Glycoconjugates of nidoꢀorthoꢀcarborane
Russ.Chem.Bull., Int.Ed., Vol. 60, No. 11, November, 2011 2363
for 6 h. Et2O (15 mL) was added with stirring. The supernatant
was decanted from the resulted oily product, which was trituratꢀ
ed with Et2O until a viscous mass was obtained. The mass was
treated several times with hot acetone until a powder was obꢀ
tained. The poweder was treated several times with MeOH, filꢀ
tered off, and dried to yield amorphous compound 8a (0.18 g,
(88%), [α]D20 +3.9 (c 1, H2O). 1H NMR, δ: 3.42—3.59 (m, 6 H);
3.61—3.86 (m, 24 H); 3.88—3.97 (m, 6 H); 3.98—4.58 (m, 17 H);
4.97—5.10 (m, 3 H, 3 H(1) Glc). MS (negative ions), m/z:
1450.3844; 724.6910. Calculated for C50H82BrN7O37, [M – H]–:
m/z 1450.3861; [M – 2H]2–: m/z 724.6894. MS (positive ions),
m/z 1474.3819. Calculated for C50H82BrN7O37, [M + Na]+:
m/z 1474.3826.
Preparation of glycoconjugates 6b—8b (general procedure).
To a solution of bromine compounds 6a—8a (0.1 mmol) in
DMSO (0.5 mL), pyridinium 1,2ꢀdicarbaꢀclosoꢀdodecaboranꢀ
1ꢀyl thiolate (1)9 (28 mg, 0.11 mmol) was added and the mixture
was kept at ~22 °C for 24 h. Et2O (10 mL) was added to the
reaction mixture. The supernatant was decanted from the preꢀ
cipitated oily product, which was triturated with Et2O until
a viscous mass was obtained.
2.18 (br.s, 1 H, HCcarb); 3.31—3.57 (m, 8 H); 3.61—3.86 (m, 24 H);
3.88—3.97 (m, 6 H); 3.98—4.57 (m, 15 H); 4.97—5.10 (m, 3 H,
3 H(1) Glc). 11B NMR, δ: –37.19 (1 B); –32.97 (1 B); –21.16
(2 B); –18.53 (1 B); –16.84 (1 B); –14.44 (1 B); –10.78 (1 B);
–9.88 (1 B). MS, (negative ions), m/z 1537.6212; 768.3068. Calꢀ
culated for С52Н94B9N7О37S, [M – H]–: m/z 1537.6247; m/z
[M – 2H]2–: m/z 768.3087.
Hydrolytic stabilities of glycoconjugates 6b—8b. A solution of
glycoconjugate 6b (31 mg, 0.05 mmol) in water (1 mL) was
heated at 37 °C for three days. The solution was diluted with
water (2 mL), applied on a column with C18 silica gel (18 mL) in
water, and eluted with water (~250 mL). The fraction eluting
together with solvent front were combined, concentrated to
~0.2 mL, and lyophilized to yield a substance (4.5 mg), which
was analyzed by electrophoresis and NMR spectroscopy. The
fractions containing a homogeneous negatively charged subꢀ
stance (electrophoresis) eluted with delay on sorbent were comꢀ
bined, concentrated to ~0.5 mL, lyophilized, and dried to yield
the starting glycoconjugate 6b (26 mg, 85%).
The hydrolytic stabilities of glycoconjugates 7b and 8b were
estimated analogously. The starting glycoconjugates 7b and 8b
were isolated in amount of ~85%.
Glycoconjugate 6b. The viscous mass was dissolved in H2O
(10 mL) and extracted with CH2Cl2 (3×4 mL). The aqueous
solution was diluted with H2O (40 mL) and concentrated until
a syrup was obtained. The syrup was dissolved in H2O (3 mL) and
applied on a column with C18 silica gel (30 mL). The column
was washed with H2O (~200 mL) before the UV absorption startꢀ
ed to grow and then with 10% aqueous MeOH (~150 mL). The
waterꢀmethanol solution containing the product was concenꢀ
trated to 2 mL, diluted with H2O (50 mL), concentrated to 3 mL
to remove pyridine, and chromatographed again under the same
conditions. The waterꢀmethanol solution was concentrated to 1 mL
and lyophilized to yield amorphous compound 6b (40 mg, 66%),
Triethylammonium salts of glycoconjugates (6c—8c). Soluꢀ
tions (1 mL each) containing glycoconjugates 6b—8b (0.01 mmol)
in D2O were iceꢀcooled, a solution of Et3N in D2O (0.5 mL,
0.025 mmol L–1) was added, and the mixtures were lyophilized.
