Synthesis of a sulfonic acid mimetic of the sulfated Lewis A pentasaccharide

Article abstract of DOI:10.1016/j.carres.2011.12.015

The first sulfonic acid mimetic of the sulfated Lewis A pentasaccharide in which the natural l-fucose unit is replaced by a d-arabinose ring was synthesized. Formation of the sulfonic acid moiety at a pentasaccharide level could be successfully achieved b

Full text of DOI:10.1016/j.carres.2011.12.015

Carbohydrate Research 350 (2012) 90–93
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Note
Synthesis of a sulfonic acid mimetic of the sulfated Lewis A pentasaccharide
a
a,b
Zsolt Jakab a, , Anikó Fekete a, Magdolna Csávás a, Anikó Borbás a, , András Lipták , Sándor Antus
⇑
a Research Group for Carbohydrates of the Hungarian Academy of Sciences, University of Debrecen, PO Box 94, H-4010 Debrecen, Hungary
b Department of Organic Chemistry, University of Debrecen, PO Box 20, H-4010 Debrecen, Hungary
a r t i c l e i n f o
a b s t r a c t
Article history:
The first sulfonic acid mimetic of the sulfated Lewis A pentasaccharide in which the natural
is replaced by a -arabinose ring was synthesized. Formation of the sulfonic acid moiety at a pentasac-
charide level could be successfully achieved by means of introduction of an acetylthio moiety into the
terminal -galactose residue and subsequent oxidation. The equatorial arrangement of the acetylthio
group linked to C-3 of the galactose ring could be obtained by double nucleophilic substitutions; efficient
formation of the gulo-triflate derivatives required low-power microwave (MW) activation. Oxidation of
the acetylthio group was carried out using Oxone in the presence of acetic acid.
Ó 2012 Elsevier Ltd. All rights reserved.
L-fucose unit
Received 12 September 2011
Received in revised form 5 December 2011
Accepted 19 December 2011
Available online 26 December 2011
D
D
Keywords:
Selectins
Sialyl Lewis A
Sulfated Lewis A pentasaccharide
Double nucleophilic displacement
Microwave activation
Sugar sulfonic acid
Selectins are calcium-dependent adhesion molecules that are
expressed on vascular endothelium and on leukocytes. Through
recognition of specific carbohydrate epitopes of their ligands, the
selectins mediate leukocytes rolling along the blood vessel wall
which is the first step of leukocyte adhesion in the inflammation
cascade.1 E-selectin is also postulated to mediate the initial interac-
tions with tumor cells and might be involved in tumor metasta-
sis.2,3 To prevent pathological recruitment of endogenous
leukocytes and to decrease the incidence of metastases, soluble
selectin ligands could be applied as antagonists binding competi-
tively to the selectins and thus inhibiting the adhesion to their nat-
ural ligands.
might show high affinity to E-selectin, moreover, it is resistant
against esterases and sulfatases. The -fucose unit was also re-
placed by a -arabinosyl moiety. It has been demonstrated by Kunz
et al. that the essential fucose unit of the sLex and sLea could be
substituted by a b- -arabinopyranoside possessing much higher
L
D
D
stability toward enzymatic degradation than the
a-L-fucosyl resi-
due (Fig. 1).12,13
Pentasaccharide backbone 6 of the planned mimetic was pre-
pared by coupling of the -arabinosyl-containing trisaccharide
D
411 to the known lactoside acceptor 514 upon NIS-TMSOTf activa-
tion and using a 1:2 ratio of the donor and the acceptor (Scheme
1). After complete conversion of the donor, formation of a main
product and decomposition were also observed (polar components
appeared on TLC). The main product could be isolated only with
moderate yield, fortunately; it proved to be the desired pentasac-
charide 6. Selective removal of the chloroacetyl (CA) group of 6
with thiourea15 resulted in 7 in a 84% yield. It is known from the
literature that 3-acetylthiogalactoside can be prepared via two
subsequent nucleophilic substitution reactions, and a strict substi-
tution pattern of the galactose is required for the successful syn-
thesis: OH-2 has to be protected by an ester group, and OH-4
and OH-6 have to be protected in the form of a benzylidene ace-
tal.16–18 Compound 7 being suitable for the introduction of the
acetylthio moiety at position 3 of the terminal galactose residue
was treated with triflic anhydride and the readily formed triflate
was reacted with tetrabutylammonium nitrite (TBANO2) affording
the gulo-compound 8 in good yield. However, the next triflation
step was very slow and did not go to completion at room temper-
ature in several days. The low reactivity of the axial hydroxyl group
The sialyl Lewis A tetrasaccharide (sLea) 1 and its positional iso-
mer sialyl Lewis X (sLex) have been identified as lead compounds
for binding to P- and E-selectins.4,5 The sulfated Lea pentasaccha-
ride 2 isolated from an ovarian cystoadenoma glycoprotein6 turned
out to be an even more potent E-selectin ligand than the sialylated
Lewis antigens,7–10 demonstrating that the sialic acid can be
advantageously substituted by a sulfate group.
