Synthesis of a glucuronic acid and glucose conjugate library and evaluation of effects on endothelial cell growth

Article abstract of DOI:10.1016/j.carres.2004.05.024

Compounds that alter endothelial cell growth are of interest in the development of angiogenesis modulators. A structurally diverse series of saccharide derivatives (glycosylamide conjugates) have been synthesized and evaluated for their effects on bovine

Full text of DOI:10.1016/j.carres.2004.05.024

Carbohydrate
RESEARCH
Carbohydrate Research 339 (2004) 1873–1887
Synthesis of a glucuronic acid and glucose conjugate library and
evaluation of effects on endothelial cell growthq
Nigel Pitt,a Rhona M. Duane,b Alan O’ Brien,a Helena Bradley,a Stephen J. Wilson,b
Kathy M. O’ Boyleb,* and Paul V. Murphya,*
aCentre for Synthesis and Chemical Biology, Department of Chemistry, Conway Institute of Biomolecular and Biomedical Research,
University College Dublin, Belfield, Dublin 4, Ireland
bDepartment of Pharmacology, Conway Institute of Biomolecular and Biomedical Research, University College Dublin,
Belfield, Dublin 4, Ireland
Received 23 April 2004; accepted 25 May 2004
Available online 25 June 2004
Abstract—Compounds that alter endothelial cell growth are of interest in the development of angiogenesis modulators. A struc-
turally diverse series of saccharide derivatives (glycosylamide conjugates) have been synthesized and evaluated for their effects on
bovine aortic endothelial cell (BAEC) growth. Heparin-albumin (HA) reduced BAEC growth by 32% at 10 lg/mL and a number of
the novel saccharide conjugates from the library were found to mimic the effect of HA as they also inhibit endothelial cell survival
under identical conditions. Two thiophene conjugates, thioglucamide (24% inhibition at 35 lM) and a related glucuronide (26%
inhibition at 33 lM) were the most potent inhibitors of BAEC growth, as determined using a methylthiazol tetrazolium (MTT)
assay. The effects of thioglucamide and HA on absolute cell number were also studied using cell counting experiments; thiogluc-
amide (47% after 24 h) was more potent than indicated by the MTT assay and initially reduced the BAEC number to a greater extent
than HA (30% after 24 h); however, its actions were over more rapidly than were HA’s as cell growth had returned to levels of the
control after 72 h where HA still caused 25% inhibition. The binding of the monosaccharide conjugates to fibroblast growth fac-
tor (FGF-2) in competition with heparin-albumin by ELISA was investigated to establish the possible mechanism by which gly-
coconjugates could alter growth but there was no general correlation between reduction in viable cell population and binding to
FGF-2. No glycoconjugate reduced the proliferation of mouse mammary epithelial cells, nor did any alter gross cell morphology,
supporting a proposal that the reduction in BAEC survival by monosaccharide conjugates such as thioglucamide is a result of the
inhibition of cell proliferation rather than being an induction of cytotoxicity. These studies indicate that cell biological studies to
determine the mechanism of action of the simple monosaccharide conjugates may be worthwhile.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Keywords: Signal transduction; Angiogenesis; Glycoconjugates; Endothelial cell growth; Carbohydrate libraries
1. Introduction
endothelial cell proliferation. In adults, angiogenesis is
tightly regulated and occurs usually only during preg-
nancy or in wound healing. However, up-regulated
angiogenesis is characteristic in rheumatoid arthritis,
diabetic retinopathy, during tumour growth and
metastasis.2 Consequently inhibitors or, in some cases,
promoters of endothelial cell survival and proliferation
are of interest as potential therapeutics. A number of
strategies3 being considered for the development of anti-
angiogenic agents include synthesis of inhibitors of the
recognition of extracellular matrix proteins to integrin
The signal transduction processes that modulate cellular
behaviour are important biological events. For example,
angiogenesis1 provides new blood vessels to growing and
developing tissue and it relies on the up-regulation of
qSupplementary data associated with this article can be found in the
online version, at 10.1016/j.carres.2004.05.024
* Corresponding authors. Tel.: +353-1-7162504; fax: +353-1-7162501;
e-mail: paul.v.murphy@ucd.ie
0008-6215/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2004.05.024

