reagents correspond to those given in Scheme 2.
OH
OH
OH
OH
O
O
OH
OH
vii
O
OH
O
OH
O
H
HO
HO
NH2
O
O
N
HO
HO
O
NO2
O
O
O
HO
(VII)
HO
(VIII) (75%)
OAc
OAc
O
OAc
viii
O
O
OAc
H
AcO
O
N
O
AcO
NO2
O
O
AcO
(IX) (78%)
OAc
AcO
OAc
AcO
COOMe
COOMe
O
ix, x
O
H
AcO
AcO
AcNH
AcO
O
O
N
O
NO2
AcNH
AcO
NHBoc
N
H
O
O
O
(X)
(XI) (78%)
Scheme 4. Synthesis of activated B derivatives (VIII) and (IX): (vii) (CH CH COOC H NO -p) , 1 : 3 DMSO–DMF; (viii)
DI
2
2
6
4
2
2
2
Ac O/Py. The synthesis of activated derivative of Neu5Ac (XI): (ix) 20% TFA/CHCl ; (x) (CH CH COOC H NO -p) , DMF,
3
2
3
2
6
4
2
2
Et N.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 28 No. 6 2002

NEOGLYCOCONJUGATES BASED ON DENDRIMER POLY(AMINOAMIDES)
475
Table 2. Chemical shifts (δ, ppm) of resonances in the 1H and 13C NMR spectra of glycoconjugates (IIc)–(Vc) and (VIIc) (D2O)
c
d
e
f
g
h
h
g
i
j
k
O
a
b
CH2CH2CNHCH2CH2NHCCH2CH2CH2CH2CNHCH2CH2CH2OBDI
CNHCH2CH2N
O
O
O
pH 3
pH 5.5
pH 10
Fragment
1H
13C
1H
13C
1H
13C
a
3.12–3.2
3.58–3.63
3.41–3.47
2.68–2.75
3.12–3.20
2.1–2.14
1.42–1.47
3.12–3.2
1.76
37.20
48.64
3.18–3.35
2.53–2.80
2.72–2.98
2.32–2.52
3.18–3.35
2.15–2.27
1.48–1.62
3.18–3.35
1.81
37.20
50.90
49.74
3.35–3.47
2.61–2.67
2.68–2.76
2.39–2.47
3.30–3.47
2.22–2.30
1.55–1.65
3.35–3.47
1.70–1.80
3.95
36.32
50.80
49.99
33.27
b
c
51.60
d
–
33.0–34.0
e + f
39.68; 39.08
36.16
39.62; 39.37
36.32
39.44; 39.28
36.19
g
h
i
25.62; 25.56
37.20
25.72; 25.68
37.20
25.59
37.16
j
29.21
29.34
29.21
k
3.81
69.01
3.92
69.10
68.45
Table 3. Chemical shifts (δ, ppm) of resonances in the 1H and 13C NMR spectra of glycoconjugates (IVd) and (Vd) (D2O)
c
d
e
f
g
h
h
g
i
j
k
O
a
b
CH2CH2CNHCH2CH2NHCCH2CH2CH2CH2CNHCH2CNHC6H4CH2O
CNHCH2CH2N
O
O
O
O
Fragment
1H
13C
a
b
c
d
g
h
j
3.3–3.42 m
3.17–3.25 m
2.9–3.05 m
39.4
50.3
49.8
2.38–2.54 m
2.13–2.3 m
31.6
36.1
1.47–1.55 m
7.32 m; 7.38 m
4.41‰; 4.64 ‰
25.5
122.4; 130.4
67.4
k
of the Neu5ÄÒα ligand (see scheme for Table 3) were
The chemical shifts of the signals of BDI and the
spacer group in the 1H and 13C NMR spectra of glyco-
dendrimers are pH-independent and correspond to the
data for the starting aminopropyl glycoside (VII) [21].
The values of chemical shifts for the signals of the
Neu5ÄÒα ligand and the spacer group in the 1H and 13C
NMR spectra of glycodendrimers correspond to the
data for the unprotected Neu5ÄÒα glycoside[22]. We
did not study their pH-dependence.
compared.
As in the case of the starting dendrimers, the chem-
ical shifts of the PAMAM matrix fragments in the spec-
tra of glycoconjugates (IIc)–(VÒ) and (VIIÒ) depend on
pH of solution (Table 2). The signals of the PAMAM
core are observed only in the spectra of conjugates
(IIÒ)–(IVÒ), with those of (IVÒ) being noted only at pH
3. This is likely takes place due to the presence of
extended conformation of the molecule. Along with the
pH growth, the maximum changes in the 1H NMR spec-
tra are observed for the signals corresponding to the
CH2 groups of the fragments b and c. In the 13C NMR
spectra, the dependence of chemical shifts on the pH
changes is less noticeable. It should be noted that the
signals corresponding to fragments d are well resolved
at pH 10, broadened at pH 5.5, and are absent at pH 3.
Conformational Analysis of PAMAM Matrices
and BDI-Glycodendrimers
To assess the distances between the carbohydrate
ligands in the synthesized dendritic glycoconjugates,
we studied the conformational and geometric charac-
teristics of BDI-glycodendrimers (IIÒ)–(VIIÒ) and the
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 28 No. 6 2002

476
TSVETKOV et al.
(‡)
(b)
(c)
(d)
(e)
(f)
Fig. 1. (a, c, e) Extended and (b, d, f) compacted conformations of (a, b) PAMAM carrier (VIIb), (c, d) of B -based conjugate
DI
(VIIc), and (e, f) of B –PAA conjugate.
DI
starting PAMAM (IIb)–(VIIb) by computer modeling
[23].
The transitions from the extended (Fig. 1a) to the
compact conformations (Fig. 1b) were found to be
characteristic of PAMAM matrices (IIb)–(VIIb). The
distances between the opposite (antipode) NH2 groups
in the compact conformations (Table 4) were approxi-
mately half of the maximum value. The time for mole-
cule compaction grows for each next generation of den-
The computational experiments for PAMAMs were
performed by the molecular dynamics method using
the vacuum approximation. The run times of 10–15 ps
were used, and all the starting molecules had the
extended conformation with stretched out chains drimers. It was also found that the surface occupied by
(Figs. 1a, 1c, 1d).
the terminal NH2 groups is only a part of the total
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 28 No. 6 2002

NEOGLYCOCONJUGATES BASED ON DENDRIMER POLY(AMINOAMIDES)
477
Table 4. Characteristics of PAMAM dendrimers obtained by the method of molecular dynamics
PAMAM
Valence
2
Dmax, Å*
Dmin, Å*
V, Å3**
S, Å2**
(I)
10
–
compacted conformation
(IIb)
4
8
15
26
7–10
4051
1620
1630
extended conformation
7–15
4142
(IIIb)
compacted conformation
14–25
39
4–16
6654
1992
3637
extended conformation
10–27
9582
(IVb)
(Vb)
16
32
64
compacted conformation
16–32
55
3–19
12873
3220
7539
extended conformation
8–25
20595
compacted conformation
30–46
62
6–13
28441
7168
extended conformation
9–30
35470
10298
(VIIb)
compacted conformation
35–49
84
7–13
50598
10111
26423
extended conformation
6–35
83367
* D
and D are the distances between terminal NH groups.
min 2
max
** The area of molecule surface (S), which is accessible for the probe with a radius of 3 Å, and the volume (V) are computed according to
[34] using the values of atomic radii reported in [35].
molecular surface for both the extended and the com- based on PAA containing 10 mol% of BDI ligands (BDI-
pacted conformations; i.e., formation of the “dense sur-
face” by the terminal groups [24] is not observed.