We have recently described the synthesis of two new sulfonic
acids containing trisaccharide mimetics of the tetrasaccharide
1.11 The present paper describes the preparation of the sulfonic
acid pentasaccharide 3, which being a stronger acid than the car-
boxylic acid-containing 1 and the sulfate ester derivative 2,8,10
⇑
Corresponding author.
E-mail address: borbas.aniko@science.unideb.hu (A. Borbás).
Present address: Department of Carbohydrate Chemistry, Chemical Research
Center, Hungarian Academy of Sciences, Pusztaszeri út 59-67, H-1025 Budapest,
Hungary.
0008-6215/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.12.015

Z. Jakab et al. / Carbohydrate Research 350 (2012) 90–93
91
OH
OH
OH
OH
OH
O
H3C
OH
OH
O
H3C
OH
OH
OH
OH
OH
O
OH
OH
OH
OH HOOC
O
O
O
HO
O
O
O
O
O
OH
NHAc
O
O
OH
NHAc
HO3SO
O
O
HO
O
HO
OH
AcHN
OH
NHAc
OH
OH
1
2
OH
OH
OH
O
OH
OH
OH
OH
OH
OH
O
O
O
O
O
O
OMe
NHAc
NaO3S
O
O
HO
OH
NHAc
OH
3
Figure 1. Sialyl Lea tetrasaccharide 1, sulfated Lea pentasaccharide 2 and sulfonic acid mimetic 3.
Conversion of 9 into the sulfonic acid derivative 10 was carried
out in a four-step synthesis involving N- and S-deacylation using
ethylenediamine (EDA) followed by selective N-acetylation, fully
debenzoylation, and subsequent oxidation of the 3-SH group of
the terminal galactose unit into the sulfonic acid salt with Oxone.19
Although TLC monitoring of the individual steps showed complete
and efficient reactions, the overall yield was rather low, probably
due to the insufficient isolation of the oxidation product from the
crude reaction mixture containing high amounts of inorganic salts.
The benzyl and benzylidene groups of 9 were removed by catalytic
hydrogenation over Pd/C to afford 3 as the first sulfonic acid ana-
logue of the sulfated Lewisa pentasaccharide 2.8,10
OBn
OBn
Ph
O
Ph
O
OBn
O
O
O
OBz
OBn
O
O
O
O
O
O
OMe
HO
BzO
SEt
O
CAO
OBz
OBz
OBz
NPhth
5
4
a
OBn
OBn
E
Ph
O
OBn
Ph
O
O
D
O
O
OBn
In conclusion, sulfonic acid-containing arabino Lewisa pentasac-
charide (3) was synthesized applying a 3+2 block synthesis. The
sulfonic acid group at position C-3D was formed at a pentasaccha-
ride level by introduction of an acetylthio moiety into the terminal
galactose residue and subsequent oxidation.
OBz
O
O
O
O
O
O
O
O
C
B
R
A
OMe
BzO
OBz
NPhth
OBz
OBz
R'
6 R= OCA, R'= H
7 R= OH, R'= H
b
c
d
8
R= H, R'= OH
9 R= SAc, R'= H
1. Experimental
e
OBn
1.1. General methods
OBn
Ph
OBn
Ph
E
O
O
D
Optical rotations were measured at room temperature with a
Perkin–Elmer 241 automatic polarimeter. Melting points were
determined on a Kofler hot-stage apparatus and are uncorrected.
All reactions were performed under anhydrous conditions and
monitored by TLC on Kieselgel 60 F254 (Merck) visualised under
UV light and charred with 5% sulfuric acid in ethanol. Column chro-
matography was performed on Silica Gel 60 (Merck 0.062–
0.200 nm). Chemicals were purchased from Aldrich and Fluka and
used without further purification. Molecular sieves were activated
by heating to 360 °C overnight and were cooled over P2O5 in vacuo.
