Synthesis, structural, and biological studies on a pseudodisaccharide containing a bicyclic, bridged carba-sugar

Article abstract of DOI:10.1016/j.tetasy.2011.07.023

Two carba-sugar containing pseudodisaccharide diastereomers have been synthesized from a racemic bicyclooctene derivative. The determination of the absolute configurations was carried out by means of CD measurements, CD calculations and X-ray diffraction.

Full text of DOI:10.1016/j.tetasy.2011.07.023

Tetrahedron: Asymmetry 22 (2011) 1404–1410
Contents lists available at SciVerse ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Synthesis, structural, and biological studies on a pseudodisaccharide
containing a bicyclic, bridged carba-sugar
Zsolt Fejes a, Tibor Kurtán b, Gerhard Raabe c, Jörg Fleischhauer c, László Szilágyi b, Attila Bényei d,
Zoltán Gál e, Wenjing Zhao f, Robert J. Linhardt f, Miroslaw Cygler g, Pál Herczegh a,
⇑
a Department of Pharmaceutical Chemistry, University of Debrecen, Debrecen H-4032, Hungary
b Department of Organic Chemistry, University of Debrecen, Debrecen H-4032, Hungary
c Institute of Organic Chemistry, RWTH Aachen University, Landoltweg 1, D-52074 Aachen, Germany
d Institute of Physical Chemistry, University of Debrecen, Debrecen H-4032, Hungary
e Oxford Diffraction Ltd, Yarnton, Oxfordshire OX5 1QU, United Kingdom
f Center for Biotechnology and Interdisciplinary Studies, Department of Biology, Rensselaer Polytechnic Institute, Troy, NY 12180, USA
g Biotechnology Research Institute, NRC, Montreal, Quebec, Canada H4P 2R2
a r t i c l e i n f o
a b s t r a c t
Article history:
Two carba-sugar containing pseudodisaccharide diastereomers have been synthesized from a racemic
bicyclooctene derivative. The determination of the absolute configurations was carried out by means
of CD measurements, CD calculations and X-ray diffraction. The compounds showed moderate compet-
itive inhibitory effects on chondroitin AC-I lyase.
Received 26 June 2011
Accepted 25 July 2011
Available online 1 September 2011
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
uronic acid part of chondroitin sulfates. The mechanism of the
cleavage is based on a b-elimination of the 4-O-substituent of the
Recently1 we have published the synthesis of a bridged analog
of the cytotoxic pericosine antibiotic in racemic form (Fig. 1).
uronic acid (Scheme 1).4
In order to obtain an inhibitor of chondroitin AC lyase, Rye and
Withers synthesized uronic acid containing thio-linked disaccha-
ride, which proved to be a weak competitive inhibitor of the en-
zyme with Ki = 45 mM.5
HO
OH
MeOOC
In order to study the role of a bridged carba-uronic acid as a D-
glucuronic acid substitute in a potential lyase enzyme inhibitor, we
decided to synthesize a pseudodisaccharide using the synthetic
intermediate rac-2 as a starting compound.
OH
OH
1
rac-
Figure 1. Pericosine analog rac-1.
2. Results and discussion
Due to the apparent similarity of rac-1 to uronic acid, rac-1 can
be regarded as a carba-uronic acid analog. Since a structural resem-
blance to the parent sugar generally induces interesting conse-
quences in the biological activity, it was obvious for us to find an
application of this compound as a precursor for the synthesis of a
pseudodisaccharide derivative. The synthesis of carba-sugars is a
developing area of carbohydrate chemistry,2 but until now, bridged
carba-analogs have not been reported.
Glycosaminoglycan-degrading enzymes, including chondroitin
AC lyases, have found widespread application for the analysis of
various polysaccharides.3,4 Chondroitin AC lyases catalyze the
cleavage of the glycosidic bond on the non-reducing end of the
The conjugate addition of the thiolate of 3 onto the activated
double bond of rac-2 led to the formation of a mixture of two dia-
stereomers 4a and 4b (Scheme 2). The latter compounds could be
obtained in pure form after column chromatography.
In the diastereomers, the absolute stereochemistry of the agly-
con could not be deduced by NMR spectroscopy from the known
absolute configuration of the sugar moiety because of the free rota-
tion about the sulfur–carbon bonds of the thioglycoside. Thus, ECD
spectroscopy was applied to determine the absolute configuration.
Diastereomers 4a and 4b gave almost mirror image ECD spectra,
which confirmed that the sugar moiety has only a minor effect
on the ECD properties. Compound 4a gave a negative n?p⁄ ECD
transition as a long plateau in the range of 275–350 nm, while
4b had a positive n?p⁄ CE (Fig. 2).
⇑
Corresponding author. Tel.: +36 52 512 900x22895; fax: +36 52 512 914.
E-mail address: herczegh.pal@pharm.unideb.hu (P. Herczegh).
0957-4166/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2011.07.023

Z. Fejes et al. / Tetrahedron: Asymmetry 22 (2011) 1404–1410
1405
OR
on the theoretical model structure 4e, in which the thioglycoside
moiety was replaced by a SMe group (Scheme 3). This simplifica-
tion could be made, since the diastereomers 4a and 4b gave nearly
mirror image ECD spectra regardless of the presence of the same
thioglycoside moiety. The ECD calculation was performed in order
to correlate the experimental ECD curves with the absolute config-
uration at C-4 and C-5 of 4d. This allowed us to assign the absolute
configuration of 4a and 4b.
RO
R'O
COO–
–
R
= H, SO3
O
O
R'
R''
= uronic acid
= hexosamine
O
OR''
HO
AcHN
OH
OR
COO–
O
chondroitin AC
lyase
RO
R'O
O
+
OR''
OH
HO
OH
AcHN
OR
Scheme 1. Proposed elimination mechanism of chondroitin AC lyase.4
HO
COOMe
O
4
NaBH4, 1,4-dioxane
5
4a
O
S
O
MeO
Ph
OBn
52 %
OAc
OAc
O
SH
O
Ph
COOMe
AcO
AcO
Et3N, CH3CN
O
AcHN
OH
+
NHAc
OAc
94 %
HO
O
O
MeO
Ph
COOMe
OAc
4c R=Bn
H2, Pd(C)
EtOAc/MeOH
71 %
Ph
4
2
3
rac-
5
O
SMe
MeO
Ph
4e
4d
R=H
OBn
COOMe
BnO
MeOOC
7
Ph
(theoretical model)
O
O
O
8
1
O
6
2
+
4
3
5
Scheme 3. Preparation of 4d and the structure of its theoretical model 4e.
O
O
R
R
MeO
Ph
OMe
Ph
Ph
Ph
4a
4b
O
S
AcO
AcO
NHAc
R =
OAc
Scheme 2. Preparation of diastereomers 4a and 4b from rac-2.
However, the n?p⁄ transition could not be used to determine
the configuration unambiguously. The application of the octant
rule is not reliable, since the orientation of the groups in the sec-
tors, especially in the front ones, cannot be estimated for certainty.
Thus the ketone carbonyl of 4a was reduced to a hydroxyl group
and its benzyl group was removed in order to simplify the mole-
cule for a TDDFT ECD calculation (4a?4d). As a result, the negative
ECD band above 270 nm disappeared, while the other regions of
the spectrum did not show any significant changes (Fig. 2). In 4d,
it is the diphenylmethylidene acetal chromophore and the C-4
and C-5 stereogenic centers of the 1,3-dioxolane ring that deter-
mine the CEs of the ECD spectrum. In order to determine the abso-
lute configuration of 4d, a TDDFT ECD calculation was carried out
Figure 3. ECD spectra of the theoretical model structure 4e calculated at the B3LYP/
TZVP//HF/6-31G⁄ level of time-dependent density functional theory (
cm2 molꢀ1).
