Synthesis of pentasaccharides corresponding to the glycoform II of the outer core region of the Pseudomonas aeruginosa lipopolysaccharide

Article abstract of DOI:10.1016/j.carres.2012.07.019

Cystic fibrosis (CF) is a congenital disease caused by a mutation in a gene responsible for the synthesis of a membrane protein called the cystic fibrosis transmembrane conductance regulator (CFTR). Resistance to Pseudomonas aeruginosa infection is closely related to the biological properties of CFTR; however, these properties have not been clearly linked to the known role of CFTR as a chloride and bicarbonate ion channel. Indeed, data indicate that CFTR is an epithelial cell receptor for P. aeruginosa, with CFTR binding to the oligosaccharide of the outer core region of the bacterial lipopolysaccharide (LPS), of which two distinct glycoforms have been identified. Binding leads to effective innate immunity to clear this pathogen in individuals with wild-type CFTR. To reveal the molecular basis of elimination of the bacterium through this interaction, the synthesis of pentasaccharides corresponding to both glycoforms of the outer core region of P. aeruginosa LPS was undertaken. Here we report the synthesis of the glycoform II. Like glycoform I, it was prepared as three pentasaccharides bearing naturally occurring N-alanyl and N-acetyl substituents in the galactosamine moiety as well as unnatural N-acetylalanine to reveal the role of the amino group in the alanyl substituent. Key features of the synthesis were two α-glucosylations with glucosyl donors bearing α-stereodirecting acyl groups at O-6 and/or O-3 and high-yielding reduction of the azido group followed by N-acylation and final O-debenzylation.

Full text of DOI:10.1016/j.carres.2012.07.019

Carbohydrate Research 360 (2012) 56–68
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of pentasaccharides corresponding to the glycoform II
of the outer core region of the Pseudomonas aeruginosa lipopolysaccharide
Bozhena S. Komarova a, Yury E. Tsvetkov a, Gerald B. Pier b, Nikolay E. Nifantiev a,
⇑
a Laboratory of Glycoconjugate Chemistry, N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky Prospect 47, 119991 Moscow, Russia
b Channing Laboratory, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Cystic fibrosis (CF) is a congenital disease caused by a mutation in a gene responsible for the synthesis of
a membrane protein called the cystic fibrosis transmembrane conductance regulator (CFTR). Resistance
to Pseudomonas aeruginosa infection is closely related to the biological properties of CFTR; however, these
properties have not been clearly linked to the known role of CFTR as a chloride and bicarbonate ion chan-
nel. Indeed, data indicate that CFTR is an epithelial cell receptor for P. aeruginosa, with CFTR binding to the
oligosaccharide of the outer core region of the bacterial lipopolysaccharide (LPS), of which two distinct
glycoforms have been identified. Binding leads to effective innate immunity to clear this pathogen in
individuals with wild-type CFTR. To reveal the molecular basis of elimination of the bacterium through
this interaction, the synthesis of pentasaccharides corresponding to both glycoforms of the outer core
region of P. aeruginosa LPS was undertaken. Here we report the synthesis of the glycoform II. Like glyco-
form I, it was prepared as three pentasaccharides bearing naturally occurring N-alanyl and N-acetyl sub-
stituents in the galactosamine moiety as well as unnatural N-acetylalanine to reveal the role of the amino
Received 29 June 2012
Accepted 30 July 2012
Available online 7 August 2012
Keywords:
Cystic fibrosis
Pseudomonas aeruginosa
Lipopolysaccharide
Outer core region
Pentasaccharide
Synthesis
group in the alanyl substituent. Key features of the synthesis were two
donors bearing -stereodirecting acyl groups at O-6 and/or O-3 and high-yielding reduction of the azido
group followed by N-acylation and final O-debenzylation.
a-glucosylations with glucosyl
a
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
ride that is produced as two glycoforms (Fig. 1).8,9 Both glycoforms
can be simultaneously expressed on the surface of a given strain of
P. aeruginosa.
Cystic fibrosis (CF) is a congenital disease1,2 in which numerous
primary and secondary defects, such as impairment of mucociliary
clearance of organisms in the lung, occur in mucosal tissues due to
lack of functional cystic fibrosis transmembrane conductance reg-
ulator (CFTR) protein.3 The hallmark of CF is chronic infection of
the affected lung tissues by Pseudomonas aeruginosa leading to
chronic and pathologic inflammation with eventual development
of chronic obstructive lung disease phenotype and eventually to
early death.4 In healthy individuals, the process of efficient P. aeru-
ginosa elimination from the lungs goes through a prerequisite step
of the bacterium interacting with CFTR,3,5,6 embedded in the
plasma membrane of lung cells, after which the ingestion of a
portion of the bacterial organisms by lung cells follows and leads
to efficient clearance of this organism from the lung. In CF patients,
the gene that codes CFTR is defective,7 leading to low levels of
mutant CFTR protein expression on lung cells and, hence, to low le-
vel of P. aeruginosa bound to and ingested by lung cells.
Three pentasaccharides corresponding to the glycoform I were
recently synthesized in our laboratory.10 Here we report the syn-
thesis of three pentasaccharides 1a–c (Fig. 2) that have a common
backbone corresponding to glycoform II. The compounds bear dif-
ferent N-acyl substituents on the galactosamine residue, which are
necessary to define the role of the free amino group on the alanine
substituent in the interaction of P. aeruginosa with wild type CFTR.
These pentasaccharides, together with the previously obtained
pentasaccharides corresponding to the glycoform I,10 will be used
for investigating the molecular basis leading to the failure to elim-
inate P. aeruginosa from the lungs of patients with CF.
2. Results and discussion
Like glycoform I,10 the structure of glycoform II comprises two
a-linked glucose residues and a vicinal 3,4-branching in the galac-
An epitope on the bacterial surface that is recognized by wild
type CFTR is the outer core region of the bacterial lipopolysaccha-
tosamine moiety. Accordingly, similar approaches were used for the
assembly of the oligosaccharide backbone of the glycoform II.
Highly stereoselective
a-glucosylation, which is not usually a
simple task,11,12 was achieved by application of glucosyl donors10
with a non-participating benzyl group at O-2 and acyl groups at
⇑
Corresponding author. Tel./fax: +7 499 135 8784.
E-mail address: nen@ioc.ac.ru (N.E. Nifantiev).
0008-6215/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2012.07.019

B. S. Komarova et al. / Carbohydrate Research 360 (2012) 56–68
57
Figure 1. Glycoforms of the outer core region of the P. aeruginosa6,7 lipopolysaccharide.
Figure 2. Structure of the target pentasaccharides.
O-6 and/or O-3 capable of anchimeric participation. The effect of re-
mote acyl groups on the stereoselectivity of glycosylation in gluco-
13–17
,
fuco-,18 xylo-,19 and altro-series20 was directly or indirectly
confirmed by multiple data obtained by different research groups.
While the nature of this effect is still controversial,21–23 it was suc-
cessfully applied by us for the synthesis of
-glucurono-,25 and -glucooligosaccharides.10,26 The most
a a
-fuco-,18,23–25 -xylo-,19
a
a
efficient leaving group upon glycosylation with partially O-acylated
donors26 was shown to be N-phenyltrifluoroacetimidoyl group.27
Such donors are more stable and less prone to decomposition under
glycosylation conditions than common trichloroacetimidates,
while retaining all the advantages of Schmidt’s donors.
The only applicable sequence of glycosylations to build the vic-
inal 3,4-branching in the galactosamine moiety proved to be initial
b-(1?3)-glucosylation followed by
the glycoform II also comprises an additional 3,6-branching that
includes an -Rha-(1?3)-b- -Glc fragment. In the case of the gly-
coform I, the -rhamnose unit was introduced at the last step of
the assembly of the pentasaccharide backbone.10 For glycoform II
it seemed to be more expedient to start the synthesis from an
-Rha-(1?3)-b- -Glc disaccharide to minimize manipulations of
the protecting group.
a
-(1?4)-glucosylation.26 But
a
-
a
L
D
Scheme 1. Retrosynthetic analysis of the pentasaccharide precursor.
a
-
L
D
Triacetate 14 and hemiacetal 15 were converted by standard
procedures into bromide 16 and trichloroacetimidate 17 respec-
tively. Coupling of these glycosyl donors with 2-azido-2-deoxyga-
lactoside 7 proceeded with low effectiveness; the best yield of
trisaccharide 18 (21%) was achieved with trichloroacetimidate 17
in the presence of Bu2BOTf. Furthermore, subsequent reductive
opening of the benzylidene acetal ring in 18 with NaBH3CN in
the presence of HCl was also not clear and afforded 6-O-benzyl
ether 19 in moderate yield and as a hardly separable mixture with
some unidentified contaminants. Multiple stages and modest
yields on the final steps made the above approach impractical for
the preparative synthesis of key trisaccharide structure 5.
Therefore, another approach toward 5 based on regioselective
glycosylation29–31 of 2,3-diol 20 was developed (Scheme 3).
AgOTf-promoted coupling of rhamnosyl bromide 8 with 20 pro-
ceeded with good regioselectivity and afforded disaccharide 21 in
66% yield along with a byproduct, which was likely the product
of bis-glycosylation. After conventional benzoylation of the only
hydroxyl group in 21, tetrabenzoate 22 was obtained.
Taking the above considerations into account, the retrosynthet-
ic scheme for the assembly of protected pentasaccharide 2 was
proposed (Scheme 1). Pentasaccharide 2 could be cleaved on the
a-glucoside linkages into a-glucosyl donors 3 and 4 and trisaccha-
ride 5 containing free 4-OH group in the GalN residue and a selec-
tively removable protecting group P1 at O-6 of b-glucose.
Trisaccharide 5 could be divided, in turn, into GalN acceptor 7
and disaccharide donor 6.
Two synthetic approaches toward a trisaccharide of general for-
mula 5 were explored. The first approach was based on the obvious
choice of di-isopropylideneglucose 9 as a precursor of the 3-O-
rhamnosylated b-glucose moiety (Scheme 2). Glycosylation of 9
with rhamnosyl bromide 8 gave disaccharide 10,28 which provided
5,6-diol 1128 upon mild acidic hydrolysis. The primary hydroxyl
group in 11 was selectively protected with a chloroacetyl group
(? 12), then the 1,2-O-isopropylidene group was removed to pro-
duce pyranose triol 13. Acetylation of 13 gave triacetate 14, and
subsequent anomeric deacetylation afforded hemiacetal 15. Both
latter steps needed strict control to prevent the reactive chloroace-
tyl group from substitution with pyridine or hydrazine.
The location of the benzoyl group at O-2 and, hence, the
rhamnosyl residue at O-3 was inferred from the low-field chemical
shift of the signal for H-2 (d 5.46) in the 1H NMR spectrum of 22.

