Synthesis of 3-aminopropyl β-glycoside of sialyl-3'-lactose and derived neoglycoconjugates as a tumor vaccine prototype and artificial antigens for the control of immune response

Article abstract of DOI:10.1007/s11172-006-0554-y

Starting from the biotechnologically available trisaccharide sialyl-3'-lactose, representing the carbohydrate portion of the tumor-associated ganglioside GM3, the corresponding 3-aminopropyl β-glycoside (1) and 3-(4-maleimidobutanoylamino)propyl glycoside were synthesized. The reaction of the latter with a thiolated derivative of the Megathura crenulata hemocyanine (KLH) afforded a carbohydrate-protein conjugate, a tumor vaccine prototype containing about 330 trisaccharide ligands attached to KLH. N-Stearoylation of ligand 1 gave the model neoglycolipid for comparative study of the activity of mono-and polyvalent immunogens and the natural ganglioside GM3. A monovalent conjugate, in which ligand 1 is linked to biotin through an oligo(ethylene glycol) spacer and a polyvalent conjugate with a polyacrylamide carrier were also prepared. These conjugates are meant as covering antigens to assess the specificity and efficiency of the immune response in the ELISA assay.

Full text of DOI:10.1007/s11172-006-0554-y

Russian Chemical Bulletin, International Edition, Vol. 55, No. 11, pp. 2095—2102, November, 2006
2095
Synthesis of 3ꢀaminopropyl βꢀglycoside of sialylꢀ3´ꢀlactose
and derived neoglycoconjugates as a tumor vaccine prototype
and artificial antigens for the control of immune response
E. A. Khatuntseva,a O. N. Yudina,a Yu. E. Tsvetkov,a A. A. Grachev,a R. N. Stepanenko,b R. Ya. Vlasenko,b
V. L. Lvov,b and N. E. Nifantieva
ꢀ
аN. D. Zelinsky Institute of Organic Chemistry of the Russian Academy of Sciences,
47 Leninsky prosp., 119991 Moscow, Russian Federation.
Fax: +7 (495) 135 8784. Eꢀmail: nen@ioc.ac.ru
bInstitute of Immunology of the Federal Department of MedicoꢀBiological and Extreme Problems,
Ministry of Health and Social Development of Russian Federation,
24/2 Kashirskoe shosse, 115478 Moscow, Russian Federation.
Starting from the biotechnologically available trisaccharide sialylꢀ3´ꢀlactose, representing
the carbohydrate portion of the tumorꢀassociated ganglioside GM3, the corresponding
3ꢀaminopropyl βꢀglycoside (1) and 3ꢀ(4ꢀmaleimidobutanoylamino)propyl glycoside were synꢀ
thesized. The reaction of the latter with a thiolated derivative of the Megathura crenulata
hemocyanine (KLH) afforded a carbohydrate—protein conjugate, a tumor vaccine prototype
containing about 330 trisaccharide ligands attached to KLH. NꢀStearoylation of ligand 1 gave
the model neoglycolipid for comparative study of the activity of monoꢀ and polyvalent immuꢀ
nogens and the natural ganglioside GM3. A monovalent conjugate, in which ligand 1 is linked
to biotin through an oligo(ethylene glycol) spacer and a polyvalent conjugate with a polyacrylꢀ
amide carrier were also prepared. These conjugates are meant as covering antigens to assess the
specificity and efficiency of the immune response in the ELISA assay.
Key words: sialylꢀ3´ꢀlactose, KLH, polyacrylamide, biotin, neoglycoconjugate, tumor
vaccine.
On the surface of tumor cells, oligosaccharides with
unique structures, the soꢀcalled tumorꢀassociated carboꢀ
hydrate antigens, which are absent on the surface of norꢀ
mal cells, are expressed.1 This feature of cancer cells can
be successfully used for typing or determination of the
stage of the disease and mortality; it can also serve as a
fundamental for the development of cancer vaccines (tuꢀ
mor vaccines). It is assumed that immunization of paꢀ
tients with the oligosaccharide antigen typical of a parꢀ
ticular kind of cancer cells may induce the formation of
cytotoxic antibodies able to recognize and destroy cancer
cells. However, the development of such vaccine is held
up by low immunogenicity of oligosaccharide antigens.2
On the one hand, this is caused by the tolerance of the
immune system to autoantigens, and on the other hand,
by inability of oligosaccharide antigens to activate one of
the components of the immune system, viz., Тꢀlymphoꢀ
cytes, which is necessary for antibody production. Thereꢀ
fore, in the development of synthetic tumor vaccines,
oligosaccharide fragments are conjugated with natural
highly immunogenic proteins capable of initiating Тꢀcell
immune response,3 such as hemocyanin from Megathura
crenulata (keyhole limpet hemocyanin, KLH) or tetanus
toxin. Previously, KLH conjugates with tumorꢀassociated
antigens such as the tetrasaccharide LeY (see Ref. 4) and
the ganglioside GD3 tetrasaccharide (see Ref. 5) have
been prepared.
The ganglioside GM3, whose carbohydrate chain is
represented by the βꢀanomer of the Nꢀacetylated trisacꢀ
charide sialylꢀ3´ꢀlactose, is a frequently encountered tuꢀ
morꢀassociated carbohydrate antigen, expressed in a large
number of tumor types, such as malignant melanoma or
neuroectodermal tumors.6 However, the ganglioside GM3
is also present on the surface of normal cells; therefore,
the use of this carbohydrate chain as a ligand for the
design of specific conjugated tumor vaccine seems probꢀ
lematic. Meanwhile, the Nꢀglycolyl analog of the ganꢀ
glioside GM3 (NꢀglycolylꢀGM3)7 expressed specifically
on tumor cells (melanoma, breast cancer, etc.)8 has not
been detected in normal tissues9 and, hence, conjugates
of trisaccharide Nꢀglycolylneuraminylꢀ3´ꢀlactose with
* Dedicated to Academician O. M. Nefedov on the occasion of
his 75th birthday.
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 11, pp. 2016—2023, November, 2006.
1066ꢀ5285/06/5511ꢀ2095 © 2006 Springer Science+Business Media, Inc.

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Russ.Chem.Bull., Int.Ed., Vol. 55, No. 11, November, 2006
Khatuntseva et al.
GM3 (R = H), NꢀglycolylꢀGM3 (R = OH)
proteins appear promising candidates for the developꢀ
ment of cancer vaccines.
the conjugation conditions for their subsequent use in the
synthesis of analogous derivatives based on the carboꢀ
hydrate chain of the NꢀglycolylꢀGM3 ganglioside. In this
study, we prepared the Nꢀstearoylated neoglycolipide 3,
a model compound for a comparative investigation of the
activities of monoꢀ and polyvalent immunogens and the
natural ganglioside GM3. In addition, we synthesized
a monovalent conjugate 4 in which ligand 1 is linked to
This study is devoted to the synthesis of the
βꢀ(3ꢀaminopropyl) glycoside of the trisaccharide sialylꢀ
3´ꢀlactose (1), which represents the carbohydrate part of
the ganglioside GM3, and its use for the preparation of
a tumorꢀvaccine prototype, a conjugate with a thiolated
derivative of KLH 2. The goal of this study is to optimize
x = 0.2, y = 0.4, z = 0.4

Sialylꢀ3´ꢀlactose neoglycoconjugates
Russ.Chem.Bull., Int.Ed., Vol. 55, No. 11, November, 2006 2097
Scheme 1
Reagents: a. N2H4•AcOH, DMF; b. Cl3CCN, DBU, CH2Cl2; c.
