A new synthesis of the 3,6-branched hexasaccharide fragment of the cell wall mannan in Candida albicans, corresponding to the antigenic factor 4

Article abstract of DOI:10.1007/s11172-011-0157-0

In the context of the project dealing with the synthesis and immunological study of the immunodominant fragments of the cell wall mannan in Candida albicans, the hexamannoside fragment corresponding to the antigenic factor 4 was assembled using the scheme

Full text of DOI:10.1007/s11172-011-0157-0

Russian Chemical Bulletin, International Edition, Vol. 60, No. 5, pp. 1004—1011, May, 2011
1004
A new synthesis of the 3,6ꢀbranched hexasaccharide fragment
of the cell wall mannan in Candida albicans,
corresponding to the antigenic factor 4*
D. A. Argunov,a A. A. Karelin,b A. A. Grachev,b Yu. E. Tsvetkov,b and N. E. Nifantievb
aHigher Chemical College of the Russian Academy of Sciences,
9 Miusskaya pl., 125047 Moscow, Russian Federation
bN. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences,
47 Leninsky prosp., 119991 Moscow, Russian Federation.
Fax: +7 (499) 135 8784. Eꢀmail: nen@ioc.ac.ru
In the context of the project dealing with the synthesis and immunological study of the
immunodominant fragments of the cell wall mannan in Candida albicans, the hexamannoside
fragment corresponding to the antigenic factor 4 was assembled using the scheme [3+2+1].
The yield of the target hexasaccharide with the (α1→3)ꢀglycosidic bond was higher than that
obtained according to the previously used assembly scheme [2+2+2].
Key words: Candida albicans, mannan, oligomannosides, synthesis.
The present work is part of the project aimed at the
are responsible for the antigen specificity of Candida. Earꢀ
lier, it has been demonstrated that the carriers of the antiꢀ
genic factor 4 are 3,6ꢀbranched structures (for example,
structures I and II).4
In this communication, we describe a new approach to
the synthesis of 3,6ꢀbranched hexamannoside 3ꢀaminoꢀ
propylglycoside 1. The presence of an amino group in the
aglycone allows subsequent covalent bonding to highꢀmoꢀ
lecularꢀweight carriers and tags of various types.
Earlier,5 we have obtained hexamannoside 1 by sucꢀ
cessively joining disaccharide units 4, 5, and 2 according
to the scheme [2+2+2] (Scheme 1, pathway a). However,
glycosylation of tetrasaccharide 3 with disaccharide 2 in
the final step of the assembly of the carbohydrate chain is
accompanied by the formation of a great amount of a byꢀ
product containing the (β1→3)ꢀbond (α : β = 2 : 1), which
synthesis and study of immunological properties of oliꢀ
gosaccharides corresponding to the mannan fragments of
the cell wall of the Candida fungi. Dimorphic yeastlike
fungi Candida albicans is part of the normal microflora of
skin, mucous membranes, and the gastrointestinal tract.
However, immunocompromised people may be vulnerꢀ
able to superficial and deep candidiases1. The Candida cell
wall is the first to interact with a host organism and is
responsible for the antigen response, adhesion, and interꢀ
cellular interactions.2
The cell wall mannan3 is a (α1→6)ꢀlinked chain with
side oligomannoside branches attached to its separate
mannose residues through (α1→2)ꢀbonds. These branchꢀ
es in turn may contain (α1→2)ꢀ, (α1→3)ꢀ, (α1→6)ꢀ, and
(β1→2)ꢀlinked mannose residues. It is the side chains that
I
II
* Dedicated to Academician of the Russian Academy of Sciences V. N. Charushin on the occasion of his 60th birthday.
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 5, pp. 980—986, May, 2011.
1066ꢀ5285/11/6005ꢀ1004 © 2011 Springer Science+Business Media, Inc.

A new synthesis of 3,6ꢀbranched oligomannoside
Russ.Chem.Bull., Int.Ed., Vol. 60, No. 5, May, 2011
1005
Scheme 1
substantially lowers the yield of the target αꢀlinked prodꢀ
uct. This reaction outcome, as well as a reported6,7 examꢀ
ple of the formation of the (β1→3)ꢀbond under nearly the
same conditions, suggests that the low stereoselectivity in
the (1→3)ꢀglycosylation step can be due not only to the
absence of the participating acyl group at the O(2) atom

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Argunov et al.
from donor 2 but also the presence of the 6ꢀOꢀmannoside
unit in the tetrasaccharide 3 under glycosylation.5 In conꢀ
nection with this, we proposed an alternative scheme
(Scheme 1, pathway b) involving triꢀ, diꢀ, and monosacꢀ
charide units 8, 2, and 6, respectively, with (1→6)ꢀglycoꢀ
sylation in the final step of the assembly of the target
hexasaccharide (synthetic scheme [3+2+1]).