58
B. S. Komarova et al. / Carbohydrate Research 360 (2012) 56–68
Scheme 2. First approach toward trisaccharide structure 5.
Scheme 3. Second approach toward trisaccharide structure 5.
Then the benzylidene acetal was subjected to the reductive open-
ing under the conditions (BH3ꢀTHF, Bu2BOTf)32 providing the
formation of a benzyl group at O-4 and a free hydroxyl group at
C-6. Conventional acetylation of resulting 23 afforded acetate 24.
The positions of signals for H-6 (d 4.54 and 4.31) and C-6 (d 62.9)
in the 1H and 13C NMR spectra of 24 confirmed the location of
the acetyl group at O-6. Thioglycoside 24 corresponds to the neces-
sary disaccharide donor of general formula 6 (Scheme 1) having
selectively removable 6-O-acetyl group. Moreover, according to
the published data,33 the presence of 4-O-benzyl group was
thought to enhance the glycosylating activity of 24 as compared
to totally acetylated counterparts 16 and 17. Indeed, NIS–TfOH-
promoted glycosylation of acceptor 7 with thioglycoside 24 affor-
ded trisaccharide 25 in 72% yield, that is, three times as high as
the best yield achieved with donors 16 or 17. Selective reductive
opening of the benzylidene ring in 25, this time under the

B. S. Komarova et al. / Carbohydrate Research 360 (2012) 56–68
59
conditions directing the benzyl group to O-6 (Me3NꢀBH3, AlCl3,
water),34 resulted in the formation of key trisaccharide 26 corre-
sponding to structure 5 (Scheme 1).
In principal, the product of 6-O-deacetylation of compound 26
could be subjected to simultaneous bis-a-glucosylation that would
directly lead to the target pentasaccharide structure. However, pos-
sible formation of a difficult to separate mixture of four stereoiso-
mers prompted us to prefer a scheme with two consecutive
4.10) groups, as well as downfield shifts of the signals for H-2 of
the GalN residue as compared to that of parent amine 31 in the
1H NMR spectra of 32 (d 3.33 ? 4.53) and 34 (d 3.33 ? 4.49)
proved their structure. Benzyl protecting groups in both 32 and
34 were efficiently removed by conventional catalytic hydrogenol-
ysis to produce N-acetylated 1a, the first of the three target penta-
saccharides, and 35 respectively. The N-Boc protection in the alanyl
moiety of 35 was quantitatively removed by treatment with neat
TFA to give the second pentasaccharide 1b. Finally, conventional
N-acetylation of 1b provided the third target compound 1c. The
homogeneity of 1a–c was ensured by HPLC purification, and their
structure was assessed by 1H and 13C NMR (see Tables 1 and 2)
and HRMS data.
a-glucosylations (Scheme 4). First glucosylation of 4-OH in the GalN
residue was carried out with N-phenyltrifluoroacetimidate 4. 6-O-
Benzoyl group in 4 is able to control the anomeric stereoselectivity;
on the other hand, it allows selective liberation of 6-OH in the b-glu-
cose unit due to stability under the conditions of acidic O-deacety-
lation.35 TMSOTf-promoted glycosylation of acceptor 26 with
imidate 4 resulted in the formation of an anomeric mixture 27 that
was subjected, without separation, to acidic O-deacetylation. Pure
To summarize, three pentasaccharide
a
-methylglycosides 1a–c
-alanyl) substitu-
bearing N-acetyl, N-( -alanyl) and N-(N-acetyl-
L
L
ents in the galactosamine residue were prepared for biological
investigations of the molecular basis of the CFTR-mediated binding
and elimination of Pseudomonas aeruginosa from the lung. A com-
bination of anomeric stereocontrol using remote acyl groups and
the N-phenyltrifluoroacetimidoyl leaving group provided glucosyl
a-anomer 28 was isolated from the resulting mixture by prepara-
tive HPLC in 76% overall yield. N-Phenyltrifluoroacetimidate 3,
which was previously shown to be the most stereoselective
a
-glucosyl donor,26 was used for final glycosylation of 28. As a re-
sult, target pentasaccharide 29 was obtained in 80% yield.
donors, which proved to be an effective tool for a-glucosylation,
After completion of the assembly of the pentasaccharide back-
bone, it was necessary to reduce the azido group, introduce various
N-acyl substituents, and remove O-acyl and benzyl protecting
groups. To this aim, protected pentasaccharide 29 was firstly
deacylated with methanolic MeONa to produce compound 30
(Scheme 5). It was shown during the synthesis of the glycoform
I10 that dithiothreitol (DTT) in the presence of di-isopropylamine
in MeCN efficiently reduced the pentasaccharide azide to the cor-
responding amine, while some other widely used methods, includ-
ing catalytic hydrogenation,36–38 did not provide good results. Here
we found that the application of aqueous MeCN as the reaction sol-
vent considerably accelerated the reduction. Reduction of azide 30
under these conditions resulted in the formation of amine 31 in
high yield. Amine 31 could be isolated and characterized; however,
direct N-acylation of crude amine proved to be more practical.
Thus, treatment of 31, obtained by simple concentration of the
reaction mixture, with Ac2O in MeOH gave acetamide 32 in 84%
overall yield.
including that of sterically and electronically demanding glycosyl
acceptors. DTT in aqueous acetonitrile in the presence of di-isopro-
pylamine provided the high-yielding transformation of the penta-
saccharide azide to the corresponding amine, a key intermediate
for final N-modification leading to three target pentasaccharides.
3. Experimental section
3.1. General experimental methods
All glycosylation reactions were carried out under dry
Ar. Molecular sieves for glycosylation reactions were crushed and
activated prior to application at 180 °C in vacuum of an oil pump
during 2 h. Acetonitrile was distilled from P2O5 and then from
CaH2 under Ar. Dichloromethane was successively distilled from
diethanolamine, P2O5, and CaH2 under Ar. For glycosylation reac-
tions, dichloromethane was freshly redistilled from CaH2. THF
was distilled from sodium benzophenone ketyl. DMF was distilled
under reduced pressure (17 mm Hg) from phthalic anhydride and
then from CaH2. Pyridine was dried by distillation from P2O5. Ana-
lytical thin-layer chromatography (TLC) was performed on Silica
Similarly, acylation of crude 31 with alanine active ester 33 pro-
vided N-alanyl derivative 34 with a yield of 91%. The presence of
signals for the acetyl (s at d 1.95) and alanyl (d at d 1.30; q at d
Scheme 4. Synthesis of protected pentasaccharide 29.

60
B. S. Komarova et al. / Carbohydrate Research 360 (2012) 56–68
Scheme 5. Synthesis of pentasaccharides 1a–c.
Gel 60 F254 aluminium sheets (Merck), and visualization was
accomplished using UV light or by charring at ꢁ150 °C with 10%
(v/v) H3PO4 in ethanol, Mostain reagent (ceric sulfate (1% w/v)
and ammonium molybdate (2.5% w/v) in 10% (v/v) aqueous
H2SO4) or a solution of ninhydrin (300 mg) in a mixture of n-buta-
nol (100 mL) and AcOH (3 mL). Column chromatography was per-
mixture was stirred for 20 min, filtered through a Celite layer, the
filtrate was diluted with dichloromethane, washed with a mixture
of satd aq NaHCO3 and 1 M Na2S2O3 (1:1), and then with water.
The organic layer was separated, concentrated, and the residue
was subjected to column chromatography (toluene–EtOAc, 17:1)
to provide disaccharide 10 (2.10 g, 77%) as an amorphous solid:
Rf 0.6 (toluene–EtOAc, 7:2). 1H NMR (500 MHz, CDCl3, for the sig-
nals of sugar ring protons see Table 1): d 8.15–7.15 (m, 15H, Ar),
1.55 (s, 3H, C–CH3), 1.45 (s, 6H, 2 C–CH3), 1.37 (s, 3H, C–CH3). 13C
NMR (125 MHz, CDCl3, for the signals of sugar ring carbons see Ta-
ble 2): d 165.8, 165.5 (C@O), 133.6–125.3 (Ar), 112.0, 109.2
(C(CH3)2), 26.8, 26.1, 25.3 (C(CH3)2). ESI HRMS: [M+Na]+ calcd for
formed on Silica Gel 60, 40–63 lm (Merck). Optical rotation values
were measured using a JASCO DIP-360 polarimeter at the ambient
temperature in solvents specified. 1H and 13C NMR spectra were re-
corded on Bruker AM-300, Bruker AMX-400, Bruker DRX-500, and
Bruker Avance spectrometers. 1H NMR chemical shifts were refer-
enced to residual signal of CHCl3 (dH 7.27) or CH3OH (dH 3.33). 13
C
chemical shifts were referenced to the central resonance of CDCl3
(dC 77.0) and CD3OD (dC 49.0). Signal assignment in 1H and 13C
NMR spectra was made using COSY, TOCSY, and 1H–13C HSQC tech-
niques. High-resolution mass spectra were acquired by electro-
spray ionization on a MicrOTOF II (Bruker Daltonics) instrument.39
C39H42O13 + Na: 741.2518, found 741.2525.
3.3. 1,2-O-Isopropylidene-3-O-(2,3,4-tri-O-benzoyl-
a-L-
rhamnopyranosyl)- -glucofuranose (11)
a-D
A solution of disaccharide 10 (1.00 g, 1.39 mmol) in 85% aq AcOH
(20 mL) was kept at 35 °C until disappearance of the starting mate-
rial (ꢁ24 h). The reaction mixture was diluted with toluene (40 mL)
and concentrated. The residue was purified by column chromatog-
raphy (toluene–acetone, 20:1 ? 5:1) to give diol 11 (0.68 g, 72%) as
an amorphous solid: Rf 0.21 (toluene–acetone, 5:1). 1H NMR
(500 MHz, CDCl3, for the signals of sugar ring protons see Table
1): d 8.05–7.05 (m, 15H, Ar), 3.03 (br s, 1H, OH), 2.81 (br s, 1H,
OH), 1.45 (c, 3H, C–CH3), 1.26 (c, 3H, C–CH3). 13C NMR (125 MHz,
CDCl3, for the signals of sugar ring carbons see Table 2): d 165.7,
3.2. 1,2:5,6-Di-O-isopropylidene-3-O-(2,3,4-tri-O-benzoyl-
a-L-
rhamnopyranosyl)- -glucofuranose (10)
a-D
A solution of diacetoneglucose 9 (0.99 g, 3.79 mmol) and bro-
mide 8 (3.07 g, 5.70 mmol) in a mixture of toluene (22 mL) and
CH2Cl2 (49 mL) was stirred at rt with powdered mol. sieve 4 Å
(500 mg) for 1 h. A solution of AgOTf (0.26 M, 22 mL) in toluene
was added to the mixture, and stirring was continued until the full
consumption of 9 (ꢁ2 h). After adding pyridine (5 mL), the reaction