(9), TMSOTf; d. NaOH
biotin through an oligo(ethylene glycol) spacer and a polyꢀ
valent conjugate with polyacrylamide carrier 5 as coverꢀ
ing antigens to control the specificity and efficiency of the
immune response in the enzymeꢀlinked immunosorbent
assay (ELISA).
stereoselectively resulting in βꢀglycoside 10 in 52% yield.
The βꢀconfiguration of the glucosidic bond in comꢀ
pound 10 was unambiguously confirmed by the correꢀ
sponding spin coupling constant (J1,2 = 8.0 Hz) in the
1H NMR spectrum. Complete deprotection of comꢀ
pound 10 by alkaline hydrolysis resulted in target aminoꢀ
propyl glycoside 1 in 88% yield.
The method used for conjugation of trisaccharide 1
with KLH was based on the addition of the thiol group to
the maleimide double bond. For the introduction of the
maleimide fragment, the amino group of trisaccharide 1
was acylated by active ester of 4ꢀmaleimidobutyric acid 11
to give maleimide derivative 12 in 85% yield (Scheme 2).
To increase the number of thiol groups in the protein
molecule able to react with maleimide, free amino groups
of the lysine residues in native KLH were modified by
2ꢀiminothiolane (13) to give thiolated KLH 14 (see
Ref. 4b). The conjugation of maleimide derivative 12 with
An anomeric mixture of sialylꢀ3´ꢀlactose peracetates
6α,β described in our previous study10 served as the startꢀ
ing compound in the preparation of aminopropyl glycoꢀ
side 1. This mixture was synthesized from the bioꢀ
technologically available trisaccharide sialylꢀ3´ꢀlactose.11
Removal of the anomeric acetyl group from peracetate
6α,β by treatment with hydrazine acetate (Scheme 1)
afforded a mixture of hemiacetals 7α,β, which was treated
with trichloroacetonitrile in the presence of DBU to give
an anomeric mixture of trichloroacetimidates 8α,β (overꢀ
all yield 69%). Glycosylation of 3ꢀtrifluoroacetamidoꢀ
propanꢀ1ꢀol (9) with the anomeric mixture of trichloroꢀ
acetimidates 8α,β in the presence of TMSOTf proceeded
Scheme 2

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Russ.Chem.Bull., Int.Ed., Vol. 55, No. 11, November, 2006
Khatuntseva et al.
Scheme 3
Scheme 4
40 mol.% of nꢀbutylamine to impart hydrophobic properꢀ
ties to the conjugate in order to enhance its adsorption
on the surface of polystyrene plates for the ELISA asꢀ
say (Scheme 4). The nonconsumed pꢀnitrophenyl ester
groups were subsequently converted into Nꢀ(2ꢀhydroxyꢀ
ethyl)amide groups by treatment with excess ethanolꢀ
amine. This gave polyacrylamide conjugate 5.
Biotin is known to form an exceptionally strong comꢀ
plex with streptavidin; therefore, biotinylation of oligosacꢀ
charide ligands is used to bind them to streptavidinꢀcoated
ELISA plates. Preliminarity, the spacer group of triꢀ
saccharide 1 was elongated by a hydrophilic tetraethylene
glycol fragment in order to ensure the optimum distance
between the oligosaccharide ligand and the plate surface,
which is essential for recognition of the carbohydrate
ligand by antibodies. For this purpose, the amino group in
compound 1 was acylated by active ester 17 prepared
from tetraethylene glycol (the synthesis of compound 17
will be published elsewhere). The removal of the Nꢀtriꢀ
fluoroacetyl group by alkaline hydrolysis gave amino deꢀ
rivative 18. Its acylation with active biotin ester 19 afꢀ
forded the target biotinylated trisaccharide 4.
thiolated protein 14 for 24 h yields conjugate 2, which was
separated from monomeric components by dialysis. Quanꢀ
titative determination of neuraminic acid in conjugate 2
was carried out by the Svennerholm method12 and of the
protein content, using the bicinchoninic acid reagent.
According to the results of analysis, conjugate 2 conꢀ
tained ~330 trisaccharide residues per protein molecule.
Acylation of the amino group in trisaccharide 1 with
pꢀnitrophenyl stearate (15) (see Ref. 13) gave neoꢀ
glycolipid 3 (Scheme 3) (yield 80%), which mimics the
natural ganglioside GM3 and was necessary for comparaꢀ
tive analysis of the immunogenicities of monoꢀ and polyꢀ
valent antigens.
To study the antibody specificity against conjugate 2
by ELISA, we synthesized two covering antigens, namely,
a polyvalent conjugate with polyacrylamide and a monoꢀ
valent biotinylated derivative starting from trisaccharide 1.
Poly(pꢀnitrophenyl acrylate) (16) (see Ref. 14) was treated
with 20 mol.% of aminopropyl glycoside 1 and then with
Immunological assays have shown that immunization
of mice by conjugate 2 induced a humoral immune reꢀ
sponse; the titers of IgG antibodies against the trisacchaꢀ
ride sialylꢀ3´ꢀlactose were essentially higher than upon
immunization with the natural ganglioside GM3.15 (Deꢀ
tailed results of the immunization by conjugate 2 and
Scheme 5

Sialylꢀ3´ꢀlactose neoglycoconjugates
Russ.Chem.Bull., Int.Ed., Vol. 55, No. 11, November, 2006 2099
(C(1)ꢀGlc, 7α); 76.3 (C(4)ꢀGlc, 7α); 76.2 (C(4)ꢀGlc, 7β); 73.6
(C(2)ꢀGlc, 7β); 73.0 (C(5)ꢀGlc, 7β); 72.5 (C(3)ꢀGlc, 7β); 72.0
(C(6)ꢀNeu, 7α,β); 71.4 (C(2)ꢀGlc, 7α, C(3)ꢀGal, 7α,β); 70.4
(C(5)ꢀGal, 7α,β); 70.1 (C(3)ꢀGlc, 7α); 69.9 (C(2)ꢀGal, 7α,β);
69.3 (C(4)ꢀNeu, 7α,β); 68.2 (C(5)ꢀGlc, 7α); 67.8 (C(7)ꢀNeu,
7α,β); 67.3 (C(4)ꢀGal, 7α,β); 66.8 (C(8)ꢀNeu, 7α,β); 62.2
(C(6)ꢀGal, 7α,β); 62.1 (C(6)ꢀGlc, 7α,β); 61.5 (C(9)ꢀNeu,
7α,β); 53.1 (COOCH3, 7α,β); 49.2 (C(5)ꢀNeu, 7α,β); 37.4
(C(3)ꢀNeu, 7α,β); 23.1—20.5 (CH3CO). Found (%): C, 49.25;
H, 5.79; N, 1.27. C44H61NO29. Calculated (%): C, 49.49;
H, 5.79; N, 1.31.
