Synthesis of oligosaccharide fragments of the Streptococcus pneumoniae type 14 capsular polysaccharide and their neoglycoconjugates with bovine serum albumin

Article abstract of DOI:10.1007/s11172-014-0462-5

2-Aminoethyl glycosides of tetra-, hexa- and octasaccharide fragments of the bacteria Streptococcus pneumoniae type 14 capsular polysaccharide were synthesized within a project directed to the development of pneumococcal conjugated vaccine based on synthetic carbohydrate ligands. Squarate method was used to obtain neoglycoconjugates of the synthesized oligosaccharides with bovine serum albumin.

Full text of DOI:10.1007/s11172-014-0462-5

Russian Chemical Bulletin, International Edition, Vol. 63, No. 2, pp. 511—521, February, 2014
511
Synthesis of oligosaccharide fragments of the Streptococcus pneumoniae
type 14 capsular polysaccharide and their neoglycoconjugates
with bovine serum albumin*
E. V. Sukhova,a D. V. Yashunsky,a,b Yu. E. Tsvetkov,a E. A. Kurbatova,c and N. E. Nifantieva
aN. 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
bV. N. Orekhovich Research Institute of Biomedical Chemistry, Russian Academy of Medical Sciences,
Str. 8, 10 ul. Pogodinskaya, 119121 Moscow, Russian Federation.
cI. I. Mechnikov Research Institute of Vaccines and Sera, Russian Academy of Medical Sciences,
5a Mal Kazennyi per., 105064 Moscow, Russian Federation
2ꢀAminoethyl glycosides of tetraꢀ, hexaꢀ and octasaccharide fragments of the bacteria
Streptococcus pneumoniae type 14 capsular polysaccharide were synthesized within a project
directed to the development of pneumococcal conjugated vaccine based on synthetic carboꢀ
hydrate ligands. Squarate method was used to obtain neoglycoconjugates of the synthesized
oligosaccharides with bovine serum albumin.
Key words: Streptococcus pneumoniae type 14, capsular polysaccharide, oligosaccharide
fragments, synthesis, neoglycoconjugates.
Bacteria Streptococcus pneumoniae cause many danꢀ
gerous infectious diseases such as pneumonia, otitis, menꢀ
ingitis, etc., and are one of the major reasons of morbidity
and mortality among children, elderly people, and immuꢀ
nocompromised patients.1—5 S. pneumoniae are gramꢀposꢀ
itive bacteria, whose cells are surrounded by a polysacchaꢀ
ride capsule. Based on the structure of the capsular
polysaccharide, bacteria S. pneumoniae are divided to more
than 90 serotypes.6 Some 20 serotypes from this number,
including type 14, are associated with more than 80%
pneumococcal diseases worldwide.7 Since the resistance
of the disease causative agents to antibiotics gradually inꢀ
creases, importance of vaccination in the battle with pneuꢀ
mococcal infections is also growing. Vaccines currently
licensed for clinical use are based on capsular polysacchaꢀ
rides. Among them are a 23ꢀvalent polysaccharide vaccine
and conjugated vaccines of various valence; all of them
include polysaccharide S. pneumoniae type 14. While
polysaccharides cause the Tꢀindependent immune
response,8 which results in the low efficiency of polyꢀ
saccharide vaccines in the vaccination of infants under
the age of two years, elderly people, and immunocomproꢀ
mised patients,9 the use of conjugated vaccines, in which
polysaccharide molecules are covalently bound to the proꢀ
tein carrier, makes it possible to induce the Tꢀdepenꢀ
dent immune response to the carbohydrate part of the
conjugate.10
The use of native capsular polysaccharides for the prepꢀ
aration of conjugated vaccines has a number of disadvanꢀ
tages related to the difficulties of culturing bacteria and
isolation, purification, and standardization of polysacchaꢀ
rides. The use of synthetic fragments of capsular polysacꢀ
charides of strictly definite structure as the carbohydrate
ligands for conjugated vaccines is free of these disadvanꢀ
tages. In the last years, this approach is under active deꢀ
velopment in many research laboratories dealing with the
studies in the field of directed oligosaccharide synthesis.
In particular, chemical and enzymatic methods were used
to synthesize a large group of oligosaccharide fragments of
the S. pneumoniae type 14 capsular polysaccharide (see
Refs 11—16), their conjugates with protein carrier CRM197
(recombinant diphtheria toxoid) were obtained, and their
immunogenicity and protecting activity against this strain
of S. pneumoniae were studied using animal models.17,18
As a result of these studies, the polysaccharide structural
elements, whose presence in the oligosaccharide is necesꢀ
sary for the corresponding conjugate to exhibit protective
activity, were identified.
To develop approaches to the design of Russian conjuꢀ
gated pneumococcal vaccines based on synthetic oligoꢀ
saccharide ligands, in the present work we describe synꢀ
thesis of three oligosaccharide fragments of the S. pneuꢀ
moniae type 14 capsular polysaccharide as 2ꢀaminoethyl
* On the occasion of the 80th anniversary of the N. D. Zelinsky
Institute of Organic Chemistry of the Russian Academy of Sciences.
Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 2, pp. 0511—0521, February, 2014.
1066ꢀ5285/14/6302ꢀ0511 © 2014 Springer Science+Business Media, Inc.

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Sukhova et al.
glycosides and their conjugates with the model protein
carrier, bovine serum albumin (BSA).
The S. pneumoniae type 14 capsular polysaccharide is
built of repeating tetrasaccharide units 1 (see Ref. 19).
Tetrasaccharide 2, hexasaccharide 3, and octasaccharꢀ
ide 4 have been chosen as the target structures.
Repeating unit 1, in turn, consists of lactose (Galꢀꢀ
(14)ꢀGlcꢀꢀ) and lactosamine (Galꢀꢀ(14)ꢀGlcNAcꢀꢀ)
disaccharide blocks bound by the (13´)ꢀglycoside linkage.
In this connection, lactose and lactosamine derivatives 7,
11, 12, and 16, whose synthesis is given in Scheme 1, were
used for the assembly of the target oligosaccharides.
Scheme 1
Phth is phthaloyl.
Reagents, conditions, and yields: i. BzCl, pyridine, 92% yield for 6, 46% yield for 14; ii. 90% aqueous TFA, CHCl3, 83% yield;
iii. TMSOTf, CH2Cl2, MS AWꢀ300, 63% yield; iv. Ac2O, pyridine, 86% yield; v. 40% aqueous HF, CH3CN, then Ac2O, pyridine, 75%
yield; vi. PdCl2, AcONa, 95% aqueous AcOH, then Me2C(OMe)2, camphorsulfonic acid; 71% yield; vii. CCl3CN, DBU, CH2Cl2,
69% yield.

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Russ.Chem.Bull., Int.Ed., Vol. 63, No. 2, February, 2014
513
The known isopropylidene derivative of 2ꢀazidoethyl
lactoside 5 (see Ref. 20) was benzoylated with the formaꢀ
tion of pentabenzoate 6, in which the acetal group was
removed by acid hydrolysis to obtain diol 7. The disaccharꢀ
ide block 7 was used as a glycosyl acceptor, the precursor
of the 2ꢀaminoethyl lactoside fragment on the reducing
end of hexaꢀ and octasaccharide. Lactosamine donor
block 11 was obtained by regioselective 4ꢀglycosylation of
3,4ꢀdiol 9 (see Ref. 21) with imidate 8 (see Ref. 22) with
subsequent acetylation of the hydroxy group at C(3) in
derivative 10. The silyl protecting group at O(6) in comꢀ
pound 10 can be selectively removed, that gives the opporꢀ
tunity to further glycosylate this position. Desilylation of
derivative 10 and acetylation of the 3,6ꢀdiol formed lead
to peracetate 12, which was used as a lactosamine donor
block in the final step of glycosylation in the synthesis of
octasaccharide. We planned to use imidate 16 as the lacꢀ
tose donor block. It is known23,24 that it can be prepared
with the use of a reaction sequence similar to that given in
Scheme 1. In the cited works, Oꢀtexyldimethylsilyl group23
or pꢀtolylthio group24 were used as a temporary protection
for the anomeric position in the glucose moiety, while we
proceeded from allyl lactoside 13 (see Ref. 25). Its benzoꢀ
ylation and subsequent removal of the anomeric allyl group
in pentabenzoate 14 upon treatment with palladium chlorꢀ
ide lead to 1ꢀOH derivative 15. The reaction of compound
15 with trichloroacetonitrile in the presence of DBU gave
rise to the target imidate 16. After glycosylation with imidꢀ
ate 16, the 3´,4´ꢀOꢀisopropylidene protecting group in the
reaction product can be easily removed, that makes it posꢀ
sible to further elongate the oligosaccharide chain at posiꢀ
tion 3 of the galactose moiety.
Having available disaccharide blocks 7, 11, 12, and
16, we proceeded with assembly of hexaꢀ and octaꢀ
saccharides, which have a lactose block on the reducꢀ
ing end. Glycosylation of diol 7 with thioglycoside 11 in
the presence of Nꢀiodosuccinimide (NIS) and trifluoroꢀ
methanesulfonic acid (TfOH) regioselectively took place
at position 3 of the galactose moiety, but was acꢀ
companied by partial acid desilylation, resulting in the
formation of a mixture of tetrasaccharides 17 and 18
(Scheme 2).
The reaction mixture without separation was subjected
to desilylation upon treatment with aqueous HF in acetoꢀ
Scheme 2
Reagents, conditions, and yields: i. NIS, TfOH, CH2Cl2, MS 4 Å, –25÷–10 C, 60% yield for 17, 80% yield for 21; ii. 40% aqueous HF,
MeCN; iii. 16, TMSOTf, CH2Cl2, MS AWꢀ300, –30÷0 C (89%); iv. 90% aqueous TFA, 87% yield.