Since the final product contains different glycosyl subꢀ
stituents at the O(3) and O(6) atoms in the branch point,
we should differentiate between these positions in the corꢀ
responding monosaccharide precursor. Thiomannoside 10
we have used earlier5 (Scheme 2) meets this requirement;
however, the trityl group is labile under glycosylation conꢀ
ditions. That is why it was replaced by the chloroacetyl
group, which remains intact during glycosylation and can
be selectively removed later in the presence of the benzoyl
groups. The presence of the 2ꢀOꢀbenzoyl group in donor
10 ensures the high αꢀselectivity of the glycosylation (no
βꢀisomer is usually formed at all).
Removal of the trityl protecting group from thioglycoꢀ
side 10 also having the tertꢀbutyl(dimethyl)silyl protecꢀ
tion was carried out under the action of iron chloride as
described8 for a similar compound containing the allyloxy
group instead of SEt at the anomeric carbon. However,
the yield was low (<50%). Fortunately, we found that the
trityl group can be removed in 1.5 h under the action
of trifluoroacetic acid in chloroform, the tertꢀbutyl(diꢀ
methyl)silyl protection persisting for several days. This
enabled selective removal of the trityl group with the silyl
protection remaining virtually intact. Then, the OH group
at the C(6) atom in thiomannoside 11 was protected with
the chloroacetyl group to give the desired donor 9.
Glycosylation of disaccharide acceptor 4 (see Ref. 9)
with donor 9 proceeded smoothly to give a trisaccharide in
high yield (96%). The latter was deprived of its silyl proꢀ
tection under the action of 90% trifluoroacetic acid, being
thereby transformed into trisaccharide mannosyl acceptor
8 (see Scheme 2).
The key step of the synthesis of the target hexamannoꢀ
side 1 (Scheme 3) is glycosylation with donor 2. Although
no sugar substituent is present at the O(6) atom in unit C,
the glycosylation gave a mixture of αꢀ and βꢀlinked penꢀ
tasaccharides 13. However, the αꢀstereoselectivity of the
reaction was appreciably higher (α : β ≈ 6 : 1, NMR data
on the intensities of the signals for the anomeric protons
showing no overlap with other signals) than that of the
earlier described5 glycosylation of tetrasaccharide 3 (α : β =
= 2 : 1). We failed to separate the anomeric pentasacchaꢀ
rides even by HPLC, either in the completely protected
(13) or dechloroacetylated form (14). Nevertheless, beꢀ
cause we have shown previously5 that an anomeric mixꢀ
ture of hexamannosides can be efficiently separated, we
used this mixture in the next steps of the synthetic seꢀ
quence and isolated the target αꢀlinked product after the
final glycosylation.
Signal assignment in the NMR spectrum is strongly
complicated by the presence of similarly protected benzylꢀ
ated monosaccharide residues and a noticeable amount of
Scheme 2
Reagents, conditions, and yields: i. TFA (90% aqueous), CHCl3, 70%; ii. Chloroacetyl chloride, pyridine, 95%; iii. NIS, TfOH,
–
–
CH2Cl2, 25— 15 °C, 96%; iv. TFA (90% aqueous), 76%.

A new synthesis of 3,6ꢀbranched oligomannoside
Russ.Chem.Bull., Int.Ed., Vol. 60, No. 5, May, 2011
1007
Scheme 3
–
–
Reagents, conditions, and yields: i. NIS, TfOH, CH2Cl2, 25— 15 °C, 70%; ii. NH2C(S)NH2, collidine, MeOH, reflux, 88%;
–
–
iii. AgOTf, CH2Cl2, 20— 10 °C, 72% for 15 and 10% for 16.
the βꢀ(1→3)ꢀisomer. That is why the assignment of the
αꢀconfiguration (for βꢀglycosidic bonds, the constants
JC(1),H(1) do not exceed 160 Hz).10
signals for the pentasaccharides is incomplete. Because
1
the vicinal coupling constants J1,2 in the H NMR specꢀ
Removal of the chloroacetyl protection by reflux with
thiourea gave a mixture of two isomeric pentasaccharides
14, which was glycosylated with benzobromomannose 6.
The resulting two hexasaccharides 15 and 16 were sepaꢀ
rated by HPLC; their yields were 72 and 10%, respectively.
The fully protected hexasaccharide 15 containing only
αꢀlinked mannose residues were debenzylated by hydroꢀ
genolysis and then the Oꢀbenzoyl and Nꢀtrifluoroacetyl
trum and the chemical shifts of the C(1) atom in the
13C NMR spectrum are not indicative of the configuration
of the glycosidic bond in mannosides, the (1→3) bond
configuration in compound 13 was determined from the
heteronuclear coupling constants JC(1),H(1). All the five
constants of the major isomer are in a range of 172—175 Hz;
i.e., all glycosidic bonds in the major product have

1008
Russ.Chem.Bull., Int.Ed., Vol. 60, No. 5, May, 2011
Argunov et al.
Scheme 4
Reagents and conditions: i. H2, Pd/C, MeOH; ii. 1) MeONa, MeOH, 2) NaOH, H2O, MeOH.
