Towards the synthesis of aryl glucuronides as potential heparanase probes. An interesting outcome in the glycosidation of glucuronic acid with 4-hydroxycinnamic acid

Article abstract of DOI:10.1016/j.carres.2005.06.029

This work describes our preliminary efforts towards the development of aryl glucuronides as potential probes for heparanase. During the course of these initial investigations, attempted glycosidation of methyl 2,3,4-tri-O-acetyl- α-d-glucopyranosyluronate trichloroacetimidate with 4-hydroxycinnamic acid gave a complex mixture of four different components. These were identified as the 1-cinnamyl glucuronate ester 13, the cinnamyl linked disaccharide 14, the glucuronate trichloroacetamide 15, and the glucuronyl α-fluoride 16. This paper rationalises the formation of each of these products, and reports our efforts in trying to optimise the formation of the α-fluoride 16.

Full text of DOI:10.1016/j.carres.2005.06.029

Carbohydrate
RESEARCH
Carbohydrate Research 340 (2005) 2077–2085
Towards the synthesis of aryl glucuronides as potential
heparanase probes. An interesting outcome in the glycosidation
of glucuronic acid with 4-hydroxycinnamic acid
Andrew G. Pearson,a Milton J. Kiefel,a Vito Ferrob and Mark von Itzsteina,*
aInstitute for Glycomics, Griffith University (Gold Coast Campus), PMB 50, Gold Coast Mail Centre, QLD 9726, Australia
bDrug Design Group, Progen Industries Limited, 2806 Ipswich Road, Darra, QLD 4076, Australia
Received 30 January 2005; accepted 22 June 2005
Available online 28 July 2005
Abstract—This work describes our preliminary efforts towards the development of aryl glucuronides as potential probes for hepa-
ranase. During the course of these initial investigations, attempted glycosidation of methyl 2,3,4-tri-O-acetyl-a-D-glucopyranosyl-
uronate trichloroacetimidate with 4-hydroxycinnamic acid gave a complex mixture of four different components. These were
identified as the 1-cinnamyl glucuronate ester 13, the cinnamyl linked disaccharide 14, the glucuronate trichloroacetamide 15,
and the glucuronyl a-fluoride 16. This paper rationalises the formation of each of these products, and reports our efforts in trying
to optimise the formation of the a-fluoride 16.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Keywords: Heparan sulfate; D-Glucuronic acid; Glycosyl fluoride; Glycoside formation; Heparanase
1. Introduction
OSO3
O
Heparanase
cleavage site
O
HO
Heparanase is an endo-b-D-glucuronidase that degrades
heparan sulfate (HS), a major component of the extra-
cellular matrix and basement membranes, and plays an
important role in a variety of biological processes
including inflammation, the metastatic potential of
tumour cells and angiogenesis.1,2 HS–glycosaminogly-
cans (HS–GAGs) are sulfated linear polysaccharides of
up to 400 sugar residues, composed of D-glucuronic acid
(GlcA) b-(1!4)-linked to N-acetyl-D-glucosamine (Glc-
NAc), with various structural modifications.3 Heparan-
ase hydrolyses the glucuronide linkage in HS–GAGs,
but only at a few sites (e.g., as indicated in Fig. 1), yield-
ing HS fragments of 10–20 saccharide units.4,5
CO2
OX
O
YHN
GlcN
O
O
O
HO
HO
CO2
OH
O3SHN
O
GlcA
O
HO
X = H or SO3
Y = Ac or SO3
OH
GlcA or IdoA
n
Figure 1. Schematic of heparan sulfate showing the heparanase
cleavage site.
The lack of a convenient functional assay has also ham-
pered progress, prompting our investigation into the
possibility of preparing compounds such as 1 that could
be used in a simple fluorometric or colourimetric assay.
The general concept is shown in Figure 2, wherein hep-
aranase cleavage of tagged compounds like 1 would
result in a measurable response. The development of
such an assay would be extremely beneficial in studies
on heparanase, including the kinetic evaluation of
Despite the important biological functions of hepa-
ranase, investigations of this enzyme have proven quite
challenging due to the difficulties in isolation and, until
recently, low yields of active recombinant protein.6
*
Corresponding author. Tel.: +617 5552 7016; fax: +617 5552 9040;
e-mail: m.vonitzstein@grifffith.edu.au
0008-6215/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2005.06.029

2078
A. G. Pearson et al. / Carbohydrate Research 340 (2005) 2077–2085
OX
O
In the work towards 5 described by Stachulski and
co-workers,11 the choice of anomeric activating group
in the glucuronide is clearly important, since glucuronyl
a-halides only gave low (ꢀ10%) yields of 5. Bearing
these issues in mind, our approach towards simple aryl
glucuronides like 2 as probes for heparanase involved
the acid-catalysed glycosidation between the trichloro-
acetimidate-activated glucuronic acid 6 and a variety
of phenols (Fig. 3). There have been a number of
approaches described for the synthesis of 6, including
using the corresponding a-bromo11,14,17 or the
anomeric acetate11,18 glucuronic acid derivatives. In
our hands, we found that the most efficient approach
was from commercially available D-glucurono-6,3-lac-
tone, wherein 6 could be obtained in ꢀ30% overall
yield over three steps. In some preliminary studies,
the BF3ÆOEt2-catalysed coupling of 6 with phenols pro-
duced consistently high yields (61–81%) of the desired
aryl glucuronides 7 to 10 (Fig. 3). Whilst compounds
813,14,19,20 and 1020,21 have been known for some time,
they have previously only been reported with limited
spectroscopic data.
cleavage
RO
HO
CO2
YHN
O
O
O
TAG
HO
OH
1
OX
O
RO
HO
CO2
TAG
+
YHN
O
O
HO
OH
OH
R = H or HS chain, X = H or SO , Y = Ac or SO3.
