Synthesis of oligosaccharide fragments of mannan from Candida albicans cell wall and their BSA conjugates

Article abstract of DOI:10.1134/S106816200701013X

3-Aminopropyl glycosides of α-D-mannopyranosyl-(1→2)-α-D- mannopyranosyl-(1→2)-α-D-mannopyranosyl-(1→2) -α-D-mannopyranose, α-D-mannopyranosyl-(1→3)-α-D- mannopyranosyl-(1→2)-α-D-mannopyranosyl-(1→2) -α-D-mannopyranose, and α-D-mannopyranosyl-(1→2)-[α-D- mannopyranosyl-(1→3)]-α-D-mannopyranosyl-(1→2) -α-D-mannopyranosyl-(1→2)-α-D-mannopyranose were efficiently synthesized starting from ethyl 2-O-acetyl(benzoyl)-3,4,6-tri-O-benzyl-1-thio- α-D-mannopyranoside, ethyl 4,6-di-O-benzyl-2-O-benzoyl-1-thio-α-D- mannopyranoside, ethyl 4,6-di-O-benzyl-2,3-di-O-benzoyl-1-thio-α-D- mannopyranoside, and 2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl bromide. The oligosaccharide chains synthesized correspond to the three structural types of side chains of mannan from Candida albicans cell wall. A conjugate of the third pentasaccharide with bovine serum albumin was prepared using the squarate method.

Full text of DOI:10.1134/S106816200701013X

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2007, Vol. 33, No. 1, pp. 110–121. © Pleiades Publishing, Inc., 2007.
Original Russian Text © A.A. Karelin, Yu.E. Tsvetkov, G. Kogan, S. Bystricky, N.E. Nifantiev, 2007, published in Bioorganicheskaya Khimiya, 2007, Vol. 33, No. 1, pp. 119–130.
SYNTHETIC
STUDIES
Synthesis of Oligosaccharide Fragments of Mannan
from Candida albicans Cell Wall and Their BSA Conjugates
A. A. Karelina, Yu. E. Tsvetkova, G. Koganb, S. Bystrickyb, and N. E. Nifantieva, 1
a Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninskii pr. 47, Moscow, 119991 Russia
b Institute of Chemistry, Slovak Academy of Sciences, Dúbravská cesta 9, 842 38 Bratislava, Slovakia
Received April 16, 2006; in final form, May 15, 2006
Abstract—3-Aminopropyl glycosides of α-D-mannopyranosyl-(1
2)-α-D-mannopyranosyl-(1
2)-α-D-
mannopyranosyl-(1
2)-α-D-mannopyranose, α-D-mannopyranosyl-(1
2)-α-D-mannopyranose, and α-D-mannopyranosyl-(1
3)-α-D-mannopyranosyl-(1
2)-
α-D-mannopyranosyl-(1
2)-[α-D-mannopyranosyl-
(1
3)]-α-D-mannopyranosyl-(1
2)-α-D-mannopyranosyl-(1
2)-α-D-mannopyranose were efficiently
synthesized starting from ethyl 2-O-acetyl(benzoyl)-3,4,6-tri-O-benzyl-1-thio-α-D-mannopyranoside, ethyl 4,6-di-
O-benzyl-2-O-benzoyl-1-thio-α-D-mannopyranoside, ethyl 4,6-di-O-benzyl-2,3-di-O-benzoyl-1-thio-α-D-man-
nopyranoside, and 2,3,4,6-tetra-O-benzoyl-α-D-mannopyranosyl bromide. The oligosaccharide chains synthesized
correspond to the three structural types of side chains of mannan from Candida albicans cell wall. A conjugate of the
third pentasaccharide with bovine serum albumin was prepared using the squarate method.
Key words: Candida albicans, mannan, neoglycoconjugates, oligomannosides, synthesis
DOI: 10.1134/S106816200701013X
INTRODUCTION
There are reported the syntheses of (α1–2)-linked
oligomannoside chains up to the octasaccharide [4, 5],
including the solid phase synthetic approach [6], and
also the type C oligosaccharides with a terminal (α1–
3)-linked mannose residue [5, 7].
The Candida albicans yeastlike fungi are the oppor-
tunistic pathogenic microorganisms capable of causing
severe infections in immunodeficient patients [1].2 The
cell surface of C. albicans is the first to interact with the
host organism. It plays the main role in the pathogen
adhesion to the host organism cells and the modulation
of immune response [1]. The external surface of
C. albicans cell wall is enriched with mannoproteins
anchored in the matrix of structural polysaccharides.
The immunogenic properties of C. albicans are in many
respects due to mannan, which is the carbohydrate part
of mannoproteins. Mannans are highly branched
polysaccharides with the backbone built up from (α1–
6)-linked mannose residues. The mannan backbone can
bear relatively short (α1–2)-oligomannoside side
chains in position 2 of mannose residues (structure A),
which can also bear short (1–4 units) (β1–2)-oligoman-
noside substituents in position 2 (structure B) or
α-mannoside residue in position 3 of the terminal man-
nose unit (structure C) [2]. The chains with (α1–3) sub-
stitution in the last but one mannose residue (structure
D) have also been shown to occur (Fig. 1) [3]. More-
over, the (β1–2)-linked chains containing 2–6 mannose
units can be attached to the side chains via the phos-
phodiester fragment [2] (not shown in Fig. 1).
However, note that, in the papers cited, the target
structures were obtained either in the form of free oli-
gosaccharides [4, 7], or as acylated allyl glycosides [5],
which substantially complicates their use in the synthe-
sis of neoglycoconjugates. The obtaining of the oli-
gosaccharide sequences A and C, included as constitu-
ents in the more complicated oligosaccharide struc-
tures, is also reported [8–11]. No synthesis of the type
→6)Man(α1→6)Man(α1→6)Man(α1→6)Man(α1→6)Man(α1→6)Man(α1→
2
2
2
2
↓
↓
↓
↓
↓
1
2
1
2
1
2
1
2
Manα
Manα
Manα
Manα
Manα
Manα
Manα
Manα
Manα
Manα
↓
↓
↓
1
2
1
2
1
2
1
2
↓
↓
↓
↓
↓
↓
↓
1
2
1
2
1
3
1
2
Manα
Manα
Man(α1→3)Manα
↓
1
2
Manα 1
Manα1
Manα 1
(Manβ1)
n
1
Corresponding author; phone/fax: +7 (495) 135-8784; e-mail:
A
B
C
D
nen@ioc.ac.ru.
2
Abbreviations: Bn, benzyl; Bz, benzoyl; NIS, N-iodosuccinimide;
Tfa, trifluoroacetyl; and Tf, trifluoromethanesulfonyl.
Fig. 1. The structure of mannan from C. albicans cell wall.
110

SYNTHESIS OF OLIGOSACCHARIDE FRAGMENTS
111
Man(α1
Man(α1
2)Man(α1
3)Man(α1
2)Man(α1
2)Man(α1
2)Man(α1
2)Man(α1
OCH2CH2CH2NH2 (I)
OCH2CH2CH2NH2 (II)
Man(α1
2)Man(α1
2)Man(α1
2)Man(α1
OCH2CH2CH2NH2 (III)
3
Manα1
OR
BnO
BnO
BnO
BnO
BnO
BnO
O
OR
BnO
OAc
O
BnO
BnO
BnO
O
BnO
i
i
HO
NHTfa
O
BnO
+
O
(IV)
NHTfa
SEt
O
NHTfa
(IV)
(V)
O
(VI) R = Ac
(VII) R = H
(VIII) R = Ac
(IX) R = H
ii
ii
Scheme 1. Reagents: (i) NIS, TfOH, molecular sieves 4Å, CH Cl ; (ii) MeONa, MeOH.
2
2
D branched oligosaccharide structure has yet been were established on the basis of NMR spectroscopy. It
reported.
is known that, in the case of mannose residues, unlike
other monosaccharides, the vicinal coupling constant
We herein report the synthesis of oligosaccharides
J
1,2 and the chemical shifts of C1 in 1ç and 13C NMR
(I)–(III) corresponding to three types of the side chain
structures (Ä, ë, and D) of the mannan from C. albi-
cans cell wall in the form of 3-aminopropyl glycosides.