[Methyl (5ꢀacetamidoꢀ4,7,8,9ꢀtetraꢀOꢀacetylꢀ3,5ꢀdideoxyꢀ
DꢀglyceroꢀαꢀDꢀgalactoꢀ2ꢀnonulopyranosyl)onate]ꢀ(2→3)ꢀ2,4,6ꢀ
triꢀOꢀacetylꢀβꢀDꢀgalactopyranosylꢀ(1→4)ꢀ2,3,6ꢀtriꢀOꢀacetylꢀ
α,βꢀDꢀglucopyranosyl trichloroacetimidate (8α,β). Trichloroꢀ
acetonitrile (0.53 mL, 5.3 mmol) was added to a solution of
hemiacetal 7α,β (0.565 g, 0.529 mmol) in anhydrous CH2Cl2
(10 mL), the mixture was cooled to –30 °C, DBU (0.08 mL)
was added, and the mixture was stirred for 1 h at –30 °C. The
mixture was applied onto a column with silica gel (50 g) preꢀ
washed with toluene containing 0.1% of triethylamine. Elution
with a toluene—acetone mixture (7 : 3, +0.1% of triethylamine)
gave 0.492 g (77%) of anomeric mixture 8α,β in a 2 : 1 ratio as a
white foam, Rf 0.35 (acetone—toluene, 2 : 3). 1H NMR (CDCl3),
δ: 6.50 (d, H(1)ꢀGlc, 8α, J1,2 = 3.3 Hz); 5.95 (d, H(1)ꢀGlc, 8β,
J1,2 = 8.2 Hz); 5.55 (dd, H(3)ꢀGlc, 8α, J3,2 = J3,4 = 9.6 Hz);
neoglycolipid 3 and study of the specificity of the immue
response using biotinylated derivative 4 and polyacrylꢀ
amide conjugate 5 will be published elsewhere.)
Experimental
Triethylamine, Nꢀhydroxysuccinimide ester of 4ꢀmaleimidoꢀ
butyric acid (Fluka), trichloroacetonitrile, TMSOTf, hydrazine
acetate (Acros), DBU (Merck), KLH, 2ꢀiminothiolane hydroꢀ
chloride, and Nꢀhydroxysuccinimide ester of biotin (Sigma) were
used as received. Dichloromethane was distilled twice from P2O5
and then from CaH2 under argon. Dimethylformamide was disꢀ
tilled in vacuo (oil pump) from phthalic anhydride and then
from CaH2. Molecular sieves (MS 4 Å) were activated by calciꢀ
nation at 180 °C in vacuo (oil pump) for 2 h. The 1H and
13C NMR spectra were recorded on Bruker DRXꢀ500 and Bruker
AMꢀ300 instruments at 25 °C. The signals were assigned by
homoꢀ and heteronuclear 2D COSY, TOCSY, and HSQC specꢀ
tra. Optical rotation was measured on a PUꢀ07 digital polariꢀ
meter (State Research and Engineering Center of Scientific Inꢀ
strument Making) at 18—25 °C. Thin layer chromatography was
carried out on silica gel Kieselgelꢀ60 plates (Merck), and comꢀ
pounds were visualized by treatment with a 10% (v/v) solution
of orthophosphoric acid in ethanol or (for amines) with a ninꢀ
hydrin solution (3 g L–1 in a butanol—acetic acid mixture, 30 : 1)
followed by heating at ~150 °C. Column chromatography was
carried out on Silica gel 60 (Merck), 0.040—0.063 mm. A Knauer
98.00 refractometer was used as the detector for gel chromatoꢀ
graphy.
[Methyl (5ꢀacetamidoꢀ4,7,8,9ꢀtetraꢀOꢀacetylꢀ3,5ꢀdideoxyꢀ
DꢀglyceroꢀαꢀDꢀgalactoꢀ2ꢀnonulopyranosyl)onate]ꢀ(2→3)ꢀ2,4,6ꢀ
triꢀOꢀacetylꢀβꢀDꢀgalactopyranosylꢀ(1→4)ꢀ2,3,6ꢀtriꢀOꢀacetylꢀ
α,βꢀDꢀglycopyranose (7α,β). A solution of acetate mixture 6α,β
(0.876 g, 0.79 mmol) and hydrazine acetate (0.109 g, 1.18 mmol)
in anhydrous DMF (10 mL) was stirred for 3 h at ~20 °C,
diluted with chloroform (50 mL), washed with water (50 mL)
and brine (20 mL), and concentrated; then DMF was coꢀevapoꢀ
rated with toluene. Column chromatography of the residue (toluꢀ
ene—methanol, 95 : 5 → 9 : 1) gave 0.786 g (89%) of anomeric
mixture 7α,β in 2 : 1 ratio as a white foam, Rf 0.18 (acꢀ
etone—toluene, 2 : 3), [α]D +18 (c 1, CHCl3). 1H NMR
(CDCl3), δ: 5.55—5.47 (m, H(8)ꢀNeu, 7α,β, H(3)ꢀGlc, 7α);
5.41 (dd, H(7)ꢀNeu, 7α,β, J7,6 = 2.7 Hz, J7,8 = 9.4 Hz); 5.37 (d,
5.50 (m, H(8)ꢀNeu, 8α,β); 5.40 (dd, H(7)ꢀNeu, 8α,β, J7,6
2.7 Hz, J7,8 = 9.4 Hz); 5.30 (dd, H(3)ꢀGlc, 8β, J3,2 = J3,4
=
=
8.9 Hz); 5.16 (d, NHꢀNeu, 8β, J5,NH = 10.0 Hz); 5.12 (d,
NHꢀNeu, 8α, J5,NH = 10.0 Hz); 5.08 (dd, H(2)ꢀGlc, 8α, J2,1
3.5 Hz, J2,3 = 10.0 Hz); 4.96 (dd, H(2)ꢀGal, 8α,β, J2,1 = J2,3
=
=
8.5 Hz); 4.90 (m, H(4)ꢀGal, 8α,β, H(4)ꢀNeu, 8α,β, H(2)ꢀGlc,
8β, H(6а)ꢀGlc, 8β); 4.68 (d, H(1)ꢀGal, 8α,β, J1,2 = 8.0 Hz);
4.52 (dd, H(3)ꢀGal, 8α,β, J2,3 = 8.5 Hz, J3,4 = 2.7 Hz);
4.46—4.39 (m, H(6a)ꢀGlc, 8α, H(9a)ꢀNeu, 8α,β); 4.23 (dd,
H(6b)ꢀGlc, 8α, J6b,6a = 12.0 Hz, J6b,5 = 4.0 Hz); 4.23 (m,
H(6b)ꢀGlc, 8β); 4.17—3.91 (m, H(4)ꢀGlc, 8α,β, H(5)ꢀGlc, 8α,
H(6а)ꢀGal, H(6b)ꢀGal, 8α,β, H(5)ꢀNeu, 8α,β, H(9b)ꢀNeu,
8α,β); 3.88—3.82 (m, H(5)ꢀGal, 8α,β, OCH3, 8α,β); 3.68 (m,
H(5)ꢀGlc, 8β); 3.64 (H(6)ꢀNeu, 8α,β); 2.58 (dd, Heq(3)ꢀNeu,
8α,β, J3eq,4 = 4.4 Hz, J3eq,3ax = 12.7 Hz); 1.82—2.30 (m,
CH3CO, 8α,β); 1.78 (dd, Hax(3)ꢀNeu, 8α,β, J3ax,3eq = J3ax,4
=
12.5 Hz). 13C NMR (CDCl3), δ: 170.0—170.8 (CH3CO,
CH3CON); 155.9 (OC(CCl3)N); 168.0 (C(1)ꢀNeu, 8α,β); 101.2
(C(1)ꢀGal, 8α,β); 96.8 (C(2)ꢀNeu, 8α,β); 96.3 (C(1)ꢀGlc, 8β);
93.2 (C(1)ꢀGlc, 8α); 75.9 (C(4)ꢀGlc, 8α,β); 72.0 (C(5)ꢀGlc,
8β, C(6)ꢀNeu, 8α,β); 71.6 (C(3)ꢀGal, 8α,β); 71.0 (C(2)ꢀGlc,
8β, C(5)ꢀGlc, 8α); 70.6 (C(5)ꢀGal, 8α,β); 70.1 (C(2)ꢀGlc, 8α);
70.0 (C(3)ꢀGlc, 8α, C(2)ꢀGal, 8α,β); 69.4 (C(4)ꢀNeu, 8α,β);
68.5 (C(3)ꢀGlc, 8β); 67.8 (C(7)ꢀNeu, 8α,β); 67.3 (C(4)ꢀGal,
8α,β); 66.8 (C(8)ꢀNeu, 8α,β); 62.2 (C(6)ꢀGal, 8α,β); 62.0
(C(9)ꢀNeu, 8α,β); 61.7 (C(6)ꢀGlc, 8α,β); 53.1 (COOCH3,
8α,β); 49.2 (C(5)ꢀNeu, 8α,β); 37.4 (C(3)ꢀNeu, 8α,β);
23.1—20.5 (CH3CO). Found (%): C, 44.69; H, 5.07; N, 2.37.