De in 1000
The general shape of the calculated CD curve of 4e (Fig. 3) nicely
reproduces the experimental CD spectrum of compound 4d in Fig-
ure 2. Thus, a very weak negative band calculated at 274 nm (not
resolved in Fig. 3) corresponds to the negative part of the experi-
mental CD curve around 260 nm. A slightly stronger positive CD
band calculated at 256 nm could be correlated with the observed
positive Cotton effect at 229 nm. Moreover, the strong negative
and the strong positive Cotton effects observed at 199 and
184 nm can be assigned to those calculated at 208 and 180 nm.
In this way, the absolute configurations (1R,2R,3R,4R,5R,7R) and
(1S,2S,3S,4S,5S,7S) have been assigned to the bicyclooctane skele-
ton of 4a and 4b, respectively. These results were supported by
X-ray diffraction measurements on 4b (Fig. 4). Single crystals were
grown from a methanol–butanol–water mixture. The asymmetric
unit contains four independent molecules of 4b in the P1 space
group resulting in a large unit cell. Solvent areas and one acetyl
group remained disordered, resulting in CheckCIF errors, but this
does not influence the overall correctness of the absolute structure
determination of 4b.
After Zemplén deacetylation of 4a, the carbonyl group was re-
duced resulting in a 6a quasi-axial alcohol with complete stereose-
Figure 2. Experimental ECD spectra of 4a, 4b, and 4d in acetonitrile. De Values are
multiplied by 10 above 225 nm.

1406
Z. Fejes et al. / Tetrahedron: Asymmetry 22 (2011) 1404–1410
HO
S
OH
HOOC
O
same steps as for
4a 9a
OH
OH
HO
HO
4b
NHAc
OH
9b
Scheme 5. Functionalization of the diastereomer 4b.
sis (Scheme 4). All of these (4a?9a) chemical transformations
were also conducted on diastereomer 4b, resulting in 9b
(Scheme 5).
Both compounds 9a and 9b showed competitive inhibition of
chondroitin AC-I lyase with Ki values of 2.4 ꢁ 10ꢀ6 and
1.7 ꢁ 10ꢀ6 M, respectively (Fig. 5). A Km value of 1.9 ꢁ 10ꢀ6
M
was determined for the enzyme without an inhibitor. The Km/Ki
values for both compounds were close to 1, indicating comparable
enzyme-binding affinities for the natural substrate and inhibitors.
Compound 9b was a slightly better inhibitor than compound 9a.
Figure 4. ORTEP view of 4b.
lectivity. The configuration of the newly formed stereogenic center
at C6 was supported by the NOEs of the H6 with H7 and the 6-OH
with 5-OMe. In the subsequent steps the diphenylmethylene acetal
protecting group was removed by acid hydrolysis and the deliber-
ated carbonyl group was stereoselectively reduced to give 7a with
a delicate stereoheptade generated step by step from the achiral
aromatic methyl gallate.1 It should be noted that during the
sodium borohydride reduction, an inversion of configuration of
the stereogenic center bearing the carboxylic residue in 7a as de-
tected. The benzyl ether and methyl ester protecting functions of
7a were removed by catalytic hydrogenation and careful hydroly-
OBn
O
NaBH4, CeCl3·7H2O
NaOMe
MeOH
COOMe
MeOH, –70 o
quant.
C
O
4a
quant.
Figure 5. Lineweaver–Burk plot showing the inhibition by 9a and 9b. Experimental
procedures are detailed in supporting information. Kinetics analysis displayed a
competitive inhibition for both compounds 9a (red square) and 9b (green triangle)
by having the same y-intercept as uninhibited enzyme (blue diamond).
O
R
MeO
Ph
Ph
5a
1. TFA, CH2Cl2
2. NaBH4
3. Conclusion
OBn
COOMe
OBn
COOMe
HO
O
THF/MeOH
HO
–15 oC
It is very interesting that both enantiomeric moieties of the 9a
and 9b pseudodisaccharide diastereomers, bridged carba-uronic
acid analogs, could mimic D-glucuronic acid almost equally as well
in binding to the enzyme. The low substrate specificity of chon-
droitin lyase AB is remarkable. These results were as expected
based on previous literature that suggests thioglycosides typically
bind to glycosidases with similar affinities as their oxygen-substi-
tuted counterparts.6
46 % (2 steps)
O
R
MeO
Ph
HO
OH
R
Ph
7a
6a
H2, Pd(C), MeOH
46 %
OH
OH
R
HO
HO
4. Experimental
4.1. General
1. NaOH, H2O
2. H+
COOMe
COOH
quant.
HO
HO
OH
OH
R
8a
9a
Solutions were concentrated in vacuo at 40 °C. Organic phases
were dried over Na2SO4. Dry MeOH was distilled over Mg. TLC
was performed on Merck Kieselgel F254 plates, spots were made
visible using UV light (254 nm) and/or spraying with acidic
(H2SO4) ammonium molybdate solution followed by heating.
Column chromatography was carried out using Merck Kieselgel
O
S
HO
R =
HO
OH
NHAc
Scheme 4. Functionalization of the diastereomer 4a.

Z. Fejes et al. / Tetrahedron: Asymmetry 22 (2011) 1404–1410
1407
60 silica (0.063–0.200 mm). NMR spectra were recorded on a Bru-
ker Avance DRX 500 or Bruker Avance 400 spectrometer, at the gi-
ven frequency and in the given solvent. Chemical shifts are given in
ppm and coupling constants in hertz. NMR assignments were
based on 2D COSY and 2D HSQC experiments. Mass spectra were
obtained on a Bruker microTOF-Q (ESI-QqTOF) spectrometer. CD
spectra were recorded with a J-810 spectropolarimeter. Melting
points were measured on a Büchi Melting Point B-540 device and
are uncorrected. X-ray data were collected on an Oxford Diffraction
SuperNova diffractometer equipped with Atlas CCD detector (Cu
c = 2.07 ꢁ 10ꢀ4): 332sh (0.21), 319 (0.28), 305sh (0.26), 268sh
(0.49), 260 (0.54), 254 (0.38), 230 (ꢀ1.18), 205sh (14.74), 197
(45.42), 182 (ꢀ42.34). 1H NMR (500 MHz, CDCl3): d 8 7.60–7.24
0
0
0
(m, 15H, Ar), 5.82 (d, 1H, J2 = 9.3, NH), 5.48 (t, 1H, J2 ꢃJ4 ꢃ10, H-
0
0
30), 5.38 (d, 1H, J2 = 10.3, H-1 ), 5.20 (t, 1H, J3 ꢃJ5 ꢃ10, H-4 ), 4.57
0
0
0
4
(t, 1H, J2ꢃ J8bꢃ3, H-3), 4.43 (d, 1H, Jgem = 11.8, OCH2Ph), 4.35
(dd, 1H, J6 a = 12.2, J5 = 5.7, H-60b), 4.35 (d, 1H, Jgem = 11.8,
0
0
OCH2Ph), 4.24 (dd, 1H, J6 b = 12.2, J5 = 2.3, H-60a), 4.14 (dt, 1H,
0
0
JNH = 9.3, J1 ꢃJ3 ꢃ10, H-20), 3.88 (ddd, 1H, J4 = 10.1, J6 b = 5.7,
0
0
0
0
J6 a = 2.3, H-50), 3.71 (dd, 1H, J8b = 8.7, J1 = 4.9, H-7), 3.71 (s, 3H,
0
K
a
radiation, k = 1.54184 Å). The structure was solved using the
COOMe), 3.47 (t, 1H, J1ꢃJ3ꢃ3, H-2), 3.24 (t, 1H, J2ꢃJ7ꢃ3.5, H-1),
2.87 (s, 3H, 5-OMe), 2.30 (d, 1H, J8b = 15.5, H-8a), 2.09 (s, 3H,
Ac), 2.08 (s, 3H, Ac), 1.90 (s, 3H, Ac), 1.89 (s, 3H, Ac), 1.69 (ddd,
1H, J8a = 15.5, J7 = 8.7, 4J3 = 2.5, H-8b). 13C NMR (125 MHz, CDCl3):
SIR-92 software7 and refined on F2 using SHELX-97 program,8 pub-
lication material was prepared with the WINGX-97 suite.9 Crystal-
lographic data (excluding structure factors) for the structure 4b in
this paper have been deposited with the Cambridge Crystallo-
graphic Data Center as supplementary publication no. CCDC
834844. Copies of the data can be obtained, free of charge, on
application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK
(fax: +44 (0)1223 336033 or e-mail: deposit@ccdc.cam.ac.uk).
