Synthesis of 3,6-branched oligomannoside fragments of the mannan from Candida albicans cell wall corresponding to the antigenic factor 4

Article abstract of DOI:10.1016/j.carres.2009.11.012

3-Aminopropyl glycosides of 3,6-branched penta- and hexamannoside fragments of the cell wall mannan from Candida albicans, corresponding to the antigenic factor 4, have been synthesized. Subsequent coupling of both oligosaccharides with BSA using the squa

Full text of DOI:10.1016/j.carres.2009.11.012

Carbohydrate Research 345 (2010) 1283–1290
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of 3,6-branched oligomannoside fragments of the mannan
from Candida albicans cell wall corresponding to the antigenic factor 4
a
a
b
b
ˇ
´
Alexander A. Karelin , Yury E. Tsvetkov , Lucia Paulovicová , Slavomír Bystricky ,
b
a,
*
ˇ
Ema Paulovicová , Nikolay E. Nifantiev
a Laboratory of Glycoconjugate Chemistry, N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky Prospect 47, 119991 Moscow, Russia
b Centre of Excellence GLYCOMED, Department of Immunochemistry of Glycoconjugates, Institute of Chemistry, Center for Glycomics, Slovak Academy of Sciences, Bratislava, Slovakia
a r t i c l e i n f o
a b s t r a c t
Article history:
3-Aminopropyl glycosides of 3,6-branched penta- and hexamannoside fragments of the cell wall mannan
from Candida albicans, corresponding to the antigenic factor 4, have been synthesized. Subsequent cou-
pling of both oligosaccharides with BSA using the squarate procedure provided corresponding
neoglycoconjugates.
Received 6 September 2009
Received in revised form 25 October 2009
Accepted 7 November 2009
Available online 12 November 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Candida albicans
Mannan
Antigenic factor 4
Oligomannosides
Neoglycoconjugates
Synthesis
1. Introduction
Herewith we report the synthesis of 3-aminopropyl glycosides of
branched penta- and hexamannosides related to the structures 1
The opportunistic pathogenic fungus Candida albicans is able to
cause severe infection in immunocompromised patients. The main
surface antigen of Candida responsible for its antigenic specificity is
mannan, which represents the carbohydrate part of the cell wall
mannoprotein.1 It has been shown that the determinants of the
antigenic factor 4 are oligosaccharide structures 1 and 2 featuring
the presence of the 3,6-branching.2 Oligomannosides correspond-
ing to the antigenic factors 1, 5, 6, 9, and some others have been
synthesized3–13 (for review see Ref. 14). Surprisingly, only one syn-
thesis of an oligosaccharide corresponding to the factor 4, viz. hep-
tasaccharide 2 as the methyl glycoside, has been described.15
and 2 and their conjugates with BSA. These products were pre-
pared within a project focused on the synthesis11,12 and glycoim-
munological studies16 of oligomannoside antigenic determinants
of Candida.
2. Results and discussion
The protected pentasaccharide 8 was prepared by glycosylation
of the known acceptor 711 with branched trisaccharide thioglyco-
side 6 (Scheme 1). The known 3,6-ditrityl ether 317 was used as a
* Corresponding author. Tel./fax: +7 499 135 8784.
E-mail address: nen@ioc.ac.ru (N.E. Nifantiev).
0008-6215/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2009.11.012

1284
A. A. Karelin et al. / Carbohydrate Research 345 (2010) 1283–1290
precursor of the 3,6-bis-glycosylated mannose unit. Conventional
benzoylation of free hydroxyl groups in 3 followed by Bredereck
bis-glycosylation of 4 with mannosyl bromide 5 as described17
smoothly afforded trisaccharide thioglycoside 6. Subsequent NIS–
TfOH-promoted coupling of 6 and 7 produced target pentamanno-
side 8.
Hexasaccharide 18 was assembled from three disaccharide syn-
thetic blocks (Scheme 2). As the target product contains different
glycosyl substituents at O-3 and O-6 in the branching point, it
was necessary to discriminate between these positions in the cor-
responding monosaccharide precursor. With this goal in mind, 6-
O-trityl thioglycoside 918 was subjected to selective silylation19
R1O
R2O
R1O
BzO
BzO
BzO
OBz
OR2
O
OBz
O
BzO
BzO
BzO
OBz
OBz
O
BzO
BzO
BzO
SEt
O
O
OBz
3 R1=Tr, R 2=H
4 R1=Tr, R 2=Bz
BzO
BzO
BzO
O
a
BzO
O
O
O
OBz
O
b
SEt
6
BzO
O
BnO
BnO
O
O
BnO
BnO
c
OH
O
BnO
BnO
BnO
BzO
BzO
BzO
OBz
O
BnO
BnO
BnO
O
O
O
O
BnO
Br
BnO
5
O(CH2)3NHCOCF3
O(CH2)3NHCOCF3
7
8
Scheme 1. Synthesis of pentasaccharide 8. Reagents and conditions: (a) BzCl, pyridine, rt, 65%; (b) AgOTf, CH2Cl2, À10 °C, 64%; (c) NIS, TfOH, CH2Cl2, À20 to À30 °C, 57%.
BzO
BzO
BzO
OBz
O
BzO
BzO
BzO
O
OBz
O
OBz
O
BzO
R1O
R3O
R2O
OR3
O
RO
BnO
BnO
O
O
OBz
O
7
5
c
O
BzO
BnO
BnO
d
TBDMSO
SEt
O
SEt
9R1=Tr, R2=R3=H
a
b
O
BnO
10R1=Tr, R2=TBDMS, R3=H
11R1=Tr, R2=TBDMS, R3=Bz
BnO
12
O(CH2)3NHCOCF3
13 R=TBDMS
14 R=H
e
BzO
OBz
O
BzO
BnO
BnO
BzO
OH
OBz
O
BzO
f
O
BzO
BnO
BnO
+
O
BnO
BzO
O
SEt
O
CCl3
NH
BnO
15
16
SEt
17
BzO
BzO
BzO
BnO
BnO
OBz
BzO
BzO
BzO
OBz
O
BzO
BzO
BzO
O
OBz
O
BzO
BzO
BzO
BnO
BnO
OBz
O
O
O
O
O
OBz
BnO
O
OBz
O
BzO
O
O
BzO
+
O
O
BnO
d
BnO
BnO
O
BnO
BnO
14 + 17
O
O
O
BnO
BnO
BnO
BnO
BnO
BnO
O
O
O
O
19
18
BnO
BnO
O(CH2)3NHCOCF3
O(CH2)3NHCOCF3
Scheme 2. Synthesis of hexasaccharide 19. Reagents and conditions: (a) TBDMSCl, imidazole, pyridine, rt; (b) BzCl, pyridine, 50 °C, 70% in two steps; (c) AgOTf, CH2Cl2,
À10 °C, 73%; (d) NIS, TfOH, CH2Cl2, À20 to À30 °C, 70% of 13, 44% of 18; (e) CF3CO2H, CHCl3, rt, 77%; (f) TMSOTf, CH2Cl2, À20 °C, 60%.

A. A. Karelin et al. / Carbohydrate Research 345 (2010) 1283–1290
1285
with tert-butyldimethylsilyl chloride to give 3-O-silyl derivative
10. Conventional benzoylation of 10 afforded desirable dibenzoate
11 with different protecting groups at O-3 and O-6. Direct glycosyl-
ation of the latter at O-6 with mannosyl bromide 5 under Brede-
reck conditions produced the necessary (1?6)-linked
disaccharide glycosyl donor 12. NIS–TfOH-promoted coupling of
disaccharide blocks 12 and 7 resulted in the formation of tetrasac-
charide 13; subsequent acidic removal of the TBDMS group gave
tetrasaccharide acceptor 14. The third, (1?2)-linked donor block
17 was obtained in acceptable yield (60%) by coupling of 2-OH
thioglycoside 1620 with imidate 15.21 It is noteworthy that
AgOTf-promoted glycosylation of 16 with bromide 5 provided
disaccharide 17 in much lower yield (ꢀ20%) due to the predomi-
nant SEt group transfer from acceptor 16 to donor 5. This side reac-
tion was also observed by TLC in the reaction of compounds 15 and
16 but to a much smaller extent (ꢀ20%, TLC). At the final step, NIS–
TfOH-promoted glycosylation of 14 with 17 produced a mixture of
the target hexasaccharide 18 and its b-anomer 19, which were iso-
lated in 44% and 14% yield, respectively. The configuration of the
(1?3)-glycoside bond in the products 18 and 19 was deduced from
groups were removed by catalytic hydrogenolysis; then O,N-deac-
ylation was achieved by treatment with either the strongly basic
anion-exchanger Amberlyst A-26 (OHÀ) or with sodium methoxide
followed by sodium hydroxide (Scheme 3).
Aminopropyl glycosides 20 and 21 were conjugated with BSA
using the squarate protocol.22–25 Reaction of 20 and 21 with
diethyl squarate at pH 7 afforded monoamides 22 and 23, which
were then coupled with BSA at pH 9 to produce target conjugates
24 and 25 (Scheme 4). MALDI-TOFMS data showed that 24 and 25
contained on average 10 and 9 oligosaccharide units per protein
molecule, respectively. Immunological investigations of obtained
oligosaccharides and glycoconjugates thereof are in the progress
and will be reported elsewhere.