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Sukhova et al.
nitrile, and the glycosylation product was isolated as diol
18 in 76% yield. Glycosylation of the latter with imidate
16 in the presence of trimethylsilyl triflate proceeded exꢀ
clusively at the primary hydroxy group and led to hexasacꢀ
charide 19 in 89% yield. The isopropylidene group in comꢀ
pound 19 was removed by mild acid hydrolysis to obtain
triol 20, which was further converted to the target free
hexasaccharide (see below), as well as was used as a glycoꢀ
syl acceptor in the synthesis of octasaccharide. The final
glycosylation of derivative 20 with thioglycoside 12 was
also selective at position 3 of the terminal galactose moiꢀ
ety and led to protected octasaccharide 21.
Unlike oligosaccharides 3 and 4, tetrasaccharide 2 conꢀ
tains Nꢀacetylglucosamine moiety on the reducing end, to
which 2ꢀaminoethyl aglycone should be added. We used
a known 2ꢀazidoethyl glycoside 22 as a precursor of the
aminosugar (see Refs 26 and 27) (Scheme 3).
which resulted in the preparation of disaccharide 23 in
good yield. The free hydroxy group in compound 23 was
acetylated to give compound 24, in which the silyl proꢀ
tecting group was removed to yield the required glycosyl
acceptor 25. The final glycosylation of compound 25 with
lactose imidate 16 smoothly led to the target tetrasacꢀ
charide 26.
The transformation of protected 2ꢀazidoethyl glycoꢀ
sides 20, 21, and 26 to the unprotected 2ꢀaminoethyl
glycosides was carried out as follows (Scheme 4). In the
first step, all the Oꢀacyl and Nꢀphthaloyl groups were reꢀ
moved upon treatment with hydrazine hydrate in refluxing
ethanol (in the case of tetrasaccharide 26, this was preꢀ
ceded by the removal of the isopropylidene group by acid
hydrolysis). The unsubstituted oligosaccharides formed
were further subjected to the exhaustive O,Nꢀacetylaꢀ
tion upon treatment with Ac2O in pyridine with subsequent
Oꢀdeacetylation with sodium methoxide in methanol.
In the final step, the azide group in the aglycone was
reduced to the amino group by catalytic hydrogenation
on a palladium catalyst. The structures of obtained
oligosaccharides 27—29 were confirmed by 1H and
13C NMR spectra. The 1H NMR spectra of comꢀ
pounds 27, 28, and 29 exhibited in the region  4.5—5.0,
respectively, six, eight, and four signals for the anoꢀ
meric protons as doublets with the spinꢀspin coupling
constants J1,2 = 7—8 Hz, that confirmed the ꢀconfigꢀ
uration of all the monosaccharide moieties in the oligoꢀ
saccharides. The 13C NMR spectra exhibited a required
number of signals for the anomeric carbon atoms in the
region  103—105, that also confirmed the number of
monosaccharide units and their ꢀconfiguration in oligoꢀ
saccharides 27—29. Besides, characteristic signals for the
2ꢀaminoethyl group were present in the 1H and 13C NMR
spectra.
Scheme 3
Conjugation of oligosaccharides 27—29 with bovine
serum albumin (BSA) was carried out by squarate methꢀ
od28—31 (Scheme 5), based on the use of diethyl squarate 30
as a linking agent. In the first step, the reaction of the
amino group of aglycone of oligosaccharides 27—29 with
30 at pH 7.0 led to squaric acid monoamides 31—33,
which in the next step reacted with the amino groups of
the BSA lysine moieties at pH 9.0 with the formation of
conjugates 34—36. The number of oligosaccharide ligands
(m) in the conjugates obtained was determined based
on the MALDI TOF mass spectrometric data and was
found to be 15, 9, and 11 for conjugates 34, 35, and 36,
respectively.
Oligosaccharides 27—29 and their conjugates with
BSA 34—36 described in the present work, as well
as synthetic polysaccharide S. pneumoniae type 14
(see Ref. 32) and lactoꢀseries oligosaccharides33,34 obꢀ
tained earlier are currently used in the immunoꢀ
logical studies, the results of which will be publishꢀ
ed later.aaa
Reagents, conditions, and yields: i. TMSOTf, CH2Cl2, MS 4 Å,
–30—0 C, 78% yield for 23, 80% yield for 26; ii. Ac2O, pyrꢀ
idine, 93% yield; iii. 40% aqueous HF, CH3CN, 89% yield.
Glycosylation of diol 22 with trichloroacetimidate 8
was also selective (cf. glycosylation of 9) at position 4,

Synthetic fragments of the capsular polysaccharide
Russ.Chem.Bull., Int.Ed., Vol. 63, No. 2, February, 2014
515
Scheme 4
Reagents, conditions, and yields: a. N2H4H2O, ethanol—water (1 : 1), 100 C; b. Ac2O, pyridine; c. MeONa, MeOH; d. H2, PdO/C,
H2O, 76% yield for 27, 58% yield for 28, 55% yield for 29; e. 90% aqueous TFA.
hydrate derivatives. For 1H NMR spectra, residual nondeuteratꢀ
ed CHCl3 (H 7.27) was used as a reference, for 13C NMR specꢀ
Experimental
tra, CDCl3 ( 77.0). Spectra of unprotected oligosaccharides
C
were recorded in heavy water (D2O); acetone was used as an
All the reactions were carried out in solvents purified acꢀ
cording to the standard procedures; TfOH, TMSOTf, DBU,
NIS (Acros), TBDMSCl (Fluka) were used without additional
purification. Thinꢀlayer chromatography was carried out on
plates with Kieselgel 60 F254 silica gel (Merck), compounds
were visualized by spraying with a solution of orcinol (180 mg)
internal standard ( 2.225,  31.45). Signals were assigned
H
C
using 2D correlation spectroscopy COSY, TOCSY, ROESY, and
HSQC. For the NMR spectra of compounds 15, 18—21, 26—29
only characteristic signals are given.
High resolution mass spectra were recorded on a Bruker
micrOTOF II instrument with electrospray ionization (ESI).
Glycosylation reactions were carried out in anhydrous solꢀ
vents under dry argon. Before carrying out reactions, molecular
sieves were activated for two hours at 180 C in vacuo of
an oil pump.
2ꢀAzidoethyl (2,6ꢀdiꢀOꢀbenzoylꢀꢀDꢀgalactopyranosyl)ꢀ(14)ꢀ
2,3,6ꢀtriꢀOꢀbenzoylꢀꢀDꢀglucopyranoside (7). Benzoyl chloride
(400 L, 3.44 mmol) was added dropwise to a solution of
2ꢀazidoethyl lactoside 5 (195 mg, 0.43 mmol) (see Ref. 20) in
pyridine (4 mL) at 4 C, and the reaction mixture was allowed to
stand for 2 h at this temperature and for 16 h at room temperaꢀ
ture. Excess of benzoyl chloride was neutralized with water
(0.3 mL), the mixture was diluted with chloroform (20 mL),
washed with 1 M H2SO4 (3×20 mL), water, and saturated aqueous
NaHCO3 (3×10 mL). The organic layer was separated and conꢀ
in
a mixture of water (85 mL), orthophosphoric acid
(10 mL), and ethanol (5 mL)) with subsequent heating at ~150 C.
Column chromatography was performed on Silica gel 60
(40—63 m, Merck), gelꢀchromatography of free oligosacchaꢀ
rides was carried out on a column with a TSK HWꢀ40(S) gel
(1.5×90 cm) in 0.1 M acetic acid, gelꢀchromatography of conjuꢀ
gates on a column with Sephadex Gꢀ15 (3×60 cm) in water;
eluates were analyzed using a Knauer Kꢀ2401 flowꢀthrough reꢀ
fractometer. Optical rotation was measured on a PUꢀ07 digital
polarimeter (Russia) at room temperature in chloroform in the
case of protected derivative (c 1) or in water in the case of free
oligosaccharides.
NMR spectra were recorded on Bruker AMꢀ300, Bruker
AMXꢀ400, Bruker DRXꢀ500, and Bruker Avance 600 at 25 C in
deuterochloroform (CDCl3) in the case of protected carboꢀ

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Sukhova et al.
Scheme 5
Reagents and conditions: i. 30, Et3N, ethanol—water (1 : 1); ii. BSA, borate buffer (pH 9).
centrated. A 90% aqueous trifluoroacetic acid (0.7 mL) was addꢀ
ed to a stirred solution of disaccharide 6 (385 mg, 0.40 mmol) in
chloroform (10 mL) with cooling. The reaction mixture was
stirred for 1.5 h, then diluted with chloroform, washed with
saturated aqueous NaHCO3, and concentrated. Compound 7
(306 mg, 83%) was isolated by column chromatography (toluꢀ
ene—acetone, 20 : 3) as a white foam, []D +36. 1H NMR
(400 MHz, CDCl3), : 8.02—7.70, 7.61—7.00 (both m, 25 H, 5 Ph);
5.69 (t, 1 H, HA(3), J = 9.4 Hz); 5.34 (dd, 1 H, HA(2), J2,3 = 9.6 Hz);
glucopyranoside (10). Molecular sieves AWꢀ300 (300 mg) were
added to a solution of 3,4ꢀdiol 9 (129 mg, 0.26 mmol) (see
Ref. 21) and trichloroacetimidate 8 (177 mg, 0.36 mmol) (see
Ref. 22) in anhydrous dichloromethane (3 mL), and the mixture
was stirred for 30 min at 20 C under dry argon. Then, the mixꢀ
ture was cooled to –30 C, followed by addition of TMSOTf
(20 L, 0.39 mmol) and stirring for 3 h, gradually elevating
temperature to 0 C. Then, the reaction mixture was neutralized
with Et3N (500 L), diluted with dichloromethane, and filtered
through a celite layer. The filtrate was washed with saturated
aqueous NaHCO3 (2×20 mL) and concentrated. Compound 10
(132 mg, 63%) was isolated by column chromatography (light
petroleum—ethyl acetate, 2 : 1) as a white foam, []D +82.