d = 197.6
(C-6),
172.5,
170.9,
170.7,
170.0,
169.5
(4ꢁCH3CO + COOMe), 143.4–125.1 (Ar), 113.8 (CPh2), 103.3 (C-5),
88.3 (C-4), 84.1 (C-10), 76.1 (C-50), 73.5 (C-30), 70.5 (C-7), 70.0
(CH2Ph), 68.7 (C-40), 62.5 (C-60), 54.2 (C-20), 52.4 (COOCH3), 50.3
(C-1), 50.2 (5-OMe), 45.8 (C-2), 43.2 (C-3), 31.3 (C-8), 23.2,
20.74, 20.67, 20.5 (4ꢁCH3CO).
4.2. (1R,2R,3R,4R,5R,7R)-7-Benzyloxy-4,5-dihydroxy-5-methoxy-
2-methoxycarbonyl-6-oxo-4,5-O-diphenylmethylidenebicyclo-
[2.2.2]octan-3-yl 20-acetamido-30,40,60-tri-O-acetyl-20-deoxy-10-
4.3. (1R,2R,3R,4R,5S,6R,7R)-7-Benzyloxy-4,5,6-trihydroxy-5-
methoxy-2-methoxy-carbonyl-4,5-O-diphenylmethylidenebi-
cyclo[2.2.2]octan-3-yl 20-acetamido-30,40,60-tri-O-acetyl-20-
thio-b-D-glucopyranoside 4a and (1S,2S,3S,4S,5S,7S)-7-benzyloxy-
4,5-dihydroxy-5-methoxy-2-methoxycarbonyl-6-oxo-4,5-O-
deoxy-10-thio-b-
D-glucopyranoside 4c
diphenylmethylidenebicyclo[2.2.2]octan-3-yl 20-acetamido-
30,40,60-tri-O-acetyl-20-deoxy-10-thio-b-
D-glucopyranoside 4b
To a solution of 4a (399 mg, 0.46 mmol) in 1,4-dioxane (10 ml)
at 15 °C, NaBH4 (24 mg, 0.63 mmol) was added. The mixture was
stirred for 3 h at 15 °C and then concentrated. The residue was
partitioned between EtOAc and an aq citric acid solution (0.3%).
The organic phase was extracted with saturated NaHCO3 solution,
dried and concentrated. The crude was purified by column chro-
matography (hexane/EtOAc 4:6) to obtain 206 mg (52%) of 4c as a
white solid. Mp: 120–123 °C. HRMS: m/z calcd for C45H51NO15SNa
A solution of 2 (1.47 g, 2.87 mmol), 2-acetamido-3,4,6-tri-O-
acetyl-2-deoxy-1-thio-b-
-glucopyranose10,11 (1.25 g, 3.44 mmol),
D
and Et3N (0.93 ml, 6.67 mmol) in MeCN (65 ml) was stirred at
room temperature for 4 h. The reaction mixture was concentrated
and the residue was subjected to column chromatography (hex-
ane/CH2Cl2/acetone 6:4:1?5:4:1?4:4:1) to furnish the two dia-
stereomers: 1.00 g (40%) 4a and 1.07 g (43%) 4b. 0.28 g (11%) of
the product was eluted as a mixture of 4a and 4b which was sep-
arated using preparative TLC (CH2Cl2/EtOAc 7:3) to furnish a fur-
ther 0.17 g (7%) of 4a and 0.10 g (4%) of 4b. Single crystals of 4b
were grown by very slow evaporation of methanol–butanol–water
mixture. Compound 4a: mp: 160–163 °C. HRMS: m/z calcd for
[M+Na]+ 900.2877, found 900.2844 (ESI-TOF). ½a 2D3
¼ ꢀ26:9 (c
ꢂ
0.13, CH2Cl2). CD (MeCN, k [nm] (
D
e
), c = 1.59 ꢁ 10ꢀ4): 268sh
(ꢀ0.84), 261 (ꢀ0.96), 254sh (ꢀ0.64), 248sh (ꢀ0.25), 228 (4.45),
214sh (ꢀ21.55), 207 (ꢀ31.00), 197 (ꢀ80.46), 183 (93.08). 1H
NMR (400 MHz, CDCl3): d = 7.55–7.19 (m, 15H, Ar); 5.83 (d, 1H,
J2 = 8.3, NH); 5.64 (dd, 1H, J2 = 10.3, J4 = 9.3, H-30); 5.26 (d, 1H,
0
0
0
45H49NO15SNa [M+Na]+ 898.2721, found 898.2750 (ESI-TOF).
J2 = 10.4, H-10); 5.17 (dd, 1H, J5 = 10.1, J3 = 9.3, H-40); 4.54 (d,
1H, J6 = 10.6, OH); 4.44 (d, 1H, Jgem = 12.0, CH2Ph); 4.40 (dd, 1H,
0
0
0
C
½
a 2D3
ꢂ
¼ ꢀ36:0 (c 0.10, CH2Cl2). CD (MeCN,
k
[nm]
(De),
c = 2.58 ꢁ 10ꢀ4): 332sh (ꢀ0.14), 318sh (ꢀ0.15), 305 (ꢀ0.18),
268sh (ꢀ0.54), 261 (ꢀ0.57), 254sh (ꢀ0.35), 232 (2.15), 216sh
(ꢀ8.89), 206sh (ꢀ17.50), 197 (ꢀ42.03), 183 (62.57). 1H NMR
J6 a = 12.2, J5 = 5.5, H-60b); 4.30 (d, 1H, Jgem = 12.0, CH2Ph); 4.29
0
0
4
(t, 1H, J2ꢃ J8bꢃ3, H-3); 4.21 (dd, 1H, J6 b = 12.2, J5 = 2.4, H-60a);
0
0
0
0
0
0
3.99 (dt, 1H, JNH = 8.3, J1 ꢃJ3 ꢃ10, H-2 ); 3.88 (ddd, 1H, J4 = 10.1,
J6 b = 5.5, J6 a = 2.4, H-50); 3.85 (d, 1H, JOH = 10.6, H-6); 3.72 (dd,
1H, J8b = 8.3, J1 = 4.4, H-7); 3.78 (s, 3H, COOMe); 3.44 (t, 1H,
J1ꢃJ3ꢃ3, H-2); 2.86 (s, 3H, 5-OMe); 2.58 (t, 1H, J2ꢃJ7ꢃ3.5, H-1);
2.07 (s, 3H, Ac); 2.03 (s, 3H, Ac); 1.97 (s, 3H, Ac); 1.90 (d, 1H,
J8b = 15.2, H-8a); 1.80 (s, 3H, Ac); 1.61 (ddd, 1H, J8a = 15.2,
J7 = 8.5, 4J3 = 2.7, H-8b). 13C NMR (100 MHz, CDCl3): d = 176.6,
170.7, 170.5, 170.4, 169.6 (4xCH3CO + COOMe); 144.6-125.1
(Ar); 112.2 (CPh2); 106.2 (C-5); 86.2 (C-4); 83.1 (C-10); 76.0 (C-
50); 74.0 (C-6); 72.9 (C-30); 72.0 (C-7); 69.6 (CH2Ph); 69.2 (C-40);
62.9 (C-60); 55.1 (C-20); 52.8 (COOCH3); 52.6 (5-OMe); 48.6 (C-
2); 44.1 (C-3); 41.4 (C-1); 30.4 (C-8); 23.3 (CH3CO); 20.7 (3C,
CH3CO).