PAA). This conjugate possesses the activity two orders
of magnitude higher than that of free BDI and was used
as a positive reference in immunological tests [26].
The study of glycodendrimers (IIÒ)–(VIIÒ) by the
molecular dynamics method has demonstrated that the
carbohydrate residues are situated on the surface of
conjugates and are not immersed into the dendrimer
matrix for both extended and compacted conformations
(Figs. 1c, 1d). Such disposal of the carbohydrate
ligands is important from the viewpoint of their acces-
sibility for recognition by proteins; evidently, it should
be stabilized additionally by a solvation. The largest
possible distances between anomeric oxygen atoms of
the β-Gal carbohydrate ligands were computed; they
are given in Table 5. In the case of 32- and 64-valent BDI
conjugates (VÒ) and (VIIÒ), the distances turned out to
be close to the distance between the Fab-fragments in
IgG (≥100 Å) [25], and one could expect an enhanced
efficiency of binding of these glycodendrimers to two
immunoglobulin sites.
The study for 15-ps run time of the molecular
dynamics of the fragment of BDI–PAA glycopolymer
consisting of 50 PAA units and containing one BDI res-
idue per 10 PAA units (Fig. 1e) demonstrated a conver-
sion of the starting extended chain into the loop-like
structure (Fig. 1f). The distance between the anomeric
oxygen atoms of the β-Gal terminal residues of BDI
ligands in this fragment can vary from 10 to 80 Å and,
in the case of the nearest neighboring BDI ligands, from
15 to 30 Å. It is hence obvious that, in the case of the
PAA glycoconjugate with a mean molecular mass of
the carrier of 40 kDa (~350 units), interligand distances
vary in a rather wide range, whereas glycodendrimers
under study are characterized by narrow intervals of
distances between carbohydrate residues. For example,
(IIÒ) and (VIIc) exhibit the maximum distances of
We compared the geometrical parameters of the about 45 and 110 Å, respectively; whereas the lowest
PAMAM matrices and glycodendrimers with the data distances in them are the same (several angstroms)
obtained for the fragment of the polymeric conjugate (Table 5).
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 28 No. 6 2002

478
TSVETKOV et al.
Table 5. The computed distances between the anomeric ox-
ygen atoms of β-Gal in BDI–êÄåÄå glycoconjugates and
the ability of these glycoconjugates to inhibit the binding of
anti-BDI–IgG to immobilized BDI–PAA
therefore, these data are not illustrated in figures. The
efficiency of glycoconjugates (IVc), (Vc), and (VIIc)
was higher than that of nonconjugated BDI, but was
nearly the same as that of BDI–PAA.
The maximum
Amount of
The effective concentrations that inhibit the binding
of IgM antibodies and the cytotoxicity of human serum
toward the PK-15 cell culture [27] (Fig. 3) were
approximately the same as in the case of the binding
inhibition of IgG antibodies. Among all the compounds
tested, only glycodendrimers (Vc) and (VIIc) were
found to inhibit the IgM binding to immobilized BDI–
PAA (Fig. 2b) at lower concentrations than monomer
(VII) did.
Compound
BDI
interligand IC50, µM**
BDI residues
distance, Å
1
~35
2
–
–
500
9
BDI-PAA*
(IÒ)
35
45
65
80
100
110
500
500
500
60
(IIÒ)
4
(IIIÒ)
(IVÒ)
(VÒ)
8
Thus, for both experimental models of inhibition of
the antibody binding and cytotoxicity, the maximum
activity was observed for BDI-dendrimers (Vc) and
(VIIc), which have the highest valence and the maxi-
mum distances between carbohydrate residues that are
close to the distance between the Fab-domains of anti-
bodies. However, the sudden change in the activity of
these conjugates was not so appreciable [1, 8], as one
could expect from the realization of two-center binding
of glycodendrimer molecules to antibodies (Fig. 4c).
16
32
64
40
(VIIÒ)
35
* Average degree of polymerization is ~350; the content of B
residues is 10 mol %.
** Concentration of 50% inhibition.
DI
Glycodendrimers containing neuraminic acid have
not been investigated by the method of molecular
dynamics, because there is no plausible estimation of
the distribution of charged carboxyl and amino groups
in the conjugate molecule. However, a rough estimation
of the maximum distance between O2 atoms of the
Neu5ÄÒα residues in the extended conformation is
possible. In the case of glycodendrimer (IVd), it is
about 80–90 Å. This value exceeds (see below, Fig. 5a)
the distance between the carbohydrate-binding sites in
HA (46 Å according to crystallographic data [28, 29])
and approximately corresponds to the range of possible
distances between carbohydrate-binding sites of neigh-
boring HA molecules, which are flexibly anchored in
the viral membrane. This range can be estimated from
the distance between the centers of HA trimers (105–
109 Å) obtained by the method of low-angle neutron
scattering [30]. The intertrimeric distance can be 60–
90 Å for various relative orientations of HA, and this
value is close to the series of possible interligand dis-
tances in dendrimer (IVd) and its homologues (Vd) and
(VId).
It seems likely that the most distant BDI residues in
conjugates (Vc) and (VIIc) are not optimally oriented
for the simultaneous incorporation into the carbohy-
drate-binding sites of antibodies, while the distances
between other pairs of carbohydrate ligands are too
small (<100 Å) for such an interaction. The dendrimers
under study were also less active than BDI–PAA in
which the orientation and relative positions of carbohy-
drate ligands necessary for the two-center binding to
antibodies are ensured by the flexibility of linear poly-
meric matrix (Fig. 4d).
A growth in the inhibitory activity along with the
growth in their valence observed in the series of
PAMAM conjugates (IIÒ)–(VIIÒ) is most likely con-
nected with the increase in the local concentration of
BDI ligands near Fab-domains rather than with the ful-
fillment of the two-center interaction (Fig. 4b). One
more explanation of the relatively low activity of den-
drimers is based on the presumption of possibility of
their cross binding to antibodies (Fig. 4e). The forma-
tion of polymolecular complexes should decrease the
entropy of the system under discussion, and the total
energy gain as a result of such multivalent interaction
may be negligible.
The Study on the Biological Activity
of Glycodendrimers
The interaction of BDI-glycodendrimers with human
blood serum containing the BDI-specific antibodies was
studied by ELISA using BDI–PAA as the immobilized
antigen [26]. Serum was preliminarily incubated with
the inhibitor taken in serial dilutions [27], and then
placed in the plate wells. The bound anti-BDI immuno-
globulins were detected using the isotype-specific anti-
bodies (Fig. 2).
It was expected that a decrease in pH of glycoden-
drimer solutions could cause changes in the conforma-
tion of the carriers due to a repulsion of the positively
charged quaternary ammonium groups generated in the
branching centers of PAMAM chains. Consequently,
the activity of dendrimer conjugates could change due
to an increase in interligand distances. However, our
attempts to increase the inhibitory activity of BDI-gly-
codendrimers (Vc) and (VIIc) by changing the medium
pH failed.