The organic solutions were dried over MgSO4, and concentrated in
vacuum. The 1H (360.13 MHz) and 13C NMR (90.54 MHz) spectra
were recorded with Bruker AM-360 spectrometer for solutions in
CDCl3. The use of a different solvent is indicated therein. Chemical
shifts are referenced to Me4Si (0.00 ppm for 1H) or to the residual
solvent signals (77.00 ppm for 13C). Microwave assisted procedures
took place in a CEM-Discover Focused Microwave Synthesis System
(2450 MHz) with a built-in infrared temperature sensor and a CEM-
Explorer computer controlled robotic sampler attaching system.
Sample was measured in a 10 mL crimp-sealed, thick-walled reac-
tion tube equipped with a magnetic stirrer. MALDI-TOF MS spectra
were recorded on a Bruker Biflex III spectrometer in positive, linear
mode using saturated 2,4,6-trihydroxy-acetophenone in water as
matrix. Elemental analyses (C, H, N, S) were performed using an
Elementar Vario MicroCube instrument.
O
O
O
OBn
OH
O
O
O
O
O
O
C
O
O
B
A
NaO3S
OMe
HO
OH
NHAc
OH
OH
10
f
3
Scheme 1. Reagents and conditions: (a) 4:5 = 1:2, NIS, TMSOTf, dry CH2Cl2, THF,
À45 °C, 2 h, then À20 °C overnight, 45%, (unreacted 5 was recovered with 48% yield)
(b) thiourea, pyridine, CH2Cl2, MeOH, 1 d, 84%; (c) Tf2O, pyridine, dry CH2Cl2, À20 °C
to rt, 1 h, then TBANO2, dry CH3CN, rt, 1 d, 75%; (d) Tf2O, pyridine, dry CH2Cl2, 0 °C,
1/2 h, then MW activation, 35 °C, 1 h, then KSAc, dry DMF, overnight, 68%; (e) EDA,
dry EtOH, reflux, 1 d, then Ac2O, MeOH, 2 h, then NaOMe, MeOH, overnight, then
Oxone, cc. AcOH, KOAc, rt, 4 h, 14% over four steps; (f) Pd(C), H2 (10 bar), EtOH,
4 days, 73%.
of 8 might arise from steric hindrance caused by the bulky phtha-
limido substituent.11 Fortunately, complete formation of the
gulo-triflate could be achieved employing low-power microwave
activation in a CEM Discover Microwave reactor. Treatment of
the obtained triflate with potassium thioacetate (KSAc) afforded
the desired protected pentasaccharide 9 in 68% overall yield for
the two steps.

92
Z. Jakab et al. / Carbohydrate Research 350 (2012) 90–93
1.2. Methyl O-(4,6-O-benzylidene-2-O-benzoyl-3-O-
chloroacetyl-b- -galactopyranosyl)-(1?3)-O-[(2,3,4-tri-O-
benzyl-b- -arabinopyranosyl)-(1?4)]-O-(6-O-benzyl-2-deoxy-
2-phthalimido-b- -glucopyranosyl)-(1?3)-O-(4,6-O-
benzylidene-2-O-benzoyl-b- -galactopyranosyl)-(1?4)-2,3,6-
tri-O-benzoyl-b- -glucopyranoside (6)
Found: C, 68.88; H, 5.36; N, 0.70. MALDI-TOF m/z calcd for
D
[M+Na]+ 2020.67. Found: 2020.64.