0
0
(500 MHz, CDCl3): d = 7.59–7.22 (m, 15H, Ar), 5.69 (d, 1H,
0
0
0
0
0
0
J2 = 9.1, NH), 5.41 (t, 1H, J2 ꢃJ4 ꢃ10, H-3 ), 5.21 (t, 1H, J3 ꢃJ5 ꢃ10,
H-40), 5.19 (d, 1H, J2 = 10.3, H-10), 4.45 (d, 1H, Jgem = 11.8, OCH2Ph),
0
4
4.42 (t, 1H, J2ꢃ J8bꢃ3, H-3), 4.39 (dd, 1H, J6 a = 12.2, J5 = 5.4, H-60b),
0
0
0
0
4.35 (d, 1H, Jgem = 11.8, OCH2Ph), 4.26 (dd, 1H, J6 b = 12.2, J5 = 2.4,
H-60a), 4.19 (dt, 1H, JNH = 9.1, J1 ꢃJ3 ꢃ10, H-2 ), 3.90 (ddd, 1H,
0
0
0
J4 = 10.1, J6 a = 5.4, J6 b = 2.4, H-50), 3.72 (dd, 1H, J8b = 8.7, J1 = 4.3,
H-7), 3.71 (s, 3H, COOMe), 3.52 (t, 1H, J1ꢃJ3ꢃ3, H-2), 3.23 (t, 1H,
J2ꢃJ7ꢃ3.5, H-1), 2.86 (s, 3H, 5-OMe), 2.12 (d, 1H, J8b = 15.7, H-8a),
2.09 (s, 3H, Ac), 2.07 (s, 3H, Ac), 1.94 (s, 3H, Ac), 1.80 (s, 3H, Ac),
1.73 (ddd, 1H, J8a = 15.7, J7 = 8.7, 4J3 = 2.5, H-8b). 13C NMR
(125 MHz, CDCl3): d = 197.4 (C-6), 172.5, 170.9, 170.7, 170.0,
169.4 (4ꢁCH3CO + COOMe), 143.4–125.0 (Ar), 113.5 (CPh2), 102.0
(C-5), 87.1 (C-4), 84.4 (C-10), 76.0 (C-50), 73.6 (C-30), 70.8 (C-7),
70.2 (CH2Ph), 68.7 (C-40), 62.6 (C-60), 54.2 (C-20), 52.4 (COOCH3),
50.4 (C-1), 50.1 (5-OMe), 48.9 (C-2), 44.6 (C-3), 31.7 (C-8), 23.1,
20.7, 20.6, 20.5 (4ꢁCH3CO).
0
0
0
4.4. (1R,2R,3R,4R,5S,6R,7R)-4,5,6,7-Tetrahydroxy-5-methoxy-2-
methoxycarbonyl-4,5-O-diphenylmethylidenebicyclo[2.2.2]-
octan-3-yl 20-acetamido-30,40,60-tri-O-acetyl-20-deoxy-10-thio-b-
D-glucopyranoside 4d
Compound 4b: mp: 125–128 °C. HRMS: m/z calcd for
C
½
45H49NO15SNa [M+Na]+ 898.2721, found 898.2753 (ESI-TOF).
To a solution of 4c (49 mg, 62 lmol) in EtOAc (2 ml) and MeOH
(10 ml), Pd(C) (43 mg, 10 % Pd) was added. The mixture was stirred
a 2D3
ꢂ
¼ þ8:3 (c 0.10, CH2Cl2). CD (MeCN,
k
[nm]
(D
e
),

1408
Z. Fejes et al. / Tetrahedron: Asymmetry 22 (2011) 1404–1410
4
under an H2 atmosphere overnight. The Pd(C) was removed by fil-
tration through Celite. The filtrate was concentrated and the crude
product was subjected to column chromatography (EtOAc) to ob-
tain 35 mg (71 %) of 4d as a white solid. Mp: 130–135 °C. HRMS:
m/z calcd for C38H45NO15SNa [M+Na]+ 810.2408, found 810.2428
(t, 1H, J2ꢃ J8bꢃ3, H-3), 4.33 (d, 1H, Jgem = 12.0, OCH2Ph), 4.27 (d,
1H, Jgem = 12.0, OCH2Ph), 4.04–3.96 (m, 3H, H-30+H-60), 3.93 (dt,
0
0
0
0
0
0
1H, JNH = 9.2, J1 ꢃJ3 ꢃ10, H-2 ), 3.83 (t, 1H, J3 ꢃJ5 ꢃ9, H-4 ), 3.64
(dd, 1H, J8b=8.8, J1 = 4.4, H-7), 3.61-3.58 (m, 1H, H-50), 3.62 (s, 3H,
COOMe), 3.41 (t, 1H, J1ꢃJ3ꢃ3, H-2), 3.19 (t, 1H, J2ꢃJ7ꢃ4, H-1),
2.83 (s, 3H, 5-OMe), 2.30 (d, 1H, J8b = 15.2, H-8a), 2.01 (s, 3H, Ac),
1.70–1.63 (m, 1H, H-8b). 13C NMR (125 MHz, CDCl3): d = 197.6
(C-6), 172.7, 172.2 (CH3CO + COOMe), 143.4–125.1 (Ar), 113.7
(CPh2), 102.2 (C-5), 88.4 (C-4), 84.9 (C-10), 80.1 (C-50), 76.0 (C-30),
70.7 (C-40), 70.1 (C-7), 69.8 (CH2Ph), 62.1 (C-60), 56.1 (C-20), 52.6
(COOCH3), 50.2 (C-1), 50.15 (5-OMe), 46.2 (C-2), 43.8 (C-3), 31.3
(C-8), 23.5 (CH3CO).
(ESI-TOF).
