Synthesis of bi- and tervalent ligands of galectins, derivatives of β-lactosylamine, with the amino group in the terminal position of the spacer

Article abstract of DOI:10.1007/s11172-008-0104-x

A method for the synthesis of glycoclusters by N-alkylation of N-glycyl-β-lactosylamine with N-chloroacetyl derivatives of β-lactosylamine and N,N'-iminodiacetyldilactosylamine has been developed. The glycoclusters obtained with two and three lactosylamine residues with the amino group in the spacer were found to be suitable for further conjugation with carboxy-containing physiologically active compounds.

Full text of DOI:10.1007/s11172-008-0104-x

Russian Chemical Bulletin, International Edition, Vol. 57, No. 3, pp. 660—664, March, 2008
660
Synthesis of biꢀ and tervalent ligands of galectins, derivatives
of βꢀlactosylamine, with the amino group in the terminal position of the spacer
L. M. Likhosherstov,a O. S. Novikova,a and G. V. Mokrovb
aN. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences,
47 Leninsky prosp., 119991 Moscow, Russian Federation.
Fax: +7 (499) 135 5328. Eꢀmail: knirel@ioc.ac.ru
bHigher Chemical College, Russian Academy of Sciences,
9 Miusskaya pl., 125047 Moscow, Russian Federation
A method for the synthesis of glycoclusters by Nꢀalkylation of Nꢀglycylꢀβꢀlactosylamine with
Nꢀchloroacetyl derivatives of βꢀlactosylamine and N,N´ꢀiminodiacetyldilactosylamine has been
developed. The glycoclusters obtained with two and three lactosylamine residues with the amino
group in the spacer were found to be suitable for further conjugation with carboxyꢀcontaining
physiologically active compounds.
Key words: glycoclusters, polyvalent ligands, Nꢀglycylꢀβꢀlactosylamine, Nꢀchloroacetylꢀβꢀlactoꢀ
sylamine.
Glycosylamines have found evergrowing application in
the preparation of glycoconjugates that are used in various
biochemical investigations associated, for example, with
the directed transport of physiologically active compounds
(PAC) to the target cells1,2 or with the quest of vaccines
against the human immunodeficiency virus.3 The syntheꢀ
sis of glycoconjugates most often consists in Nꢀacylation
of glycosylamine, which leads either to the target products
or to derivatives with functional groups suitable for further
conjugation. From Nꢀacyl derivatives, Nꢀglycylꢀβꢀglycoꢀ
sylamines derivatives from monoꢀ and oligosaccharides,
the synthesis of which has been described earlier (see Ref.
4 and references cited therein), are suitable for the prepaꢀ
ration of glycoconjugates. These compounds are used for
both the direct synthesis of glycoconjugates4—9 and the
preliminary modification of the spacer, i.e., a bridge conꢀ
necting a carbohydrate and PAC. A spacer can be varied in
length and hydrophilicity10 with subsequent yield of glyꢀ
coconjugates.11 As follows from the literature data,12,13
the carbohydrate fragments of glycoconjugates with the
optimally selected spacer can possess significantly higher
binding ability to the receptors of a target cell and, thereꢀ
fore, enhanced efficiency of delivery of PAC. Yet higher
efficiency of interaction of glycoconjugates with receptors
can be usually achieved by multiple binding of glycoconꢀ
jugates containing few identical carbohydrate residues,
clusters (the soꢀcalled multivalent or cluster effect). Mulꢀ
tivalent glycoconjugates are obtained from compounds
containing several reaction centers, for example, by the
reaction of Nꢀglycylꢀβꢀglycosylamines, derivatives of oliꢀ
gosaccharides, with activated polyacrylic esters.6 For the
preparation of multivalent glycoconjugates from PAC with
one reaction center, a necessity arises in a preliminary
synthesis of a glycocluster with a functional group in
the spacer appropriate for the conjugation.
In continuation of our research on the synthesis of
glycoconjugates of polyhedral boron compounds (see Ref. 11
and references cited therein), the present work deals with
the development of a method for the synthesis of glycoꢀ
clusters containing up to three residues of βꢀlactosylꢀ
amine with the amino group in the terminal position of
the spacer. The selection of the disaccharide lactose with
βꢀgalactose at the nonreducing end in the glycocluster
structures is connected with the hyperexpression on
the surface of tumor cells of galectins, i.e., proteins,
specifically binding the terminal βꢀgalactose residues.14
One can expect that the conjugates of the glycoclusters
with PAC will efficiently bind to these cells.