The presence of the amino group in the aglycones of the
synthetic oligosaccharides allows their subsequent
covalent binding to high-molecular carriers (synthetic
or protein) and labels of various types. In particular, we
have obtained a conjugate of oligosaccharide (III) with
BSA for further immunological studies. Such neogly-
coconjugates and molecular probes are valuable tools
for glycobiological studies and can be a basis for the
creation of vaccines of a new generation.
spectra, respectively, do not characterize the configura-
tion of glycoside bonds. At the same time, the chemical
shift of H5 can be a rather reliable indicator of the ano-
meric configuration of mannosides [13]. The signals of
H5 of the glycosylation products (VI) and (VIII) are
exhibited in the region of δ 3.70–4.00 ppm, which is in
a good agreement with the data for 3,4,6-tri-é-bensy-
lated α-mannosides [6, 14], whereas the H5 protons of
β-linked mannose residues resonate in the region of
[delta] 3.30–3.40 ppm [14]. It should be noted that the
presence of similarly protected benzylated monosac-
charide units in disaccharides (VIII) and (IX) and
trisaccharides (see below) results in a significant over-
lap of the signals of ring protons H3–H6 of mannose
RESULTS AND DISCUSSION
1
units in the ç NMR spectra, substantially complicat-
The glycosylating agents bearing permanent protec-
tive groups (benzyl or benzoyl) in positions 3, 4, and 6
and an acetyl group in position 2, which provides a high
stereoselectivity of α-mannosylation due to participa-
tion and can be easily removed from the glycosylation
product keeping the other protective groups intact, are
usually used for the design of (α1–2)-linked mannooli-
gosaccarides. In this study, we used the well-known thi-
omannoside (IV) as such a glycosylating agent [12].
Glycosylation of 3-trifluoroacetamidopropanol (V)
with thioglycoside (IV) in the presence of NIS and
TfOH smoothly resulted in glycoside (VI), which was
deacetylated to give monosaccharide glycosyl acceptor
(VII) (Scheme 1). The repetition of the operations of
glycosylation and deacetylation yielded disaccharide
acceptor (IX).
ing their reliable assignment. Therefore, when report-
ing the NMR data for (1−2)-linked oligosaccharides
(see the Experimental section), we give the full assign-
ment of signals only for the terminal nonreducing
monosaccharide residue that was introduced upon gly-
cosylation or was deacetylated to obtain a new glycosyl
acceptor. Only the data for the anomeric H and C atoms
are given for the other monosaccharide units.
Glycosylation of disaccharide acceptor (IX) with
thiomannoside (IV) resulted in a mixture of the
expected α-linked trisaccharide (X) and its β-anomer at
the nonreducing unit C (XI) with the latter compound
prevailing (Scheme 2). In the case of trisaccharide (X),
the signal of H5 of the terminal nonreducing mannose
residue is at δ 3.96 ppm, which confirms its α-configu-
ration, whereas in the case of derivative (XI), the signal
Structures of the glycosylation products, first of all, of H5 of the β-connected mannose residue is shifted
the configuration of the newly formed glycoside bonds, upfield (δ 3.33 ppm). Taking into consideration a pro-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 33 No. 1 2007

112
KARELIN et al.
OAc
O
C
O
OAc
O
BnO
BnO
BnO
BnO
BnO
BnO
BnO
BnO
BnO
BnO
BnO
BnO
O
BnO
BnO
BnO
BnO
BnO
BnO
O
O
i
B
(IX) + (IV)
+
O
O
O
O
A
NHTfa
O
NHTfa
O
(X)
(XI)
Scheme 2. Reagents: (i) NIS, TfOH, molecular sieves 4Å, CH Cl .
2
2
OBz
O
BnO
BnO
BnO
i, ii
(IV)
SEt
(XII)
OBz
O
BzO
BzO
BzO
D
OR
O
BnO
BnO
BnO
OBz
O
BzO
BzO
BzO
O
BnO
BnO
BnO
O
C
O
BnO
BnO
BnO
BnO
Br
O
iii
(XV)
(IX) + (XII)
iv
O
O
BnO
BnO
BnO
BnO
BnO
BnO
O
B
O
BnO
BnO
O
NHTfa
O
O
A
(XIII) R = Bz
(XIV) R = H
NHTfa
O
i
(XVI)
Scheme 3. Reagents: (i) MeONa, MeOH; (ii) BzCl, Py; (iii) NIS, TfOH, molecular sieves 4Å, CH Cl ; (iv)AgOTf, molecular sieves
2
2
4Å, CH Cl .
2
2
nounced tendency of mannopyranose glycosyl donors
to stereoselective α-glycosylation even in the case of
nonparticipating group at O2, e.g., glycosyl [4, 5, 8–
10], this result appeared to be rather unexpected.
Removal of benzoyl group in trisaccharide (XIII)
resulted in acceptor (XIV). Since we did not plan the
further extension of the oligosaccharide chain, we used
a more available benzobromomannose (XV) as the
mannosyl donor at the final step rather than thioglyco-
side (IV). The reaction of bromide (XV) with acceptor
We have substituted benzoyl group in mannosyl
donor (IV) for acetyl group and obtained thioglycoside (XIV) in the presence of silver triflate resulted in the
target tetrasaccharide (XVI) in 68% yield (Scheme 3).
The α-configuration of the terminal mannose unit D
unambiguously followed from the low-field position of
the H5 signal (δ 4.65 ppm) in the 1H NMR spectrum of
(XVI).
(XII) to increase α-stereoselectivity of mannosylation
of disaccharide acceptor (IX) [15] (Scheme 3). In fact,
the use of benzoylated derivative (XII) allowed us to
minimize the formation of β-anomer; the target α-
trisaccharide (XIII) was obtained in 64% yield. The
higher stereoselectivity of mannosylation in the case of
donor (XII) can be explained by a lesser tendency of
phenyl-substituted 1,2-dioxolenium intermediate to
isomerisation as compared with methyl-substituted
analogue in the case of acetylated donor (IV).
The [2 + 2] scheme was used to synthesize tetrasac-
charide (II). Glycosylation of disaccharide acceptor
(IX) with the derivative of (α1–3)-mannobiose was the
key step of the synthesis. The known 4,6-di-O-benzyl
thioglycoside (XVII) [16], which was selectively ben-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 33 No. 1 2007

SYNTHESIS OF OLIGOSACCHARIDE FRAGMENTS
113
OBz
O
BzO
OH
O
OBz
O
BnO
BnO
HO
BnO
BnO
BzO
OBz
O
BnO
BnO
+ (XV)
iii
BzO
i, ii
HO
O
SEt
SEt
(XVII)
(XVIII)
(XIX)
SEt
OBz
O
BzO
BzO
BzO
OBz
O
BnO
BnO
C
E
O
O
BnO
BnO
BnO
BnO
BnO
BnO
+ (IX)
iv
O
B
O
O
A
NHTfa
O
(XX)
Scheme 4. Reagents: (i) PhC(OMe) , camphorsulfonic acid, MeCN; (ii) 80% aqueous AcOH; (iii) AgOTf, molecular sieves 4Å,
3
CH Cl ; (iv) NIS, TfOH, molecular sieves 4Å, CH Cl .
2
2
2
2
OBz
O
BzO
BzO
BzO
D
E
OBz
BzO
BzO
BzO
O
OR
O
BnO
BnO
RO
O
BnO
BnO
O
C
O
OBz
O
BnO
BnO
BzO
+ (IX)
ii
+ (XV)
iv
O
BnO
BnO
BnO
BnO
BnO
BnO
O
BnO
BnO
BnO
BnO
BnO
BnO
(XVII)
i
O
O
B
SEt
O
O
(XXI)
O
O
A
NHTfa
O
NHTfa
O
(XXII) R = Bz
(XXIII) R = H
(XXIV)
iii
Scheme 5. Reagents: (i) BzCl, Py; (ii) NIS, TfOH, molecular sieves 4Å, CH Cl ; (iii) MeONa, MeOH; (iv)AgOTf, molecular sieves
2
2
4Å, CH Cl .
2
2
zoylated at O2 via the intermediate formation of cyclic ration of the resulting glycoside bond was confirmed by
2,3-orthobenzoate (Scheme 4), was the starting com-
pound in the synthesis of (α1–3)-linked dimannoside
block. The reaction of the resulting monobenzoate
(XVIII) with bromide (XV) in the presence of silver
triflate resulted in disaccharide thioglycoside (XIX) in
64% yield, which can be directly used in the subsequent
glycosylation. The low-field position of the H5 signal δ
4.50 ppm) of tetra-é-benzoylated nonreducing man-
the chemical shift of H5 (δ 4.15 ppm) of the monosac-
charide unit C in the 1H NMR spectrum of (XX).