Highꢀresolution mass spectra were measured on a Bruker
micrOTOF II instrument (ESI) in the positive (capillary voltage
4500 V) or negative ion mode (capillary voltage 3200 V) for the
scan range m/z 50—3000 D; external or internal calibration was
performed with an Electrospray Calibrant Solution (Fluka). Samꢀ
ples were dissolved in acetonitrile and infused through syringes
(flow rate 3 μL min–1, nitrogen as a spraying gas (4 L min–1),
interface temperature 180 °C).
groups were removed by successive treatment with MeONa
in methanol and aqueous NaOH (Scheme 4). The target
hexasaccharide 1 was obtained in 85% yield and is comꢀ
pletely identical with an authentic sample.5
As expected, the scheme [3+2+1] for the synthesis of
hexamannoside 1 is superior to the previously proposed
scheme [2+2+2] because of the substantially higher stereꢀ
oselectivity of the (1→3)ꢀglycosylation.
Glycosylation was carried out in dehydrated solvents under
dry argon. Before the reaction, molecular sieves MS 4 Å were
activated in an oil pump vacuum at 180 °C for 2 h.
Experimental
Ethyl 2,4ꢀdiꢀOꢀbenzoylꢀ3ꢀOꢀ[tertꢀbutyl(dimethyl)silyl]ꢀ1ꢀ
thioꢀαꢀDꢀmannopyranoside (11). A 90% aqueous solution of triꢀ
fluoroacetic acid (0.7 mL) was added to a stirred solution of
monosaccharide 10 (see Ref. 5) (385 mg, 0.49 mmol) in chloroꢀ
form (10 mL). The reaction mixture was stirred for 1.5 h, diluted
with chloroform, washed with a saturated solution of NaHCO3,
and concentrated. Compound 11 was isolated by column chroꢀ
matography with light petroleum—ethyl acetate as an eluent.
The yield was 190 mg (70%), transparent syrup. 1H NMR
(500 MHz, CDCl3), δ: 8.11—8.04, 7.61—7.54, 7.48—7.42 (m,
10 H, 2 Ph); 5.59 (t, 1 H, H(4), J3,4 = J4,5 = 9.8 Hz); 5.46 (br.s,
1 H, H(1)); 5.44 (d, 1 H, H(2), J2,3 = 3.3 Hz); 4.38 (dd, 1 H,
H(3), J2,3 = 3.3 Hz, J3,4 = 9.8 Hz); 4.24 (m, 1 H, H(5)); 3.76—3.68
(m, 2 H, H(6a), H(6b)); 2.76—2.62 (m, 2 H, SCH2Me);
1.32 (t, 3 H, SCH2CH3); 0.61 (s, 9 H, Me3CSi); 0.03 (s, 3 H,
MeSi); –0.09 (s, 3 H, MeSi). 13C NMR (125.75 MHz), δ:
166.4, 165.9 (PhCO); 133.5, 133.2, 129.9—128.4 (Ph); 82.6
(C(1)); 74.4 (C(2)); 71.4 (C(5)); 70.2 (C(4)); 69.2 (C(3));
61.6 (C(6)); 25.7 (SCH2Me); 25.2 (3 C, (CH3)3C); 17.6 (1 C,
Me3C); 14.8 (SCH2CH3); –4.8, –5.1 (2 C, MeSi). Found (%):
C, 61.36; H, 7.04. C28H38O7SSi. Calculated (%): C, 61.51;
H, 7.01.
Solvents were purified according to standard procedures;
TfOH, NIS (Acros), AgOTf (Merck), TBDMSCl (Fluka), colliꢀ
dine (Fluka), chloroacetyl chloride (Acros), and thiourea (Reaꢀ
khim) were used without further purification. Thinꢀlayer chroꢀ
matography was carried out on Kieselgel 60 F254 plates (Merck);
spots were visualized by spraying the plates with a solution of
orcinol (180 mg) in a mixture of water (85 mL), H3PO4 (10 mL),
and ethanol (5 mL) followed by heating at ~150 °C. Column
chromatography was carried out on Silica gel 60 (40—63 μm,
Merck). For HPLC, a Knauer G27ꢀ185 column packed with
Silica gel 60 (5 μm) was used. Gel chromatography was carried
out on a column (1.5×90 cm) filled with gel TSK HWꢀ40(S) in
0.1 M acetic acid; the eluates were analyzed with a Knauer 88 00
flow refractometer.
NMR spectra were recorded at 25 °C on Bruker DRXꢀ500
and Bruker Avance 600 instruments in CDCl3 (δC 77.0) for proꢀ
tected sugar derivatives. The residual nondeuterated chloroform
(δ 7.27) and CDCl3 (δ 77.0) were used as the internal stanꢀ
H
C
dards for 1H and 13C NMR spectra, respectively. The spectra
of free oligosaccharides were recorded in D2O with acetone
(δ 2.225, δ 31.45) as the internal standard. The signals were
Ethyl 2,4ꢀdiꢀOꢀbenzoylꢀ3ꢀOꢀ[tertꢀbutyl(dimethyl)silyl]ꢀ6ꢀOꢀ
chloroacetylꢀ1ꢀthioꢀαꢀDꢀmannopyranoside (9). Pyridine (110 μL,
1.40 mmol) and chloroacetyl chloride (55 μL, 0.70 mmol) were
added to a solution of compound 11 (190 mg, 0.35 mmol) in
anhydrous CH2Cl2 (4 mL). After 30 min, the reaction mixture
H
C
assigned by 2D correlation spectroscopic techniques (COSY,
TOCSY, ROESY, and HSQC). Elemental analysis was performꢀ
ed at the Laboratory of Analytical Chemistry of the N. D. Zelinsky
Institute of Organic Chemistry, Russian Academy of Sciences.