3
Figure 2. Heparanase cleavage of the chromogenic or fluorogenic tag
from 1 generates a measurable response.
potential inhibitors and the correlation of heparanase
activity with various disease states.
2. Results and discussion
Our initial approach towards compounds such as 1
involved the preparation of a series of aryl glucuronides
of the general structure 2, which could serve as potential
substrates for heparanase after deprotection. Several
methods are available for the synthesis of glycosides of
glucuronic acids, and these can be placed into two broad
categories involving either oxidation of the correspond-
ing glucoside7,8 or by carrying out the glycosidation on
an activated glucuronic acid.9–14 In relation to com-
pounds like 2, we were particularly interested in the pre-
viously reported methods for the synthesis of aryl
glucuronides, including the 20-methoxycarbonyl deriva-
tive 3,10,15 the p-nitrophenyl glycoside 416 and the
7-hydroxycoumarin glucuronide 5.11
Interestingly, the attempted BF3ÆOEt2-catalysed
glycosidation of 6 with 4-hydroxycinnamic acid (11, 1–
3 equiv) did not give the desired glycoside 12, but
instead gave a complex mixture from which the four
products 13–16 (Fig. 4) were identified, with yields for
each product shown in parentheses.
The formation of 13 and 14 can be rationalised as
shown in Figure 5. The isolation of both 13 and 14 from
attempted glycosidation between 6 and 11 suggests that,
due to the resonance structures of 11, 4-hydroxycin-
namic acid (11) preferentially reacts initially through
the carboxylate group to give 13, which then subse-
quently reacts with another molecule of 6 to give 14.
The enhanced nucleophilicity of the carboxylate group
in other phenolic carboxylic acid species has been noted
previously.22 The formation of the trichloroacetamide
byproduct 15 during attempted coupling of 6 with 11
arises from the acid-catalysed rearrangement of 6. The
acid-catalysed rearrangement of trichloroacetimidate-
activated glucose and galactose derivatives to the corre-
sponding trichloroacetamides has been noted previously
during their attempted coupling with relatively unreac-
tive acceptors such as 2-thio-glucosides or enol silyl
ethers.23–25
CO2Me
CO2Me
O
CO2Me
O
AcO
AcO
AcO
AcO
OR
O
OAc
OAc
3
2
R = aryl
CO2Me
O
CO2Me
O
AcO
NO2 AcO
O
O
O
O
AcO
OAc
OAc
4
5
CO2Me
O
CO2Me
AcO
AcO
ROH, BF3•OEt2, CH2Cl2
4Å sieves, –15˚C to rt, 16 h
O
AcO
AcO
OR
AcO
O
CCl3
OAc
7 R = 3-NO2-C6H4 (63%)
6
NH
8 R = 4-NO2-C6H4 (81%)
9 R = 3,5-(CO2Me)2-C6H3 (75%)
10 R = 4-methylumbelliferyl (61%)
Figure 3. Synthesis of some aryl glucuronides.

A. G. Pearson et al. / Carbohydrate Research 340 (2005) 2077–2085
2079
CO2Me
O
AcO
HO
O
AcO
+
6
CO2H
CO2H
OAc
11
12
BF3•OEt2, CH2Cl2
4Å sieves, –15˚C to rt, 16 h
O
CO2Me
O
O
CO2Me
O
AcO
AcO
AcO
AcO
O
OAc
+
O
AcO
O
OAc
OAc
CO2Me
OAc
O
OH
13 (14%)
14 (22%)
CO2Me
O
CO2Me
O
H
N
AcO
AcO
AcO
AcO
CCl3
+
+
OAc
15 (13%)
AcO
O
F
16 (14%)
Figure 4. Attempted glycosidation of the donor 6 with 4-hydroxycinnamic acid (11).
CO2Me
O
O
AcO
AcO
+
HO
OH
AcO
O
CCl3
6
11
NH
OAc
O
CO2Me
O
OAc
CO2Me
AcO
O
AcO
AcO
O
+
Cl3C
O
OAc
6
OH
13
NH
O
CO2Me
O
AcO
AcO
O
OAc
OAc
AcO
O
OAc
CO2Me
O
14
Figure 5. Proposed mechanism for the formation of 13 and 14.
The isolation of the glucuronosyl ester 13 from the
attempted glycosidation of 6 with 11, albeit as a minor
component of a complex mixture of products, prompted
us to investigate this reaction further, perhaps as an ave-
nue into novel glucuronosyl ester derivatives. Reaction
of 6 with 11 in the presence of TMSOTf under condi-
tions analogous to those employed above using
BF3ÆOEt2, gave the glucuronosyl ester 13 (38%), to-
gether with the reducing sugar 1714 (27%) (Fig. 6). It is
noteworthy that the dimeric product 14 was only formed
in trace amounts (<10%) in this instance, compared with
its being obtained as the major product (22%) when
BF3ÆOEt2 was used as the Lewis acid (Fig. 4). Stirring
6 in the presence of 11 for 16 h without the addition
of a Lewis acid catalyst failed to furnish any of the prod-
uct 13, returning predominantly unreacted 6 together
with a small amount (ꢀ20%) of the reducing sugar 17.
This suggests that 11 is not acidic enough to catalyse
its own reaction with 6. Interestingly, treatment of 6
with the known protected cinnamic acid derivative
1826 in the presence of BF3ÆOEt2 gave the glucuronosyl
ester 19 in 54% yield (Fig. 6), together with small
amounts of the reducing sugar 17 (ꢀ20%) as well as
the glucuronosyl fluoride 16 (ꢀ12%).