1H NMR (400 MHz, CDCl3), : 7.87—7.63 (m, 4 H, Phth); 5.33
(d, 1 H, HB(4), J3,4 = 3.3 Hz); 5.28 (d, 1 H, HA(1), J1,2 = 10.4 Hz);
5.20 (t, 1 H, HB(2), J = 8.0 Hz); 4.96 (dd, 1 H, HB(3), J2,3 = 10.4 Hz,
J3,4 = 3.3 Hz); 4.60 (d, 1 H, HB(1), J1,2 = 8.0 Hz); 4.41 (t, 1 H,
HA(3), J = 8.9 Hz); 4.15 (t, 1 H, HA(2), J = 10.4 Hz); 4.08—4.00
(m, 2 H, HB(6a), HB(6b)); 3.95 (br.t, 1 H, HB(5) J = 7.0 Hz);
3.85 (dd, 1 H, HA(6a), J6a,5 = 1.2 Hz, J6a,6b = 11.6 Hz); 3.73 (dd,
1 H, HA(6b), J6b,5 = 3.5 Hz); 3.65 (t, 1 H, HA(4), J = 8.9 Hz);
3.50 (m, 1 H, HA(5)); 2.70—2.52 (m, 2 H, SCH2CH3); 1.19 (t, 3 H,
SCH2CH3, J = 7.4 Hz); 0.91 (s, 9 H, (CH3)3CSi); 0.11, 0.08
(both s, 6 H, SiCH3). 13C NMR (100 MHz), : 170.4, 170.1,
170.0, 169.2 (CH3CO), 134.1, 123.6—123.1 (Ph), 101.7 (CB(1)),
82.0 (CA(4)), 81.7 (CA(1)), 80.7 (CA(5)), 71.3 (CB(5)), 70.9
5.20 (dd, 1 H, HB(2), J2,3 = 9.6 Hz); 4.63 (d, 1 H, HA(1), J1,2
=
= 7.8 Hz); 4.54 (d, 1 H, HB(1), J1,2 = 8.3 Hz); 4.52 (dd, 1 H,
HB(6a), J5,6a = 1.7 Hz); 4.43 (dd, 1 H, HB(6b), J5,6b = 4.6 Hz,
J6a,6b = 12.1 Hz); 4.08 (t, 1 H, HA(4), J = 9.3 Hz); 4.00—3.90
(dd, 1 H, HB(6a), J5,6a = 6.4 Hz, J6a,6b = 11.3 Hz); 3.83 (m, 1 H,
OCH2CH2N); 3.77 (m, 1 H, HA(5)); 3.72 (d, 1 H, HB(4)); 3.61
(dd, 1 H, HB(3), J3,4 = 3.5 Hz); 3.57—3.49 (m, 2 H, HB(6b),
OCH2CH2N); 3.41 (br.t, 1 H, HB(5)); 3.28, 3.15 (both m, 2 H,
OCH2CH2N). 13C NMR (100 MHz), : 166.6, 166.2, 166.0,
165.9, 165.3 (PhCO), 133.2, 129.6, 128.4 (Ph), 100.9 (CA(1)),
(CB(1)), 75.9 (CA(4)), 73.7 (CB(2)), 73.1 (CA(5)), 72.9 (CB(3)),
72.5 (CB(3)), (CB(5)), 71.5 (CA(2)), 68.4 (OCH2CH2N, CB(4)),
62.4 (CA(6)), 61.8 (CB(6)), 50.5 (OCH2CH2N). Found: m/z
954.2695 [M + Na]+. C49H45N3NaO16. Calculated: M + Na =
= 954.2698.
Ethyl (2,3,4,6ꢀtetraꢀOꢀacetylꢀꢀDꢀgalactopyranosyl)ꢀ(14)ꢀ
6ꢀOꢀ(tertꢀbutyldimethylsilyl)ꢀ2ꢀdeoxyꢀ2ꢀphthalimidoꢀ1ꢀthioꢀꢀDꢀ

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Russ.Chem.Bull., Int.Ed., Vol. 63, No. 2, February, 2014
517
(CB(3)), 70.6 (CA(3)), 68.9 (CB(2)), 66.9 (CB(4)), 61.7 (CA(6)),
61.5 (CB(6)), 55.3 (CA(2)), 25.9 (3 C, (CH3)3C), 23.7 (SCH2CH3),
20.7, 20.6, 20.5, 20.3 (4 C, O(O)CCH3), 17.6 ((CH3)3C), 14.9
(SCH2CH3), –4.8, –5.1 (2 C, SiCH3). Found: m/z 820.2641
[M + Na]+. C36H51NNaO15SSi. Calculated: M + Na = 820.2642.
Ethyl (2,3,4,6ꢀtetraꢀOꢀacetylꢀꢀDꢀgalactopyranosyl)ꢀ(14)ꢀ
3ꢀOꢀacetylꢀ6ꢀOꢀ(tertꢀbutyldimethylsilyl)ꢀ2ꢀdeoxyꢀ2ꢀphthalimidoꢀ
1ꢀthioꢀꢀDꢀglucopyranoside (11). Monohydroxy derivative 10
(130 mg, 0.16 mmol) was acetylated with acetic anhydride
(800 L) in pyridine (1.5 mL) at room temperature for 12 h.
Then, the mixture was concentrated, the residue was dissolved
in toluene, and the solvent was evaporated (3×5 mL). Comꢀ
pound 11 (118 mg, 86%) was isolated by column chromatograꢀ
phy (light petroleum—ethyl acetate, 4 : 1) as a foam, []D +64.
1H NMR (400 MHz, CDCl3), : 7.87—7.64 (m, 4 H, Phth); 5.70
(dd, 1 H, HA(3), J3,4 = 9.2 Hz); 5.40 (d, 1 H, HA(1), J1,2 = 10.5 Hz);
5.30 (d, 1 H, HB(4)); 5.05 (dd, 1 H, HB(2), J2,3 = 10.4 Hz); 4.91
(dd, 1 H, HB(3), J3,4 = 3.4 Hz); 4.70 (d, 1 H, HB(1), J1,2 = 8.0 Hz);
4.21 (t, 1 H, HA(2), J = 10.4 Hz); 4.06 (m, 2 H, HB(6a), HB(6b));
Allyl (2,6ꢀdiꢀOꢀbenzoylꢀ3,4ꢀOꢀisopropylideneꢀꢀDꢀgalactoꢀ
pyranosyl)ꢀ(14)ꢀ2,3,6ꢀtriꢀOꢀbenzoylꢀꢀDꢀglucopyranoside (14).
Benzoyl chloride (5 mL, 43.20 mmol) was added dropwise to
a solution of allyl lactoside 13 (2.40 g, 5.68 mmol) (see Ref. 25) in
pyridine (15 mL) at 4 C, and the reaction mixture was allowed
to stand for 2 h at this temperature and for 16 h at room temperꢀ
ature. Methanol (1 mL) was added, the mixture was diluted with
chloroform (100 mL), washed with 1 M aqueous H2SO4 (3×50 mL),
water, and saturated aqueous NaHCO3 (3×50 mL). The organic
layer was separated and concentrated. Compound 14 (2.50 g, 46%)
was isolated by column chromatography (light petroleum—ethyl
acetate, 2 : 1), []D +54. 1H NMR (400 MHz, CDCl3),
: 8.12—7.84, 7.60—7.11, (both m, 25 H, 5 Ph); 5.65 (m, 1 H,
OCH2CH=CH2); 5.64 (t, 1 H, HA(3), J = 9.3 Hz); 5.36 (dd, 1 H,
HA(2), J2,3
OCH2CH=CH2); 4.63 (d, 1 H, HA(1), J1,2 = 7.8 Hz); 4.52 (m, 2 H,
HB(1), HA(6a)); 4.39 (dd, 1 H, HA(6b) J6b,5 = 4.5 Hz, J6b,6a
= 9.6 Hz); 5.10—4.93 (m, 3 H, HB(2),
=
= 12.1 Hz); 4.21—4.10 (m, 4 H, HB(6a), HA(4), HB(4), HB(3));
4.00—3.90 (m, 2 H, OCH2CH=CH2); 3.73 (m, 2 H, HA(5),
HB(5)); 3.60 (dd, 1 H, HB(6b), J6b,5 = 7.5 Hz, J6b,6a = 11.4 Hz); 1.41,
1.15 (both s, 6 H, C(CH3)2). 13C NMR (100 MHz), : 166.1—164.9
(PhCO), 133.4—133.0, 130.6—128.2 (OCH2CH=CH2, Ph), 117.7
(OCH2CH=CH2), 100.3 (CB(1)), 99.8 (CA(1)), 77.2 (CB(3)),
75.5 (CA(4)), 73.8 (CB(2)), 73.2, (CB(4)), 73.1 (CB(5)), 72.8 (CA(3)),
72,0 (CA(2)), 71.5 (CA(5)), 70.1 (OCH2CH=CH2), 62.9 (CB(6)),
62.7 (CA(6)), 27.4, 26.2 (both s, C(CH3)2). Found: m/z 965.2993
[M + Na]+. C53H50NaO16. Calculated: M + Na = 965.2997.
(2,6ꢀDiꢀOꢀbenzoylꢀ3,4ꢀOꢀisopropylideneꢀꢀDꢀgalactopyranoꢀ
syl)ꢀ(14)ꢀ2,3,6ꢀtriꢀOꢀbenzoylꢀDꢀglucopyranose (15). A suspenꢀ
sion of allyl glycoside 14 (985 mg, 1.04 mmol), palladium chlorꢀ
ide (210 mg, 1.18 mmol), and sodium acetate (206 mg, 2.52 mmol)
in 95% aqueous acetic acid (30 mL) was heated for 3 h at 70 C.
The reaction mixture was diluted with chloroform (20 mL), filꢀ
tered through celite, and the filtrate was concentrated. The resiꢀ
due was dissolved in toluene, the solvent was evaporated (3×10 mL).