A new synthesis of 3,6ꢀbranched oligomannoside
Russ.Chem.Bull., Int.Ed., Vol. 60, No. 5, May, 2011
1009
was diluted with chloroform, washed with 1 M HCl and a satuꢀ
rated solution of NaHCO3, and concentrated. Compound 9 was
isolated by column chromatography with light petroleum—ethyl
acetate (10 : 1) as an eluent. The yield was 200 mg (95%), white
C87H97ClF3NO20Si. Calculated (%): C, 65.42; H, 6.12; N, 0.88.
Found: m/z 1613.6347 [M + NH4]+. C87H101ClF3N2O20Si. Calꢀ
culated: [M + NH4] = 1613.6352.
3ꢀTrifluoroacetamidopropyl (2,4ꢀdiꢀOꢀbenzoylꢀ6ꢀOꢀchloroꢀ
acetylꢀαꢀDꢀmannopyranosyl)ꢀ(1→2)ꢀ(3,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀ
mannopyranosyl)ꢀ(1→2)ꢀ3,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannopyranoꢀ
side (8). Trisaccharide 12 (288 mg, 0.18 mmol) was dissolved in
90% trifluoroacetic acid (6 mL). The resulting solution was stirred
for 2 h and concentrated. The residue was concentrated with
toluene. Compound 8 was isolated by column chromatography
with toluene—ethyl acetate (6 : 1) as an eluent. The yield was
200 mg (76%), syrup. 1H NMR (600 MHz, CDCl3), δ: 8.19,
8.04, 7.71—7.15, 6.98 (all m, 40 H, Ph); 6.80 (br.s, 1 H, NH);
5.56 (s, 1 H, H(2)C); 5.54 (t, 1 H, H(4)C, J3,4 = J4,5 = 9.9 Hz);
5.30 (s, 1 H, H(1)B); 5.09 (s, 1 H, H(1)C); 4.94—4.90 (m, 2 H,
H(1)A, PhCH2); 4.84 (d, 1 H, PhCH2, J = 10.8 Hz); 4.69—4.53
(m, 10 H, PhCH2); 4.45 (m, 1 H, H(3)C); 4.41—4.34 (m, 2 H,
1
foam. H NMR (600 MHz, CDCl3), δ: 8.18—8.06, 7.66—7.59,
7.54—7.46 (all m, 10 H, 2 Ph); 5.72 (t, 1 H, H(4), J3,4 = J4,5
=
= 9.7 Hz); 5.48 (br.s, 2 H, H(1), H(2)); 4.56 (m, 1 H, H(5)); 4.45
(dd, 1 H, H(6a), J5,6 = 5.1 Hz, J6a,6b = 12.0 Hz); 4.38—4.32
(m, 2 H, H(3), H(6b)); 4.11 (d, 1 H, C(O)CH2Cl, Ja,b = 15.0 Hz);
4.07 (d, 1 H, C(O)CH2Cl, Ja,b = 15.0 Hz); 2.80—2.68 (m, 2 H,
SCH2Me); 1.38 (t, 3 H, SCH2CH3); 0.66 (s, 9 H, Me3CSi); 0.06
(s, 3 H, MeSi); –0.09 (s, 3 H, MeSi). 13C NMR (150 MHz), δ:
167.0, 165.8, 165.4 (2 PhCO, C(O)CH2Cl); 133.4, 133.3,
129.8—128.4 (Ph); 82.7 (C(1)); 74.2 (C(2)); 74.2 (C(4)); 69.8
(C(3)); 69.0 (C(5)); 64.5 (C(6)); 40.6 (C(O)CH2Cl); 25.8
(SCH2Me); 25.2 (3 C, (CH3)3C); 17.6 (1 C, Me3C); 14.9
(SCH2CH3); –4.8, –5.1 (2 C, MeSi). Found (%): C, 58.00;
H, 6.57. C30H39ClO8SSi. Calculated (%): C, 57.82; H, 6.31.