3. Experimental
3.1. General methods
NMR spectra were recorded on Bruker DRX-500 and Bruker
AM-300 instruments. Spectra of protected oligosaccharides were
measured for solutions in CDCl3, and 1H NMR chemical shifts
were referenced to the residual signal of CHCl3. NMR spectra of
free oligosaccharides were measured for solutions in D2O using
acetone (dH 2.225, dC 31.45) as internal standard. Monosaccha-
ride residues in oligosaccharides are numbered by the Roman
numerals starting from the reducing end. MALDI-TOF mass spec-
tra were obtained on a Bruker Ultraflex mass spectrometer with
2,5-dihydroxybenzoic acid as the matrix in the positive reflector
mode. HRESIMS were obtained on Finnigan LTQ FT (Thermo
Scientific) and MicrOTOF II (Bruker Daltonics) instruments. Opti-
cal rotations were measured using a JASCO DIP-360 polarimeter
at 18–22 °C in CHCl3 in the case of the protected and partially
protected derivatives and in water in the case of free oligosac-
charides. TLC was performed on Silica Gel 60 F254 plates
(E. Merck), and visualization was accomplished using UV light
or by charring with 10% H3PO4 in EtOH. Column chromatography
corresponding 1JC1,H1 coupling constants values (173 Hz for
mer 18 and 155 Hz for b-anomer 19).
a-ano-
Protected oligomannosides 8 and 18 were converted into the
corresponding free sugars 20 and 21 in two steps: first benzyl
HO
HO
HO
OH
O
HO
HO
HO
OR
O
O
OH
O
HO
O
HO
O
a
O
HO
8 / 18
HO
HO
HO
O
O
was carried out on Silica Gel 60 (40–63
meation chromatography of protected oligosaccharides was per-
formed on Bio-Beads SX-3 (Bio-Rad Laboratories) column
lm, E. Merck). Gel-per-
HO
O(CH2)3NH2
a
20 R = H
21 R = α-D-Man
(13 Â 450 mm) in toluene. Gel-permeation chromatography of
free oligosaccharides was performed on a column of TSK HW-
40 (S) gel (25 Â 800 mm) in 0.1 M AcOH. All reactions involving
air- or moisture-sensitive reagents were carried out using dry
solvents under dry argon.
p
Scheme 3. Synthesis of free oligomannosides 20 and 21. Reagents and conditions:
(a) (1) H2, Pd/C, MeOH, rt; (2) Amberlyst A-26 (OHÀ), water, or MeONa, MeOH then
NaOH, aq MeOH, rt, 57% of 20, 65% of 21.
HO
HO
HO
OR
O
OH
O
HO
HO
HO
OR
O
OH
O
HO
HO
HO
HO
HO
HO
O
O
OH
OH
O
HO
O
HO
O
O
a
b
HO
O
HO
20 / 21
O
O
HO
O
HO
HO
HO
HO
HO
O
HO
HO
O
O
O
O
O
O
O
HO
HO
O(CH2)3NH
NH
BSA
9-10
O(CH2)3NH
OEt
22 R = H
23 R = α-D-Man
24 R = H
25 R = α-D-Man
p
p
Scheme 4. Synthesis of oligomannoside–BSA conjugates 24 and 25. Reagents and conditions: (a) diethyl squarate, water, EtOH, pH 7, rt, 89% of 22, 80% of 23; (b) BSA, 350 mM
KHCO3, 70 mM Na2B4O7Á10H2O, pH 9, rt.

1286
A. A. Karelin et al. / Carbohydrate Research 345 (2010) 1283–1290
3.2. Ethyl 2,4-di-O-benzoyl-1-thio-3,6-di-O-trityl-
mannopyranoside (4)
a
-
D
-
was quenched with pyridine, diluted with CHCl3, and filtered
through a Celite layer. The filtrate was washed with 1 M aq Na2S2O3
soln and water, concentrated, and toluene was twice evaporated
from the residue. Column chromatography of the residue (15:1 tolu-
ene–EtOAc) afforded pentasaccharide 8 (73 mg, 57%) as a colorless
BzCl (0.76 mL, 6.52 mmol) was added to soln of 3 (1.15 g,
1.64 mmol) in pyridine (4 mL). The mixture was kept for 24 h at
room temperature, diluted with CHCl3, washed with satd NaHCO3,
concentrated, and toluene was evaporated twice from the residue.
Column chromatography (8:1 light petroleum–EtOAc) provided 4
foam; [a D
]
À17.4 (c 0.5, CHCl3). 1H NMR (500 MHz, CDCl3): d 8.41,
8.17–6.78 (m, 80H, 16Ph-H), 6.80 (m, 1H, NH), 6.13 (t, 1H,
J3,4 = J4,5 = 10.0 Hz, H-4IV), 6.12 (t, 1H, J3,4 = J4,5 = 10.0 Hz, H-4V), 6.02
(t, 1H, J3,4 = J4,5 = 9.9 Hz, H-4III), 5.99 (dd, 1H, J2,3 = 3.0, J3,4 = 10.2 Hz,
H-3V), 5.92 (br s, 1H, H-2V), 5.82 (br s, 1H, H-2III), 5.76 (dd, 1H,
J2,3 = 2.9, J3,4 = 10.1 Hz, H-3IV), 5.42 (s, 1H, H-1II), 5.41 (br s, 1H, H-
2IV), 5.37 (s, 1H, H-1IV), 5.12 (s, 1H, H-1III), 5.10 (s, 1H, H-1V), 4.41
(d, 1H, J = 10.9 Hz, PhCH2), 4.88 (s, 1H, H-1I), 4.85 (d, 1H,
J = 11.0 Hz, PhCH2), 4.83 (d, 1H, J = 10.7 Hz, PhCH2), 4.71 (dd, 1H,
J2,3 = 3.2, J3,4 = 9.9 Hz, H-3III), 4.68 (d, 1H, J = 11.1 Hz, PhCH2), 4.65–
4.56(m, 5H, 5PhCH2), 4.56–4.45 (m, 5H, H-5III, H-5IV, H-6aIV, 2 PhCH2),
4.43 (m, 1H, H-6aV), 4.39 (d, 1H, J = 12.2 Hz, PhCH2), 4.36 (m, 1H,
H-5V), 4.26 (dd, 1H, J5,6 = 3.7, J6a,6b = 12.2 Hz, H-6bV), 4.22 (s,
1H, H-2I), 4.12–4.08 (m, 2H, H-2II, H-6bIV), 4.01–3.94 (m, 3H, H-3II,
H-4II, H-6aIII), 3.92–3.86 (m, 2H, H-3I, H-5II), 3.74–3.70 (m, 3H, H-5I,
(976 mg, 65%) as a colorless foam; [
a]
+11.1 (c 1, CHCl3). 1H
D
NMR (500 MHz, CDCl3): d 8.29–8.05, 7.37–6.99 (m, 40H, 8Ph-H),
6.01 (t, 1H, J3,4 = J4,5 = 9.7 Hz, H-4), 5.37 (s, 1H, H-1), 4.48 (s, 1H,
H-2), 4.19 (m, 1H, H-5), 4.08 (dd, J2,3 = 2.6, J3,4 = 9.7 Hz, 1H, H-3),
3.14 (m, 2H, H-6a, H-6b), 2.52–2.64 (m, 2H, SCH2CH3), 1.35 (t, 3H,
SCH2CH3); 13C NMR (126 MHz, CDCl3): d 165.7, 165.2 (PhCO),
144.0, 143.7, 133.3, 132.8, 130.6–126.0 (Ph), 86.4 (Ph3C), 81.6 (C-
1), 73.9 (C-2), 71.3 (C-5), 71.2 (C-3), 68.3 (C-4), 62.6 (C-6), 25.4
(SCH2CH3), 14.8 (SCH2CH3). Anal. Calcd for C60H52O7S: C, 78.58;
H, 5.71. Found: C, 78.51; H, 5.79.
3.3. Ethyl 2,3,4,6-tetra-O-benzoyl-
[2,3,4,6-tetra-O-benzoyl- -mannopyranosyl-(1?6)]-2,4-di-O-
benzoyl-1-thio- -mannopyranoside (6)
a-D-mannopyranosyl-(1?3)-
H-6a , H-6bIII), 3.70–3.60 (m, 3H, H-4I, H-6b , H-6aII), 3.59–3.52 (m,
I
I
a-D
II
a-
D
2H, H-6b
,
OCH2CH2CH2N), 3.27–3.21 (m, 2H, OCH2CH2CH2N,
OCH2CH2CH2N), 3.18 (m, 1H, OCH2CH2CH2N), 1.58 (m, 1H,
OCH2CH2CH2N), 1.49 (m, 1H, OCH2CH2CH2N); 13C NMR (126 MHz,
CDCl3): d 166.1–164.7 (PhCO), 138.4–138.0, 133.6–132.8, 130.3–
127.4 (Ph), 100.4 (1C, C-1II), 99.3 (2C, C-1I, C-1IV), 99.2 (1C, C-1III),
97.2 (1C, C-1V), 80.0 (1C, C-3I), 78.5 (1C, C-3II), 77.5 (1C, C-2II), 76.1
(1C, C-3III), 75.4 (1C, C-2I), 75.2 (2C, PhCH2), 75.0 (1C, C-4I), 74.4
(1C, C-4II), 73.3, 72.2, 72.7 (3C, PhCH2), 72.0 (2C, C-5I, PhCH2), 71.9
(1C, C-5II), 71.7 (1C, C-2III), 70.7 (1C, C-3V), 70.2 (1C, C-2IV), 70.0
(1C, C-2V), 69.6 (1C, C-5IV), 69.5 (1C, C-3IV), 69.4 (1C, C-6I), 69.3 (1C,
C-5III), 68.8 (2C, C-5V, C-6II), 68.0 (1C, C-4III), 66.3 (1C, C-4V), 66.1
(1C, C-4IV), 65.9 (1C, C-6III), 65.6 (1C, OCH2CH2CH2N), 62.5 (1C, C-
6V), 61.8 (1C, C-6IV), 37.6 (1C, OCH2CH2CH2N), 27.9 (1C,
OCH2CH2CH2N). Anal. Calcd for C147H134F3NO37: C, 68.87; H, 5.27;
N, 0.55. Found: C, 68.57; H, 5.58; N, 0.53.