Others have shown that the BF3ÆOEt2-catalysed cou-
pling of 18 with peracetylated glucosyl fluoride gives
the corresponding b-glucosyl ester in 67% yield.27 This
same work also attempts to define the difference in reac-
tivity between the phenol group and the carboxylic acid
group in cinnamic acid derivatives.27 Thus, treatment of
a 1:1 mixture of 18 and the methyl ester of 11 with
BF3ÆOEt2 in the presence of peracetylated glucosyl
fluoride gave a 73% yield of the glycoside 20 and a
10% yield of the glucosyl ester 21 (Fig. 7).27 Our
isolation of both 13 and 14 from coupling between 6 and
11, together with our failure to observe the formation

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A. G. Pearson et al. / Carbohydrate Research 340 (2005) 2077–2085
Lewis Acid, CH2Cl2
RO
+
6
CO2H
4Å sieves, –15 ºC to rt, 16 h
11 R = H
18 R = Ac
O
CO2Me
O
CO2Me
AcO
AcO
O
AcO
AcO
O
+
OH
OAc
OAc
OR
17
13 R = H
19 R = Ac
Figure 6. Alternative approach towards glucuronosyl esters like 13 and 19.
OAc
O
HO
AcO
+
AcO
AcO
+
F
CO2Me
CO2H
AcO
18
BF3•OEt2, DTBMP
CH2Cl2, rt, 1 h
OAc
OAc
OAc
O
O
AcO
AcO
AcO
AcO
O
+
O
AcO
CO2Me
AcO
O
20 (73%)
21 (10%)
Figure 7. Reported competitive reactivity of phenols versus carboxylic acids.27
of any of the glycoside 12 (Fig. 4), suggests that in our
system a different reactivity preference is operating.
Turning our attention back to the original BF3ÆOEt2-
catalysed attempted glycosidation of 6 with 11 shown in
Figure 4, we were also interested in the isolation of
the glucuronosyl fluoride 16, obtained exclusively as the
a-anomer. That the product obtained was indeed the
a-fluoride was evident from examination of the 1H
NMR spectrum of 16. Most notably, the signal due to
H-1 (d 5.81) was present as a doublet of doublets, due
to coupling to H-2 (J = 2.7 Hz) and to fluorine
(J = 52.5 Hz).28 The magnitude of the coupling between
H-1 and H-2 in 16 (2.7 Hz) is clearly consistent with an
equatorial–axial relationship between H-1 and H-2 and
supports the fluoride in 16 being in the a-configuration.
To the best of our knowledge, this is the first time that
exclusively the a-anomer of 16 has been reported.
Others have reported the formation of exclusively the b-
glucuronosyl fluoride, from the corresponding hemi-
acetal, by treatment with hydrogen fluoride–pyridine29
or diethyl-aminosulfur trifluoride (DAST).30,31 The syn-
thesis of a 1:9 a:b-mixture of 16 has also been described
by treatment of the hemiacetal with a-fluoroenamine,
although there is limited spectroscopic data reported
for the a-fluoride 16 in this mixture.28
only source of fluoride in the glycosidation reaction
was BF3ÆOEt2, and this was present in up to 0.5 molar
equiv. Glycosyl fluorides have been used for over
20 years as anomeric activating groups in glycosida-
tions.28,29,32–35 Whilst reagents like HF or HF–pyridine,
DAST, and metal fluorides (e.g., AgF2) are commonly
used to prepare glycosyl fluorides,35 to the best of our
knowledge glycosyl fluorides have never been prepared
previously using BF3ÆOEt2 as the source of fluoride. Per-
haps the closest example is a modification of the Mitsun-
obu reaction wherein the mannosyl fluoride 22 is
generated by exposure of 23 to DEAD and triethyloxo-
nium tetrafluoroborate, which is believed to proceed
through the stabilised carbonium ion 24 (Fig. 8).36
Although not completely clear, we suggest that in our
case it is possible that reaction of the trichloroacetim-
idate 6 with BF3ÆOEt2 initially forms the stabilised glu-
curonosyl cation intermediate 25 (Fig. 9). Presumably
the fluoride that is required to form the product 16 arises
from the boron adduct [(F3B–OR)ꢁ] generated during
the initial formation of the intermediate 25.
Whilst anomerically pure glycosyl fluorides are pre-
ferred, many of the common fluorination methods lead
to anomeric mixtures.35,–40 Based upon our rationale
above (Fig. 9), we wondered if we could promote the
formation of the a-fluoride 16 by reacting 6 with
BF3ÆOEt2 in the absence of an aglycon. Unfortunately,
The isolation of the a-fluoride 16 from coupling
between 6 and 11 was somewhat surprising, since the

A. G. Pearson et al. / Carbohydrate Research 340 (2005) 2077–2085
2081
O
O
O
CMe2
CMe2
CMe2
Me2C
Me2C
Me2C
O
O
O
O
O
O
O
O
O
O
BF4
O
O
OH
F
24
23
22
Figure 8. Reported synthesis of mannosyl fluoride 22.36
CO2Me
CO2Me
CO2Me
O
AcO
O
O
AcO
AcO
AcO
AcO
AcO
AcO
F3B OR
AcO
O
CCl3
AcO
F
25
16
F3B
NH
Figure 9. Possible rationale for the formation of the a-fluoride 16.
as can be seen by the results shown in Table 1, an
increase in BF3ÆOEt2 from 0.3 (entries 1–3) to 1.0 molar
equiv (entry 4) failed to increase the yield of 16, which
remained generally around 24%. Indeed, the increase
in BF3ÆOEt2 appeared to have little effect on the overall
outcome of the reaction, with products 15 and 17 also
being isolated in relatively consistent yields. Similarly,
the use of either commercially available BF3ÆOEt2
(entries 1 and 2) or freshly distilled BF3ÆOEt2 (entries
3–5) had no discernible effect on the yields or respective
ratios of products obtained (Table 1). Increasing the
amount of BF3ÆOEt2 to greater than 5 molar equiv gen-
erally resulted in substantial decomposition of 6, with
the formation of 15 and the methyl glucuronide
(a:b ꢀ2:1) as minor products upon quenching the reac-
tion with methanol. At this stage we are yet to determine
why increasing the amount of BF3ÆOEt2 fails to improve
the yield of the a-fluoride 16. As expected, exposure of 6
to TMSOTf without any aglycon present afforded a mix-
ture of the reducing sugar 17 and the rearranged trichlo-
roacetamide derivative 15.
