The synthesis of linear trilactosamine

Article abstract of DOI:10.1134/S1068162008050129

TrilactosamineGalβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1- 3Galβ1-4GlcNAcβ-sp, where sp = O(CH₂)₃NH ₂ is a spacer, was synthesized. The tetrasaccharide fragment Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ-sp was obtained by successive glycosylation using elongation by one monosaccharide residue at a time; and the tetrasaccharide was then transformed into a hexasaccharide with a disaccharide glycosyl donor. A 2,2,2-trichloroethoxycarbonyl group was used for the protection of the glucosamine amino group.

Full text of DOI:10.1134/S1068162008050129

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2008, Vol. 34, No. 5, pp. 625–631. © Pleiades Publishing, Ltd., 2008.
Original Russian Text © G.V. Pazynina, V.V. Severov, N.V. Bovin, 2008, published in Bioorganicheskaya Khimiya, 2008, Vol. 34, No. 5, pp. 696–730.
The Synthesis of Linear Trilactosamine
G. V. Pazynina, V. V. Severov, and N. V. Bovin1
Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,
ul. Miklukho-Maklaya 16/10, Moscow, 117997 Russia
Received August 1, 2007; in final form, January 11, 2008
Abstract—TrilactosamineGalβ1–4GlcNAcβ1–3Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAcβ-sp, where sp = O(CH2)3NH2
is a spacer, was synthesized. The tetrasaccharide fragment Galβ1–4GlcNAcβ1–3Galβ1–4GlcNAcβ-sp was
obtained by successive glycosylation using elongation by one monosaccharide residue at a time; and the tet-
rasaccharide was then transformed into a hexasaccharide with a disaccharide glycosyl donor. A 2,2,2-trichloro-
ethoxycarbonyl group was used for the protection of the glucosamine amino group.
Key words: N-acetyllactosamine, oligosaccharide synthesis, polylactosamine
DOI: 10.1134/S1068162008050129
INTRODUCTION
lactosamine or glucosamine were used as glycosyl
donors because the N-Troc protection provided β-ste-
reoorientation of glycosylation, but at the same time
transformation of the Troc-NH fragment into the target
Ac-NH did not affect other protective groups [14]. The
presence of 6-OH benzyl groups in the intermediate
products increased their solubility in organic solvents,
made chromatographic separation easier and, what is
most important, enabled the use of other protective
groups for release of this or that hydroxyl group.
Oligolactosamine and polylactosamine fragments
are specific structural elements of complex N- and
O-chains of glycoproteins and some glycolipids [1, 2].2
One of the functions of oligolactosamines is specific
interaction with proteins of the galectine family [3].
The topicality of detailed studies of galectine carbohy-
drate specificity is explained by their involvement in
various biological processes, for example, they mediate
cell adhesion, proliferation, and apoptosis [4–7]. For
studying galectine specificity directly on the cell sur-
face we needed to have oligolactosamines and fluores-
cein-labeled glycoconjugates on their basis. Although
chemical and enzymatic synthesis of oligolactosamines
was described earlier [8–11], we propose another
method providing intermediate properly protected
derivatives for the synthesis of substituted oligolac-
tosamines, such as sulfated and fucosylated oligosac-
charides.
Glycosylation of glucosamine derivative (II) [15] at
the 4-OH group with glycosyl bromide (I) [16] yielded
61% of disaccharide (III) (Scheme). The formation of
a β-glycoside bond was supported by a characteristic
value of J1b,2b 7.9 Hz.
Successive deacetylation of lactosamine derivative
(III), orthoether protection, acetylation, and opening of
the orthoether cycle [17] rsulted in disaccharide (IV) in
a total yield of 59%. The presence of an OH group at
Earlier, we reported the synthesis of short and the C3 galactose fragment was supported by a down-
branched oligolactosamines [12]. In this work, we field shift of the H3b resonance (δ 3.54–3.68) if com-
describe the synthesis of linear hexasaccharide, which pared with acetylated analogue (III) (δ 4.870 ppm,
is currently considered to be one of the most potent H3b).
affinity ligands of most human galectines [13].
Trisaccharide (IV) was obtained in a yield of 65%
by glycosylation of disaccharide (IV) with bromide
(V). In addition, the corresponding α anomer at the ter-
RESULTS AND DISCUSSION
minal residue was isolated (15%). In the presence of
The main goal of this work was the preparation of
trilactosamine in the form of a 3-aminopropyl deriva-
tive suitable for preparation of a fluorescein-labeled
probe. N-Trichloroethoxycarbonyl (Troc) derivatives of
zinc in a mixture of acetic acid and acetic anhydride,
the Troc protection of compound (VI) was substituted
by a N-acetyl group to give 77% of derivative (VII).
The glycosylation site and β configuration of the glu-
cosamine terminal residue was confirmed by 1H NMR
spectroscopy (δ 3.69 ppm, H3b, J1Ò,2Ò 7.9 Hz). Trisac-
charide (VII) was deacetylated, benzylidenated, and
acetylated (see the scheme) to obtain derivative (VIII)
in a total yield of 70%. The use of sodium cyanoboro-
1
Corresponding author; phone: +7 (495) 330-7138; e-mail:
bovin@carbohydrate.ru.
2
Abbreviations: Bn, benzyl; LacNAc, N-acetyllactosamine; MS,
mass-spectrum; AgOTf, silver triflate; TMM, tetramethylurea;
Troc, 2,2,2-trichlorethoxycarbonyl.
625

626
PAZYNINA et al.