B. S. Komarova et al. / Carbohydrate Research 360 (2012) 56–68
61
Table 1
1H NMR chemical shifts (d, ppm) and coupling constants (J, Hz) for compounds 10–35, 1a–c
Compound
Monosaccharide unit
H-1 (J1,2
)
H-2 (J2,3
)
H-3 (J3,4
)
H-4 (J4,5
)
H-5 (J5,6a
)
H-6a (J5,6b
)
H-6b (J6a,6b)
10
a
a
a
a
a
a
a
a
a
-
-
-
-
-
-
-
-
-
L
-Rha-(1?3)
-Glc-f
-Rha-(1?3)
-Glc-f
-Rha-(1?3)
-Glc-f
-Rha-(1?3)
-GlcOH
-Rha-(1?3)
-GlcOH
-Rha-(1?3)
-GlcOAc
-Rha-(1?3)
-GlcOAc
-Rha-(1?3)
-GlcOH
-Rha-(1?3)
-GlcOH
-Glc-Br
-Rha-(1?3)
-Glc-OC(NH)CCl3
-Rha-(1?3)
-GalN-OMe
-Glc-(1?3)
5,23
6,02 (3,6)
5,24
6,01 (3,8)
5,22
5,98 (3,6)
5,41
5,30 (3,0)
5,43
4,69 (8,0)
5.19
6.38 (3.6)
5.13
5.68
5.19
5.56 (3.5)
5.17
4.70 (9.0)
6,71 (4,0)
5,24
6,64 (3,6)
5,24
4,92 (3,4)
4,79 (8,0)
5,09
5.54
4.43 (9.8)
5.09
4.67 (10.1)
5.35
4.66 (10.0)
5.34
4.60 (10.1)
5.33
4.96 (7.6)
4.85 (3.3)
4.78
4.86 (7.9)
5.36
4.82 (3.5)
4.86
5.39
5.01
5.06
4.85
5.32
5.20 (2.9)
4.81
4.88 (3.6)
4.48 (8.0)
5.44
4.76
5.01 (3.3)
4.71
5,65
4,65
5,64 (3,5)
4,66 (3,7)
5,62 (3,4)
4,65
5,82
3,78 (9,0)
5,82
3,58
5.57
5.19
5.49
5.30
5.63
4.99 (10.0)
5.56
5.02
4,93 (9,7)
5,62
5,24
5,62
3,91
5,81 (10,2)
4,50 (3,1)
5,79 (10,2)
4,51 (2,9)
5,78 (10,2)
4,50 (2,9)
5,83
3,92 (9,0)
5,83
3,69
5.7–5.58
4.19
5.7–5.58
4.01
5.70–5.62
4.31
5.70–5.62
4.01
5,74 (9,9)
4,21 (9,1)
5,70 (10,0)
4,26 (9,1)
5,64 (9,8)
4,26 (9,2)
5,70
3,54 (9,0)
5,70
3,60
4,66
4,55 (6,2)
4,52
4,15 (3,1)
4,48
4,31
4,53
4,11
4,51
3,61
4.16
4.06
4.16
3.77
4.19
4.23
4.19
3.71
4,26 (4,6)
4,16 (6,2)
4,18
4,21 (6,2)
3,66
3,62
4,15
4.53 (6.2)
3.56 (0)
4.45
3.65
4.39 (6.1)
3.61
4.33 (6.1)
3.74
4.35 (6.1)
3.75
3.68
3.97
3.76
4.34 (6.1)
4.28
1,37
D
4,31 (5,9)
1,38 (6,2)
4,01 (5,2)
1,34 (6,2)
4,66
1,37
4,49
1,37
4,54
1.30
4.33
1.30
4.36
1.31
4.36
1.31
4.36
4,44
1,34
4,38
1,35
4,26
4,38
1,26
1.01
4.38 (10.5)
1.36
4.45
1.07
4.04
1.05
4.54
1.08
4.77
4.27
3.79
4.58 (4.5)
1.09
3.91
4.00
1.09
5.34 (11,7)
3.72
4.04
1.07
4.21
5.11
3.84
3.76
1.09
3.72
3.95
3.86
3.79
1.09
3.73
3.96
3.82
3.76
1.07
3.73
3.95
3.82
3.75
1.07
3.75
3.93
3.91
3.92
1.26
3.89
3.85
3,26 (8,3)
3,87 (11,3)
4,41
11
12
13
L
D
L
D
-
D
L
a-Isomer
D
13
b-Isomer
14
L
b-
D
4,46
4.21
4.24
4.26
a
a
a
-
-
-
L
a-Isomer
D
5.25
5.24
5.11
14
b-Isomer
15
L
b—
D
a-
a-
a-
L
a-Isomer
D
15
b-Isomer
16
L
b-
D
5.11
5,31 (9,7)
5,70
5,33
5,70
4.26
4,27 (13,1)
a
a
a
a
a
-
D
4,33 (9,4)
5,65
4,36
5,68
4,10
-
-
-
-
L
17
18
D
4,26
L
D
4,37
5,17
4,11
4,26
b-
D
5,26
5,46
3,94
5,57
a-
L
-Rha-(1?3)
-Rha-(1?3)
5,62 (9,5)
5.57 (10.1)
3.73 (4.9)
5.46
3.87 (9.0)
5.54 (10.0)
3.88
5.52 (10.1)
3.83 (9.6)
5.54
3.91 (9.5)
4.48
4.17
3.81
5.54
4.34
4.21 (9,5)
5.53 (10,1)
3.82 (9,6)
4.26
3.88
5.47
21
22
23
24
25
a-
L
5.74 (3.4)
3.70 (9.1)
5.39 (3.6)
5.48 (9.5)
5.52 (3.5)
5.46 (9.6)
5.50 (3.3)
5.46 (9.5)
5.57
5.54
3.81 (10.7)
3.61
5.48 (8.7)
5.53 (3.2)
3.61
5.57
5.56 (3,3)
3.55
3.60
5.43
5.48 (3.1)
3.43
3.59
3.78
3.38
5.80 (10.1)
4.02 (9.5)
5.76 (10.1)
4.26 (9.2)
5.82 (10.2)
4.29 (9.1)
5.81 (10.2)
4.27 (9.2)
5.83 (10.1)
4.29
4.09 (2.9)
3.96
4.29 (9.3)
5.82 (10.1)
3.96
b-
D
-Glc-SEt
-Rha-(1?3)
-Glc-SEt
-Rha-(1?3)
-Glc-SEt
-Rha-(1?3)
-Glc-SEt
-Rha-(1?3)
-Glc-(1?3)
3.81
a-L
b-
D
3.86
a-
L
b-
D
3.88
a-
L
b-
D
4.31 (11.8)
a-
L
b-
D
4.24
4.12
3.72
4.25 (11.9)
a-
D
-GalN-OMe
-GalN-OMe
26
a-
D
b-
D
-Glc-(1?3)
-Rha-(1?3)
-GalN-OMe
-Glc-(1?3)
-Rha-(1?3)
a-
L
28
a-
D
3.53
3.95
a-Isomer
b-
D
4.29
3.61
a
a
a
-
-
-
L
5.85 (10,1)
4.11 (9,4)
4.05
4.23 (9.4)
5.74 (10.0)
5.58 (9.6)
4.10
4.14 (2.7)
3.80
3.76
4.00 (9.3)
3.92 (9.4)
3.87
3.80
3.76
3.97
3.92
4.02
3.76
3.75
3.96
3.97
4.06
4.43 (5,1)
4.53 (10,1)
3.39
3.82
4.30 (6.1)
4.02
4.50
4.04
3.59
3.86 (6.2)
3.68
D
-Glc-(1?4)
-GalN-OMe
-Glc-(1?3)
4.73
3.67
29
30
31
32
34
35
D
b-
D
a
a
a
a
-
L-Rha-(1?3)
-
-
-
D
D
D
-Glc-(1?6)
-Glc-(1?4)
-GalN-OMe
3.41
3.83
4.27
3.43
3.40
3.32
3.58
4.24
4.13
4.63
3.62
3.61
b-
D
-Glc-(1?3)
a
a
a
a
-
L-Rha-(1?3)
4.03
3.34
3.38
3.33
3.41
4.00
3.32
-
-
-
D
D
D
-Glc-(1?6)
-Glc-(1?4)
-GalN-OMe
3.53
3.70
3.64
3.57
4.19
4.02
b-
D
-Glc-(1?3)
4.45 (7.9)
5.43
4.81
5.00 (3.2)
4.73
4.41
5.42
4.80
4.99
4.84
a
a
a
a
-
L-Rha-(1?3)
3.85 (6.2)
3.72
4.21
4.03
3.59
3.84
3.70
4.21
4.04
3.58
3.84 (6.2)
3.68
4.19
4.04
3.61
4.01 (6,3)
3.73
-
-
-
D
D
D
-Glc-(1?6)
-Glc-(1?4)
-GalN-OMe
3.31
3.57
4.19
3.40
3.38
3.29
3.53
4.16
3.43
3.38
3.30
3.52
4.28
3.53
3.47
3.43
3.52
3.49
3.71
3.65
3.64
4.53
3.35
3.97
3.34
3.37
4.52
3.35
3.96
3.34
3.39
4.47 (11.2)
3.32
4.05
b-
D
-Glc-(1?3)
a
a
a
a
-
L-Rha-(1?3)
-
-
-
D
D
D
-Glc-(1?6)
-Glc-(1?4)
-GalN-OMe
3.51
3.67
3.63
3.69
b-
D
-Glc-(1?3)
4.43 (8.0)
5.41
4.74
3.77
3.73
3.99
3.98
4.13
3.63
3.81
3.71
a
a
a
a
-
L-Rha-(1?3)
-
-
-
D
D
D
-Glc-(1?6)
-Glc-(1?4)
-GalN-OMe
3.53
3.67
5.00
4.85 (3.6)
4.51 (7.8)
5.18
5.02 (3.5)
4.99
b-
D
-Glc-(1?3)
4.51
a
a
a
-
-
-
L-Rha-(1?3)
D
-Glc-(1?6)
-Glc-(1?4)
3.59
3.52
5.02
4.99
D
3.87
4.21
(continued on next page)