The preparation of the glycoclusters includes a stepꢀ
wise introduction of lactosylamine residues by the iteraꢀ
tion of the Nꢀalkylation reactions of the amino group in
Nꢀglycylꢀβꢀlactosylamine followed by the Nꢀacylation of
the formed imino groups (Scheme 1). A similar approach
was described earlier for the synthesis of the soꢀcalled Nꢀglyꢀ
copeptoids in which the residues of Nꢀsubstituted glycine
are present (see Ref. 8 and references cited therein). The
major differences of our synthetic scheme consists in
the use of unprotected carbohydrates and in that both comꢀ
ponents of the Nꢀalkylation contain carbohydrate residues.
NꢀGlycylꢀβꢀlactosylamine (1) and Nꢀchloroacetylꢀβꢀ
lactosylamine (2) were used as the starting compounds
for the synthesis of the bivalent ligand (see Scheme 1).
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 3, pp. 647—651, March, 2008.
1066ꢀ5285/08/5703ꢀ660 © 2008 Springer Science+Business Media, Inc.

NꢀGlycyl(lactosylamine) glycoclusters
Russ.Chem.Bull., Int.Ed., Vol. 57, No. 3, March, 2008
661
Scheme 1
carbodiimide (DCC) in the presence of Nꢀhydroxysucꢀ
cinimide (NHS) in DMSO—DMF (10 : 1) at 10 °C.
Small amount of Oꢀacyl groups in the Nꢀacylated product
5 was removed by treatment with Et3N in aq. MeOH.
After removal of the Nꢀprotecting group (hydrogenolysis
over Pd/C) in compound 5, the bivalent ligand 6 with
the elongated (as compared to 1) spacer and the terminal
amino group was obtained.
For the synthesis of a precursor of the tervalent ligand,
Nꢀchloroacetylation of imino compound 3 with chloroꢀ
acetic anhydride (7) (a twofold excess) in anhydrous
DMF was carried out. Small amount of Oꢀacyl groups in
the Nꢀacylated product was removed by treatment with
Et3N in aq. MeOH to obtain compound 8. The Nꢀalkylaꢀ
tion of amino compound 1 with Nꢀchloroacetyl comꢀ
pound 8 led to imino compound 9 with three lactosylꢀ
amide residues. The elongation of the spacer in comꢀ
pound 9 was carried out as described above for comꢀ
pound 3 to obtain compound 10, hydrogenolysis of which
afforded the target tervalent ligand 11.
Structures of the synthesized compounds were conꢀ
firmed by the elemental analysis and data from NMR
spectroscopy. In the 1H NMR spectra of all the comꢀ
pounds with two and three lactosylamine residues, sigꢀ
nals for the protons H(2)—H(6) of the galactose and
glucose residues in the region of δ 3.40—4.00 are present,
which we did not study in detail. Some variations should
be noted in the number and character of the observed
H(1) proton signals in the spectra. Thus for compound 5
the signals for the protons H(1) of the βꢀgalactose residue
manifest themselves as two close doublets (∆δ 0.01 ppm),
while for compounds 6 and 11 as broadened doublets.
More noticeable distinctions in the signals for the proꢀ
tons H(1) are observed for the βꢀglucose residues of all
the compounds, except for compound 3. In the spectra of
compounds 5, 6, and 8 containing two βꢀglucose residues
two doublets each with ∆δ from 0.04 to 0.06 ppm are
present. In the spectra of compounds containing three
βꢀglucose residues a doublet and a doublet with a twofold
larger intensity with ∆δ 0.03 ppm (compound 9) or mulꢀ
tiplets with ∆δ up to 0.12 ppm (compounds 10 and 11) are
present.
Lac = DꢀGalp(β1—4)DꢀGlcpβ
Z = PhCH2OCO
Reagents and conditions: a. Pri2EtN, MeOH—H2O (3 : 2); b. DCC,
NHS, DMSO—DMF (10 : 1); c. H2, Pd/C, H2O; d. DMF.