Glycoconjugates (Ic)–(IIIc) bound the IgG class
xenoantibodies (Fig. 2a) like monomeric BDI (VII);
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NEOGLYCOCONJUGATES BASED ON DENDRIMER POLY(AMINOAMIDES)
Cytotoxicity, %
479
Inhibition, %
100
100
(‡)
80
80
60
40
20
0
60
40
20
0
10000
1000
100
10
1
0.1
[BDI], µM
100
80
60
40
20
(b)
Fig. 3. Inhibition of cytotoxicity of human serum toward the
PK15 cell culture by glycodendrimers. Serum was incu-
bated with serial dilutions of (᭹) B , (᭿) glycodendrimer
DI
(IVc), (ᮀ) glycodendrimer (Vc), and (᭡) B –PAA and then
DI
examined using the cytotoxicity test [27]. Cytotoxicity of
serum in the absence of inhibitors is taken as 100%.
drimers (IVd) and (VId) one and two orders of magni-
tude, respectively, in comparison with that of
Neu5ÄÒα-OBzl (Table 6).
0
10000 1000
100
10
1
0.1
0
In the case of influenza virus, the multivalent inter-
action of Neu5ÄÒα–PAMAM conjugates with HA can
be realized in two variants. The first consists in a simul-
taneous interaction of the molecule of one conjugate
with all the carbohydrate-recognizing sites of one HA
trimer. This variant seems to be improbable because of
a number of reasons. Although the distances between
the Neu5ÄÒα ligands in the glycodendrimers under
study presumably allow the intratrimer binding
(Fig. 5a), the necessary orientation of carbohydrate
moieties (dictated by the direction of their interaction
with receptors) and their real orientation (dictated by
the geometry of the molecule of conjugate) may differ
from one another. Moreover, own carbohydrate chains
of HA can create a steric hindrance to this interaction.
Therefore, the mechanism involving the simultaneous
interaction of one conjugate with various HA trimers,
i.e., intertrimer binding (Fig. 5b), seems to be more
probable.
[BDI], µM
Fig. 2. (a, b) Inhibition of binding of human anti-B immu-
DI
noglobulins (a, IgG; b, IgM) in ELISA by B conjugates:
DI
(᭿) glycodendrimer (IVc), (ᮀ) glycodendrimer (VÒ) (᭞)
glycodendrimer (VIIc), (᭹) B (reference), and (᭢) B
–
DI
DI
PAA.
The sialodendrimers (IVd)–(VId), we synthesized,
were studied as the inhibitors of influenza virus adhe-
sion in ELISA. The method is based on the competitive
interaction of peroxidase-labeled fetuin and the inhibi-
tor with viral particles adsorbed on the surface of poly-
styrene plates [29]. The dissociation constants for
virus–inhibitor complexes are given in Table 6; the
smaller constant corresponds to a higher inhibition
degree.
In the primary test, the samples of sialodendrimers
(IVd) and (Vd) demonstrated a low inhibitory activity
close to that of monomeric Neu5ÄÒα α-glycoside. As
a possible explanation of this fact, we speculated that
the negatively charged Neu5ÄÒα residues interact with
the positively charged ammonium groups of the carrier.
This process should result in shielding the ligands and
hindering the contacts of dendrimers with the viral sur-
face. If this assumption were true, a change in the pH
value of dendrimer solutions should result in enhance-
ment of their inhibitory properties. In fact, during the
test, we have established that the incubation for 18 h at
4°ë in 0.1 M aqueous ammonia and subsequent neu-
tralization have enhanced the activities of glycoden-
Table 6. Dissociation constants for virus–sialoside complexes.
Virus strain A/NIB/44/90M
Sialoside
Kd, µM
Kd, µM**
Neu5AcαBzl
(IVd)
0.1
0.2
0.1
–
0.1
0.01*
–
(Vd)
(VId)
<0.001*
* Data for samples preincubated in 0.1 M ammonia.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 28 No. 6 2002

480
TSVETKOV et al.
(‡)
(b)
(c)
(d)
Monovalent
binding
Monovalent
binding
Multivalent
directed
binding
Multivalent
statistical
binding
BDI–PAA
BDI
B
DI–PAMAM
logG
logG
Kddendr
BDI–PAMAM
logG
logG
Kdpol
Kdmon
≥
Kddendr
>>
≥
(e)
BDI–PAMAM
logG
logG
logG
Fig. 4. Neutralization of xenoantibodies with mono- and multivalent B ligands (K is the dissociation constant for the ligand–
DI
d
antibody complex).
Neu5Ac-PAMAM
(b)
(‡)
Hemagglutinin
60–90 Å
105–109 Å
46 Å
Fig. 5. An analysis of probable variants of interaction of sialodendrimers with viral surface: (a) intratrimer and (b) intertrimer bind-
ing.
Some increase in antiviral activity that was observed from the crosslinked PAMAM molecules (as well as
PAA conjugates) can also be explained by the possible
realization of several intertrimer interactions and also
by the blockade of virus with bulky glycoconjugate
molecules, which sterically hinder the virus approach-
ing to its receptor [31].
for conjugate (IVd) after its incubation in aqueous
ammonia can be explained by the intratrimer interac-
tion. This corresponds to the previous presumption of
the necessity of favorable orientation of ligands, in
addition to the necessary correlation of the distances
between the O2 atoms of Neu5ÄÒα residues in the
extended conformation and the range of the possible
distances between carbohydrate-binding sites of HA
trimers (Fig. 5). At the same time, a high activity of
Unfortunately, the detailed study of the properties of
sialoglycoconjugates (IVd)–(VId) was complicated by
their lability in solution, especially in the case of (VId).
Thus, the solutions of glycodendrimers coagulated
conjugate (VId) on the basis of the carrier constructed when being stored at neutral or alkaline pH values. A
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NEOGLYCOCONJUGATES BASED ON DENDRIMER POLY(AMINOAMIDES)
481
comparison of the characteristics of sialodendrimers panol (25 ml). Column chromatography of the residue
reported previously [11] with those of our conjugates (elution with 96% aqueous ethanol) yielded 0.9 g
(IVd)–(VId) allows one to suppose that the reological (82%) of (IIa) as a colorless syrup, Rf 0.56 (MeOH) and
0.3 (3 : 1 : 1 n-BuOH–AcOH–H2O); 1H NMR (CDCl3):
3.57 (12 H, s, CH3O), 2.66 (8 H, t, éë(é)ëH2ëH2),
2.34 (8 H, t, éë(é)ëH2ëH2), 2.30 (4 H, t,
(ëH2)2NëH2), and 1.15–1.35 (8 H, m, (ëH2)4); 13C
NMR (CDCl3): 173.0 (ëO), 51.3 (CH3O), 32.2
(éë(é)ëH2ëH2), 49.0 (éë(é)ëH2ëH2), 53.5
((ëH2)2NëH2), and 26.9 and 27.0 ((ëH2)4).
instability of glycodendrimer solutions may be caused
by the processes associated with the presence of the
hydrophobic aromatic nucleus in the spacer group
instead of the short aliphatic spacer in the case of sialo-
dendrimers previously described [11]. Note that we
used such spacer group intentionally, as the α-glyco-
sides of Neu5ÄÒα with an aromatic aglycone are
known to inhibit the adhesion of this influenza virus
strain more effectively than the corresponding aliphatic
spacer-armed glycosides [22].