½
a 2D3
ꢂ
¼ ꢀ18:1 (c 0.24, CH2Cl2). 1H NMR (400 MHz,
0
CDCl3): d = 7.58–7.17 (m, 10H, Ar), 5.85 (d, 1H, J2 = 8.8, NH), 5.41
(dd, 1H, J2 = 10.3, J4 = 9.3, H-30), 5.17 (d, 1H, J2 = 10.5, H-10), 5.15
0
0
0
(dd, 1H, J5 = 10.0, J3 = 9.3, H-40), 4.51 (d, 1H, J6 = 10.7, OH), 4.43
0
0
4
(dd, 1H, J6 a = 12.2, J5 = 5.6, H-60b), 4.38 (t, 1H, J2ꢃ J8bꢃ3, H-3),
4.20–4.09 (m, 2H, H-20+H-60a), 4.01 (dd, 1H, J8b = 8.6, J1 = 4.4, H-
7), 3.85 (d, 1H, J6-OH = 10.6, H-6), 3.81 (s, 3H, COOMe), 3.79 (ddd,
0
0
1H, J4 = 10.0, J6 b = 5.6, J6 a = 2.6, H-50), 3.44 (t, 1H, J1ꢃJ3ꢃ3, H-2),
2.85 (s, 3H, 5-OMe), 2.48 (t, 1H, J2ꢃJ7ꢃ3.5, H-1), 2.07 (s, 3H, Ac),
2.05 (s, 3H, Ac), 1.96 (s, 3H, Ac), 1.95 (s, 3H, Ac), 1.81 (ddd, 1H,
J8a = 15.8, J7 = 8.6, 4J3 = 2.5, H-8b), 1.55 (d, 1H, J8b = 15.8, H-8a).
13C NMR (100 MHz, CDCl3): d = 176.1, 170.9, 170.6, 170.4, 169.4
(4xCH3CO + COOMe), 144.5-125.1 (Ar), 112.5 (CPh2), 106.0 (C-5),
85.6 (C-4), 83.1 (C-10), 76.0 (C-50), 74.0 (C-6), 73.3 (C-30), 68.8 (C-
40), 65.8 (C-7), 62.5 (C-60), 54.1 (C-20), 53.0 (COOCH3), 52.5 (5-
OMe), 48.0 (C-2), 44.2 (C-3), 43.6 (C-1), 34.3 (C-8), 23.2, 20.7,
20.65, 20.6 (4C, CH3CO).
0
0
0
4.6. (1R,2R,3R,4R,5S,6R,7R)-7-Benzyloxy-4,5,6-trihydroxy-5-
methoxy-2-methoxycarbonyl-4,5-O-diphenylmethylidenebi-
cyclo[2.2.2]octan-3-yl 20-acetamido-20-deoxy-10-thio-b-
D-
glucopyranoside 6a and (1S,2S,3S,4S,5R,6S,7S)-7-benzyloxy-
4,5,6-trihydroxy-5-methoxy-2-methoxycarbonyl-4,5-O-
diphenylmethylidenebicyclo[2.2.2]octan-3-yl 20-acetamido-20-
deoxy-10-thio-b-
D-glucopyranoside 6b
To a solution of CeCl3ꢄ7H2O (1.47 g, 3.95 mmol) in MeOH
(30 ml) at ꢀ30 °C was added NaBH4 (121 mg, 3.20 mmol). The mix-
ture was stirred for 40 min at ꢀ30 °C. After cooling the mixture to
ꢀ70 °C, a solution of 5a/5b (608 mg, 0.81 mmol) in MeOH (9 ml)
was added dropwise over 20 min. The reaction mixture was stirred
at ꢀ70 °C for 2 h, then allowed to warm up to room temperature
and evaporated. The residue was separated between CH2Cl2
(100 ml) and a 0.25% aq citric acid solution (40 ml). The organic
phase was extracted with a saturated NaHCO3 solution. The com-
bined aq extracts were shaken with CH2Cl2, then the combined or-
ganic extracts were dried, filtered, and evaporated. The crude
product was purified by column chromatography (CH2Cl2/MeOH
93:7) to furnish 6a/6b (612 mg, quant./523 mg, 86%) as a white so-
lid. Compound 6a: mp: 144–147 °C. HRMS: m/z calcd for
4.5. (1R,2R,3R,4R,5R,7R)-7-Benzyloxy-4,5-dihydroxy-5-methoxy-
2-methoxycarbonyl-6-oxo-4,5-O-diphenylmethylidenebicyclo-
[2.2.2]octan-3-yl 20-acetamido-20-deoxy-10-thio-b-
D-glucopyr-
anoside 5a and (1S,2S,3S,4S,5S,7S)-7-benzyloxy-4,5-dihydroxy-5-
methoxy-2-methoxycarbonyl-6-oxo-4,5-O-diphenylmethyl-
idenebicyclo[2.2.2]octan-3-yl 20-acetamido-20-deoxy-10-thio-b-
D-glucopyranoside 5b
To a solution of 4a/4b (710 mg, 0.81 mmol) in dry MeOH
(30 ml), NaOMe solution (1.0 ml, c = 0.15 M in MeOH) was added
and the mixture was stirred at room temperature for 3 h. The
reaction mixture was neutralized using a Serdolit-Red cation-ex-
changer. The resin was filtered off, washed with MeOH and the
filtrate was concentrated. The residue was coevaporated with
CH2Cl2/hexane to furnish 5a/5b (608/613 mg, quant.) as an off-
white powder, which was used in the next step without purifi-
cation. Analytically pure product can be obtained by column
chromatography (CH2Cl2/MeOH 94:6). Compound 5a: mp: 143–
146 °C. HRMS: m/z calcd for C39H43NO12SNa [M+Na]+ 772.2404,
C
39H45NO12SNa [M+Na]+ 774.2560, found 774.2571 (ESI-TOF).
½
a 2D3
ꢂ
¼ ꢀ37:2 (c 0.31, CH2Cl2). CD (MeCN,
k
[nm]
(De),
c = 2.47 ꢁ 10ꢀ4): 268sh (ꢀ0.71), 261 (ꢀ0.78), 254sh (ꢀ0.45), 229
(4.04), 207sh (ꢀ3.75), 197 (ꢀ56.10), 186 (52.60). 1H NMR
(500 MHz, CDCl3): d = 7.60–7.09 (m, 16H, Ar+NH), 5.06 (d, 1H,
J2 = 9.1, H-10), 4.48 (d, 1H, J6 = 10.8, 6-OH), 4.37 (d, 1H, Jgem = 12.1,
0
OCH2Ph), 4.28 (br s, 1H, H-3), 4.26 (d, 1H, Jgem = 12.1, OCH2Ph), 4.05
(br s, 2H, H-60), 4.01–3.90 (m, 2H, H-20+H-30), 3.90–3.82 (m, 1H, H-
40), 3.82 (d, 1H, J6-OH = 10.8, H-6), 3.70 (s, 3H, COOMe), 3.68 (dd, 1H,
J8b = 8.7, J1 = 4.3, H-7), 3.59–3.51 (m, 1H, H-50), 3.47 (t, 1H, J1ꢃJ3ꢃ3,
H-2), 2.81 (s, 3H, 5-OMe), 2.54 (t, 1H, J2ꢃJ7ꢃ4, H-1), 1.94 (s, 3H, Ac),
1.79 (d, 1H, J8b = 15.1, H-8a), 1.65–1.56 (m, 1H, H-8b). 13C NMR
(125 MHz, CDCl3): d = 176.5, 172.2 (CH3CO + COOMe), 144.5–
125.2 (Ar), 112.2 (CPh2), 106.0 (C-5), 85.9 (C-4), 84.7 (C-10), 79.9
(C-50), 75.7 (C-30), 74.0 (C-6), 71.8 (C-7), 70.4 (C-40), 69.5 (CH2Ph),
61.9 (C-60), 55.9 (C-20), 53.1 (COOCH3), 52.5 (5-OMe), 48.9 (C-2),
45.4 (C-3), 41.2 (C-1), 30.7 (C-8), 23.4 (CH3CO).
found 772.2433 (ESI-TOF). ½a D23
¼ ꢀ52:6 (c 0.31, CH2Cl2). CD
ꢂ
(MeCN,
k
[nm]
(D
e
), c = 2.39 ꢁ 10ꢀ4): 332sh (ꢀ0.18), 318
(ꢀ0.23), 305sh (ꢀ0.21), 268sh (ꢀ0.39), 261 (ꢀ0.42), 255sh
(ꢀ0.23), 231 (1.97), 214sh (ꢀ6.63), 197 (ꢀ33.56), 187 (33.94).