In the spectra of the synthesized compounds, the sigꢀ
nals for the protons of the CH2 groups of the spacer are
found in a broad region (δH 3.39—5.20). A part of these
signals (mainly in the form of broadened singlets) in
the spectra of compounds 3, 5, 6, 8, and 9 were assigned
to particular CH2 groups (see Experimental) based on
both the literature data4,11,15 and the appearance or disꢀ
appearance of signals as a result of certain reactions. No
assignment of signals for particular CH2 groups in comꢀ
pounds 10 and 11 were made (except for the CH2Ph
group) since the greater part of the protons for the CH2
groups gave complicated spectra with numerous signals,
while the lesser part of the protons was hidden among the
These have been obtained by us earlier4,15 from βꢀlactosyꢀ
lamine, the synthesis of which in the present work was
carried out by a new and more efficient method.16 The
Nꢀalkylation of amino compound 1 with chloroacetꢀ
amide 2 in aqueous MeOH in the presence of Pri2EtN
leads to N,N'ꢀiminodiacetyldilactosylamine (3). Imino
compound 3 was further used for both the elongation of
the spacer and the synthesis of the tervalent ligand.
The elongation of the spacer was carried out by the conꢀ
densation of compound 3 with a twofold excess of Nꢀbenꢀ
zyloxycarbonylglycylglycine (4) and N,N'ꢀdicyclohexylꢀ

662
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Likhosherstov et al.
was filtered off, washed with 70% aq. MeOH, MeOH, and Et2O,
and dried to give amorphous compound 3 (1.58 g). The mother
liquor and a solution after washing with 70% aq. MeOH were
combined and concentrated nearly to dryness. The residue was
dissolved in H2O (30 mL), anionite Dowex 1w×8 (OH–) (30 mL)
was added, the mixture was stirred for 10 min and diluted with H2O
(100 mL). The formed Pri2EtN was evaporated as an azeotrope with
H2O, concentrating the suspension to 60 mL. The resin was filtered
off and washed with H2O (300 mL). The filtrate and washings were
concentrated to 100 mL, cationite Dowex 50w×8 (H+) (15 mL) was
added, and the mixture was stirred for 1 h. The resin was filtered off,
washed with H2O (150 mL) and 0.5 M Py (110 mL). The latter
elutate was concentrated to dryness. The residue was dissolved in
H2O (2.3 mL), MeOH (5 mL) was added with stirring and
the mixture was kept for 16 h at 5 °C. The pricipitate that formed was
filtered off, washed with 70% aq. MeOH, MeOH, and Et2O, and
dried to give additionally amorphous compound 3 (0.38 g, total
proton signals of lactosylamide residues. The presence in
the spectra of compounds 10 and 11 of numerous signals
for the protons of the CH2 groups and multiplets for
the protons H(1) of the glucose residues, apparently, is
caused by the formation of rotamers due to the presence
of two disubstituted amide groups (see Ref. 8 and referꢀ
ences cited therein).
The 13C NMR spectra of the target glycoclusteres 6 and
11 gave additional confirmation of their structures. In
the spectra of these compounds, signals for 12 carbon
atoms of the galactose and glucose residues are present
the chemical shifts of which are practically identical for
both compounds 6 and 11 and compounds with one lacꢀ
tosylamide residue.11 In addition, signals of low intensity
of the C atoms of the CH2 and CO groups are present in
these spectra: three signals for four CH2 groups for comꢀ
pound 6, four signals for six CH2 groups for compound
11, three signals for four CO groups, and five signals for
six CO groups for compounds 6 and 11, respectively.
In conclusion, we have developed a method of
the synthesis of biꢀ and tervalent ligands of galectins,
derivatives of βꢀlactosylamine, without the use of the Oꢀproꢀ
tecting groups. The elongation of the spacer by the introꢀ
duction of a diglycine and the presence of the terminal
amino group make the synthesized compounds suitꢀ
able for the conjugation with carboxyꢀcontaining PAC.
The synthesized glycoclusters allow us to continue our
further research on the synthesis of new polyvalent glycoꢀ
conjugates of polyhedral boron compounds, which are of
interest as potential agents for the boron neutron capture
cancer therapy.
25
yield 50%), [α]D +5.0 (c 0.5, H2O). Found (%): C, 42.09;
H, 6.24; N, 5.08; H2O, 2.58. C28H49N3O22•H2O. Calculated (%):
C, 42.15; H, 6.44; N, 5.27; H2O, 2.26. 1H NMR, δ: 3.43 (s, 4 H,
2 CH2N); 3.45—3.60 (m, 4 H); 3.62—3.84 (m, 16 H); 3.88—3.94
(m, 4 H); 4.45 (d, 2 H, 2 H(1) Gal, J = 7.5 Hz); 5.02 (d, 2 H, 2 H(1)
Glc, J = 9.0 Hz).