OBn
AcO
OBn
AcO
OBn
OBn
a
O
O
O
O
HO
AcO
O
+
O
NHCOCF3
O
NHCOCF3
(III) (61%)
AcO
AcO
AcO
OAc
AcO
NHAc
Br
NHAc
(I)
(II)
b, c, d, e
OBn
OBn
OAc
AcO
O
O
O
+
AcO
AcO
O
O
NHCOCF3
(IV) (59%)
AcO
HO
OAc
NHAc
Br
O
TrocNH
(V)
a
OBn
AcO
OBn
OBn
OAc
O
O
AcO
AcO
O
O
O
NHCOCF3
AcO
OAc
NHAc
NHR
(VI), R = Troc (65%)
(VII), R = Ac (77%)
b, g, d
f
OR1
OBn
OBn
AcO
O
O
O
R2O
AcO
O
(I) +
O
O
NHCOCF3
AcO
OAc
NHAc
NHAc
(VIII), R1 + R2 = CHPh (70%)
(IX), R1 = Bn, R2 = H (91%)
a
h
OBn
OBn
AcO
OBn
AcO
O
O
O
O
O
O
O
AcO
O
NHCOCF3
AcO
OAc
AcO
NHAc
NHAc
OAc
(X) (47%)
b, c, d, e
OBn
AcO
OAc
AcO
OBn
O
OAc
OBn
OBn
AcO
O
O
O
O
O
O
AcO
O
AcO
O
+
O
O
NHCOCF3
AcO
OAc
AcO
HO
Br
NHAc
NHAc
TrocNH
OAc
OAc
(XII)
(XI) (48%)
a
OBn
OBn
OBn
OAc
AcO
OBn
OAc
O
AcO
AcO
O
O
O
O
O
O
OAc
O
AcO
O
O
NHAc
O
NHCOCF3
O
AcO
AcO
AcO
OAc
NHAc
NHR
OAc
(XIII), R = Troc (47%)
(XIV), R = Ac (70%)
i, d, b, j
f
Galb1–4GlcNAcb1–3Galb1–4GlcNAcb1–3Galb1–4GlcNAcb-O(CH ) NH (XV) (89%)
2 3
2
Scheme. a: AgOTf, TMM, MS-4 Å, CH Cl ; b: MeONa/MeOH; c: MeC(OEt) , TsOH, MeCN;
2
2
3
d: Ac O/Py; e: 80% aqueous AcOH; f: AcOH, Ac O, Zn, NEt ; g: PhCH(OMe) , TsOH, MeCN;
2
2
3
2
h: NaCNBH , THF, HCl/Et O; i: 10% Pd/C, H , MeOH; j: NaOH/H O.
3
2
2
2
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 5 2008

THE SYNTHESIS OF LINEAR TRILACTOSAMINE
627
hydride [18] provided selective opening of the ben-
(1) sulfated at C-6 galactose or glucosamine resi-
(2) sulfated at C-3 galactose residues;
zylidene cycle to give 91% oligosaccharide (IX) bear- dues;
ing an OH group at C4c and a benzyl group at C6c,
which was supported by correlation of proton reso-
nances in the 1H NMR spectra at C4c (δ 3.58–3.70 ppm)
and C6c (δ 3.872 and 4.027 ppm) with proton reso-
nances at C4 and C6 (δ 3.70–3.81 ppm) of compound
(II).
(3) α-fucosyl and α-galactosyl derivatives at galac-
tose positions 3 and 6;
(4) oligolactosamines branched at C-6 galactose res-
idue.
Hexasaccharide (XV) was converted into a fluores-
cein-labeled polyacrylamide derivative (OS-PAA-fluo)
with a molecular mass of ~30 kDa using the earlier
described procedure [20]. This derivative as well a
series of analogous probes obtained from simpler oli-
golactosamines were used for studying specificity and
functioning of galectines-1 and -3 composing a cell
membrane [21].
Tetrasaccharide (X) in 47% yield was prepared by
glycosylation of trisacchardie (IX) with 6-OBn-aceto-
bromogalactose (I). The formation of a β1–4 bond fol-
lowed from the 1H NMR data: J1d,2d 7.9 Hz; proton res-
onances of C4c and C4a (δ 3.885 and 3.902 ppm), C5c
and C5a (δ 3.50–3.60 ppm), as well as C6c and C6a
(δ 3.66–3.74 ppm).
Derivative (XI) with one OH group in the terminal
galactose residue (δ 3.708 3.708 ppm, H3d) was
obtained in a total yield of 48% by successive deacety-
lation, introduction of the orthoether protection, acety-
lation, and opening of the orthoether cycle of dilac-
tosamine derivative (XI). Glycosylation of the latter
with glycosyl bromide of N-Troc lactosamine deriva-
tive (XII) resulted in 47% hexasaccharide (XIII). The
Troc protective group was substituted by an N-acetyl
one as described above to obtain derivative (XIV) in a
yield of 70% (J1e,2e ~ 8 Hz).
EXPERIMENTAL
Optical rotation was measured on a digital polarim-
eter Jasco DIP-360 at 25°ë. 1H NMR spectra (in CDCl3
if not stated otherwise) were registered on a Bruker WM
spectrometer 500 MHz; 13C NMR on a Bruker 600 MHz
at 303K. Chemical shifts (δ for characteristic proton
resonances are given in ppm and coupling constants J,
1
in Hz. The resonances in H NMR spectra were
assigned using a technique of spin–spin decoupling
(double resonance) and 2D-1H,1H- COSY experiments.
The resonances in 13C NMR spectra were assigned
using DEPT-135 experiments. Mass spectra were regis-
tered on a MALDI-TOF Vision-2000 spectrometer
(Thermo Bioanalysis Corp., England). Dihydroxyben-
zoic acid was used as a template. TLC was performed
on Kieselgel-60 plates (Merck, Germany); the com-
pounds were developed with 5% orthophosphoric acid
at 150°ë (carbohydrates) or ninhydrin solution (3 g/l in
a 30 : 1 butanol–acetic acid mixture) (amines). Column
chromatography was performed on Silica gel 60
(0.040–0.063 mm, Merck, Germany); gel-chromatog-
raphy, on Sephadex LH-20 columns (Pharmacia, Swe-
den). Solvents were evaporated in vacuum at 30–40°ë.
Solvents for glycoside syntheses were dried and stored
with molecular sieves; solid reagents were dried for two
hours in vaccum (0.1 mm Hg) at 20–40°ë. For the syn-
theses of bromides (V) and (XII), ethyl-3,4,6-tri-é-acetyl-
2-deoxy-2-(2,2,2-trichloroethoxycarbonylamido)-1-thio-β-
D-glucopyranoside and ethyl-3,6-di-é-acetyl-2-deoxy-
2-(2,2,2-trichloroethoxycarbonylamido)-4-(2,3,4,6-tetra-
é-acetyl-β-D-galactopyranosyl)-1-thio-β-D-glucopyra-
noside obtained by acetylation of commercial ethylth-
ioglycosides of N-Troc substituted glucosamine and
lactosamine (GlycoRex, Sweden), respectively, were
used.