1H NMR (500 MHz, CDCl3): d = 7.60–7.14 (m, 16H, Ar+NH), 5.78
(br s, 1H, 30-OH), 5.32 (br s, 1H, 40-OH), 5.14 (d, 1H, J2 = 9.8,
0
4
H-10), 4.41 (t, 1H, J2ꢃ J8bꢃ3, H-3), 4.37 (d, 1H, Jgem = 12.0,
OCH2Ph), 4.31 (d, 1H, Jgem = 12.0, OCH2Ph), 4.10–3.82 (m, 4H,
0
0
0
0
0
0
H-2 +H-3 +H-6 ), 4.01 (t, 1H, J3 ꢃJ5 ꢃ9.5, H-4 ), 3.87 (br s, 1H,
50-OH), 3.68 (dd, 1H, J8b = 8.9, J1 = 4.5, H-7), 3.66–3.60 (m, 1H,
H-50), 3.63 (s, 3H, COOMe), 3.56 (t, 1H, J1ꢃJ3ꢃ3, H-2), 3.22 (t,
1H, J2ꢃJ7ꢃ4, H-1), 2.85 (s, 3H, 5-OMe), 2.12 (d, 1H, J8b = 15.3,
H-8a), 1.96 (s, 3H, Ac), 1.72 (ddd, 1H, J8a = 15.3, J7 = 8.9,
4J3 = 2.5, H-8b). 13C NMR (125 MHz, CDCl3): d = 197.7 (C-6),
172.9, 172.2 (CH3CO + COOMe), 143.4–125.1 (Ar), 113.6 (CPh2),
102.0 (C-5), 87.3 (C-4), 84.8 (C-10), 79.9 (C-50), 75.8 (C-30), 70.6
(C-7), 70.3 (C-40), 70.1 (CH2Ph), 61.8 (C-60), 55.9 (C-20), 52.7
(COOCH3), 50.4 (C-1), 50.2 (5-OMe), 49.0 (C-2), 44.8 (C-3), 31.8
(C-8), 23.4 (CH3CO).
Compound 6b: mp: 155–157 °C. HRMS: m/z calcd for
C
39H45NO12SNa [M+Na]+ 774.2560, found 774.2578 (ESI-TOF).
½
a 2D3
ꢂ
¼ þ5:6 (c 0.30, CH2Cl2). CD (MeCN,
k
[nm]
(De),
c = 2.42 ꢁ 10ꢀ4): 268sh (0.64), 261 (0.77), 254sh (0.50), 230
(ꢀ3.00), 210sh (17.30), 197 (70.20), 186 (ꢀ44.92). 1H NMR
(500 MHz, CDCl3): d = 7.57–7.11 (m, 15H, Ar), 6.87 (d, 1H,
J2 = 6.5, NH), 5.17 (d, 1H, J2 = 9.7, H-10), 4.54 (d, 1H, J6 = 10.8, 6-
0
0
4
OH), 4.35 (t, 1H, J2ꢃ J8bꢃ2, H-3), 4.32 (d, 1H, Jgem = 12.1, OCH2Ph),
4.21 (d, 1H, Jgem = 12.1, OCH2Ph), 3.99 (br s, 2H, H-60), 3.90–3.79
(m, 4H, H-6+H-20+H-30+H-40), 3.70 (s, 3H, COOMe), 3.65 (dd, 1H,
J8b = 8.6, J1 = 4.4, H-7), 3.59–3.53 (m, 1H, H-50), 3.24 (t, 1H,
J1ꢃJ3ꢃ3, H-2), 2.82 (s, 3H, 5-OMe), 2.53 (t, 1H, J2ꢃJ7ꢃ4, H-1),
1.88 (s, 3H, Ac), 2.09 (d, 1H, J8b = 14.7, H-8a), 1.62–1.54 (m, 1H,
Compound 5b: mp: 150–153 °C. HRMS: m/z calcd for
C
39H43NO12SNa [M+Na]+ 772.2404, found 772.2429 (ESI-TOF).
½
a 2D3
ꢂ
¼ þ15:8 (c 0.31, CH2Cl2). 1H NMR (500 MHz, CDCl3):
d = 7.60-7.12 (m, 16H, Ar+NH), 5.29 (d, 1H, J2 = 10.2, H-10), 4.54
0

Z. Fejes et al. / Tetrahedron: Asymmetry 22 (2011) 1404–1410
1409
H-8b). 13C NMR (125 MHz, CDCl3): d = 176.7, 172.3 (CH3CO+-
COOMe), 144.5–125.2 (Ar), 112.6 (CPh2), 106.3 (C-5), 87.5 (C-4),
85.2 (C-10), 79.8 (C-50), 76.7 (C-6; from 2D-HSQC), 73.8 (C-30),
71.4 (C-7), 70.7 (C-40), 69.2 (CH2Ph), 62.1 (C-60), 56.1 (C-20), 52.9
(COOCH3), 52.5 (5-OMe), 45.4 (C-2), 44.9 (C-3), 41.2 (C-1), 29.9
(C-8), 23.3 (CH3CO).
40), 71.4 (CH2Ph), 67.0 (C-6), 63.1 (C-60), 57.0 (C-20), 52.6
(COOCH3), 43.8 (C-3), 45.8 (C-2), 42.9 (C-1), 36.2 (C-8), 23.2
(CH3CO).
4.8. (1R,2S,3R,4R,5R,6R,7R)-4,5,6,7-Tetrahydroxy-2-methoxy-
carbonyl-bicyclo[2.2.2]octan-3-yl 20-acetamido-20-deoxy-10-
thio-b-
tetrahydroxy-2-methoxycarbonyl-bicyclo[2.2.2]octan-3-yl 20-
acetamido-20-deoxy-10-thio-b-
-glucopyranoside 8b
D-glucopyranoside 8a and (1S,2R,3S,4S,5S,6S,7S)-4,5,6,7-
4.7. (1R,2S,3R,4R,5R,6R,7R)-7-Benzyloxy-4,5,6-trihydroxy-2-
methoxycarbonyl-bicyclo[2.2.2]octan-3-yl 20-acetamido-20-
D
deoxy-10-thio-b-
D-glucopyranoside 7a and (1S,2R,3S,4S,5S,6S,
7S)-7-benzyloxy-4,5,6-trihydroxy-2-methoxycarbonyl-
bicyclo[2.2.2]octan-3-yl 20-acetamido-20-deoxy-10-thio-b-
glucopyranoside 7b
To a solution of 7a/7b (128 mg, 0.23 mmol) in MeOH (15 ml)
Pd(C) (94 mg, 10 % Pd) was added. After flushing the flask with
argon the suspension was hydrogenized using a H2-filled balloon
for 24 h. The mixture was filtered through Celite to remove Pd(C)
and then concentrated. The crude product was purified by column
chromatography (CH2Cl2/MeOH 6:4) to furnish 45 mg (42%) of 8a/
8b (45 mg, 42%/55 mg, 54%) as a colorless film, which was then
lyophilized to obtain a hygroscopic white substance. Compound
8a: HRMS: m/z calcd for C18H29NO11SNa [M+Na]+ 490.1359, found
D
-
To a solution of 6a/6b (390 mg, 0.52 mmol) in CH2Cl2 (20 ml),
TFA (0.2 ml) was added and stirred at room temperature for 1 h.