Aminoamide (IIb). EDA (2 ml, 30 mmol) was
added to aminoester (IIa) (0.68 g, 1.5 mmol), and the
reaction mixture was stirred for 24 h at 20°ë. The reac-
tion mixture was evaporated, and the traces of EDA
were removed in a vacuum by coevaporation with water
(2 × 20 ml). The residue was purified by gel filtration of
TSK HW-40F (2.5 × 90 cm) in 0.5% aqueous ammonia.
Fractions containing the front peak were evaporated to
give 0.57 g (67%) of aminoamide (IIb); a light yellow
oil; Rf 0.32 (3 : 1 MeOH–H2é); 13C NMR (D2O): 176.5
(ëO), 42.8 (NH2ëH2), 41.0 (ëH2NHë(é)), 33.8
(NHë(é)ëH2ëH2), 50.0 (NHë(é)ëH2ëH2), 53.8
((ëH2)2NëH2), and 26.9 and 27.9 ((ëH2)4).
Thus, the use of polyvalent (polydentate) PAMAM
carriers offers promise for obtaining glycoconjugates
as molecular probes with high content of active ligands.
The synthesis of medium-sized PAMAM dendrimers
bearing up to 32 terminal NH2 groups is not trouble-
some, whereas the obtaining of more bulky molecules
is a considerable synthetic problem and leads to a com-
plicated analysis of the conjugates based on them.
Moreover, it should be taken into account that the con-
formation and, consequently, biological activity of gly-
coconjugates based on PAMAM carriers depends on
the presence of charge of the carbohydrate ligand.
Aminoester (IIIa). Methyl acrylate (1.9 ml,
20.9 mmol) was added to a solution of aminoamide
(IIb) (0.17 g, 0.3 mmol) in absolute MeOH (1 ml). The
reaction mixture was stirred for 24 h at 20°ë and evap-
orated in a vacuum. The traces of methyl acrylate were
removed in a vacuum by coevaporation with isopro-
panol (25 ml). Column chromatography of the residue
(elution with 96% aqueous ethanol) resulted in 0.23 g
(62%) of (IIIa); colorless oil; Rf 0.24 (10 : 1 åÂéç–
EXPERIMENTAL
Reagents from Merck (Germany) and Fluka (Swit-
zerland) were used. PAMAM-G4, 64-dentate den-
drimer (VIIb), was purchased from Dendritech Inc.
(United States). Solvents and reagents were purified
according to the procedures reported previously in [32].
Methyl acrylate was stabilized after distillation by addi-
tion of 0.01% di-(tert-butyl)phenol to avoid its possible
radical polymerization. The reactions were monitored
by TLC on Silica gel 60 (Merck, Germany) precoated
plates. The spots were visualized by charring with 2%
H2SO4 (~150°C), ninhydrin, or chlorine–toluidine
reagent or an exposure to iodine vapor [33]. Solvents
from the reaction mixtures were removed in a vacuum
at temperature below 40°ë. Column chromatography
was performed on Silica gel 60 (32–63 µm, Merck,
Germany). Gel chromatography was carried out on
TSK gels HW-40F, HW-50F, and HW-55F (TOYO
SODA Manufacturing Co., Japan). NMR spectra were
registered on an AM-300 (Bruker, Germany) spectrom-
eter. The experiments on the two-dimension correlation
NMR spectroscopy were performed according to the
standard procedures on a DRX-500 (Bruker, Germany)
instrument. The values of chemical shifts (ppm) and
coupling constants (Hz) for the characteristic signals in
the NMR spectra are given.
1
H2é) and 0.7 (40 : 1 MeOH—25% ammonia); H
NMR (CDCl3): 7.15 (4 H, br. s, NH), 3.63 (24 H, s,
CH3O), 2.40 (16 H, br. t, éë(é)ëH2ëH2), 2.32 (8 H,
m, NHë(é)ëH2), 2.71 (24 H, m, éë(é)ëH2ëH2 and
NHë(é)ëH2ëH2), 2.50 (8 H, m, (ëH2)2NëH2ëH2NH),
3.24 (8 H, m, CH2NHë(é)), and 1.15–1.45 (8 H, m,
(ëH2)4); 13C NMR (CDCl3): 172.0 (C(O)O), 172.9
(C(O)NH), 53.4 (ëH2N(ëH2ëH2ë(é)NH)2), 53.1
(ëH2N(ëH2ëH2ë(é)O)2), 51.4 (CH3O), 49.6
(N(ëH2ëH2ë(é)NH)2), 49.3 (N(ëH2ëH2ë(é)O)2),
37.1 (ë(é)NHëH2), 33.6 (ëH2ë(é)NH), 32.7
(ëH2ë(é)é), 26.8, and 26.8 and 27.5 ((ëH2)4).
Aminoamide (IIIb) was obtained from aminoester
(IIIa) (0.98 g, 0.78 mmol) and EDA (2 ml, 30 mmol) as
described for (IIb); yield: 0.88 g (74%); light yellow
oil, Rf 0.4 (3 : 2 MeOH–25% ammonia); 13C NMR
(D2O): 176.7 and 176.5 (C=O), 53.3 and 51.9
(NëH2ëH2NH), 49.7 and 49.4 (NëH2ëH2C(O)), 42.1
(C(O)NHëH2ëH2NH2), 40.4 (C(O)NHëH2ëH2NH2),
Aminoester (IIa). Methyl acrylate (8.8 ml, 98 mmol)
was added to 1,6-diaminohexane (I) (1.1 g, 9.8 mmol).
The mixture was stirred for 24 h at 20°ë and evaporated
in a vacuum. The traces of methyl acrylate were
37.4
(NëH2ëH2ë(é)NH), and 27.4 and 26.3 ((ëH2)4).
Aminoester (IVa) was obtained from aminoamide
(C(O)NHëH2),
33.5
and
33.3
removed in a vacuum by coevaporation with isopro- (IIIb) (0.19 g, 0.12 mmol) and methyl acrylate
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 28 No. 6 2002

482
TSVETKOV et al.
(0.22 ml, 2.5 mmol) as described for (IIa); yield: 0.123 g (C(O)NHëH2ëH2NH2), 40.4 (C(O)NHëH2ëH2NH2),
(24%); yellow oil; Rf 0.5 (60 : 1 MeOH–25% ammo-
39.2 and 37.6 (C(O)NHëH2), and 33.6 and 33.4
nia); 1H NMR (ëDCl3): 3.62 (48 H, s, OCH3), 3.25 (24 (NëH2ëH2ë(é)NH).
H, m, C(O)NHëH2ëH2N), 2.73 and 2.70 (56 H, m,
PAMAM polymer (VIb). EDA (58 µl, 0.88 mmol)
was added to a solution of aminoester (Va) (200 mg,
0.033 mmol) in MeOH (0.5 ml). After 24 h, the reaction
mixture was evaporated, and the traces of EDA were
removed in a vacuum by coevaporation with water (2 ×
20 ml). The residue was purified by gel filtration on
TSK HW-55F (2.5 × 90 cm). The factions correspond-
ing to the retention volume of ~130 ml were concen-
trated and lyophilized to yield 8 mg (8%) of (VIb). 1H
NMR (D2O): 3.35 and 3.17 (br. m, C(O)NHCH2), 2.83
(br. m, (ëH2)2NëH2ëH2NH and CH2NH2), 2.63 (br. m,
NHC(O)CH2CH2N),
NCH2CH2NHC(O)),
MeOC(O)CH2CH2N),
NHC(O)CH2CH2N), and 1.45–1.10 (8 H, m, (CH2)4);
13C NMR (CDCl3): 172.8 and 172.2 (C=O), 52.36
(NCH2CH2NHC(O)), 51.5 (OCH3), 49.7 and 49.0
(NHC(O)CH2CH2N), 36.97 (NCH2CH2NHC(O)), 33.6,
33.2, and 32.5 (NHC(O)CH2CH2N), and 27.3 and 26.3
((ëH2)4).
2.5
2.38
2.3
(24
(32
(24
H,
H,
H,
m,
m,
m,
Aminoamide (IVb). EDA (180 µl, 2.5 mmol) was
added to a solution of aminoester (IVa) (0.86 g,
(ëH2)2NëH2ëH2NH),
and
2.45
(br.
m,
0.03 mmol) in absolute MeOH. The reaction mixture NCH2CH2C(O)N); 13C NMR (D2O): 176.9 (C=O), 53.9
was stirred for 48 h at 20°ë and treated as described for
(IIb) to isolate 0.74 g (74%) of aminoamide (IVb);
light yellow oil, Rf 0.75 (3 : 1 MeOH–25% ammonia);
13C NMR (D2O): 175.7 and 175.3 (C=O), 53.5, 53.5,
and 51.1 (NëH2ëH2NH), 50.0 and 49.7
(NëH2ëH2C(O)), 42.1 (C(O)NHëH2ëH2NH2), 40.6
(C(O)NHëH2ëH2NH2), 39.1 and 37.6 (C(O)NHëH2),
33.7 and 33.6 (NëH2ëH2ë(é)NH), and 27.4 and 26.3
((ëH2)4).
((ëH2)2NëH2ëH2NH),
((ëH2)2NëH2ëH2NH),
(C(O)NHCH2), and 35.1 ((ë(é)ëH2ëH2)2N).
52.4
42.0
and
(CH2NH2),
51.5
39.2
a
b
(3-(Aminopropyl) 3-O- -D-galactopyranosyl- -
D-galactopyranoside (VII) was obtained in 62% yield
by the modified method [21] using AgOSO2CF3 as the
1
promoter; H NMR (D2O): 5.14 (1 H, d, J2, 1 4, H1 in
Galα), 4.47 (1 H, d, J2, 1 8, H1 in Galβ), 4.18 (2 H, m,
H4 in Galβ, H5 in Galα), 4.05 (1 H, m, OCH), 4.01 (1
H, br. d, H4 in Galα), 3.94 (1 H, dd, J2, 3 10, J4, 3 3.5, H3
in Galα), 3.86 (1 H, dd, J3, 2 10, H2 in Galα), 3.80 (m,
OCH’), 3.65 (1 H, dd, H2 in Galβ), 3.09 (2 H, t, CH2N),
and 1.97 (2 H, m, CH2).
Aminoester (Va). Methyl acrylate (0.29 ml,
3.2 mmol) was added to a solution of aminoamide
(IVb) (0.13 g, 0.04 mmol) in absolute MeOH. The
reaction mixture was stirred for 48 h at 20°ë and
treated as described for (IIa) to isolate 0.14 g (58%) of
aminoester (Va); yellow oil; Rf 0.5 (MeOH); 1H NMR
(ëDCl3): 3.62 (96 H, s, OCH3), 3.29 (56 H, m,
CH2NHC(O)), 2.75 (64 H, t, C(O)CH2CH2N), 2.52 (32
H, t, C(O)NHëH2ëH2N), 2.41 (64 H, t,
OC(O)CH2CH2N), 2.35 (m, NHC(O)CH2), and 1.25 (m,
3-[(4-Nitrophenyloxy)carbonylpentanoylamido]-
propyl 3-O-(a-D-galactopyranosyl)-b-D-galactopy-
ranoside (VIII). A solution of bis(4-nitrophenyl)adi-
pate (287 mg, 0.74 mmol) in DMF (1.7 ml) was added
at vigorous stirring to a solution of disaccharide (VII)
(74 mg, 0.19 mmol) in DMSO (0.5 ml). The reaction
mixture was stirred for 0.5 h at 20°ë and applied onto
a Sephadex LH-20 (1.7 × 50 cm) column eluted with
1 : 1 MeCN–H2O. Concentration and lyophilization of
the corresponding fractions yielded 90 mg (75%) of
(VIII); Rf 0.67 (4 : 3 : 2 isopropanol–EtOAc–H2O); 1H
NMR (D2O): 8.38 and 7.43 (2 × 2H, 2 d, J 9.5, 4-nitro-
phenyloxy), 5.19 (1 H, d, J1, 2 4, H1 in Galα), 4.48 (1 H,
d, J1, 2 8, H1 in Galβ), 4.23 (1 H, br. t, H5 in Galα), 4.20
(1 H, br. d, H4 in Galβ), 4.06 (1 H, br. d, H4 in Galα),
4.01 (1H, m, OCH), 4.00 (1 H, dd, H3 in Galα), 3.91
(1 H, dd, J3, 2 10, H2 in Galα), 3.78 (2 H, m, 2 H6 in
Galα), 3.76 (1 H, m, OCH’), 3.69 (1 H, t, J3, 2 9, H2 in
Galβ), 3.36 (2 H, m, CH2N), 2.77 (2 H, m, NHCOCH2),
2.36 (2 H, t, CH2COO), 1.89 (2 H, m, CH2), 1.78 (4 H,
(CH2)4); 13C NMR (CDCl3): 173.0 (C(O)O) and 172.3
(C(O)N),
51.5 (NH2CH2CH2NHC(O)),
52.36
(NCH2CH2NHC(O)),
49.7 and 49.0
(NHC(O)CH2CH2N), 36.97 (NCH2CH2NHC(O)), 33.6
and 32.5 (NHC(O)CH2CH2N), and 27.3 and 26.2
((ëH2)4).
Aminoamide (Vb). EDA (780 µl, 11.7 mmol) was
added to aminoester (Va) (0.44 g, 0.073 mmol) dis-
solved in absolute MeOH. The reaction mixture was
stirred for 48 h at 20°ë and evaporated. The traces of
EDA were removed in a vacuum by coevaporation with
water (2 × 20 ml). The residue was purified by gel fil-
tration on TSK HW-40F (2.5 × 90 cm) in 0.5% aqueous
ammonia and subsequent rechromatography on TSK
HW-50F (2.5 × 90 cm) in the same solvent. The frac-
tions containing aminoamide (Vb) were concentrated
and dried in a vacuum to yield 0.38 g (74%) of (Vb);
light yellow oil; Rf 0.45 (3 : 2 MeOH–25% ammonia);
m, CH2CH2); 13C NMR (DMSO-d6): 171.8 (CO), 171.1
(CO), 155.4 (C4, 4-nitrophenyloxy), 144.9 (C1,
4-nitrophenyloxy), 125.2 (C3 and C5, 4-nitropheny-
loxy), 123.2 (C2 and C6, 4-nitrophenyloxy), 103.1 (C1,
Galβ), 96.2 (C1, Galα), 60.3 and 60.2 (C6, Gal), 35.8
13C NMR (D2O): 176.8 and 176.2 (C=O), 52.1, 51.2,
and 49.7 (NëH2ëH2NH), 49.8 (NëH2ëH2C(O)), 40.8
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 28 No. 6 2002

NEOGLYCOCONJUGATES BASED ON DENDRIMER POLY(AMINOAMIDES)
483
(CH2N), 35.0 (CH2COO), 33.2 (NHCOCH2), 29.4
(CH2CH2CH2), and 24.6 and 23.8 (CH2CH2).