Trilactosamine peracetate was obtained in 80%
yield by hydrogenation of derivative (XIV) followed by
acetylation. Its deacetylation and removal of the N-trif-
luoroacetyl group resulted in 89% of target trilac-
tosamine (XV). The analysis of 13C NMR spectral data
[19] indicated the presence of an aminopropyl spacer
(δ 26.96, 37.60, and 67.96 ppm) and nonglycosylated
C6-OH groups (δ 59.85–61.03 ppm). The lack of upfield
resonances of methylene groups (δ 68–69 ppm) unam-
biguously indicated the absence of 1–6 glycoside
bonds. The presence of a double-carbon resonance
(δ 82.08 ppm) confirmed the presence of two β1–3 gly-
cosylated galactose residues and the presence of a
group of signals (δ 78.15, 78.17, and 78.47 ppm), of
three β1–4 glycosylated glucosamine residues. The
presence in the anomeric area (δ 101.16–102.91 ppm)
of three resonance groups of six anomeric carbon atoms
completely corresponded to the proposed structure con-
taining three β-galactose residues, one terminal and two
internal residues of β-glucosamine. The obtained
13C NMR data and their comparison with the published
data for trilactosamine [11] and dilactosamine deriva-
tive [9] unequivocally confirmed the structure of the
synthesized hexasaccharide (XV).
The structures of the prepared compounds were
1
confirmed by mass spectral data and H NMR data
based on 2D-1H and1H-COSY experiments.
Deacetylation was carried out using the Zemplen
The proposed synthetic scheme enables the control reaction in dry methanol by adding 2 M sodium meth-
of regio- and stereoselectivity of successive elongation ylate in methanol to the acyl derivative up to pH 8–9.
of the oligosaccharide chain and the use of intermediate After the reaction completion Na+ ions were removed
compounds for the synthesis of more complex oligolac- on a resin Dowex 50X-400 (H+) (Acros, Belgium) and
tosamine derivatives, particularly:
the solution was evaporated.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 5 2008

628
PAZYNINA et al.
Glycosyl bromides (I), (V), and (XII). A solution of (1 H, dd, J6',6'' 11.9, J5,6'' 4.2, H6''c), 4.311 (1 H, dd, J6',6''
the corresponding thioglycoside (270 mg, 0.332 mmol) in 11.9, J5,6'' 2.2, H6'c), 4.345 and 4.361 (2 ꢀ 2 1 H, 2 d,
dry dichloromethane (10 ml) was cooled to 0°ë and J1,2 7.9, H1a, H1b), 4.468, 4.490, 4.514, and 4.703 (4 ꢀ
bromine (18 µl, 0.37 mmol) was added. The reaction 1 H, 4 d,JA,B 12.0, 2 × CH2Ph), 4.939 (1 H, dd, J1,2 7.9,
mixture was kept for 1 h at 0°ë, evaporated, coevapo- J2,3 10.3, H2b), 4.980 (1 H, dd ≈ t, J3,4 8.8, J2,3 9.7,
rated with toluene, and used without additional purifi- H3a), 5.000 (1 H, d, J1,2 7.9, H1c), 5.029 (1 H, dd ≈ t,
cation.
General glycosylation procedure. A solution of
J3,4 9.5, J4,5 9.9, H4c), 5.393 (1 H, dd ≈ d, J3,4 3.4, J4,5 <
1, H4b), 5.421 (1 H, d, J2,NH 7.9, NHAc-c), 5.508 (1 H,
dd ≈ t, J2,3 10.3, J3,4 9.5, H3c), 5.794 (1 H, d, J2,NH 8.8,
NHAc-a), 7.25–7.39 (10 H, m, 2 ꢀ Ph), 7.491 (1 H, m,
NHCOCF3).
glycosyl acceptor (0.1 mmol), silver triflate (0.2 mmol),
tetramethylurea (0.2 mmol), and freshly calcinated
molecular sieves 4 Å in dry dichloromethane (5 ml)
were stirred at room temperature and in darkness for
30 min. Another portion of sieves 4 Å (100 mg) was
added, and a solution of glycosyl bromide (0.2 mmol)
in dry dichloromethane (2 ml) was added. The mixture
was filtered in 15–20 h, the solvent was evaporated, and
the product was isolated by silica gel chromatography.
(3-Trifluoroacetamidopropyl)-2-acetamido-3-O-
acetyl-6-O-benzyl-2-deoxy-4-O-(2,4-di-O-acetyl-6-
O-benzyl-b-D-galactopyranosyl)-b-D-glucopyrano-
side (IV). Disaccharide obtained by Zemplen deacety-
lation of lactosamine derivative (III) (2.74 g, 3.1 mmol)
was dissolved in dry acetonitrile (50 ml). Triethy-
lorthoacetate (0.6 ml, 4.57 mmol) and toluenesulfonic
acid (80 mg) were added, and the mixture was stirred at
room temperature for 1 h. After neutralization with
pyridine and evaporation the resulting orthoacetate was
acetylated with a 2 : 1 mixture of pyridine and acetic
anhydride (30 ml) and 80% acetic acid (40 ml) was
added. The mixture was kept for 2 h and coevaporated
with toluene. After silica gel chromatography (elution
with 100 : 5 : 0.5 chloroform–isopropanol–pyridine)
lactosamine derivative (IV) (1.53 g, 59%) was isolated;
Rf 0.37 (10% isopropanol in chloroform), [α]D –54.9
(Ò1, CHCl3); MS, m/z: 865 (842 + 23) (M+ + Na+).