Column chromatography (toluene—acetone, 20 : 8) was used to
isolate a mixture containing a hydrolysis product of the isoꢀ
propylidene group (900 mg). 2,2ꢀDimethoxypropane (19 mL,
0.15 mol) and camphorsulfonic acid (50 mg, 0.21 mmol) were
added to a solution of this mixture in acetone (5 mL). After 30 min,
the mixture was neutralized with Et3N and concentrated. Colꢀ
umn chromatography (light petroleum—ethyl acetate, 2 : 1) was
used to isolate hemiacetal 15 (670 mg, 71%) as an anomeric
mixture with the ratio  :  = 2.8 : 1. 1H NMR (400 MHz,
CDCl3), : 6.00 (t, 1 H, HA(3), J = 9.6 Hz); 5.68 (t, 0.4 H,
J = 9.6 Hz, HA(3)); 5.51 (d, 1 H, HA(1), J1,2 = 3.8 Hz); 5.18 (dd,
0.4 H, HA(2)), J2,3 = 9.8 Hz); 5.12 (dd, 1 H, HA(2), J2,3 =9.3 Hz);
4.82 (t, 0.4 H, HA(1), J1,2 = 8.0 Hz); 4.63 (d, 0.4 H, HB(1),
J1,2 = 8.0 Hz); 4.61 (d, 1 H, HB(1), J1,2 = 7.6 Hz); 1.50, 1.19
(both s, 6 H, C(CH3)2). 13C NMR (100 MHz), : 100.3 (CB(1)),
100.2 (CB(1)), 95.6 (CA(1)), 90.3 (CA(1)), 27.4, 26.3 (both s,
C(CH3)2). Found: m/z 925.2679 [M + Na]+. C50H46NaO16. Calꢀ
culated: M + Na = 925.2684.
3.96 (t, 1 H, HA(4), J = 9.4 Hz); 3.92 (dd, 1 H, HA(6a), J6a,5
=
= 1.2 Hz); 3.84 (dd, 1 H, HA(6b), J6b,5 = 3.0 Hz, J6a,6b = 11.8 Hz);
3.80 (br.t, 1 H, HB(5), J = 6.8 Hz); 3.50 (m, 1 H, HA(5));
2.69—2.51 (m, 2 H, SCH2CH3); 2.08, 2.04, 1.98, 1.91, 1.85 (all
s, 15 H, 5 CH3C(O)O); 1.15 (t, 3 H, SCH2CH3, J = 7.4 Hz);
0.92 (s, 9 H, (CH3)3CSi), 0.12, 0.10 (both s, 6 H, SiCH3). 13C NMR
(100 MHz), : 170.3, 170.2, 170.1, 170.0, 168.8, (CH3CO), 134.3,
123.6—123.1 (Ph), 100.4 (CB(1)), 80.4 (CA(1)), 79.5 (CA(5)),
74.9 (CA(4)), 71.8 (CA(3)), 71.2 (CB(3)), 70.7 (CB(5)), 69.3
(CB(2)), 67.0 (CB(4)), 61.2 (CA(6)), 61.1 (CB(6)), 54.1 (CA(2)),
25.9 (3C, (CH3)3C), 23.6 (SCH2CH3), 21.0, 20.7, 20.6, 20.5,
20.3 (5 C, O(O)CCH3), 18.3 ((CH3)3C), 14.2 (SCH2CH3),
–4.8, –5.1 (2 C, SiCH3). Found: m/z 862.2747 [M + Na]+.
C38H53NNaO16SSi. Calculated: M + Na = 862.2746.
Ethyl (2,3,4,6ꢀtetraꢀOꢀacetylꢀꢀDꢀgalactopyranosyl)ꢀ(14)ꢀ
3,6ꢀdiꢀOꢀacetylꢀ2ꢀdeoxyꢀ2ꢀphthalimidoꢀ1ꢀthioꢀꢀDꢀglucopyranoꢀ
side (12). A 40% aqueous HF (100 L) was added to a solution of
monohydroxy derivative 10 (288 mg, 0.36 mmol) in acetonitrile
(2 mL). The reaction mixture was allowed to stand for 20 min at
room temperature, diluted with dichloromethane, washed with
saturated aqueous NaHCO3 (2×20 mL), and concentrated. The
dihydroxy derivative obtained was acetylated with acetic anhyꢀ
dride (1 mL) in pyridine (2 mL) at room temperature for 12 h.
The mixture was concentrated, the residue was dissolved in toluꢀ
ene, and the solvent was evaporated (3×5 mL). Compound 12
(208 mg, 75%) was isolated by column chromatography (light
petroleum—ethyl acetate, 2 : 1), []D +39. 1H NMR (500 MHz,
CDCl3), : 7.09—7.64 (m, 4 H, Phth); 5.77 (t, 1 H, HA(3),
J = 10.4 Hz); 5.47 (d, 1 H, HA(1), J1,2 = 10.6 Hz); 5.32 (d, 1 H,
HB(4), J3,4 = 3.2 Hz); 5.11 (t, 1 H, HB(2), J = 9.6 Hz); 4.95 (dd, 1 H,
HB(3), J2,3 = 9.6 Hz); 4.53 (d, 1 H, HB(1), J1,2 = 8.4 Hz); 4.50
(m, 1 H, HB(6a)); 4.26 (t, 1 H, HA(2), J = 10.4 Hz); 4.13 (m, 3 H,
HB(6b)); 4.11—4.00 (m, 2 H, HA(6a), HA(6b)); 3.89—3.77 (m, 3 H,
HA(4), HA(5), HB(5)); 2.69—2.57 (m, 2 H, SCH2CH3); 2.12, 2.05,
2.02, 1.95, 1.88 (all s, 18 H, 6 CH3C(O)O); 1.19 (t, 3 H, SCH2CH3,
J = 7.5 Hz). 13C NMR (125 MHz), : 170.1, 169.9—169.0, 167.7
(CH3CO), 134.3, 123.6—123.1 (Ph), 101.0 (CB(1)), 81.4 (CA(1)),
77.0 (CA(5)), 76.9 (CA(4)), 71.9 (CA(3)), 71.0 (CB(3)), 70.6
(CB(5)), 69.1 (CB(2)), 66.5 (CB(4)), 62.5 (CB(6)), 60.7 (CA(6)),
54.0 (CA(2)), 24.5 (SCH2CH3), 20.7, 20.6, 20.5, 20.4 (6 C,
O(O)CCH3), 14.2 (SCH2CH3). Found: m/z 790.1987 [M + Na]+.
C34H41NNaO17S. Calculated: M + Na = 790.1993.
(2,6ꢀDiꢀOꢀbenzoylꢀ3,4ꢀOꢀisopropylideneꢀꢀDꢀgalactopyranoꢀ
syl)ꢀ(14)ꢀ(2,3,6ꢀtriꢀOꢀbenzoylꢀꢀDꢀglucopyranosyl)trichloroꢀ
acetimidate (16). Trichloroacetonitrile (745 L, 7.42 mmol) and
DBU (33 L, 0.22 mmol) were added to a solution of hemiacetal 15
(640 mg, 0.74 mmol) in anhydrous dichloromethane (10 mL)
under dry argon at –30 C. The temperature of the reaction
mixture was elevated to 20 C within 2 h. The reaction mixture
was applied on a column with silica gel and subjected to

518
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Sukhova et al.
chromatography in the system light petroleum—ethyl acetate
(2 : 1) with addition of Et3N (1%, v/v) to give trichloroacetimiꢀ
date 16 (536 mg, 69%), []D +84. 1H NMR (500 MHz, CDCl3),
: 8.56 (s, 1 H, OC(NH)CCl3); 8.12—7.94, 7.68—7.04 (both m,
25 H, Ph); 6.71 (d, 1 H, HA(1), J1,2 = 3.8 Hz); 6.11 (t, 1 H,
HA(3), J = 9.3 Hz); 5.51 (dd, 1 H, HA(2), J2,3 = 9,3 Hz); 5.18
(t, 1 H, HB(2), J = 7.0 Hz); 4.72 (d, 1 H, HB(1), J1,2 = 7.5 Hz); 4.61
(3ꢀOꢀacetylꢀ2ꢀdeoxyꢀ2ꢀphthalimidoꢀꢀDꢀglucopyranosyl)ꢀ(13)ꢀ
(2,6ꢀdiꢀOꢀbenzoylꢀꢀDꢀgalactopyranosyl)ꢀ(14)ꢀ2,3,6ꢀtriꢀOꢀ
benzoylꢀꢀDꢀglucopyranoside (19). Molecular sieves AWꢀ300
(200 mg) were added to a solution of diol 18 (92 mg, 0.06 mmol)
and trichloroacetimidate 16 (91 mg, 0.09 mmol) in anhydrous
dichloromethane (2 mL), and the mixture was stirred for 30 min
at 20 C under dry argon. After cooling the mixture to –30 C,
TMSOTf (5 L, 0.03 mmol) was added. The mixture was stirred
for 3 h, gradually elevating temperature to 0 C. The reaction
mixture was neutralized with Et3N (500 L), diluted with
dichloromethane, and filtered through a celite layer. The filtrate
was washed with saturated aqueous NaHCO3 (2×20 mL) and
concentrated. Column chromatography of the residue (light
petroleum—ethyl acetate, 2 : 1) gave compound 19 (127 mg,
89%) as a syrup, []D +13. 1H NMR (400 MHz, CDCl3),
: 8.07—7.01 (m, 54 H, Phth, Ph); 5.74 (t, 1 H, HA(3), J = 9.6 Hz);
5.63 (t, 1 H, HE(3), J = 9.2 Hz); 5.49 (t, 1 H, HC(3), J = 8.6 Hz);
5.45 (d, 1 H, (HC(1), J1,2 = 8.4 Hz); 5.20 (t, 1 H, HB(2), J = 8.7 Hz);
5.19 (d, 1 H, HD(4)); 4.96 (dd, 1 H, HD(2), J2,3 = 10.4 Hz); 4.90
(dd, 1 H, HD(3), J3,4 = 3.4 Hz); 4.81 (d, 1 H, HC(1), J1,2 = 7.9 Hz);
4.69, 4.65 (both d, 2 H, HA(1), J1,2 = 6.7 Hz, HE(1), J1,2 = 7.7 Hz);
(d, 1 H, HA(6a), J6a,6b = 11.9 Hz); 4.55 (dd, 1 H, HA(6b), J6b,5
=
= 3.2 Hz); 4.36—4.28 (m, 4 H, HA(5), HA(4), HB(3), HB(6a));
4.14 (d, 1 H, HB(4), J3,4 = 3.1 Hz); 3.83 (m, 2 H, HB(5), HB(6b));
1.50, 1.29 (both s, 6 H, C(CH3)2). 13C NMR (125 MHz), : 165.9,
165.7, 165.5, 165.3, 165.0 (PhCO), 133.7—133.0, 129.3—128.2
(Ph), 100.8 (CB(1)), 93.1 (CA(1)), 77.1 (CB(3)), 75.1 (CA(4)),
73.7 (CB(3)), 72.9 (CB(4)), 71.3 (CA(5)), 70.7 (CA(2)), 70.1
(CA(3)), 62.8 (CB(6)), 62.1 (CA(6)), 27.2, 26.0 (both s, C(CH3)2).
Found: m/z 1068.1774 [M + Na]+. C52H46Cl3NNaO16. Calcuꢀ
lated: M + Na = 1068.1780.
2ꢀAzidoethyl (2,3,4,6ꢀtetraꢀOꢀacetylꢀꢀDꢀgalactopyranosyl)ꢀ
(14)ꢀ3ꢀOꢀacetylꢀ2ꢀdeoxyꢀ2ꢀphthalimidoꢀꢀDꢀglucopyranosylꢀ
(13)ꢀ(2,6ꢀdiꢀOꢀbenzoylꢀꢀDꢀgalactopyranosyl)ꢀ(14)ꢀ2,3,6ꢀ
triꢀOꢀbenzoylꢀꢀDꢀglucopyranoside (18). Molecular sieves 4 Å
(800 mg) were added to a solution of acceptor 7 (260 mg,
0.31 mmol) and thioglycoside 11 (262 mg, 0.28 mmol) in anꢀ
hydrous CH2Cl2 (10 mL), the mixture was stirred for 30 min and
cooled to –20 C, NIS (93 mg, 0.41 mmol) was added. After 10 min,
the temperature was decreased to –30 C, and TfOH (32 L,
0.37 mmol) was added. The mixture was stirred for 45 min,
keeping temperature within the range from –25 to –10 C, then
neutralized with pyridine (10 L), diluted with chloroform, and
filtered through a celite layer. The filtrate was washed with a 1 M
aqueous Na2S2O3 (60 mL) and saturated aqueous NaHCO3
(60 mL). The organic layer was concentrated, and the residue
was subjected to column chromatography (light petroleum—ethyl
acetate, 2 : 3) to give a mixture of 17 and diol 18 (500 mg).
A 40% aqueous HF (100 L) was added to the solution of this
mixture in acetonitrile (3 mL), the solution was allowed to stand
for 20 min at room temperature, diluted with dichloromethane,
washed with saturated aqueous NaHCO3 (2×20 mL), and conꢀ
centrated. Compound 18 (269 mg, 60%) was isolated by column
chromatography of the residue (gradient elution with light peꢀ
troleum—ethyl acetate 2 : 1  ethyl acetate), []D +48. 1H NMR
(400 MHz, CDCl3), : 8.01—7.82, 7.63—7.00 (both m, 29 H,
Phth, Ph); 5.60 (t, 1 H, HA(3), J = 9.3 Hz); 5.53 (t, 1 H, HC(3),
J = 9.5 Hz); 5.49 (d, 1 H, HC(1), J1,2 = 8.5 Hz); 5.32 (dd, 1 H,
HA(2), J2,3 = 9.6 Hz); 5.30 (d, 1 H, HD(4)); 5.23 (dd, 1 H,
HB(2), J2,3 = 9.8 Hz); 5.05 (dd, 1 H, HD(2), J2,3 = 10.4 Hz); 4.98
(dd, 1 H, HD(3), J3,4= 3.4 Hz); 4.62 (d, 1 H, HA(1), J1,2 = 7.7 Hz);
4.43 (d, 1 H, HD(1), J1,2 = 7.7 Hz); 4.39 (d, 1 H, HB(1), J1,2
=
= 8.7 Hz); 3.32, 3.19 (both m, 2 H, OCH2CH2N); 2.06, 1.99, 1.95,
1.83 1.72 (all s, 15 H, 5 O(O)CCH3); 1.57, 1.31 (both s, 6 H,
C(CH3)2). 13C NMR (100 MHz), : 170.1, 169.7, 169.6, 168.7
(CH3CO), 165.9, 165.8, 165.4, 165.3, 165.1, 165.0, 163.9
(PhCO), 133.6, 129.6—128.3 (Ph), 101.2 (CA(1)), 100.8 (both s,
C(1)E, CC(1)), 100.5 (CB(1)), 100.3 (CD(1)), 98.3 (CC(3)), 63.2,
62.7, 62.4, 62.2, 60.7 (CA(6), CB(6), CD(6), C(6)E, CC(6)), 54.5
(CC(2)), 50.4 (OCH2CH2N), 27.3, 26.0 (both s, C(CH3)2), 20.5,
20.4, 20.3 (5 C, O(O)CCH3). Found: m/z 2501.7172 [M + Na]+.
C129H122N4NaO47. Calculated: M + Na = 2501.7177.
2ꢀAzidoethyl (2,6ꢀdiꢀOꢀbenzoylꢀꢀDꢀgalactopyranosyl)ꢀ(14)ꢀ
2,3,6ꢀtriꢀOꢀbenzoylꢀꢀDꢀglucopyranosylꢀ(16)ꢀ[2,3,4,6ꢀtetraꢀ
OꢀacetylꢀꢀDꢀgalactopyranosylꢀ(14)]ꢀ(3ꢀOꢀacetylꢀ2ꢀdeoxyꢀ2ꢀ
phthalimidoꢀꢀDꢀglucopyranosyl)ꢀ(13)ꢀ(2,6ꢀdiꢀOꢀbenzoylꢀꢀDꢀ
galactopyranosyl)ꢀ(14)ꢀ2,3,6ꢀtriꢀOꢀbenzoylꢀꢀDꢀglucopyranoꢀ
side (20). A 90% aqueous trifluoroacetic acid (0.1 mL) was addꢀ
ed to a solution of hexasaccharide 19 (28 mg, 0.01 mmol) in
anhydrous dichloromethane (2 mL) using ice for cooling. The
mixture was allowed to stand for 20 min at room temperature
and concentrated. The residue was dissolved in toluene and the
solvent was evaporated (3×5 mL). Compound 20 (23 mg, 87%)
was isolated by column chromatography (toluene—acetone,
20 : 8), []D +42. 1H NMR (600 MHz, CDCl3), : 8.10—7.00
(m, 54 H, Phth, Ph); 5.47 (d, 1 H, H(1), J1,2 = 8.3 Hz); 4.85
(d, 1 H, H(1), J1,2 = 7.9 Hz); 4.71 (d, 1 H, H(1), J1,2 = 6.8 Hz); 4.65
(d, 1 H, H(1), J1,2 = 7.6 Hz); 4.43 (d, 1 H, H(1), J1,2 = 7.6 Hz);
4.39 (d, 1 H, H(1), J1,2 = 7.9 Hz); 3.33, 3.19 (both m, 2 H,
OCH2CH2N); 3.16 (br.s, 1 H, OH); 2.91 (s, 1 H, OH); 2.86
(br.s, 1 H, OH); 2.08, 2.00, 1.95, 1.85, 1.73 (all s, 15 H,
5 O(O)CCH3). 13C NMR (150 MHz, : 170.2, 170.1, 169.8, 169.6,
168.5 (5 CH3CO), 166.6, 166.3, 166.1, 165.9 (2 C), 165.5, 165.4,
165.2, 165.1, 164.0 (10 PhCO), 101.2 (2 C), 100.8, 100.5, 100.4,
98.2 (6 C(1)), 63.5, 62.5, 62.3, 61.4, 60.6 (5 C(6)), 54.5 (C(2)GlcN),
50.5 (OCH2CH2N), 20.6 (3 C), 20.5, 20.3 (5 O(O)CCH3).
Found: m/z 2456.7305 [M + NH4]+. C126H122N5O47. Calculatꢀ
ed: M + NH4 = 2456.7310.
4.60 (d, 1 H, HD(1), J1,2 = 7.8 Hz); 4.49 (d, 1 H, HB(1), J1,2
=
= 7.9 Hz); 4.38 (dd, 1 H, HB(6a), J6a,6b = 12.1 Hz, J6a,5 = 2.0 Hz);
4.25 (dd, 1 H, HB(6b), J6b,5 = 4.7 Hz); 3.19, 3.07 (both m, 2 H,
OCH2CH2N); 3.15 (br.s, 1 H, OHB(4)); 2.60 (br.t, 1 H, OHC(6));
2.00, 1.95, 1.90, 1.87, 1.69 (all s; 15 H, 5 O(O)CCH3). 13C NMR
(100 MHz), : 170.1, 170.1, 169.9, 169.0, 169.4 (CH3CO), 166.1,
165.9, 165.5, 165.3, 164.2 (PhCO), 133.7, 129.6—128.1 (Ph),
101.0 (CD(1)), 100.7 (CA(1)), 100.6 (CB(1)), 98.8 (CC(1)), 81.3
(CB(3)), 62.5, 62.2, 60.8, 60.3 (CA(6), CB(6), CC(6), CD(6)), 54.5
(CC(2)), 50.5 (OCH2CH2N), 20.6, 20.5, 20.3 (5 C, O(O)CCH3).
Found: m/z 1617.4491 [M + Na]+. C79H78N4NaO32. Calculatꢀ
ed: M + Na = 1617.4497.