3ꢀTrifluoroacetamidopropyl (2,4ꢀdiꢀOꢀbenzoylꢀ3ꢀOꢀ[tertꢀ
butyl(dimethyl)silyl]ꢀ6ꢀOꢀchloroacetylꢀαꢀDꢀmannopyranosyl)ꢀ
(1→2)ꢀ(3,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→2)ꢀ3,4,6ꢀ
triꢀOꢀbenzylꢀαꢀDꢀmannopyranoside (12). Molecular sieves MS 4 Å
(400 mg) were added to a solution of acceptor 4 (see Ref. 9)
(207 mg, 0.20 mmol) and thiomannoside 9 (185 mg, 0.30 mmol)
in anhydrous CH2Cl2 (4 mL). The mixture was stirred for 30 min
and then NIS (135 mg, 0.6 mmol) was added at –15 °C. After
10 min, the mixture was cooled to –30 °C and TfOH (6 μL,
60 μmol) was added. The temperature was maintained within
–25 to –10 °C throughout the reaction. After 45 min, the reacꢀ
tion mixture was neutralized with pyridine (10 μL), diluted with
chloroform, and filtered through Celite. The filtrate was washed
with a small amount of 1 M HCl and a saturated solution of
NaHCO3 and concentrated. Compound 12 was isolated by colꢀ
umn chromatography with toluene—ethyl acetate (10 : 1) as an
eluent. The yield was 308 mg (96%), transparent syrup. 1H NMR
(500 MHz, CDCl3), δ: 8.19, 8.00, 7.63—7.12, 6.95 (all m, 40 H,
Ph); 6.80 (br.s, 1 H, NH); 5.64 (t, 1 H, H(4)C, J3,4 = J4,5 = 9.6
Hz); 5.48 (br.s, 1 H, H(2)C); 5.22 (d, 1 H, H(1)B, J1,2 = 1.4 Hz);
H(5)C, H(6a)C); 4.21 (dd, 1 H, H(6b)C, J5,6 = 4.3 Hz, J6a,6b
=
= 11.8 Hz); 4.08 (d, 1 H, ClCH2CO, J = 14.9 Hz); 4.06—4.03
(m, 2 H, H(2)A, H(2)B); 4.02 (d, 1 H, C(O)CH2Cl, J = 14.9 Hz);
4.01—3.97 (m, 2 H, H(3)B, H(5)B); 3.89 (t, 1 H, H(4)B, J3,4
=
= J4,5 = 9.5 Hz); 3.84 (m, 1 H, H(3)A); 3.78 (t, 1 H, H(6a)B,
J5,6 = J6a,6b = 9.8 Hz); 3.77—3.68 (m, 6 H, H(4)A, H(5)A, H(6a)A,
H(6b)A, H(6b)B, OCH2CH2CH2N); 3.46 (m,
1
H,
OCH2CH2CH2N); 3.37—3.27 (m, 2 H, OCH2CH2CH2N,
OCH2CH2CH2N), 1.78 (m, 2 H, OCH2CH2CH2N). 13C NMR
(150 MHz, CDCl3), δ: 166.9, 166.8, 165.7 (3 C, 2 PhCO,
C(O)CH2Cl); 138.3—138.0, 133.7, 133.6, 130.0—127.5 (Ph);
100.6 (1 C, C(1)B); 99.1 (2 C, C(1)A, C(1)C); 79.7 (1 C, C(3)A);
78.8 (1 C, C(3)B); 77.0 (1 C, C(2)B); 75.8 (1 C, C(2)A); 75.1 (2 C,
PhCH2); 74.9 (1 C, C(4)A); 74.6 (1 C, C(4)B); 73.4 (1 C, PhCH2);
73.3 (1 C, PhCH2); 72.8 (1 C, C(2)C); 72.5 (1 C, PhCH2); 72.4
(1 C, PhCH2); 72.2 (2 C, C(5)A, C(5)B); 70.1 (1 C, C(4)C); 69.6
(1 C, C(6)B); 69.3 (1 C, C(6)A); 69.0 (1 C, C(5)C); 68.8 (1 C,
C(3)C); 65.9 (1 C, OCH2CH2CH2N); 64.1 (1 C, C(6)C); 40.6
(1 C, C(O)CH2Cl); 38.0 (1 C, OCH2CH2CH2N); 28.2 (1 C,
OCH2CH2CH2N). Found (%): C, 64.97; H, 5.69; N, 0.93.
C81H83ClF3NO20. Calculated (%): C, 65.60; H, 5,64; N, 0.94.
Found: m/z 1504.5047 [M + Na]+. C81H83ClF3NNaO36. Calꢀ
culated: [M + Na] = 1504.5041.