1H NMR: 1.845 (2 H, m, ëç2), 1.965, 1.984, 2.036,
and 2.080 (4 × 3 ç, 4 s, 4Ac), 2.416 (1 H, s, OH), 3.265
(1 H, m, CHN), 3.441 (1 H, dd, J6',6'' 9.2, J5,6'' 7.2,
H6''b), 3.515 (2 H, m, J6',6'' 9.2, J5,6' 5.5, H6'b, H5a),
3.54–3.68 (4 H, m, CHN, CHO, H5b, H3b), 3.713 (1 H,
dd, J6',6'' 10.7, J5,6'' 2.0, H6''a), 3.768 (1 H, dd, J6',6'' 10.7,
J6',5 4.0, H6'a), 3.929 (2 H, m, CHO, J3,4 8.8, J4,5 8.6,
H4a), 4.039 (1 H, ddd, J2,NH 8.8, J1,2 8.1, J2,3 9.7, H2a),
4.384 (1 H, d, J1,2 8.1, H1a), 4.407 (1 H, d, J1,2 7.9,
H1b), 4.443, 4.494, 4.524, and 4.693 (4 × 1 H, 4 d, JA,B
11.9, 2 × CH2Ph), 4.785 (1 H, dd, J1,2 7.9, J2,3 10.1, H2b),
5.000 (1 H, dd ≈ t, J2,3 9.7, J3,4 8.8, H3a), 5.335 (1 H,
dd ≈ d, J3,4 3.5, J4,5 < 1, H4b), 5.824 (1 H, d, J2,NH, 8.8,
NHAc), 7.28–7.39 (10 H, m, 2 × Ph), 7.470 (1 H, m,
NHCOCF3).
Acetylation was performed with a 2 : 1 pyridine–
acetic anhydride mixture at 20°ë for 12–24 h; the
reagents were coevaporated with toluene.
Hydrogenolysis was carried out over 10% Pd/C
(Merck, Germany) at a substrate–catalyst ratio of 1 : 1
(w/w) in methanol at atmospheric pressure for 1–3 h.
Removal of the Troc group followed by N-acety-
lation. Freshly activated Zn dust (4 g) and then triethy-
lamine (0.5 ml) were added to a solution of Troc deriv-
ative (0.5 mmol) in acetic acid (40 ml) and acetic anhy-
dride (2 ml). In 20–30 h the mixture was filtered,
evaporated, and chromatographed on a LH-20 column
eluted with a 1 : 1 chloroform–methanol mixture.
Deacetylation and removal of N-trifluoroacetyl
protection. A solution of 2 M sodium methylate in
methanol (50 µl) was added to a solution of oligosac-
charide peracetate (0.05 mmol) in dry methanol (2 ml),
the solution was evaporated in 1 h, water (2 ml) was
added, the solution was kept for 3 h and chromato-
graphed on a Dowex-ç+ column (elution with 1 M
ammonia). The eluate was evaporated and lyophilized.
(3-Trifluoroacetamidopropyl)-2-acetamido-3-O-
acetyl-6-O-benzyl-2-deoxy-4-O-(2,3,4-tri-O-acetyl-
6-O-benzyl-b-D-galactopyranosyl)-b-D-glucopyra-
noside (III). Glycosylation of glucosamine derivative
(II) (2.6 g, 5.14 mmol) with glycosyl bromide (I)
obtained from the corresponding thioglycoside (4.52 g,
10.28 mmol) gave after twofold purification by silica
gel chromatography (elution with 0–6% MeOH in
chloroform, then 4 : 2 : 1 hexane–chloroform–isopro-
panol) the starting monosaccharide (II) (409 mg, 16%)
and disaccharide (III) (2.78 g, 61%), Rf 0.4 (10%
(3-Trifluoroacetamidopropyl)-2-acetamido-3-O-
acetyl-6-O-benzyl-2-deoxy-4-O-[2,4-di-O-acetyl-6-
O-benzyl–3-O-(2-acetamido-3,4,6-tri-O-acetyl-2-
deoxy)-b-D-glucopyranosyl]-b-D-glucopyranoside
(VII). Lactosamine derivative (IV) (1.53 g, 1.82 mmol)
was glycosylated with glycosyl bromide (V) obtained
from the corresponding thioglycoside (1.98 g,
3.64 mmol), the mixture was chromatographed on sil-
1
MeOH in chloroform)), [α]D –48.7 (Ò1, CHCl3), ç
NMR: 1.840 (2 H, m, CH2), 1.900, 1.959, 1.972, 1.995,
2.019, 2.029, 2.053, and 2.060 (5 × 3 ç, 5 Ò, 5 ÄÒ),
3.260 (1 H, m, CHN), 3.303 (1 H, ddd, J2,NH 7.9, J1,2
7.9, J2,3 10.3, H2c), 3.439 (1 H, dd, J6',6'' 9.0, J5,6'' 7.9
H6''b), 3.489 (2 H, m, H5a, H6'b), 3.559 (1 H, m,
ica gel (elution with 3
10%) isopropanol in chloro-
form) to give starting disaccharide (IV) (310 mg, 20%),
α-glycosylation product (355 mg, 15%), and trisaccha-
ride (VI) (1.53 g, 65%), Rf 0.28 (10% isopropanol in
CHN), 3.613 (2 H, m, CHO, H5b), 3.65–3.74 (4 H, chloroform). Troc derivative (VI) was treated with zinc
H6'a, H6''a, H3b, H5c), 3.917 (2 H, m, CHO, H4a), in acetic acid and the product was chromatographed on
4.032 (1 H, ddd, J1,2 7.9, J2,NH 8.6, J2,3 9.7, H2a), 4.144 silica gel (elution with 10
20% isopropanol in
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 5 2008

THE SYNTHESIS OF LINEAR TRILACTOSAMINE