62
B. S. Komarova et al. / Carbohydrate Research 360 (2012) 56–68
Table 1 (continued)
Compound
Monosaccharide unit
-GalN-OMe
-Glc-(1?3)
-Rha-(1?3)
H-1 (J1,2
)
H-2 (J2,3
)
H-3 (J3,4
)
H-4 (J4,5
)
H-5 (J5,6a
)
H-6a (J5,6b
)
H-6b (J6a,6b)
1a
a
b-
-
D
D
4.83 (3.6)
4.49 (8.1)
5.17
5.01 (3.7)
4.98 (3.5)
4.84 (3.7)
4.50 (8.1)
5.16
5.09 (3.7)
4.98 (3.6)
4.80 (3,6)
4.51 (8.0)
5.17
4.45 (11.2)
3.32 (8.6)
4.04
3.59
3.51
4.47 (11.3)
3.30 (8.6)
4.02
3.57
3.52
4.45 (11.3)
3.31 (8.5)
4.04
3.57
3.52
4.08 (2.5)
3.59
3.80 (9.7)
3.70 (9.3)
3.85
4.16 (2.8)
3.56
3.79
3.70 (9.6)
3.83
4.14 (2.7)
3.60
3.79
3.71
4.27
3.51
3.46
3.42
3.52
4.29
3.51
3.45
3.41
3.52
4.27
3.51
3.46
3.42
3.52
4.03
3.62
4.00 (6.3)
3.74
4,21
4.03
3.61
3.96 (6.3)
3.71
4.20
4.03
3.90
3.90
1,3
4.49
5,01
a
a
a
a
-
L
-
-
-
D-Glc-(1?6)
D-Glc-(1?4)
D-GalN-OMe
3.87
3.83
3.89
3.90
1.25
3.87
3.82
3.88
3.91
1.27
3.87
3.82
1b
1c
b-
D
-Glc-(1?3)
4.50
5.09
a
a
a
a
-
L
-Rha-(1?3)
-
-
-
D
D
D
-Glc-(1?6)
-Glc-(1?4)
-GalN-OMe
b-
D
-Glc-(1?3)
3.62
4.00 (6.3)
3.73
4.51
5.01
a
a
a
-
-
-
L
-Rha-(1?3)
-Glc-(1?6)
-Glc-(1?4)
D
5.01 (3.7)
4.99 (3.5)
D
3.84
4.20
165.6 (CO), 133.6–128.3 (Ar), 112.0 (C(CH3)2), 26.7, 26.1 (C(CH3)2).
Anal. Calcd for C36H38O13: C, 63.71; H, 5.64. Found: C, 63.93; H, 5.68.
poured into a mixture of crushed ice and satd aq NaHCO3. The
aqueous solution was extracted four times with EtOAc and com-
bined organic extracts were washed successively with water, 1 M
HCl, and water. The organic layer was concentrated, and the resi-
due was subjected to column chromatography (toluene–methyl
tert-butyl ether, 11:1) to give triacetate 14 (768 mg, 76%) as an
amorphous solid: 1H NMR (500 MHz, CDCl3, for the signals of sugar
ring protons see Table 1): d 8.10–7.10 (m, Ar), 4.16–4.14 (m,
ClCH2), 2.34–2.10 (cluster of s, CH3CO). 13C NMR (125 MHz, CDCl3,
for the signals of sugar ring carbons see Table 2): d 169.6, 169.5,
169.2, 168.7 (CH3CO), 167.2 (ClCH2CO), 165.8, 165.3, 165.2 (PhCO),
40.7 (ClH2CCO), 21.1, 21.0, 21.0, 20.9, 20.7, 20.5 (CH3CO). ESI
HRMS: [M+Na]+ calcd for C41H41ClO17 + Na: 863.1924, found
863.1927.
3.4. 6-O-Chloroacetyl-1,2-O-isopropylidene-3-O-(2,3,4-tri-O-
benzoyl-a-L-rhamnopyranosyl)-a-D-glucofuranose (12)
To a solution of diol 11 (1.1 g, 1.6 mmol) in dry CH2Cl2 (10.8 mL)
were added chloroacetyl chloride (144 L, 1.6 mmol) and pyridine
(130 L,
L, 1.6 mmol) at ꢂ15 °C. After 30 min, more pyridine (30
0.4 mmol) was added and the mixture was stirred for 1 h. To bring
the reaction to completion, more chloroacetyl chloride (30 L,
0.38 mmol) and pyridine (30 L, 0.4 mmol) were added and stir-
l
l
l
l
l
ring was continued for next 30 min. After quenching with water
(4 mL), the reaction mixture was poured into 1 M HCl (80 mL)
and extracted with CH2Cl2. Combined organic extracts were
washed with water and concentrated. Column chromatography
of the residue (toluene–EtOAc, 10:1) afforded disaccharide 12
(1.10 g, 90%) as an amorphous solid: Rf 0.54 (toluene–acetone,
4:1). 1H NMR (500 MHz, CDCl3, for the signals of sugar ring protons
see Table 1): d 8.11–7.10 (15H, m, Ar), 4,35 (2H, s, ClCH2), 1,50 (3H,
s, C–CH3), 1,33 (c, 3H, C–CH3). 13C NMR (125 MHz, CDCl3, for the
signals of sugar ring carbons see Table 2): d 167.9 (ClCH2CO),
165.8, 165.7, 165.6 (PhCO), 133.6, 133.3, 133.2 (ipso-C, Bz), 129.9,
129.7, 129.6, 129.2, 129.1, 129.0, 128.4, 128.3, 128.2 (Ph), 112.2
(C(CH3)2), 40.8 (ClCH2), 26.7, 26.2 (C(CH3)2). Anal. Calcd for
C38H39ClO14: C, 60.44; H, 5.21. Found: C, 60.51; H, 5.39.
3.7. 2,4-Di-O-acetyl-6-O-chloroacetyl-3-O-(2,3,4-tri-O-benzoyl-
b-L
-rhamnopyranosyl)-D-glucopyranose (15)
To a solution of compound 14 (251 mg, 0.30 mmol) in DMF
(4.3 mL) was added N2H4ꢀAcOH (41 mg, 0.45 mmol). After 2.5 h,
the reaction mixture was poured into satd aq NaHCO3 and the
aqueous solution was extracted four times with EtOAc. Combined
organic extracts were washed with water, dried with Na2SO4,
and concentrated. The product 15 (198 mg, 83%) was isolated by
column chromatography (toluene–acetone, 5:1): 1H NMR
(500 MHz, CDCl3, for the signals of sugar ring protons see Table
1): d 8.11–7.21 (m, Ar), 4.19 (s, ClCH2), 2.39, 2.32, 2.31, 2.19 (4 s,
CH3CO). 13C NMR (125 MHz, CDCl3, for the signals of sugar ring car-
bons see Table 2): d 170.8, 169.7 (CH3CO), 167.3 (ClCH2CO), 165.8,
165.6, 165.3 (PhCO), 133.6, 133.5, 133.1 (ipso-C, Ph), 129.8, 129.7,
129.7, 129.2, 129.1, 129.0, 128.6, 128.4, 128.2 (Ph), 40.7 (ClCH2CO),
21.1, 20.8, 20.7 (CH3CO). Anal. Calcd for C39H39ClO16: C, 58.61; H,
4.92. Found: C, 59.01; H, 4.69.
3.5. 6-O-Chloroacetyl-3-O-(2,3,4-tri-O-benzoyl-
a-L-
rhamnopyranosyl)- -glucopyranose (13)
D
Aq CF3CO2H (90%, 18 mL) was added to a chilled (+4 °C) solution
of 12 (1.0 g, 1.4 mmol) in CH2Cl2 (2 mL). The mixture was kept at rt
for 1 h, diluted with toluene (50 mL) and concentrated in vacuum.
Residual CF3CO2H was removed by coevaporation with toluene.
Triol 13 (933 mg, 97%) was purified by column chromatography
(toluene–acetone, 4:1): Rf 0.23 (toluene–acetone, 4:1). 1H NMR
(500 MHz, CDCl3, for the signals of sugar ring protons see Table
1): d 8.05–7.12 (m, Ar), 4.12 (s, ClCH2). 13C NMR (125 MHz, CDCl3,
for the signals of sugar ring carbons see Table 2): d 166.1
(ClCH2CO), 165.8, 165.7 (PhCO), 133.6, 133.3 (ipso-C, Bz), 129.9,
129.7, 129.0, 128.8, 128.6, 128.4, 128.3 (Ph), 40.8 (ClCH2CO). ESI
HRMS: [M+Na]+ calcd for C35H35ClO14 + Na: 737.1608, found
737.1614.
3.8. 2,4-Di-O-acetyl-6-O-chloroacetyl-3-O-(2,3,4-tri-O-benzoyl-
a-L
-rhamnopyranosyl)-a-D-glucopyranosyl bromide (16)
A solution of HBr in AcOH (33% wt, 0.45 mL) was added to a
solution of 14 (68 mg, 0.081 mmol) in CH2Cl2 (0.50 mL). The reac-
tion mixture was kept for 2 h at rt, diluted with CH2Cl2 (50 mL) and
poured into ice water. The organic layer was separated and the
aqueous layer was extracted with CH2Cl2 (3 ꢃ 20 mL), combined
organic extracts were washed with satd aq NaHCO3, and concen-
trated. Column chromatography (gradient elution, toluene–methyl
tert-butyl ether, 25:1 ? 50:3) of the residue afforded bromide 16
(58 mg, 83%) as an amorphous solid. 1H NMR (500 MHz, CDCl3,
for the signals of sugar ring protons see Table 1): d 8.12–7.20
(15H, m, Ar), 4.18 (2H, s, ClCH2), 2.31, 2.19 (6H, 2 s, CH3CO). 13C
NMR (125 MHz, CDCl3 for the signals of sugar ring carbons see
3.6. 1,2,4-Tri-O-acetyl-6-O-chloroacetyl-3-O-(2,3,4-tri-O-
benzoyl-a-L-rhamnopyranosyl)-D-glucopyranose (14)
A solution of triol 13 (856 mg, 1.20 mmol) in Ac2O (17 mL) and
pyridine (17 mL) was kept for 6 h, then the reaction mixture was