Nꢀ(NꢀBenzyloxycarbonyldiglycyl)ꢀNꢀdi{Nꢀ[4ꢀOꢀ(βꢀDꢀgalactoꢀ
pyranosyl)ꢀβꢀDꢀglucopyranosyl]carbamoylmethyl}amine (5). A mixꢀ
ture of Nꢀdi{Nꢀ[4ꢀOꢀ(βꢀDꢀgalactopyranosyl)ꢀβꢀDꢀglucopyranoꢀ
syl]carbamoylmethyl}amine (3) (0.45 g, 0.56 mmol), Nꢀbenzyloxyꢀ
carbonylglycylglycine (4) (0.315 g, 1.18 mmol), and NHS (0.162 g,
1.4 mmol) were dissolved with heating at 50 °C in anhydrous
DMSO (2 mL) and DMF (0.2 mL). The solution was cooled to
10 °C and DCC (0.313 g, 1.52 mmol) was added. The reaction
mixture was stirred for 45 min and kept for 70 h at 18 °C. A pricipitate
of N,N'ꢀdicyclohexylurea was filtered off and washed with DMSO
(2×0.5 mL). The filtrate was added with stirring to toluene (40 mL)
and the liquid after clearing was separated by decanting from the oily
pricipitate. The pricipitate was triturated several times with toluene
(10 mL each time) until a viscous substance was obtained, which
further was treated several times with hot acetone (10 mL each time)
until a powder was obtained. The powder was filtered off, washed
with Et2O, dried, and dissolved in a mixture of H2O (2.5 mL) and
MeOH (2 mL), Et3N (0.5 mL) was added to the mixture and it was
kept for 3 h at 20 °C. The solution was diluted with MeOH (15 mL)
and concentrated. The procedure was repeated twice and concenꢀ
trated to dryness. The residue was washed with hot PriOH (4×7 mL)
and Et2O and dried. The residue was dissolved in H2O (10 mL),
cationite Dowex 50w×2 (H+) (8 mL) was added to the mixture and
it was stirred for 1 h. The resin was filtered off and washed with H2O
(90 mL). The solution was concentrated to 5 mL and passed
through a filter with silica gel Cꢀ18 (10 g) in H2O. Silica gel was
washed with H2O (200 mL), 10% aq. MeOH (80 mL), and 20% aq.
MeOH (200 mL). The fractions containing the target product were
concentrated to 4 mL and liophilized to obtain amorphous comꢀ
Experimental
1
H and 13C NMR spectra were recorded on a Bruker WMꢀ250
1
spectrometer in D2O at 24 °C (250 MHz for H and 62.7 MHz
for 13C) with acetone as the external standard. Optical rotation was
determined on a PUꢀ07 polarimeter (Russia). The course of
the reactions and control over isolation of products were carried out
by electrophoresis (30 V cm–1, 1 h) on Filtrak FN1 paper in
a pyridinium acetate buffer (0.05 M with respect to Py, pH 4.5).
Substances were visualized by the ninhydrin reagent and by
the sequence of reagents KIO4—AgNO3—KOH.17 Determination
of water was performed by the Fischer method. Monitoring of
the elution of compounds during chromatography on silica gel Cꢀ18
was performed by the UV absorption at 206 or 260 nm.
NꢀDi{Nꢀ[4ꢀOꢀ(βꢀDꢀgalactopyranosyl)ꢀβꢀDꢀglucopyranosyl]ꢀ
carbamoylmethyl}amine (3). A mixture of 4ꢀOꢀ(βꢀDꢀgalactopyranoꢀ
syl)ꢀNꢀglycylꢀβꢀDꢀglucopyranosylamine (1) (2.46 g, 6.2 mmol)
and 4ꢀOꢀ(βꢀDꢀgalactopyranosyl)ꢀNꢀchloroacetylꢀβꢀDꢀglucopyraꢀ
nosylamine (2) (2.58 g, 6.2 mmol) in 60% aq. MeOH (18 mL) and
Pri2EtN (1.12 mL, 6.6 mmol) was heated in a screwꢀcapped test
tube for 20 h at 70 °C. The reaction mixture was diluted with MeOH
(4 mL) and kept for 5 h at 20 °C. A gelꢀlike pricipitate formed was
filtered off and washed with 70% aq. MeOH. The precipitate was
dissolved in H2O (10 mL), MeOH (23 mL) was added with stirring,
and the mixture was kept for 16 h at 5 °C. The pricipitate that formed
25
pound 5 (0.38 g, 62%), [α]D +5.0 (c 0.5, H2O). Found (%):
C, 46.15; H, 6.34; N, 6.14; H2O, 5.37. C40H61N5O26•3H2O.