The reaction mixture was diluted with dry toluene (20 ml) and
evaporated, then a further 10 ml of dry toluene was evaporated
from the residue. The crude product was dissolved in THF/MeOH
(9:1, 20 ml), cooled to ꢀ15 °C, then NaBH4 (85 mg, 2.25 mmol)
was added. After stirring the reaction mixture at ꢀ15 °C for 30
minutes, it was diluted with MeOH (15 ml) and neutralized using
Serdolit-Red cation-exchanger at room temperature. The resin
was filtered off and washed with MeOH. To the filtrate aq. AcOH
(96%, 0.3 ml) was added and evaporated to a volume of approxi-
mately 10 ml. The residue was then coevaporated with MeOH
(20 ml) to a volume of approximately 5 ml. Traces of AcOH were
removed by coevaporating the residue with 2 ꢁ 20 ml dry tolu-
ene. The crude product was purified by column chromatography
(CH2Cl2/MeOH 83:17) to furnish 7a/7b (133 mg, 46%/162 mg,
56%) as a white powder if coevaporated with CH2Cl2/hexane.
Compound 7a: mp: 180–190 °C (dec). HRMS: m/z calcd for
490.1356 (ESI-TOF).
½
a 2D3
ꢂ
¼ ꢀ41:8 (c 0.12, MeOH).1H NMR
(500 MHz, CD3OD): d = 4.69 (d, 1H, J2 = 10.2, H-10), 3.87 (dd, 1H,
0
4
J2=8.3, J8b = 2.5, H-3), 3.95–3.88 (m, 3H, H-6+H-7+H-60a), 3.76 (t,
0
0
0
1H, J1 ꢃJ3 ꢃ10, H-2 ), 3.69 (s, 3H, COOMe), 3.65 (d, 1H, J6 = 8.4, H-
0
0
0
0
0
5), 3.64 (dd, 1H, J6 a = 12.1, J5 = 6.1, H-6 b), 3.45 (t, J2 ꢃJ4 ꢃ9, 1H,
H-30), 3.40–3.32 (m, 2H, H-40+H-50), 2.98 (dd, 1H, J3 = 8.3, J1 = 1.9,
H-2), 2.47–2.45 (m, 1H, H-1), 1.96 (s, 3H, Ac), 1.75 (ddd, 1H,
J8a = 14.1, J7 = 9.7, 4J3 = 2.5, H-8b), 1.63 (dd, 1H, J8b = 14.1, J7 = 5.1,
H-8a). 13C NMR (125 MHz, CD3OD): d = 176.7, 173.9 (CH3CO + -
COOMe), 87.8 (C-10), 82.3 (C-50), 77.4 (C-30), 72.7 (C-4), 72.0 (C-
40), 71.3 (C-5), 67.0 (C-6), 64.7 (C-7), 63.1 (C-60), 56.6 (C-20), 52.6
(COOCH3), 47.0 (C-3), 46.5 (C-1), 45.6 (C-2), 39.0 (C-8), 23.1
(CH3CO).
C
25H35NO11SNa [M+Na]+ 580.1829, found 580.1810 (ESI-TOF).
Compound 8b: HRMS: m/z calcd for C18H29NO11SNa [M+Na]+
½
a 2D3
ꢂ
¼ ꢀ24:5 (c 0.20, MeOH). CD (MeCN,
k
[nm]
(
D
e
),
490.1359, found 490.1347 (ESI-TOF). ½a D23
ꢂ
¼ þ28:2 (c 0.12, MeOH).
c = 3.10 ꢁ 10ꢀ4): 236 (ꢀ0.18), 213 (2.43). 1H NMR (500 MHz,
1H NMR (500 MHz, CD3OD): d = 4.86 (d, 1H, J2 = 10.5, H-10), 4.11
0
4
CD3OD): d = 7.36–7.24 (m, 5H, Ar), 4.66 (d, 1H, J2 = 10.5, H-10),
(dd, 1H, J2 = 7.4, J8b = 2.4, H-3), 3.96 (ddd, 1H, J8b = 9.7, J8a = 4.6,
0
4
4.49 (br s, 2H, OCH2Ph), 3.93 (dd, 1H, J2 = 7.9, J8b = 2.3, H-3),
J1=2.6, H-7), 3.90 (dd, 1H, J5 = 8.7, J1 = 3.7, H-6), 3.77 (dd, 1H,
3.90 (dd, 1H, J5 = 8.6, J1 = 3.7, H-6), 3.89 (dd, 1H, J6 b = 12.1,
J6 a = 12.1, J5 = 2.5, H-60a), 3.71–3.65 (m, 2H, H-20+H-60b), 3.67 (s,
0
0
0
0
0
0
0
0
0
0
J5 =2.2, H-6 a), 3.79 (t, 1H, J1 ꢃJ3 ꢃ9, H-2 ), 3.75–3.70 (m, 1H, H-
3H, COOMe), 3.60 (d, 1H, J6 = 8.7, H-5), 3.43 (t, 1H, J2 ꢃJ4 ꢃ9, H-
0 0 0
0 0
7), 3.69 (s, 3H, COOMe), 3.66 (d, 1H, J6 = 8.6, H-5), 3.63 (dd, 1H,
3 ), 3.38 (m, 1H, J3 ꢃJ5 ꢃ9, H-4 ), 3.24-3.19 (m, 2H, H-2+H-5 ),
2.45–2.43 (m, 1H, H-1), 1.97 (s, 3H, Ac), 1.86 (dd, 1H, J8b = 13.7,
J7 = 4.6, H-8a), 1.71 (ddd, 1H, J8a = 13.7, J7 = 9.7, 4J3 = 2.4, H-8b).
13C NMR (125 MHz, CD3OD): d = 177.3, 173.9 (CH3CO + COOMe),
86.0 (C-10), 81.5 (C-50), 77.9 (C-30), 73.6 (C-4), 71.7 (C-40),
71.8 (C-5), 66.9 (C-6), 65.1 (C-7), 62.9 (C-60), 57.0 (C-20), 52.5
(COOCH3), 44.1 (C-3), 46.4 (C-1), 45.4 (C-2), 38.1 (C-8), 23.2
(CH3CO).
0
0
0
0
0
0
J6 a = 12.1, J5 = 5.9, H-6 b), 3.43 (t, 1H, J2 ꢃJ4 ꢃ9, H-3 ), 3.32 (t,
1H, J3 ꢃJ5 ꢃ9, H-40), 3.39–3.33 (m, 1H, H-50), 2.99 (dd, 1H,
J3 = 7.9, J1 = 2.1, H-2), 2.69 (m, 1H, H-1), 1.95 (s, 3H, Ac), 1.79
(dd, 1H, J8b = 14.1, J7 = 4.9, H-8a), 1.74 (ddd, 1H, J8a = 14.1,
J7 = 9.2, 4J3 = 2.3, H-8b). 13C NMR (125 MHz, CD3OD): d = 176.6,
174.0 (CH3CO + COOMe), 139.8, 129.5, 128.9, 128.8 (Ar), 87.6 (C-
10), 82.3 (C-50), 77.3 (C-30), 72.7 (C-4), 72.3 (C-7), 71.9 (C-40),
71.5 (C-5), 71.4 (CH2Ph), 66.7 (C-6), 63.0 (C-60), 56.4 (C-20), 52.6
(COOCH3), 46.4 (C-3), 46.2 (C-2), 42.9 (C-1), 37.0 (C-8), 23.1
(CH3CO).