629
chloroform) to give N-acetyl derivative (VII) (1.06 g, syl]-b-D-glucopyranoside (IX). A mixture of ben-
77%), Rf 0.23 (10% methanol in chloroform), [α]D zylidene derivative (VIII) (780 mg, 0.664 mmol) and
–21.2 (c1, CHCl3); MS, m/z: 1194 (1171 + 23) (M+ +
freshly calcinated molecular sieves 3 Å (2.5 g) in dry
tetrahydrofuran (20 ml) was stirred for 30 min. Another
portion of sieves (1 g) and sodium cyanoborohydride
(420 mg, 6.67 mmol) in portions were added and then
saturated HCl in dry Et2O up to pH 2–3 was added for
1 h. After 1 h the mixture was neutralized with triethy-
lamine, filtered, the residue was washed with chloro-
form and the united filtrate was evaporated. The dry res-
idue was dissolved in chloroform (50 ml), washed with
water (2 × 50 ml), dried with sodium sulfate, and evap-
orated. Trisaccharide (IX) (714 mg, 91%) was isolated
by silica gel chromatography (elution with 10% isopro-
panol in ethyl acetate), Rf 0.30 (elution with 10% iso-
propanol in ethyl acetate); [α]D –24.8 (Ò0.5, CHCl3–
1
Na+). H NMR: 1.840 (2 H, m, CH2), 1.900, 1.959,
1.972, 1.995, 2.019, 2.029, 2.053, and 2.060 (8 × 3 H,
8 s, 8 Ac), 3.260 (1 H, m, CHN), 3.303 (1 H, ddd, J2,NH
7.9, J1,2 7.9, J2,3 10.3, H2c), 3.439 (1 H, dd, J6',6'' 9.7,
J5,6'' 6.2, H6''b), 3.489 (2 H, m, H5a, H6'b), 3.559 (1 H,
m, CHN), 3.613 (2 H, m, CHO, H5b), 3.65–3.74 (4 H,
H6'a, H6''a, H3b, H5c), 3.917 (2 H, m, CHO, H4a),
4.032 (1 H, ddd, J2,NH 8.8, J1,2 7.9, J2,3 9.1, H2a), 4.144
(1 H, dd, J6',6'' 11.9, J5,6'' 4.2, H6''c), 4.311 (1 H, dd, J6',6''
11.9, J5,6' 2.2, H6'c), 4.345, and 4.361 (2 ꢀ 1 H, 2 d, J1,2
7.9, H1a, H1b), 4.468, 4.490, 4.514, and 4.703 (4 × 1 H,
4 d, JA,B 12.0, 2 × CH2Ph), 4.939 (1 H, dd, J1,2 7.9, J2,3
10.3, H2b), 4.980 (1 H, dd ≈ t, J3,4 ≈ J2,3 ≈ 9.1, H3a),
5.000 (1 H, d, J1,2 7.9, H1c), 5.029 (1 H, dd ≈ t, J3,4 9.5,
J4,5 9.9, H4c), 5.393 (1 H, dd ≈ d, J3,4 3.5, J4,5 < 1, H4b),
5.421 (1 H, d, J2,NH 7.9, NHAc-c), 5.508 (1 H, dd ≈ t,
J2,3 10.3, J3,4 9.5, H3c), 5.794 (1 H, d, J2,NH 8.8, NHAc-
a), 7.25–7.39 (10 H, m, 2 × Ph), 7.491 (1 H, m,
NHCOCF3).
1
CH3OH); MS, m/z: 1177 (1201 + 23) (M+ + Na+). H
NMR (3 : 1 CDCl3–CD3OD): 1.962 (2 H, m CH2),
2.006, 2.076, 2.093, 2.151, 2.199, and 2.205 (6 × 3 H,
6 s, 6 Ac), 3.395 (1 H, m, CHN), 3.564 (1 H, dd, J6',6''
9.7, J5,6'' 6.4, H6''b), 3.58–3.70 (7 H, m, CHN, CHO,
H5a, H6'b, H2c, H4c, H5c), 3.719 (1 H, ddd ≈ t, J5,6'
5.9, J5,6'' 6.4, J4,5 < 1, H5b), 3.814 (1 H, dd, J2,3 10.0, J3,4
3.5, H3b), 3.852 (2 H, m, H6'a, H6''a), 3.872 (1 H, dd,
J6',6'' 10.8, J5,6'' 6.1, H6''c), 4.027 (3 H, m, CHO, H4a,
H6'c), 4.085 (1 H, dd, J1,2 8.4, J2,3 10.3, H2a), 4.417
(1 H, d, J1,2 8.1, H1b), 4.544 (1 H, d, J1,2 8.3, H1a),
4.593, 4.603, 4.639, 4.729, 4.775, and 4.857 (6 × 1 H,
6 d, JA,B 11.9, 3 × CH2Ph), 4.785 (1 H, d, J1,2 8.3, H1c),
4.986 (1 H, dd, J1,2 8.1, J2,3 10.0, H2b), 5.121 (1 H, dd,
J3,4 9.2, J2,3 10.3, H3a), 5.215 (1 H, dd, J2,3 10.6, J3,4 8.6,
H3c), 5.616 (1 H, dd ≈ d, J3,4 3.5, J4,5 < 1, H4b), 7.462
(15 H, m, 3 × Ph).
(3-Trifluoroacetamidopropyl)-2-acetamido-3-O-
acetyl-6-O-benzyl-2-deoxy-4-O-[2,4-di-O-acetyl-6-
O-benzyl-3-O-(2-acetamido-3-O-acetyl-4,6-O-ben-
zylidene-2-deoxy)-b-D-glucopyranosyl]-b-D-galac-
topyranosyl]-b-D-glucopyranoside VIII. To trisac-
charide obtained by the Zemplen deacetylation of com-
pound (VII) (1.06 g, 0.905 mmol) dry acetonitrile (20 ml),
α,α-dimethoxymethylbenzene (235 µl, 1.57 mmol), and
toluene sulfonic acid (35 mg) were added, and the mix-
ture was stirred for 3 h at room temperature. The mix-
ture was neutralized with pyridine, evaporated, and the
resulting benzylidene derivative was acetylated with a
2 : 1 pyridine–acetic anhydride mixture (18 ml). After
silica gel chromatography (elution with 7–15% isopro-
panol in ethyl acetate) derivative (VIII) (780 mg, 70%)