Diol (XVII) was benzoylated to give dibenzoate
(XXI), which was used as the precursor of the 2,3-bis-
glycosylated mannose residue in the pentasaccaride
(III) (Scheme 5). The reaction of thioglycoside (XXI)
with disaccharide acceptor (IX) in the presence of NIS
and TfOH resulted in trisaccharide derivative (XXII) in
95% yield, with the α-configuration of the terminal
mannoside bond being confirmed by the chemical shift
(δ 4.10 ppm) of H5 of the ring C in the 1H NMR spec-
1
nose unit (the future residue E) in the H NMR spec-
trum of (XIX) proved its α-configuration. Glycosyla-
tion of disaccharide acceptor (IX) with thioglycoside
(XIX) in the presence NIS and TfOH resulted in the tar-
get tetrasaccharide (XX) in 65% yield. The α -configu- trum. The subsequent debenzoylation of (XXII)
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 33 No. 1 2007

114
KARELIN et al.
OR1
O
HO
HO
R2O
(I) R1 = α-D-Man, R2 = H
(II) R1 = H, R2 = α-D-Man
(III) R1 = R2 = α-D-Man
O
HO
HO
HO
O
i, ii
(XVI), (XX), (XXIV)
HO
O
O
HO
HO
NH2
O
OH
HO
HO
HO
O
HO
i, ii
O
(VIII)
O
HO
HO
NH2
O
(XXV)
–
Scheme 6. Reagents: (i) H , Pd(OH) /C, MeOH; (ii) Amberlyst A-26 (OH ), water.
2
2
H
N
H
N
H
N
EtO
O
OEt
O
O
OEt
O
O
BSA
R
R
i
BSA
(III), (XXV) +
ii
O
O
O
n
(XXVI)
(XXVII), (XXIX)
(XXVIII), (XXX)
(XXVII), (XXVIII) R = Man(α1
(XXIX), (XXX) R = Man(α1
2)Man(α1
2)Man(α1
2) Man(α1
2)Man(α1
3
Manα1
Scheme 7. Reagents: (i) Et N, 50% aqueous ethanol; (ii) borate buffer, pH 9.0.
3
resulted in diol (XXIII), which was bisglycosylated step, the reaction of oligosaccharide (XXV) with
with benzobromomannose (XV) to give the target pro- diethyl squarate (XXVI) at pH 7 resulted in monosub-
1
tected pentasaccaride (XXIV) in 85% yield.
stituted adduct (XXVII) (Scheme 7).The H NMR
spectrum of the carbohydrate part of (XXVII) was
practically indistinguishable from that of starting disac-
charide (XXV) except for the signal of H1 of the man-
nose unit A, which appeared as two closely located sin-
glets of half intensity, which is likely due to the isomer-
ism of the vinylogous amide group in the squarate
fragment [17, 19]. The same reason caused the dou-
bling of the signals of the spacer propyl group and the
ethoxy group. The subsequent reaction of derivative
(XXVII) with free amino groups of BSA proceeded at
pH 9 and resulted in conjugate (XXVIII). Under the
The protected oligosaccharides (XVI), (XX), and
(XXIV) were first subjected to hydrogenolysis to
remove benzyl groups; then benzoyl and N-trifluoro-
acetyl groups were simultaneously removed by the
treatment with anion exchanger Amberlyst A-26 (OH–)
to give the target free oligosaccharides (I)–(III)
(Scheme 6). Similarly, protected derivative (VIII) was
converted into free 3-aminopropyl glycoside (XXV),
which was used for obtaining of the model conjugate
with BSA.
The conjugation of derivative (XXV) with BSA was same conditions, pentasaccharide (III) was trans-
performed by the squarate method [17–19]. At the first formed to monoamide (XXIX), which was coupled
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 33 No. 1 2007

SYNTHESIS OF OLIGOSACCHARIDE FRAGMENTS
115
with BSA to give conjugate (XXX). According to
66275
MALDI TOF mass spectrometry [18] (Fig. 2), conju-
gates (XXVIII) and (XXX) contained, on the average,
20 oligosaccharide residues (n = 20) at an initial oli-
gosaccharide–BSA molar ratio of 20 : 1.
Thus, 3-aminopropyl glycosides of the oligosaccha-
rides corresponding to the three types of side chains of
the mannoprotein from C. albicans cell wall were syn-
thesized. Conjugates of disaccharide (XXV) and pen-
tasaccharide (III) with BSA were obtained using the
squarate method.
(‡)
(b)
(c)
71143
EXPERIMENTAL
The procedures for solvent purification are similar
to those described in [20]. TfOH, camphorsulfonic
acid, ethyl orthobenzoate, diethyl squarate (Fluka), NIS
(Acros), and AgOTf (Merck) were used without addi-
tional purification. 1ç and 13C NMR spectra were reg-
istered on Bruker DRX-500 and Bruker AM-300 spec-
trometers at 25°ë in CDCl3 in the case of the protected
derivatives and in D2O in the case of free oligosaccha-
rides. The signals in the NMR spectra were assigned
using COSY, TOCSY, ROESY, and HSQC two-dimen-
sional correlation spectroscopy experiments. The des-
ignations of monosaccharide residues used in the
assigning of NMR spectra, are shown in schemes.
MALDI-TOF spectra were registered on a Bruker
Ultraflex mass spectrometer with the double time-of-
flight analyzer in a linear mode with registration of pos-
itive ions. The source had panoramic delay of ion
extraction, and an accelerating voltage of 20 kV were
used. 2,5-Dihydroxybenzoic acid was used as a matrix.
The values of optical rotation were measured on a Pol-
yarimetr Universal’nyi PU-7 polarimeter (Russia) at
18–22°ë in chloroform in the case of the protected and
partially protected derivatives and in water in the case
of free oligosaccharides at Ò = 1%. TLC was carried out
on silica gel 60 (Merck) precoated plates. The spots
were visualized by spraying with 10 vol % orthophos-
phoric acid in ethanol and the subsequent heating at
~150°ë. Column chromatography was carried out on
silica gel 60 (0.040–0.063 mm) (Merck). For gel chro-
matography, a TSK HW-40 (S) column (1.5 × 90 cm) in
0.1 M acetic acid was used; the eluate was analyzed by
a Knauer 88 00 flow-through refractometer. Molecular
sieves 4Å (Fluka) were activated before reaction by
heating at 200°ë for 2 h in a vacuum (1 mm Hg). Gly-
cosylation was achieved in anhydrous solvents in the
atmosphere of dry argon.
85148
60000
80000
m/z
100000
120000
Fig. 2. MALDI TOF mass spectra of (a) initial BSA and
conjugates (b) (XXVIII) and (c) (XXX).
the reaction mixture was maintained within a range
from –25°C to –30°C.After the starting glycosyl accep-
tor was exhausted (according to TLC), the reaction
mixture was quenched with pyridine, diluted with chlo-
roform, and filtered through a Celite layer. The filtrate
was washed with 1 M sodium thiosulfate and water, the
solvent was removed, and the residue was twice
coevaporated with toluene.
A general procedure for deacylation (procedure
B). A 1 M sodium methylate in MeOH was added a
solution of a starting compound in anhydrous methanol
in the amount necessary to provide the final concentra-
tion of MeONa of 0.05–0.1 M. After the deacylation
was over (TLC monitoring); cation exchange resin KU-
2 (H+) was added to the reaction mixture until the neu-
tral reaction, then the cation exchanger was filtered off
and washed with MeOH, and the filtrate was concen-
trated in a vacuum.
A general procedure for glycosylation with
thioglycosides (procedure A). Molecular sieves 4Å
were added to a solution of a glycosyl acceptor and a
glycosyl donor in dichloromethane; the mixture was
A general procedure for glycosylation with ben-
stirred for 30 min at room temperature and cooled to the zobromomannose (procedure C). Molecular sieves
temperature in a range from –10 to –15°C. NIS was 4Å were added to a solution of a glycosyl acceptor and
added, and, after 10 min, the temperature of the reac- benzobromomannose in dichloromethane, the mixture
tion mixture was decreased to a value from –30 to was stirred for 30 min at room temperature, cooled to
−35°C and TfOH was added. Then the temperature of the temperature in a range from –40 to –50°C, and
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116
KARELIN et al.
AgOTf was added. The resulting mixture was stirred at (180 µl, 1.99 mmol) according to procedure A. Column
a temperature within a range from –25 to –30°C until chromatography (10 : 1 toluene–ethyl acetate) led to
the disappearance of the starting glycosyl acceptor 3.50 g (82%) of mannobioside (VIII) as a syrup; Rf
1
(TLC monitoring), quenched with pyridine, diluted
0.55 (3 : 1 toluene–ethyl acetate); [α]D = +20.5°; H
with chloroform, and filtered through a Celite layer.
The filtrate was washed with 1 M sodium thiosulfate
and water, the solvent was removed, and the residue
was twice coevaporated with toluene.