Molecular sieves 4 Å (350 mg) were added to a soln of thioglyco-
side 4 (93 mg, 101 mol) and mannosyl bromide 5 (267 mg,
405 mol) in CH2Cl2 (3 mL), the mixture was stirred for 30 min at
room temperature, cooled to À20 °C, and then AgOTf (156 mg,
608
until disappearance of 4 (TLC monitoring), quenched with triethyl-
amine, diluted with CHCl3, and filtered through a Celite layer. The
filtrate was washed with 1 M aq Na2S2O3, water, and the solvent
was evaporated. The residue was subjected to column chromatogra-
phy (20:1 toluene–EtOAc) to give 6 (102 mg, 64%) as a colorless
l
l
l
mol) was added. The resulting mixture was stirred at À10 °C
foam; [
a
]
À36.0 (c 0.5, CHCl3). 1H: NMR (500 MHz, CDCl3): d
D
8.30–7.11 (m, 50H, 10Ph-H), 6.10 (t, 1H, J3,4 = J4,5 = 10.1 Hz, H-4III),
5.98 (t, 1H, J3,4 = J4,5 = 10.0 Hz, H-4II), 5.92 (dd, 1H, J2,3 = 3.2,
J3,4 = 10.1 Hz, H-3III), 5.89 (t, 1H, J3,4 = J4,5 = 9.9 Hz, H-4I), 5.79 (dd,
1H, J1,2 = 1.0, J2,3 = 3.3 Hz, H-2I), 5.72 (dd, 1H, J1,2 = 1.8, J2,3 = 2.9 Hz,
H-2III), 5.67 (dd, 1H, J2,3 = 3.0, J3,4 = 10.1 Hz, H-3II), 5.61 (s, 1H, H-
1I), 5.34 (s, 1H, H-1II), 5.33 (d, 1H, J2,3 = 3.0 Hz, H-2II), 5.10 (d, 1H,
J1,2 = 1.8 Hz, H-1III), 4.70 (m, 1H, H-5I), 4.60 (dd, 1H, J2,3 = 3.4,
J3,4 = 9.9 Hz, H-3I), 4.58–4.52 (m, 2H, H-6aII, H-6aIII), 4.49–4.44 (m,
2H, H-5II, H-5III), 4.37–4.30 (m, 2H, H-6bII, H-6bIII), 4.17 (dd, 1H,
3.5. 3-Aminopropyl
pyranosyl-(1?6)]-
anosyl-(1?2)- -mannopyranoside (20)
a
-
D
-mannopyranosyl-(1?3)-[ -manno-
a
-
D
a
-
D
-mannopyranosyl-(1?2)-a-D-mannopyr-
a-D
Pd(OH)2/C (60 mg) was added to a soln of pentamannoside 8
(61 mg, 24 mol) in MeOH (2 mL) and EtOAc (1 mL). The mixture
l
I
I
J5,6 = 6.8, J6a,6b = 10.8 Hz, H-6a ), 3.75 (d, 1H, J6a,6b = 10.8 Hz, H-6b ),
2.83 (m, 2H, SCH2CH3), 1.43 (t, 3H, SCH2CH3); 13C NMR (126 MHz,
CDCl3): d 166.1–164.6 (PhCO), 133.6–132.9, 130.1–128.1 (Ph), 99.5
(1C, C-1II), 97.4 (1C, C-1III) 81.8 (1C, C-1I), 76.7 (1C, C-3I), 73.4 (1C,
C-2I), 70.2 (1C, C-2III), 70.1 (1C, C-3III), 70.0 (2C, C-5I, C-2II), 69.6
(1C, C-5II), 69.2 (1C, C-3II), 68.8 (1C, C-4I), 68.8 (1C, C-5III), 66.9 (1C,
C-6I), 66.5 (1C, C-4III), 66.4 (1C, C-4II), 62.6 (2C, C-6II, C-6III), 25.5
(1C, SCH2CH3), 14.9 (SCH2CH3). Anal. Calcd for C90H76O25S: C,
68.00; H, 4.82. Found: C, 68.39; H, 5.22
was stirred under hydrogen (1 atm) at room temperature for 20 h
and then filtered through a Celite layer. The catalyst was carefully
washed with MeOH, and the combined filtrates were concentrated.
The residue was dissolved in water (2 mL) and treated with anion-
exchange resin Amberlyst A-26 (OHÀ) (1.5 mL) for 12 h. The resin
was filtered off, and the filtrate was concentrated. The residue
was subjected to gel chromatography, appropriate fractions were
collected, and lyophilized to give 20ÁAcOH (12 mg, 57%) as a white
amorphous powder; [a]
+83.7 (c 0.3, H2O). 1H NMR (500 MHz,
D
D2O): d 5.21 (s, 1H, H-1II), 5.12 (s, 1H, H-1IV), 5.06 (s, 1H, H-1I),
5.01 (s, 1H, H-1III), 4.90 (s, 1H, H-1V), 4.20 (br s, 1H, H-2III), 4.10
(br s, 1H, H-2II), 4.05 (br s, 1H, H-2IV), 4.00–3.95 (m, 2H, H-2V,
H-6aIII), 3.95–3.85 (m, 11H, H-2I, H-3I, H-3II, H-3III, H-3IV, H-4III,
3.4. 3-Trifluoroacetamidopropyl 2,3,4,6-tetra-O-benzoyl-
mannopyranosyl-(1?3)-[2,3,4,6-tetra-O-benzoyl- -manno-
pyranosyl-(1?6)]-2,4-di-O-benzoyl- -mannopyranosyl-
(1?2)-3,4,6-tri-O-benzyl- -mannopyranosyl-(1?2)-3,4,6-tri-
O-benzyl- -mannopyranoside (8)
a-D-
a
-D
a-D
H-5III, H-6a , H-6a
,
H-6a
,
H-6aV), 3.85–3.80 (m, 2H, H-3V,
I
II
IV
a-
D
OCH2CH2CH2N), 3.80–3.68 (m, 8H, H-4I, H-5II, H-5IV, H-6b , H-6b
,
I
II
a-
D
H-6bIII, H-6bIV, H-6bV), 3.68–3.61 (m, 4H, H-4II, H-4IV, H-4V, H-5V),
3.61–3.56 (m, 2H, H-5I, OCH2CH2CH2N), 3.11 (m, 2H, OCH2CH2-
CH2N), 1.98 (m, 2H, OCH2CH2CH2N), 1.90 (s, 3H, CH3CO2H); 13C
NMR (126 MHz, D2O): d 103.7 (1C, C-1IV), 103.6 (1C, C-1III), 102.2
(1C, C-1II), 100.7 (1C, C-1V), 99.7 (1C, C-1I), 80.4 (2C, C-2I, C-2II),
79.7 (1C, C-3III), 75.0 (1C, C-5II), 74.8 (1C, C-5IV), 74.3 (1C, C-5I),
74.2 (1C, C-5V), 72.8 (1C, C-5III), 72.0 (1C, C-3V), 71.8 (1C, C-3IV),
71.7 (1C, C-3I), 71.5 (2C, C-2IV, C-2V), 71.4 (1C, C-3II), 71.0 (1C,
Molecular sieves 4 Å (150 mg) were added to a soln of acceptor 7
(52 mg, 50 mol) and donor 6 (87 mg, 55 mol) in CH2Cl2 (3 mL); the
mixture was stirred for 30 min at room temperature and cooled to
À10 °C. NIS (23 mg, 100 mol) was added, the mixture was stirred
for 10 min, then the temperature was decreased to À30 °C and TfOH
(2 L, 20 mol) was added. The reaction mixture was stirred at
À20 °C to À30 °C until TLC showed disappearance of 7. The reaction
l
l
l
l
l

A. A. Karelin et al. / Carbohydrate Research 345 (2010) 1283–1290
1287
C-2III), 68.5, 68.4, 68.3, 68.2 (4C, C-4I, C-4II, C-4IV, C-4V), 67.1 (1C, C-
3.8. 3-Trifluoroacetamidopropyl 2,3,4,6-tetra-O-benzoyl-
mannopyranosyl-(1?6)-2,4-di-O-benzoyl-3-O-(tert-butyldi-
methylsilyl)- -mannopyranosyl-(1?2)-3,4,6-tri-O-benzyl-
-mannopyranosyl-(1?2)-3,4,6-tri-O-benzyl-
mannopyranoside (13)
a
-
D
-
4III), 66.6 (1C, C-6III), 66.4 (1C, OCH2CH2CH2N), 62.7, 62.5, 62.4 (4C,
C-6I, C-6II, C-6IV, C-6V), 38.9 (1C, OCH2CH2CH2N), 28.1 (1C,
OCH2CH2CH2N). HRESIMS: found m/z 886.3416 [M+H]+; calcd for
a-D
a
-
D
a-D-
C33H60NO26 886.3404.