C46H61Cl3N2O29. Calculated (%): C, 45.57; H, 5.07; N, 2.31.
3ꢀТrifluoroacetamidopropyl [methyl (5ꢀacetamidoꢀ4,7,8,9ꢀ
tetraꢀOꢀacetylꢀ3,5ꢀdideoxyꢀDꢀglyceroꢀαꢀDꢀgalactoꢀ2ꢀnonuloꢀ
pyranosyl)onate]ꢀ(2→3)ꢀ2,4,6ꢀtriꢀOꢀacetylꢀβꢀDꢀgalactopyranoꢀ
sylꢀ(1→4)ꢀ2,3,6ꢀtriꢀOꢀacetylꢀβꢀDꢀglucopyranoside (10). A soluꢀ
tion of imidate 8α,β (0.482 g, 0.398 mmol) and 3ꢀtrifluoroꢀ
acetamidopropanꢀ1ꢀol 9 (0.136 g, 0.795 mmol) in anhydrous
H(1)ꢀGlc, 7α, J1,2 = 3.5 Hz); 5.22 (dd, H(3)ꢀGlc, 7β, J3,2
=
J3,4 = 9.5 Hz); 5.12 (d, NHꢀNeu, 7α,β, J5,NH = 10.0 Hz); 4.94
(dd, H(2)ꢀGal, 7α,β, J2,1 = 8.0 Hz, J2,3 = 10.0 Hz); 4.92—4.87
(m, H(4)ꢀGal, 7α,β, H(4)ꢀNeu, 7α,β); 4.86 (dd, H(2)ꢀGlc, 7α,
J2,1 = 3.5 Hz, J2,3 = 10.5 Hz); 4.80 (dd, H(2)ꢀGlc, 7β, J2,1
=
J2,3 = 9.5 Hz); 4.73 (d, H(1)ꢀGlc, 7β, J1,2 = 9.5 Hz); 4.67 (d,
H(1)ꢀGal, 7α,β, J1,2 = 8.0 Hz); 4.52 (m, H(3)ꢀGal, 7α,β); 4.47
(d, H(6a)ꢀGlc, 7β); 4.43 (m, H(6a)ꢀGlc, 7α, H(9a)ꢀNeu, 7α,β);
4.25—4.14 (m, H(5)ꢀGlc, 7α, H(6b)ꢀGlc, 7α,β); 4.08—3.97
(m, H(5)ꢀNeu, 7α,β, H(6a)ꢀGal, H(6b)ꢀGal, 7α,β, H(9b)ꢀNeu,
7α,β); 3.88—3.82 (m, H(4)ꢀGlc, 7α,β, H(5)ꢀGal, 7α,β, OCH3
7α,β); 3.66 (m, H(5)ꢀGlc, 7β); 3.64 (m, H(6)ꢀNeu, 7α,β); 2.58
(dd, Heq(3)ꢀNeu, 7α,β, J3eq,4 = 4.4 Hz, J3eq,3ax = 12.7 Hz);
1.82—2.30 (m, CH3CO, 7α,β); 1.78 (dd, Hax(3)ꢀNeu, 7α,β,
J3ax,3eq = J3ax,4 = 12.5 Hz). 13C NMR (CDCl3), δ: 170.0—170.8
(CH3CO, CH3CON); 168.0 (C(1)ꢀNeu, 7α,β); 101.0 (C(1)ꢀGal,
7α,β); 96.8 (C(2)ꢀNeu, 7α,β); 95.2 (C(1)ꢀGlc, 7β); 90.2

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Russ.Chem.Bull., Int.Ed., Vol. 55, No. 11, November, 2006
Khatuntseva et al.
CH2Cl2 (3 mL) with activated molecular sieves MSꢀ4А (100 mg)
was stirred for 2 h under argon and cooled to –20 °C. Then
TMSOTf (0.1 mL, 0.54 mmol) was added and the mixture was
stirred for 2 h at this temperature. After completion of the reacꢀ
tion, Et3N (0.05 mL) was added, the temperature was brought
to ~20 °C, and the mixture was filtered through Celite and conꢀ
centrated. The residue was chromatographed on silica gel
(toluene→toluene—acetone, 1 : 1) to give 250 mg (52%) of comꢀ
pound 10 as a white foam, [α]D –7.8 (c 1, CHCl3), Rf 0.13
H(8)ꢀNeu, H(9b)ꢀNeu, H(3)ꢀGlc, H(4)ꢀGlc, H(5)ꢀGlc);
3.24 (dd, 1 H, H(2)ꢀGlc, J2,3 = 10.0 Hz); 3.07 (t, 2 H,
OCH2CH2CH2NH2, J = 7.0 Hz); 2.66 (dd, 1 H, Heq(3)ꢀNeu,
J3e,3a = 4.5 Hz, J3e,4 = 12.5 Hz); 1.94 (s, 3 H, CH3CO); 1.92 (m,
2 H, OCH2CH2CH2NH2); 1.70 (t, 1 H, Hax(3)ꢀNeu, J3a,3e
=
J3a,4 = 12.5 Hz). 13C NMR (D2O), δ: 176.2 (CH3CON); 175.0
(C(1)ꢀNeu); 103.8 (C(1)ꢀGal); 103.3 (C(1)ꢀGlc); 101.0
(C(2)ꢀNeu); 79.4 (C(4)ꢀGlc); 76.6 (C(3)ꢀGal); 76.3 (C(5)ꢀGal);
75.9 (C(8)ꢀNeu); 75.4 (C(5)ꢀGlc); 74.0 (C(3)ꢀGlc); 73.9
(C(2)ꢀGlc); 72.9 (C(6)ꢀNeu); 70.5 (C(2)ꢀGal); 69.4
(C(4)ꢀNeu); 69.3 (C(7)ꢀNeu); 69.0 (OCH2CH2CH2NH2); 68.7
(C(4)ꢀGal); 63.8 (C(9)ꢀNeu); 62.1 (C(6)ꢀGal); 61.2 (C(6)ꢀGlc);
52.9 (C(5)ꢀNeu); 40.8 (C(3)ꢀNeu); 38.7 (OCH2CH2CH2NH2);
27.8 (OCH2CH2CH2NH2); 23.2 (CH3CON).