NMR: 2.17 (3 ç, s, ëç3ëé), 4.92 (1 H, br s, H1-A),
5.08 (1 H, br s, H1-B), 5.55 (1 H, br s, H2-B), 3.99 (H3-
B), 3.80 (H4-B), 4.00 (H5-B), 3.72, 3.78 (2 H6-B),
4.45–4.92 (12 H, a cluster of d, 6 PhCH2), 7.18–7.40
(30 H, m, arom.); 13C NMR: 21.1 (ëç3ëé), 170.0
(CH3CO), 98.9 (C1-A), 99.6 (C1-B), 68.7 (C2-B), 78.0
(C3-B), 74.3 (C4-B), 71.9 (C5-B), 69.5 (C6-B). Found,
%: C 67.68, H 6.19. Calculated for C61H66O13F3N, %:
C 67.95, H 6.17.
3-Trifluoroacetamidopropyl 2-O-acetyl-3,4,6-tri-
O-benzyl-a-D-mannopyranoside (VI). 3-Trifluoroac-
etamidopropanol (V) (3.45 g, 20.2 mmol) was glycosy-
lated with thioglycoside (IV) (5.40 g, 10.1 mmol) in
30 ml of dichloromethane in the presence of molecular
sieves 4Å (6 g), NIS (4.55 g, 20.2 mmol) and TfOH
(33 µl, 0.37 mmol) according to procedure A. Column
chromatography (8 : 1 toluene–ethyl acetate) resulted
in 4.10 g (62%) of mannoside (VI) as a syrup; Rf 0.57
(3 : 2 toluene–ethyl acetate); [α]D = +40.4°; 1H NMR:
2.18 (3 ç, Ò, ëç3ëé), 4.85 (1 H, br s, H1), 5.35 (1 H,
br s, H2), 3.94 (1 H, dd, J3,2 3.3 Hz, J3,4 8.8 Hz, H3),
3.83 (H4), 3.79 (H5), 3.74 (2 H6), 4.45–4.90 (6 H, a
cluster of d, 3 PhCH2), 7.10–7.42 (15 H, m, arom.). The
spectrum also contained the signals of 3-trifluoroaceta-
midopropyl aglycone: 1.89 (2 H, m, ëç2ëç2ëç2),
3.42 and 3.51 (2 H, 2 m, ëç2N), 3.57 and 3.83 (2 H, 2
m, ëç2é);3 13C NMR: 21.0 (ëç3ëé), 170.4
3-Trifluoroacetamidopropyl 3,4,6-tri-O-benzyl-
a-D-mannopyranosyl-(1
2)-3,4,6-tri-O-benzyl-a-
D-mannopyranoside (IX). Dimannoside (VIII)
(1.50 g, 1.39 mmol) was deacetylated with 1 M
MeONa (0.5 ml) in MeOH (5 ml) according to method
B. Column chromatography (5 : 1 toluene–ethyl ace-
tate) resulted in 955 mg (66%) of acceptor (VII) as a
syrup; Rf 0.44 (3 : 1 toluene–ethyl acetate); [α]D +25.6°;
1H NMR: 4.79 (1 H, br s, H1-A), 4.94 (1 H, br s, H1-
B), 4.00 (1 H, br s, H2-B), 3.74 (H3-B), 3.66 (H4-B),
3.83 (H5-B), 3.62 (2 H6-B), 4.36 - 4.74 (12 H, a cluster
of d, 6 PhCH2), 7.05–7.27 (30 H, m, arom.); 13C NMR:
(CH3CO), 98.0 (C1), 68.6 (C2), 78.2 (C3), 74.2 (C4), 98.7 (C1-B), 101.6 (C1-B), 68.1 (C2-B), 79.3 (C3-B),
71.9 (C5), 69.0 (C6). The spectrum also contained the 74.2 (C4-B), 71.8 (C5-B), 69.4 (C6-B).
signals of 3-trifluoroacetamidopropyl aglycone: 28.2
3-Trifluoroacetamidopropyl 2-O-acetyl-3,4,6-tri-
(ëç2ëç2ëç2), 38.0 (ëç2N), 66.2 (CH2O).4 Found,
O-benzyl-a-D-mannopyranosyl- and -b-D-man-
%: C 64.80, H 6.45. Calculated for C34H38O7F3N, %: C
nopyranosyl-(1
nopyranosyl-(1
nopyranosides (X) and (XI). Glycosylation of accep-
tor (XI) (65 mg, 0.062 mmol) with thioglycoside (IV)
(57 mg, 0.106 mmol) in dichloromethane (6 ml) in the
presence of MS 4Å (50 mg), NIS (48 mg, 0.214 mmol),
2)-3,4,6-tri-O-benzyl-a-D-man-
2)-3,4,6-tri-O-benzyl-a-D-man-
64.86, H 6.08.
3-Trifluoroacetamidopropyl 3,4,6-tri-O-benzyl-
a-D-mannopyranoside (VII). Mannoside (VI)
(4.10 g, 6.3 mmol) was deacetylated with 1 M MeONa
(3 ml) in MeOH (30 ml) according to procedure B. Col-
umn chromatography (3 : 1 toluene–ethyl acetate) and TfOH (3.9 µl, 0.044 mmol) was performed accord-
resulted in 2.40 g (61%) of acceptor (VII) as amor-
phous substance; Rf 0.29 (3 : 1 toluene–ethyl acetate);
[α]D +40.6°; 1H NMR: 3.64 (3 H, m, H5, 2 H6), 3.74 (2
H, m, H3, H4), 3.93 (1 H, br s, H2), 4.42–4.78 (6 H, a
cluster of d, 3 PhCH2), 4.78 (1 H, br s, H1), 7.08–7.32
(15 H, m, arom.).
ing to method A. Column chromatography (10 : 1 tolu-
ene–ethyl acetate) resulted in 27 mg (28%) of α-isomer
(ï) and 40 mg (42%) of β-isomer (XI). Compound (ï),
a syrup; Rf 0.58 (4 : 1 toluene–ethyl acetate); [α]D =
+32.2°; 1H NMR: 2.15 (3 ç, Ò, ëç3ëé), 4.95 (1 H, br
s, H1-A), 5.20 (1 H, br s, H1-B), 5.07 (1 H, br s, H1-C),
3-Trifluoroacetamidopropyl 2-é-acetyl-3,4,6-tri- 5.55 (1 H, br s, H2-C), 4.02 (H3-C), 3.93 (H4-C), 3.96
é-benzyl-a-D-mannopyranosyl-(1
2)-3,4,6-tri-é-
(H5-C), 3.72 and 3.78 (2 H6-C), 4.33–4.89 (18 H, a
cluster of d, 9 PhCH2), 7.15–7.39 (45 H, m, arom.); 13C
NMR: 21.0 (ëç3ëé), 170.5 (CH3CO), 99.0 (C1-A),
100.7 (C1-B), 99.3 (C1-C), 68.8 (C2-C), 78.0 (C3-C),
74.2 (C4-C), 72.2 (C5-C), 69.9 (C6-C). Compound
(XI), a syrup; Rf 0.40 (4 : 1 toluene–ethyl acetate); [α]D
benzyl-a-D-mannopyranoside (VIII). Acceptor
(VII) (2.40 g, 3.98 mmol) was glycosylated with
thioglycoside (IV) (3.20 g, 5.97 mmol) in dichlo-
romethane (25 ml) in the presence of molecular sieves
4Å (4.50 g), NIS (2.69 g, 12.0 mmol), and TfOH
–3.1°; 1H NMR: 2.11 (3 ç, Ò, ëç3ëé), 4.99 (1 H, br s,
H1-A), 5.15 (1 H, d, J1,2 3.1 Hz, H1-B), 4.75 (1 H, br s,
H1-C), 5.53 (1 H, d, J2,3 2.3 Hz, H2-C), 3.44 (1 H, dd,
3
Similar signals with negligible alterations in chemical shifts
( 0.1 ppm) were present in the spectra of all protected oligosac-
charides described below. Therefore, the signals of 3-trifluoroace-
1
tamidopropyl group are not given in the further assignment of H
J
3,4 9.2 Hz, H3-C), 3.78 (H4-C), 3.33 (1 H, m, H5-C),
NMR spectra.
4
Similar signals ( 0.5 ppm) were present in the spectra of all pro-
3.69 (2 H, H6-C), 4.23–4.90 (18 H, a cluster of d, 9
PhCH2), 7.13–7.44 (45 H, m, arom.); 13C NMR: 21.0
(ëç3ëé), 170.5 (CH3CO), 99.2 (C1-A), 100.3 (C1-B),
tected oligosaccharides described below. Therefore, the signals of
3-trifluoroacetamidopropyl group are not given in the further
13
assignment of C NMR spectra.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 33 No. 1 2007

SYNTHESIS OF OLIGOSACCHARIDE FRAGMENTS
117
96.1 (C1-C), 68.1 (C2-C), 80.1 (C3-C), 74.4 (C4-C), (1 H, d, J1,2 1.2 Hz, H1-B), 4.91 (1 H, d, J1,2 1.5 Hz, H1-
75.4 (C5-C), 69.2 (C6-C). Found, %: C 69.37, H 6.35.
C), 3.95 (H2-C), 3.76 (1 H, dd, J3,2 2.9 Hz, J3,4 9.1 Hz,
H3-C), 3.53 (H4-C), 3.78 (H5-C), 3.46 (H6-C), 4.31–
4.70 (18 H, a cluster of d, 9 PhCH2), 7.00–7.25 (45 H,
Calculated for C88H94O18F3N, %: C 69.97, H 6.27.
Ethyl 3,4,6-tri-O-benzyl-2-O-benzoyl-1-thio-a-
D-mannopyranoside (XII). A solution of thioglyco- m, arom.); 13C NMR: 98.6 (C1-A), 100.6 (C1-B), 101.5
side (IV) (200 mg, 0.373 mmol) in MeOH (4 ml) was (C1-C), 68.1 (C2-C), 79.0 (C3-C), 74.8 (C4-C), 71.7
treated with 1 M MeONa (0.2 ml) according to proce- (C5-C), 69.1 (C6-C). Found, %: C 69.58, H 6.15. Cal-
dure B. The resulting monohydroxy derivative was dis- culated for C86H92O17F3N, %: C 70.33, H 6.31.
solved in pyridine (4 ml), and benzoyl chloride
(0.13 ml, 1.11 mmol) was added. When benzoylation
3-Trifluoroacetamidopropyl 2,3,4,6-tetra-é-ben-
zoyl-a-D-mannopyranosyl-(1
zyl-a-D-mannopyranosyl-(1
zyl-a-D-mannopyranosyl-(1
2)-3,4,6-tri-é-ben-
2)-3,4,6-tri-é-ben-
2)-3,4,6-tri-é-ben-
was over, the reaction mixture was poured in ice-cold
water; the product was extracted with chloroform
(100 ml). The extract was washed with the saturated
sodium hydrocarbonate and water, the solvent was
removed, and the residue was twice coevaporated with
toluene. Column chromatography of the residue (25 : 1
toluene–ethyl acetate) resulted in 200 mg (90%) of ben-
zoate (XII) as a syrup; Rf 0.86 (4 : 1 toluene–ethyl ace-
zyl-a-D-mannopyranoside (XVI). Acceptor (XIV)
(157 mg, 0.107 mmol) was glycosylated with bromide
(XV) (106 mg, 0.161 mmol) in dichloromethane (4 ml)
in the presence of molecular sieves 4Å (50 mg) and
AgOTf (47 mg, 0.118 mmol) according to procedure C.
The product was isolated by column chromatography
(10 : 1 toluene–ethyl acetate) to yield 150 mg (68%) of
tetrasaccharide (XVI) as a syrup; Rf 0.58 (4 : 1 toluene–
1
tate); [α]D +27.3°; H NMR: 1.29 (3 H, t, J 7.5 Hz,
ëç2ëç3), 2.65 (2 H, m, ëç2ëç3), 3.75 (1 H, d, J6,6'
10.8 Hz, H6), 3.92 (1 H, dd, J6',5 3.8 Hz, H6'), 4.02 (1 H,
dd, J3,2 3.0 Hz, J3,4 9.2 Hz, H3), 4.13 (1 H, t, J4,5 9.7 Hz,
H4), 4.49–4.89 (6 H, a cluster of d, 3 PhCH2), 5.43 (1
H, br s, H1), 5.69 (1 H, br s, H2), 7.19–8.17 (20 H, m,
arom.).
1
ethyl acetate); [α]D +56.2°; H NMR: 5.03 (1 H, br s,
H1-A), 5.29 (1 H, br s, H1-B), 5.40 (1 H, br s, H1-C),
5.19 (1 H, br s, H1-D), 5.96 (1 H, br s, H2-D), 6.07 (1
H, dd, J2,3 3.3 Hz, J3,4 10.0 Hz, H3-D), 6.21 (1 H, t, H4-
D), 4.65 (H5-D), 4.32 and 4.57 (2 H6-D), 4.47–5.01 (18
H, a cluster of d, 9 PhCH2), 7.10–8.16 (65 H, m, arom.);
3-Trifluoroacetamidopropyl 3,4,6-tri-O-benzyl-
13C NMR: 99.2 (C1-A), 101.1 (C1-B), 100.5 (C1-C),
99.0 (C1-D), 70.6 (C2-D), 70.2 (C3-D), 66.9 (C4-D),
69.5 (C5-D), 62.5 (C6-D), 165.1, 165.3, 165.5, and
166.1 (4 PhCO). Found, %: C 70.25, H 5.67, N 0.78.
Calculated for C120H118O26F3N, %: C 70.40, H 5.81, N
0.68.
2-O-benzoyl-a-D-mannopyranosyl-(1
2)-3,4,6-
2)-3,4,6-
tri-é-benzyl-a-D-mannopyranosyl-(1
tri-é-benzyl-a-D-mannopyranoside (XIII). Accep-
tor (IX) (1.75 g, 1.69 mmol) was glycosylated with
thioglycoside (XII) (1.50 g, 2.51 mmol) in dichlo-
romethane (20 ml) in the presence of molecular sieves
4Å (3 g), NIS (1.12 g, 4.98 mmol), and TfOH (60 µl,
0.68 mmol) by procedure A. Column chromatography
(15 : 1 toluene–ethyl acetate) resulted in 1.70 g (64%)
of trimannoside (XIII) as a syrup; Rf 0.43 (6 : 1 tolu-
Ethyl 4,6-di-O-benzyl-2-O-benzoyl-1-thio-a-D-
mannopyranoside (XVIII). Methyl orthobenzoate
(3.2 ml, 18.8 mmol) and the catalytic amount of cam-
phorsulfonic acid were added to a solution of diol
(XVII) (2.4 g, 5.9 mmol) in acetonitrile (15 ml). The
mixture was kept for 30 min, then 80% acetic acid was
added. After 10 min, the reaction mixture was diluted
with chloroform and washed with saturated sodium
hydrocarbonate and water. The solvent was removed,
and the residue was chromatographed (5 : 1 toluene–
ethyl acetate) to give 1.53 g (51%) of (XVIII); [α]D =
ene–ethyl acetate); [α]D +16.3°; 1H NMR: 4.98 (1 H, d,
J1,2 1.5 Hz, H1-A), 5.27 (1 H, d, J1,2 1.5 Hz, H1-B), 5.16
(1 H, d, J1,2 1.6 Hz, H1-C), 5.80 (1 H, br s, H2-C), 4.15
(H3-C), 4.15 (H4-C), 4.03 (H5-C), 3.76 and 3.82 (2
H6-C), 4.42–4.93 (18 H, a cluster of d, 9 PhCH2), 7.10–
13
8.17 (50 H, m, arom.); C NMR: 99.1 (C1-A), 100.8
(C1-B), 99.5 (C1-C), 69.1 (C2-C), 78.0 (C3-C), 74.4
(C4-C), 72.3 (C5-C), 70.0 (C6-C), 165.5 (PhCO).
Found, %: C 71.90, H 6.38. Calculated for
C93H96O18F3N, %: C 71.02, H 6.15.
1
+46.3°; H NMR: 1.26 (3 H, t, J 7.4 Hz, SCH2CH3),
2.62 (2 H, m, SCH2CH3), 3.73 (1 H, d, J6,6' 9.9 Hz, H6),
3.89 (1 H, dd, J6',5 3.6 Hz, H6'), 4.01 (1 H, t, J4,5 9.5 Hz,
H4), 4.14 (1 H, dd, J3,2 2.9 Hz, J3,4 9.4 Hz, H3), 4.17 (1
H, m, H5), 4.49, 4.60, 4.69, and 4.82 (4 H, 4 d, J 11.0–
12.0 Hz, 2 PhCH2), 5.39 (2 H, br s, H1, H2), 7.20–8.04
(15 H, m, arom.). Found, %: C 68.66, H 6.49. Calcu-
lated for C29H32O6S, %: C 68.48, H 6.34.