D
D
1.4. Methyl O-(4,6-O-benzylidene-2-O-benzoyl-b-
gulopyranosyl)-(1?3)-O-[(2,3,4-tri-O-benzyl-b-
arabinopyranosyl)-(1?4)]-O-(6-O-benzyl-2-deoxy-2-
phthalimido-b- -glucopyranosyl)-(1?3)-O-(4,6-O-benzylidene-
2-O-benzoyl-b- -galactopyranosyl)-(1?4)-2,3,6-tri-O-benzoyl-
b- -glucopyranoside (8)
D-
D
D-
D
D
A mixture of donor 4 (200 mg, 0.16 mmol), acceptor 5 (272 mg,
2 equiv), and 4 Å molecular sieves in dry CH2Cl2 (5 mL) was stirred
for 2 h. Then, the temperature was decreased to À60 °C and NIS
D
D
(46 mg, 1.3 equiv) and TMSOTf (6
l
L, 0.25 equiv) in dry THF
To a solution of the starting compound 7 (330 mg, 0.17 mmol)
in dry CH2Cl2 (1 mL) and pyridine (150 L) was added dropwise
Tf2O (57
L, 0.27 mmol, 1.6 equiv) at À30 °C and the mixture was
(1 mL) was added to the mixture via syringe under Ar and stirred
L) was
l
at À45 °C for 2 h, then at À20 °C overnight. Pyridine (50
l
l
added and the mixture was diluted with CH2Cl2 and filtered
through Celite. The filtrate was washed with satd aq Na2S2O3,
water, satd aq NaHCO3, again with water, dried, and concentrated.
Column chromatography of the residue (9:1 CH2Cl2–EtOAc) gave
compound 6 (147 mg, 45%) as a syrup. Unreacted 5 was also recov-
stirred for 1 h. The mixture was diluted with CH2Cl2, washed with
satd aq NaHCO3, and with water, dried, and concentrated. The
crude triflate was dried under high vacuum for 3 h. To a solution
of the crude triflate in dry acetonitrile (2 mL) was added TBANO2
(150 mg, 3 equiv) and stirred overnight. When TLC indicated the
disappearance of triflate the acetonitrile was evaporated from the
mixture. The residue was diluted with EtOAc, washed three times
with water, dried, and concentrated. The crude product was puri-
fied by column chromatography (96:4 CH2Cl2–acetone) gave com-
ered (130 mg, 48%). Compound 6: [a]
D +8.9 (c 0.18, CHCl3); 1H NMR
(360 MHz): d (ppm) 7.90–7.10 (m, 59H, arom.), 5.61 (t, 1H, J
8.9 Hz), 5.49 (dd, 1H, J 10.3 Hz, J 8.2 Hz), 5.44 (s, 1H), 5.22–5.11
(m, 3H), 5.05–4.99 (m, 2H), 4.88 (d, 1H, J 11.5 Hz), 4.78 (t, 1H, J
9.9 Hz), 4.60 (d, 3H, J 12.4 Hz), 4.51–4.31 (m, 6H), 4.30–4.14 (m,
7H), 4.08–3.60 (m, 18H), 3.54 (dd, 2H, J 23.8 Hz, J 8.9 Hz), 3.36–
3.25 (m, 5H), 3.14 (s, 1H), 2.68 (s, 1H); 13C NMR (90 MHz): d
(ppm) 166.81 (COCH2Cl), 165.34, 165.17, 164.87, 164.59, 164.02
(5 Â PhCO), 139.25, 139.09, 138.49, 138.03, 137.50, 137.21 (arom.