2ꢀAzidoethyl (2,3,4,6ꢀtetraꢀOꢀacetylꢀꢀDꢀgalactopyranosyl)ꢀ
(14)ꢀ(3,6ꢀdiꢀOꢀacetylꢀ2ꢀdeoxyꢀ2ꢀphthalimidoꢀꢀDꢀglucopyrꢀ
anosyl)ꢀ(13)ꢀ(2,6ꢀdiꢀOꢀbenzoylꢀꢀDꢀgalactopyranosyl)ꢀ(14)ꢀ
(2,3,6ꢀtriꢀOꢀbenzoylꢀꢀDꢀglucopyranosyl)ꢀ(16)ꢀ[2,3,4,6ꢀtetraꢀ
2ꢀAzidoethyl (2,6ꢀdiꢀOꢀbenzoylꢀ3,4ꢀOꢀisopropylideneꢀꢀDꢀ
galactopyranosyl)ꢀ(14)ꢀ2,3,6ꢀtriꢀOꢀbenzoylꢀꢀDꢀglucopyranoꢀ
sylꢀ(16)ꢀ[2,3,4,6ꢀtetraꢀOꢀacetylꢀꢀDꢀgalactopyranosylꢀ(14)]ꢀ

Synthetic fragments of the capsular polysaccharide
Russ.Chem.Bull., Int.Ed., Vol. 63, No. 2, February, 2014
519
OꢀacetylꢀꢀDꢀgalactopyranosylꢀ(14)]ꢀ(3ꢀOꢀacetylꢀ2ꢀdeoxyꢀ2ꢀ
phthalimidoꢀꢀDꢀglucopyranosyl)ꢀ(13)ꢀ(2,6ꢀdiꢀOꢀbenzoylꢀꢀDꢀ
galactopyranosyl)ꢀ(14)ꢀ2,3,6ꢀtriꢀOꢀbenzoylꢀꢀDꢀglucopyranoꢀ
side (21). Molecular sieves 4 Å (300 mg) were added to a solution
of acceptor 20 (66 mg, 0.03 mmol) and thioglycoside 12 (35 mg,
0.05 mmol) in anhydrous CH2Cl2 (3 mL), the mixture was stirred
for 30 min at room temperature, then cooled to –20 C, folꢀ
lowed by addition of NIS (15 mg, 0.06 mmol). After 10 min,
the temperature was decreased to –30 C, and TfOH (6 L,
0.07 mmol) was added. The mixture was stirred for 45 min,
keeping temperature within the range from –25 to –10 C. Pyrꢀ
idine (10 L) was added, the mixture was diluted with chloroꢀ
form and filtered through a celite layer. The filtrate was washed
with a 1 M aqueous Na2S2O3 (40 mL) and saturated aqueous
NaHCO3 (30 mL). The organic layer was concentrated, and the
residue was subjected to column chromatography (gradient eluꢀ
tion with light petroleum—ethyl acetate (2 : 1)  ethyl acetate)
to give compound 21 (68 mg, 80%), []D +48. 1H NMR
(400 MHz, CDCl3), : 7.95—6.89 (m, 58 H, Phth, Ph); 5.47
(d, 1 H, H(1), J1,2 = 8.5 Hz); 5.29 (d, 1 H, H(1), J1,2 = 8.3 Hz); 4.61
(d, 1 H, H(1), J1,2 = 8.0 Hz); 4.55 (d, 1 H, H(1), J1,2 = 7.7 Hz);
4.45 (d, 1 H, H(1), J1,2 = 6.5 Hz); 4.44 (d, 1 H, H(1), J1,2 = 7.8 Hz);
4.27 (d, 1 H, H(1), J1,2 = 7.3 Hz); 4.26 (d, 1 H, H(1), J1,2 = 7.9 Hz);
3.23, 3.09 (both m, 2 H, OCH2CH2N) 2.67, 2.61 (both br.s, 2 H,
2 OH); 2.04, 1.97, 1.96, 1.94 (2 H), 1.88 (2 H), 1.83, 1.72, 1.69,
1.60 (all s, 33 H, 11 CH3C(O)O). 13C NMR (100 MHz),
: 170.5, 170.4, 170.3 (2 C), 170.2 (2 C), 169.9, 169.8, 169.7, 169.2,
168.9 (11 CH3CO), 166.1 (2 C), 165.9 (2 C), 165.7, 165.4 (2 ),
165.3, 164.4, 164.1 (10 PhCO), 101.3, 101.2 (2 C), 101.0, 100.8,
100.5, 98.5, 98.4 (8 C(1)), 63.4, 62.8, 62.7, 62.2, 61.9, 60.9, 60.8
(7 C(6)), 54.7, 54.6 (2 C(2)GlcN), 50.4 (OCH2CH2N), 21.4, 20.6
CB(6)), 56.0 (CA(2)), 50.5 (OCH2CH2N), 25.8 ((CH3)3C), 20.7,
20.6, 20.5, 20.3 (4 C, O(O)CCH3), 18.2 ((CH3)3C), –4.8, –5.1
(2 C, SiCH3). Found: m/z 845.2883 [M + Na]+. C36H50N4NaO16Si.
Calculated: M + Na = 845.2889.
2ꢀAzidoethyl (2,3,4,6ꢀtetraꢀOꢀacetylꢀꢀDꢀgalactopyranosyl)ꢀ
(14)ꢀ3ꢀOꢀacetylꢀ6ꢀOꢀ(tertꢀbutyldimethylsilyl)ꢀ2ꢀdeoxyꢀ2ꢀ
phthalimidoꢀꢀDꢀglucopyranoside (24). Monohydroxy derivative
23 (95 mg, 0.12 mmol) was acetylated with acetic anhydride
(400 L) in pyridine (1.5 mL) at room temperature for 12 h. The
mixture was concentrated, the residue was dissolved in toluene,
and the solvent was evaporated (3×5 mL). Column chromatoꢀ
graphy (toluene—acetone, 5 : 1) was used to isolate compound 24
(93 mg, 93%) as syrup, []D +15. 1H NMR (400 MHz, CDCl3),
: 7.87—7.71 (m, 4 H, Phth); 5.70 (t, 1 H, HA(3), J = 9.9 Hz);
5.42 (d, 1 H, HA(1), J1,2 = 8.5 Hz); 5.34 (d, 1 H, HB(4)); 5.08
(dd, 1 H, HB(2), J2,3 = 10.4 Hz); 4.95 (dd, 1 H, HB(3), J3,4 = 3.5 Hz);
4.73 (d, 1 H, HB(1), J1,2 = 8.0 Hz); 4.18 (dd, 1 H, HA(2), J2,3
=
= 10.7 Hz); 4.12—4.08 (m, 2 H, HB(6a), HB(6b)); 4.00—3.86
(m, 3 H, HA(6a), HA(6b), OCH2CH2N); 3.83 (br.t, 1 H, HB(5)
J = 6.7 Hz); 3.60 (m, 1 H, OCH2CH2N); 3.53 (m, 1 H, HA(5));
3.32, 3.15 (both m, 2 H, OCH2CH2N); 2.12, 2.06, 2.05 1.96,
1.89 (all s, 15 H, 5 CH3C(O)O); 0.96 (s, 9 H, (CH3)3CSi); 0.15,
0.12 (both s, 6 H, SiCH3). 13C NMR (100 MHz), : 170.2, 170.1,
169.7, 169.1 (CH3CO), 134.1, 123.4 (Ph), 100.9 (CB(1)), 98.1
(CA(1)), 75.5 (CA(4)), 74.9 (CA(5)), 71.3 (CA(3)), 71.0 (CB(3)),
70.6 (CB(5)), 69.3 (CB(2)), 68.4 (OCH2CH2N), 66.7 (CB(4)),
60.8 (CB(6)), 60.4 (CA(6)), 55.0 (CA(2)), 50.4 (OCH2CH2N),
26.0 ((CH3)3C), 20.6, 20.5, 20.3 (5 C, O(O)CCH3), 18.4 ((CH3)3C),
–4.9, –5.2 (both s, SiCH3). Found: m/z 887.2989 [M + Na]+.
C38H52N4NaO17Si. Calculated: M + Na = 887.2994.
2ꢀAzidoethyl (2,3,4,6ꢀtetraꢀOꢀacetylꢀꢀDꢀgalactopyranosyl)ꢀ
(14)ꢀ3ꢀOꢀacetylꢀ2ꢀdeoxyꢀ2ꢀphthalimidoꢀꢀDꢀglucopyranoꢀ
side (25). A 40% aqueous HF (100 L) was added to a solution of
derivative 24 (90 mg, 0.11 mmol) in acetonitrile (3 mL). The
reaction mixture was allowed to stand for 20 min at room temꢀ
perature, diluted with dichloromethane, washed with saturated
aqueous NaHCO3 (2×20 mL), and concentrated. Column
chromatography (gradient elution with light petroleum—ethyl
acetate (2 : 3)  ethyl acetate) was used to isolate compound 25
(69 mg, 89%), []D +4. 1H NMR (300 MHz, CDCl3), : 7.88—7.69,
(m, 4 H, Phth); 5.72 (dd, 1 H, HA(3), J3,4 = 8.9 Hz); 5.47 (d, 1 H,
HA(1), J1,2 = 8.5 Hz); 5.32 (d, 1 H, HB(4)); 5.10 (dd, 1 H, HB(2),
J2,3 = 10.4 Hz); 5.00 (dd, 1 H, HB(3), J3,4 = 3.4 Hz); 4.66 (d, 1 H,
HB(1), J1,2 = 7.8 Hz); 4.18 (dd, 1 H, HA(2), J2,3 = 10.7 Hz);
4.11—3.75 (m, 7 H, HB(6a), HB(6b), HA(4), HB(6a), HB(6b),
OCH2CH2N, HB(5)); 3.69—3.57 (m, 2 H, OCH2CH2N, HA(5));
3.32, 3.18 (both m, 2 H, OCH2CH2N); 2.11, 2.07, 2.05 1.97,
1.89 (all s, 15 H, 5 CH3C(O)O). 13C NMR (75 MHz), : 170.2,
170.1, 169.7, 169.1, 167.9 (CH3CO), 134.1, 123.4 (Ph), 100.9
(CB(1)), 98.1 (CA(1)), 75.5 (CA(4)), 74.9 (CA(5)), 71.3 (CA(3)),
71.0 (CB(3)), 70.6 (CB(5)), 69.3 (CB(2)), 68.4 (OCH2CH2N),
66.7(CB(4)), 60.8 (CB(6)), 60.4 (CA(6)), 55.0 (CA(2)), 50.4
(OCH2CH2N), 20.6, 20.5, 20.3 (5 C, O(O)CCH3). Found: m/z
815.2231 [M + Na]+. C34H40N4NaO18Si. Calculated: M + Na =
= 815.2235.