Calculated (%): C, 44.40; H, 6.24; N, 6.47; H2O, 5.00. 1H NMR,
δ: 3.43—3.60 (m, 4 H); 3.61—3.86 (m, 16 H); 3.86—4.01 (m, 6 H);
4.14 (br.s, 2 H, CH2N); 4.17 (br.s, 2 H, CH2N); 4.36 (br.s, 2 H,
CH2N); 4.44 (d, 1 H, H(1) Gal, J = 7.5 Hz); 4.45 (d, 1 H, H(1) Gal,
J = 7.5 Hz); 5.02 (d, 1 H, H(1) Glc, J = 9.0 Hz); 5.08 (d, 1 H, H(1)
Glc, J = 9.0 Hz); 5.16 (s, 2 H, CH2Ph); 7.44 (br.s, 5 H, Ph).

NꢀGlycyl(lactosylamine) glycoclusters
Russ.Chem.Bull., Int.Ed., Vol. 57, No. 3, March, 2008
663
NꢀDi{Nꢀ[4ꢀOꢀ(βꢀDꢀgalactopyranosyl)ꢀβꢀDꢀglucopyranosyl]ꢀ
carbamoylmethyl}ꢀNꢀ(diglycyl)amine (6). 10% Palladium on actiꢀ
vated carbon (0.18 g) was added to a solution of compound 5
(0.35 g, 0.32 mmol) in H2O (9 mL) under argon and hydrogenation
was carried out in a weak flow of H2 under vigorous stirring for 8 h
at 20 °C. The catalyst was filtered off and washed with H2O
(3×5 mL). The solution was concentrated to 7 mL and filtered
(0.45 µm filter), cationite Dowex 50w×2 (H+) (3 mL) was added
and the mixture was stirred for 1 h. The resin was filtered off, washed
with H2O (40 mL) and 0.5 M NH4OH (30 mL). The alkaline
fraction was concentrated to 5 mL, MeOH (20 mL) was added, and
this was concentrated to 5 mL, the resulting solution with a formed
precipitate was kept for 16 h at 5 °C. A gelꢀlike precipitate was
filtered off, washed with 90% aq. MeOH, MeOH, and Et2O, and
eluate (150 mL) containing the target product was concentrated to
dryness. The residue was dissolved in H2O (10 mL), the solution
was filtered (0.45 µm filter) and concentrated to dryness. The
residue was three times treated with boiling MeOH (5 mL each
time) and Et2O and dried to obtain amorphous compound 9 (0.73 g,
25
87%), [α]D +7.8 (c 0.5, H2O). Found (%): C, 43.06; H, 6.51;
N, 5.44. C44H75N5O34. Calculated (%): C, 43.38; H, 6.21; N,
5.75. 1H NMR, δ: 3.44 (s, 2 H, CH2NH); 3.44—3.61 (m, 8 H);
3.61—3.86 (m, 24 H); 3.90—4.00 (m, 6 H); 4.18 (br.s, 2 H,
CH2N); 4.30 (s, 2 H, CH2N); 4.45 (d, 3 H, 3 H(1) Gal, J = 7.5 Hz);
5.01 (d, 2 H, 2 H(1) Glc, J = 9.0 Hz); 5.04 (d, 1 H, H(1) Glc,
J = 9.0 Hz).
Nꢀ(NꢀBenzyloxycarbonyldiglycyl)ꢀNꢀ{Nꢀ[4ꢀOꢀ(βꢀDꢀgalactopyꢀ
ranosyl)ꢀβꢀDꢀglucopyranosyl]carbamoylmethyl}ꢀNꢀ(Nꢀ{Nꢀbis[4ꢀOꢀ
(βꢀDꢀgalactopyranosyl)ꢀβꢀDꢀglucopyranosyl]carbamoylmethyl}ꢀ
carbamoylmethyl)amine (10) was obtained as described for comꢀ
pound 5, from compound 9 (0.23 g, 0.19 mmol), dipeptide 4 (0.1 g,
0.38 mmol), NHS (0.046 g, 0.4 mmol), and DCC (0.087 g,
0.42 mmol) in anhydrous DMSO (1.5 mL) and anhydrous DMF
(0.15 mL). The yield of amorphous compound 9 was 0.16 g (58%),
25
dried to give amorphous compound 6 (0.24 g, 80%), [α]D +6.4
(c 0.5, H2O). Found (%): C, 41.49; H, 6.55; N, 7.44; H2O, 3.28.