B. S. Komarova et al. / Carbohydrate Research 360 (2012) 56–68
63
Table 2
13C NMR chemical shifts (d, ppm) for compounds 10–35, 1a–c
Compound
Monosaccharide residue
C-1
C-2
C-3
C-4
C-5
C-6
10
a
a
a
a
a
a
a
a
a
-
-
-
-
-
-
-
-
-
L
-Rha-(1?3)
-Glc-f
-Rha-(1?3)
-Glc-f
-Rha-(1?3)
-Glc-f
-Rha-(1?3)
-GlcOH
-Rha-(1?3)
-GlcOH
-Rha-(1?3)
-GlcOAc
-Rha-(1?3)
-GlcOAc
-Rha-(1?3)
-GlcOAc
-Rha-(1?3)
-GlcOAc
-Glc-Br
-Rha-(1?3)
-Glc-OC(NH)CCl3
-Rha-(1?3)
-GalN-OMe
-Glc-(1?3)
94,9
105,4
96,0
105,2
95,9
105,2
99,0
92,4
98,8
96,6
99.7
89.2
99.8
91.9
99.9
90.2
99.9
95.9
87,1
99,8
93,1
99,6
99,6
101,7
99,6
97.5
87.3
97.8
84.4
97.9
83.8
97.9
83.7
97.7
101.9
99.7
98.9
101.4
97.8
99.2
104.0
98.1
99.0
98.4
103.4
98.1
96.6
98.9
100.4
106.3
102.6
98.2
98.8
98.2
107.2
102.5
101.0
98.7
100.1
106.4
102.5
98.2
99.1
98.1
105.7
102.4
100.0
99.3
99.8
105.6
102.3
99.5
100.6
99.9
71,1
81,9
71,1
82,0
71,0
81,9
70,7
71,4
70,6
74,3
70.7
70.6
70.7
70.6
70.7
72.7
70,0
77,0
69,9
79,1
69,8
78,7
70,1
83,6
71,6
81,0
71,5
79,6
71,5
78,9
71,3
69,0
71,3
73,5
67,1
72,0
67,7
68,5
67,2
67,7
67,6
69,3
67,6
69,0
67.9
70.0
68.0
72.7
67.8
67.5
17,2
68,3
17,5
64,4
17,5
68,4
17,5
65,0
17,5
65,0
17.5
63.1
17.4
63.1
17.6
63.6
17.6
63.5
62,6
17,6
63,1
17,6
69,0
63,3
17,4
17.0
68.7
16.8
68.7
17.3
61.7
17.3
62.9
17,3
62.0
69.1
69.4
62.4
17.3
67.7
61.4
17.3
62.9
69.3
67.2
17.3
63.3
63.5
71.2
67.9
18.1
62.6
62.1
71.2
68.1
18.1
62.8
62.0
71.4
68.0
18.1
62.8
62.3
71.3
67.8
18.1
62.7
62.3
62.0
67.7
18.0
62.2
61.6
62.0
D
11
12
13
L
D
L
D
L
a-Isomer
D
13
b-Isomer
14
L
70,2
85,0
b-
D
a
a
a
-
-
-
L
71.1 or 69.7a
78.2
a-Isomer
D
68.7
68.9
69.3
14
b-Isomer
15
L
71.1 or 69.7a
81.6
b-D
a
a
a
-
-
-
L
71.3 or 69.8a
78.2
a-Isomer
D
15
b-Isomer
16
L
71.1 or 69.7a
81.1
b-
D
74.6
72,4
70,6
71,4
70,6
59,2
71,5
70,9
70.7
74.2
70.5
72.9
70.7
73,0
70.7
72.8
70,7
71.8
58.9
59.0
73.8
70.6
59.0
73.5
70.6
80.8
59.3
73.7
70.5
77.4
81.1
61.4
76.4
72.4
81.6
82.0
52.3
76.6
72.4
81.8
81.9
50.7
76.2
72.4
81.6
82.1
50.8
76.5
72.3
81.6
82.1
50.7
75.2
71.8
72.9
73.4
50.5
69.3
67,8
72.0
72,3
67,9
70,2
67,9
63,0
72,0
67,9
66.3
71.1
66.6
71.2
67.3
80.1
67.5
77.2
67,4
73.5
63.0
68.8
73.6
67.5
73.0
76.2
67.2
69.5
70.5
76.2
67.2
68.9
69.3
72.1
75.7
70.3
72.5
72.8
72.5
a
a
a
a
a
-
D
78,3
-
-
-
-
L
69,7 or 71,2a
77,9
17
18
D
68,5
L
71,2 or 69,7a
75,1
D
75,7
69,6
71,2
71.7
7.9
b-
D
81,9
69,6
70.2
77.5
69.5
77.1
69.4
79.4
69.3
79.4
69,5
77.9
75.4
77.8
77.5
69.3
78.8
77.8
69.4
81.9
77.9
77.9
69.3
73.2
81.9
78.2
82.0
72.4
74.8
82.9
82.2
82.0
72.4
74.8
83.0
78.1
81.6
72.3
74.8
83.0
77.2
81.5
72.4
74.8
83.0
77.4
83.3
71.7
a-
L
L
-Rha-(1?3)
-Rha-(1?3)
21
22
23
24
25
a-
b-
D
-Glc-SEt
-Rha-(1?3)
-Glc-SEt
-Rha-(1?3)
-Glc-SEt
-Rha-(1?3)
-Glc-SEt
-Rha-(1?3)
-Glc-(1?3)
a-
L
71.6
79.0
71.7
76.4
71.7
77.0
74,0
76.7
75.6
68.5
76.7
71.6
77.2
75.6
71.9
78.0
77.7
77.1
71.9
76.0
78.2
78.3
78.8
74.2
80.1
79.3
77.7
b-
D
a-
L
b-
D
a-
L
b-
D
a-
L
b-
D
a-
D
-GalN-OMe
-GalN-OMe
26
a-
D
b-
D
-Glc-(1?3)
-Rha-(1?3)
-GalN-OMe
-Glc-(1?3)
-Rha-(1?3)
-Glc-(1?4)
-GalN-OMe
-Glc-(1?3)
-Rha-(1?3)
-Glc-(1?6)
-Glc-(1?4)
-GalN-OMe
-Glc-(1?3)
-Rha-(1?3)
-Glc-(1?6)
-Glc-(1?4)
-GalN-OMe
-Glc-(1?3)
-Rha-(1?3)
-Glc-(1?6)
-Glc-(1?4)
-GalN-OMe
-Glc-(1?3)
-Rha-(1?3)
-Glc-(1?6)
-Glc-(1?4)
-GalN-OMe
-Glc-(1?3)
-Rha-(1?3)
-Glc-(1?6)
-Glc-(1?4)
-GalN-OMe
-Glc-(1?3)
-Rha-(1?3)
-Glc-(1?6)
-Glc-(1?4)
-GalN-OMe
a-
L
28
a-
D
a-Isomer
b-
D
a
a
a
-
-
-
L
D
29
30
31
32
34
D
b-
D
a
a
a
a
-
L
-
-
-
D
D
D
b-
D
a
a
a
a
-
L
-
-
-
D
D
D
b-
D
a
a
a
a
-
L
70.3
72.4
72.6
72.2
75.8
70.2
72.6
72.8
72.1
76.1
70.2
72.7
72.9
73.3
75.9
70.3
73.4
72.9
73.4
-
-
-
D
D
D
80.3
79.3
78.6
78.8
74.0
80.1
79.4
78.9
78.7
74.1
80.0
79.4
73.5
77.4
73.5
71.1
70.7
77.4
b-
D
a
a
a
a
-
L
-
-
-
D
D
D
b-
D
a
a
a
a
-
L
-
-
-
D
D
D
b-
D
35
a
a
a
a
-
L
-
-
-
D
D
D
74.7
74.1
78.3
(continued on next page)

64
B. S. Komarova et al. / Carbohydrate Research 360 (2012) 56–68
Table 2 (continued)
Compound
Monosaccharide residue
b- -Glc-(1?3)
-Rha-(1?3)
C-1
C-2
C-3
C-4
C-5
C-6
D
106.2
102.3
99.4
100.5
99.5
105.7
102.2
99.5
100.4
99.8
75.0
71.8
72.9
73.4
51.0
7.0
71.7
72.9
73.4
50.8
75.1
71.8
72.9
73.4
83.6
71.6
74.7
74.1
77.2
83.3
71.8
74.7
74.1
77.2
83.5
71.7
74.8
74.2
69.9
73.4
71.1
70.7
77.2
69.8
73.4
71.1
70.7
77.4
69.9
73.5
71.1
70.7
75.7
70.3
73.4
72.7
73.2
75.9
70.3
73.3
72.8
73.3
75.9
70.3
73.4
72.8
67.8
18.0
62.3
61.6
62.0
67.8
18.0
62.3
61.7
62.0
67.8
18.0
62.3
61.7
1a
1b
a
a
a
a
a
a
a
a
a
-L
-
-
-
-
-
-
-
-
D
D
D
D
-Glc-(1?6)
-Glc-(1?4)
-GalN-OMe
-Glc-(1?3)
L
-Rha-(1?3)
D-Glc-(1?6)
D-Glc-(1?4)
D-GalN-OMe
b-D
-Glc-(1?3)
105.6
102.3
99.5
1c
a
a
a
-L-Rha-(1?3)
-
-
D
-Glc-(1?6)
-Glc-(1?4)
D
100.5
a
Assignment of these signals may be interchanged.
Table 2):
d
170.3, 169.4, (CH3CO), 167.1 (ClCH2CO), 165.7,
3.11. Methyl 2,3,4-tri-O-benzoyl-
2,4-di-O-acetyl-6-O-chloroacetyl-b-
azido-6-O-benzyl-2-deoxy- -galactopyranoside (19)
a
-
L
-rhamnopyranosyl-(1?3)-
165.6, 165.4 (PhCO), 133.6, 133.4, 133.1 (ipso-C, Ph), 40.6
D-glucopyranosyl-(1?3)-2-
(ClCH2CO), 21.0, 20.6 (CH3CO). ESI HRMS: [M+Na]+ calcd for
a-D
C39H38BrClO15 + Na: 883.0975, found 883.0983.
NaBH3CN (4 mg, 0.064 mmol) and 4 M solution of HCl in
dioxane (16 L, 0.061 mmol) were consecutively added to a
3.9. 2,4-Di-O-acetyl-6-O-chloroacetyl-3-O-(2,3,4-tri-O-benzoyl-
-rhamnopyranosyl)- -glucopyranosyl
trichloroacetimidate (17)
l
a-
L
a-
D
mixture of trisaccharide 18 (10 mg, 0.009 mmol) and mol. sieve
0
4 ÅA (28 mg) in dry THF (0.5 mL) at +4 °C. The resulting mixture
was stirred for 1.5 h at rt, then made neutral by adding satd aq
NaHCO3. The solution was extracted with EtOAc, the combined
extracts were washed with water and concentrated to give a
chromatographically homogeneous product with Rf 0.27 (tolu-
ene–acetone, 11:2). NMR examination revealed this product to
be a mixture of 19 and, presumably, its positional isomer with
a 4-O-benzyl group in a ratio of about 1:1. 1H and 13C NMR
spectra of this mixture showed the absence of signals corre-
sponding to CH of the benzylidene group (dH 5.59, dC
100.5 ppm). The 13C NMR spectrum of the mixture contained
Cl3CCN (76 lL, 0.754 mmol) and DBU (6 lL, 0.038 mmol) were
added to a solution of hemiacetal 15 (100 mg, 0.126 mmol) in
dry CH2Cl2 (1.5 mL) at ꢂ18 °C. The reaction mixture was allowed
to attain rt within 1.5 h and applied onto a silica gel column. Elu-
tion with toluene–methyl tert-butyl ether (25:2) provided trichlo-
roacetimidate 17 (88 mg, 74%): Rf 0.2 (toluene–methyl tert-butyl
ether, 35:2). 1H NMR (500 MHz, CDCl3, for the signals of sugar ring
protons see Table 1): d 8.78 (1H, s, Cl3CC(NH)O), 8.11–7.15 (15H,
m, Ar), 4.17 (m, ClCH2), 2.23, 2.18 (6H, 2 s, CH3CO). 13C NMR
(125 MHz, CDCl3, for the signals of sugar ring carbons see Table
2): d 170.4, 169.5 (CH3CO), 167.1 (ClCH2CO), 165.8, 165.6, 165.3
(PhCO), 160.7 (Cl3CC(NH)), 133.6, 133.4, 133.1 (ipso-C, Ph), 129.8,
129.7, 129.3, 129.2, 129.1, 129.0, 128.6, 128.5, 128.3 (Ar), 40.1
(ClCH2CO), 21.0, 20.5 (CH3CO). ESI HRMS: [M+Na]+ calcd for
signals of double intensities for C1 of the
a-GalN3 and a-Rha
units (d 99.1 and 99.8) and two signals for C1 of the b-Glc res-
idue (d 101.8 and 101.6).
3.12. Ethyl 2,3,4-tri-O-benzoyl-a-L-rhamnopyranosyl-(1?3)-
C41H39Cl4NO16 + Na: 964.0915, found 964.0936.
4,6-O-benzylidene-1-thio-b- -glucopyranoside (21)
D
3.10. Methyl 2,3,4-tri-O-benzoyl- -rhamnopyranosyl-(1?3)-
2,4-di-O-acetyl-6-O-chloroacetyl-b- -glucopyranosyl-(1?3)-2-
azido-4,6-O-benzylidene-2-deoxy- -D-galactopyranoside (18)
a
-
L
A solution of donor 8 (1.15 g, 2.13 mmol, 1.14 equiv) and
acceptor 20 (0.58 g, 1.86 mmol) in dry CH2Cl2 (12 mL) was added
to a suspension of AgOTf (0.55 g, 2.14 mmol) and 2,6-di-tret-bu-
tyl-4-methylpyridine (0.31 g, 1.47 mmol, 0.98 equiv) in hexane
(12.5 mL) at ꢂ12 °C under argon. The mixture was stirred at this
temperature for 2 h, then the reaction was quenched with Et3N
(0.5 mL). The resulting mixture was diluted with CH2Cl2 (75 mL),
washed consecutively with a mixture of 1 M Na2S2O3 and satd
aq NaHCO3 (1:1), water, 1 M HCl, and water. The organic phase
was concentrated and the residue was subjected to silica gel col-
umn chromatography (toluene–acetone, 50:1?20:1) to yield
disaccharide 21 (0.93 g, 66%) as a colorless foam, Rf 0.37 (tolu-
D
a
One molar solution of Bu2BOTf in CH2Cl2 (4 lL) was added to a
stirred mixture of donor 17 (33 mg, 0.035 mmol), acceptor 7 (9 mg,
0.029 mmol), and powdered mol. sieve AW-300 (55 mg) in freshly
distilled CH2Cl2 (750
l
L) at ꢂ40 °C. The resulting mixture was stir-
red at ꢂ30 °C for 1 h, diluted with CH2Cl2 (20 mL), and filtered
through a Celite pad. The filtrate was washed with satd aq NaHCO3,
water, and concentrated. Purification of the residue by column
chromatography (CHCl3–acetone, 55:1 ? 42:1) gave trisaccharide
18 (7 mg, 21%) as an amorphous solid. 1H NMR (500 MHz, CDCl3,
for the signals of sugar ring protons see Table 1): d 8.08–7.20
(20H, m, Ar), 5.58 (1H, s, PhCH), 4.08 (2H, m, ClCH2), 3.44 (OCH3),
2.25 (3H, s, CH3CO), 2.10 (3H, s, CH3CO). 13C NMR (125 MHz, CDCl3,
for the signals of sugar ring carbons see Table 2): d 169.7 (CH3CO),
167.1 (ClCH2CO), 165.8, 165.7, 165.1 (PhCO), 137.7 (ipso-C, PhCH),
133.6, 133.4, 133.0 (ipso-C, Bz), 129.8, 129.7, 129.2, 128.8,
128.6, 128.4, 128.2, 128.1 (Ar), 100.5 (PhCH), 55.6 (OCH3), 40.7
(ClCH2CO), 21.2, 20.8 (CH3CO). ESI HRMS: [M+Na]+ calcd for
ene–acetone, 15:1), [a]
12.0 (c 0.94, CHCl3). 1H NMR (500 MHz,
D
CDCl3, for the signals of sugar ring protons see Table 1): d 8.01–
7.12 (20H, m, Ar); 5.64 (1H, s, PhCH); 2.75 (2H, m, SCH2CH3);
1.33 (3H, t, SCH2CH3). 13C NMR (125 MHz, CDCl3, for the signals
of sugar ring carbons see Table 2): d 165.7; 165.6 (PhCO); 137.2
(ipso-C, PhCH); 133.3; 133.1; 133.0 (ipso-C, Bz); 129.9; 129.7;
129.6; 129.5; 129.4; 129.3; 129.0; 128.5; 128.3; 128.2; 128.1;
126.2 (Ph); 101.7 (PhCH); 24.7 (SCH2CH3); 15.4 (SCH2CH3).
Anal. Calcd for C42H42O12S: C, 65.45; H, 5.49. Found: C, 65.44; H,
5.49.
C53H54ClN3O20 + Na: 1110.2881, found 1110.2880.