was isolated, Rf 0.42 (10% isopropanol in ethyl ace-
tate), [α]D –55.2 (Ò1, CHCl3); MS, m/z: 1175 (1175)
(3-Trifluoroacetamidopropyl)-2-acetamido-3-O-
acetyl-6-O-benzyl-2-deoxy-4-O-{2,4-di-O-acetyl-6-
O-benzyl-3-O-[2-acetamido-3-O-acetyl-6-O-benzyl-
2-deoxy-4-O-(2,3,4-tri-O-acetyl-6-O-benzyl-b-D-
galactopyranosyl)-b-D-glucopyranosyl]-b-D-galac-
topyranosyl}-b-D-glucopyranoside (X). Trisaccha-
ride (IX) (709 mg, 0.60 mmol) was glycosylated with
glycosyl bromide (I) obtained from the corresponding
thioglycoside (528 mg, 1.20 mmol) [16], the mixture
was chromatographed on silica gel (elution with
1
(M+). H NMR: 1.839 (2 H, m, CH2), 1.916, 1.961,
1.974, 2.003, 2.059, and 2.091 (6 × 3 H, 6 s, 6 Ac),
3.259 (1 H, m, CHN), 3.433 (1 H, dd, J6',6'' 9.4, J5,6'' 6.4,
H6''b), 3.47–3.74 (11 H, CHN, CHO, H5a, H6'a, H6''a,
H3b, H5b, H6'b, H2c, H5c, and H6''c), 3.829 (1 H,
dd ≈ t, J3,4 ≈ J4,5 ≈ 10.5, H4c), 3.922 (2 H, m, CHO,
H4a), 4.041 (1 H, ddd, J2,NH 8.8, J1,2 8.3, J2,3 9.7, H2a),
4.356 (3 H, m, H1a, H1b, H6'c), 4.461, 4.494, 4.517,
10
20%) isopropanol in ethyl acetate) to give tet-
rasaccharide (X) (442 mg, 47%), Rf 0.50 (10% isopro-
panol in ethyl acetate), [α]D –31.9 (Ò1, CHCl3); MS,
m/z: 1555 (1579 + 23) (M+ + Na+). H NMR: 1.841
1
(2 H, m, CH2), 1.900, 1.961 (×5), 1.986, 2.032, and
and 4.699 (4 × 1 H, 4 d, JA,B 12.0, 2 × CH2Ph), 4.742 2.077 (9 × 3 H, 9 s, 9 Ac), 3.258 (1 H, m, CHN), 3.36–
(1 H, d, J1,2 7.9, H1c), 4.969 (2 H, m, H3a, H2b), 5.365 3.75 (16 H, m, CHN, CHO, H5a, H6'a, H6''a, H3b,
(1 H, dd ≈ t, J2,3 ≈ J3,4 ≈ 9.9, H3c), 5.399 (2 H, m, H4b, H5b, H6'b, H6''b, H2c, H5c, H6'c, H6''c, H5d, H6'd,
NHAc-c), 5.513 (1 H, s, CHPh), 5.828 (1 H, d, J2,NH 8.8, and H6''d), 3.86–3.95 (3 H, m, CHO, H4a and H4c),
NHAc-a), 7.29–7.45 (15 H, m, 3 × Ph), 7.493 (1 H, m, 4.037 (1 H, ddd, J1,2 7.9, J2,3 9.2, J2,NH 8.8, H2a), 4.301
NHCOCF3).
(1 H, d, J1,2 7.9, H1b), 4.347 (1 H, d, J1,2 7.9, H1a),
4.395, 4.457, 4.476, 4.501, 4.521, 4.545, 4.681, and
4.725 (8 × 1 H, 8 d, JA,B 12.0, 4 × CH2Ph), 4.485 (1 H,
d, J1,2 7.9, H1d), 4.577 (1 H, d, J1,2 7.9, H1c), 4.913
(1 H, dd, J2,3 10.5, J3,4 3.5, H3d), 4.946 (1 H, dd, J1,2 7.9,
(3-Trifluoroacetamidopropyl)-2-acetamido-3-O-
acetyl-6-O-benzyl-2-deoxy-4-O-[2,4-di-O-acetyl-6-
O-benzyl-3-O-(2-acetamido-3-O-acetyl-6-O-benzyl-
2-deoxy-b-D-glucopyranosyl)-b-D-galactopyrano-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 5 2008

630
PAZYNINA et al.
ethyl acetate); MS, m/z: 2155 (2131 + 23) (M++Na+). 1H
5.019 (1 H, dd, J1,2 7.9, J2,3 10.5, H2d), 5.104 (1 H, dd, NMR: 1.836 (2 H, m, CH2), 1.894, 1.907, 1.941, 1.958
J2,3 9.7, J3,4 8.4, H3c), 5.357 (1 H, d, J2,NH 8.4, NHAc-c), (×2), 1.978, 1.983, 2.003, 2.059 (×2), 2.067 (×2), 2.073
J2,3 9.7, H2b), 4.964 (1 H, dd ≈ t, J2,3 ≈ J3,4 ≈ 9.2, H3a),
(×2), 2.154 (15 × 3 H, 15 c, 15 Ac), 3.258 (1 H, m,
CHN), 3.37–3.75 (19 H, m, CHN, CHO, H5a, H6'a,
H6''a, H3b, H5b, H6'b, H6''b, H2c, H5c, H6'c, H6''c,
H3d, H5d, H6'd, H6''d, H2e, and H5e), 3.791 (1 H, dd ≈
t, J3,4 ≈ J4,5 ≈ 8.9, H4e), 3.891 (4 H, m, CHO, H4a, H4c,
and H5f), 4.037 (2 H, m, H2a and H6'e), 4.114 (2 H, m,
H6'f and H6''f), 4.298 (1 H, d, J1,2 7.8, H1b), 4.345
(1 H, d, J1,2 7.6, H1a), 4.390 (1 H, d, J1,2 8.1, H1d),
4.42–4.59 (8 H, 8 d, 3 × CH2Ph, H1c, H1f), 4.63–4.77
(4 H, m, CH2Ph, H1e and H6'e), 4.960 (4 H, m, H3a,
H2b, H2d, and H3f), 5.078 (1 H, dd ≈ t, J2,3 ≈ J3,4 ≈ 9.2,
H3c), 5.122 (1 H, dd, J1,2 7.9, J2,3 10.3, H2f), 5.195 (1 H,
dd ≈ t, J2,3 ≈ J3,4 ≈ 9.1, H3e), 5.34–5.41 (4 H, m, H4d,
H4f, NHAc-c, NHAc-e), 5.456 (1 H, dd ≈ d, J3,4 3.5,
J4,5 < 1, H4b), 5.825 (1 H, d, J2,NH 8.7, NHAc-a), 7.27–
7.39 (20 H, m, 4 × Ph), 7.509 (1 H, m, NHCOCF3).
5.423 (1 H, dd ≈ d, J3,4 3.5, J4,5 < 1, H4b), 5.460 (1 H,
dd ≈ d, J3,4 3.5, J4,5 < 1, H4d), 5.817 (1 H, d, J2,NH 8.8,
NHAc-a), 7.27–7.39 (20 H, m, 4 × Ph), 7.506 (1 H, m,
NHCOCF3).