C32H55N5O24·2 H2O. Calculated (%): C, 41.33; H, 6.40; N, 7.53;
1
H2O, 3.87. H NMR, δ: 3.39 (br.s, 2 H, CH2NH2); 3.43—3.58
(m, 4 H); 3.61—3.85 (m, 16 H); 3.88—3.96 (m, 4 H); 4.14, 4.17,
4.36 (all br.s, 2 H each, CH2NH, 2 CH2N); 4.44 (br.d, 2 H, 2 H(1)
Gal, J = 7.5 Hz); 5.00 (d, 1 H, H(1) Glc, J = 9.0 Hz); 5.06 (d, 1 H,
H(1) Glc, J = 9.0 Hz). 13C NMR, δ: 41.9 (CH2NH2); 44.2
(CH2NH); 52.9 (br., CH2NCH2); 60.9 (C(6)Glc); 62.1 (C(6)Gal);
69.6 (C(4)Gal): 72.0 (C(2)Gal); 72.5 (C(2)Glc); 73.5 (C(3)Gal);
76.0 (C(3)Glc); 76.4 (C(5)Gal); 77.5 (C(5)Glc); 78.8 (C(4)Glc);
80.2 (C(1)Glc); 103.9 (C(1)Gal); 172.6; 173.1; 175.9 (CO).
25
[α]D + 4.8 (c 0.5, H2O). Found (%): C, 42.88; H, 6.51; N, 6.35;
H2O, 5.06. C56H87N7O38•5H2O. Calculated (%): C, 43.21;
1
H, 6.28; N, 6.30; H2O, 5.79. H NMR, δ: 3.44—3.54 (m, 3 H);
3.54—3.60 (m, 3 H); 3.61—3.86 (m, 24 H); 3.88—3.98 (m, 9 H);
4.07 (br.s, 1 H); 4.13 (br.s, 1 H); 4.17 (br.s, 1 H); 4.21 (br.s, 1 H);
4.22—4.41 (m, 4 H); 4.42—4.59 (m, 4 H); 4.98—5.10 (m, 3 H,
3 H(1) Glc); 5.18 (s, 2 H, CH2Ph); 7.46 (br.s, 5 H, Ph).
NꢀDi{Nꢀ[4ꢀOꢀ(βꢀDꢀgalactopyranosyl)ꢀβꢀDꢀglucopyranosyl]ꢀ
carbamoylmethyl}ꢀNꢀ(chloroacetyl)amine (8). A suspension of comꢀ
pound 3 (0.6 g, 0.75 mmol) in anhydrous DMF (2.8 mL) was
cooled in ice followed by the addition of chloroacetic anhydride (7)
(0.154 g, 0.9 mmol) and stirring for for 4 h at 0 °C. Another portion
of chloroacetic anhydride (0.13 g, 0.76 mmol) was added to
the reaction mixture with stirring, and the mixture was kept for 16 h
at 5 °C. The obtained suspension was diluted with MeOH (5 mL),
a gelꢀlike precipitate was filtered off, washed several times with
MeOH (until neutral pH) and Et2O and dried. The residue was
dissolved in H2O (2.5 mL) followed by addition of MeOH containꢀ
ing 20% of Et3N (2.5 mL), and kept for 3 h at 20 °C. The precipitate
that formed was filtered off, washed with 70% aq. MeOH, then
MeOH and Et2O and dried to give amorphous compound 8 (0.5 g,
Nꢀ{Nꢀ[4ꢀOꢀ(βꢀDꢀGalactopyranosyl)ꢀβꢀDꢀglucopyranosyl]carꢀ
bamoylmethyl}ꢀNꢀ(Nꢀ{Nꢀbis[4ꢀOꢀ(βꢀDꢀgalactopyranosyl)ꢀβꢀDꢀ
glucopyranosyl]carbamoylmethyl}carbamoylmethyl)ꢀNꢀ(diglycyl)ꢀ
amine (11). 10% Palladium on activated carbon (0.12 g) was added
to a solution of compound 10 (0.234 g, 0.16 mmol) in H2O (7 mL)
under argon and hydrogenation was carried out in a weak flow of H2
under vigorous stirring for 8 h at 20 °C. The catalyst was filtered off
and washed with H2O (3×5 mL). The solution was concentrated to
3.5 mL and passed through a filter with Cꢀ18 silica gel (2 g) in H2O.