B. S. Komarova et al. / Carbohydrate Research 360 (2012) 56–68
65
3.13. Ethyl 2,3,4-tri-O-benzoyl-
O-benzoyl-4,6-O-benzylidene-1-thio-b-
a
-
L
-rhamnopyranosyl-(1?3)-2-
-glucopyranoside (22)
(2H, m, SCH2CH3); 2.12 (3H, s, CH3CO); 1.22 (3H, t, SCH2CH3). 13C
NMR (150 MHz, CDCl3, for the signals of sugar ring carbons see Ta-
ble 2): d 170.6 (CH3CO); 165.7; 165.3; 165.0; 164.7 (PhCO); 137.2
(ipso-C, PhCH2); 133.2; 133.1; 133.0 (ipso-C, Bz); 130.2; 129.6;
129.4; 129.2; 129.0; 128.9; 128.7; 128.4; 128.2; 127.9; 127.4
(Ph); 75.4 (PhCH2), 24.1 (SCH2CH3); 20.9 (CH3CO); 14.8 (SCH2CH3).
Anal. Calcd for C51H50O14S: C, 66.65; H, 5.48. Found: C, 66.69; H,
5.52.
D
Benzoyl chloride (0.82 mL, 7.03 mmol) and DMAP (40 mg,
0.33 mmol) were added to a solution of alcohol 21 (0.77 g,
1.00 mmol) in a mixture of CH2Cl2 (5 mL) and pyridine (10 mL) at
+4 °C. The reaction mixture was kept at rt for 72 h, diluted with
EtOAc (250 mL) and washed successively with satd aq NaHCO3,
water, 1 M HCl, and finally with water. The organic phase was con-
centrated and the residue was purified by column chromatography
(toluene–methyl tert-butyl ether, 50:1) to afford product 22
(0.80 g, 91%) as a colorless foam, Rf 0.22 (toluene–methyl tert-butyl
3.16. Methyl 2,3,4-tri-O-benzoyl-
6-O-acetyl-2-O-benzoyl-4-O-benzyl-b-
a
-
L
-rhamnopyranosyl-(1?3)-
-glucopyranosyl-
D-D
(1?3)-2-azido-4,6-O-benzylidene-2-deoxy-a-D-
galactopyranoside (25)
ether, 40:1), [a]
D 80.6 (c 1, CHCl3). 1H NMR (500 MHz, CDCl3, for the
signals of sugar ring protons see Table 1): d 8.01–7.12 (25H, m, Ar);
5.66 (1H, s, PhCH); 2.75 (2H, m, SCH2CH3); 1.33 (3H, t, SCH2CH3).
13C NMR (125 MHz, CDCl3, for the signals of sugar ring carbons
see Table 2): d 165.6; 165.3; 165.0; 164.5 (PhCO); 137.0 (ipso-C,
PhCH); 133.2; 133.11; 133.07; 133.0 (ipso-C, Bz); 130.0; 129.7;
129.4; 129.3; 129.2; 129.0; 128.4; 128.3; 128.2; 128.1 (Ph);
102.0 (PhCH); 24.1 (SCH2CH3); 14.8 (SCH2CH3). Anal. Calcd for
A mixture of donor 24 (250 mg, 0.27 mmol), acceptor 7 (74 mg,
0.24 mmol), and powdered mol. sieve AW-300 (900 mg) in dry
CH2Cl2 (8 mL) was stirred for 1 h under argon. Then NIS (65 mg,
0.27 mmol) and TfOH (3
and stirring was continued for 1 h. The reaction was quenched
by adding di-isopropylamine (100 L), the mixture was diluted
l
L, 0.011 mmol) were added at ꢂ20 °C,
l
C49H46O13S: C, 67.26; H, 5.30. Found: C, 67.49; H, 5.42.
with CH2Cl2 (20 mL) and filtered through a pad of Celite. The fil-
trate was washed with a mixture of satd aq NaHCO3 and 1 M
Na2S2O3 (1:1), and the solvent was evaporated. Silica gel column
chromatography of the residue (toluene–methyl tert-butyl ether,
6:1 ? 5.5:1) gave trisaccharide 25 (201 mg, 72%) as a syrup, Rf
3.14. Ethyl 2,3,4-tri-O-benzoyl-
a-L
-rhamnopyranosyl-(1?3)-2-
O-benzoyl-4-O-benzyl-1-thio-b-
D
-glucopyranoside (23)
A solution of Bu2BOTf (0.85 M, 0.65 mL, 0.55 mmol) in CH2Cl2
was added dropwise to a solution of 22 (0.42 g, 0.50 mmol) and
H3BꢀTHF (8.4 mmol) in THF (4.5 mL) at +4 °C. The mixture stirred
was for 2 h at the same temperature, then the excess of BH3 and
Bu2BOTf was destroyed by adding MeOH (1.5 mL) and Et3N
(4.0 mL). If the used solution of H3BꢀTHF did not contain any stabi-
lizer, the reaction mixture could be simply concentrated in a vac-
uum. If a commercial solution of H3BꢀTHF containing a stabilizer
was used, the reaction mixtures should be washed with aq. NaH-
CO3 before concentration. MeOH (3 ꢃ 5 mL) was evaporated from
the residue obtained after concentration of the solution, and pure
23 (0.38 g, 86%) was isolated by silica gel column chromatography
(toluene–methyl tert-butyl ether, 11:1 ? 10:1), Rf 0.50 (toluene–
0.40 (toluene–acetone, 20:2.8), [
a]
89.0 (c 1, CHCl3). 1H NMR
D
(300 MHz, CDCl3, for the signals of sugar ring protons see Table
1): d 8.10–7.20 (30H, m, Ar); 5.64 (1H, s, PhCH); 5.07 (1H, d,
Jgem = 11.2 Hz, PhCH2); 4.77 (1H, d, Jgem = 11.2 Hz, PhCH2); 3.41
(3H, s, CH3O), 2.09 (3H, s, CH3CO). 13C NMR (75 MHz, CDCl3, for
the signals of sugar ring carbons see Table 2): d 170.5 (CH3CO);
165.7; 165.3; 164.9; 165.7 (PhCO); 137.9; 137.3 (ipso-C, PhCH,
PhCH2); 133.2; 133.1; 133.0 (ipso-C, Bz); 129.9; 129.7; 129.6;
129.5; 129.3; 129.2; 128.7; 128.5; 128.4; 128.3; 128.2; 128.1;
128.0; 127.4; 126.1 (Ph); 75.4 (PhCH2), 20.9 (CH3CO). Anal. Calcd
for C63H61N3O19: C, 65.00; H, 5.28; N, 3.61. Found: C, 64.73; H,
5.25; N, 3.83.
acetone, 10:1) as a syrup, [
a]
D
53.9 (c 1, CHCl3). 1H NMR
3.17. Methyl 2,3,4-tri-O-benzoyl-
6-O-acetyl-2-O-benzoyl-4-O-benzyl-b-
2-azido-6-O-benzyl-2-deoxy- -galactopyranoside (26)
a
-L
-rhamnopyranosyl-(1?3)-
(600 MHz, CDCl3, for the signals of sugar ring protons see Table
1): d 8.10–7.20 (25H, m, Ar); 5.07 (1H, d, Jgem = 11.3 Hz, PhCH2);
4.91 (1H, d, Jgem = 11.3 Hz, PhCH2); 2.79 (2H, m, SCH2CH3); 1.29
(3H, t, SCH2CH3). 13C NMR (150 MHz, CDCl3, for the signals of sugar
ring carbons see Table 2): d 165.7; 165.2; 165.0; 164.7 (PhCO);
137.6 (ipso-C, PhCH2); 133.2; 133.1; 133.0 (ipso-C, Bz); 130.0;
129.7; 129.6; 129.5; 129.3; 129.2; 128.4; 128.2; 127.8; 127.4
(Ph); 75.3 (PhCH2), 24.2 (SCH2CH3); 14.8 (SCH2CH3). Anal. Calcd
for C49H48O13S: C, 67.11; H, 5.52. Found: C, 67.21; H, 5.54.
D-glucopyranosyl)-(1?3)-
a-D
To a solution of benzylidene acetal 25 (1.38 g, 1.2 mmol) in
dry THF (75 mL) were added Me3NꢀBH3 (0.35 g, 4.7 mmol) and
AlCl3 (0.94 g, 7.0 mmol), then water (46 lL, 2.6 mmol) was added
to the resulting mixture at +4 °C. The reaction mixture was
stirred at rt for 20 h, then 1 M HCl (500 mL) and water
(500 mL) were added. The mixture was extracted with EtOAc
(3 ꢃ 170 mL), and the combined extracts were washed with satd
aq NaHCO3 and brine. The organic phase was dried over Na2SO4
and concentrated. Pure 26 (1.26 g, 91%) was isolated as a syrup
by silica gel column chromatography (toluene–EtOAc, 80:12 ?
3.15. Ethyl 2,3,4-tri-O-benzoyl-a-L-rhamnopyranosyl-(1?3)-6-
O-acetyl-2-O-benzoyl-4-O-benzyl-1-thio-b-D-glucopyranoside
(24)
80:16), Rf 0.22 (petroleum ether–EtOAc, 7:3), [
a
]
D
100 (c 1,
Disaccharide 23 (1.39 g, 1.59 mmol) was acetylated with Ac2O
(15 mL) in pyridine (15 mL). When the reaction ended, the mixture
was diluted with CHCl3 (150 mL) and poured into cold water, the
organic phase was separated, and the aqueous phase was extracted
with CHCl3 (3 ꢃ 30 mL). The combined organic extracts were suc-
cessively washed with satd aq NaHCO3 (three times), water, 1 M
HCl, and water (200 mL each time). The solvent was evaporated,
and the residue was dried in vacuum to give disaccharide 24
CHCl3). 1H NMR (600 MHz, CDCl3, for the signals of sugar ring
protons see Table 1): d 8.08–7.20 (30H, m, Ar); 5.10 (1H, d,
Jgem = 11.2 Hz, PhCH2); 4.79 (1H, d, Jgem = 11.1 Hz, PhCH2); 4.65
(1H, d, Jgem = 11.9 Hz, PhCH2); 4.58 (1H, d, Jgem = 12.0 Hz, PhCH2);
3.41 (3H, s, CH3O), 2.11 (3H, s, CH3CO). 13C NMR (150 MHz,
CDCl3, for the signals of sugar ring carbons see Table 2): d
170.6 (CH3CO); 165.7; 165.3; 165.0; 164.7 (PhCO); 138.1; 137.0
(ipso-C, PhCH, PhCH2); 133.2; 133.1; 133.0; 132.9 (ipso-C, Bz);
129.9; 12.7; 129.6; 129.3; 129.2; 129.1; 128.5; 128.4; 128.2;
128.1; 128.0; 127.7 (Ph); 75.5, 73.6 (PhCH2), 20.8 (COCH3). Anal.
Calcd for C63H63N3O19: C, 64.88; H, 5.45; N, 3.60. Found: C,
64.65; H, 5.59; N, 3.56.
(1.44 g, 97%), Rf 0.66 (toluene–aceton, 20:2.8) as a syrup, [a]
D
73,8 (c 1, CHCl3). 1H NMR (600 MHz, CDCl3, for the signals of sugar
ring protons see Table 1): d 8.02–7.15 (25H, m, Ar); 5.05 (1H, d,
Jgem = 11.1 Hz, PhCH2); 4.74 (1H, d, Jgem = 11.1 Hz, PhCH2); 2.70