(3-Trifluoroacetamidopropyl)-2-acetamido-3-O-
acetyl-6-O-benzyl-2-deoxy-4-O-{2,4-di-O-acetyl-6-
O-benzyl-3-O-[2-acetamido-3-O-acetyl-6-O-benzyl-
2-deoxy-4-O-(2,4-di-O-acetyl-6-O-benzyl-b-D-galacto-
pyranosyl)-b-D-glucopyranosyl]-b-D-galactopyr-
anosyl}-b-D-glucopyranoside (XI). To tetrasaccha-
ride obtained by deacetylation by the Zemplen method
of dilactosamine derivative (X) (442 mg, 0.284 mmol),
dry acetonitrile (20 ml), triethyl orthoacetate (0.1 ml,
0.53 mmol), and toluene sulfonic acid (30 mg) were
added, and the mixture was stirred for 3 h at room tem-
perature. The mixture was neutralized with pyridine,
evaporated, and the resulting orthoacetate was acety-
lated with a 2 : 1 pyridine–acetic anhydride mixture
(6 ml). After 80% acetic acid (10 ml) was added, the
mixture was kept for 1 h and coevaporated with tolu-
ene. Silica gel chromatography (elution with 8 : 1 :
(3-Aminopropyl)-2-acetamido-2-deoxy-4-O-{3-
O-[2-acetamido-2-deoxy-4-O-(3-O-{2- acetamido-2-
deoxy-4-O-[b-D-galactopyranosyl]-b-D-glucopyrano-
syl}-b-D-galactopyranosyl)-b-D-glucopyranosyl]-[b-
D-galactopyranosyl}-b-D-glucopyranoside (XV).
Hydrogenolysis of hexasaccharide (XIV) (93 mg,
0.044 mmol) followed by acetylation with a pyridine–
acetic anhydride mixture (2 : 1. 3) and silica gel chro-
matography (elution with 10–25% isopropanol in chlo-
roform) gave 68 mg (80%) of trilactosamine peracetate,
Rf 0.17 (10% isopropanol in ethyl acetate). é-Deacety-
lation of the latter and removal of N-trifluoroacetamide
protection yielded 36.4 mg (89%) of hexasaccharide
(XV), Rf 0.46 (4 : 1 : 1 : 1 ethanol–water–pyridine–ace-
tic acid), [α]D –5.0 (Ò0.5, H2O), MS, m/z: 1194 (1171 +
23) (M+ + Na+). 1H NMR (D2O): 1.966 (2 H, m, CH2),
2.050 (×2), 2.062 (3 × 3 H, 3 c, 3 Ac), 3.101 (1 H, m,
CH2N), 3.53–3.64 (6 H, m, H2b, H2d, H2f, H5a, H5c,
and H5e), 3.66–3.83 (25 H, m), 3.93–4.07 (5 H, m,
CHO, H4f, H6'a, H6'c, and H6'e), 4.172 (2 H, m, H4b
and H4d), 4.476, 4.485, and 4.497 (3 H, 3 d, J1,2 7.8,
0.05
5 : 1 : 0.05 chloroform–isopropanol–pyri-
dine) allowed derivative (XI) (205 mg, 48%), Rf 0.39
(10% isopropanol in ethyl acetate), [α]D –25.8 (Ò1,
1
CHCl3); MS, m/z: 1537 (1513 + 23) (M+ + Na+). H
NMR: 1.844 (2 H, m, CH2), 1.903, 1.952 (×2), 1.965,
1.995, 2.017, and 2.075 (×2) (8 × 3 H, 8 c, 8 Ac), 3.272
(1 H, m, CHN), 3.38–3.80 (17 H, m, CHN, CHO, H5a,
H6'a, H6''a, H3b, H5b, H6'b, H6''b, H2c, H5c, H6'c,
H6''c, H3d, H5d, H6'd, and H6''d), 3.900 (3 H, m, CHO,
H4a and H4c), 4.052 (1 H, ddd, H2a), 4.302 (1 H, d, J1,2
8.0, H1b), 4.396 (1 H, d, J1,2 7.8, H1a), 4.40–4.58 (7 H,
7 d, JA,B ≈ 12, 3 × CH2Ph, H1d), 4.597 (1 H, d, J1,2 7.8,
H1c), 4.685, 4.796 (2 H, 2 d, JA,B 12.1, CH2Ph), 4.796
(1 H, dd, J1,2 8.0, J2,3 9.8, H2b), 4.939 (1 H, dd ≈ t, J2,3
≈
J3,4 ≈ 8.8, H3a), 4.996 (1 H, dd ≈ t, J1,2 ≈ J2,3 ≈ 9.0,
H2d), 5.128 (1 H, dd ≈ t, J2,3 ≈ J3,4 ≈ 9.2, H3c), 5.351
(1 H, dd ≈ d, J3,4 3.5, J4,5 < 1, H4b), 5.467 (1 H, dd ≈ d, H1b, H1d, and H1f), 4.531 (1 H, d, J1,2 8.0, H1a), 4.723
13
(2 H, 2 d, J1,2 8.3, H1e, H1c). C NMR (D2O): 26.96
(OCH2CH2CH2Nç), 37.6 (CH2N), 55.00, 55.14, and
55.18 (3 C2, GlcN), 59.85, and 59.98 (2 C6, GlcN),
60.95, and 61.03 (2 C6, Gal), 67.96 (OCH2), 68.28,
68.30, and 68.54 (3 C4, Gal), 69.94 (2 C2, Gal), 70.96
(C2, Gal), 72.16, 72.18, and 72.20 (3 C3, GlaN), 72.50
(C3f), 74.54 (2 C5, GlcN), 74.71 (C5, GlcN), 74.87
(2 C5, Gal), 75.35 (C5, Gal), 78.15, and 78.17 (2 C4,
GlcN), 78.47 (C4a), 82.08 (2 C3, Gal), 101.16 (C1a),
102.75 (2 C1, GlcN), 102.86, 102.89, and 102.91 (3 C1,
Gal).
J3,4 3.5, J4,5 < 1, H4d), 5.529 (1 H, d, J2,NH 8.9, NHAc-
c), 6.055 (1 H, d, J2,NH 8.5, NHAc-a), 7.30 (20 H, m, 4 ×
Ph), 7.585 (1 H, m, NHCOCF3).