(7 C), 20.5, 20.4, 20.3 (11 O(O)CCH3). Found: m/z 1602.9201
2+
[M + Na + K]
= 1602.9203.
C
H153KN5NaO64. Calculated: M + K + Na =
158
2ꢀAzidoethyl (2,3,4,6ꢀtetraꢀOꢀacetylꢀꢀDꢀgalactopyranosyl)ꢀ
(14)ꢀ6ꢀOꢀ(tertꢀbutyldimethylsilyl)ꢀ2ꢀdeoxyꢀ2ꢀphthalimidoꢀꢀDꢀ
glucopyranoside (23). Molecular sieves 4 Å (200 mg) were added
to a solution of 3,4ꢀdiol 22 (109 mg, 0.22 mmol) and trichloroꢀ
acetimidate 8 (77 mg, 0.16 mmol) in anhydrous dichloromethane
(2 mL) under dry argon, and the mixture was stirred for 30 min at
20 C and cooled to –30 C. After addition of TMSOTf (5 L,
0.03 mmol), the mixture was stirred for 3 h, gradually elevating
temperature to 0 C. The reaction mixture was neutralized with
Et3N (500 L), diluted with dichloromethane, and filtered
through a celite layer. The filtrate was washed with saturated
aqueous NaHCO3 (2×20 mL) and concentrated. Column chromꢀ
atography (toluene—acetone, 5 : 1) was used to isolate 23 (100 mg,
1
78%) as a white foam, []D +4. H NMR (500 MHz, CDCl3),
: 7.82—7.68 (m, 4 H, Phth); 5.35 (d, 1 H, HB(4)); 5.28 (d, 1 H,
HA(1), J1,2 = 8.5 Hz); 5.21 (dd, 1 H, HB(2), J2,3 = 10.4 Hz); 4.97
(dd, 1 H, HB(3), J3,4 = 3.2 Hz); 4.60 (d, 1 H, HB(1), J1,2 = 8.1 Hz);
4.40 (t, 1 H, HA(3), J = 9.4 Hz); 4.10 (t, 1 H, HA(2), J = 10.4 Hz);
4.10—4.02 (m, 2 H, HB(6a), HB(6b)); 3.95 (m, 2 H, HB(5),
OCH2CH2N); 3.87 (d, 1 H, HA(6a), J6a,6b = 11.6 Hz); 3.77 (dd,
1 H, HA(6b), J6b,5 = 3.5 Hz); 3.65—3.57 (m, 2 H, HA(4),
OCH2CH2N); 3.51 (m, 1 H, HA(5)); 3.31, 3.15 (both m, 2 H,
OCH2CH2N); 2.11, 2.08, 1.96, 1.87 (all s, 12 H, 4 CH3C(O)O);
0.92 (s, 9 H, (CH3)3CSi); 0.11, 0.08 (both s, 6 H, SiCH3).
13C NMR (125 MHz), : 170.4, 170.1, 169.9, 169.0 (CH3CO),
133.9, 123.6—123.4 (Ph), 101.8 (CA(1)), 98.0 (CB(1)), 82.0
(CA(4)), 75.0 (CA(5)), 71.3 (CB(5)), 71.0 (CB(3)), 69.7 (CA(3)),
68.9 (CB(2)), 68.1 (OCH2CH2N), 66.9 (CB(4)), 61.6 (CA(6),
2ꢀAzidoethyl (2,6ꢀdiꢀOꢀbenzoylꢀ3,4ꢀOꢀisopropylideneꢀꢀDꢀ
galactopyranosyl)ꢀ(14)ꢀ2,3,6ꢀtriꢀOꢀbenzoylꢀꢀDꢀglucopyranoꢀ
sylꢀ(16)ꢀ[2,3,4,6ꢀtetraꢀOꢀacetylꢀꢀDꢀgalactopyranosylꢀ(14)]ꢀ
3ꢀOꢀacetylꢀ2ꢀdeoxyꢀ2ꢀphthalimidoꢀꢀDꢀglucopyranoside (26).
Molecular sieves AWꢀ300 (200 mg) were added to a solution of
monohydroxy derivative 25 (51 mg, 0.07 mmol) and trichloroꢀ
acetimidate 16 (107 mg, 0.1 mmol) in anhydrous dichloroꢀ

520
Russ.Chem.Bull., Int.Ed., Vol. 63, No. 2, February, 2014
Sukhova et al.
methane (2 mL) under dry argon. The mixture was stirred for
30 min at 20 C and cooled to –30 C, then TMSOTf (6 L,
0.03 mmol) was added. The mixture was stirred for 3 h, gradually
elevating temperature to 0 C. The reaction mixture was neuꢀ
tralized with Et3N (500 L), diluted with dichloromethane, and
filtered through a celite layer. The filtrate was washed with satuꢀ
rated aqueous NaHCO3 (2×20 mL) and concentrated. Column
chromatography (light petroleum—ethyl acetate, 2 : 1) was used
to isolate 26 (89 mg, 80%) as a foam, []D +54. 1H NMR
(600 MHz, CDCl3), : 8.11—7.32 (m, 29 H, Phth, Ph); 5.79 (t, 1 H,
HB(3), J = 8.7 Hz); 5.64 (dd, 1 H, HA(3), J2,3 = 10.7, J3,4 = 8.4 Hz);
5.46 (dd, 1 H, HB(2), J2,3 = 8.9 Hz); 5.31 (d, 1 H, HA(1),
J1,2 = 8.5 Hz); 5.22 (d, 1 H, HB(4), J3,4 = 3.5 Hz); 5.20 (t, 1 H,
HC(2), J = 7.4 Hz); 5.04 (dd, 1 H, HD(2), J2,3 = 10.5 Hz); 4.90
(dd, 1 H, HD(3), J3,4= 3.5 Hz); 4.89 (d, 1 H, HB(1), J1,2 =7.8 Hz);
D2O), : 176.1 (N(O)CCH3); 104.2 (2 C), 104.0, 103.9, 103.8,
103.2 (6 C(1)), 69.0 (C(6)GlcN), 67.0 (OCH2CH2N), 62.2 (3 C),
61.4, 61.3 (5 C(6)), 56.5 (C(2)GlcN), 40.7 (OCH2CH2N), 23.5
(N(O)CCH3). Found: m/z 1097.3855 [M + Na]+. C40H70N2NaO31
.
Calculated: M + Na = 1097.3860.
2ꢀAminoethyl ꢀDꢀgalactopyranosylꢀ(14)ꢀ2ꢀacetamidoꢀ2ꢀ
deoxyꢀꢀDꢀglucopyranosylꢀ(13)ꢀꢀDꢀgalactopyranosylꢀ(14)ꢀ
ꢀDꢀglucopyranosylꢀ(16)ꢀ[ꢀDꢀgalactopyranosylꢀ(14)]ꢀ2ꢀ
acetamidoꢀ2ꢀdeoxyꢀꢀDꢀglucopyranosylꢀ(13)ꢀꢀDꢀgalactopyrꢀ
anosylꢀ(14)ꢀꢀDꢀglucopyranoside (28). Octasaccharide 21
(80 mg, 25 mol) was subjected to N,Oꢀdeacylation, Nꢀacetylation,
and hydrogenation as described for derivative 20. Octasaccharꢀ
ide 28 (21 mg, 58%) was isolated after chromatography on a TSK
HWꢀ40(S) gel in 0.1 M aqueous AcOH and subsequent lyoꢀ
philization, []D +2. 1H NMR (600 MHz, D2O), : 4.742 (d, 1 H,
H(1), J1,2 = 8.3 Hz); 4.738 (d, 1 H, H(1), J1,2 = 8.2 Hz); 4.56
(d, 1 H, H(1), J1,2 = 8.1 Hz); 4.55 (d, 1 H, H(1), J1,2 = 7.8 Hz); 4.54
(d, 1 H, H(1), J1,2 = 7.6 Hz); 4.50 (d, 1 H, H(1), J1,2 = 7.9 Hz);
4.46 (d, 1 H, H(1), J1,2 = 7.9 Hz); 4.45 (d, 1 H, H(1), J1,2 = 7.8 Hz);
3.29 (t, 2 H, OCH2CH2N, J = 5.3 Hz); 2.05 (s, 6 H, 2 N(O)CCH3).
13C NMR (150 MHz, D2O), : 176.0 (N(O)CCH3), 104.1 (3 C),
104.0 (2 C), 103.9, 103.8, 103.1 (8 C(1)), 68.8 (C(6)GlcN), 67.0
(OCH2CH2N), 62.2 (5 C), 61.3, 61.1 (7 C(6)), 56.4 (2 C(2)GlcN),
40.6 (OCH2CH2N), 23.2 (2 N(O)CCH3). Found: m/z 1462.5177
[M + Na]+. C54H93N3NaO41. Calculated: M + Na = 1462.5182.
2ꢀAminoethyl ꢀDꢀgalactopyranosylꢀ(14)ꢀꢀDꢀglucopyranoꢀ
sylꢀ(16)ꢀ[ꢀDꢀgalactopyranosylꢀ(14)]ꢀ2ꢀacetamidoꢀ2ꢀdeoxyꢀ
ꢀDꢀglucopyranoside (29). A 90% aqueous trifluoroacetic acid
(0.1 mL) was added to a solution of tetrasaccharide 26 (65 mg,
0.04 mmol) in anhydrous dichloromethane (2 mL) with cooling
by ice, the mixture was allowed to stand for 20 min at room
temperature and concentrated. Excessive trifluoroacetic acid was
removed by coevaporation with toluene (3×5 mL). Column
chromatography (toluene—acetone, 20 : 8) was used to isolate
the diol (55 mg, 87%), which was subjected to N,Oꢀdeacylation,
Nꢀacetylation, and hydrogenation as described for derivative 20.