1
(acetone—toluene, 2 : 3). H NMR (CDCl3), δ: 5.52 (m, 1 H,
H(8)ꢀNeu); 5.33 (dd, 1 H, H(7)ꢀNeu, J7,6 < 1, J7,8 = 9.1 Hz);
5.18 (d, 1 H, NHꢀNeu, J5,NH = 8.0 Hz); 5.15 (dd, 1 H, H(3)ꢀGlc,
J3,2 = J3,4 = 9.3 Hz); 4.90 (dd, 1 H, H(2)ꢀGal, J2,1 = 8.0 Hz,
J2,3 = 9.8 Hz); 4.84 (d, 1 H, H(4)ꢀGal, J3,4 = 2.7 Hz, J4,5 < 1 Hz);
4.82 (m, 1 H, H(4)ꢀNeu); 4.80 (dd, 1 H, H(2)ꢀGlc, J2,3
9.3 Hz); 4.66 (d, 1 H, H(1)ꢀGal, J1,2 = 8.0 Hz); 4.46—4.51 (m,
2 H, H(3)ꢀGal, H(6a)ꢀGlc); 4.43 (d, 1 H, H(1)ꢀGlc, J1,2
8.0 Hz); 4.38 (dd, 1 H, H(9a)ꢀNeu, J9a,8 = 2 Hz, J9a,9b
12.5 Hz); 4.13 (dd, 1 H, H(6b)ꢀGlc, J6a,6b = 12.0 Hz, J6b,5
=
3ꢀ(4ꢀMaleimidobutanoylamino)propyl [(5ꢀacetamidoꢀ3,5ꢀ
dideoxyꢀDꢀglyceroꢀαꢀDꢀgalactoꢀ2ꢀnonulopyranosyl)onic acid]ꢀ
(2→3)ꢀβꢀDꢀgalactopyranosylꢀ(1→4)ꢀβꢀDꢀglucopyranoside (12).
Active ester 11 (44 mg, 0.158 mmol, 2.1 equiv.) was added to a
suspension of aminopropyl glycoside 1 (51 mg, 0.074 mmol) in
anhydrous DMF (2 mL), the mixture was stirred for 10 min, and
a 2% solution of Et3N in anhydrous DMF (0.8 mL) was added
in portions. The reaction mixture was concentrated in vacuo (oil
pump) at ~20 °C, and the residue was subjected to gel chromaꢀ
tography on a column with Sephadex Gꢀ15 (45×2.5 cm) in
water. Freezeꢀdrying of carbohydrateꢀcontaining fractions from
=
=
=
4.9 Hz); 3.89—4.40 (m,
4 H, H(5)ꢀNeu, H(6a)ꢀGal,
H(6b)ꢀGal, H(9b)ꢀNeu); 3.80—3.87 (m, 3 H, H(4)ꢀGlc,
HaꢀOCH2CH2CH2N, H(5)ꢀGal); 3.79 (s, 3 H, COOCH3); 3.67
(m, 1 H, HbꢀOCH2CH2CH2N); 3.61 (d, 1 H, H(6)ꢀNeu,
J6,7 < 1 Hz, J6,5 = 10.8 Hz); 3.57 (m, 1 H, H(5)ꢀGlc);
3.45 (m,
1 H, HaꢀOCH2CH2CH2N); 3.35 (m, 1 H,
1
HbꢀOCH2CH2CH2N); 2.54 (dd, 1 H, Heq(3)ꢀNeu, J3eq,4
=
water gave 54 mg (85%) of compound 12. H NMR (D2O), δ:
4.4 Hz, J3eq,3ax = 12.7 Hz); 1.70—2.20 (m, 33 H, CH3CO); 1.82
(m, 2 H, OCH2CH2CH2N); 1.62 (dd, 1 H, Hax(3)ꢀNeu,
J3ax,3eq = J3ax,4 =12.5 Hz). 13C NMR (CDCl3), δ: 169.6—170.5
(CH3CO, CH3CON); 167.9 (C(1)ꢀNeu); 100.9 (C(1)ꢀGal);
100.6 (C(1)ꢀGlc); 96.8 (C(2)ꢀNeu); 75.9 (C(4)ꢀGlc); 73.2
(C(3)ꢀGlc); 73.0 (C(5)ꢀGlc); 71.9 (C(2)ꢀGlc); 71.7 (C(6)ꢀNeu);
71.3 (C(3)ꢀGal); 70.5 (C(5)ꢀGal); 69.9 (C(2)ꢀGal); 69.3
(C(4)ꢀNeu); 68.1 (OCH2CH2CH2N); 67.7 (C(8)ꢀNeu); 67.2
(C(4)ꢀGal); 67.0 (C(7)ꢀNeu); 62.3 (C(9)ꢀNeu); 61.9
(C(6)ꢀGlc); 61.4 (C(6)ꢀGal); 53.1 (COOCH3); 49.0
(C(5)ꢀNeu); 37.8 (OCH2CH2CH2N); 37.4 (C(3)ꢀNeu); 28.3
(OCH2CH2CH2N); 23.1—20.5 (CH3CO). Found (%): C, 47.64;
H, 5.53; N 2.32. C49H67F3N2O30. Calculated (%): C, 48.20;
H, 5.53; N, 2.29.