Conjugate (Ic). A solution containing 0.4 mg
(3.4 µmol) of 1,6-diaminohexane (I) in DMF was
added to a solution of active ester (VIII) (5.1 mg,
8.0 µmol) in DMF (0.1 ml). After 24 h, the reaction
mixture was evaporated, and the residue was applied
onto a Lichrosorb RP-8 (4 × 250 mm) column eluted
with 80% aqueous MeCN. The fractions containing
glycoconjugate (IÒ) were concentrated and lyophilized
to yield 3.1 mg (79%) of (Ic), Rf 0.50 (3 : 1 MeOH–25%
ammonia) and 0.22 (3 : 1 : 1 n-BuOH–AcOH–H2O); 1H
NMR (D2O): 5.12 (2 H, d, J1, 2 4, H1 in Galβ), 4.41
(2 H, d, J1, 2 8, H1 in Galα), 4.20 (2 H, br. t, H5 in
Galα), 4.18 (2 H, br. d, H4 in Galβ), 4.06 (2 H, br. d, H4
in Galα), 3.93 (4 H, br. t, OCH2CH2CH2N), 3.26 (4 H,
t, CH2N), 3.15 (4 H, br. m, CH2N), 2.24 (8 H, m,
NHC(O)CH2), 2.16 (4 H, t, OCH2CH2CH2N), 1.57
(8 H, m, NHC(O)CH2CH2), 1.45 (4 H, br. m,
NHCH2CH2CH2CH2), and 1.27 (8 H, br. m, (CH2)4).
3-[(4-Nitrophenyloxy)carbonylpentanoylamido]-
propyl 3-O-(2,3,4,6-tetra-O-acetyl-a-D-galactopyr-
anosyl)-2,4,6-tri-O-acetyl-b-D-galactopyranoside (IX).
A solution of (VIII) (30 mg, 0.046 mmol) in a mixture
of Ac2O (1 ml) and Py (0.2 ml) was kept for 18 h, evap-
orated in a vacuum, and traces of the reagents were
removed by coevaporation with toluene. Column chro-
matography in 40 : 1 CHCl3–MeOH yielded 34 mg
(78%) of (IX); Rf 0.53 (15 : 1 CHCl3–isopropanol); 1H
NMR (CDCl3): 8.22 and 7.25 (2 × 2H, 2 d, J 8.9, p-
C6H4–NO2), 6.08 (1 H, br. t, NHCO), 5.40 and 5.33 (2 ×
1 H, 2 br. d, H4 in Galα and H4 in Galβ), 5.24 (1 H, dd,
J1, 2 3.5, J2, 3 10.5, H2 in Galα), 5.21 (1 H, d, J1, 2 3.5,
H1 in Galα), 5.17 (1 H, dd, J1, 2 8, J3, 2 10.5, H2 in
Galβ), 5.11 (1 H, dd, J3, 4 3.3, H3 in Galα), 4.33 (1 H,
d, J1, 2 8, H1 in Galβ), 4.26 (1 H, br. t, H5 in Galα), 4.14
(1 H, dd, J5, 6 7, H6 in Galα), 4.13 (1 H, dd, H6 in Galβ),
4.09 (1 H, J5, 6' 7, J6, 6' 11, H6' in Galβ), 4.01 (1 H, dd,
J5, 6' 6.5, J6, 6' 11.5, H6' in Galα), 3.96 (1 H, m, OCH),
3.88 (1 H, dd, J4, 3 3.3, H3 in Galβ), 3.80 (1 H, br. t, H5
in Galβ), 3.51 (1 H, m, OCH'), 3.40 and 3.27 (2 × 1 H,
m, CH2N), 2.60 (2 H, t, CH2CO), 2.26 (2 H, m,
CH2CO), 2.11, 2.10 (2), 2.02, 2.01, 2.00, and 1.91 (7 ×
3 H, 7 s, 7 COCH3), and 1.75 (6 H, m, 3 CH2).
Conjugate (IIc). A solution of aminoamide (IIb)
(2.9 mg, 5.1 µmol) in DMSO (100 µl) was added to a
solution of (VIII) (15 mg, 23.1 µmol) in DMF (150 µl).
After 24 h, the reaction mixture was concentrated in a
vacuum. The residue was dissolved in water (1 ml) and
applied onto a Lichrosorb RP-8 (4 × 250 mm) column,
which was eluted for 40 min with a 5–85% gradient of
MeCN in H2O containing 0.01% TFA. The fractions
containing glycodendrimer (IIÒ) were concentrated and
lyophilized to give 6.7 mg (51%) of (IIc); Rf 0.35 (3 : 1
MeOH—25% ammonia) and 0.05 (3 : 1 : 1 n-BuOH–
AcOH–H2O); 1H NMR (D2O): 5.12 (4 H, d, J1, 2 4, H1
in Galβ), 4.41 (4 H, d, J1, 2 8, H1 in Galα), 4.20 (4 H,
br. t, H5 in Galα), 3.50 (8 H, br. s,
Ligand (XI). Trifluoroacetic acid (2.5 ml) was
added to a solution of Boc-glycoside (X) (375 mg,
0.655 mmol) obtained by the procedure [22]. The mix-
ture was kept for 1 h at 20°C and coevaporated with tol-
uene (5 ml). The residue was dried in a vacuum and dis-
solved in THF (7 ml). A solution of bis(4-nitrophe-
nyl)adipate (975 mg, 2.5 mmol) in THF (14 ml) and
Et3N (0.6 ml) was added. The mixture was stirred for
24 h at 20°C and evaporated to dryness. The target
amorphous (XI) was isolated by column chromatogra-
phy on silica gel (elution with 20 : 1 CHCl3–MeOH);
(NHC(O)CH2CH2)2N), 3.62–3.40 (24
CH2NHC(O)), 2.71–2.60 (8 H,
H,
br.
m,
s,
(NHC(O)CH2CH2)2N), 2.32–2.15 (16 H, br. s,
NHC(O)CH2(ëH2)3), 1.80 (8 H, br. t, OCH2CH2CH2N),
1.62–1.51 (16 H, br. s, NHC(O)CH2CH2(ëH2)2), and
1.27 (4 H, br. m, (CH2)4).
1
yield 351 mg (78%); Rf 0.6 (7 : 1 CHCl3–MeOH); H
Conjugate (IIIc). (a) A solution of aminoamide
(IIIb) (2.0 mg, 1.34 µmol) in DMSO (200 µl) was
added to a solution of ester (IX) (11.2 mg, 11.8 µmol)
in DMF (150 µl). After 24 h, the reaction mixture was
concentrated in a vacuum, dissolved in MeOH (1 ml),
and treated with 1 N aqueous NaOH (300 µl). After 2 h,
the reaction mixture was concentrated; the residue was
dissolved in water (1 ml), applied on a TSK HW-40F
(1 × 50 cm) column, and eluted with 0.5% ammonia.