3.6. Ethyl 2,4-di-O-benzoyl-3-O-(tert-butyldimethylsilyl)-1-
thio-6-O-trityl- -mannopyranoside (11)
Molecular sieves 4 Å (150 mg) were added to a soln of acceptor
7 (72 mg, 70 mol) and donor 12 (87 mg, 77 mol) in CH2Cl2
(3 mL); the mixture was stirred for 30 min at room temperature
and cooled to À10 °C. NIS (35 mg, 150 mol) was added, the mix-
ture was stirred for 10 min, then the temperature was decreased to
L, 30 mol) was added. The reaction mixture
a
-D
l
l
Trityl chloride (171 mg, 614
DMAP were added to a soln of ethyl 1-thio-
(110 mg, 491 mol) in pyridine (5 mL). The mixture was stirred at
80 °C overnight and cooled to room temperature. Then TBDMSCl
(82 mg, 543 mol) and imidazole (65 mg, 980 mol) were added,
l
mol) and a catalytic amount of
l
a
-D
-mannopyranoside
l
À25 °C and TfOH (3
l
l
was stirred at À20 °C to À30 °C until TLC showed disappearance of
starting 7. The reaction was quenched with a drop of pyridine, di-
luted with CHCl3, and filtered through a Celite layer. The filtrate
was washed with 1 M aq Na2S2O3 soln and water, concentrated,
and toluene was twice evaporated from the residue. Column chro-
matography of the residue (10:1 toluene–EtOAc) afforded 13
l
l
the mixture was kept at room temperature for 16 h and concen-
trated. Residual pyridine was removed by coevaporation with
toluene, the residue was filtered through a silica gel layer in tolu-
ene–EtOAc (20:1), and the filtrate was concentrated to give crude
10. To a solution of 10 in pyridine (4 mL) was added BzCl (230
lL,
(103 mg, 70%) as a colorless foam; [a]
+2.3 (c 1, CHCl3). 1H NMR
D
1.97 mmol), the resulting mixture was stirred at 50 °C for 16 h, di-
luted in CHCl3, washed with satd NaHCO3, concentrated and toluene
was twice evaporated from the residue. Column chromatography of
the residue (15:1 petroleum ether–EtOAc) provided 11 (270 mg,
(500 MHz, CDCl3): d 8.23, 7.99, 7.93, 7.81, 7.59–7.16, 6.88 (60H,
12Ph-H), 6.74 (m, 1H, NH), 6.06 (t, 1H, J3,4 = J4,5 = 10.0 Hz, H-4IV),
5.93 (dd, 1H, J2,3 = 3.1, J3,4 = 10.1 Hz, H-3IV), 5.86 (br s, 1H, H-2IV),
5.81 (t, 1H, J3,4 = J4,5 = 9.9 Hz, H-4III), 5.56 (br s, 1H, H-2III), 5.36 (s,
1H, H-1II), 5.09 (s, 1H, H-1IV), 5.02 (s, 1H, H-1III), 4.86 (s, 1H, H-
1I), 4.84 (d, 1H, J = 10.7 Hz, PhCH2), 4.81 (d, 1H, J = 10.7 Hz, PhCH2),
4.79 (d, 1H, J = 11.2 Hz, PhCH2), 4.67–4.56 (m, 6H, PhCH2), 4.54 (m,
1H, H-3III), 4.50–4.45 (m, 2H, H-5III, PhCH2), 4.40 (m, 2H, PhCH2),
4.31 (dd, 1H, J5,6 = 2.1, J6,6 = 12.2 Hz, H-6aIV), 4.26 (m, 1H, H-5IV),
4.20–4.16 (m, 2H, H-2I, H-6bIV), 4.08 (br s, 1H, H-2II), 3.97–3.83
(m, 5H, H-3I, H-3II, H-4II, H-5II, H-6aIII), 3.72–3.66 (m, 2H, H-5I, H-
70%) as white foam; [a]
+10.3 (c 1, CHCl3). 1H NMR (300 MHz,
D
CDCl3): d 8.23–7.06 (m, 25H, 5Ph-H), 5.72 (t, 1H, J3,4 = J4,5 = 9.8 Hz,
H-4), 5.53 (s, 1H, H-1), 5.47 (d, 1H, J2,3 = 3.3 Hz, H-2), 4.44 (m, 1H,
H-5), 4.30 (dd, 1H, J2,3 = 3.3, J3,4 = 9.8 Hz, H-3), 3.29 (m, 2H, H-6a,
H-6b), 2.81 (m, 2H, SCH2CH3), 1.43 (t, 3H, SCH2CH3), 0.62 (s, 9H,
tert-Bu–Si), 0.02 (s, 3H, CH3–Si), À0.17 (s, 3H, CH3–Si); 13C NMR
(75.47 MHz, CDCl3): d 166.0, 164.3 (PhCO), 143.7, 134.5–132.8,
130.5–126.7 (Ph), 82.2 (1C, C-1), 74.6 (1C, C-2), 71.1 (1C, C-5), 70.1
(1C, C-4), 69.8 (1C, C-3), 62.7 (1C, C-6), 25.4 (SCH2CH3), 25.2 (3C,
(CH3)3C), 14.8 (SCH2CH3), À4.9, À5.2 (2C, Si-CH3). Anal. Calcd for
6a ), 3.66–3.59 (m, 4H, H-4I, H-6aII, H-6aIII, H-6b ), 3.56 (m, 1H,
OCH2CH2CH2N), 3.47 (m, 1H, H-6bII), 3.27–3.21 (m, 2H,
OCH2CH2CH2N, OCH2CH2CH2N), 3.17 (m, 1H, OCH2CH2CH2N),
1.59 (m, 1H, OCH2CH2CH2N), 1.49 (m, 1H, OCH2CH2CH2N), 0.67
(s, 9H, tert-Bu–Si), 0.07 (s, 3H, CH3–Si), À0.10 (s, 3H, CH3–Si); 13C
NMR (126 MHz, CDCl3): d 166.0–164.9 (6C, PhCO), 138.4–138.2,
133.4–132.9, 130.0–127.5 (Ph), 100.7 (1C, C-1II), 99.3 (2C, C-1I, C-
1III), 97.2 (1C, C-1IV), 79.8 (1C, C-3I), 78.8 (1C, C-3II), 76.7 (1C, C-
2II), 75.9 (1C, C-2I), 75.2 (2C, PhCH2), 75.0 (1C, C-4I), 74.4 (1C, C-
4II), 73.3 (2C, PhCH2), 72.7 (1C, C-2III), 72.6 (1C, PhCH2), 72.0 (1C,
C-5I), 71.9 (1C, PhCH2), 71.7 (1C, C-5II), 70.6 (1C, C-3IV), 70.1 (1C,
C-2IV), 69.4 (2C, C-4III, C-6I), 69.2 (1C, C-5III), 69.0 (1C, C-3III), 68.7
(1C, C-5IV), 68.5 (1C, C-6II), 66.3 (1C, C-4IV), 66.1 (1C, C-6III), 65.6
(1C, OCH2CH2CH2N), 62.5 (1C, C-6IV), 37.6 (1C, OCH2CH2CH2N),
27.9 (1C, OCH2CH2CH2N) 25.4 (3C, (CH3)3C), À4.8, À5.1 (2C, Si-
CH3). Anal. Calcd for C119H122F3NO28Si: C, 68.08; H, 5.86; N, 0.67.
Found: C, 68.10; H, 6.15; N, 0.65.