Cs), 133.80–125.50 (arom. Cs), 101.17, 101.03, 99.89, 99.67,
99.61, 98.51 (double int.) (2 Â PhCH, C-1A, C-1B, C-1C, C-1D, C-1E),
78.02, 76.03 (double int.), 75.62, 75.26 (double int.), 75.07, 73.74
(triple int.), 73.31, 73.13, 72.75, 72.41, 72.06, 70.77, 68.27, 66.48,
66.00 (skeleton Cs), 74.88, 72.69, 71.45, 70.36 (4 Â PhCH2), 68.77,
67.73, 67.54 (C-6B, C-6C, C-6D), 62.06 (C-6A), 60.60 (C-5E), 56.60,
55.82 (OMe, C-2C), 40.18 (COCH2Cl). Anal. Calcd for C117H108O32NCl
(2075.55): C, 67.71; H, 5.24; N, 0.67. Found: C, 67.41; H, 5.26; N,
0.68. MALDI-TOF m/z calcd [M+Na]+ 2096.64. Found: 2097.96.
pound 8 (250 mg, 75%) as a syrup: [
a
]
D
À2.4 (c 0.17, CHCl3); 1H
NMR (360 MHz): d (ppm) 7.93 (d, 2H, J 7.7 Hz, arom.), 7.87 (t,
4H, J 7.2 Hz, arom.), 7.79 (d, 2H, J 7.8 Hz, arom.), 7.62–7.02 (m,
51H, arom.), 5.64 (t, 1H, J 9.0 Hz), 5.43 (s, 1H, PhCH), 5.27–5.10
(m, 4H), 5.06 (d, 1H, J 8.4 Hz), 4.95 (d, 1H, J 3.2 Hz), 4.90–4.82
(m, 2H), 4.67 (d, 1H, J 8.6 Hz), 4.60 (t, 2H, J 12.3 Hz), 4.50 (d, 1H,
J 7.9 Hz), 4.47–4.41 (m, 2H), 4.39–4.16 (m, 6H), 4.13–3.92 (m,
5H), 3.91–3.81 (m, 5H), 3.80–3.71 (m, 3H), 3.70–3.48 (m, 5H),
3.37 (s, 2H), 3.32–3.25 (m, 4H, OMe), 2.70 (s, 1H); 13C NMR
(90 MHz): d (ppm) 165.40 (double int.), 165.27, 164.72, 164.14
(5 Â PhCO), 139.28, 139.15, 138.61, 138.11, 137.65, 137.57 (arom.
Cs), 133.70–122.70 (arom. Cs), 101.16 (double int.), 100.12, 99.59,
99.19, 98.61, 95.95 (2 Â PhCH, C-1A, C-1B, C-1C, C-1D, C-1E), 78.13,
77.16, 76.08, 75.90, 75.71, 75.26 (double int.), 75.16, 73.91,
73.82, 72.52, 72.10, 71.88, 70.89, 70.81, 68.94, 66.57, 65.77 (skele-
ton Cs), 74.86, 72.83, 71.54, 70.43 (4x PhCH2), 69.16, 68.10, 67.64
(C-6B, C-6C, C-6D), 62.07 (C-6A), 60.84 (C-5E), 56.69, 56.05 (OMe,
C-2C). Anal. Calcd for C115H107O31N (1999.07): C, 69.09; H, 5.39;
N, 0.70. Found: C, 69.21; H, 5.31; N, 0.70. MALDI-TOF m/z calcd
for [M+Na]+ 2020.67. Found: 2020.56.
1.3. Methyl O-(4,6-O-benzylidene-2-O-benzoyl-b-
D-
galactopyranosyl)-(1?3)-O-[(2,3,4-tri-O-benzyl-b-
D
-
arabinopyranosyl)-(1?4)]-O-(6-O-benzyl-2-deoxy-2-
phthalimido-b- -glucopyranosyl)-(1?3)-O-(4,6-O-benzylidene-
2-O-benzoyl-b- -galactopyranosyl)-(1?4)-2,3,6-tri-O-benzoyl-
b- -glucopyranoside (7)
D
D
D
1.5. Methyl O-(3-S-acetyl-4,6-O-benzylidene-2-O-benzoyl-b-
galactopyranosyl)-(1?3)-O-[(2,3,4-tri-O-benzyl-b-
arabinopyranosyl)-(1?4)]-O-(6-O-benzyl-2-deoxy-2-
phthalimido-b- -glucopyranosyl)-(1?3)-O-(4,6-O-benzylidene-
2-O-benzoyl-b- -galactopyranosyl)-(1?4)- 2,3,6-tri-O-benzoyl-
b- -glucopyranoside (9)
D-
To a solution of compound 6 (411 mg, 0.2 mmol) in CH2Cl2
D-
(6 mL), MeOH (8 mL), and pyridine (2 mL) was added thiourea
(74 mg, 5 equiv) and stirred overnight. The mixture was diluted
with CH2Cl2, washed twice with water, dried, and concentrated.