5.04 (d, 1 H, H(1)C, J1,2 = 1.7 Hz); 4.90 (d, 1 H, H(1)A, J1,2
=
= 1.5 Hz); 4.85 (d, 1 H, PhCH2, J = 10.9 Hz); 4.81 (d, 1 H,
PhCH2, J = 10.9 Hz); 4.64—4.49 (m, 10 H, PhCH2); 4.43 (dd, 1 H,
H(3)C, J2,3 = 3.3 Hz, J3,4 = 9.6 Hz); 4.23 (m, 1 H, H(5)C);
4.21—4.18 (m, 2 H, H(6a)C, H(6b)C); 4.05 (m, 1 H, H(2)B);
4.03 (d, 1 H, C(O)CH2Cl, J = 15.1 Hz); 4.00 (br.s, 1 H, H(2)A);
3.99—3.91 (m, 3 H, H(3)B, H(5)B, C(O)CH2Cl); 3.83—3.78
(m, 2 H, H(3)A, H(4)B); 3.74—3.62 (m, 7 H, H(4)A, H(5)A, H(6a)A,
H(6b)A, H(6a)B, H(6b)B, OCH2CH2CH2N); 3.41 (m, 1 H,
OCH2CH2CH2N); 3.31—3.22 (m, 2 H, OCH2CH2CH2N,
OCH2CH2CH2N); 1.72 (m, 2 H, OCH2CH2CH2N); 0.63 (s, 9 H,
ButSi); 0.03 (s, 3 H, MeSi); –0.12 (s, 3 H, MeSi). 13C NMR
(125 MHz, CDCl3), δ: 166.9, 166.5, 165.4 (3 C, 2 PhCO,
C(O)CH2Cl); 138.2—138.0, 133.3, 129.9—127.5 (Ph); 100.7
(1 C, C(1)B); 99.2 (1 C, C(1)C); 99.0 (1 C, C(1)A); 79.5 (1 C,
C(3)A); 79.0 (1 C, C(3)B); 76.6 (1 C, C(2)B); 75.8 (1 C, C(2)A);
75.1 (2 C, PhCH2); 74.8 (1 C, C(4)A); 74.6 (1 C, C(4)B); 73.3
(2 C, PhCH2); 72.5 (2 C, C(2)C, PhCH2); 72.1 (2 C, C(5)A,
PhCH2); 72.0 (1 C, C(5)B); 69.5 (2 C, C(5)C, C(6)A); 69.3 (2 C,
C(4)C, C(6)B); 68.6 (1 C, C(3)C); 65.8 (1 C, OCH2CH2CH2N);
64.3 (1 C, C(6)C); 40.6 (1 C, C(O)CH2Cl); 37.9 (1 C,
OCH2CH2CH2N); 28.2 (1 C, OCH2CH2CH2N); 25.3 (3 C, But);
–4.9, –5.1 (2 C, MeSi). Found (%): C, 64.29; H, 6.43; N, 0.96.
Mixture of 3ꢀtrifluoroacetamidopropyl (2,3,4,6ꢀtetraꢀOꢀ
benzoylꢀαꢀDꢀmannopyranosyl)ꢀ(1→2)ꢀ(3,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀ
mannopyranosyl)ꢀ(1→3)ꢀ(2,4ꢀdiꢀOꢀbenzoylꢀ6ꢀOꢀchloroacetylꢀαꢀ
Dꢀmannopyranosyl)ꢀ(1→2)ꢀ(3,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannopyraꢀ
nosyl)ꢀ(1→2)ꢀ3,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannopyranoside and
3ꢀtrifluoroacetamidopropyl (2,3,4,6ꢀtetraꢀOꢀbenzoylꢀαꢀDꢀmanꢀ
nopyranosyl)ꢀ(1→2)ꢀ(3,4,6ꢀtriꢀOꢀbenzylꢀβꢀDꢀmannopyranosyl)ꢀ
(1→3)ꢀ(2,4ꢀdiꢀOꢀbenzoylꢀ6ꢀOꢀchloroacetylꢀαꢀDꢀmannopyranoꢀ
syl)ꢀ(1→2)ꢀ(3,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→2)ꢀ
3,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannopyranoside (13). Molecular sieves
MS 4 Å (370 mg) were added to a solution of acceptor 8 (177 mg,
0.12 mmol) and donor 2 (see Ref. 5) (193 mg, 0.18 mmol) in
anhydrous CH2Cl2 (4 mL). The mixture was stirred for 35 min
and then NIS (81 mg, 0.36 mmol) was added at –15 °C. After
20 min, the mixture was cooled to –35 °C and TfOH (3.5 μL,
0.036 mmol) was added. The reaction temperature was mainꢀ
tained within –25 to –15 °C. After 1 h, the reaction mixture was
neutralized with pyridine (10 μL), diluted with chloroform, and
filtered through Celite. The filtrate was washed with a small
amount of 1 M HCl and a saturated solution of NaHCO3 and
concentrated. Compound 13 was isolated by column chromatoꢀ

1010
Russ.Chem.Bull., Int.Ed., Vol. 60, No. 5, May, 2011
Argunov et al.