I
I
C47H52O7SSi: C, 71.54; H, 6.64. Found: C, 71.60; H, 6.69
3.7. Ethyl 2,3,4,6-tetra-O-benzoyl- -mannopyranosyl-(1?6)-
a-D
2,4-di-O-benzoyl-3-O-(tert-butyldimethylsilyl)-1-thio-a-D-
mannopyranoside (12)
Molecular sieves 4 Å (500 mg) were added to a soln of 11
(194 mg, 246 mol) and mannosyl bromide 5 (327 mg, 511 mol)
in CH2Cl2 (5 mL), the mixture was stirred for 30 min at room tem-
mol)
l
l
perature, cooled to À20 °C, and then AgOTf (156 mg, 608
l
was added. The resulting mixture was stirred at À10 °C until disap-
pearance of 11 (TLC monitoring), quenched with triethylamine, di-
luted with CHCl3, and filtered through a Celite layer. The filtrate
was washed with 1 M aq Na2S2O3, water, and the solvent was
evaporated. The residue was subjected to silica gel column chro-
matography (20:1 toluene–EtOAc) and then to gel chromatography
3.9. 3-Trifluoroacetamidopropyl 2,3,4,6-tetra-O-benzoyl-
mannopyranosyl-(1?6)-2,4-di-O-benzoyl- -mannopyran-
osyl-(1?2)-3,4,6-tri-O-benzyl- -mannopyranosyl-(1?2)-
3,4,6-tri-O-benzyl- -mannopyranoside (14)
a-D-
to give 12 (202 mg, 73%) as a colorless foam; [
a
]
D À0.6 (c 1, CHCl3).
a-D
1H NMR (500 MHz, CDCl3): d 8.17–7.23 (m, 30H, 6Ph-H), 6.08 (t,
1H, J3,4 = J4,5 = 10.1 Hz, H-4II), 5.89 (dd, 1H, J2,3 = 3.3, J3,4 = 10.1 Hz,
H-3II), 5.71 (t, 1H, J3,4 = J4,5 = 9.8 Hz, H-4I), 5.67 (br. s, 1H, H-2II),
5.50 (br. s, 2H, H-2I, H-1I), 5.06 (s, 1H, H-1II), 4.63 (m, 1H, H-5I),
4.53 (dd, 1H, J5,6 = 2.1, J6a,6b = 12.2 Hz, H-6aII), 4.42–4.37 (m, 2H,
H-3I, H-5II), 4.30 (dd, 1H, J5,6 = 6.8, J6a,6b = 12.2 Hz, H-6bII), 4.09
a-D
a-D
Tetramannoside 13 (85 mg, 41 lmol) was dissolved in CHCl3
(2 mL) and 90% aq CF3COOH (0.5 mL) was added. The mixture
was kept until TLC showed disappearance of starting 13, diluted
with CHCl3, washed with water, satd NaHCO3, and concentrated.
Column chromatography of the residue (7:1 toluene–EtOAc) pro-
I
(dd, 1H, J5,6 = 6.8, J6a,6b = 10.7 Hz, H-6a ), 3.67 (dd, 1H, J5,6 = 1.6,
I
J6a,6b = 10.7 Hz, H-6b ), 2.82 (m, 2H, SCH2CH3), 1.44 (t, 3H,
SCH2CH3), 0.62 (s, 9H, tert-Bu–Si), 0.04 (s, 3H, CH3–Si), À0.11 (s,
3H, CH3–Si). 13C NMR (126 MHz, CDCl3): d 166.0–165.1 (6C, PhCO),
133.4–133.0, 130.4–128.3 (PhCO), 97.3 (1C, C-1II), 82.1 (1C, C-1I),
74.2 (1C, C-2I), 70.2 (1C, C-2II), 70.0 (1C, C-4I), 69.9 (2C, C-5I, C-
3II), 69.7 (1C, C-3I), 68.7 (1C, C-5II), 67.0 (1C, C-6I), 66.5 (1C, C-
4II), 62.5 (1C, C-6II), 25.5 (SCH2CH3), 25.3 (3C, (CH3)3C), 14.9
(SCH2CH3) À4.9, À5.1 (2C, Si-CH3). Anal. Calcd for C62H64O16SSi:
C, 66.17; H, 5.73. Found: C, 66.09; H, 5.95.
vided 14 (65 mg, 77%) as a foam; [
a]
+12.0 (c 0.5, CHCl3). 1H
D
NMR (500 MHz, CDCl3): d 8.27, 8.03–7.93, 7.83, 7.61–7.14, 6.91
(60H, 12Ph-H), 6.71 (m, 1H, NH), 6.08 (t, 1H, J3,4 = J4,5 = 10.0 Hz, H-
4IV), 5.98 (dd, 1H, J2,3 = 3.1, J3,4 = 10.1 Hz, H-3IV), 5.85 (br s, 1H, H-
2IV), 5.79 (t, 1H, J3,4 = J4,5 = 9.9 Hz, H-4III), 5.64 (br s, 1H, H-2III),
5.35 (s, 1H, H-1II), 5.11 (s, 1H, H-1III), 5.04 (s, 1H, H-1IV), 4.92–4.88
(m, 2H, H-1I, PhCH2), 4.81 (d, 1H, J = 10.8 Hz, PhCH2), 4.77 (d, 1H,
J = 11.5 Hz, PhCH2), 4.68–4.55 (m, 6H, PhCH2), 4.54–4.45 (m, 5H,

1288
A. A. Karelin et al. / Carbohydrate Research 345 (2010) 1283–1290
H-3III, H-5III, 3PhCH2), 4.29 (dd, 1H, J5,6 = 1.7, J6a,6b = 12.2 Hz, H-6aIV),
4.15–4.10 (m, 2H, H-2I, H-5IV), 4.09–4.03 (m, 2H, H-2II, H-6bIV),
4.01–3.89 (m, 4H, H-3II, H-4II, H-5II, H-6aIII), 3.87 (dd, 1H, J2,3 = 2.7,
was stirred for 10 min, then the temperature was decreased to
À25 °C and TfOH (1 L, 10 mol) was added. The reaction mixture
l
l
was stirred at À20 °C to À25 °C until TLC showed disappearance of
starting 14. The reaction was quenched with a drop of pyridine, di-
luted with CHCl3, and filtered through a Celite layer. The filtrate
was washed with 1 M aq Na2S2O3 soln and water, concentrated,
and toluene was twice evaporated from the residue. Column chro-
matography of the residue (10:1 toluene–EtOAc) afforded 18
(38 mg, 44%) and 19 (12 mg, 14%) as a colorless foam.
I
III
J3,4 = 8.8 Hz, H-3I), 3.73–3.58 (m, 8H, H-4I, H-5I, H-6a , H-6aII, H-6a
,
I
H-6b , H-6bII, OCH2CH2CH2N), 3.34–3.27 (m, 2H, OCH2CH2CH2N,
OCH2CH2CH2N), 3.15 (m, 1H, OCH2CH2CH2N), 1.70–1.54 (m, 2H,
OCH2CH2CH2N); 13C NMR (126 MHz, CDCl3): d 166.9, 165.9, 165.3,
165.1 (6C, PhCO), 138.4–138.1, 133.6–132.9, 130.1–127.5 (Ph),
100.5 (1C, C-1II), 99.3 (2C, C-1I, C-1III), 97.3 (1C, C-1IV), 79.9 (1C, C-
3I), 78.8 (1C, C-3II), 77.2 (1C, C-2II), 75.5 (1C, C-2I), 75.1 (2C, PhCH2),
75.0 (1C, C-4I), 74.4 (1C, C-4II), 73.4 (1C, PhCH2), 73.3 (1C, PhCH2),
72.9 (1C, C-2III), 72.5 (1C, PhCH2), 72.3 (1C, PhCH2), 72.1 (2C, C-5I,
C-5II), 70.3 (1C, C-3IV), 70.1 (1C, C-2IV), 70.0 (1C, C-4III), 69.5 (1C,
C-6I), 69.2 (1C, C-3III), 69.0 (1C, C-5III), 68.9 (2C, C-5IV, C-6II), 66.4
(1C, C-4IV), 65.8 (1C, C-6III), 65.7 (1C, OCH2CH2CH2N), 62.2 (1C, C-
6IV), 37.7 (1C, OCH2CH2CH2N), 28.1 (1C, OCH2CH2CH2N). Anal. Calcd
for C113H108F3NO28: C, 68.37; H, 5.48; N, 0.71. Found: C, 68.08; H,
5.43; N, 0.69.