0
0
4.9. (1R,2S,3R,4R,5R,6R,7R)-4,5,6,7-Tetrahydroxy-2-carboxy-
bicyclo[2.2.2]octan-3-yl 20-acetamido-20-deoxy-10-thio-b-
D-
Compound 7b: mp: 180–190 °C (dec). HRMS: m/z calcd for
glucopyranoside 9a and (1S,2R,3S,4S,5S,6S,7S)-4,5,6,7-tetra-
C
½
25H35NO11SNa [M+Na]+ 580.1829, found 580.1838 (ESI-TOF).
ꢂ
hydroxy-2-carboxybicyclo[2.2.2]octan-3-yl 20-acetamido-20-
a 2D3
¼ þ26:1 (c 0.20, MeOH). 1H NMR (500 MHz, CD3OD):
deoxy-10-thio-b-
D-glucopyranoside 9b
d = 7.37–7.24 (m, 5H, Ar), 4.84 (d, 1H, J2 = 10.5, H-10), 4.51 (br
0
4
s, 2H, OCH2Ph), 4.11 (dd, 1H, J2 = 7.1, J8b = 2.5, H-3), 3.87 (dd,
To a solution of 8a/8b (5 mg, 11
lmol) in H2O (200 ll), aq NaOH
0
0
1H, J5 = 8.6, J1 = 3.7, H-6), 3.79 (dd, 1H, J6 b = 12.1, J5 = 2.5, H-
60a), 3.73 (ddd, 1H, J8b = 9.6, J8a = 4.2, J1 = 2.7, H-7), 3.71–3.65
(m, 2H, H-20+H-60b), 3.67 (s, 3H, COOMe), 3.61 (d, 1H, J6 = 8.6,
solution (210 l, 0.1 M) was added. After stirring for 1.5 h, the reac-
l
tion mixture was neutralized using a Serdolit-Red cation-exchanger.
The resin was filtered off, washed with H2O, and the filtrate was
lyophilized to obtain 9a/9b (5 mg/5 mg, quant.) as a hygroscopic
substance. Compound 9a: HRMS: m/z calcd for C17H27NO11SNa
0
0
0
0
0
0
H-5), 3.44 (t, 1H, J2 ꢃJ4 ꢃ9, H-3 ), 3.37 (t, 1H, J3 ꢃJ5 ꢃ9, H-4 ),
3.25 (ddd, 1H, J4 = 9.5, J6 b = 5.5, J6 a = 2.5, H-50), 3.14 (dd, 1H,
J3 = 7.1, J1 = 2.5, H-2), 2.66 (m, 1H, H-1), 1.98 (dd, 1H, J8b=14.0,
J7 = 4.2, H-8a), 1.97 (s, 3H, Ac), 1.70 (ddd, 1H, J8a = 14.0, J7=9.6,
4J3 =2.5, H-8b). 13C NMR (125 MHz, CD3OD): d = 177.0, 173.9
(CH3CO + COOMe), 139.8, 129.5, 128.9, 128.8 (Ar), 86.1 (C-10),
81.6 (C-50), 77.7 (C-30), 73.5 (C-4), 72.6 (C-7), 71.9 (2C, C-5+C-
0
0
0
[M+Na]+ 476.1203, found 476.1192 (ESI-TOF). ½a 2D3
ꢂ
¼ ꢀ51:8 (c
0.18, MeOH). 1H NMR (400 MHz, CD3OD): d = 4.65 (d, 1H, J2 = 10.5,
H-10), 3.94–3.85 (m, 3H, H-6+H-7+H-60a), 3.83–3.74 (m, 2H, H-
3+H-20), 3.68–3.60 (m, 2H, H-40+H-60b), 3.42–3.32 (m, 3H, H-5+H-
30+H-50), 3.04 (br d, 1H, J3ꢃ9, H-2), 2.17 (m, 1H, H-1), 1.99 (s, 3H,
0

1410
Z. Fejes et al. / Tetrahedron: Asymmetry 22 (2011) 1404–1410
Ac), 1.71 (ddd, 1H, J8a = 13.9, J7 = 9.2, 4J3 = 2.2, H-8b), 1.60 (dd, 1H,
J8b = 13.9, J7 = 5.0, H-8a). 13C NMR (100 MHz, CD3OD): d = 182.8,
174.0 (CH3CO + COOH), 87.1 (C-10), 82.3 (C-50), 78.0 (C-30), 73.0 (C-
4), 72.5 (C-40), 72.0 (C-5), 68.1 (C-6), 66.2 (C-7), 63.0 (C-60), 56.5
(C-20), 50.2 (C-2), 49.1 (C-3; from 2D-HSQC), 44.7 (C-1), 38.3 (C-8),
23.1 (CH3CO).
bance at 232 nm was monitored at 37 °C over a 5 min period of time.
Initial reaction rates were calculated using an extinction coefficient
of 3800 Mꢀ1 cmꢀ1 for the enzymatic disaccharide products obtained
from chondroitin sulfate A.
Acknowledgments
Compound 9b: HRMS: m/z calcd for C17H27NO11SNa [M+Na]+
476.1203, found 476.1199 (ESI-TOF). ½a D23
ꢂ
¼ þ35:4 (c 0.18, MeOH).
The work was supported by the TÁMOP 4.2.1/B-09/1/KONV-
2010-0007 project. The project was co-financed by the European
Union and the European Social Fund. The authors also acknowl-
edge the support of the Hungarian Research Foundation for grant
OTKA K79126 and also grants HL62244, GM38060 and HL101721
(to R.J.L.) from the US National Institutes of Health.
1H NMR (400 MHz, CD3OD): d = 4.79 (d, 1H, J2 = 10.4, H-10), 3.94–
0
3.79 (m, 4H, H-3+H-6+H-7+H-60a), 3.71–3.59 (m, 3H, H-5+H-
0
0
0
0
0
0
2 +H-6 b), 3.50 (t, 1H, J2 ꢃJ4 ꢃ9, H-3 ), 3.33–3.25 (m, 2H, H-4 +H-
50; partially covered by CHD2OD), 3.00 (br d, 1H, J3ꢃ9, H-2), 2.15
(br s, 1H, H-1), 1.98 (s, 3H, Ac), 1.77–1.67 (m, 2H, H-8). 13C NMR
(100 MHz, CD3OD): d = 182.7, 173.9 (CH3CO+COOH), 84.8 (C-10),
82.1 (C-50), 77.7 (C-30), 73.7 (C-4), 72.7 (C-40), 72.0 (C-5), 68.2 (C-
6), 66.3 (C-7), 63.0 (C-60), 57.1 (C-20), 49.1 (C-2; from 2D-HSQC),
46.1 (C-3), 44.6 (C-1), 37.9 (C-8), 23.2 (CH3CO).
References
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4.10. Enzyme inhibitory tests
Chondroitin AC-I lyase from Flavobacterium heparinum12 was
prepared as a recombinant enzyme from Escherichia coli in Professor
Cygler0s laboratory at the Biotechnology Research Institute in Mon-
treal as previously described.13 Assays were performed in quartz
cuvettes (1 cm path length) using a total solution volume of
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500
ll. Mixtures containing the desired amount of chondroitin sul-
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fate A substrate and inhibitor 9a or 9b (6
phosphate buffer, pH 6.0 were pre-incubated at 25 °C for 10 min.
Chondroitin AC lyase was then added to a final concentration of
0.01 lM. The reaction solution was quickly mixed and the absor-
l
M) in 100 mM sodium