6.83 (s, 2 H, CH=CH); 4.51 (d, 1 H, H(1)ꢀGal, J2,1 = 7.8 Hz);
4.45 (d, 1 H, H(1)ꢀGlc, J2,1 = 7.9 Hz); 4.09 (dd, 1 H, H(3)ꢀGal,
J3,2 = 9.9 Hz, J3,4 = 2.6 Hz); 3.97 (dd, 1 H, H(6а)ꢀGlc, J6a,6b
=
12.0 Hz, J6a,5 < 1 Hz); 3.94 (d, 1 H, H(4)ꢀGal, J3,4 = 3.1 Hz,
J4,5 < 1 Hz); 3.92 (m, 1 H, HаꢀOCH2CH2CH2NH); 3.89—3.76
(m, 5 H, H(5)ꢀNeu, H(9a)ꢀNeu, HbꢀOCH2CH2CH2NH,
H(5)ꢀGal, H(6b)ꢀGlc); 3.76—3.60 (m, 9 H, H(4)ꢀNeu,
H(6)ꢀNeu, H(8)ꢀNeu, H(9b)ꢀNeu, H(3)ꢀGlc, H(4)ꢀGlc,
H(5)ꢀGlc, H(6a)ꢀGal, H(6b)ꢀGal); 3.60—3.54 (m, 2 H,
H(7)ꢀNeu, H(2)ꢀGal); 3.51 (t, 2 H, NHCOCH2CH2CH2N,
J = 6.8 Hz); 3.29 (dd, 1 H, H(2)ꢀGlc, J2,3 = J2,1 = 7.9 Hz); 3.22
(m, 2 H, OCH2CH2CH2NH); 2.74 (dd, 1 H, Heq(3)ꢀNeu,
J3e,3a
=
12.4 Hz, J3e,4
=
4.5 Hz); 2.23 (t,
2
H,
NHCOCH2CH2CH2N, J = 7.0 Hz); 2.02 (s, 3 H, CH3CO);
3ꢀAminopropyl [(5ꢀacetamidoꢀ3,5ꢀdideoxyꢀDꢀglyceroꢀαꢀDꢀ
galactoꢀ2ꢀnonulopyranosyl)onic acid]ꢀ(2→3)ꢀβꢀDꢀgalactopyranoꢀ
sylꢀ(1→4)ꢀβꢀDꢀglucopyranoside (1). A solution of protected deꢀ
rivative 10 (240 mg, 0.196 mmol) in a mixture of methanol
(8.3 mL) and 3 M aqueous NаOH (1.7 mL) was kept at room
temperature for 6 h, AcOH was added to рH 7, and the mixture
was concentrated. The residue was subjected to gel chromatogꢀ
raphy on a column with TSK HWꢀ40(S) gel (1.5×90 cm) in
0.05 M NH4HCO3, the carbohydrateꢀcontaining fractions were
concentrated and freezeꢀdried from water to give 120 mg (88%)
of compound 1, [α]D +2.0 (c 1, H2O), Rf 0.43 (butanol—proꢀ
panol—hydrochloric acid—acetonitrile—methanol—water,
1.88 (m, 2 H, NHCOCH2CH2CH2N); 1.79 (m, 2 H,
OCH2CH2CH2NH); 1.78 (dd, 1 H, Hax(3)ꢀNeu, J3a,3e = J3a,4
=
12.5 Hz). 13C NMR (D2O), δ: 176—172 (CH3CON, C(1)ꢀNeu,
COCH=CHCO, NHCOCH2CH2CH2N); 135.6 (CH=CH);
103.9 (C(1)ꢀGal); 103.3 (C(1)ꢀGlc); 101.0 (C(2)ꢀNeu); 79.6
(C(4)ꢀGlc); 76.7 (C(3)ꢀGal); 76.4 (C(5)ꢀGal); 76.0 (C(8)ꢀNeu);
75.6 (C(5)ꢀGlc); 74.1 (C(3)ꢀGlc); 74.1 (C(2)ꢀGlc); 73.0
(C(6)ꢀNeu); 70.6 (C(2)ꢀGal); 69.5 (C(4)ꢀNeu); 69.4
(C(7)ꢀNeu); 69.1 (OCH2CH2CH2NH); 68.7 (C(4)ꢀGal); 63.9
(C(9)ꢀNeu); 62.2 (C(6)ꢀGal); 61.4 (C(6)ꢀGlc); 52.9
(C(5)ꢀNeu); 40.9 (C(3)ꢀNeu); 38.2 (OCH2CH2CH2NH); 34.4
(NHCOCH2CH2CH2N); 37.6 (NHCOCH2CH2CH2N); 29.6
(OCH2CH2CH2NH); 24.9 (NHCOCH2CH2CH2N); 23.3
(CH3CO).
1
2 : 2 : 2 : 3 : 3 : 3). H NMR (D2O), δ: 4.43 (d, 1 H, H(1)ꢀGal,
J2,1 = 10.0 Hz); 4.41 (d, 1 H, H(1)ꢀGlc, J2,1 = 10.0 Hz); 4.01
(dd, 1 H, H(3)ꢀGal, J3,2 = 9.9 Hz, J3,4 = 2.5 Hz); 3.95 (m, 1 H,
Thiolation of KLH. 2ꢀIminothiolane hydrochloride 13
(27 mg) was added under argon to a mixture of a solution of
KLH (7 mL, ~37.1 mg of protein) in a PBS buffer (5 mL)
(containing 1 mM MgCl2 and 15 mM NaN3, pH 7.2) and a
buffer (7 mL) containing 50 mM triethanolamine, 0.15 mM
NaCl, and 5 mM EDTA (pH 8.0). The mixture was kept for 2 h
at ~20 °C and dialyzed under Ar against a buffer solution
HаꢀOCH2CH2CH2NH2); 3.90 (dd, 1 H, H(6а)ꢀGlc, J6a,6b
=
12.0 Hz, J6a,5 < 1); 3.87 (d, 1 H, H(4)ꢀGal, J3,4 = 2.5 Hz,
J4,5 < 1 Hz); 3.82—3.68 (m, 5 H, H(5)ꢀNeu, H(6)ꢀNeu,
H(9a)ꢀNeu, HbꢀOCH2CH2CH2NH2, H(6а)ꢀGlc, H(6b)ꢀGlc);
3.68—3.58 (m,
5 H, H(4)ꢀNeu, H(2)ꢀGal, H(5)ꢀGal,
H(6a)ꢀGal, H(6b)ꢀGal); 3.58—3.44 (m, 6 H, H(7)ꢀNeu,

Sialylꢀ3´ꢀlactose neoglycoconjugates
Russ.Chem.Bull., Int.Ed., Vol. 55, No. 11, November, 2006 2101
(250 mL) containing 0.1 M sodium phosphate, Na2H2edta
(0.1 mol), NaCl (0.15 mol), and 0.01% NaN3 (buffer soluꢀ
tion A, pH 7.2). The dialysis was repeated three times to give a
solution of thiolated KLH 14, which was used directly for conꢀ
jugation with maleimide derivative 12.
HWꢀ40(S) in 0.05 M NH4HCO3. Freezeꢀdrying of carbohydrateꢀ
containing fractions gave 17 mg (85%) of amino derivative 17,
Rf 0.38 (butanol—propanol—hydrochloric acid—acetoniꢀ
trile—methanol—water, 2 : 2 : 2 : 3 : 3 : 3). H NMR (D2O), δ:
4.50 (d, 1 H, H(1)ꢀGal, J2,1 = 7.5 Hz); 4.45 (d, 1 H, H(1)ꢀGlc,
1
Conjugation of maleimide 12 with thiolated KLH 14. A soluꢀ
tion of maleimide 12 (14.5 mg, 0.017 mmol) in buffer solution A
(1 mL) was added with gentle shaking under Ar to the obtained
solution of thiolated protein 14. The mixture was kept for 24 h at
~20 °C and dialyzed under Ar against a PBS buffer (250 mL)
containing 0.01% NaN3. Dialysis was repeated three times to
give a solution of conjugate 2. The content of neuraminic acid in
conjugate 2 was determined by the Svennerholm method and
the protein content was determined by spectrophotometry using
the bicinchoninic acid reagent (BCA Protein assay kit, Pierce)
according to the manufacturer´s protocol.