Compound 18: [
a]
À4.5 (c 0.8, CHCl3). 1H NMR (500 MHz,
D
CDCl3): d 8.29, 8.09, 7.98–7.90, 7.84–7.74, 7.61–6.93, 6.83 (95H,
19Ph-H), 6.73 (m, 1H, NH), 6.06 (t, 1H, J3,4 = J4,5 = 10.0 Hz, H-4VI),
6.03–5.95 (m, 3H, H-3VI, H-4III, H-4V), 5.87–5.82 (m, 2H, H-2VI, H-
3V), 5.75 (br s, 2H, H-2III, H-2V), 5.38 (s, 1H, H-1II), 5.35 (s, 1H, H-
1IV), 5.13 (s, 1H, H-1III), 4.98 (s, 1H, H-1VI), 4.91–4.87 (m, 2H,
H-1I, PhCH2), 4.82 (d, 1H, J = 11.4 Hz, PhCH2), 4.79–4.73 (m, 3H,
H-1V, 2 PhCH2), 4.71 (dd, 1H, J2,3 = 2.9, J3,4 = 9.6 Hz, H-3III), 4.66–
4.54 (m, 7H, PhCH2), 4.52–4.41 (m, 6H, H-5III, 5PhCH2), 4.34–4.28
(m, 3H, H-6aVI, 2PhCH2), 4.28–4.19 (m, 2H, H-5V, H-5VI), 4.19–
4.13 (m, 4H, H-2I, H-4IV, H-6aV, H-6bVI), 4.12 (br s, 1H, H-2II),
4.01–3.88 (m, 5H, H-3II, H-4II, H-5II, H-5IV, H-6aV), 3.88–3.79 (m,
4H, H-2IV, H-3I, H-3IV, H-6aIII), 3.72–3.52 (m, 10H, H-4I, H-5I, H-
3.10. Ethyl 2,3,4,6-tetra-O-benzoyl- -mannopyranosyl-
a-
D
(1?2)-3,4,6-tri-O-benzyl-1-thio- -D-mannopyranoside (17)
a
I
I
Molecular sieves 4 Å (140 mg) were added to a soln of thiogly-
coside 16 (43 mg, 87 mol) and imidate 15 (78 mg, 105 mol) in
CH2Cl2 (2 mL). The mixture was stirred for 30 min at room temper-
L, 35 mol) was
6a , H-6b , H-6aII, H-6bII, H-6aIV, H-6bIV, H-6bIII, OCH2CH2CH2N),
3.29–3.18 (m, 2H, OCH2CH2CH2N, OCH2CH2CH2N), 3.09 (m, 1H,
OCH2CH2CH2N), 1.58 (m, 1H, OCH2CH2CH2N), 1.51 (m, 1H, OCH2-
CH2CH2N); 13C NMR (126 MHz, CDCl3): d 166.0–164.7 (PhCO),
139.0–138.0, 133.6–132.9, 130.2–127.0 (Ph), 100.5 (1C, C-1II),
100.4 (1C, C-1IV), 99.4 (1C, C-1III), 99.2 (1C, C-1I), 99.1 (1C, C-1V),
97.4 (1C, C-1VI), 79.9 (1C, C-3I), 79.3 (1C, C-3IV), 78.9 (1C, C-3II),
76.9 (2C, C-2II, C-2IV), 75.4 (1C, C-2I), 75.2 (2C, PhCH2), 75.0 (1C,
PhCH2), 74.8 (1C, C-4I), 74.4 (1C, C-4II), 74.1 (1C, C-3III), 73.7 (1C,
C-4IV), 73.3 (2C, PhCH2), 73.0 (1C, PhCH2), 72.8 (1C, C-5IV), 72.6
(1C, PhCH2), 72.4 (1C, PhCH2), 72.1 (2C, C-5I, PhCH2), 72.0 (1C, C-
5II), 71.9 (1C, C-2III), 70.5 (1C, C-3VI), 70.1 (2C, C-2V, C-2VI), 69.9
(1C, C-3V), 69.4 (1C, C-6I), 69.3 (1C, C-6II), 69.2 (1C, C-5III), 69.0
(1C, C-4III), 68.8 (2C, C-5V, C-5VI), 68.1 (1C, C-6IV), 66.4 (2C, C-4V,
C-4VI), 65.8 (1C, C-6III), 65.6 (1C, OCH2CH2CH2N), 62.4 (1C, C-6VI),
61.9 (1C, C-6V), 37.6 (1C, OCH2CH2CH2N), 28.0 (1C, OCH2CH2CH2N).
Anal. Calcd for C174H162F3NO42: C, 69.75; H, 5.45; N, 0.47. Found: C,
69.88; H, 5.70; N, 0.52.
l
l
ature, cooled to À50 °C, and then TMSOTf (6.8
l
l
added. The resulting mixture was stirred at À20 °C until disappear-
ance of starting 16 (TLC monitoring), quenched with a drop of tri-
ethylamine, diluted with CHCl3, and filtered through a Celite layer.
The filtrate was washed with water and concentrated. The residue
was subjected first to column chromatography (20:1 toluene–
EtOAc) and then to gel chromatography to give 17 (56 mg, 60%)
as a colorless foam. [
a
]
D
+2.0 (c 1, CHCl3). 1H NMR (300 MHz,
CDCl3): d 8.17–7.83, 7.37–6.99 (m, 35H, Ph-H), 6.11 (t, 1H,
J3,4 = J4,5 = 9.9 Hz, H-4II), 6.01 (dd, 1H, J2,3 = 2.7, J3,4 = 9.9 Hz, H-3II),
5.94 (br s, 1H, H-2II), 5.51 (s, 1H, H-1I), 5.28 (s, 1H, H-1II), 4.92 (d,
1H, J = 11.0 Hz, PhCH2), 4.79–4.63 (m, 5H, H-5II, H-6aII, 3PhCH2),
4.63–4.56 (m, 2H, PhCH2), 4.48 (dd, 1H, J5,6 = 4.8, J6a,6b = 12.6 Hz,
H-6bII), 4.22 (m. 1H, H-5I), 4.16 (br s, 1H, H-2I), 4.09 (t, 1H,
J3,4 = J4,5 = 9.3 Hz, H-4I), 3.92 (dd, 1H, J2,3 = 2.0, J3,4 = 9.3 Hz, H-3I),
3.86 (dd, 1H, J5,6 = 4.8, J6a,6b = 10.7 Hz, H-6a ), 3.77 (m, 1H, H-6b ),
2.65–2.52 (m, 2H, SCH2CH3), 1.35 (t, 3H, SCH2CH3); 13C NMR
(75.47 MHz, CDCl3): d 166.2, 165.5, 165.3, 165.1 (PhCO), 138.4,
138.3, 133.3, 133.0, 129.9–127.5 (Ph), 99.4 (1C, C-1II), 83.7 (1C, C-
1I), 80.1 (1C, C-3I), 78.2 (1C, C-2I), 75.2 (1C, PhCH2), 75.0 (1C, C-
4I), 73.2 (1C, PhCH2), 72.6 (1C, PhCH2), 72.1 (1C, C-5I), 70.4 (1C,
C-2II), 70.1 (1C, C-3II), 69.4 (1C, C-5II), 69.1 (1C, C-6I), 67.0 (1C, C-
4II), 63.1 (1C, C-6II), 25.5 (SCH2CH3), 14.9 (SCH2CH3). Anal. Calcd
for C63H60O14S: C, 70.51; H, 5.64. Found: C, 70.46; H, 5.54.
Compound 19: [a]
À18.6 (c 0.8, CHCl3). 1H NMR (500 MHz,
I
I
D
CDCl3): d 8.13, 8.04, 8.02–7.96, 7.90, 7.86, 7.78–7.72, 7.70, 7.59–
7.52, 7.49–7.00, 6.89 (95 H, Ph-H), 6.71 (m, 1H, NH), 6.12 (t, 1H,
J3,4 = J4,5 = 9.9 Hz, H-4VI), 6.04–5.95 (m, 3H, H-2VI, H-3VI, H-4V),
5.84 (br s, 1H, H-2V), 5.77 (br s, 1H, H-2III), 5.63 (t, 1H,
J3,4 = J4,5 = 9.9 Hz, H-4III), 5.54 (dd, 1H, J2,3 = 3.0, J3,4 = 10.2 Hz, H-
3V) 5.37 (s, 1H, H-1II), 5.15 (s, 1H, H-1III), 5.09 (s, 1H, H-1VI), 5.02
(s, 1H, H-1V), 4.89 (s, 1H, H-1I), 4.88–4.77 (m, 4H, PhCH2), 4.77–
4.68 (m, 2H, PhCH2), 4.68–4.65 (m, 2H, H-1IV, PhCH2), 4.63–4.59
(m, 3H, H-3III, 2PhCH2), 4.59–4.47 (m, 6H, H-5V, H-6V, 4PhCH2),
4.47–4.39 (m, 6H, H-5III, H-5VI, H-6aVI, 3PhCH2), 4.37 (d, 1H,
J = 12.2 Hz, PhCH2), 4.26 (dd, 1H, J5,6 = 3.0, J6a,6b = 12.0 Hz, H-6bVI),
4.20–4.13 (4H, H-2I, H-2II, H-2IV, PhCH2), 4.01 (dd, 1H, J2,3 = 2.3,
J3,4 = 8.6 Hz, H-3II), 3.98–3.90 (m, 4H, H-4IV, H-5II, H-6aIII, H-6bV),
3.89–3.84 (m, 2H, H-3I, H-4II), 3.71–3.56 (9H, H-3IV, H-4I, H-5I, H-
3.11. 3-Trifluoroacetamidopropyl 2,3,4,6-tetra-O-benzoyl-
mannopyranosyl-(1?2)-3,4,6-tri-O-benzyl- -mannopyran-
osyl-(1?3)-[2,3,4,6-tetra-O-benzoyl- -mannopyranosyl-
(1?6)]-2,4-di-O-benzoyl- -mannopyranosyl-(1?2)-3,4,6-tri-
O-benzyl- -mannopyranosyl-(1?2)-3,4,6-tri-O-benzyl-
mannopyranoside (18) and 3-trifluoroacetamidopropyl 2,3,4,6-
tetra-O-benzoyl- -mannopyranosyl-(1?2)-3,4,6-tri-O-
benzyl-b- -mannopyranosyl-(1?3)-[2,3,4,6-tetra-O-benzoyl-
-mannopyranosyl-(1?6)]-2,4-di-O-benzoyl- -mannopyran-
osyl-(1?2)-3,4,6-tri-O-benzyl- -mannopyranosyl-(1?2)-
3,4,6-tri-O-benzyl- -mannopyranoside (19)
a-D-
a-D
a
-D
a-D
a-
D
a-D-
I
I
II
6a , H-6b , H-6aII, H-6bIII, H-6aIV, H-6bIV), 3.56–3.50 (m, 2H, H-6b
,
a
-D
OCH2CH2CH2N), 3.40 (m, 1H, H-5IV), 3.24–3.18 (m, 2H,
OCH2CH2CH2N, OCH2CH2CH2N), 3.06 (m, 1H, OCH2CH2CH2N), 1.58
(m, 1H, OCH2CH2CH2N), 1.49 (m, 1H, OCH2CH2CH2N); 13C NMR
(126 MHz, CDCl3): d 166.2–164.9 (PhCO), 138.6–138.0, 133.3–
132.6, 130.7–127.2 (Ph), 100.7 (1C, C-1II), 99.4 (2C, C-1III, C-1V),
99.3 (1C, C-1I), 97.8 (1C, C-1IV), 97.7 (1C, C-1VI), 82.3 (1C, C-3IV),
79.9 (1C, C-3I), 79.2 (1C, C-3II), 77.2 (2C, C-2I, C-2IV), 76.1 (1C, C-
5IV), 75.7 (1C, C-2II), 75.1 (4C, C-4I, 3PhCH2), 74.7 (2C, C-4II, C-
4IV), 73.4 (3C, PhCH2), 73.3 (1C, PhCH2), 72.7 (1C, PhCH2), 72.3
(2C, C-3III, PhCH2), 72.1 (2C, C-5I, C-5II), 70.9 (1C, C-3V), 70.8 (1C,
D
a-
D
a-D
a-D
a-D
Molecular sieves 4 Å (100 mg) were added to a soln of acceptor
14 (58 mg, 29 mol) and donor 17 (47 mg, 44 mol) in CH2Cl2
(3 mL); the mixture was stirred for 30 min at room temperature
and cooled to À10 °C. NIS (15 mg, 66 mol) was added, the mixture
l
l
l

A. A. Karelin et al. / Carbohydrate Research 345 (2010) 1283–1290
1289
C-3VI), 70.5 (2C, C-2V, C-2VI), 69.7 (1C, C-6IV), 69.5 (2C, C-5III, C-6I),
69.2 (1C, C-2III), 69.1 (1C, C-5VI), 68.9 (1C, C-6II), 68.4 (1C, C-5V),
67.3 (1C, C-4III), 66.5 (1C, C-6III), 66.4 (1C, C-4VI), 66.0 (2C, C-4V),
65.6 (1C, OCH2CH2CH2N), 62.7 (1C, C-6VI), 62.5 (1C, C-6V), 37.6
(1C, OCH2CH2CH2N), 28.0 (1C, OCH2CH2CH2N). Anal. Calcd for
3.63–3.56 (m, 3H, H-5I, OCH2CH2CH2N, OCH2CH2CH2N), 1.94 (m,
2H, OCH2CH2CH2N), 1.45 (m, 3H, OCH2CH3); 13C NMR (126 MHz,
D2O): d 103.7 (1C, C-1IV), 103.6 (1C, C-1III), 102.3 (1C, C-1II), 100.8
(1C, C-1V), 99.8, 99.6 (1C, C-1I), 80.3 (1C, C-2I), 79.9 (1C, C-2II),
79.7 (1C, C-3III), 74.9 (1C, C-5II), 74.8 (1C, C-5IV), 74.3 (1C, C-5I),
74.2 (1C, C-5V), 72.9 (1C, C-5III), 72.1 (2C, C-3V, OCH2CH3), 71.8
(2C, C-2IV, C-3I), 71.5 (3C, C-2V C-3II, C-3IV), 71.0 (1C, C-2III), 68.4
(2C, C-4I, C-4II), 68.3 (1C, C-4V), 68.2 (1C, C-4IV), 67.1 (1C, C-4III),
66.5 (1C, C-6III), 66.3, 66.2 (1C, OCH2CH2CH2N), 62.6, 62.5, 62.4
(4C, C-6I, C-6II, C-6IV, C-6V), 43.4, 43.1 (1C, OCH2CH2CH2N), 30.7,
30.5 (1C, OCH2CH2CH2N), 16.6 (1C, OCH2CH3). HRESIMS: found
m/z 1032.3341 [M+Na]+; calcd for C39H63NNaO29 1032.3383.
C174H162F3NO42: C, 69.75; H, 5.45; N, 0.47. Found: C, 69.62; H,
5.37; N, 0.49.
3.12. 3-Aminopropyl
a-
D
-mannopyranosyl-(1?2)-
a
-D
-
mannopyranosyl-(1?3)-[
a
-D-mannopyranosyl-(1?6)]-a-D-
mannopyranosyl-(1?2)-
a-
D
-mannopyranosyl-(1?2)-
a
-D
-
mannopyranoside (21)
3.14. 3-(3,4-Dioxo-2-ethoxycyclobut-1-enylamino)propyl a-D-
Pd(OH)2/C (90 mg) was added to a soln of hexamannoside 18
(85 mg, 28 mol) in MeOH (2 mL) and EtOAc (1 mL). The mixture
mannopyranosyl-(1?2)- -mannopyranosyl-(1?3)-[
mannopyranosyl-(1?6)]- -mannopyranosyl-(1?2)-
mannopyranosyl-(1?2)- -D-mannopyranoside (23)
a
a
a
-
-
D
a
-
D
D
-
-
l
D
a-
was stirred under H2 (1 atm) at room temperature for 20 h and
then filtered through a Celite layer. The catalyst was carefully
washed with MeOH and the combined filtrates were concentrated.
The residue was dissolved in MeOH (4 mL) and 1 M MeONa
(0.4 mL) was added. The mixture was kept for 3 h, water (0.5 mL)
was added, and after 24 h the solvents were evaporated. The resi-
due was subjected to gel chromatography, appropriate fractions
were collected, and lyophilized to give 21ÁAcOH (19 mg, 65%) as
Compound 23 was obtained from amine 21 (8.1 mg, 7.5
and diethyl squarate (1.3 L, 9.1 mol) in the presence of Et3N
(1.1 L) as described above for 22. Yield 7 mg (80%); [ +68.3 (c
lmol)
l
l
l
a]
D
0.5, H2O). 1H NMR (500 MHz, D2O): d 5.38 (s, 1H, H-1IV), 5.19 (s,
1H, H-1II), 5.05–5.03 (m, 2H, H-1I, H-1V), 5.02 (s, 1H, H-1III), 4.92
(s, 1H, H-1VI), 4.75 (m, 2H, OCH2CH3), 4.21 (br s, 1H, H-2III), 4.09
(br s, 2H, H-2II, H-2IV), 4.06 (m, 1H, H-2V), 4.02–3.97 (m, 3H, H-
2VI, H-3IV, H-6aIII), 3.95–3.82 (m, 13H, H-2I, H-3I, H-3II, H-3III, H-
a white amorphous powder. [
a
]
+77.0 (c 0.3, H2O). 1H NMR
D
(500 MHz, D2O): d 5.38 (s, 1H, H-1IV), 5.23 (s, 1H, H-1II), 5.17 (s,
1H, H-1I), 5.04 (s, 1H, H-1V), 5.01 (s, 1H, H-1III), 4.92 (s, 1H, H-
1VI), 4.21 (br s, 1H, H-2III), 4.10 (br s, 2H, H-2II, H-2IV), 4.07 (br s,
1H, H-2V), 4.00–3.97 (m, 3H, H-2VI, H-3IV, H-6aIII), 3.97–3.81 (m,
3V, H-3VI, H-4III, H-5III, H-6a , H-6aII, H-6aIV, H-6aV, H-6aVI), 3.81
I
(m, 1H, OCH2CH2CH2N), 3.79–3.71 (m, 9H, H-5II, H-5IV, H-5V, H-
6b , H-6bII, H-6bIV, H-6bV, H-6bVI, OCH2CH2CH2N), 3.70 (m, 1H, H-
I
14H, H-2I, H-3I, H-3II, H-3III, H-3V, H-3VI, H-4III, H-5III, H-6a , H-6a
,
I
II
6bIII), 3.69–3.60 (m, 7H, H-4I, H-4II, H-4IV, H-4V, H-4VI, H-5VI,
OCH2CH2CH2N), 3.60–3.53 (m, 2H, H-5I, OCH2CH2CH2N), 1.94 (m,
2H, OCH2CH2CH2N), 1.44 (m, 3H, OCH2CH3); 13C NMR (126 MHz,
D2O): d 103.7 (1C, C-1V), 103.6 (1C, C-1III), 102.3 (1C, C-1II), 102.2
(1C, C-1IV), 100.9 (1C, C-1VI), 99.9, 99.7 (1C, C-1I), 80.4 (1C, C-2I),
80.2 (1C, C-3III), 80.1 (2C, C-2II, C-2IV), 75.0 (1C, C-5II), 74.8 (2C, C-
5IV, C-5V), 74.4 (1C, C-5I), 74.2 (1C, C-5VI), 72.9 (1C, C-5III), 72.2
(3C, C-3V, C-3VI, OCH2CH3), 71.9 (1C, C-2VI), 71.5 (4C, C-2V, C-3I,
C-3II, C-3IV), 71.1 (1C, C-2III), 68.6 (3C, C-4I, C-4IV, C-4VI), 68.3 (2C,
C-4II, C-4V), 67.1 (1C, C-4III), 66.6 (1C, C-6III), 66.4, 66.2 (1C,
OCH2CH2CH2N), 62.7–62.3 (5C, C-6I, C-6II, C-6IV, C-6V, C-6VI), 43.4,
43.2 (1C, OCH2CH2CH2N), 30.7, 30.6 (1C, OCH2CH2CH2N), 16.6
(1C, OCH2CH3). HRESIMS: found m/z 1194.3940 [M+Na]+; calcd
for C45H73NNaO34 1194.3912.
H-6aIV, H-6aV, H-6aVI, OCH2CH2CH2N), 3.80–3.69 (m, 9H, H-5II, H-
5IV, H-5V, H-6b , H-6bII, H-6bIII, H-6bIV, H-6bV, H-6bVI), 3.69–3.56
I
(m, 8H, H-4I, H-4II, H-4IV, H-4V, H-4VI, H-5I, H-5VI, OCH2CH2CH2N),
3.11 (m, 2H, OCH2CH2CH2N), 1.99 (m, 2H, OCH2CH2CH2N), 1.90
(s, 3H, CH3COOH); 13C NMR (126 MHz, D2O): d 103.8 (1C, C-1V),
103.5 (1C, C-1III), 102.1 (2C, C-1II, C-1IV), 100.7 (1C, C-1VI), 99.7
(1C, C-1I), 80.2 (4C, C-2I, C-2II, C-2IV, C-3III), 75.0 (1C, C-5II), 74.7
(2C, C-5IV, C-5V), 74.3 (1C, C-5VI), 74.2 (1C, C-5I), 72.8 (1C, C-5III),
72.0 (1C, C-3V), 71.8 (1C, C-3VI), 71.7 (1C, C-3I), 71.5 (2C, C-2VI, C-
3II), 71.4 (2C, C-2V,C-3IV), 71.0 (1C, C-2III), 68.6–68.2 (5C, C-4I, C-
4II, C-4IV, C-4V, C-4VI), 67.1 (1C, C-4III), 66.6 (1C, C-6III), 66.4 (1C,
OCH2CH2CH2N), 62.7–62.4, (5C, C-6I, C-6II, C-6IV, C-6V, C-6VI), 38.9
(1C, OCH2CH2CH2N), 28.1 (1C, OCH2CH2CH2N). HRESIMS: found
m/z 1048.3931 [M+H]+; calcd for C39H70NO31 1048.3932.
3.15. BSA–pentasaccharide conjugate (24)
3.13. 3-(3,4-Dioxo-2-ethoxycyclobut-1-enylamino)propyl a-D-
mannopyranosyl-(1?3)-[
a
-
-
D
D
-mannopyranosyl-(1?6)]-
a-D-
A soln of 22 (8 mg, 7.3 lmol) and BSA (16 mg, 0.240 lmol)
mannopyranosyl-(1?2)-
a
-mannopyranosyl-(1?2)-a-D-
in 2 mL of the buffer soln (350 mM KHCO3 and 70 mM
Na2B4O7Á10H2O, pH 9) was kept for three days at room tempera-
ture. The resulting mixture was subjected to gel chromatography
on a Sephadex G-15 column (350 Â 25 mm) in water to give, after
lyophilization, conjugate 24 (18 mg, 98%) as a white amorphous
powder. MALDI-TOFMS showed a broad peak with a maximum
at m/z 76121 (ꢀ10 pentasaccharide residues per BSA molecule).
mannopyranoside (22)
Diethyl squarate (1.6
added to a soln of pentasaccharide 20 (8.1 mg, 8.9
lL, 10.7
l
mol) and Et3N (1.2
lL) were
lmol) in 50%
aq EtOH (2 mL); the mixture was kept for 24 h at room tempera-
ture and then concentrated. The residue was dissolved in water
and applied on a Sep-Pak C-18 cartridge. The cartridge was washed
with water (10 mL); then the product was eluted with aq MeOH in
2-mL portions, increasing the concentration of MeOH from 5% to
20%. Concentration of the eluate and subsequent lyophilization
from water afforded 22 (8 mg, 89%) as a white amorphous powder;
3.16. BSA–hexasaccharide conjugate (25)
Conjugate 25 (13 mg, 95%) was obtained similarly from 23
(7 mg, 5.5 lmol) and BSA (12 mg, 0.181 lmol). MALDI-TOFMS
[a]
+65.3 (c 0.5, H2O), 1H NMR (500 MHz, D2O): d 5.19 (s, 1H, H-
D
1II), 5.13 (s, 1H, H-1IV), 5.04–5.01 (m, 2H, H-1I, H-1III), 4.91 (s, 1H,
H-1V), 4.75 (m, 2H, OCH2CH3), 4.21 (br s, 1H, H-2III), 4.10 (br s,
1H, H-2II), 4.06 (br s, 1H, H-2IV), 4.03–3.98 (m, 2H, H-2V, H-6aIII),
3.95–3.82 (m, 12H, H-2I, H-3I, H-3II, H-3III, H-3IV, H-3V, H-4III, H-
showed a broad peak with a maximum at m/z 75955 (ꢀ9 hexasac-
charide residues per BSA molecule).
References
5III, H-6a , H-6aII, H-6aIV, H-6aV), 3.79 (m, 1H, OCH2CH2CH2N),
I
1. Masuoka, J. Clin. Microbiol. Rev. 2004, 17, 281–310.
2. Shibata, N.; Ikuta, K.; Imai, T.; Satoh, Y.; Satoh, R.; Suzuki, A.; Kojima, C.;
Kobayashi, H.; Hisamichi, K.; Suzuki, S. J. Biol. Chem. 1995, 270, 1113–1122.
3.78–3.68 (m, 8H, H-5II, H-5IV, H-6b , H-6bII, H-6bIII, H-6bIV, H-6b
,
I
V
OCH2CH2CH2N), 3.68–3.63 (m, 5H, H-4I, H-4II, H-4IV, H-4V, H-5V),

1290
A. A. Karelin et al. / Carbohydrate Research 345 (2010) 1283–1290
15. Zhu, Y.; Chen, L.; Kong, F. Chin. J. Chem. 2001, 19, 1289–1295.
3. Ogawa, T.; Yamamoto, H. Carbohydr. Res. 1982, 104, 271–283.
4. Ogawa, T.; Yamamoto, H. Carbohydr. Res. 1985, 137, 79–87.
5. Grathwohl, M.; Schmidt, R. R. Synthesis 2001, 2263–2272.
6. Zhu, Y.; Kong, F. Chin. J. Chem. 2001, 19, 120–122.
7. Nitz, M.; Bundle, D. R. J. Org. Chem. 2001, 66, 8411–8423.
8. Mathew, F.; Mach, M.; Hazen, K. C.; Fraser-Reid, B. Synlett 2003, 1319–1322.
9. Crich, D.; Banerjee, A.; Yao, Q. J. Am. Chem. Soc. 2004, 126, 14930–14934.
10. Wu, X.; Bundle, D. R. J. Org. Chem. 2005, 70, 7381–7387.
ˇ
´
ˇ
16. Paulovicová, L.; Bystricky, S.; Paulovicová, E.; Karelin, A. A.; Tsvetkov, Y. E.;
Nifantiev, N. E. FEMS Immunol. Med. Microbiol., in press.
17. Backinowsky, L. V.; Abronina, P. I.; Nepogodiev, S. A.; Grachev, A. A.; Kochetkov,
N. K. Pol. J. Chem. 1999, 73, 955–966.
18. Ottoson, H. Carbohydr. Res. 1990, 197, 101–107.
19. Du, Y.; Zhang, M.; Kong, F. Tetrahedron 2001, 57, 1757–1763.
20. Paulsen, H.; Heume, M.; Neurnberger, H. Carbohydr. Res. 1990, 200, 127–166.
21. Bien, F.; Ziegler, T. Tetrahedron: Asymmetry 1998, 9, 781–790.
22. Tietze, L. F.; Arlt, M.; Beller, M.; Glüsenkamp, K.-H.; Jähde, E.; Rajewsky, M.
Chem. Ber. 1991, 124, 1215–1221.
´
11. Karelin, A. A.; Tsvetkov, Y. E.; Kogan, G.; Bystricky, S.; Nifantiev, N. E. Russ. J.
Bioorg. Chem. 2007, 33, 110–121.
12. Karelin, A. A.; Tsvetkov, Y. E.; Paulovicová, L.; Bystricky, S.; Paulovicová, E.;
Nifantiev, N. E. Carbohydr. Res. 2009, 344, 29–35.
ˇ
´
ˇ
23. Kamath, V. P.; Diedrich, P.; Hindsgaul, O. Glycoconjugate J. 1996, 13, 315–319.
ˇ
13. Collot, M.; Sendid, B.; Fievez, A.; Savaux, C.; Standaert-Vitse, A.; Tabouret, M.;
Drucbert, A. S.; Danzé, P. M.; Poulain, D.; Mallet, J.-M. J. Med. Chem. 2008, 51,
6201–6210.
24. Chernyak, A.; Karavanov, A.; Ogawa, Y.; Kovác, P. Carbohydr. Res. 2001, 330,
479–486.
ˇ
25. Hou, S.-J.; Saksena, R.; Kovác, P. Carbohydr. Res. 2008, 343, 196–210.
14. Collot, M.; Loukou, C.; Mallet, J.-M. In Progress in the Synthesis of Complex
Carbohydrate Chains of Plant and Microbial Polysaccharides; Nifantiev, N. E., Ed.;
Transworld Research Network: Kerala, 2009; pp 373–399.