During the course of this work, it was also noted that
the workup procedure had a significant effect on the iso-
lation of the a-fluoride 16. Initially, the workup con-
sisted of simply adding methanol to the reaction
mixture and then filtering prior to concentration in
vacuo (Table 1, entries 1–4). However, when an aqueous
workup was employed (dilution of the reaction mixture
with CH2Cl2, filtration to remove the molecular sieves,
and washing with aqueous Na2CO3 prior to concentra-
tion in vacuo) the yield of the a-fluoride 16 was signifi-
cantly lower (Table 1, entry 5).
The desired cinnamyl glycoside of glucuronic acid was
ultimately obtained by use of the known41 methyl ester
26 as the glycosyl acceptor. Compound 26 was conve-
niently synthesised in excellent yield (96%) by esterifica-
tion of 11 with methanolic HCl [prepared in situ by the
addition of trimethyl orthoformate and thionyl chloride
to anhydrous methanol. (Potter, J. J.; von Itzstein, M.
unpublished work.)] The glycosidation of 6 with 26
using BF3ÆOEt2 as catalyst provided the fully protected
glycoside 27 in 83% yield (Fig. 10). Interestingly, 1H
Table 1. Attempted formation of the a-fluoride 16
CO2Me
CO2Me
CO2Me
H
BF3•OEt2, CH2Cl2
O
O
O
AcO
AcO
AcO
AcO
N
CCl3
OH
+
+
6
AcO
AcO
–15 ºC to rt, 16 h
OAc
OAc
AcO
O
F
17
15
16
Entry
Scale 6 (mg)
BF3ÆOEt2 (equiv)
Products isolateda
15 (%)
16 (%)
17 (%)
1
2
3
4
5d
35
381
234
238
294
0.3b
0.3b
0.3c
1.0c
0.5c
6
15
11
15
12
16
26
24
24
6
57
20
ND
31
45
a ND = not determined.
b Commercially available BF3ÆOEt2.
c Distilled BF3ÆOEt2.
d Alternative workup procedure.

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A. G. Pearson et al. / Carbohydrate Research 340 (2005) 2077–2085
CO2Me
BF3•OEt2, CH2Cl2
O
AcO
AcO
+ HO
6
O
4Å sieves, –15˚C
CO2Me
CO2Me
OAc
26
27
i) LiOH, MeOH, H2O
ii) Amberlite IR-120 (H+)
iii) NaOH (to pH 7.2)
CO2Na
O
O
HO
HO
CO2Na
OH
28
Figure 10. Synthesis of target compound 28 via glucuronide 27.
NMR examination of the product 27 in CDCl3 showed
several overlapped peaks, which resulted in a complex,
second-order spin system for H-5 of the glucuronic acid
portion.14 Changing the NMR solvent to either C6D6 or
CD3CN (see Section 3.6) resolved many of the overlap-
ping signals and also simplified the second-order spin
systems (in the case of CD3CN).
ono-6,3-lactone by a three-step procedure that involved
the initial generation of methyl 1,2,3,4-tetra- O-acetyl-D-
glucopyranosyluronate,44 selective removal of the ano-
meric acetate using benzylamine45 and subsequent intro-
duction of the trichloroacetimidate group following the
method described.18
Interestingly, the glycosidation of 6 with 26 using
TMSOTf as a Lewis acid catalyst furnished the desired
glucuronide 27 in lower yield (ꢀ40%) as well as small
amounts (ꢀ10%) of the known42 1-O-TMS glucuronide
together with some of the reducing sugar 17. Finally,
deprotection of 27 (dil aq LiOH in MeOH) gave the
desired compound 28 (Fig. 10). A series of glucuronic
acid aryl glycosides like 1 and 28 are currently being
evaluated as potential substrates for heparanase.
3.2. General procedure for glycosidations
A suspension of 6 (1.0 equiv) and alcohol (1.1 equiv) in
˚
anhydrous CH2Cl2 under Ar over 4 A sieves was stirred
at rt for 60 min, then cooled to ꢁ15 ꢁC before the addi-
tion of BF3ÆOEt2 (0.5 equiv) in CH2Cl2. The reaction
mixture was allowed to warm to rt and was stirred for
16 h at rt before being diluted with CH2Cl2, filtered
through Celiteꢂ and washed with aq Na2CO3. The
organic phase was dried over Na2SO4, the solvent was
removed in vacuo, and the residue were purified by flash
chromatography on silica gel. In this way the com-
pounds listed in Sections 3.2.1, 3.2.2, 3.2.3 and 3.2.4
were prepared.
3. Experimental
3.1. General methods
All solvents and reagents were either freshly distilled or
of analytical grade. Reactions were monitored by thin-
layer chromatography (TLC) using Merck precoated
silica gel GF254 aluminium-backed plates that were vis-
ualised under UV light (254 nm), followed by dipping
in H2SO4 in EtOH (5% v/v) and heating to ꢀ200 ꢁC.