(3-Trifluoroacetamidopropyl)-2-acetamido-3-O-
acetyl-6-O-benzyl-2-deoxy-4-O-{2,4-di-O-acetyl-6-O-
benzyl-3-O-[2-acetamido-3-O-acetyl-6-O-benzyl-2-
deoxy-4-O-(2,4-di-O-acetyl-6-O-benzyl-3-O-{2-aceta-
mido-3,6-di-O-acetyl-2-deoxy-4-O-2,3,4,6-tetra-O-
acetyl-b-D-galactopyranosyl)-b-D-glucopyranosyl]-
b-D-galactopyranosyl}-b-D-glucopyranoside (XIV).
Tetrasaccharide (XI) (203 mg, 0.134 mmol) was glyc-
osylated with glycosyl bromide (XII) obtained from
the corresponding thioglycoside, the mixture was chro-
Fluorescein-labeled glycoconjugate [19]. A solu-
tion of amino spacer-bearing fluorescein (A-10466,
Molecular Probes, United States) (0.112 mg,
0.171 mmol) in DMSO (50 µl), a solution of polynitrophe-
nyl acrylate (3.299 mg, 17.08 µg-eq) in DMSO (165 µl),
matographed on silica gel (elution with 8
15%)
isopropanol in chloroform) to give hexasaccharide
(XIII) (141 mg, 47%), Rf 0.50 (10% isopropanol in
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 5 2008

THE SYNTHESIS OF LINEAR TRILACTOSAMINE
631
9. Sherman, A.A., Synthesis of Oligosaccharide Chains of
Galectin Receptors, Cand. Sci. (Chem.) Dissertation,
Moscow: Zelinsky Institute of Organic Chemistry, 2001.
10. Mong, T.K.-K., Huang, C.-Y., and Wong, C.-H, J. Org.
Chem., 2003, vol. 68, pp. 2135–2142.
11. Vasiliu, D., Razi, N., Zhang, Y., Jacobsen, N., Allin, K.,
Liu, X., Hoffmann, J., Bohorov, O., and Blixt, O., Car-
bohydr. Res., 2006, vol. 341, pp. 1447–1457.
12. Severov, V.V., Belyanchikov, I.M., Razynina, G.V., and
Bovin, N.V., Bioorg. Khim., 2007, vol. 33, pp. 131–147;
Rus. J. Bioorg. Chem., 2007, vol. 33, pp. 122–138.
and triethylamine (2 µl, 2 vol %) were added to a solu-
tion of hexasaccharide (XIV) (4 mg, 3.416 µl) in
DMSO (350 µl). The mixture was kept for 25 h at 40°ë,
cooled to room temperature, and ethanolamine (57 µl,
10 vol %) was added, the mixture was kept for 24 h at
room temperature, and the solution was loaded onto a
Sephadex LH-20 column. The conjugate was eluted
with a 1 : 1 acetonitrile–water, the target fractions were
evaporated, and the residue was lyophilized from water
to give glycoconjugate (5.66 mg, 97%).
13. http://www.functionalglycomics.org/glycomics/public-
data/selectedScreens.jsp.
ACKNOWLEDGMENTS
We thank M.L. Maisel for registration of NMR
spectra.
14. Windholz, T.M. and Jonston, B.R., Tetrahedron Lett.,
1967, p. 2555.
The work was supported by the NIH grant 5 U54
GM062116-05 and Russian Foundation for Basic
Research, project nos. 03-04-48926, 07-04-00630, and
07-04-00969.
15. Bovin, N.V., Zemlyanukhina, T.V., Chagiashvili, C.N.,
and Khorlin, A.Ya., Khim. Prir. Soedin., 1988, pp. 777–
785 [Chem. Nat. Compd. (Engl. Transl.), 1988, vol. 24,
p.659].
16. Pazynina, G.V., Tyrtysh, T.V., and Bovin, N.V., Men-
deleev Commun., 2002, pp. 143–145.
REFERENCES
17. Mukhopadhyay, B. and Field, R.A., Carbohydr. Res.,
1. Huges, R.C., Glycoproteins, New York: Chapman and
2003, vol. 338, pp. 2149–2152.
Halls, 1983.
18. Garegg, P. and Hultberg, H., Carbohydr. Res., 1982,
2. Renkonen, O., Cell. Mol. Life Sci., 2000, vol. 57,
vol. 108, pp. 97–101.
pp. 1423–1439.
19. Lipkind, G.M., Shashkov, A.S., Knirel, Y.A., Vinogra-
dov, E.V., and Kochetkov, N.K., Carbohydr. Res., 1988,
vol. 175, pp. 59–75.
20. Bovin, N.V., Korchagina, E.Yu., Zemlyanukhina, T.V.,
Byramova, N.E., Galanina, O.E., Zemlyakov, A.E.,
Ivanov, A.E., Zubov, V.P., and Mochalova, L.V., Glyco-
conjugate J., 1993, vol. 10, pp. 142–151.
21. Rapoport, E.M., Kurmyshkina, O.V., Pochechueva, T.V.,
Severov, V.V., Pazynina, G.V., Gabius, H.-J., and
Bovin, N.V.,: Carbohydrate Binding Specificity of
Galectin-1 and Galectin-3 in Composition of Cellular
Membrane, Glycobiology (in press).
3. Liu, F.-T., Int. Arch. Allergy Immunol., 2005, vol. 136,
pp. 385–400.
4. Perillo, N.L., Marcus, M.E., and Baum, L.G., J. Mol.
Med., 1998, vol. 76, pp. 402–412.
5. Hsu, D.K., Yang, R.-Y., and Liu, F.-T., Methods Enzy-
mol., 2006, vol. 417, pp. 256–273.
6. Hughes, R.C., Biochimie, 2001, vol. 83, pp. 667–676.
7. Liu, F.-T., Patterson, R.J., and Wang, J.L., Biochim. Bio-
phys. Acta, 2002, vol. 1572, pp. 263–273.
8. Mirsa, A.K., Fukuda, M., and Hindsgaul, O., Bioorg.
Med. Chem. Lett., 2001, vol. 11, pp. 2667–2669.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 5 2008