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The residues were dissolved in D2O (0.5 mL) and the H NMR
spectra were recorded. The solutions were lyophilized to yield
salts 6c—8c. The 1H NMR spectra of the salts contained the
proton signals for the Et3ND+ group in addition to the proton
signals analogous to the corresponding starting glycoconjugates
6b—8b, δ: 1.25 (t, 9 H, 3 CH3, J = 6.0 Hz); 3.17 (q, 6 H, 3 CH2,
J = 6.0 Hz).
Inhibition of hemagglutination was performed according to
the standard procedure.15 A suspension of galectin RCA120 (2 mg)
in 0.9% aqueous NaCl (1.5 mL) was stirred at room temperature
for 1.5 h. The undissolved protein was separated by centrifugaꢀ
tion and the supernatant was stored at 5 °C for no more than
3 days. A portion of the galectin solution was diluted with 0.9%
NaCl (~10—20ꢀfold) and the lectin concentration that upon fourꢀ
fold dilution of the galecting solution did not cause agglutination
of a 2% suspension of red cells of human blood group O was
determined.
Solutions of salts 6c—8c (4 mmol L–1) in 0.9% NaCl were
used for the analysis.
Solutions of NaCl (0.9%, 25 μL each) were placed to roundꢀ
bottom wells of the enzyme immunoassay plate (96 wells) and
a solution of the glycoconjugate salt (25 μL) was added to the
first well and stirred. The resulted solution (25 μL) was taken and
placed to the second well and, analogously, the solution from
the second well (25 μL) was taken and placed to the third well
and so on until the tenth well. Galectin (25 μL) was added to
each well and the mixture was stirred and kept at ~20 °C for 1 h.
A 2% suspension of red blood cells in a NaCl solution (25 μL
each) was added and stirred and, after 3—5 h, emergence or
absence of agglutination was observed.
[α]D +3.8 (c 1, H2O). 1H NMR, δ: 2.19 (br.s, 1 H, HCcarb);
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3.36, 3.58 (both d, 1 H each, CH2S, J = 14.5 Hz); 3.42—3.56
(m, 2 H); 3.61—3.83 (m, 8 H); 3.88—3.95 (m, 2 H); 4.00, 4.10,
and 4.04, 4.12 (both AB systems, 1 H each, CH2, J = 25.5 Hz,
J = 9.5 Hz); 4.44 (d, 1 H, H(1) Gal, J = 7.5 Hz); 5.02 (d, 1 H,
H(1) Glc, J = 9.0 Hz). 11B NMR, δ: –37.25 (1 B); –32.94 (1 B);
–21.27 (2 B); –18.68 (1 B); –16.76 (1 B); –14.50 (1 B); –11.15
(1 B); –9.91 (1 B). MS (negative ions), m/z 604.3050. Calculatꢀ
ed for C18H39B9N2O12S, [M – H]–: m/z 604.3039.
Glycoconjugate 7b. The viscous mass was triturated several
times with acetone until a powder was obtained. The powder was
triturated in H2O (50 mL), the resulted mixture was evaporated
to dryness, and this procedure was repeated until pyridine disꢀ
appeared completely (UV control at 260 nm). The residue was
chromatographed as described for compound 6b to yield amorꢀ
25
1
phous compound 7b (77 mg, 70%), [α]D +3.0 (c 1, H2O). H
NMR, δ: 2.18 (s, 1 H, HCcarb); 3.37, 3.59 (both d, 1 H each,
CH2S, J = 14.5 Hz); 3.42—3.57 (m, 4 H); 3.60—3.87 (m, 16 H);
3.88—3.96 (m, 4 H); 4.02, 4.10 (AB system, 2 H, CH2, J = 15.0 Hz);
4.17 (m, 4 H, 2 CH2); 4.37 (br.s, 2 H, CH2); 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);
5.05 (d, 1 H, H(1) Glc, J = 9.0 Hz). 11B NMR, δ: –37.26 (1 B);
–32.93 (1 B); –21.23 (2 B); –18.64 (1 B); –16.78 (1 B); –14.53
(1 B); –10.99 (1 B); –9.94 (1 B). MS (negative ions), m/z
1099.4770. Calculated for С36Н68B9N5О25S, [M – H]–: m/z
1099.4752.
The authors are grateful to V. A. Lapuk (IOC RAS) for
the provided galectin RCA120. This work was financially
supported by the Russian Foundation for Basic Research
(Project No. 07ꢀ03ꢀ00120).
Glycoconjugate 8b was prepared analogously to compound 7b
in a yield of 94 mg (61%), [α]D25 +3.2 (c 1, H2O). 1H NMR, δ: