Synthesis of a heptasaccharide fragment of the mannan from Candida guilliermondii cell wall and its conjugate with BSA

Article abstract of DOI:10.1016/j.carres.2008.09.016

The 3-aminopropyl glycoside of a heptasaccharide fragment of the cell wall mannan from Candida guilliermondii 18, which corresponds to the antigenic Factor 9, has been synthesized by a convergent approach based on glycosylation of a tetrasaccharide accept

Full text of DOI:10.1016/j.carres.2008.09.016

Carbohydrate Research 344 (2009) 29–35
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of a heptasaccharide fragment of the mannan from
Candida guilliermondii cell wall and its conjugate with BSA
a
a
b
ˇ
Alexander A. Karelin , Yury E. Tsvetkov , Lucia Paulovicová ,
b
b
a,
*
´
ˇ
Slavomír Bystricky , 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 Institute of Chemistry, 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:
Received 10 August 2008
Received in revised form 6 September 2008
Accepted 17 September 2008
Available online 24 September 2008
The 3-aminopropyl glycoside of a heptasaccharide fragment of the cell wall mannan from Candida guil-
liermondii 18, which corresponds to the antigenic Factor 9, has been synthesized by a convergent
approach based on glycosylation of a tetrasaccharide acceptor with a trisaccharide donor as the key step
to give a protected heptasaccharide 17. Subsequent two-step deprotection of 17 afforded the heptaman-
noside 18, which was then conjugated with BSA using the squarate procedure.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Candida guilliermondii
Mannan
Oligomannosides
Neoglycoconjugates
Synthesis
1. Introduction
chains includes the sequence b-D-Man-(1?2)-a-D-Man-(1?3)-a-
D
-Man and corresponds to the antigenic factor 9. This structural
The yeast-like fungi of the genus Candida are opportunistic
pathogenic microorganisms capable of causing severe infections
in immunocompromised patients. The cell surface of Candida is
the immediate point of contact between the fungus and host,
and plays the main role in adhesion and modulation of the immune
response. The main surface antigen of Candida is mannan, which
represents the carbohydrate part of the cell wall mannoprotein.1
Extensive structural investigations of Candida mannans in the past
two decades have revealed the details of their structure.2 These
glycopolymers have a comb-like structure with a backbone com-
motif has been found in the mannan from Candida guilliermondii.2h
Mannooligosaccharides corresponding to the antigenic factors 1, 4,
5, and 6, and some others have been synthesized5, while the syn-
thesis of oligosaccharides corresponding to factor 9 has not been,
to the best of our knowledge, described so far.
We now report the synthesis of the 3-aminopropyl glycoside of
heptamannoside 18 comprising alternating
a-(1?2)- and a-
(1?3)-linked mannose units capped with a b-(1?2)-linked man-
nose residue. This oligosaccharide was isolated from C. guilliermon-
dii mannan by acetolysis. We have also obtained a conjugate of
oligosaccharide 18 with BSA for further immunological investiga-
tions. Synthetic yeast oligomannosides and neoglycoconjugates
thereof can be used as molecular probes to assess the carbohydrate
specificity of mannan-binding proteins6 in the diagnostic of fungal
infections7 and Crohn’s disease,8 and for the design of vaccines of a
new generation.5f,i,9
posed of
containing both
through -(1?2)-linkages. These side chains are responsible for
a-(1?6)-mannan, to which relatively short side chains
a- and b-linked mannose residues are attached
a
the antigenic specificity of Candida species. For example, it has
been shown that the determinants of antigenic factors 1 and 4
are linear (1?2)-linked2f and 3,6-branched
a
-oligomannosides,2g
respectively. On the other hand, the determinants of antigenic fac-
tors 5 and 6 are represented by linear b-(1?2)-oligomers2d and by
2. Results and discussion
one or two b-D-Man-(1?2)-residues attached to short a-(1?2)-
linked chains,3 respectively. The former oligomers are bound to
the side chains via a phosphodiester bond and are known as the
acid-labile mannan.4 The third type of b-mannose-containing side
A convergent approach based on glycosylation of a tetrasaccha-
ride glycosyl acceptor with a trisaccharide glycosyl donor as the
key step was applied for the preparation of the target heptamannos-
ide. A critical b-mannosylation step was planned at early stages of
the assembly of the trisaccharide donor to simplify isolation of the
individual b-anomer, because separation of the anomers would be
* Corresponding author. Tel./fax: +7 499 135 8784.
E-mail address: nen@ioc.ac.ru (N. E. Nifantiev).
0008-6215/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2008.09.016

30
A. A. Karelin et al. / Carbohydrate Research 344 (2009) 29–35
easier for disaccharides than for higher oligosaccharides. Stereose-
lective construction of b-mannosides is one of the challenges in the
glycoside synthesis, and was extensively investigated in the past
decade. As a result, diverse approaches to this goal have been
developed.5f–i,10 Glycosylation with easily accessible ethyl thiom-
annoside 111 under the conditions proposed by Crich et al.12 has
been chosen for the stereoselective introduction of the b-(1?2)-
linked mannosyl residue. Glycosylation of acceptor 213 with thio-
glycoside 1 in the presence of Tf2O and 1-benzenesulfinylpyrroli-
dine resulted in the formation of b-disaccharide 3 in 69% yield
(Scheme 1). The b-configuration of the nonreducing mannosyl res-
which was used as the donor to construct the nonreducing trisac-
charide sequence of the target heptamannoside.
The tetrasaccharide acceptor block was assembled as follows.
The a-(1?3)-linked disaccharide 10 was obtained by glycosylation
of thiomannoside 7 with the known chloride 914 in the presence of
AgOTf (Scheme 2). Subsequent NIS-TfOH-promoted coupling of 10
with our previously described disaccharide acceptor 135j afforded
smoothly tetrasaccharide 14. Selective removal of an acetyl group
in the presence of benzoyl groups is reported to be easily achieved
by mild acidic methanolysis.15 However, the acetyl group at O-2 of
the terminal mannose unit in 14 proved to be unusually stable un-
der these conditions, and its removal required a prolonged reaction
time and was accompanied by a partial loss of the N-trifluoroacetyl
and benzoyl groups. As a result, target tetrasaccharide acceptor 16
was obtained in poor yield. To circumvent this difficulty, the acetyl
group in thioglycoside 10 was replaced by a chloroacetyl one.
Treatment of 10 with HCl in MeOH provided the deacetylated
product 11 in an acceptable yield of 54%; its further conventional
chloroacetylation afforded the needed disaccharide donor 12. Cou-
pling of 12 with acceptor 13 in the presence of NIS and TfOH
yielded tetrasaccharide 15; subsequent removal of the chloroacetyl
1
idue in disaccharide 3 was confirmed by the corresponding JC1,H1
(157 Hz) coupling constant value.
The acid-labile benzylidene group in 3 was replaced by stable
benzoyl groups by treatment with aq CF3CO2H followed by con-
ventional benzoylation with BzCl in pyridine to produce dibenzo-
ate 4. Removal of the anomeric allyl group from 4 with PdCl2 and
subsequent reaction of hemiacetal 5 with trichloroacetonitrile in
the presence of DBU gave disaccharide donor 6.
Glycosylation of thioglycoside 75j with trichloroacetimidate 6
resulted in the formation of the trisaccharide thioglycoside 8,
BzO
BzO
BnO
OBn
O
OBn
O
Ph
O
O
OBn
O
BnO
BnO
BnO
Ph
OH
O
O
O
BnO
b
a
O
O
BnO
BnO
BnO
BnO
BnO
O
O
e
BnO
SEt
BnO
OAll
1
2
OR
OAll
4 R = All
5 R = H
3
c
d
BzO
BzO
BnO
OBn
6 R = C(=NH)CCl3
O
O
BnO
BnO
BnO
O
BnO
BnO
HO
OBz
O
BnO
BnO
OBz
O
O
SEt
7
8
SEt
Scheme 1. Synthesis of trisaccharide donor 8. Reagents and conditions: (a) (i) 1-benzenesulfinyl-pyrrolidine, 2,6-di-tert-butylpyridine, Tf2O, CH2Cl2, À78 °C; (ii) 2,
À78 °C?rt, 69%; (b) (i) CF3CO2H 90%, CHCl3, rt; (ii) BzCl, pyridine, rt, 71%; (c) PdCl2, MeOH, rt, 67%; (d) CCl3CN, DBU, CH2Cl2, À50 °C, 99%; (e) TMSOTf, CH2Cl2, À20 °C, 45%.
BnO
BnO
BnO
OR
BnO
BnO
HO
BnO
BnO
BnO
OBz
O
OAc
O
O
a
BnO
BnO
OBz
O
O
SEt
Cl
9
7
10 R = Ac
b
c
SEt
11 R = H
12 R = CAc
BnO
BnO
BnO
OR
O
BnO
BnO
OBz
O
O
BnO
BnO
BnO
OH
O
BnO
BnO
BnO
O
10 or 12
O
BnO
BnO
BnO
d
O
O
BnO
BnO
BnO
O
O
O(CH2)3NHCOCF3
14 R = Ac
15 R = CAc
16 R = H
13
O(CH2)3NHCOCF3
b
e
Scheme 2. Synthesis of tetrasaccharide acceptor 16. Reagents and conditions: (a) AgOTf, CH2Cl2, –10 °C, 61%; (b) HCl, MeOH, rt, 54% of 11,% of 16; (c) CAcCl, pyridine, CH2Cl2
rt, 82%; (d) NIS, TfOH, CH2Cl2, –20 to –15 °C, 59%; (e) thiourea, 2,4,6-collidine, MeOH, reflux, 83%.

A. A. Karelin et al. / Carbohydrate Research 344 (2009) 29–35
31
HO
HO
HO
OH
O
O
HO
HO
HO
O
HO
HO
OH
O
RO(CH2)3NH2
≡
BzO
BzO
BnO
OBn
O
O
O
HO
HO
HO
O
O
BnO
BnO
BnO
HO
HO
OH
O
O
BnO
BnO
OBz
O
O
b
O
HO
O
O
HO
BnO
BnO
BnO
O
HO
O
a
BnO
BnO
8
16
OBz
O
HO
O
O
HO
O
HO
BnO
BnO
BnO
O
O(CH2)3NH2
O
18
BnO
BnO
BnO
O
O
O(CH2)3NHCOCF3
17
Scheme 3. Synthesis of heptamannoside 18. Reagents and conditions: (a) NIS, TfOH, CH2Cl2, –20 to –15 °C, 67%; (b) (i) H2, Pd/C, MeOH, rt; (ii) Amberlyst A-26 (OHÀ), water, rt,
60%.
O
O
O
O
a
b
RO(CH2)3NH2
BSA
RO(CH2)3NH
NH
RO(CH2)3NH
OEt
19
18
19
20
Scheme 4. Synthesis of heptasaccharide-BSA conjugate 20. Reagents and conditions: (a) diethyl squarate, water, EtOH, pH 7, rt, 95%; (b) BSA, KHCO3, Na2B4O7, water, pH 9, rt,
80%.
group with thiourea proceeded smoothly producing the expected
tetrasaccharide acceptor 16 in high yield.
in oligosaccharides are numbered by Roman numerals starting
from the reducing end. MALDI-TOF mass spectra were obtained
on a Bruker Ultraflex mass spectrometer with 2,5-dihydroxyben-
zoic acid as the matrix in the positive reflector mode. HRESIMS
were obtained on a Finnigan LTQ FT (Thermo Electrom Corp.)
instrument. Optical 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
oligosaccharides. 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
Finally, heptamannoside 17 was obtained by NIS-TfOH-pro-
moted coupling of the trisaccharide donor 8 and the tetrasaccharide
acceptor 16 in 67% yield. The protected oligosaccharide 17 was first
subjected to hydrogenolysis to remove benzyl groups; then benzoyl
and N-trifluoroacetyl groups were simultaneously removed by
treatment with the anion-exchange resin Amberlyst A-26 (OHÀ)
to give the target free heptamannoside 18 (Scheme 3).
The squarate procedure16 was employed for conjugation of hep-
tasaccharide 18 with BSA. First, treatment of 18 with diethyl squa-
rate at pH 7 resulted in the formation of the monosubstituted
adduct 19 (Scheme 4). Subsequent reaction of 19 with BSA at pH
9 afforded the target conjugate 20. According to MALDI-TOFMS
data, the conjugate contained on average 19 oligosaccharide copies
per protein molecule.
was carried out on Silica Gel 60 (40–63 lm, E. Merck). Gel-perme-
ation 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 car-
ried out using dry solvents under dry argon.
3.2. Allyl 2,3-di-O-benzyl-4,6-O-benzylidene-b-D-
mannopyranosyl-(1?2)-3,4,6-tri-O-benzyl-a-D-
3. Experimental
mannopyranoside (3)
3.1. General methods
Molecular sieves 4 Å (400 mg) were added to a soln of thiogly-
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. Monosaccharide residues
coside 1 (201 mg, 0.408 mmol), 1-benzenesulfinylpyrrolidine12
(88 mg, 0.45 mmol), and 2,6-di-tert-butylpyridine (180
0.49 mmol) in CH2Cl2 (4 mL). The mixture was cooled to À78 °C,
stirred for 40 min, then Tf2O (83 L, 0.49 mmol) was added fol-
lowed by a soln of acceptor 2 (200 mg, 0.408 mmol) in CH2Cl2
lL,
l

32
A. A. Karelin et al. / Carbohydrate Research 344 (2009) 29–35
(4 mL). The resulting mixture was stirred at À78 °C for 30 min, and
was then allowed to warm to room temperature. The mixture was
diluted with CHCl3, filtered through a Celite layer, the filtrate was
washed with satd NaHCO3 and water, and the solvent was evapo-
rated. Column chromatography of the residue (20:1 toluene–
3.4. 4,6-Di-O-benzoyl-2,3-di-O-benzyl-b-
D-mannopyranosyl-
(1?2)-3,4,6-tri-O-benzyl- -mannopyranose (5)
D
PdCl2 (54 mg, 0.304 mmol) was added to a soln of 4 (316 mg,
0.304 mmol) in dry MeOH (3 mL). The mixture was vigorously stir-
red until disappearance of starting 4 (TLC monitoring). The mixture
was diluted with EtOAc, filtered through a silica gel layer, the fil-
trate was made neutral with NEt3, and the solvent was evaporated.
The residue was subjected to column chromatography (5:1 tolu-
EtOAc) gave 3 (258 mg, 69%) as a colorless foam; [
a
]
À36.7 (c 1,
D
CHCl3). 1H NMR (500 MHz, CDCl3): d 7.58–7.05 (m, 30H, 6Ph),
5.88 (m, 1H, OCH2CH@CH2), 5.59 (s, 1H, PhCH), 5.26 (d, 1H, J
17.2 Hz, OCH2CH@CH2), 5.18 (d, 1H, J 10.3 Hz, OCH2CH@CH2),
5.09 (d, 1H, J 11.7 Hz, PhCH2), 4.96 (s, 1H, H-1I), 4.85 (d, 2H, J
11.4 Hz, PhCH2), 4.78 (d, 1H, J 10.8 Hz, PhCH2), 4.67 (d, 1H, J
12.6 Hz, PhCH2), 4.64 (s, 1H, H-1II), 4.62 (d, 1H, J 12.6 Hz, PhCH2),
4.59 (d, 1H, J 12.1 Hz, PhCH2), 4.52 (d, 1H, J 11.2 Hz, PhCH2), 4.45
(d, 1H, J 12.1 Hz, PhCH2), 4.32 (d, 1H, J 10.9 Hz, PhCH2), 4.29 (br
s, 1H, H-2I), 4.24–4.20 (m, 3H, H-6aII, H-4II, OCH2CH@CH2), 4.03
(d, 1H, J2,3 3.0 Hz, H-2II), 4.00 (m, 1H, OCH2CH@CH2), 3.97 (m, 1H,
H-3I), 3.91 (t, 1H, J3,4 = J4,5 9.4 Hz, H-4I), 3.88 (t, 1H, J5,6 = J6,6
10.2 Hz, H-6IbI), 3.78 (m, 1H, H-5I), 3.72 (m, 1H, H-6Ia), 3.67
ene–EtOAc) to give 5 (204 mg, 67%) as an
a
,b-mixture in a ratio
of ꢀ3:1; [
a
]
D À46.8 (c 1, CHCl3). 1H NMR for
a-5 (500 MHz, CDCl3):
d 7.98–7.03 (m, 35H 7 Ph), 5.75 (t, 1H, J3,4 = J4,5 9.7 Hz, H-4II), 5.34
(s, 1H, H-1I), 5.08 (d, 1H, J 11.8 Hz, PhCH2), 4.88–4.82 (m, 2H,
PhCH2), 4.74 (d, 1H, J 10.8 Hz, PhCH2), 4.71 (d, 1H, H-1II), 4.57 (m,
1H, H-6IaI), 4.55–4.43 (m, 3H, PhCH2), 4.42–4.35 (m, 3H, H-2I,
H-6II, PhCH2), 4.29 (d, 1H, J 11.1 Hz, PhCH2), 4.09(d, 1H, J2,3
b
2.7 Hz, H-2II), 4.03–3.96 (m, 2H, H-3I, H-5I), 3.82 (m, 1H, H-5II),
3.76 (t, 1H, J3,4 = J4,5 9.2 Hz, H-4I), 3.63–3.58 (m, 3H, H-3II, H-6aI ,
H-6IbI); 13C (126 MHz, CDCl3): d 166.1, 165.3 (PhCO), 138.2–137.5,
133.1, 132.8, 129.8–129.6, 128.4–127.2 (Ph), 99.4 (1C, C-1II), 91.8
(1C, C-1I), 77.9 (1C, C-3II), 77.3 (1C, C-3I), 74.6 (1C, PhCH2), 74.2
(2C, C-4I, PhCH2), 73.5 (1C, C-2II), 73.4 (1C, PhCH2), 72.7 (1C,
C-5II), 72.1 (1C, C-2I), 71.4 (1C, C-5I), 70.4, 70.3 (2C, PhCH2), 69.4
(1C, C-6I), 69.0 (1C, C-4II), 63.8 (1C, C-6II). Anal. Calcd for
C61H60O13: C, 73.18; H, 6.04. Found: C, 73.03; H, 6.25.
(m, 1H, H-6I ), 3.58 (dd, J2,3 3.1, J3,4 9.9 Hz, H-3II), 3.32 (m, 1H,
b
H-5II); 13C NMR (126 MHz, CDCl3):
d
138.4–137.6. 133.7,
133.6, 129.1–127.4, 126.1, 125.3 (Ph, OCH2CH@CH2), 117.6 (1C,
OCH2CH@CH2), 101.4 (1C, JC1,H1 = 157 Hz, C-1II), 100.4 (1C, PhCH),
96.5 (1C, JC1,H1 168 Hz, C-1I), 78.4 (1C, C-4II), 78.1 (1C, C-3I), 77.2
(1C, C-3II), 76.2 (1C, C-2II), 75.0, 74.9 (2C, PhCH2), 74.3 (1C, C-4I),
73.3 (1C, PhCH2), 72.8 (1C, C-2I), 71.8 (1C, PhCH2), 71.5 (1C, C-5I),
70.7 (1C, PhCH2), 69.0 (1C, C-6I), 68.5 (1C, C-6II), 68.1 (1C,
OCH2CH@CH2), 67.7 (1C, C-5II). Anal. Calcd for C57H60O11: C,
74.33; H, 6.57. Found: C, 74.17; H, 6.73.
3.5. 4,6-Di-O-benzoyl-2,3-di-O-benzyl-b-
(1?2)-3,4,6-tri-O-benzyl-a-D-mannopyranosyl
D-mannopyranosyl-
trichloroacetimidate (6)
3.3. Allyl 4,6-di-O-benzoyl-2,3-di-O-benzyl-b-D-
mannopyranosyl-(1?2)-3,4,6-tri-O-benzyl-
mannopyranoside (4)
a-
D-
To a soln of 5 (69 mg, 0.069 mmol) and trichloroacetonitrile
(69 L, 0.69 mmol) in CH2Cl2 (2 mL) was added a catalytic amount
l
of DBU at À50 °C. After being stirred for 20 min, the mixture was
allowed to warm up to room temperature and the residue was sub-
jected to column chromatography (10:1 toluene–EtOAc) to give 6
90% aq CF3CO2H (1 mL) was added to a soln of 3 (407 mg,
0.442 mmol) in CHCl3 (8 mL). After 30 min, the mixture was di-
luted with CHCl3, washed with satd NaHCO3 and water, and the
solvent was evaporated. The residue was dissolved in pyridine
(1 mL), BzCl (0.31 mL, 2.6 mmol) was added and the mixture
was allowed to stand overnight. The mixture was diluted with
CHCl3, washed with satd NaHCO3 and water, concentrated, and
the residual pyridine was removed by coevaporation with toluene.
Column chromatography of the residue (15:1 toluene–EtOAc) pro-
(78 mg, 99%) as a white foam; [
a
]
D
À26.6 (c 1, CHCl3). 1H NMR
(500 MHz, CDCl3): d 8.46 (s, 1H, NH), 7.98–7.06 (m, 35 H, 7 Ph),
6.37 (d, 1H, J1,2 1.9 Hz, H-1I), 5.74 (t, 1H, J3,4 = J4,5 9.7 Hz, H-4II),
5.08 (d, 1H, J 11.8 Hz, PhCH2), 4.88 (d, 1H, J 11.3 Hz, PhCH2), 4.84
(d, 1H, J 11.8 Hz, PhCH2), 4.81 (s, 1H, H-1II), 4.78 (d, 1H, J 10.8 Hz,
PhCH2), 4.60–4.56 (m, 2H, H-6IaI, PhCH2), 4.56–4.52 (m, 2H, H-2I,
PhCH2), 4.45 (d, 2H, J 11.8 Hz, PhCH2), 4.41 (dd, 1H, J5,6 6.2, J6,6
vided 4 (328 mg, 71%) as a white solid; [
a]
D À41.7 (c 1, CHCl3). 1H
12.0 Hz, H-6II), 4.36 (d, 1H, J 10.8 Hz, PhCH2), 4.29 (d, 1H, J
b
NMR (500 MHz, CDCl3): d 7.78–7.03 (m, 35H, 7Ph), 5.84 (m, 1H,
12.6 Hz, PhCH2), 4.11 (d, 1H, J2,3 2.7 Hz, H-2II), 4.03 (t, 1H,
J3,4 = J4,5 9.2 Hz, H-4I), 3.98–3.93 (m, 2H, H-3I, H-5II), 3.90 (m, 1H,
OCH2CH@CH2), 5.74 (t, 1H, J3,4 = J4,5 9.7 Hz, H-4II), 5.23 (dd,
1H,
J
17,2,
J
1.4 Hz, OCH2CH@CH2), 5.15 (d, 1H,
J
10.4 Hz,
H-5II), 3.74 (dd, 1H, J5,6 3.9, J6,6 11.0 Hz, H-6I ), 3.68 (dd, 1H, J5,6
a
OCH2CH@CH2), 5.10 (d, 1H, J 11.7 Hz, PhCH2), 4.96 (d, 1H, J1,2
1,5 Hz, H-1I), 4.87 (d, 1H, J 11.9 Hz, PhCH2), 4.84 (d, 1H, J
12.3 Hz, PhCH2), 4.74 (d, 1H, J 10.8 Hz, PhCH2), 4.71 (s, 1H, H-
1II), 4.59 (d, 1H, J 12.1 Hz, PhCH2), 4.54 (dd, J5,6 3.2, J6,6 12.0 Hz,
1.6, J6,6 11.0 Hz, H-6I ), 3.64 (dd, J2,3 3.0, J3,4 9.7 Hz, H-3II); 13C
b
NMR (126 MHz, CDCl3): d 166.1, 165.4 (PhCO), 160.5 (C@NH),
138.6–137.5, 133.1, 132.8, 129.8–129.6, 128.4–127.3 (Ph), 99.3
(1C, C-1II), 95.4 (1C, C-1I), 77.7 (1C, C-3II), 76.9 (1C, C-3I), 75.0,
74.3 (2C, PhCH2), 74.2 (1C, C-5I), 73.4 (1C, C-2II), 73.3 (2C, C-4I,
PhCH2), 72.8 (1C, C-5II), 70.4, 70.1 (2C, PhCH2), 69.9 (1C, C-2I),
69.0 (1C, C-4II), 68.6 (1C, C-6I), 63.9 (1C, C-6II). Anal. Calcd for
C63H60Cl3NO13: C, 66.06; H, 5.28; N, 1.22. Found: C, 65.81; H,
5.27; N, 1.32.
H-6II), 4.53 (d, 1H, J 12.7 Hz, PhCH2), 4.44 (d, 1H, J 12.1 Hz, PhCH2),
a
4.41–4.36 (m, 3H, H-2I, H-6bII, PhCH2), 4.28 (d, 1H, J 12.7 Hz,
PhCH2), 4.27 (d, 1H, J 10.8 Hz, PhCH2), 4.17 (m, 1H, OCH2CH@CH2),
4.03 (d, 1H, J2,3 2.9 Hz, H-2II), 3.98–3.92 (m, 2H, H-3I,
OCH2CH@CH2), 3.88 (t, 1H, J3,4 = J4,5 9.5 Hz, H-4I), 3.84 (m, 1H,
H-5II), 3.70 (m, 1H, H-5I), 3.76 (dd, 1H, J5,6 4.3, J6,6 9,6 Hz, H-6Ia),
3.67 (dd, 1H, J5,6 1.9, J6,6 9.6 Hz, H-6I ), 3.62 (dd, J2,3 2.9, J3.4
3.6. Ethyl 4,6-di-O-benzoyl-2,3-di-O-benzyl-b-
mannopyranosyl-(1?2)-3,4,6-tri-O-benzyl-
mannopyranosyl-(1?3)-2-O-benzoyl-4,6-di-O-
benzyl-1-thio- -mannopyranoside (8)
D-
b
9.7 Hz, H-3II); 13C NMR (126 MHz, CDCl3): d 166.2, 165.3 (PhCO),
138.8–137.6. 133.7, 133.6, 133.0, 132.8, 129.8, 129.6, 128.4–
127.4, 126.1, 127.2 (Ph, OCH2CH@CH2), 117.5 (1C, OCH2CH@CH2),
99.3 (1C, C-1I), 96.1 (1C, C-1II), 77.9 (1C, C-3I), 77.7 (1C, C-3II),
74.8, 74.3 (2C, PhCH2), 74.0 (1C, C-4I), 73.6 (1C, C-2II), 73.3 (1C,
PhCH2), 72.7 (1C, C-5II), 71.8 (1C, C-2I), 71.4 (1C, C-5I), 70.3, 70.1
(2C, PhCH2), 69.1 (1C, C-6I), 69.0 (1C, C-4II), 68.1 (1C, OCH2CHCH2),
63.9 (1C, C-6II). Anal. Calcd for C64H64O13: C, 73.83; H, 6.20. Found:
C, 73.69; H, 6.20.
a-D-
a-D
Molecular sieves 4 Å (150 mg) were added to a soln of thiogly-
coside acceptor 7 (35 mg, 0.068 mmol) and donor 6 (78 mg,
0.68 mmol) in CH2Cl2 (2 mL). The mixture was stirred for 30 min
at room temperature, cooled to À50 °C, and then TMSOTf (4.6
lL,
0.024 mmol) was added. The resulting mixture was stirred at –15

A. A. Karelin et al. / Carbohydrate Research 344 (2009) 29–35
33
to À20 °C until disappearance of starting 7 (TLC monitoring),
quenched with triethylamine, diluted with CHCl3, and filtered
through a Celite layer. The filtrate was washed with water and con-
centrated. Column chromatography of the residue (20:1 toluene–
mixture was kept for 3 days at ambient temperature, diluted with
CHCl3, washed with satd NaHCO3 and water, and concentrated.
Column chromatography of the residue (10:1 toluene–EtOAc) gave
11 (120 mg, 54%) as a colorless foam; [a]
+38.6 (c 1, CHCl3). 1H
D
EtOAc) afforded 8 (46 mg, 45%) as a colorless foam; [
a
]
À17.5 (c
NMR (500 MHz, CDCl3): d 8.02–7.08 (m, 30H, 6Ph), 5.53 (dd, 1H,
J1,2 1.9, J2,3 3.1 Hz, H-2I), 5.42 (d, 1H, J1,2 1.9, H-1I), 5.19 (s, 1H, H-
1II), 4.73–4.62 (m, 4H, PhCH2), 4.56–4.42 (m, 6H, PhCH2), 4.24
(dd, 1H, J2,3 3.1, J3,4 9.1 Hz H-3I), 4.19 (m, 1H, H-5I), 4.15 (t, 1H,
J3,4 = J4,5 9.1 Hz, H-4I) 3.91–3.83 (m, 3H, H-6Ia, H-2II H-4II), 3.79
(m, 1H, H-5II), 3.73–3.67 (m, 2H, H-6bI , H-3II), 3.59 (dd, 1H, J5,6
D
1, CHCl3). 1H NMR (500 MHz, CDCl3): d 8.09–6.95 (m, 50H, 10Ph),
5.57 (t, 1H, J3,4 = J4,5 9.8 Hz, H-4III), 5.54 (br s, 1H, H-2I), 5.44 (s,
1H, H-1I), 5.33 (s, 1H, H-1II), 5.03 (d, 1H, J 11.6 Hz, PhCH2), 4.75–
4,62 (m, 7H, PhCH2), 4.57–4.45 (m, 4H, PhCH2), 4.39 (dd, 1H, J2,3
3.1, J3,4 9.9 Hz, H-3I) 4.33 (dd, 1H, J5,6 3.2, J6,6 12.0 Hz H-6IaII),
4.25–4.18 (m, 4H, H-2II, H-4I, H-5I, PhCH2), 4.15 (d, 2H, J 10.1 Hz,
PhCH2), 4.13 (dd, 1H, J5,6 5.2, J6,6 12.0 Hz, H-6IbII), 4.03 (s, 1H, H-
1III), 3.93 (m, 1H, H-5II), 3.91(br s, 1H, H-2III), 3.87 (t, 1H, J3,4 = J4,5
9.0 Hz, H-4II) 3.84 (dd, 1H, J5,6 3.2, J6,6 10.6 Hz, H-6Ia), 3.76 (dd,
J2,3 3.1, J3.4 8.9 Hz, H-3II), 3.71–3.66 (m, 3H, H-6Ib, H-6aII, H-6IbI),
3.11 (dd, J2,3 3.0, J3,4 9.8 Hz, H-3III), 2.73 (m, 1H, H-5III), 2.65 (m,
2H, SCH2CH3), 1.30 (t, 3H, SCH2CH3); 13C NMR (126 MHz, CDCl3):
d 166.1, 165.7, 165.4 (PhCO), 139.0–138.0, 133.4–133.0, 130.2–
127.3, 126.1–125.9 (Ph), 99.1 (1C, JC1,H1 155 Hz, C-1III), 99.1 (1C,
JC1,H1 170 Hz, C-1II), 82.6 (1C, JC1,H1 168 Hz, C-1I) 77.6 (1C, C-3II),
76.9 (1C, C-3III), 76.1 (1C, C-3I), 75.5 (1C, PhCH2), 74.7–74.1 (C-2I,
C-4I, PhCH2), 73.8–73.4 (C-4II, PhCH2), 73.3 (1C, C-5II), 72.2 (1C,
C-5I), 72.0 (3C, C-2II, C-2III, C-5III), 70.1, 69.7 (2C, PhCH2), 68.7 (1C,
C-4III), 68.6 (2C, C-6I, C-6II), 63.5 (1C, C-6III), 25.6 (1C, SCH2CH3),
14.9 (1C, SCH2CH3). Anal. Calcd for C90H90O18S: C, 72.46; H, 6.08.
Found: C, 72.10; H, 6.23.
3.4, J6,6 10.9 Hz, H-6II), 3.56 (d, 1H, J5,6 1.8, J6,6 10.7 Hz, H-6IbI),
a
2.61 (m, 2H, SCH2CH3), 1.27 (t, 3H, SCH2CH3); 13C NMR
(126 MHz, CDCl3): d 165.6, (PhCO), 138.5 138.3, 137.9. 133.1,
129.8, 128.9–127.3 (Ph), 101.4 (1C, C-1II), 82.3 (1C, C-1I), 79.6
(1C, C-3II) 77.9 (1C, C-3I), 75.1 (1C, PhCH2), 75.0 (1C, C-4I), 74.5
(1C, PhCH2), 74.2 (1C, C-2I), 73.9 (1C, C-4II), 73.4 (2C, 2 PhCH2),
72.0 (3C, C-5I, C-5II, PhCH2), 68.9 (1C, C-6I), 68.8 (1C, C-2II), 68.4
(1C, C-6II), 25.6 (1C, SCH2CH3), 14.9 (1C, SCH2CH3). Anal. Calcd for
C56H60O11S: C, 71.47; H, 6.43. Found: C, 71.23; H, 6.68.
3.9. Ethyl 3,4,6-tri-O-benzyl-2-O-chloroacetyl-
mannopyranosyl-(1?3)-2-O-benzoyl-4,6-di-O-benzyl-1-
thio- -mannopyranoside (12)
a-D-
a-D
To a soln of disaccharide 11 (93 mg, 0.10 mmol) in dry CH2Cl2
(2 mL) was added pyridine (32 L, 0.40 mmol) followed by chloro-
acetyl chloride (16 L, 0.20 mmol). After 6 h, the mixture was di-
l
l
3.7. Ethyl 2-O-acetyl-3,4,6-tri-O-benzyl-
(1?3)-2-O-benzoyl-4,6-di-O-benzyl-1-thio-a-D-
mannopyranoside (10)
a
-
D
-mannopyranosyl-
luted with CHCl3 and washed successively with 1 M HCl, satd
NaHCO3 and water. The solvent was evaporated and the residue
was chromatographed (15:1 toluene–EtOAc) to give 12 (83 mg,
82%) as a yellowish foam; [
a]
D
+28.6 (c 1, CHCl3). 1H NMR
Molecular sieves 4 Å (2.0 g) were added to a soln of a thioglyco-
side 7 (716 mg, 1.41 mmol) and chloride 9 (1.10 g, 2.15 mmol) in
CH2Cl2 (8 mL), the mixture was stirred for 30 min at room temper-
ature, cooled to –50 °C, and then AgOTf (830 mg, 3.22 mmol) was
added. The resulting mixture was stirred at –15 to –20 °C until dis-
appearance of 7 (TLC monitoring), quenched with triethylamine,
diluted with CHCl3, and filtered through a Celite layer. The filtrate
was washed with 1 M aq Na2S2O3, water, and the solvent was
evaporated. Column chromatography of the residue (20:1 tolu-
(500 MHz, CDCl3): d 8.03–7.05 (m, 30H, 6Ph), 5.50 (dd, 1H, J1,2
1.4, J2,3 2.7 Hz, H-2I), 5.40 (d, 1H, J1,2 1.4, H-1I), 5.36 (br s, 1H, H-
2II), 5.17 (d, 1H, J1,2 1.4, H-1II), 4.72 (d, 2H, J 11.1 Hz, PhCH2), 4.68
(d, 1H, J 10.0 Hz, PhCH2), 4.64 (d, 1H, J 12.1 Hz, PhCH2), 4.57 (d,
1H, J 10.9, PhCH2), 4.49 (d, 1H, J 11.9 Hz, PhCH2), 4.47 (d, 1H, J
11.1 Hz, PhCH2), 4.41 (m, 2H, PhCH2), 4.31 (d, 1H, J 11.1 Hz, PhCH2),
4.25–4.19 (m, 3H, H-3I, H-4I, H-5I), 4.07 (d, 1H, J 15.2, ClCH2CO),
4.01 (d, 1H, J 15.2, ClCH2CO), 3.90 (dd, 1H, J5.6 2.2, J6,6 10.8 Hz,
H-6Ia), 3.87–3.80 (m, 3H, H-3II, H-4II, H-5II), 3.71 (d, 1H, J6,6 10.9,
H-6I ), 3.63 (dd, 1H, J5,6 2.4, J6,6 10.7 Hz, H-6II), 3.58 (d, 1H, J6,6
ene–EtOAc) provided 10 (846 mg, 61%) as a colorless foam; [a]
D
b
a
+27.6 (c 1, CHCl3). 1H NMR (500 MHz, CDCl3): d 8.03–7.05 (m,
30H, 6 Ph-H), 5.52 (br s, 1H, H-2I), 5.42 (s, 1H, H-1I), 5.36 (br s,
1H, H-2II), 5.17 (s, 1H, H-1II), 4.75 (d, 2H, J 11.0 Hz, PhCH2), 4.71
(d, 1H, J 12.5 Hz, PhCH2), 4.68 (d, 1H, J 12.9 Hz, PhCH2), 4.54 (d,
1H, J 10.8, PhCH2), 4.48 (d, 2H, J 12.3 Hz, PhCH2), 4.41 (m, 2H,
PhCH2), 4.32 (d, 1H, J 11.1 Hz, PhCH2), 4.25–4.18 (m, 3H, H-3I, H-
4I, H-5I), 3.89 (m, 2H, H-4II, H-6aI ), 3.82 (m, 2H, H-3II, H-5II), 3.71
10.7 Hz, H-6II), 2.61 (m, 2H, SCH2CH3), 1.27 (t, 3H, SCH2CH3); 13C
b
NMR (126 MHz, CDCl3): d 166.6 (ClCH2CO), 165.6, (PhCO), 138.4,
–137.6. 133.2, 129.8, 128.5–127.3 (Ph), 99.1 (1C, C-1II), 82.3 (1C,
C-1I), 77.6 (1C, C-3II), 77.4 (1C, C-3I), 75.2 (1C, PhCH2), 75.0 (1C,
C-4I), 74.5 (1C, PhCH2), 74.0 (1C, C-2I), 73.7 (1C, C-4II), 73.4 (2C, 2
PhCH2), 72.3 (1C, C-5II), 72.1 (2C, C-5I, PhCH2), 70.7 (1C, C-2II),
68.8 (1C, C-6I), 68.2 (1C, C-6II), 40.7 (1C, ClCH2CO), 25.6 (1C,
SCH2CH3), 14.9 (1C, SCH2CH3). Anal. Calcd for C58H61ClO12S: C,
68.46; H, 6.04. Found: C, 68.26; H, 6.18.
(d, 1H, J6,6 10.8, H-6I ), 3.62 (dd, 1H, J5.6 3.1, J6.6 10.7 Hz, H-6IaI),
b
3.56 (d, 1H, J6.6 10.7 Hz, H-6II), 2.61 (m, 2H, SCH2CH3), 2.08 (s,
b
3H, CH3CO), 1.26 (t, 3H, SCH2CH3); 13C NMR (126 MHz, CDCl3): d
170.2 (CH3CO), 165.6, (PhCO), 138.6, 138.3, 137.9. 133.2, 129.8,
128.9–127.3 (Ph), 99.6 (1C, C-1II), 82.2 (1C, C-1I), 77.7 (2C, C-3I,
C-3II), 75.2 (1C, PhCH2), 75.0 (1C, C-4I), 74.5 (1C, PhCH2), 74.1
(1C, C-2I), 73.9 (1C, C-4II), 73.4 (2C, 2 PhCH2), 72.3 (1C, C-5II),
72.0 (1C, C-5I), 71.8 (1C, PhCH2), 69.0 (1C, C-2II), 68.8 (1C, C-6I),
68.3 (1C, C-6II), 25.7 (1C, SCH2CH3), 21.0 (1C, CH3CO), 15.0 (1C,
SCH2CH3). Anal. Calcd for C58H62O12S: C, 70.85; H, 6.36. Found: C,
70.20; H, 6.48.
3.10. 3-Trifluoroacetamidopropyl 3,4,6-tri-O-benzyl-2-O-
chloroacetyl-
a-D-mannopyranosyl-(1?3)-2-O-benzoyl-4,6-di-
O-benzyl- -mannopyranosyl-(1?2)-3,4,6-tri-O-benzyl-a-D-
a
-D
mannopyranosyl-(1?2)-3,4,6-tri-O-benzyl-a-D-
mannopyranoside (15)
Molecular sieves 4 Å (700 mg) were added to a soln of accep-
tor 13 (300 mg, 0.290 mmol) and donor 12 (360 mg, 0.344 mmol)
in CH2Cl2 (2 mL); the mixture was stirred for 30 min at room
temperature and cooled to À15 °C. NIS (155 mg, 0.644 mmol)
was added, the mixture was stirred for 10 min, then the temper-
3.8. Ethyl 3,4,6-tri-O-benzyl-
a-
D-mannopyranosyl-(1?3)-2-O-
benzoyl-4,6-di-O-benzyl-1-thio-
a
-D
-mannopyranoside (11)
ature was decreased to À35 °C and TfOH (11
lL, 0.12 mmol) was
added. The reaction mixture was stirred at À25 °C to À30 °C until
Anhyd HCl in MeOH (1 M, 1.6 mL), obtained by adding AcCl
(114 L, 1.6 mmol) to chilled MeOH (1.5 mL) was added to a soln
of 10 (233 mg, 0.237 mmol) in dry CH2Cl2 (0.4 mL). The resulting
l
TLC showed disappearance of starting 13. The reaction was
quenched with pyridine (19 lL, 0.24 mmol), diluted with CHCl3,

34
A. A. Karelin et al. / Carbohydrate Research 344 (2009) 29–35
and filtered through a Celite layer. The filtrate was successively
washed with 1 M aq Na2S2O3 soln, 1 M HCl and water, and con-
centrated. Column chromatography of the residue (9:1 toluene–
3.12. 3-Trifluoroacetamidopropyl 4,6-di-O-benzoyl-2,3-
di-O-benzyl-b- -mannopyranosyl-(1?2)-3,4,6-tri-O-benzyl-
-mannopyranosyl-(1?3)-2-O-benzoyl-4,6-di-O-benzyl-
-mannopyranosyl-(1?2)-3,4,6-tri-O-benzyl-
D
a
-
D
D
EtOAc) afforded 15 (406 mg, 59%); colorless foam, [
a]
+16.7 (c
a-
a-D-
D
1, CHCl3). 1H NMR (500 MHz, CDCl3): d 8.03–6.85 (m, 60H, 12
Ph), 5.55 (br s, 1H, H-2III), 5.43 (br s, 1H, H-2IV), 5.22 (s, 2H, H-
1II, H-1IV), 5.10 (s, 1H, H-1III), 4.91 (s, 1H, H-1I), 4.86 (d, 1H, J
10.8 Hz, PhCH2), 4.78 (d, 1H, J 10.8 Hz, PhCH2), 4.71–4.44 (m,
16H, PhCH2), 4.39–4.31 (m, 4H, H-3III, PhCH2), 4.22 (d, 1H, J
12.6 Hz, PhCH2), 4.18 (t, 1H, J3,4 = J4,5 9.5 Hz, H-4III), 4.08 (d,
1H, J 15.3 Hz, ClCH2CO), 4.02 (br s, 1H, H-2II), 4.00 (d, 1H, J
15.3 Hz, ClCH2CO), 3.99–3.89 (m, 5H, H-2I, H-3II, H-4IV, H-5III, H-
5II), 3.87 (dd, 1H, J2,3 2.8, J3,4 8.5 Hz, H-3IV), 3.83 (m, 1H, H-5IV),
3.80–3.69 (m, 5H, H-3I, H-4II, H-6IaI, H-6bII, H-6aIII), 3.69–3.64 (m,
4H, H-41, H-51, H-6aI , H-6bI ), 3.60 (m, 1H, OCH2CH2CH2N), 3.54
(d, 2H, J 10.8 Hz, H-6III, H-6IV), 3.47 (d, 1H, J6,6 10.6 Hz, H-6IbV),
3.36 (m, 1H, OCH2CH2CH2N), 3.26–3.15 (m, 2H, OCH2CH2CH2N,
OCH2CH2CH2N), 1.70 (m, 2H, OCH2CH2CH2N). 13C NMR (126
MHz, CDCl3): d 166.6 (ClCH2CO), 165.4, (PhCO), 138.5–137.6,
133.2, 129.8, 129.0, 128.5–127.4 (Ph), 100.7, (1C, C-1II), 99.2
(1C, C-1III), 99.1 (1C, C-1IV), 98.9 (1C, C-1I), 79.5 (1C, C-3I), 78.9
(1C, C-3II), 77.8 (1C, C-3IV), 76.9 (1C, C-3III), 76.5 (1C, C-2II),
75.3–75.0 (C-2I, C-4II, PhCH2), 74.8–74.5 (C-4I, C-4III, PhCH2),
73.6 (1C, C-4IV), 73.4–73.2 (PhCH2), 72.3–71.8 (C-2III, C-5I, C-5II,
C-5III, C-5IV, PhCH2), 70.8 (1C, C-2IV), 69.9 (1C, C-6II), 69.3 (1C,
C-6I), 68.8 (1C, C-6III), 68.0 (1C, C-6IV), 65.8 (1C, OCH2CH2CH2N),
40.8 (1C, ClCH2CO), 38.0 (1C, OCH2CH2CH2N), 28.0 (1C,
OCH2CH2CH2N). Anal. Calcd for C115H119ClF3NO24: C, 69.35; H,
6.02; N, 0.70. Found: C, 69.02; H, 6.04; N, 0.78.
mannopyranosyl-(1?3)-2-O-benzoyl-4,6-di-O-benzyl-
a-D
-mannopyranosyl-(1?2)-3,4,6-tri-O-benzyl-
a-D-
mannopyranosyl-(1?2)-3,4,6-tri-O-benzyl-a-D-
mannopyranoside (17)
Molecular sieves 4 Å (100 mg) were added to a soln of 16
(30 mg, 0.016 mmol) and (43 mg, 0.029 mmol) in CH2Cl2
8
(2 mL), and the resulting mixture was stirred for 30 min at room
temperature. After cooling to À15 °C, NIS (13 mg, 0.058 mmol)
was added, and, after 10 min, the temperature of the reaction mix-
ture was decreased to À35 °C, and then TfOH (1
lL, 0.01 mmol)
was added. The stirring was continued for further 5 h at À15 °C
to À20 °C. 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 and water, concentrated, and tol-
uene was twice evaporated from the residue. Chromatographic
purification of the residue (10:1 toluene–EtOAc) produced 17
b
a
(36 mg, 67%) as a colorless foam; [
a
]
À2.3 (c 1, CHCl3). 1H
D
(500 MHz, CDCl3): d 8.21–6.92 (m, 110H, 22Ph), 5.63 (br s, 1H, H-
2III), 5,55 (br s, 1H, H-2V), 5.52 (t, 1H, J3,4 = J4,5 9.8 Hz, H-4VII),
5.39 (s, 1H, H-1IV), 5.33 (s, 1H, H-1VI), 5.22 (s, 1H, H-1II), 5.17 (s,
1H, H-1III), 5.02 (m, 2H, H-1V, PhCH2), 4.91 (d, 1H, J1,2 1.2 Hz, H-
1I), 4.84–4.76 (m, 3H, PhCH2), 4.71–4.39 (m, H-3V, PhCH2), 4.39–
4.35 (m, 2H, H-3III, PhCH2), 4.30–4.24 (m, 2H, H-6VII, PhCH2),
a
4.20–4.03 (m, H-2II, H-2VI, H-4III, H-4V, H-5V, H-6bVII, PhCH2), 4.03–
3.83 (m, 12H, H-1VII, H-2I, H-2IV, H-2VII, H-3II, H-3IV, H-4IV, H-4VI,
H-5II, H-5III, H-5IV, H-5VI), 3.78–3.69 (m, 4H, H-3I, H-3VI, H-4II, H-
6I), 3.68–3.48 (m, 13H, H-4I, H-5I, H-6bI , H-6aII, H-6bII, H-6aIII, H-6IbII,
H-6IV, H-6IV, H-6V, H-6VI, H-6VI, OCH2CH2CH2N), 3.44 (d, 1H, J
3.11. 3-Trifluoroacetamidopropyl 3,4,6-tri-O-benzyl-
a-D-
mannopyranosyl-(1?3)-2-O-benzoyl-4,6-di-O-benzyl-
a-D-
mannopyranosyl-(1?2)-3,4,6-tri-O-benzyl-
mannopyranosyl-(1?2)-3,4,6-tri-O-benzyl-
mannopyranoside (16)
a
a
-
-
D
-
-
a
b
a
a
b
D
10.8 Hz, H-6V), 3.31 (m, 1H, OCH2CH2CH2N), 3.20–3.14 (m, 2H,
b
OCH2CH2CH2N, OCH2CH2CH2N), 3.01 (dd, J2,3 2.8, J3,4 9.7 Hz, H-
3
VII), 2.61 (m, 1H, H-5VII), 1.63 (m, 2H, OCH2CH2CH2N); 13C NMR
Tetrasaccharide 15 (379 mg, 0.191 mmol) was dissolved in a
mixture of MeOH (10 mL) and CHCl3 (2 mL), then 2,4,6-collidine
(27 lL, 0.20 mmol) and thiourea (77 mg, 1.01 mmol) were added.
(50.32 MHz, CDCl3): d 165.9, 165.4, 165.4, 165.1 (4C, PhCO), d
139.0–137.7. 133.1, 132.7, 129.9–127.0, 125.6 (Ph), 101.8 (1C,
C-1II), 100.5 (1C, C-1IV), 99.7 (1C, C-1V), 99.2 (1C, C-1III), 98.9 (2C,
C-1I, C-1VII), 98.7 (1C, C-1VI), 79.5 (1C, C-3I), 79.2 (1C, C-3II), 77.8
(2C, C-3IV, C-3VI), 77.2 (1C, C-3III), 76.8 (1C, C-3VII), 75.8 (1C, C-
2II), 75.5–74.6 (C-2I, C-3V, C-4I, C-4II, C-4III, C-4V, PhCH2), 74.6-
74.0 (C-4IV, PhCH2), 73.7–72.8 (C-2IV, C-2VI, C-4VI, PhCH2), 72.5–
The mixture was boiled under reflux for 3 days, cooled, and taken
to dryness. A soln of the residue in CHCl3 was washed with 1 M HCl
and satd NaHCO3, and concentrated. The residue was purified by
column chromatography (5:1 toluene–EtOAc) to provide 16
(304 mg, 83%) as a white foam; [
a]
+24.5 (c 1, CHCl3). 1H NMR
71.4 (C-2III, C-2V, C-2VII, C-5I, C-5II, C-5III, C-5IV, C-5V, C-5VI, C-5VII
,
D
(500 MHz, CDCl3): d 8.20–7.09 (m, 60 H, 12 Ph), 5.70 (br s, 1H,
H-2III), 5.37 (s, 1H, H-1II), 5.32 (s, 1H, H-1IV), 5.27 (s, 1H, H-1III),
5.04 (s, 1H, H-1I), 4.96 (d, 1H, J 10.9 Hz, PhCH2), 4.91 (d, 1H, J
10.8 Hz, PhCH2), 4.88 (d, 1H, J 11.3 Hz, PhCH2), 4.81 (d, 1H, J
11.1 Hz, PhCH2), 4.78–4.57 (m, 16H, PhCH2), 4.49–4.56 (m, 2H, H-
3III, PhCH2), 4.38 (d, 1H, J 11.5 Hz, PhCH2), 4.25 (t, 1H, J3,4 = J4,5
7.3 Hz, H-4III), 4.22 (br s, 1H, H-2II), 4.14–4.00 (m, 6H, H-2I, H-2IV,
H-3II, H-4IV, H-5II, H-5III), 3.95–3.91 (m, 2H, H-3I, H-5IV), 3.91–
3.78 (m, 9H, H-3IV, H-4I, H-4II, H-5I, H-6aI , H-6Ib, H-6aII, H-6IbI,
H-6III), 3.74 (m, 1H, OCH2CH2CH2N), 3.70–3.66 (m, 2H, H-6III,
PhCH2), 70.0 (2C, C-61, PhCH2), 69.5 (PhCH2), 69.3 (1C, C-6II), 68.8
(1C, C-6III), 68.7–68.2 (4C, C-4VII, C-6IV, C-6V, C-6VI), 65.8 (1C,
OCH2CH2CH2N), 63.4 (1C, C6VII), 38.0 (1C, OCH2CH2CH2N), 28.0
(1C, OCH2CH2CH2N). Anal. Calcd for C201H202F3NO41: C, 72.18; H,
6.09; N, 0.42. Found: C, 72.23; H, 6.24; N, 0.46.
3.13. 3-Aminopropyl b-D-mannopyranosyl-(1?2)-a-D-
mannopyranosyl-(1?3)-
mannopyranosyl-(1?3)-
mannopyranosyl-(1?2)-
a
a
a
-D
-D
-D
-mannopyranosyl-(1?2)-
-mannopyranosyl-(1?2)-
-mannopyranoside, acetate (18)
a
a
-
-
D
-
-
D
a
b
H-6aIV), 3.61 (d, 1H, J6,6 = 9.4 Hz, H-6IbV), 3.44–3.34 (m, 3H,
OCH2CH2CH2N, 2 OCH2CH2CH2N), 1.86 (m, 2H, OCH2CH2CH2N);
13C NMR (126 MHz, CDCl3): d 138.9–138.0, 129.9, 128.3–127.4
(Ph), 101.8 (1C, C-1IV), 100.7, (1C, C-1II), 99.2 (1C, C-1III), 99.0 (1C,
C-1I), 79.7 (1C, C-3IV), 79.6 (1C, C-3I), 79.1 (1C, C-3II), 77.3 (1C,
C-3III), 76.4 (1C, C-2II), 75.4–74.9 (C-2I, C-4I, C-4II, PhCH2), 74.7
(1C, C-4III), 74.5 (1C, PhCH2), 73.9 (1C, C-4IV), 73.7–73.3 (PhCH2),
72.5 (1C, C-2III), 72.3–71.8 (C-5I, C-5II, C-5III, C-5IV, PhCH2), 70.0
(1C, C-6II), 69.5 (1C, C-6I), 69.1 (1C, C-6III), 69.0 (1C, C-2IV), 68.7
(1C, C-6IV), 65.3 (1C, OCH2CH2CH2N), 37.4 (1C, OCH2CH2CH2N),
28.3 (1C, OCH2CH2CH2N). Anal. Calcd for C113H118ClF3NO23: C,
70.87; H, 6.21; N, 0.73. Found: C, 70.88; H, 6.38; N, 0.78.
Pd(OH)2/C (20%, 40 mg) was added to a soln of heptamannoside
17 (82 mg, 0.025 mmol) in MeOH (3 mL). The mixture was stirred
under H2 (1 atm) at room temperature for 16 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 re-
sin Amberlyst A-26 (OHÀ) (1.5 mL) for 16 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 18ÁAcOH (18 mg, 60%) as a white amorphous
powder; [a]
+68.4 (c 1, water). 1H NMR (500 MHz, D2O): d 5,38
D

A. A. Karelin et al. / Carbohydrate Research 344 (2009) 29–35
35
(s, 1H, H-1IV), 5.28 (s, 1H, H-1II), 5.25 (1H, H-1VI), 5.08 (s, 1H, H-1I),
5.03 (s, 1H, H-1V), 5.02 (s, 1H, H-1III), 4.76 (s, 1H, H-1VII), 4.27 (br s.
1H, H-2VI), 4.22 (d, 1H, J2,3 2.0 Hz, H-2V), 4.21 (d, 1H, J2,3 2.2 Hz, H-
2III), 4.10 (br s, 2H, H-2II, H-2IV), 4.04 (d, 1H, J2,3 2.9 Hz, H-2VII), 3.99
(dd, 1H, J2,3 3.1, J3,4 9.6 Hz, H-3IV), 3.97–3.84 (m, 13H, H-2I, H-3I, H-
3II, H-3III, H-3V, H-3VI, 7 H-6), 3.83 (m, 1H, OCH2CH2CH2N), 3.81–
3.62 (m, 19H, H-3VII, H-4I, H-4II, H-4III, H-4IV, H-4V, H-4VI, H-5II,
H-5III, H-5IV, H-5V, H-5VI, 7 H-6), 3.61–3.54 (m, 3H, H-4VII, H-5I,
OCH2CH2CH2N), 3.37 (m, 1H, H-5VII), 3.11 (m. 2H, OCH2CH2CH2N),
1.97 (m, 2H, OCH2CH2CH2N), 1.90 (s, 3H, CH3CO2H). 13C NMR
(126 MHz, D2O): d 103.5 (2C, C-1III, C-1V), 102.0 (1C, C-1II), 101.9
(1C, C-1IV), 101.3 (1C, C-1VI), 99.9 (1C, C-1VII), 99.5 (1C, C-1I), 80.0
(1C, C-2I), 79.7 (2C, C-2II, C-2IV), 79.4 (2C, C-3III, C-3V), 78.2 (1C,
C-2VI), 77.5 (1C, C-5VII), 74.5 (5C, C-5II, C-5III, C-5IV, C-5V, C-5VI),
74.0 (1C, C-3VII), 73.9 (1C, C-5I), 71.9 (1C, C-2VII), 71.3 (1C, C-3I),
71.2 (1C, C-3IV), 71.1 (1C, C-3II), 70.9 (1C, C-3VI), 70.7 (2C, C-2III,
C-2V), 68.2 (3C, C-4II, C-4IV, C-4VI), 68.1 (1C, C-4I), 67.9 (1C, C-
B4O7Á10H2O, pH 9) was kept for 7 days at room temperature. 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 20 (3.0 mg, 80%) as a white amorphous
powder. MALDI-TOFMS showed a broad peak with a maximum
at m/z 91640.
References
1. Masuoka, J. Clin. Microbiol. Rev. 2004, 17, 281–310.
2. (a) Shibata, N.; Fukasawa, S.; Kobayashi, H.; Tojo, M.; Yonezu, T.; Ambo, A.;
Ohkubo, Y.; Suzuki, S. Carbohydr. Res. 1989, 187, 239–253; (b) Kobayashi, H.;
Shibata, N.; Mitobe, H.; Ohkubo, Y.; Suzuki, S. Arch. Biochem. Biophys. 1989, 272,
364–375; (c) Kobayashi, H.; Kojimahara, T.; Takahashi, K.; Takikawa, M.;
Takahashi, S.; Shibata, N.; Okawa, Y.; Suzuki, S. Carbohydr. Res. 1991, 214, 131–
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Kobayashi, H.; Suzuki, S. Infect. Immun. 1992, 60, 4100–4110; (e) Kobayashi, H.;
Matsuda, K.; Ikeda, T.; Suzuki, M.; Takahashi, S.; Suzuki, A.; Shibata, N.; Suzuki,
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Infect. Immun. 1994, 62, 4425–4431; (g) 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; (h) Shibata, N.; Akagi, R.; Hosoya, T.; Kawahara,
K.; Suzuki, A.; Ikuta, K.; Kobayashi, H.; Hisamichi, K.; Okawa, Y.; Suzuki, S. J.
Biol. Chem. 1996, 271, 9259–9266; (i) Shibata, N.; Kobayashi, H.; Okawa, Y.;
Suzuki, S. Eur. J. Biochem. 2003, 270, 2565–2575; (j) Oyamada, H.; Ogawa, Y.;
Shibata, N.; Okawa, Y.; Suzuki, S.; Kobayashi, H. Arch. Microbiol. 2008, 189, 483–
490.
4
VII), 67.3 (1C, C-4V), 67.2 (1C, C-4III), 66.2 (1C, OCH2CH2CH2N),
62.4–61.8 (7C, C-6), 38.8 (1C, OCH2CH2CH2N), 28.0 (1C,
7
OCH2CH2CH2N), 24.6 (1C, CH3CO2H); HRESIMS: found m/z
1210.4446 [M+H]+; calcd for C45H80NO36 1210.4460.
3.14. 3-(3,4-Dioxo-2-ethoxycyclobut-1-enylamino)propyl b-D-
mannopyranosyl-(1?2)-
mannopyranosyl-(1?2)-
mannopyranosyl-(1?2)-
mannopyranoside (19)
a-
a-
a-
D
D
D
-mannopyranosyl-(1?3)-
-mannopyranosyl-(1?3)-
-mannopyranosyl-(1?2)-
a
a
a
-D
-D
-D
-
-
-
3. Kobayashi, H.; Shibata, N.; Suzuki, S. Infect. Immun. 1992, 60, 2106–2109.
4. Shibata, N.; Ichikawa, T.; Tojo, M.; Takahashi, M.; Ito, N.; Okubo, Y.; Suzuki, S.
Arch. Biochem. Biophys. 1985, 243, 338–348.
5. (a) Ogawa, T.; Yamamoto, H. Carbohydr. Res. 1982, 104, 271–283; (b)
Ogawa, T.; Yamamoto, H. Carbohydr. Res. 1985, 137, 79–87; (c) Grathwohl,
M.; Schmidt, R. R. Synthesis 2001, 2263–2272; (d) Zhu, Y.; Kong, F. Chin. J. Chem.
2001, 19, 120–122; (e) Zhu, Y.; Chen, L.; Kong, F. Chin. J. Chem. 2001, 19, 1289–
1295; (f) Nitz, M.; Bundle, D. R. J. Org. Chem. 2001, 66, 8411–8423; (g) Mathew,
F.; Mach, M.; Hazen, K. C.; Fraser-Reid, B. Synlett 2003, 1319–1322; (h) Crich,
D.; Banerjee, A.; Yao, Q. J. Am. Chem. Soc. 2004, 126, 14930–14934; (i) Wu, X.;
Bundle, D. R. J. Org. Chem. 2005, 70, 7381–7388; (j) Karelin, A. A.; Tsvetkov, Y.
E.; Kogan, G.; Bystricky, S.; Nifantiev, N. E. Russ. J. Bioorg. Chem. 2007, 33, 110–
121.
6. Kohatsu, L.; Hsu, D. K.; Jegalian, A. G.; Liu, F.-T.; Baum, L. G. J. Immunol. 2006,
177, 4718–4726.
7. Sendid, B.; Tabouret, M.; Poirot, J. L.; Mathieu, D.; Fruit, J.; Poulain, D. J. Clin.
Microbiol. 1999, 37, 1510–1517.
8. Vandewalle-El Khoury, P.; Colombel, J.-F.; Joossens, S.; Standaert-Vitse, A.;
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Targan, S. R.; Chamaillard, M.; Mallet, J.-M.; Sendid, B.; Poulain, D. Am. J.
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13531.
Diethyl squarate (1.5
l
L, 10.1
lmol) and Et3N (0.5
lL) were
added to a soln of heptasaccharide 18 (8.0 mg, 6.7
l
mol) 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 19 (8 mg, 95%) as a white amorphous powder;
[a]
+56.5 (c 0.5, water). 1H NMR (selected assignments for the
D
spacer and squarat moieties, 500 MHz, D2O): d 4.75 (q, 2H, J
7.2 Hz, OCH2CH3), 3.81 (m, 1H, OCH2CH2CH2N), 3.72 (m, 1H,
OCH2CH2CH2N), 3.59 (m, 2H, OCH2CH2CH2N, OCH2CH2CH2N),
1.92 (m, 2H, OCH2CH2CH2N), 1.44 (t, 3H, OCH2CH3); 13C NMR (se-
lected assignments for the spacer and squarat moieties, 126 MHz,
D2O): d 72.1 (1C, OCH2CH3), 66.4, 66.2 (1C, OCH2CH2CH2N), 43.1,
43.3 (1C, OCH2CH2CH2N), 30.7, 30.5 (1C, OCH2CH2CH2N), 16.4
(1C, OCH2CH3); the carbohydrate parts of the 1H and 13C NMR spec-
tra of 19 were essentially the same as those in the spectra of the
starting 3-aminopropyl glycoside 18.
10. De Meo, C.; Kamat, M. N.; Demchenko, A. V. Eur. J. Org. Chem. 2005, 706–711
and references sited therein.
11. Garreg, P. J.; Kvarnstroem, I.; Niklasson, G.; Svensson, S. C. T. J. Carbohydr. Chem.
1993, 12, 933–954.
12. Crich, D.; Banerjee, A.; Li, W.; Yao, Q. J. Carbohydr. Chem. 2005, 24, 415–424.
13. Ogawa, T.; Nukada, T. Carbohydr. Res. 1985, 136, 135–152.
14. Ogawa, T.; Katano, K.; Sasajima, K.; Matsui, M. Tetrahedron 1981, 37, 2779–
2786.
15. Byramova, N. E.; Ovchinnikov, M. V.; Backinowsky, L. V.; Kochetkov, N. K.
Carbohydr. Res. 1983, 124, C8.
16. (a) Tietze, L. F.; Arlt, M.; Beller, M.; Glüsenkamp, K.-H.; Jähde, E.; Rajewsky, M.
Chem. Ber. 1991, 124, 1215–1221; (b) Kamath, V. P.; Diedrich, P.; Hindsgaul, O.
Glycoconjugate J. 1996, 13, 315–319; (c) Chernyak, A.; Karavanov, A.; Ogawa, Y.;
3.15. BSA—heptasaccharide conjugate (20)
A sol of 19 (3.7 mg, 7.6
lmol) and BSA (3.2 mg, 0.048 lmol) in
ˇ
ˇ
Kovác, P. Carbohydr. Res. 2001, 330, 479–486; (d) Hou, S.-j.; Saksena, R.; Kovác,
P. Carbohydr. Res. 2008, 343, 196–210.
2 mL of the buffer soln (350 mM KHCO3 and 70 mM Na2-