3-Trifluoroacetamidopropyl 3,4,6-tri-O-benzyl-
a-D-mannopyranosyl-(1
2)-3,4,6-tri-é-benzyl-
2)-3,4,6-tri-é-benzyl-a-
a-D-mannopyranosyl-(1
D-mannopyranoside (XIV). A solution of trimanno-
side (XIII) (1.65 g, 1.05 mmol) in MeOH (20 ml) was
treated with 1 M MeONa (1.0 ml) according to proce-
dure B. Column chromatography (10 : 1 toluene–ethyl
acetate) resulted in 950 mg (62%) of acceptor (XIV) as
white foam; Rf 0.50 (4 : 1 toluene–ethyl acetate); [α]D =
Ethyl 2,3,4,6-tetra-O-benzoyl-a-D-mannopyra-
nosyl-(1
3)-4,6-di-O-benzyl-2-é-benzoyl-1-thio-
a-D-mannopyranoside
(XIX).
Thioglycoside
(XVIII) (135 mg, 0.266 mmol) was glycosylated with
bromide (XV) (263 mg, 0.40 mmol) in dichlo-
+33.4°; 1H NMR: 4.76 (1 H, d, J1,2 1.4 Hz, H1-A), 5.04
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 33 No. 1 2007

118
KARELIN et al.
romethane (3 ml) in the presence of molecular sieves 1.34 (3 H, t, J 7.3 Hz, ëç2ëç3), 2.70 (2 H, m,
4Å (300 mg) andAgOTf (102 mg, 0.4 mmol) according
to procedure C. The product was isolated by column
chromatography (3 : 1 petroleum ether–ethyl acetate) to
give 186 mg (64%) of (XIX) as white foam; [α]D –
ëç2ëç3), 3.80 (1 H, d, J6,6' 9.9 Hz, H6), 4.01 (1 H, dd,
6',5 9.9 Hz, H6'), 4.38 (1 H, m, H5), 4.43 (1 H, t, J4,5 9.7
Hz, H4), 4.57 (2 H, d, J 11.8 Hz, PhCH2), 4.68 (1 H, d,
J 10.9 Hz, PhCH2), 4.80 (1 H, d, J 11.9 Hz, PhCH2),
5.51 (1 H, br s, H1), 5.70 (1 H, dd, J3,2 3.1 Hz, J3,4 8.5
Hz, H3), 5.74 (1 H, br s, H2), 7.07–8.22 (20 H, m,
arom.).
J
17.5°; 1H NMR: 1.31 (3 H, t, J 7.2 Hz, SCH2CH3), 2.67
(2 H, m, SCH2CH3), 3.79 (1 H, d, J6,6' 11.0 Hz, H6-C),
3.99 (1 H, dd, J6',5 3.2 Hz, H6'-C), 4.29 (1 H, m, H5-C),
4.35 (1 H, t, J4,5 9.2 Hz, H4-C), 4.38 (2 H, m, H3-C, H6-
E), 4.50 (1 H, m, H5-E), 4.57, 4.73, 4.77, and 5.02 (4
H, 4 d, J 11.5–12.0 Hz, 2 PhCH2), 4.60 (1 H, dd, H6'-
E), 5.47 (1 H, br s, H1-E), 5.52 (1 H, br s, H1-C), 5.70
(1 H, br s, H2-C), 5.80 (1 H, br s, H2-E), 5.82 (1 H, dd,
3-Trifluoroacetamidopropyl 4,6-di-O-benzyl-2,3-
di-O-benzoyl-a-D-mannopyranosyl-(1
2)-3,4,6-
2)-3,4,6-
tri-é-benzyl-a-D-mannopyranosyl-(1
tri-é-benzyl-a-D-mannopyranoside (XXII). Glyco-
sylation of acceptor (IX) (122 mg, 0.118 mmol) with
thioglycoside (XXI) (108 mg, 0.176 mmol) was carried
out in dichloromethane (3 ml) in the presence of molec-
ular sieves 4Å (250 mg), NIS (79 mg, 0.35 mmol), and
TfOH (3 µl, 0.035 mmol) according to procedure A.
Column chromatography (5 : 1 toluene–ethyl acetate)
yielded 177 mg (95%) of (XXII); [α]D +8.5°; 1H NMR:
4.96 (1 H, d, J1,2 1.7 Hz, H1-A), 5.23 (1 H, d, J1,2 1.6 Hz,
H1-B), 5.17 (1 H, d, J1,2 1.7 Hz, H1-C), 5.82 (1 H, dd,
J2,3 3.2 Hz, H2-C), 5.84 (1 H, dd, J3,4 9.2 Hz, H3-C),
4.35 (1 H, t, H4-C), 4.10 (H5-C), 3.58 (1 H, dd, J6,5 1.4
Hz, J6,6' 11.0 Hz, H6-C), 3.78 (1 H, dd, J6',5 3.2 Hz, H6'-
C), 4.38–4.89 (16 H, a cluster of d, 8 PhCH2), 7.07–
J3,2 2.8 Hz, J3,4 10.1 Hz, H3-E), 6.09 (1 H, t, J4,5 9.8 Hz,
H4-E), 7.17–8.25 (35 H, m, arom.); 13C NMR: 14.9
(SCH2CH3), 25.7 (SCH2CH3), 82.3 (C1-C), 74.2 (C2-
C), 79.2 (C3-C), 75.0 (C4-C), 72.1 (C5-C), 68.8 (C6-
C), 99.7 (C1-E), 70.2 (C2-E), 70.3 (C3-E), 66.3 (C4-E),
69.6 (C5-E), 62.7 (C6-E), 165.1, 165.4, 166.0, and
166.1 (5 PhCO). Found, %: C 69.38, H 5.59. Calculated
for C63H58O15S, %: C 69.60, H 5.38.
3-Trifluoroacetamidopropyl 2,3,4,6-tetra-O-ben-
zoyl-
-mannopyranosyl-
-di- -benzyl-
3)-4,6
a-D
(1
O
-benzoyl-
-mannopyranosyl-
-tri-
2-é a-D
(1
2)-3,4,6
-benzyl-
benzyl-
-mannopyranosyl-
-mannopyranoside (XX). Acceptor (IX)
-tri- -
2)-3,4,6
é
a-D
(1
é
8.10 (50 H, m, arom.); 13C NMR: 99.0 (C1-A), 100.7
(C2-B), 99.4 (C1-C), 70.8 (C2-C), 72.5 (C3-C), 73.2
(C4-C), 72.1 (C5-C), 68.8 (C6-C). Found,%: C 69.82,
H 6.32, N 0.88. Calculated for C93H94O19F3N, %: C
70.40, H 5.97, N 0.88.
a-D
(84 mg, 0.081 mmol) was glycosylated with thioglyco-
side (XIX) (106 mg, 0.098 mmol) in dichloromethane
(3 ml) in the presence of molecular sieves 4Å (350 mg),
NIS (44 mg, 0.0196 mmol), and TfOH (2.9 µl, 0.032
mmol) by procedure A. The product was isolated by
column chromatography (5 : 1 toluene–ethyl acetate) to
3-Trifluoroacetamidopropyl 4,6-di-O-benzyl-a-
D-mannopyranosyl-(1
D-mannopyranosyl-(1
2)-3,4,6-tri-é-benzyl-a-
2)-3,4,6-tri-é-benzyl-a-
1
yield 109 mg (65%) of (XX); [α]D +0.45°; H NMR:
4.93 (1 H, br s, H1-A), 5.24 (1 H, br s, H1-B), 5.18 (1
H, br s, H1-C), 5.72 (1 H, br s, H2-C), 4.49 (H3-C),
4.35 (1 H, t, J 9.6 Hz, H4-C), 4.15 (H5-C), 3.62 (H6-C),
3.83 (1 H, dd, J6,6' 11.0 Hz, J6,5 3.2 Hz, H6'-C), 5.42 (1
H, br s, H1-E), 4.41–5.04 (16 H, a cluster of d, 8
PhCH2), 7.00–8.25 (65 H, m, arom.); 13C NMR: 99.0
(C1-A), 100.8 (C1-B), 99.8 (C1-E), 99.3 (C1-C), 72.3
(C2-C), 78.9 (C3-C), 74.5 (C4-C), 72.2 (C5-C), 68.9
(C6-C), 165.1, 165.6, and 165.8 (5 PhCO). Found, %:
D-mannopyranoside (XXIII). Compound (XXII)
(177 mg, 0.112 mmol) was debenzoylated by treatment
with 1 M MeONa (0.2 ml) in MeOH (2 ml) according
to procedure B. Column chromatography (3 : 1 tolu-
ene–ethyl acetate) resulted in 100 mg (61%) of diol
1
(XXIII); [α]D +34.7°; H NMR: 4.85 (1 H, d, J1,2 1.6
Hz, H1-A), 5.12 (1 H, d, J1,2 1.5 Hz, H1-B), 5.09 (1 H,
br s, H1-C), 3.90 (H2-C), 3.65 (H3-C), 4.24, 4.80 (16
H, a cluster of d, 8 PhCH2), 7.11–7.29 (40 H, m, arom.);
C
70.34,
H
5.83,
N
0.66. Calculated for
13C NMR: 98.7 (C1-A), 100.7 (C1-B), 101.7 (C1-C).