Column chromatography of the residue (94:6 CH2Cl2–acetone)
D
D
D
gave compound 7 (333 mg, 84%) as a syrup: [
a
]
À13.2 (c 0.05,
D
CHCl3); 1H NMR (360 MHz): d (ppm) 7.90–7.75 (m, 6H, arom.),
7.62–7.08 (m, 53H, arom.), 5.61 (t, 1H, J 8.9 Hz), 5.46 (s, 1H, PhCH),
5.25–5.14 (m, 4H), 5.02 (d, 1H, J 8.2 Hz), 4.99 (d, 1H, J 2.8 Hz), 4.87
(d, 1H, J 11.6 Hz), 4.82 (t, 1H, J 9.8 Hz), 4.63–4.55 (m, 2H), 4.50–
4.30 (m, 5H), 4.29–4.16 (m, 4H), 4.14 (d, 1H, J 8.1 Hz), 4.08–3.72
(m, 14H), 3.68–3.55 (m, 4H), 3.50 (d, 1H, J 9.8 Hz), 3.40 (s, 1H),
3.33–3.18 (m, 5H, OMe), 3.04 (s, 1H), 2.69 (s, 1H); 13C NMR
To a solution of compound 8 (249 mg, 0.13 mmol) in dry CH2Cl2
(2 mL) and pyridine (1 mL) in a CEM equipped vessel was added
dropwise Tf2O (32 L, 1.6 equiv) at 0 °C and the mixture was al-
l
lowed to reach rt and stirred for half an hour. The mixture was acti-
vated under MW conditions (t = 1 h, T = 35 °C, power = 10 kW,
pressure = 20 PSI). The mixture was diluted with CH2Cl2, washed
twice with water, dried, and concentrated. The crude triflate was
left under high vacuum for 3 h. To a solution of the crude triflate
in dry DMF (5 mL) was added KSAc (44 mg, 3 equiv) and stirred
at 70 °C for 2 h. The residue was diluted with EtOAc, washed three
times with water, dried, and concentrated. Column chromatogra-
phy of the residue (96:4 CH2Cl2–acetone) gave compound 9
(90 MHz):
d (ppm) 166.39, 165.34, 165.19, 164.64, 164.06
(5 Â PhCO), 139.18, 139.02, 138.47, 138.00, 137.49, 137.27 (arom.
Cs), 133.80–125.50 (arom. Cs), 101.18, 101.02, 99.87 (double int.),
99.40, 98.69, 98.48 (2 Â PhCH, C-1A, C-1B, C-1C, C-1D, C-1E), 78.06,
76.27, 76.10, 75.62, 75.28, 75.20 (double int.), 75.06, 73.72 (double
int.), 72.60, 72.39, 72.00, 71.80, 71.53, 70.76, 66.47, 66.36 (skeleton
Cs), 74.84, 72.69, 71.48, 70.40 (4 Â PhCH2), 68.82, 67.80, 67.53 (C-
6B, C-6C, C-6D), 62.03 (C-6A), 60.65 (C-5E), 56.62, 55.87 (OMe, C-2C).
Anal. Calcd for C115H107O31N (1999.07): C, 69.09; H, 5.39, N, 0.70.
(180 mg, 68%) as a syrup: [
a
]
D
+8.1 (c 0.09, CHCl3); 1H NMR
(360 MHz): d (ppm) 7.88 (dd, 4H, J 7.2 Hz, J 1.1 Hz, arom.), 7.79
(d, 2H, J 7.9 Hz, arom.), 7.70–7.08 (m, 53H), 5.64 (t, 1H, J 8.9 Hz,
H-3A), 5.46 (s, 1H, PhCH), 5.29–5.11 (m, 4H, H-2D, H-2A, H-2B,

Z. Jakab et al. / Carbohydrate Research 350 (2012) 90–93
93
PhCH), 5.06–4.96 (m, 2H, H-1C, H-1E), 4.87 (d, 1H, J 11.5 Hz, PhCH2),
4.76 (t, 1H, J 9.8 Hz, H-3C), 4.65–4.55 (m, 2H, H-5E, PhCH2), 4.57–
4.42 (m, 4H, H-1A, H-1B, PhCH2), 4.42–4.31 (m, 2H, H-6Aa, PhCHH),
4.31–4.21 (m, 3H, H-6Da, H-1D, PhCH2), 4.20–4.11 (m, 2H, H-2C, H-
6Ab), 4.11–3.93 (m, 5H, H-4B, H-2E, H-3E, H-4A, H-6Db), 3.92–3.70
(m, 6H, PhCH2, H-6Ca, H-6Cb, H-4C, H-3B), 3.69–3.53 (m, 6H, H-
6Ba, H-6Bb, H-5Eb, H-5A, H-3D, H-5C), 3.40–3.25 (m, 6H, OMe, H-
4D, H-4E, H-5D), 2.68 (s, 1H, H-5B), 1.99 (s, 3H, SCOCH3); 13C NMR
(90 MHz): d (ppm) 194.32 (SCOCH3), 165.45, 165.27, 165.11,
164.68, 164.08 (5 Â PhCO), 139.32, 139.21, 138.60, 138.13,
137.55, 137.27 (arom. Cs), 133.70–125.50 (arom. Cs), 101.24 (dou-
ble int.), 101.16 (C-1A, C-1B, C-1D), 100.03 (double int.) (2 Â PhCH),
98.71, 98.56 (C-1C, C-1E), 78.09, 76.33, 76.09, 75.73, 75.41 (double
int.), 75.33, 75.15, 73.79 (double int.), 73.00, 72.56, 72.18, 70.82,
68.37, 68.25, 66.55 (skeleton Cs), 74.93, 72.79, 71.51, 70.44 (4x
PhCH2), 68.95, 67.91, 67.61 (C-6B, C-6C, C-6D), 62.15 (C-6A), 60.62
(C-5E), 56.70 (OMe), 55.85 (C-2C), 46.83 (C-3D), 30.22 (SCOCH3).