NMR (CDCl3): 8.586 (1 H, s, NHAr), 7.221 and 8.208
(2 × 2 H, 2 d, J 9, p-C6H4NO2), 7.243 and 7.450 (2 × 2
H, 2 d, J 8.5, p-C6H4NO2), 6.616 (1 H, t,
COCH2NHCO), 5.406 (1 H, ddd, H8 in Neu5Ac),
5.359 (1 H, d, J5 9.7, NH), 5.307 (1 H, dd, J8, 7 8.4, J6, 7
2.3, H7 in Neu5Ac), 4.839 (1 H, ddd, J5, 4 10.2, J3ax, 4
12.3, J3eq, 4 4.6, H4 in Neu5Ac), 4.343 and 4.722 (2 × 1
H, 2 d, J 12, OCH2Ar), 4.298 (1 H, dd, J9a, J9b 12.5, J8, 9b
2.9, H9b in Neu5Ac), 4.111 (1 H, dd, J5, 6 10.7, J7, 6 2.3, The fractions containing glycoconjugate (IIIc) were
H6 in Neu5Ac), 4.074 (2 H, d, JNH 5.5, COCH2NHCO),
lyophilized to give 2.6 mg (35%) of (IIIc); Rf 0.20
1
4.062 (1 H, dd, J8, 9 6, H9a in Neu5Ac), 4.049 (1 H, ddd, (2 : 1 MeOH–25% ammonia); H NMR (D2O): 5.15
H5 in Neu5Ac), 3.688 (2 H, t, J 4.7, CH2CH2CONH), (8 H, d, J1, 2 4, H1 in Galβ), 4.46 (8 H, d, J1, 2 8, H1 in
3.645 (3 H, s, COOCH3), 2.604 (1 H, dd, H3eq in Galα), 4.18 (16 H, m, H5 in Galα and H4 in Galβ),
3.35–3.25 (56 H, m, CH2NHC(O)), 2.87–2.80 (24 H, t,
Neu5Ac), 2.601 (2 H, t, J 6, CH2CH2C), 2.335 and
2.393 (2 × 1 H, m, CH2CH2CONH), 1.843, 1.984, 2.00,
2.100, and 2.117 (5 × 3 H, 5 s, 5 Ac), 1.966 (1 H, dd,
H3ax in Neu5Ac), and 1.774 (2 H, m, CH2CH2COO).
(NHC(O)CH2CH2)2N),
C(O)NHCH2CH2N),
(NHC(O)CH2CH2)2N),
2.69–2.63
2.44–2.39
(8
(24
H,
H,
(32H,
t,
t,
2.28–2.20
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 28 No. 6 2002

484
TSVETKOV et al.
NHC(O)CH2(ëH2)3), 1.82 (16 H, m, OCH2CH2CH2N), C(O)NHCH2CH2N), 2.45–2.40 (60 H, br. t,
1.62–1.55 (32H, NHC(O)CH2CH2(ëH2)2), and 1.40– (NHC(O)CH2CH2)2N), 2.30–2.22 (128 H, br. s,
1.32 (4 H, (CH2)2); 13C NMR (D2O): 103.4 (C1, Galα),
96.0 (C1, Galβ), 50.1 ((NHë(é)ëH2ëH2)2N), 39.5 and
NHC(O)CH2(ëH2)3),
OCH2CH2CH2N), and 1.68–1.53 (128 H, br. s,
39.3 (NHCH2), 37.1 ((ëH2)2NëH2ëH2NH), 36.3 NHC(O)CH2CH2(ëH2)2); 13C NMR (D2O): 103.6 (C1,
(NHC(O)CH2(ëH2)3), 33.2 ((NHë(é)ëH2ëH2)2N), 29.3 Galα), 96.4 (C1, Galβ), 49.9 ((ë(é)ëH2ëH2)2N), 39.8
(OCH2CH2CH2N), 25.7 (NHC(O)CH2CH2(ëH2)2), 15.4 and 37.5 (NHCH2), 36.6 ((ëH2)2NëH2ëH2NH), 33.3
1.87–1.80
(64
H,
m,
((CH2)2).
((ë(é)ëH2ëH2)2N),
(NHC(O)CH2CH2(ëH2)2).
and
26.0
(b) Aminoamide (IIIb) (2.6 mg, 1.8 µmol) in water
(0.1 ml) was added to a solution of disaccharide (VIII)
(12.5 mg, 19.3 µmol) in DMSO (0.4 ml). After 18 h, the
reaction mixture was dried in a vacuum. The residue
was dissolved in water (1 ml), applied on a TSK HW-
40F (1 × 50 cm) column, and eluted with 0.5% ammo-
nia. The fractions containing glycoconjugate (IIIÒ)
were concentrated and lyophilized to yield 7.8 mg
(74%) of (IIIÒ), Rf 0.22 (2 : 1 MeOH–25% ammonia).
Conjugate (VIIc). A solution of PAMAM-G4
(VIIb) (6.0 mg, 0.42 µmol) in 1 : 2 H2O–DMSO
(150 µl) was added to a solution of ester (VIII) (20.0
mg, 30.8 µmol) in DMF (100 µl). After 18 h, the reac-
tion mixture was dried in a vacuum; the residue was
dissolved in water (1 ml), applied onto a TSK HW-55F
(2.5 × 90 cm) column, and eluted with 0.5% ammonia.
The fractions containing glycoconjugate (VIIÒ) were
concentrated and lyophilized to yield 14.9 mg (76%) of
Conjugate (IVc). A solution of PAMAM (IVb)
(7.2 mg, 2.2 µmol) in water (50 µl) was added to a solu-
tion of ester (VIII) (30.0 mg, 46.2 µmol) in DMF
(250 µl). After 48 h, the reaction mixture was dried in a
vacuum. The residue was dissolved in water (1 ml),
applied on a TSK HW-55F (2.5 × 90 cm) column, and
eluted with 0.5% ammonia. The fractions containing
glycoconjugate (IVÒ) were concentrated and lyo-
philized to yield 24.5 mg (98%) of (IVÒ); Rf 0.12 (2 : 1
1
(VIIÒ); H NMR (D2O): 5.13 (32 H, d, J1, 2 4, H1 in
Galβ), 4.42 (32 H, d, J1, 2 8, H1 in Galα), 4.17 (64 H,
m, H5 in Galα and H4 in Galβ), 3.40–3.20 (248 H, m,
CH2NHC(O)),
3.0–2.8
(60
H,
br.
t,
(NHC(O)CH2CH2)2N), 2.78–2.65 (28 H, br. t,
C(O)NHCH2CH2N), 2.51–2.35 (60 H, br. t,
(NHC(O)CH2CH2)2N), 2.30–2.15 (128 H, br. s,
NHC(O)CH2(ëH2)3),
1.87–1.80
(64
H,
m,
MeOH–25% ammonia); 1H NMR (D2O): 5.16 (16 H, d,
J1, 2 4, H1 in Galβ), 4.45 (16 H, d, J1, 2 8, H1 in Galα),
4.19 (32 H, m, H5 in Galα and H4 in Galβ), 3.35–3.25
(120 H, m, CH2NHC(O)), 2.87–2.76 (56 H, t,
OCH2CH2CH2N), and 1.60–1.53 (128 H, br. s,
NHC(O)CH2CH2(ëH2)2); 13C NMR (D2O): 176.1,
175.8, and 173.9 (C=O), 102.3 (C1, Galα), 94.9 (C1,
Galβ), 51.1 and 48.8 ((ë(é)ëH2ëH2)2N), 38.2 and
36.8 (NHCH2), 35.1 ((ëH2)2NëH2ëH2NH), 31.7
(NHC(O)CH2CH2)2N), 2.69–2.63
C(O)NHCH2CH2N), 2.48–2.39
(28
(56
H,
H,
t,
t,
((ë(é)ëH2ëH2)2N),
and
24.5
(NHC(O)CH2CH2)2N), 2.32–2.20 (64 H, br. s,
(NHC(O)CH2CH2(ëH2)2).