Found, %: C 69.28, H 6.91, N 0.83. Calculated for
C79H86O17F3N, %: C 68.83, H 6.29, N 1.02.
C120H116O27F3N, %: C 69.93, H 5.67, N 0.68.
Ethyl 4,6-di-O-benzyl-2,3-di-O-benzoyl-1-thio-
a-D-mannopyranoside (XXI). Benzoyl chloride
(0.61 ml, 5.2 mmol) was added to a solution of diol
(XVII) (352 mg, 0.87 mmol) in pyridine (4 ml). The
reaction mixture was stirred for 50 min at room temper-
ature, diluted with chloroform, and washed with a satu-
rated sodium hydrocarbonate and water. The solvent
was removed; the residue was twice coevaporated with
toluene. The product was isolated by column chroma-
tography (10 : 1 toluene–ethyl acetate) to yield 458 mg
3-Trifluoroacetamidopropyl 2,3,4,6-tetra-O-ben-
zoyl-a-D-mannopyranosyl-(1
3)-[2,3,4,6-tetra-
é-benzoyl-a-D-mannopyranosyl-(1
O-benzyl-a-D-mannopyranosyl-(1
é-benzyl-a-D-mannopyranosyl-(1
2)]-4,6-di-
2)-3,4,6-tri-
2)-3,4,6-O-
benzyl-a-D-mannopyranoside (XXIV). Diol (XXIII)
(85 mg, 0.058 mmol) was glycosylated with bromide
(XV) (114 mg, 0.174 mmol) in dichloromethane (4 ml)
in the presence of molecular sieves 4Å (350 mg) and
AgOTf (53 mg, 0.21 mmol) according to procedure B.
1
(86%) of dibenzoate (XXI); [α]D –16.6°; H NMR:
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 33 No. 1 2007

SYNTHESIS OF OLIGOSACCHARIDE FRAGMENTS
119
The product was isolated by column chromatography
3-Aminopropyl a-D-mannopyranosyl-(1
3)-
(10 : 1 toluene–ethyl acetate) to yield 126 mg (85%) of a-D-mannopyranosyl-(1
2)-a-D-mannopyrano-
pentasaccaride (XXIV); [α]D +5.4°; 1H NMR: 4.96 (1
H, br s, H1-A), 5.18 (1 H, br s, H1-B), 5.61 (1 H, br s,
H1-C), 5.07 (1 H, br s, H1-D), 5.88 (1 H, br s, H2-D),
6.12 (1 H, dd, J2,3 2.5 Hz, J3,4 10.3 Hz, H3-D), 6.31 (1
H, t, J4,5 10.3 Hz, H4-D), 4.97 (H5-D), 4.42 and 4.70 (2
H6-D), 5.47 (1 H, br s, H1-E), 6.07 (1 H, br s, H2-E),
6.26 (1 H, dd, J2,3 2.3 Hz, J3,4 10.4 Hz, H3-E), 6.40 (1
H, t, J4,5 10.0 Hz, H4-E), 4.80 (H5-E), 4.75 (2 H6-E),
4.37–4.93 (16 H, a cluster of d, 8 PhCH2), 6.80–8.20
(80 H, m, arom.); 13C NMR: 99.0 (C1-A), 100.8 (C1-
B), 99.4 (C1-C), 98.2 (C1-D), 70.8 (C2-D), 70.4 (C3-
D), 66.4 (C4-D), 69.4 (C5-D), 62.7 (C6-D), 100.6 (C1-
E), 70.7 (C2-E), 70.9 (C3-E), 66.3 (C4-E), 69.9 (C5-E),
62.6 (C6-E). Found, %: C 69.50, H 5.52, N 0.55. Cal-
culated for C147H138O35F3N, %: C 69.63; H 5.49; N
0.55.
syl-(1
2)-a-D-mannopyranoside (II). Derivative
(XX) was deprotected according to procedure D to give
free tetrasaccharide (II) in 90% yield; [α]D = +57.2°;
1H NMR: 5.26 (1 H, s, H1-B), 5.12 (1 H, s, H1-E), 5.07
(1 H, s, H1-A), 5.01 (1 H, s, H1-C), 4.20 (1 H, br s, H2-
C), 4.09 (1 H, br s, H2-B), 4.05 (1 H, br s, H2-E), 3.93
(H2-A), 3.92 (H3-B, H3-C), 3.87 (H3-A), 3.86 (H3-E),
3.73 (H4-C), 3.66 (H4-A), 3.64 (H4-B), 3.62 (H4-E),
3.72–3.79 (H5-B, H5-C, H5-E), 3.58 (H5-A), 3.69–
3.77 (H6-A, H6-B, H6-C, H6-E), 3.85–3.92 (H6'-A,
H6'-B, H6'-C, H6'-E), 3.82 and 3.57 (CH2O), 3.10 (2 H,
m, CH2N), 1.97 (2 H, m, CH2CH2CH2); 13C NMR: 103.3
(×2) (C1-C, C1-E), 101.9 (C1-B), 99.4 (C1-A), 80.0
(C2-A), 79.7 (C2-B), 79.1 (C3-C), 74.5 (×3) (C5-B, C5-
C, C5-E), 74.1 (C5-A), 71.5, 71.3, and 71.2 (×2) (C2-E,
C3-A, C3-B, C3-E), 70.8 (C2-C), 68.3, 68.1 (×2)
(C4-A, C4-B, C4-E), 67.4 (C4-C), 62.4, 62.3, and 62.2
(×2) (C6-A, C6-B, C6-C, C6-E), 66.2 (CH2O), 38.6
(CH2N), 27.8 (CH2CH2CH2).
A general procedure for removal of protective
groups (procedure D). Palladium hydroxide on carbon
(20%, Aldrich) in the amount equal to the mass of oli-
gomannoside to be deprotected was added to the solu-
tion of the protected oligosaccharide in methanol. A
mixture was stirred for 16 h in the atmosphere of hydro-
gen at room temperature and then filtered through a
Celite layer, the catalyst was carefully washed with
methanol, and the combined filtrates were concen-
trated. The residue was dissolved in water and treated
with anion exchange resin Amberlyst A-26 (éç–)
(Fluka) for 16 h. Then the resin was filtered off, and the
filtrate was concentrated. Free oligosaccharides (I)–
(III) were isolated by gel chromatography on a TSK
HW-40 (S) column in the form of acetates and were
lyophilized from water.
3-Aminopropyl a-D-mannopyranosyl-(1
2)-
[a-D-mannopyranosyl-(1
syl-(1
3)]-a-D-mannopyrano-
2)-a-D-man-
2)-a-D-mannopyranosyl-(1
nopyranoside (III) was obtained from the protected
pentasaccaride (XXIV) by procedure D; yield 85%;
[α]D +58.8°; 1H NMR: 5.25 (1 H, s, H1-C), 5.23 (1 H,
s, H1-B), 5.15 (1 H, s, H1-E), 5.10 (1 H, s, H1-D), 5.06
(1 H, s, H1-A), 4.22 (1 H, br s, H2-C), 4.07 (1 H, br s,
H2-B), 4.03 (1 H, br s, H2-E), 3.98 (1 H, br s, H2-D),
3.92 (H2-A), 4.02 (H3-C), 3.90 (H3-B), 3.85 (H3-A),
3.82 (H3-D), 3.73 (H3-E), 3.77 (H4-C), 3.60–3.67 (H4-
A, H4-B, H4-D, H4-E), 3.71–3.77 (H5-B, H5-C, H5-
D), 3.65 (H5-E), 3.56 (H5-A), 3.68–3.76 (H6-A, H6-B,
H6-C, H6-D, H6-E), 3.82–3.89 (H6'-A, H6'-B, H6'-C,
H6'-D, H6'-E), 3.82 and 3.56 (CH2O), 3.09 (2 H, m,
3-Aminopropyl a-D-mannopyranosyl-(1
a-D-mannopyranosyl-(1 2)-a-D-mannopyrano-
syl-(1 2)-a-D-mannopyranoside (I) was obtained
2)-
CH2N), 1.95 (2 H, m, CH2CH2CH2); 13C NMR: 103.3
(C1-E), 102.8 (C1-D), 101.8 (C1-B), 101.7 (C1-C),
99.3 (C1-A), 80.1 (C2-A), 79.8 (C2-B), 78.5 (C2-C),
78.3 (C3-C), 74.8, 74.6, 74.5, 74.4 (C5-B, C5-C, C5-D,
C5-E), 74.0 (C5-A), 71.7, 71.5, 71.3 (×2), and 71.1 (×2)
(C2-D, C2-E, C3-A, C3-B, C3-D, C3-E), 68.3, 68.1,
and 67.9 (×3) (C4-A, C4-B, C4-C, C4-D, C4-E), 62.4
and 62.2 (×2), and 62.1 (×2) (C6-A, C6-B, C6-C, C6-
D, C6-E), 66.2 (CH2O), 38.6 (CH2N), 27.8
(CH2CH2CH2).