graphy with toluene—ethyl acetate (10 : 1) as an eluent. The
yield was 208 mg (70%), white foam. 1H NMR (600 MHz,
CDCl3), δ: 8.22—7.80, 7.70—7.00 (both m, Ph); 6.80 (br.s, 1 H,
NH); 6.07 (t, 1 H, H(4)E, J3,4 = J4,5 = 10 Hz); 5.88 (m, 1 H,
H(3)E); 5.75 (br.s, 1 H, H(2)E); 5.74 (t, 1 H, H(4)C, J = 10 Hz);
5.70 (br.s, 1 H, H(2)C); 5.38 (s, 1 H, H(1)D, JC(1),H(1) = 172 Hz);
5.32 (s, 1 H, H(1)B, JC(1),H(1) = 172 Hz); 5.29 (s, H(1)β); 5.18 (s,
H(1)β); 5.15 (s, 1 H, H(1)C, JC(1),H(1) = 175 Hz); 5.08 (s, H(1)β);
4.94 (s, 1 H, H(1)A, JC(1),H(1) = 173 Hz); 4.72 (br.s, 1 H, H(1)E,
JC(1),H(1) = 172 Hz). 13C NMR (150 MHz, CDCl3), δ: 166.9—164.7
(PhCO, C(O)CH2Cl); 138.9—138.0, 133.7—132.9, 130.1—127.1
(Ph); 100.5 (C(1)B); 100.4 (C(1)D); 99.7 (C(1)β); 99.5 (C(1)β);
99.2 (C(1)C); 99.1 (C(1)E); 99.0 (C(1)A); 97.9 (C(1)β); 79.3
(C(2)D); 75.7 (C(2)A); 72.0 (C(2)C); 70.2 (C(2)E); 70.1 (C(3)E);
69.1 (C(5)E); 68.8 (C(4)C); 66.5 (C(4)E); 65.8 (OCH2CH2CH2N);
64.1 (C(6)C); 61.8 (C(6)E); 40.6 (C(O)CH2Cl); 37.9
(OCH2CH2CH2N); 28.2 (OCH2CH2CH2N). Found (%):
C, 68.50; H, 5.43; N, 0.50. C142H137ClF3NO34. Calculated (%):
C, 68.38; H, 5,54; N, 0.56. Found: m/z 2509.9003 [M + NH4]+.
C142H141ClF3N2O34. Calculated: [M + NH4] = 2509.9001.
Mixture of 3ꢀtrifluoroacetamidopropyl (2,3,4,6ꢀtetraꢀOꢀ
benzoylꢀαꢀDꢀmannopyranosyl)ꢀ(1→2)ꢀ(3,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀ
mannopyranosyl)ꢀ(1→3)ꢀ(2,4ꢀdiꢀOꢀbenzoylꢀαꢀDꢀmannopyranoꢀ
syl)ꢀ(1→2)ꢀ(3,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→2)ꢀ
3,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannopyranoside and 3ꢀtrifluoroacetamidoꢀ
propyl (2,3,4,6ꢀtetraꢀOꢀbenzoylꢀαꢀDꢀmannopyranosyl)ꢀ(1→2)ꢀ
(3,4,6ꢀtriꢀOꢀbenzylꢀβꢀDꢀmannopyranosyl)ꢀ(1→3)ꢀ(2,4ꢀdiꢀOꢀ
benzoylꢀαꢀDꢀmannopyranosyl)ꢀ(1→2)ꢀ(3,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀ
mannopyranosyl)ꢀ(1→2)ꢀ3,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannopyranoꢀ
side (14). Thiourea (47 mg, 0.62 mmol) and collidine (10 μL,
0.075 mmol) were added to a solution of pentasaccharides 13
(154 mg, 0.062 mmol) in anhydrous methanol (8 mL). The mixꢀ
ture was refluxed for 24 h. After the reaction was completed, the
solvent was removed in vacuo. The residue was suspended in
chloroform; the undissolved precipitate was filtered off and
washed with chloroform. The filtrate was washed with 1 M HCl
and a saturated solution of NaHCO3 and concentrated. Comꢀ
pound 14 was isolated by column chromatography with toluꢀ
ene—ethyl acetate (5 : 1) as an eluent. The yield was 122 mg
(81%), white foam. 1H NMR (600 MHz, CDCl3), δ: 8.20—7.79,
7.68—7.02 (both m, Ph); 6.86 (br.s, 1 H, NH); 6.1 (t, 1 H,
H(4)E, J3,4 = J4,5 = 9.9 Hz); 5.91 (dd, 1 H, H(3)E, J2,3 = 2.9 Hz,
J3,4 = 9.9 Hz); 5.78 (br.s, 1 H, H(2)E); (br.s, 1 H, H(2)C);
5.58 (t, 1 H, H(4)C, J3,4 = J4,5 = 9.7 Hz); 5.41 (s, 1 H, H(1)D);
5.32 (s, 1 H, H(1)B); 5.25 (s, H(1)β); 5.23 (s, 1 H, H(1)C);
5.17 (s, H(1)β); 4.93 (br.s, 1 H, H(1)A); 4.76 (s, 1 H, H(1)E).
13C NMR (125 MHz, CDCl3), δ: 166.0—164.74 (PhCO);
138.9—137.8, 133.7—132.5, 130.6—126.8 (Ph); 100.4 (C(1)B);
100.3 (C(1)D); 99.6 (C(1)β); 99.2 (C(1)C); 99.0 (C(1)E); 98.9
(C(1)A); 97.5 (C(1)β); 76.0 (C(2)B); 72.0 (C(2)C); 70.2 (C(2)E);
70.0 (C(3)E); 66.6 (C(4)E); 65.8 (OCH2CH2CH2N); 62.0 (C(6)E);
61.4 (C(6)C); 37.9 (OCH2CH2CH2N); 28.1 (OCH2CH2CH2N).