Anal. Calcd for C117H109O31NS (2057.17): C, 68.31; H, 5.34; N,
0.68; S, 1.56. Found: C, 68.38; H, 5.30; N, 0.68; S, 1.53. MALDI-
TOF m/z calcd for [M+Na]+ 2078.66. Found: 2078.62.
NSNa (1476.52): C, 60.20; H, 5.87; N, 0.95; S, 2.17. Found: C,
60.38; H, 5.89; N, 0.96; S, 2.15. MALDI-TOF m/z calcd for [M+Na]+
1498.49. Found: 1498.79.
1.7. Methyl O-(3-deoxy-3-sodiumsulfonato-b-
galactopyranosyl)-(1?3)-O-[(b- -arabinopyranosyl)-(1?4)]-O-
(2-acetamido-2-deoxy-b- -glucopyranosyl)-(1?3)-O-
(b- -galactopyranosyl)-(1?4)-b- -glucopyranoside (3)
D-
D
D
D
D
To a solution of compound 10 (18 mg, 0.01 mmol) in ethanol–
water (5 mL, 4:1) was added 10% Pd/C (30 mg) and stirred under
H2 (10 bar) for 6 d. The mixture was filtered through Celite and
the filtrate was concentrated. Column chromatography of the res-
idue (1:1 CH2Cl2–MeOH) gave compound 3 (8 mg, 73%) as an
amorphous product: [
a
]
À55 (c 0.04, 1:1 MeOH–water); 1H
D
NMR (360 MHz, MeOD): d (ppm) 5.07 (d, 1H, J 3.5 Hz, H-1E), 4.76
(d, 2H, J 8.6 Hz, 2 Â H-1), 4.59 (d, 1H, J 7.4 Hz), 4.37 (d, 1H, J
7.5 Hz), 4.23–4.18 (m, 2H), 4.10 (t, 1H, J 9.5 Hz), 4.05 (d, 1H, J
2 Hz), 3.99 (dd, 1H, J 10.6 Hz, J 7.6 Hz), 3.94–3.66 (m, 13H), 3.66–
3.36 (m, 14H, incl. OMe), 3.22 (t, 1H, J 8.4 Hz), 2.84 (dd, 1H, J
10.9 Hz, J 2 Hz, H-3D), 1.98 (s, 3H, NHCOCH3); 13C-NMR (90 MHz,
MeOD): d (ppm) 173.15 (NHCOCH3), 103.85, 103.61 (double int.),
102.34, 98.91 (C-1A, C-1B, C-1C, C-1D, C-1E), 82.26, 79.06, 77.63,
76.09, 75.87, 75.24, 75.07, 74.99, 73.30, 72.51, 70.16, 69.66,
68.95, 68.92, 68.45, 66.74, 65.47, 63.64 (skeleton Cs), 63.97,
61.07 (double int.), 60.43, 59.90 (C-6A, C-6B, C-6C, C-6D, C-5E),
56.26 (C-2C), 55.92 (OMe), 21.88 (NHCOCH3). Anal. Calcd for
1.6. Methyl O-(4,6-O-benzylidene-3-deoxy-3-sodiumsulfonato-
b-
arabinopyranosyl)-(1?4)]-O-(2-acetamido-6-O-benzyl-2-
deoxy-b- -glucopyranosyl)-(1?3)-O-(4,6-O-benzylidene-b-
galactopyranosyl)-(1?4)-b- -glucopyranoside (10)
D-galactopyranosyl)-(1?3)-O-[(2,3,4-tri-O-benzyl-b-D-
D
D-
D
To a solution of compound 9 (89 mg, 0.04 mmol) in anhydr. eth-
anol (5 mL) was added EDA (1 mL) and refluxed for 1 day. The
amine was detected by ninhydrin and the mixture was concen-
trated and coevaporated twice with toluene. The residue was dis-
solved in MeOH (3 mL) and treated with Ac2O (1 mL). After 2 h
the mixture was concentrated, coevaporated twice with toluene,
and dried. The residue was solved again in MeOH (3 mL) and
C32H54O27NSNa (939.82): C, 41.63; H, 5.86; N, 2.86; S, 3.27. Found:
C, 41.47; H, 5.88; N, 2.85; S, 3.25. MALDI-TOF m/z calcd for [M+Na]+
962.24. Found: 962.47.
Acknowledgements
This work was supported by the TÁMOP 4.2.1/B-09/1/KONV-
2010-0007 project. The project is co-financed by the European
Union and the European Social Fund. Financial support of the
Hungarian Research Fund (K 62802) is also acknowledged.
30 lL 30% NaOMe in MeOH was added and the mixture was stirred
overnight. The mixture was neutralized with Amberlite IR-120 H+
cation exchange resin, filtered, and concentrated to dryness. To a
suspension of the residue in glacial acetic acid (1 mL) was added
KOAc (85 mg, 20 equiv) and Oxone (66 mg, 2.5 equiv) and stirred
vigorously for 2 h under Ar. TLC analysis indicated the completion
of the reaction. The reaction mixture was neutralized by adding
satd aq and solid NaHCO3 and washed three times with EtOAc.
The collected organic phase was washed with water, dried, and
concentrated. Twofold column chromatography of the residue in
LH-20 (1:1 CH2Cl2–MeOH) gave compound 10 (11 mg, 14% over
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four steps) as an amorphous product: [
a
]
D
À27.3 (c 0.03, MeOH);
1H NMR (360 MHz, CDCl3, MeOD): d (ppm) 7.81 (d, 2H, J 9.8 Hz,
arom.), 7.59 (dd, 2H, J 6.6 Hz, J 2.7 Hz, arom.), 7.54–7.28 (m, 26H,
arom.), 5.82 (s, 1H, PhCH), 5.59 (s, 1H, PhCH), 5.17 (d, 1H, J
1.4 Hz, H-1E), 5.01–4.87 (m, 3H, H-1A, H-5Ea, PhCH2), 4.81 (d, 1H,
J 7.7 Hz, H-1C), 4.78–4.62 (m, 5H, PhCH2, H-2D, H-1D), 4.56–4.50
(m, 2H, H-3C, PhCH2), 4.40–3.38 (m, 7H, PhCH2, H-2C, H-1B, H-
6Ba,H-6Bb, H-6Da, H-4D), 4.21–4.15 (m, 1H, H-2A), 4.13–3.45 (m,
24H, H-6Ca, H-6Aa, H-6Ab, H-2E, H-3A, H-6Cb, PhCH2, H-4C, H-5C,
H-3E, H-5Eb, H-4E, H-4A, H-2B, H-3B, H-5A, H-5B, H-4B, OMe,
H-5D), 3.23 (dd, 1H, J 10.8 Hz, J 2.9 Hz, H-3D), 2.15 (s, 3H,
NHCOCH3); 13C NMR (90 MHz, CDCl3, MeOD): d (ppm) 172.56
(NHCOCH3), 138.39, 138.14, 137.87, 137.60, 137.24, 137.04 (arom.
Cs), 127.80–125.25 (arom. Cs), 103.07 (C-1B), 102.95 (C-1C), 102.56
(C-1D), 101.42 (C-1A), 99.98 (PhCH), 98.96 (PhCH), 97.75 (C-1E),
79.76, 77.78, 76.84, 76.49, 74.90, 74.84, 74.76, 74.30, 74.17,74.03,
73.80, 72.93, 72.66, 72.57, 72.45, 68.34, 67.94, 65.97 (skeleton
Cs) 73.80, 72.45, 70.89, 69.75 (4 Â PhCH2), 68.51, 67.94, 67.45
(C-6B, C-6C, C-6D), 62.24 (C-3D), 60.22 (C-5E), 59.58 (C-6A), 55.46
(OMe), 55.07 (C-2C), 21.30 (NHCOCH3). Anal. Calcd for C74H86O27
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acid formed in situ in the reaction of potassium peroxomonosulfate and acetic
acid was used as the reagent for the oxidation of the masked thiol group.