from the protected tetrasaccharide (XVI) according to
procedure D in 87% yield; [α]D +74.3°; 1H NMR: 5.30
and 5.27 (2 H, 2 c, H1-B, H1-C), 5.09 (1 H, c, H1-A),
5.04 (1 H, c, H1-D), 4.10 and 4.09 (2 H, 2 br s, H2-B,
H2-C), 4.07 (1 H, 2 br s, H2-D), 3.95 (H2-A), 3.94 (H3-
B, H3-C), 3.89 (H3-C), 3.84 (H3-D), 3.62–3.70 (H4-A,
H4-B, H4-C, H4-D), 3.73–3.79 (H5-B, H5-C, H5-D),
3.61 (H5-A), 3.72–3.79 (H6-A, H6-B, H6-C, H6-D),
3.86–3.92 (H6'-A, H6'-B, H6'-C, H6'-D), 3.85 and 3.59
(CH2O), 3.12 (2 H, m, CH2N), 1.97 (2 H, m,
3-Aminopropyl a-D-mannopyranosyl-(1
2)-
a-D-mannopyranoside (XXV)[bold] was obtained
from disaccharide (VIII) by procedure D; yield 95%;
[α]D = +53°; 1H NMR: 5.10 (1 H, s, H1-A), 5.02 (1 H,
s, H1-B), 4.07 (1 H, br s, H2-B), 3.98 (1 H, br s, H2-A),
3.90 (H3-A), 3.84 (H3-B), 3.68 (H4-A), 3.61 (H4-B),
3.77 (H5-B), 3.62 (H5-A), 3.70–3.79 (H6-A, H6-B),
3.88–3.92 (H6'-A, H6'-A), 3.61, 3.85 (CH2O), 3.13 (2
CH2CH2CH2); 13C NMR: 103.4 (C1-D), 101.8 (×2)
(C1-B, C1-C), 99.4 (C1-A), 80.1, 79.9, and 79.7 (C2-A,
C2-B, C2-C), 74.5 and 74.4 (×2), 74.1 (C5-A, C5-B,
C5-C, C5-D), 71.5 (C3-D), 71.4 and 71.2 (×3) (C2-D,
C3-A, C3-B, C3-C), 68.4, 68.3, 68.2, and 68.0 (C4-A,
C4-B, C4-C, C4-D), 62.2, 62.3, and 62.4 (×2) (C6-A,
C6-B, C6-C, C6-D), 66.2 (CH2O), 38.7 (CH2N), 27.8
(CH2CH2CH2).
13
H, m, CH2N), 1.99 (2 H, m, CH2CH2CH2); C NMR:
103.5 (C1-B), 99.4 (C1-A), 79.9 (C2-A), 74.5 (C5-B),
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 33 No. 1 2007

120
KARELIN et al.
1
74.1 (C5-A), 71.5 (C3-B), 71.4 (C3-A), 71.1 (C2-B),
68.8 (×2) (C4-A, C4-B), 62.2 and 62.4 (H6-A, H6-B),
66.2 (CH2O), 38.7 (CH2N), 27.8 (CH2CH2CH2).
9.3 mg (84%) of adduct (XXIX), [α]D +45.0°. H
NMR: 1.44 (3 H, m, éëç2ëç3), 1.92 (2 H, m,
éëç2ëç2ëç2N), 3.52 and 3.73 (2 H, 2 m,
OCH2CH2Cç2N), 3.57 and 3.81 (2 H, 2 m,
OCç2CH2CH2N), 4.71 and 4.75 (2 H, 2 q, J 7.0 Hz,
OCç2CH3), 5.02 and 5.04 (1 H, 2 s, H1-A); the carbo-
hydrate part of the spectrum was practically the same as
that in the spectrum of the starting 3-aminopropyl gly-
coside (III), except for signal H1-A; 13C NMR: 16.4
and 16.5 (1 C, OCH2CH3), 30.5 and 30.7 (1 C,
OCH2CH2CH2N), 43.0 and 43.2 (1 C, OCH2CH2CH2N),
66.2 and 66.3 (1 C, OCH (1 C, OCH2CH2CH2N), 71.9
and 72.0 (1 C, OCH2CH3), 99.5 and 99.6 (1 C, C1-A),
174.7, 178.3, 184.5, and 190.3 (cyclobutenedione); the
carbohydrate part of the spectrum was practically the
same as that in the spectrum of the starting 3-aminopro-
pyl glycoside (III), except for signal C1-A.
3-(3,4-Dioxo-2-ethoxycyclobut-1-enylamino)pro-
pyl a-D-mannopyranosyl-(1
2)-a-D-mannopyr-
anoside (XXVII). Diethyl squarate (XXVI) (4 µl,
0.027 mmol) and triethylamine (3 µl) were added to a
solution of disaccharide (XXV) (10 mg, 0.022 mmol)
in 50% aqueous ethanol (0.8 ml); the mixture was kept
for 16 h at room temperature and then concentrated in
a vacuum. The residue was dissolved in water and
applied on a Sep-Pak C-18 cartridge. The cartridge was
washed with water (10 ml); then the product was eluted
with aqueous methanol in 2-ml portions, increasing the
concentration of methanol from 5 to 20%. Concentra-
tion of the eluate and the subsequent lyophilization
1
resulted in 13 mg (90%) of monoamide (XXVII); H
NMR: 1.43 (3 H, m, éëç2ëç3), 1.92 (2 H, m,
BSA–pentasaccaride conjugate (XXX). A solu-
tion of derivative (XXIX) (4.5 mg, 4.5 µmol) and BSA
(15.2 mg) in 3 ml of the buffer solution (pH 9) was kept
for 2 days at room temperature. The conjugate was iso-
lated by gel chromatography on a Sephadex G-15 col-
umn in water and lyophilized to give 9 mg (46%) of
conjugate (XXX). For MALDI TOF mass spectrum see
Fig. 2.
éëç2ëç2ëç2N), 3.60, 3.72 (2 H,
OCH2CH2CH2N), 3.56, 3.81 (2 H,
2
2
m,
m,
OCH2CH2CH2N), 4.70 and 4.74 (2 H, 2 q, J 7.2 Hz,
OCH2CH3), 5.04 and 5.06 (1 H, 2 s, H1-A). The carbo-
hydrate part of the spectrum was practically the same as
that in the spectrum of the starting 3-aminopropyl gly-
coside (XXV), except for the signal of H1-A; 13C
NMR: 16.0 and 16.3 (1 C, OCH2CH3), 30.3 and 30.4 (1
C, OCH2CH2CH2N), 43.1 and 43.2 (1 C,
OCH2CH2CH2N), 66.1 and 66.2 (1 C, OCH2CH2CH2N),
71.9 (1 C, OCH2CH3), 174.7 (cyclobutenedione); the
carbohydrate part of the spectrum was practically the
same as that in the spectrum of the starting 3-aminopro-
pyl glycoside (XXV).
ACKNOWLEDGMENTS
We are grateful to A.A. Grachev for the registration
of NMR spectra and to I.J. Toropygin for the registra-
tion of MALDITOF mass spectra.
The work was supported by the Russian Foundation
for Basic Research (project no. 05-03-08107).
BSA–disaccharide conjugate (XXVIII). A solu-
tion of monoamide (XXVII) (4 mg, 7.6 µmol) and BSA
(25 mg, 0.37 µmol) in 6 ml of the buffer solution
(250 ml of water, 8.8 g of KHCO3, 6.7 g of Na2B4O7 ·
10H2O, pH 9) was kept for 3 days at room temperature.
The conjugate was isolated by gel chromatography on
a Sephadex G-15 column in water and lyophilized from
water to give 17 mg (59%) of conjugate (XXVIII). For
MALDI TOF mass spectrum see Fig. 2.
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