Column chromatography was carried out following the
procedure described,43 using Scharlau Silica Gel 60 (230–
400 mesh). 1H and 13C NMR spectra were recorded
using a Bruker Avance 300 spectrometer, and low-reso-
lution mass spectra were obtained in the electrospray
ionisation mode on a Bruker Esquire 3000 mass spec-
trometer. High-resolution mass spectra (HRMS) were
recorded by the mass spectrometry service at the
Department of Chemistry (University of Queensland)
or within the School of Science (Griffith University), in
the positive-ion electrospray-ionisation mode. Combus-
tion analyses were carried out by the microanalysis ser-
vice of the Department of Chemistry, University of
Queensland. Compound 6 was prepared from D-glucur-
3.2.1. Methyl (30-nitrophenyl 2,3,4-tri-O-acetyl-b-D-
glucopyranosid)uronate (7). Yield 63%; Rf 0.2 in 2:1
hexanes–EtOAc. H NMR (300 MHz, CDCl3): d 7.96
1
(ddd, 1H, J4 ;5 8.2, J4 ;2 2.2, J4 ;6 0.9 Hz, H-40); 7.86
0
0
0
0
0
0
(t, 1H, J2 ;6 2.2 Hz, H-20); 7.48 (t, 1H, J5 ;6 8.2 Hz, H-
50); 7.34 (ddd, 1H, H-60); 5.40–5.24 (m, 4H, H-1, H-2,
H-3, H-4); 4.26 (AX m, 1H, H-5); 3.73 (s, 3H, CO2Me);
2.08, 2.06, 2.05 (3 · s, 9H, 3 · OAc). 13C NMR
0
0
0
0
(75 MHz, CDCl3):
d
170.0, 169.3, 169.2 (3 ·
OC(O)CH3); 166.6 (C-6); 156.8 (C-10); 149.1 (C-30);
130.3 (C-50); 123.6 (C-60); 118.4 (C-40); 111.9 (C-20);
98.7 (C-1); 72.6 (C-5); 71.6, 70.9, 68.8 (C-2, C-3, C-4);
53.1 (CO2CH3); 20.6, 20.6, 20.5 (3 · OC(O)CH3).
ESIMS (m/z): 477.8 [M+Na+]. HRMS Calcd for
C19H21NNaO12 [M+Na+] 478.0961. Found 478.0967.
3.2.2. Methyl (40-nitrophenyl 2,3,4-tri-O-acetyl-b-D-
glucopyranosid)uronate (8). Yield 81%; Rf 0.2 in 2:1
hexanes–EtOAc.13,14 Recrystallisation of the column-
purified product from hexanes–ethyl acetate gave 8 as

A. G. Pearson et al. / Carbohydrate Research 340 (2005) 2077–2085
2083
white needles: mp 155–157 ꢁC (lit.13 153–154 ꢁC). 1H
NMR (300 MHz, CDCl3): d 8.19 (AX m, 2H, H-30, H-
50); 7.07 (AX m, 2H, H-20, H-60); 5.38–5.28 (m, 4H,
H-1, H-2, H-3, H-4); 4.26 (AX m, 1H, H-5); 3.70 (s,
3H, CO2Me); 2.06, 2.05, 2.04 (3 · s, 9H, 3 · OAc). 13C
ene–acetone, followed by 1:1 hexanes–EtOAc) to give a
mixture of four products, 13–16 data for which are pro-
vided in Sections 3.3.1–3.3.4.
3.3.1. Methyl 1-O-[20(E)-30-(400-hydroxyphenyl)-20-prop-
enoyl] 2,3,4-tri-O-acetyl-b-D-glucopyranosyluronate (13).
Yield 14%; Rf 0.2 in 3:1 toluene–acetone. 1H NMR
NMR (75 MHz, CDCl3):
d 170.0, 169.3, 169.1
(3 · OC(O)CH3); 166.6 (C-6); 161.0 (C-10); 143.3 (C-
40); 125.8 (C-30, C-50); 116.7 (C-20, C-40); 98.0 (C-1);
72.6 (C-5); 71.4, 70.8, 68.7 (C-2, C-3, C-4); 53.1
(CO2CH3); 20.6, 20.6, 20.5 (3 · OC(O)CH3). ESIMS
(m/z): 477.8 [M+Na+].
(300 MHz, CDCl3): d 7.60 (d, 1H, J3 ;2 16.0 Hz, H-30);
0
0
7.36 (d, 2H, J2 ;3 8.4 Hz, H-200); 6.82, (d, 2H, H-300);
00 00
6.13 (d, 1H, H-20); 5.89 (d, 1H, J1,2 7.8 Hz, H-1); 5.40–
5.23 (m, 3H, H-2, H-3, H-4); 4.24, (d, 1H, J5,4 9.0 Hz,
H-5); 3.74 (s, 3H, CO2Me); 2.06, 2.05, 2.02 (3 · s, 9H,
3 · OAc). 13C NMR (75 MHz, CDCl3): d 170.0, 169.7,
169.6 (3 · OC(O)Me); 167.5 (C-6); 165.1 (C-10); 159.2
(C-400); 147.6 (C-30); 130.5 (C-200); 126.0 (C-100); 116.0
(C-300); 112.5 (C-20); 91.3 (C-1); 72.7 (C-5); 71.8, 70.1,
69.1 (C-2, C-3, C-4); 53.2 (CO2CH3); 20.6, 20.6, 20.5
(3 · OC(O)CH3). ESIMS (m/z): 503.1 [M+Na+]. Anal.
Calcd for C22H24O12ÆH2O: C, 53.01; H, 5.26. Found:
C, 53.01; H, 4.92.
3.2.3. Methyl (30,50-dimethoxycarbonyl 2,3,4-tri-O-acet-
yl-b-D-glucopyranosid)uronate (9). Yield 75%; Rf 0.2 in
2:1 hexanes–EtOAc. 1H NMR (300 MHz, CDCl3): d
8.37 (t, 1H, J4 ;2 ¼ J4 ;6 1.45 Hz, H-40); 7.82 (d, 2H,
H-20, H-60); 5.39–5.26 (m, 4H, H-1, H-2, H-3, H-4);
4.26 (AX m, 1H, H-5); 3.92 (s, 6H, 2 · CO2Me); 3.70
(s, 3H, CO2Me); 2.06, 2.04, 2.03 (3 · s, 9H, 3 · OAc).