NHC(O)CH2(ëH2)3),
OCH2CH2CH2N),
1.90–1.82
and 1.66–1.53
(32
H,
(64
m,
H,
Conjugate (IVd). A solution of aminoamide (IVb)
(4.2 mg, 0.3 µmol) in H2O (10 µl) was added to a solu-
tion of ester (XI) (20.0 mg, 25.0 µmol) in DMF
(200 µl). After 18 h, the reaction mixture was dried in a
vacuum. The residue was dissolved in MeOH (1 ml),
and 1 N NaOH (0.5 ml) was added. After 2 h, the result-
ing solution was concentrated in a vacuum; the residue
was dissolved in water (1 ml), applied onto a TSK HW-
40F (1 × 50 cm) column successively attached to a TSK
HW-50F (1 × 50 cm) column, and eluted with 0.5%
ammonia. The fractions containing glycoconjugate
(IVd) were concentrated and lyophilized to yield
10.5 mg (67%) of (IVd); 1H NMR (D2O): 7.39 (64 H,
m, 4-aminophenyl), 4.72 and 4.50 J 12, ArCH2O), 4.01
(32 H, s, COCH2NH), 3.26 (NëH2ëH2NHC(O)), 3.05
(24 H, m, (ëH2)2NëH2ëH2NH), 2.97 (24 H, m,
NHC(O)CH2CH2(ëH2)2); 13C NMR (D2O): 103.4 (C1,
Galα), 96.0 (C1, Galβ), 50.9 ((ëH2)2NëH2ëH2NH),
49.9 ((ë(é)ëH2ëH2)2N), 39.4 and 39.3 (NHCH2), 37.1
((ëH2)2NëH2ëH2NH), 33.3 ((ë(é)ëH2ëH2)2N), 36.1
(NHC(O)CH2(ëH2)3), 29.2 (OCH2CH2CH2N), and 25.6
(NHC(O)CH2CH2(ëH2)2).
Conjugate (Vc). A solution of aminoamide (Vb)
(2.1 mg, 0.3 µmol) in H2O (10 µl) was added to a solu-
tion of ester (VIII) (7.0 mg, 10.8 µmol) in DMF
(80 µl). After 72 h, the reaction mixture was concen-
trated in a vacuum; the residue was dissolved in water
(1 ml), applied on a TSK HW-55F (2.5 × 90 cm) col-
umn, and eluted with 0.5% ammonia. The fractions
containing glycoconjugate (VÒ) were concentrated and
lyophilized to yield 3.2 mg (46%) of (VÒ); Rf 0.08 (2 : (ëH2)2NëH2ëH2NH), 2.76 (16 H, dd, H3eq), 2.51 (32
1
H, br. t, NCH2CH2C(O)N), 2.34 and 2.23 (64 H, s,
CH2CH2CH2CONH), 2.04 (48 H, NHC(O)CH3), 1.67
(16 H, dd, J2, 3ax, 12.5, H3ax), and 1.65–1.56 (64 H, br.
s, CH2CH2CH2C(O)N); 13C NMR (D2O): 178.2, 177.8,
176.3, 174.6, 174.2, and 171.0 (C=O), 137.7, 135.5,
1 MeOH—25% aqueous ammonia); H NMR (D2O):
5.14 (32 H, d, J2 4, H1, in Galβ), 4.43 (32 H, d, J2 8, H1,
in Galα), 4.17 (64 H, m, H5 in Galα and H4 in Galβ),
3.35–3.25 (248 H, m, CH2NHC(O)), 2.87–2.82 (60 H,
br. t, (NHC(O)CH2CH2)2N), 2.58–2.53 (28 H, br. t,
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 28 No. 6 2002

NEOGLYCOCONJUGATES BASED ON DENDRIMER POLY(AMINOAMIDES)
485
130.7, and 122.8 (4-aminophenyl), 102.2 (C1), 67.7
(OCH2Ar), 50.3 (ëH2)2NëH2ëH2NH), 49.8
(ëH2)2NëH2ëH2NH), 44.3 (C(O)CH2NH), 41.8 (C3),
40.1 and 39.7 (C(O)NHCH2), 36.7 and 36.3
(CH2CH2C(O)N), 31.7 ((ë(é)ëH2ëH2)2N), 26.0 and
25.8 (ë(é)ëH2ëH2), and 23.3 (CH3C(O)).
ACKNOWLEDGMENTS
This work was supported by INTAS (grant nos. 94-
4606 and 97-32036), Russian Foundation for Basic
Research (project nos. 00-03-32815a and 99-04-
48064), and the Foundation for Assistance to Domestic
Science.
Conjugate (Vd). A solution of aminoamide (Vb)
(3.83 mg, 5.5 µmol) in H2O (30 µl) was added to a solu-
tion of ester (XI) (17.5 mg, 19.5 µmol, 1.1 equiv) in
DMF (200 µl). After 24 h, the reaction mixture was
concentrated in a vacuum. The residue was dissolved in
MeOH (1 ml), and 1 N NaOH (1 ml) was added. After
2 h, the resulting solution was concentrated in a vac-
uum; the residue was dissolved in water (1 ml), applied
onto a TSK HW-40F (1 × 50 cm) column successively
attached to a TSK HW-50F (1 × 50 cm) column, and
eluted with 0.5% ammonia. The fractions containing
glycoconjugate (Vd) were concentrated and lyo-
REFERENCES
1. Kiessling, L. and Pohl, N., Chem. Biol., 1996, vol. 3,
pp. 71–77.
2. Zanini, D. and Roy, R., Carbohydrate Mimics: Concepts
and Methods, Chapleur, Y., Ed., Weinheim: Verlag Che-
mie, 1998.
3. Bovin, N., Glycoconjugate J., 1998, vol. 15, pp. 431–
436.
4. Newkome, G.R., Moorefeld, C.N., and Vogtle, F., Den-
dritic Molecules. Concepts. Syntheses. Perspectives,
Weinheim: Verlag Chemie, 1996.
1
philized to yield 8.1 mg (46%) of (Vd); H NMR
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((ëH2)2NëH2ëH2NH),
3.20
(ëH2NHCO), 3.16–2.75 ((ëH2)2NëH2ëH2NH), 2.70
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and 2.18 (br. s, CH2CH2CH2CONH), 1.98 (s,
NHC(O)CH3), 1.60 (dd, H3ax), and 1.55 (br. s,
CH2CH2CH2CONH); 13C NMR (D2O): 177.6, 177.5,
177.0, 175.9, and 174.3 (C=O); 137.4, 135.0, 130.4,
and 122.4 (4-aminophenyl); 101.8 (C2); 73.6 (C6);
72.6 (C8); 69.2 (C7); 69.0 (C4); 67.4 (OCH2Ar); 63.4
(C9); 52.8 (C5); 50.3 (ëH2)2NëH2ëH2NH), 49.2
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