66
B. S. Komarova et al. / Carbohydrate Research 360 (2012) 56–68
3.18. Methyl 2,3,4-tri-O-benzoyl-
a
-
L
-rhamnopyranosyl-(1?3)-
4.96 (3H, m, H-1I, PhCH2), 4.92–4.80 (7H, m, PhCH2, H-1II, H-1V);
2-O-benzoyl-4-O-benzyl-b-
benzoyl-2,3,4-tri-O-benzyl-
6-O-benzyl-2-deoxy- -galactopyranoside (28)
D
-glucopyranosyl)-(1?3)-[6-O-
4.67, 4.54–4.44 (6H, m, H-5V, PhCH2), 3.48 (OCH3), 2.05–2.00 (6H,
2 s, 2 CH3CO). 13C NMR (125 MHz, CDCl3, for the signals of sugar
ring carbons see Table 2): d 170.5, 169.5 (CH3CO); 166.1, 165.7,
165.0, 164.5, 164.3 (PhCO); 138.6, 138.4, 138.3, 137.7, 137.4
(ipso-C, Bz); 133.1, 133.0, 132.8, 132.7, 132.6 (ipso-C, PhCH2);
130.3, 129.7, 129.6, 129.3, 129.0, 128.5, 128.4, 128.3, 128.2,
128.1, 128.0, 127.9, 127.8, 127.6, 127.5, 127.3 (Ph); 75.7, 75.4,
74.8, 74.0, 73.8, 72.8, 72.1 (PhCH2); 55.2 (OCH3); 21.0, 20.7
(CH3CO). Anal. Calcd for C119H119N3O31: C, 68.48; H, 5.75; N, 2.01.
Found: C, 68.40; H, 5.83; N, 2.10.
a
-D
-glucopyranosyl-(1?4)]-2-azido-
a-D
Dry toluene (2 ꢃ 5 mL) was evaporated from a mixture of donor
3 (0.68 g, 0.93 mmol) and acceptor 26 (0.98 g, 0.84 mmol), the res-
idue was dissolved in dry CH2Cl2 (30 mL) and mol. sieve AW-300
(3.5 g) was added. The mixture was stirred for 1 h at rt, cooled to
ꢂ30 °C, and TMSOTf (10
lL, 0.055 mmol) was added. Stirring was
continued for 1.5 hours, and then the reaction was quenched by
adding di-isopropylamine (0.2 mL). The mixture was diluted with
CH2Cl2 (30 mL), filtered through a pad of Celite, and the filtrate
was washed with satd aq NaHCO3 and water. The organic layer
was separated, and the aqueous layer was extracted with CH2Cl2
(3 ꢃ 30 mL). Combined organic solutions were concentrated and
the residue was subjected to chromatographic purification (tolu-
ene–CH3CN, 19:1 ? 17:1) to yield tetrasaccharide 27 (1.22 g,
90%) as an anomeric mixture, Rf 0.42 (toluene–CH3CN, 10:1). To a
solution of 27 in a mixture of CH2Cl2 (35 mL) and MeOH (35 mL)
was added methanolic hydrogen chloride (35 mL) prepared by
addition of AcCl (8 mL) to MeOH (35 mL). After being kept for 3 h
at rt, the mixture was diluted with CH2Cl2 (150 mL) and washed
with satd aq NaHCO3 solution. Aqueous layer was extracted back
with CH2Cl2 (3 ꢃ 70 mL), and the combined organic solutions were
concentrated. The residue was firstly purified by conventional sil-
ica gel column chromatography (toluene–CH3CN, 30:4), and then
3.20. Methyl
glucopyranosyl-(1?6)]-4-O-benzyl-b-
[2,3,4-tri-O-benzyl- -glucopyranosyl-(1?4)]-2-amino-6-O-
benzyl-2-deoxy- -galactopyranoside (31)
a
-L
-rhamnopyranosyl-(1?3)-[2,4-di-O-benzyl-
a-D-
D-glucopyranosyl)-(1?3)-
a
-D
a-D
To a solution of 29 (495 mg, 0.24 mmol) in a mixture of CH2Cl2
(10 mL) and MeOH (10 mL) was added 1 M methanolic MeONa
(3 mL). After being kept for 24 h, the mixture was neutralized with
Amberlyte IRA-120 (H+). The resin was filtered off, washed with
MeOH, and the combined filtrates were concentrated. Silica gel col-
umn chromatography (toluene–EtOH, 17:1) of the residue pro-
vided 30 (109.5 mg, 91%), Rf 0.2 (toluene–EtOH, 10:1). Deacylated
pentasaccharide 30 was dissolved in a mixture of CH3CN (2.0 mL)
and water (0.4 mL), and then di-isopropylamine (100 lL) and
DTT (30.3 mg, 0.196 mmol) were added. The mixture was kept at
rt until complete transformation of the starting azide, the solvents
were evaporated, and pure amine 31 (92.7 mg, 86%) was isolated as
a syrup by silica gel column chromatography (CHCl3–MeOH, 50:1
the anomers were separated by preparative HPLC on a 5
gel column (250 ꢃ 21.2 mm) in toluene–CH3CN (60:4) to provide
pure -tetrasaccharide 28 (0.96 g, 76% over two steps) as a syrup,
Rf 0.23 (toluene–CH3CN, 15:1), [
36.2 (c 1, CHCl3). 1H NMR
l silica
a
a
]
? 10:1), Rf 0.29 (CHCl3–MeOH, 7:1), [a]
77.0 (c 1, MeOH). 1H
D
D
(600 MHz, CDCl3, for the signals of sugar ring protons see Table
1): d 8.12–7.18 (55H, m, Ar); 5.11 (1H, m, PhCH2); 5.05–5.01;
4.97–4.91; 4.87–4.84; 4.71 (m, PhCH2); 3.39 (3H, s, CH3O); 3.13
(1H, br d, OH). 13C NMR (150 MHz, CDCl3, for the signals of sugar
ring carbons see Table 2): d 166.3; 165.7; 165.1; 164.5 (PhCO);
138.6; 138.4; 138.3; 138.1; 138.0 (ipso-C, PhCH2); 133.1; 133.0;
132.8; 132.6 (ipso-C, Bz); 130.4; 129.9; 129.8; 129.6; 129.3;
128.5; 128.4; 128.3; 128.2; 128.1; 128.0; 127.6; 127.4 (Ph), 75.7,
75.3, 74.9, 74.1 (PhCH2), 55.3 (CH3O). Anal. Calcd for
C95H93N3O24: C, 68.70; H, 5.64; N, 2.53. Found: C, 68.52; H, 5.80;
N, 2.71.
NMR (600 MHz, CD3OD, for the signals of sugar ring protons see
Table 1): d 7.42–7.09 (30H, m, Ar), 4.93–4.89, 4.84–4.63, 4.59–
4.53, 4.39–3.99 (PhCH2), 3.38 (OCH3). 13C NMR (150 MHz, CD3OD,
for the signals of sugar ring carbons see Table 2): d 139.9, 139.7,
139.5 (ipso-C, Ph), 129.6, 129.5, 129.4, 129.3, 129.2, 129.0, 128.9,
128.8, 128.7, 128.6 (Ph), 78.8, 76.3, 76.1, 75.8, 75.6, 74.7, 74.6,
74.4, 74.0 (PhCH2 and C4, C5 of b-D-Glc, C4 of a-L-Rha), 55.8
(OCH3). ESI HRMS: [M+H]+ calcd for C80H97NO24 + H: 1456.6479,
found 1456.6473.
3.21. Methyl
glucopyranosyl-(1?6)]-4-O-benzyl-b-
[2,3,4-tri-O-benzyl- -glucopyranosyl-(1?4)]-2-acetamido-6-
O-benzyl-2-deoxy- -galactopyranoside (32)
a
-L
-rhamnopyranosyl-(1?3)-[2,4-di-O-benzyl-
a-D-
D-glucopyranosyl)-(1?3)-
3.19. Methyl 2,3,4-tri-O-benzoyl-
a-L
-rhamnopyranosyl-(1?3)-
-glucopyranosyl-(1?6)]-2-
a-D
a-D
[3,6-di-O-acetyl-2,4-di-O-benzyl-
a-
D
O-benzoyl-4-O-benzyl-b-
benzoyl-2,3,4-tri-O-benzyl-
6-O-benzyl-2-deoxy- -galactopyranoside (29)
D
-glucopyranosyl)-(1?3)-[6-O-
a
-D
-glucopyranosyl-(1?4)]-2-azido-
Azide 30 (74.0 mg, 0.05 mmol) was reduced with DTT as de-
scribed in the previous experiment, the reaction mixture was con-
centrated, and toluene (2 ꢃ 2.0 mL) was evaporated from the
residue. A solution of the residue in MeOH (1.0 mL) was treated
a-D
A solution of donor 2 (404.0 mg, 0.66 mmol) and acceptor 28
(794 mg, 0.48 mmol) in CH2Cl2 (17 mL) was added to powdered
mol. sieve AW-300 (2.3 g), the resulting mixture was stirred at rt
with Ac2O (9.4 lL, 0.10 mmol) and Et3N (7.0 lL, 0.05 mmol) for
1 h, then the resulting mixture was taken to dryness. Column chro-
matography (toluene–EtOH, 60:1 ? 10:1) of the residue gave acet-
amide 32 (63.1 mg, 84% over two steps from azide 30) as a syrup, Rf
for 1 h, and then cooled to ꢂ20 °C. A solution of TMSOTf (25
0.07 mmol), prepared by mixing of 25 L TMSOTf and 0.5 mL of
CH2Cl2, was added. The same solution was added three times (each
time 10 L) within next 3 h. When TLC revealed full conversion of
lL,
l
0.18 (toluene–EtOH, 10:1). [
a]
71.6 (c 1, MeOH), 1H NMR
D
l
(600 MHz, CD3OD, for the signals of sugar ring protons see Table
1): d 7.42–7.18 (30H, m, Ar), 4.91, 4.89, 4.83 (3H, 3 d, PhCH2),
4.76–4.54 (6H, m, H-1I, PhCH2, H-21), 4,40 (1H, s, PhCH2), 4,28
(1H, d, Jgem = 11,1 Hz, PhCH2), 4,23 (1H, d, Jgem = 11,1 Hz, PhCH2);
3.34 (OCH3); 1.95 (CH3CON). 13C NMR (150 MHz, CD3OD, for the
signals of sugar ring carbons see Table 2): d 174.4 (CO); 140.2,
120.3, 139.9, 139.8, 139.6, 139.5 (ipso-C, PhCH2); 129.6, 129.5,
129.4, 129.4, 129.3, 129.0, 128.8, 128.7, 128.6, 128.5 (Ph); 76.4,
76.2, 76.0, 75.9, 74.6, 74.4, 74.2 (PhCH2); 55.7 (OCH3); 23.0
(CH3CON). ESI HRMS: [M+Na]+ calcd for C82H99NO25 + Na
1520.6404, found 1520.6415.
acceptor 28, the reaction mixture was diluted with CH2Cl2
(150 mL) and filtered through a pad of Celite. The filtrate was
washed with satd aq NaHCO3 and concentrated. The residue was
purified by silica gel column chromatography (toluene–CH3CN,
16:1). The isolated mixture (0.83 g) of pentasaccharide anomers
was separated by preparative HPLC on a 5
(250 ꢃ 21.2 mm) in toluene–CH3CN (735:40) to yield pure
mer 29 (0.80 g, 80%) as a syrup, Rf 0.16 (toluene–CH3CN, 3:0.2),
82.1 (c 1, CHCl3). 1H NMR (500 MHz, CDCl3, for the signals of
l
silica gel column
a
-iso-
[a]
D
sugar ring protons see Table 1): d 8.09–7.16 (50H, m, Ar); 5.07–