13C NMR (75 MHz, CDCl3): d 170.0, 169.3, 169.2
(3 · OC(O)CH3); 166.7 (C-6); 165.6 (2 · CO2CH3);
156.5 (C-10); 132.1 (C-30, C-50); 125.6 (C-40); 122.1 (C-
20, C-60); 98.5 (C-1); 72.6 (C-5); 71.6, 71.0, 68.9 (C-2,
C-3, C-4); 53.0 (CO2CH3); 52.5 (2 · CO2CH3); 20.6,
20.6, 20.5 (3 · OC(O)CH3). ESIMS (m/z): 548.9
[M+Na+]. HRMS Calcd for C23H26NaO14 [M+Na+]
549.1220. Found 549.1220.
0
0
0
0
3.3.2. Methyl (40-{300-[methyl (2000,3000,4000-tri-O-acetyl-b-D-
glucopyranosyl)uronate prop-200(E)-enoate]}phenyl 2,3,4-
tri-O-acetyl-b-D-glucopyranosid)uronate
(14). Yield
22%; Rf 0.1 in 3:1 toluene–acetone. 1H NMR
00 00
(300 MHz, CDCl3): d 7.69 (d, 1H, J3 ;2 16.0 Hz, H-
300); 7.48 (d, 2H, J3 ;2 8.8 Hz, H-30); 7.00, (d, 2H, H-
0
0
20); 6.28 (d, 1H, H-200); 5.89 (d, 1H, J1
7.6 Hz, H-
000;2000
3.2.4. Methyl (40-methylumbelliferyl 2,3,4-tri-O-acetyl-b-
D-glucopyranosid)uronate (10). Yield 61%; Rf 0.2 in 2:3
hexanes–EtOAc.20,21 1H NMR (300 MHz, CDCl3): d
1000); 5.39–5.19 (m, 7H, H-1, H-2, H-3, H-4, H-2000, H-
3000, H-4000); 4.24–4.20, (m, 2H, H-5, H-5000); 3.72, 3.71
(2 · s, 6H, 2 · CO2Me); 2.05, 2.04, 2.04, 2.04, 2.03,
2.01 (6 · s, 18H, 6 · OAc). 13C NMR (75 MHz, CDCl3):
d 169.9, 169.8, 169.3, 169.2, 169.2, 169.1 (6 · OC(O)Me);
166.7, 166.7 (C-6, C-6000); 164.4 (C-100); 158.4 (C-10);
146.5 (C-300); 130.0 (C-30); 129.0 (C-40); 117.0 (C-20);
114.9 (C-200); 98.2 (C-1); 91.4 (C-1000); 72.9, 72.5, 71.7,
71.6, 70.8, 70.0, 68.9, 68.8 (C-2, C-3, C-4, C-5, C-2000,
C-3000, C-4000, C-5000); 52.9, 52.9 (2 · CO2CH3); 20.6,
20.5, 20.5, 20.4, 20.4, 20.3 (6 · OC(O)CH3). ESIMS
(m/z): 819.2 [M+Na+]. Anal. Calcd for C35H40O21: C,
52.77; H, 5.06. Found: C, 52.92; H, 5.08.
7.51 (d, 1H, J5 ;6 9.5 Hz, H-50); 6.94–6.90 (m, 2H, H-
0
0
60, H-80); 6.18 (q, 1H, J3 ;Me 1.2 Hz, H-30); 5.37–5.23
(m, 4H, H-1, H-2, H-3, H-4); 4.24 (AX m, 1H, H-5);
3.73 (s, 3H, CO2Me); 2.39 (d, 3H, Me0); 2.06, 2.05,
2.05 (3 · s, 9H, 3 · OAc). 13C NMR (75 MHz, CDCl3):
d 169.8, 169.3, 169.1 (3 · OC(O)Me); 166.7 (C-6); 160.6
(C-20); 158.9 (C-70); 154.5, 152.3 (C-40, C-8a0); 125.7 (C-
50); 115.4 (C-4a0); 113.7 (C-60); 112.9 (C-30); 103.8 (C-80);
97.8 (C-1); 72.2 (C-5); 71.5, 70.6, 68.8 (C-2, C-3, C-4);
52.9 (CO2CH3); 20.5, 20.5, 20.3 (3 · OC(O)CH3); 18.5
(C-40 CH3). ESIMS (m/z): 515.0 [M+Na+]. HRMS
Calcd for C23H26NaO14 [M+Na+] 515.1165. Found
515.1172.
0
0
3.3.3.