Found (%): C, 69.59; H, 5.78; N, 0.65. C140H136F3NO33. Calcuꢀ
lated (%): C, 69.55; H, 5,67; N, 0.58. Found: m/z 2433.9284
[M + NH4]+. C140H140F3N2O33. Calculated: [M + NH4] =
= 2433.9285.
Dꢀmannopyranoside (15) and 3ꢀtrifluoroacetamidopropyl (2,3,4,6ꢀ
tetraꢀOꢀbenzoylꢀαꢀDꢀmannopyranosyl)ꢀ(1→2)ꢀ(3,4,6ꢀtriꢀOꢀ
benzylꢀβꢀDꢀmannopyranosyl)ꢀ(1→3)ꢀ[2,3,4,6ꢀtetraꢀOꢀbenzoylꢀ
αꢀDꢀmannopyranosylꢀ(1→6)]ꢀ(2,4ꢀdiꢀOꢀbenzoylꢀαꢀDꢀmannoꢀ
pyranosyl)ꢀ(1→2)ꢀ(3,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ
(1→2)ꢀ3,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannopyranoside (16). Molecuꢀ
lar sieves MS 4 Å (165 mg) were added to a stirred solution of an
anomeric mixture of pentasaccharides 14 (109 mg, 0.045 mmol)
and benzobromomannose 6 (45 mg, 0.068 mmol) in anhydrous
CH2Cl2 (4 mL). The mixture was stirred for 40 min and solidꢀ
state AgOTf (26 mg, 0.10 mmol) was added at –40 °C. The
reaction temperature was maintained within –20 to –15 °C.
After 1.5 h, the reaction mixture was neutralized with a drop of
Et3N, diluted with chloroform, and filtered through Celite. The
filtrate was washed with a small amount of 1 M HCl and a satuꢀ
rated solution of NaHCO3, concentrated, and subjected to
column chromatography (toluene—ethyl acetate, 8 : 1). Then
isomeric compounds 15 and 16 were separated by HPLC in light
petroleum—acetone (2 : 1). The yields of products 15 and 16
were 97 mg (72%) and 14 mg (10%), respectively. Their NMR
spectra are identical with the literature data.5
Compound 15. Found (%): C, 68.55; H, 5.29; N, 0.56.
C174H162F3NO42. Calculated (%): C, 69.75; H, 5,45; N, 0.47.
Found: m/z 3012.0839. [M + NH4]+. C174H166F3N2O42. Calcuꢀ
lated: [M + NH4] = 3012.0862.
Compound 16. Found (%): C, 69.66; H, 5.36; N, 0.47.
C174H162F3NO42. Calculated (%): C, 69.75; H, 5,45; N, 0.47.
Found: m/z 3012.0872 [M + NH4]+. C174H166F3N2O42. Calcuꢀ
lated: [M + NH4] = 3012.0862.
3ꢀAminopropyl αꢀDꢀmannopyranosylꢀ(1→2)ꢀαꢀDꢀmannopyrꢀ
anosylꢀ(1→3)ꢀ[αꢀDꢀmannopyranosylꢀ(1→6)]ꢀαꢀDꢀmannopyraꢀ
nosylꢀ(1→2)ꢀαꢀDꢀmannopyranosylꢀ(1→2)ꢀαꢀDꢀmannopyranoside
(1). A catalyst Pd/C (90 mg) was added to a stirred solution of
protected hexasaccharide 15 (90 mg, 0.030 mmol) in methaꢀ
nol—ethyl acetate (1 : 1, 2 mL). The mixture was stirred under
hydrogen at room temperature for 20 h. After the reaction was
completed, the reaction mixture was diluted with methanol and
filtered through Celite. The filtrate was concentrated and the
residue was dissolved in methanol (3.5 mL). Then a solution of
1 M MeONa in methanol (0.3 mL) was added. After 40 min,
a drop of water was added and the mixture was left for 16 h,
neutralized with acetic acid, and concentrated. Product 1 was
isolated by gel chromatography in the form of acetate and lyophilꢀ
ized from water. The yield was 26.5 mg (85%).
The NMR spectra of compound 1 are fully identical with
those published earlier.5 Found: m/z 1048.3913 [M + H]+.
C39H69NO31. Calculated: [M + H] = 1048.3926.
This work was financially supported by the Council on
Grants at the President of the Russian Federation (State
Support Program for Leading Scientific Schools of the
Russian Federation and young Ph.D. scientists, Grant
MKꢀ5544.2010.3).
References
3ꢀTrifluoroacetamidopropyl (2,3,4,6ꢀtetraꢀOꢀbenzoylꢀαꢀDꢀ
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(1→6)]ꢀ(2,4ꢀdiꢀOꢀbenzoylꢀαꢀDꢀmannopyranosyl)ꢀ(1→2)ꢀ(3,4,6ꢀ
triꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→2)ꢀ3,4,6ꢀtriꢀOꢀbenzylꢀαꢀ
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Received February 11, 2011;
in revised form April 13, 2011