B. S. Komarova et al. / Carbohydrate Research 360 (2012) 56–68
67
3.22. Methyl
(1?6)] -b-
(1?4)]-2-acetamido-2-deoxy-
a
D
-
L
-rhamnopyranosyl-(1?3)-[
-glucopyranosyl-(1?3)-[ -glucopyranosyl-
-galactopyranoside (1a)
a
-
D
-glucopyranosyl-
Table 2): 172.5 (CO of Ala), 50.9 (CH of Ala), 18.0 (CH3 of Ala).
ESI HRMS: [M+Na]+ calcd for C34H60N2O25 + Na 919.3383, found
919.3374.
D-
a-D
a
-D
Partially benzylated derivative 32 (61.2 mg, 0.041 mmol) was
subjected to catalytic hydrogenolysis in MeOH (3 mL) in the pres-
ence of Pd(OH)2/C (33 mg). When the reaction was complete, the
mixture was filtered through a pad of Celite, the catalyst was
washed with MeOH, and the resulting filtrate was concentrated.
Product 1a (35.0 mg, 99%) was isolated by reversed phase C18
HPLC (250 ꢃ 10 mm column) in H2O–CH3CN (99:1), Rf 0.23
(n-butanol–EtOH–H2O–NH4OH, 5:5:4:1)as an amorphous powder,
3.25. Methyl
(1?6)]-b- -glucopyranosyl-(1?3)-[
2-(N-acetyl- -alanylamino)-2-deoxy-a-D
a
-
L
-rhamnopyranosyl-(1?3)-[
-glucopyranosyl-(1?4)]-
-galactopyranoside (1c)
a-D-glucopyranosyl-
D
a-D
L
Derivative 1c (26.0 mg, 80%) was prepared by N-acetylation of
1b (34.9 mg, 0.035 mmol) as described for 1a. Purification of the
product was accomplished by gel-permeation chromatography
on a TSK HW-40(S) column in 0.1 M AcOH. For 1c Rf 0.31 (n-buta-
[
a]
D 87.3 (c 1, H2O). 1H NMR (600 MHz, D2O, for the signals of sugar
nol–EtOH–H2O–NH4OH, 5:5:4:1), [
a
]
D
62.0 (c 1, H2O). 1H NMR
ring protons see Table 1): d 3.42 (OCH3); 2.02 (CH3CON). 13C NMR
(150 MHz, D2O, for the signals of sugar ring carbons see Table 2): d
176.1 (CO), 50.5 (OCH3), 23.6 (CH3CO). ESI HRMS: [M+Na]+ calcd
for C33H57NO25 + Na 890.3117, found 890.3112.
(600 MHz, D2O, for the signals of sugar ring protons see Table 1):
d 4.32 (1H, m, JCH,CH3 = 7,2 Hz, CH of Ala), 4.71 (OCH3), 2.04 (3H,
s, CH3CON), 1.39 (3H, d, JCH3,CH = 7,1 Hz, CH3 of Ala). 13C NMR
(150 MHz, D2O, for the signals of sugar ring carbons see Table 2):
d 177.0 (CH3CO); 175.5 (CO of Ala); 51.4 (CH of Ala); 48.2
(OCH3); 23.2 (CH3CON); 18.0 (CH3 of Ala). ESI HRMS: [M+Na]+ calcd
for C36H62N2O26 + Na 961.3494, found 961.3489.
3.23. Methyl
(1?6)] -b - -glucopyranosyl-(1?3)-[
(1?4)]-2-[N-(tert-butoxycarbonyl)- -alanylamino]-2-deoxy-
-galactopyranoside (35)
a
-
L
-rhamnopyranosyl-(1?3)-[
a-D-glucopyranosyl-
D
a-D-glucopyranosyl-
L
a-
D
Acknowledgments
Azide 30 (22.6 mg, 0.015 mmol) was reduced with DTT as de-
The authors are grateful to Dr. A. A. Grachev and Dr. A. O. Chiz-
hov for recording of NMR and mass spectra respectively.
scribed for 31, the reaction mixture was concentrated and coevap-
orated twice with toluene (1.0 mL). To a solution of the residue in
dry DMF (0.5 mL) alanine active ester 33 (6.5 mg, 0.023 mmol) and
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matography (toluene–EtOH, 50:1), Rf 0.29 (toluene–EtOH, 8.5:1).
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s, (CH3)3CO), 1.30 (3H, d, CH3 of Ala). 13C NMR (150 MHz, CD3OD,
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EtOH–H2O–NH4OH, 5:5:4:1),
[
a]
73.6 (c 1, H2O). 1H NMR
D
(600 MHz, D2O, for the signals of sugar ring protons see Table 1):
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(1?6)] -b- -glucopyranosyl-(1?3)-[
(1?4)]-2-( -alanylamino)-2-deoxy-a-D
a
-
L
-rhamnopyranosyl-(1?3)-[
-glucopyranosyl-
-galactopyranoside (1b)
a-D-glucopyranosyl-
D
a-D-D
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L
Anhydrous TFA (2.0 mL) was added to lyophilized and dried
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13C NMR (150 MHz, D2O, for the signals of sugar ring carbons see

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