Methyl
2,3,4-tri-O-acetyl-b-D-glucopyranos-
yluronate trichloroacetamide (15). Yield 13%; Rf 0.3
1
in 3:1 toluene–acetone. H NMR (300 MHz, CDCl3):
3.3. Attempted glycosidation of 6 with 11
d 7.61 (br d, 1H, JNH,1 8.8 Hz, NH); 5.43 (dd, 1H, J3,4
9.5 Hz, J3,2 9.5 Hz, H-3); 5.25–5.15 (m, 2H, H-1, H-4);
5.09 (dd, 1H, J2,1 9.5 Hz, H-2); 4.20 (d, 1H, J5,4
9.7 Hz, H-5); 3.74 (s, 3H, CO2Me); 2.06, 2.06, 2.04
(3 · s, 9H, 3 · OAc). 1H NMR (300 MHz, C6D6): d
7.85 (br d, 1H, JNH,1 9.0 Hz, NH); 5.47 (dd, 1H, J3,4
9.5 Hz, J3,2 9.2 Hz, H-3); 5.40 (dd, 1H, J4,5 9.5 Hz,
H-4); 5.27 (dd, 1H, J1,2 9.2 Hz, H-1); 5.15 (dd, 1H, H-
2); 3.74 (d, 1H, H-5); 3.24 (s, 3H, CO2Me); 1.82, 1.69,
1.58 (3 · s, 9H, 3 · OAc). 13C NMR (75 MHz, CDCl3):
d 171.0, 169.7, 169.5 (3 · OC(O)CH3); 166.8 (C-6); 162.1
To a stirred suspension of 6 (144 mg, 0.30 mmol) in
anhyd CH2Cl2 (5 mL) under Ar over 4 A sieves at
˚
ꢁ15 ꢁC was added 11 (98 mg, 0.90 mmol). After
30 min at ꢁ15 ꢁC, BF3ÆOEt2 (20 lL, 0.16 mmol) was
added, the reaction was allowed to warm to rt, and
the mixture was stirred overnight. MeOH (0.5 mL)
was added, and the mixture was filtered through cotton
wool. The solvents were removed in vacuo, and the
product was purified by flash chromatography (3:1 tolu-

2084
A. G. Pearson et al. / Carbohydrate Research 340 (2005) 2077–2085
(NHC(O)CCl3); 91.5 (CCl3); 79.8 (C-1); 74.2 (C-5); 71.3
(C-3); 70.0 (C-2); 69.5 (C-4); 53.1 (CO2CH3); 20.6, 20.5,
20.4 (3 · OC(O)CH3). 13C NMR (75 MHz, C6D6):
d170.7, 169.7, 169.4 (3 · OC(O)CH3); 166.9 (C-6);
162.2 (NHC(O)CCl3); 92.5 (CCl3); 80.0 (C-1); 74.2
(C-5); 72.1 (C-3); 70.8 (C-2); 69.7 (C-4); 52.2 (CO2CH3);
20.2, 20.1, 20.0 (3 · OC(O)CH3). ESIMS (m/z): 501.7
[M+Na+].
0.27 mmol) was stirred at rt under N2 for 24 h. Addi-
tional portions of CH(OMe)3 (840 lL) and SOCl2
(20 lL) were added, and the mixture stirred for a further
24 h. The solvent was removed in vacuo, and the residue
azeotroped with toluene to give a pale purple solid. The
product was purified by flash chromatography (3:2 hex-
anes–EtOAc, Rf 0.3) to afford 21 as a white solid (1.05 g,
96%), which exhibited spectroscopic data in agreement
with those in the literature.46
3.3.4. Methyl 2,3,4-tri-O-acetyl-a-D-glucopyranosyl-
uronate fluoride (16). Yield 14%; Rf 0.4 in 3:1 toluene–
acetone. H NMR (300 MHz, CDCl3): d 5.80 (dd, 1H,
3.6. Methyl {40-[300-(methyl prop-200(E)-enoate)]phenyl
2,3,4-tri-O-acetyl-b-D-glucopyranosid}uronate (27)
1
J1,F 52.5 Hz, J1,2 2.7 Hz, H-1); 5.54 (dd, 1H, J3,2
9.9 Hz, J3,4 9.9 Hz, H-3); 5.22 (dd, 1H, J4,5 9.9 Hz,
H-4); 4.96 (ddd, 1H, J2,F 24.0 Hz, H-2); 4.45 (d, 1H,
H-5); 3.75 (s, 3H, CO2Me); 2.09, 2.03, 2.03 (3 · OAc).
13C NMR (75 MHz, CDCl3): d 169.9, 169.7, 169.4
(3 · OC(O)Me); 166.9 (C-6); 103.5 (d, J1,F 230 Hz,
C-1); 69.9 (d, J5,F 4 Hz, C-5); 69.9 (d, J2,F 24 Hz, C-2);
68.6 (C-4); 68.5 (C-3); 53.1 (CO2CH3); 20.6, 20.5, 20.4
(3 · OC(O)CH3). ESIMS (m/z): 359.0 [M+Na+].
Compound 22 was obtained in 83% yield by the cou-
pling of 6 and 26 using the general procedure for glycos-
idation (Rf 0.2 in 3:2 hexanes–EtOAc). 1H NMR
00 00
(300 MHz, CDCl3): d 7.63 (d, 1H, J3 ;2 16.0 Hz, H-
300); 7.47 (d, 2H, J3 ;2 8.7 Hz, H-30); 6.99, (d, 2H, H-
20); 6.33 (d, 1H, H-200); 5.38–5.25 (m, 3H, H-2, H-3,
H-4); 5.19 (d, 1H, J1,2 6.9 Hz, H-1); 4.20 (AX m, 1H,
H-5); 3.79, 3.71 (2 · s, 6H, CO2Me, CO2Me0); 2.05,
2.05, 2.04 (3 · s, 9H, 3 · OAc). 1H NMR (300 MHz,
0
0
3.4. Methyl 1-O-[20(E)-30-(400-acetoxyphenyl)-20-prop-
enyl] 2,3,4-tri-O-acetyl-b-D-glucopyranosyluronate (19)
CD3CN): d 7.62 (d, 1H, J3 ;2 16.0 Hz, H-300); 7.58 (d,
00 00
2H, J3 ;2 8.7 Hz, H-30); 7.04, (d, 2H, H-20); 6.43 (d,
1H, H-200); 5.41 (dd, 1H, J3,2 9.6 Hz, J3,4 9.6 Hz, H-3);
5.40 (d, 1H, J1,2 7.7 Hz, H-1); 5.22 (dd, 1H, H-2); 5.19
(dd, 1H, J4,5 9.8 Hz, H-4); 4.38 (d, 1H, H-5); 3.72,
3.65 (2 · s, 6H, CO2Me, CO2Me0); 1.99, 1.99, 1.98
(3 · s, 9H, 3 · OAc). 1H NMR (300 MHz, C6D6): d
0
0
To a stirred suspension of 6 (171 mg, 0.36 mmol) in
˚
anhyd CH2Cl2 (5 mL) under Ar over 4 A sieves at rt
was added 4-acetoxycinnamic acid (18,26 102 mg,
0.50 mmol). After 60 min, the mixture was cooled to
ꢁ15 ꢁC, BF3ÆOEt2 (14 lL, 0.11 mmol) in CH2Cl2
(1 mL) was added, the reaction mixture was allowed to
warm to rt, and the mixture was stirred overnight.