3ꢀ(Octadecanoylamino)propyl [(5ꢀacetamidoꢀ3,5ꢀdideoxyꢀDꢀ
glyceroꢀαꢀDꢀgalactoꢀ2ꢀnonulopyranosyl)onic acid]ꢀ(2→3)ꢀβꢀDꢀ
galactopyranosylꢀ(1→4)ꢀβꢀDꢀglucopyranoside (3). pꢀNitrophenyl
stearate 15 (3.2 mg, 0.008 mmol) and Et3N (0.01 mL) were
added to a solution of trisaccharide 1 (5.9 mg, 0.0078 mmol) in
anhydrous DMF (0.4 mL), and the resulting mixture was stirred
for 16 h at ~20 °C. The solvent was evaporated in vacuo (oil
pump), and the residue was chromatographed on a column with
Sephadex LHꢀ20 (2×45 cm) in methanol. Carbohydrateꢀconꢀ
taining fractions were concentrated, and the residue was dissolved
in water (1.5 mL) and applied onto the cartridge SepꢀPak Cꢀ18.
The cartridge was washed with water (10 mL) and methanol
(10 mL). The methanolic eluate was concentrated, and the resiꢀ
due was freezeꢀdried from water to give 6 mg (80%) of amide 3,
Rf 0.68 (CHCl3—MeOH—water, 5 : 5 : 1). 1H NMR (D2O),
δ: 2.15 (t, 2 H, NHCOCH2, J = 7.4 Hz); 1.57 (m, 2 H,
NHCOCH2CH2); 1.26 (m, 30 H, 15 CH2); 0.87 (t, 3 H, CH3,
J = 7.1 Hz); the carbohydrate part of the spectrum did not
virtually differ from the spectrum of starting 1.
Conjugate of trisaccharide 1 with polyacrylamide (5). A soluꢀ
tion of poly(pꢀnitrophenyl acrylate) 16 (10 mg, 0.052 mmol
of nitrophenyl residues), aminopropyl glycoside 1 (7 mg,
0.01 mmol), and Et3N (0.015 mL) in dry DMF (0.3 mL) was
stirred for 24 h at 40 °C, then BuNH2 (1.5 mg, 0.02 mmol) was
added, the mixture was stirred for 48 h at ~20 °C, ethanolamine
(0.02 mL) was added, and the mixture was stirred for an addiꢀ
tional 48 h at 40 °C. The reaction mixture was concentrated at
40 °C in vacuo (oil pump). The product was isolated by chromaꢀ
tography on a column with Sephadex LHꢀ20 in aqueous MeCN
(1 : 1). Freezeꢀdrying gave 9.5 mg of functionalized polyacrylꢀ
amide 5.
3ꢀ(14ꢀAminoꢀ3,6,9,12ꢀtetraoxatetradecanoylamino)propyl
[(5ꢀacetamidoꢀ3,5ꢀdideoxyꢀDꢀglyceroꢀαꢀDꢀgalactoꢀ2ꢀnonuloꢀ
pyranosyl)onic acid]ꢀ(2→3)ꢀβꢀDꢀgalactopyranosylꢀ(1→4)ꢀβꢀ
Dꢀglucopyranoside (18). A solution of pentafluorophenyl
14ꢀtrifluoroacetamidoꢀ3,6,9,12ꢀtetraoxatetradecanoate (17)
(28 mg, 0.054 mmol) in anhydrous DMF (0.2 mL) and Et3N
(87 mg, 0.86 mmol) in anhydrous DMF (0.1 mL) were added
with stirring to a suspension of aminopropyl glycoside 1 (15 mg,
0.022 mmol) in anhydrous DMF (0.2 mL). After complete disꢀ
solution of the starting 1, water (0.2 mL) was added, and the
solution was concentrated in vacuo (oil pump) at ~20 °C. The
residue was dissolved in 0.5 M NaOH (1.2 mL) and kept for
10 min, then AcOH was added to pH 6.5—7, and the solution
was concentrated and chromatographed on a column with TSK
J2,1 = 8.0 Hz); 4.08 (dd, 1 H, H(3)ꢀGal, J3,2 = 9.9 Hz, J3,4
2.9 Hz); 4.05 (s, 2 H, NHCOCH2O); 3.95 (dd, 1 H, H(6a)ꢀGlc,
J6a,6b 12.7 Hz, J6a,5 1.1 Hz); 3.94 (m, H,
=
=
=
1
OCH2CH2CH2NH); 3.93 (d, 1 H, H(4)ꢀGal, J3,4 = 3.2 Hz,
J4,5 < 1 Hz); 3.88—3.79 (m, 3 H, H(5)ꢀNeu, H(9a)ꢀNeu,
H(5)ꢀGal); 3.79 (dd, 1 H, H(6b)ꢀGlc, J6b,6a = 12.7 Hz,
J6b,5 = 4.8); 3.75—3.65 (m, 20 H, OCH2CH2O, OCH2CH2NH2,
OCH2CH2CH2NH, H(6a)ꢀGal, H(6b)ꢀGal, H(4)ꢀNeu,
H(6)ꢀNeu); 3.65—3.58 (m, 6 H, H(3)ꢀGlc, H(4)ꢀGlc,
H(8)ꢀNeu, H(9b)ꢀNeu); 3.52—3.58 (m, 4 H, H(2)ꢀGal,
H(5)ꢀGl), H(7)ꢀNeu); 3.34 (t, 2 H, OCH2CH2CH2NH); 3.28
(dd, 1 H, H(2)ꢀGlc, J2,3 = J2,1 = 8.5 Hz); 3.12 (t, 2 H,
OCH2CH2NH2); 2.73 (dd, 1 H, Heq(3)ꢀNeu, J3e,3a = 12.5 Hz,
J3e,4 = 4.6 Hz); 2.00 (s, 3 H, CH3CON); 1.84 (m, 2 H,
OCH2CH2CH2NH); 1.78 (dd, 1 H, Hax(3)ꢀNeu, J3a,3e = J3a,4
=
12.5 Hz). 13C NMR (D2O), δ: 176—172 (CH3CON, CH2CON,
C(1)ꢀNeu); 103.9 (C(1)ꢀGal); 103.3 (C(1)ꢀGlc); 101.0
(C(2)ꢀNeu); 79.8 (C(4)ꢀGlc); 76.7 (C(3)ꢀGal); 76.4 (C(5)ꢀGal);
76.0 (C(8)ꢀNeu); 75.6 (C(5)ꢀGlc); 74.1 (C(3)ꢀGlc); 74.1
(C(2)ꢀGlc); 73.0 (C(6)ꢀNeu); 73.0—68.5 (C(8), OCH2CH2O,
OCH2CH2NH2, OCH2CH2CH2NH, C(4)ꢀNeu); 71.3
(NHCOCH2O); 70.6 (C(2)ꢀGal); 69.4 (C(7)ꢀNeu); 68.5
(C(4)ꢀGal); 63.9 (C(9)ꢀNeu); 62.2 (C(6)ꢀGal); 61.4 (C(6)ꢀGlc);
52.9 (C(5)ꢀNeu); 40.9 (C(3)ꢀNeu); 40.3 (OCH2CH2NH2); 38.2
(OCH2CH2CH2NH); 30.4 (OCH2CH2CH2NH); 23.3
(CH3CO).