Tetrasaccharide 29 (17 mg, 55%) was obtained after chromatoꢀ
graphy on a TSK HWꢀ40(S) gel in 0.1 M aqueous AcOH and
subsequent lyophilization, []D –15. 1H NMR (600 MHz, D2O),
: 4.61 (d, 1 H, H(1), J1,2 = 8.4 Hz); 4.57 (d, 1 H, H(1),
J1,2 = 8.1 Hz); 4.55 (d, 1 H, H(1), J1,2 = 7.8 Hz); 4.47 (d, 1 H,
H(1), J1,2 = 7.8 Hz); 4.07, 3.93 (both m, 2 H, OCH2CH2N);
3.27, 3.22 (both m, 2 H, OCH2CH2N); 2.07 (s, 3 H, N(O)CCH3).
13C NMR (150 MHz, D2O), : 104.2, 104.0, 103.7, 102.3 (4 C(1)),
68.6 (C(6)GlcN), 67.1 (OCH2CH2N), 62.3 (2 C), 61.3 (3 C(6)),
56.2 (C(2)GlcN), 40.7 (OCH2CH2N), 23.8 (N(O)CCH3). Found:
m/z 773.2798 [M + Na]+. C28H50N2NaO21. Calculated: M + Na =
= 773.2804.
4.67 (d, 1 H, HC(1), J1,2 =7.7 Hz); 4.65 (dd, 1 H, HB(6a), J6a,5
=
= 2.2 Hz, J6a,6b = 12.3 Hz); 4.53 (dd, 1 H, HB(6b), J6b,5 = 4.2 Hz);
4.40 (d, 1 H, HD(1), J1,2 =7.9 Hz); 3.32 (m, 1 H, OCH2CH2N);
3.05, 2.80 (both m, 2 H, OCH2CH2N); 2.10, 2.06, 2.03, 1.95
1.84 (all s, 15 H, 5 CH3C(O)O); 1.55, 1.28 (both s, 6 H,
C(CH3)2). 13C NMR (150 MHz), : 170.2, 170.1, 169.8, 169.7,
168.9 (CH3CO), 165.9, 165.6, 165.5, 165.1, 164.9 (PhCO), 133.7,
129.6—128.1 (Ph), 100.9 (CD(1)), 100.8 (CB(1)), 100.6 (CC(1)),
97.6 (CA(1)), 62.8, 62.6, 60.8 (CB(6), CC(6), CD(6)), 54.9
(CC(2)), 50.2 (OCH2CH2N), 27.4, 26.1 (both s, C(CH3)2), 20.6,
20.5, 20.3 (5 C, O(O)CCH3). Found: m/z 1657.4804 [M + Na]+.
C82H82N4NaO32. Calculated: M + Na = 1657.4810.
2ꢀAminoethyl ꢀDꢀgalactopyranosylꢀ(14)ꢀꢀDꢀglucopyranoꢀ
sylꢀ(16)ꢀ[ꢀDꢀgalactopyranosylꢀ(14)]ꢀ2ꢀacetamidoꢀ2ꢀdeoxyꢀ
ꢀDꢀglucopyranosylꢀ(13)ꢀꢀDꢀgalactopyranosylꢀ(14)ꢀꢀDꢀ
glucopyranoside (27). Hydrazine hydrate (0.2 mL) was added to
a solution of hexasaccharide 20 (62 mg, 25 mol) in a mixture of
ethanol—water (1 : 1, 2 mL), and the mixture was refluxed for
3 h. The solvent was evaporated and the residue was dried in vacuo
at +50 C for 30 min. The product obtained was acetylated with
Ac2O (0.8 mL) in pyridine (1.5 mL) at room temperature for 12 h.
The mixture was concentrated, traces of pyridine and Ac2O were
removed by coevaporation with toluene (3×5 mL). Acetylated
oligosaccharide was isolated by column chromatography (chloroꢀ
form—methanol, 20 : 1). A 0.1 M sodium methoxide in methaꢀ
nol (200 L) was added to a solution of the latter oligosaccharide
in anhydrous methanol (2 mL). After 12 h, aqueous MeOH
(2 mL, 3 : 1) and cationite Amberlit IRꢀ120 (H+) (2 mL) were addꢀ
ed, and the mixture was allowed to stand for 20 min, the resin
was filtered off, washed with methanol (10 mL), and the filtrate
was concentrated. The hexasaccharide 2ꢀazidoethyl glycoside
(21 mg, 78%) was isolated by chromatography of the residue on
a TSK HWꢀ40(S) column in 0.1 M aqueous AcOH (mass spectroꢀ
metric data: found m/z 1123.3761 [M + Na]+; C40H68N4NaO31
;
Preparation of the conjugate of hexasaccharide with BSA 34.
Diethyl squarate 30 (1.6 L, 11 mol) and Et3N (1.5 L) were
added to a solution of hexasaccharide 27 (9.3 mg, 8.7 mol) in
50% aqueous ethanol (800 L), the mixture was allowed to stand
for 4 h at room temperature and then concentrated. The residue
was dissolved in water and applied on a SepꢀPak Cꢀ18 cartridge
(Waters, USA). The cartridge was washed with water (10 mL),
then the product was eluted with aqueous MeOH in 2ꢀmL porꢀ
tions, increasing concentration of methanol from 5 to 20%. Comꢀ
pound 31 (9 mg, 95%) was obtained by evaporation of the aqueꢀ
ous methanol eluent and subsequent lyophilization from water,
a white amorphous powder (mass spectrometric data: found m/z
1221.4015 [M + Na]+; C46H74N2NaO34; calculated M + Na =
= 1221.3982). A solution of compound 31 (9 mg, 7.5 mol) and
calculated M + Na = 1123.3765). A 10% PdO/C (20 mg) and
AcOH (5 L) were added to a solution of the product obtained in
water (1 mL), and the mixture was stirred for 3 h under hydroꢀ
gen. The catalyst was filtered off using a layer of celite and
washed with aqueous methanol (30 mL), the filtrate was conꢀ
centrated. 2ꢀAminoethyl glycoside 27 (19 mg, 98%) was obꢀ
tained by chromatography on a TSK HWꢀ40(S) column in a 0.1 M
aqueous AcOH with subsequent lyophilization, []D –9. 1H NMR
(600 MHz, D2O), : 4.74 (d, 1 H, H(1), J1,2 = 8.4 Hz); 4.56 (d, 1 H,
H(1), J1,2 = 7.9 Hz); 4.55 (d, 1 H, H(1), J1,2 = 7.7 Hz); 4.54 (d, 1 H,
H(1), J1,2 = 7.3 Hz); 4.47 (d, 1 H, H(1), J1,2 = 7.8 Hz); 4.45 (d, 1 H,
H(1), J1,2 = 7.9 Hz); 4.14, 3.97 (both H, OCH2CH2N); 3.29 (t, 2 H,
OCH2CH2N); 2.05 (s, 3 H, N(O)CCH3). 13C NMR (150 MHz,

Synthetic fragments of the capsular polysaccharide
Russ.Chem.Bull., Int.Ed., Vol. 63, No. 2, February, 2014
521
BSA (25 mg, 0.380 mol) in the buffer solution (190 L) (350 mM
KHCO3 and 70 mM Na2B4O7 10H2O, pH 9) was allowed to
stand for 48 h at room temperature. The mixture obtained was
subjected to gelꢀchromatography on a column with Sephadex
Gꢀ15 in water, conjugate 34 (34 mg, 98%) was obtained after
lyophilization as a white amorphous powder. The MALDIꢀTOF
mass spectrum exhibits a broad peak with the maximum at
m/z 84342, that corresponds to on average 15 hexasaccharide
moieties included in the conjugate.
Preparation of the conjugate of octasaccharide with BSA (35).
The reaction of octasaccharide 28 (5.0 mg, 4 mol) with diethyl
squarate 30 (0.7 L, 5 mol) in the presence of Et3N (1.0 L) in
50% aqueous ethanol (500 L) and subsequent isolation of the
product were carried out as described above for compound 31.
Compound 32 (5.4 mg, 96%) was obtained as a white amorphous
powder (mass spectrometric data: found m/z 1586.5337 [M + Na]+;
C60H97N3NaO44; calculated M + Na = 1586.5343). Conjugaꢀ
tion of 32 (5.4 mg, 4 mol) with BSA (12 mg, 0.173 mol) in the
buffer solution (190 L) and isolation of the conjugate were carꢀ
ried out as described above for 34. Conjugate 35 (17 mg, 98%)
was obtained as a white amorphous powder. The MALDIꢀTOF
mass spectrum of the conjugate exhibits a broad peak with the
maximum at m/z 80503, that corresponds to on average nine
octasaccharide moieties included in the conjugate.
8. E. C. Beuvery, F. Van Rossem, J. Nagel, Infect. Immun.,
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9. G. Ada, D. Isaacs, Clin. Microbiol. Infect., 2003, 9, 79—85.
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Preparation of the conjugate of tetrasaccharide with BSA (36).
The condensation of tetrasaccharide 29 (9.0 mg, 12 mol) with
diethyl squarate 30 (2.0 L, 15 mol) in the presence of Et3N
(1.5 L) in 50% aqueous ethanol (800 L) and isolation of the
product were carried out similarly to those described for comꢀ
pound 31. Compound 33 (10 mg, 96%) was obtained as a white
amorphous powder (mass spectrometric data: found m/z 897.2959
[M + Na]+; C34H54N2NaO24; calculated M + Na = 897.2964).
Compound 33 (10 mg, 11 mol) was conjugated with BSA (37 mg,
0.560 mol) in the buffer solution (190 L), and the conjugation
product was isolated as described for compound 34. Conjugate
36 (47 mg, 98%) was obtained as a white amorphous powder.
The MALDIꢀTOF mass spectrum exhibits a broad peak with the
maximum at m/z 75674, that corresponds to on average 11 tetraꢀ
saccharide moieties included in the conjugate.
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Received January 10, 2014