The silica gel was washed with H2O (25 mL) and the solution was
concentrated. Methanol (5 mL) was added to the residue, the formed
precipitate was filtered off, washed with MeOH and Et2O and dried
25
to give amorphous compound 11 (0.19 g, 88%), [α]D + 5.2 (c 0.5,
25
73%), [α]D +6.0 (c 0.5, H2O). Found (%): C, 39.39; H, 6.50;
H2O). Found (%): C, 42.30; H, 6.52; N, 7.38; H2O, 1.96.
C48H81N7O36•H2O. Calculated (%): C, 42.70; H, 6.20; N, 7.26;
H2O, 1.33. 1H NMR, δ: 3.43—3.51 (m, 5 H); 3.52—3.59 (m, 3 H);
3.61—3.84 (m, 26 H); 3.88—3.96 (m, 7 H); 4.05—4.55 (m, 9 H);
4.44 (d, 3 H, 3 H(1) Gal, J = 7.5 Hz); 4.97— 5.07 (m, 3 H, 3 H(1)
Glc). 13C NMR, δ: 41.7 (CH2NH2); 43.9 (CH2NH); 52.2
(CH2NCH2); 52.6 (CH2NCH2); 60.9 (C(6)Glc); 62.0 (C(6)Gal);
69.5 (C(4)Gal): 71.9 (C(2)Gal); 72.5 (C(2)Glc); 73.5 (C(3)Gal);
76.0 (C(3)Glc); 76.3 (C(5)Gal); 77.4 (C(5)Glc); 78.8 (C(4)Glc);
80.2 (C(1)Glc); 103.9 (C(1)Gal); 172.1; 172.5; 172.8; 173.2;
175.0 (CO).
Cl, 4.60; N, 4.67; H2O, 5.37. C30H50ClN3O23•3H2O. Calculatꢀ
ed (%): C, 39.58; H, 6.20; Cl, 3.90; N, 4.62; H2O, 5.94. 1H NMR,
δ: 3.40—3.61 (m, 4 H); 3.61—3.86 (m, 16 H); 3.86—3.98 (m, 4 H);
4.20 (br.s, 2 H, CH2Cl); 4.37 (s, 2 H, CH2N); 4.40 (s, 2 H,
CH2N); 4.45 (d, 2 H, 2 H(1) Gal, J = 7.5 Hz); 5.01 (d, 1 H, H(1)
Glc, J = 9.0 Hz); 5.05 (d, 1 H, H(1) Glc, J = 9.0 Hz).
Nꢀ{Nꢀ[4ꢀOꢀ(βꢀDꢀGalactopyranosyl)ꢀβꢀDꢀglucopyranosyl]carꢀ
bamoylmethyl}ꢀNꢀ(Nꢀ{Nꢀbis[4ꢀOꢀ(βꢀDꢀgalactopyranosyl)ꢀβꢀDꢀ
glucopyranosyl]carbamoylmethyl}carbamoylmethyl)amine (9). A mixꢀ
ture of 4ꢀOꢀ(βꢀDꢀgalactopyranosyl)ꢀNꢀglycylꢀβꢀDꢀglucopyranosyꢀ
lamine (1) (0.545 g, 1.37 mmol), chloroacetyl compound 8 (0.63 g,
0.69 mmol) in anhydrous DMSO (4 mL) and Pri2EtN (0.117 mL,
0.69 mmol) was heated in a screwꢀcapped test tube for 13 h at 70 °C.
The reaction mixture was diluted with Et2O (50 mL) with stirring
and the liquid after clearing was decanted from the oily precipitate.
The precipitate was triturated several times with Et2O (10 mL each
time) until a viscous substance was obtained, which was dissolved in
H2O (45 mL). Cationite Dowex 50w×2 (H+) (30 mL) was added to
the solution followed by stirring for 1 h. The resin was filtered off,
washed with H2O (300 mL) and 0.5 M Py (250 mL). The latter
This work was financially supported by the Russian
Foundation for Basic Research (Project No. 07ꢀ03ꢀ00120).
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Received November 21, 2007