3ꢀ(14ꢀBiotinylaminoꢀ3,6,9,12ꢀtetraoxatetradecanoylamiꢀ
no)propyl [(5ꢀacetamidoꢀ3,5ꢀdideoxyꢀDꢀglyceroꢀαꢀDꢀgalactoꢀ2ꢀ
nonulopyranosyl)onic acid]ꢀ(2→3)ꢀβꢀDꢀgalactopyranosylꢀ(1→4)ꢀ
βꢀDꢀglucopyranoside (4). Triethylamine (0.03 mL) was added to
a solution containing compound 18 (9.4 mg, 0.010 mmol) and
active ester 19 (5 mg, 0.014 mmol) in anhydrous DMF (0.3 mL),
the mixture was stirred for 10 min and concentrated in vacuo (oil
pump) at ~20 °C. The residue was chromatographed on a colꢀ
umn with TSK HWꢀ40(S) in 0.05 M NH4HCO3. Freezeꢀdrying
of carbohydrateꢀcontaining fractions gave 9.4 mg (82%) of
biotinylated product 5. Rf 0.58 (butanol—propanol—hydrochloꢀ
ric acid—acetonitrile—methanol—water, 2 : 2 : 2 : 3 : 3 : 3).
1H NMR (D2O), δ: 4.58 (dd, 1 H, H(2)ꢀbiotin, J2,3 = 7.8 Hz,
J2,1cis < 1, J2,1trans = 4.2 Hz); 4.50 (d, 1 H, H(1)ꢀGal, J2,1
=
7.5 Hz); 4.44 (d, 1 H, H(1)ꢀGlc, J2,1 = 7.8 Hz); 4.40 (dd, 1 H,
H(3)ꢀbiotin, J3,2 = 7.8 Hz, J3,4 = 4.5 Hz); 4.08 (dd, 1 H,
H(3)ꢀGal, J3,2 = 9.9 Hz, J3,4 = 2.7 Hz); 4.04 (s, 2 H,
NHCOCH2O); 3.99—3.89 (m, 3 H, H(4)ꢀGal, H(6a)ꢀGlc,
OCH2CH2CH2NH); 3.89—3.76 (m, 6 H, H(5)ꢀGal, H(5)ꢀNeu,
H(6)ꢀNeu, H(9a)ꢀNeu, H(6b)ꢀGlc); 3.51—3.76 (m, 25 H,
OCH2CH2O, OCH2CH2NH, OCH2CH2CH2NH, H(2)ꢀGal,
H(6a)ꢀGal, H(6b)ꢀGal, H(4)ꢀNeu, H(7)ꢀNeu, H(8)ꢀNeu,
H(9b)ꢀNeu, H(3)ꢀGlc, H(4)ꢀGlc, H(5)ꢀGlc); 3.39—3.26 (6 H,
OCH2CH2NH, H(2)ꢀGlc, H(4)ꢀbiotin); 2.96 (dd, 1 H,
H(1)ꢀtransꢀbiotin, J1cis,1trans = 13.0 Hz, J1trans,2 = 4.2 Hz); 2.75
(dd, 1 H, H(1)ꢀcisꢀbiotin, J1cis,1trans = 13.0 Hz, J1cis,2 < 1 Hz);
2.73 (dd, 1 H, Heq(3)ꢀNeu, J3e,3a = 12.5 Hz, J3e,4 = 4.3 Hz);
2.25 (dd, 2 H, H(8)ꢀbiotin, J7,8 = J7,6 = 7.1 Hz, J2,1cis < 1 Hz);
2.00 (s, 3 H, CH3CON); 1.84 (m, 2 H, NHCOCH2CH2CH2N);
1.78 (dd, 1 H, Hax(3)ꢀNeu, J3a,3e = J3a,4 = 12.5 Hz); 1.73—1.50

2102
Russ.Chem.Bull., Int.Ed., Vol. 55, No. 11, November, 2006
Khatuntseva et al.
(m, 2 H, H(5)ꢀbiotin, H(3)ꢀbiotin); 1.38 (m, 1 H, H(6)ꢀbiotin).
13C NMR (D2O), δ: 178.0 (OCH2CH2NCO); 176.2 (CH3CON);
174.8 (C(1)ꢀNeu); 173.5 (NCOCH2O); 151.2 (HNC(O)NH);
103.9 (C(1)ꢀGal); 103.3 (C(1)ꢀGlc); 101.0 (C(2)ꢀNeu); 79.7
(C(4)ꢀGlc); 76.8 (C(3)ꢀGal); 76.3 (C(6)ꢀNeu); 76.0 (C(5)ꢀGlc);
75.6 (C(8)ꢀNeu); 74.2 (C(2)ꢀGal, C(3)ꢀGal); 73.0 (C(5)ꢀGal);
71.5—70.1 (C(10), OCH2CH2O, OCH2CH2NH, C(2)ꢀGal,
NHCOCH2O); 69.5 (C(4)ꢀNeu); 69.4 (C(7)ꢀGal); 69.0
(OCH2CH2CH2NH); 68.7 (C(4)ꢀGlc); 63.9 (C(9)ꢀNeu); 63.3
(C(3)ꢀbiotin); 62.2 (C(6)ꢀGal); 61.4 (C(6)ꢀGlc, C(2)ꢀbiotin);
56.5 (C(4)ꢀbiotin); 52.9 (C(5)ꢀNeu); 40.9 (C(3)ꢀNeu,
C(1)ꢀbiotin); 40.1 (OCH2CH2NH); 37.2 (OCH2CH2CH2NH);
36.7 (C(8)ꢀbiotin); 29.7 (OCH2CH2CH2NH); 29.1
(C(6)ꢀbiotin); 28.9 (C(5)ꢀbiotin); 26.3 (C(7)ꢀbiotin);
23.3 (CH3CO).
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This work was financially supported by the Russian
Foundation for Basic Research (Project No. 05ꢀ03ꢀ
08107).
14. N. V. Bovin, E. Yu. Korchagina, T. V. Zemlyanukhina,
N. E. Byramova, O. E. Galanina, A. E. Zemlyakov, A. E.
Ivanov, V. P. Zubov, and L. V. Mochalova, Glycoconjugate J.,
1993, 10, 142.
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Tsvetkov, E. A. Khatuntseva, R. Ya. Vlasenko, and I. K.
Verner, VI S"ezd allergologov i immunologov SNG [VI Conꢀ
gress of Allegologists and Immunologisys of the CIS], Abstrs.
Allergologiya i immunologiya [Allergology and Immunology],
2006, 7, 342 (in Russian).
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Received June 21, 2006;
in revised form September 15, 2006