MeOH (0.5 mL) was added, and the mixture was filtered
through Celiteꢂ. The solvents were removed in vacuo,
and the product was purified by flash chromatography
(2:1 hexanes–EtOAc, Rf 0.2) to afford the title com-
7.73 (d, 1H, J3 ;2 16.0 Hz, H-300); 6.96 (d, 2H, J3 ;2
00 00
0
0
8.7 Hz, H-30); 6.75, (d, 2H, H-20); 6.32 (d, 1H, H-200);
5.59–5.48 (m, 3H, H-2, H-3, H-4); 4.83 (AX m, 1H,
H-1); 3.87 (d, 1H, J5,4 9.2 Hz, H-5); 3.49, 3.23 (2 · s,
6H, CO2Me, CO2Me); 1.69, 1.68, 1.68 (3 · s, 9H,
3 · OAc). 13C NMR (75 MHz, CDCl3): d 169.9, 169.2,
169.1 (3 · OC(O)Me); 167.3, 166.7 (C-6, CO2Me);
157.9 (C-10); 143.8 (C-30); 129.6 (C-40); 129.5 (C-30);
117.0 (C-20); 116.7 (C-200); 98.4 (C-1); 72.5, 71.6, 70.9,
68.9, (C-2, C-3, C-4, C-5); 52.9, 51.6 (2 · CO2CH3);
20.5, 20.5, 20.4, (3 · OC(O)CH3). ESIMS (m/z): 517.0
[M+Na+]. HRMS Calcd for C23H26NaO12 [M+Na+]
517.1322. Found 517.1317.
1
pound 19 (100 mg, 54%). H NMR (300 MHz, CDCl3):
d 7.71 (d, 1H, J3 ;2 16.0 Hz, H-30); 7.53 (d, 2H, J2 ;3
8.4 Hz, H-200); 7.11 (d, 2H, H-300); 6.34 (d, 1H, H-20);
5.88 (d, 1H, J1,2 7.8 Hz, H-1); 5.39–5.20 (m, 3H, H-2,
H-3, H-4); 4.23 (d, 1H, J5,4 9.6 Hz, H-5); 3.71 (s, 3H,
CO2Me); 2.29 (s, 3H, PhOAc); 2.02, 2.02, 2.00 (3 · s,
9H, 3 · OAc). 13C NMR (75 MHz, CDCl3): d 169.9,
169.5, 169.3, 169.1 (4 · O C(O)Me); 166.8 (C-6); 164.4
(C-10); 152.6 (C-400); 140.5 (C-30); 131.5 (C-100); 129.6
(C-200); 122.3 (C-300); 116.2 (C-20); 91.5 (C-1); 73.0 (C-
5); 71.7 (C-3); 70.1 (C-2); 69.0 (C-4); 53.1 (CO2CH3);
21.1 (PhOC(O)CH3); 20.6, 20.6, 20.5 (3 · OC(O)CH3).
ESIMS (m/z): 545.1 [M+Na+]. HRMS Calcd for
C24H26NaO13 [M+Na+] 545.12656. Found 545.12810.
0
0
00 00
3.7. Sodium {40-[300-(sodium prop-200(E)-enoate)]phenyl
b-D-glucopyranosid}uronate (28)
A solution of 0.1 M LiOH (6 equiv) in MeOH–H2O was
added to 27 (239 mg, 0.48 mmol), and the mixture was
stirred at 0 ꢁC for 20 h. The solution was then diluted
with H2O, and the pH was adjusted to 4.0 with Amber-
lite IR120 (H+). The resin was removed by filtration, the
solution was neutralised to pH 7.2 with dilute NaOH
and the solvents were removed in vacuo. The residue
was then purified by flash chromatography (7:2:1
EtOAc–MeOH–H2O) to give 23 (130 mg, 70%) after
3.5. Methyl 4-hydroxycinnamate (26)41
A
solution of 4-hydroxycinnamic acid (1.00 g,
6.09 mmol) in dry MeOH (20 mL) containing
CH(OMe)3 (840 lL, 7.64 mmol) and SOCl2 (20 lL,
1
lyophilisation. H NMR (300 MHz, D2O): d 7.59 (d,

A. G. Pearson et al. / Carbohydrate Research 340 (2005) 2077–2085
2085
2H, J3 ;2 8.8 Hz, H-30); 7.35 (d, 1H, J3 ;2 16.1 Hz, H-300);
7.14 (d, 2H, H-20); 6.43 (d, 1H, H-200); 5.15 (d, 1H, J1,2
7.4 Hz, H-1); 3.91 (d, 1H, J5,4 9.3 Hz, H-5); 3.63 (m,
3H, H-2, H-3, H-4). 13C NMR (75 MHz, D2O): d
175.2, 175.1 (C-6, CO2Na); 157.6 (C-10); 141.1 (C-300);
129.8 (C-40); 129.4 (C-30), 122.7 (C-200), 116.7 (C-20);
99.7 (C-1), 76.2 (C-5), 75.2, 72.6, 71.6 (C-2, C-3, C-4).
ESIMS (m/z): 406.8 [M+Na+].
0
0
00 00
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