1874
N. Pitt et al. / Carbohydrate Research 339 (2004) 1873–1887
receptors,4 matrix metalloprotease inhibitors5 as well as
natural products,6 collagenase inhibitors, COX inhibi-
tors, kinase inhibitors, aminopeptidase inhibitors7 and
inhibitors of growth factors and their receptors. Several
growth factors, such as FGF-2, VEGF and PDGF are
involved in the stimulation of angiogenesis, and so the
blockage of one factor alone might not be sufficient to
inhibit angiogenesis for a prolonged period, as the other
factors might be up-regulated to compensate. Thus, it is
likely that in chemotherapy more than one agent will be
needed for treatments and that the continuing develop-
ment of such agents will be necessary. Aside from their
angiogenic properties, FGFs display a range of activities
such as cell growth, differentiation and wound healing.
The cellular receptors for FGFs are tyrosine kinases
(FGFR) and these are activated by ligand-induced
dimerization, requiring heparin or heparan sulfate pro-
teoglycans (HSPG’s) as co-receptors (1, 2).8 The con-
sequences of exposing cells to growth factors in the
presence of heparin/HSPG’s can be cell movement, dif-
ferentiation, proliferation or protection from death. It is
widely believed that highly sulfated oligosaccharides
containing at least six saccharide units comprised of
iduronic acid are required to promote signaling.8 A re-
port by Ornitz et al. showed that non-sulfated disac-
charides and trisaccharides (e.g., 3, 4), with structural
features found in heparin, were active in a number of
FGF-2 dependent assays:9 they displayed binding to
FGF-2 in competition with 125I heparin, enhanced the
binding of FGF-2 to its receptor and, in the presence of
FGF-2, promoted proliferation of cell lines expressing
this receptor. These workers determined 3D structures
of their oligosaccharides binding to FGF-2 and also
performed cross-linking experiments; suggesting that the
oligosaccharides could promote the formation of FGF-2
clusters or aggregates that are responsible for the ob-
served activity (Chart 1). The 3D structure showed
occupation of other binding sites, not normally associ-
ated with heparin binding that may be physiologically
relevant.10 Some of the compounds were potent stimu-
lators of F32 cell proliferation in the presence of FGF-2
even though the binding affinity of the oligosaccharides,
measured in competition with heparin for FGF-2, was
moderate. Some oligosaccharides did not show affinity
in the binding assay but were also able to stimulate
proliferation of these cells. We were interested to explore
an approach in trying to identify compounds reduced in
carbohydrate character (monosaccharides) that might
be mimetics of disaccharides 3 or 4 or identify com-
pounds that would inhibit cell growth. Herein, we de-
scribe the synthesis and biological evaluation of a
library of monosaccharide derivatives, which has led to
identification of novel saccharide conjugates for inhibi-
tion of endothelial cell growth and are thus of interest as
potential angiogenesis or signal transduction inhibitors.
2. Results and discussion
2.1. Design and synthesis of the saccharide conjugate
library
Recent studies by Hindsgaul and co-workers (carbohy-
brids targeting oligosaccharide receptors)11 and Wong
and co-workers (targeting RNA)12 and by a number of
researchers in the selectin area13 have shown that it is
possible to replace parts of oligosaccharide-like struc-
tures by non-carbohydrate groups and retain biological
activity giving rise to glycomimetics reduced in carbo-
hydrate character that are still biologically active. With
a view to discovery of carbohybrids (Chart 2) which
possibly would display inhibition or promotion of FGF-
2 mediated processes, we synthesized a collection of
structurally diverse glycoconjugates (Chart 3) where D-
glucuronic acid and -glucopyranoside are attached to a
D
diverse range of non-carbohydrate groups. Such com-
OSO3
O
O2C
OSO3
O
O
OH
O
(
HO
O
O
HO
O3SHN
(
n
O2C
O
HO
O3SHN
O3SO
(
n
O
(
OSO3
2
1
FGF
Domain 1
OH
O
OH
O
HO2C
O
O
O
HO
O
HO
HO
HO
HO
HO
OH
CO2H
OH
AcHN
HO2C
AcHN
OMe
O
HO
O
OMe
3
OH
FGF Domain 2
4
Chart 1. Structures of 1–4. Evidence from 3D structures determined by crystallography and cross-linking experiments suggests that 3 and 4 induce
dimers/oligomers of FGF that can activate signal transduction.

N. Pitt et al. / Carbohydrate Research 339 (2004) 1873–1887
1875
FGF binding site
FGF binding site
HO2C
OH
O
HO2C
HO
HO
O
O
HO
O
HO
HO
G
OH
OH
AcHN
OMe
Disaccharide. Binds to FGF
(and is mitogenic towards F32 cell line)
Monosaccharide conjugates. Retain
glucuronic acid and replace GlcNAc
with non-carbohydrate groups G
to probe FGF binding sites (and
effects on cell growth)
Chart 2. Basis for choosing monosaccharide conjugates for synthesis and biological evaluation.
pounds were selected for synthesis based on a hypothesis
that they might bind in regions where the disaccharides
3 and 4 have previously been shown to bind (Chart 2).
The synthetic sequences used for preparation of 5–29
were based on methods described in the literature
(Scheme 1). The diversity of O-glycosides that could be
prepared by glycosidation reactions from the imidate 41
or bromide 42 was limited due to low reactivity of these
donors.14 However, the synthesis of the glycosyl amides
(e.g., 7–29) could be achieved more easily and from a
wide range of carboxylic acids. The glucuronic acid
conjugate precursors (Scheme 1) were condensed with
acid chlorides (from 43 and promoted by PPh3) or carb-
oxylic acids (from 44 using amide coupling reagents, e.g.,
DCC) and the protecting groups were then removed to
give the b-glucuronic acid conjugates. The carboxylic
acid derivatives used for conjugation to the glucuronic
acid were selected to have maximum diversity based
on their partial atomic charge, atomic r-charge, atomic
p-charge, hydrogen bonding, topological characteristics
and on the ‘Lipinksi rule of 5’ (Chart 2).15
2.2. Biological evaluation of saccharides and discussion
The effects of compounds 5–40 on bovine aortic endo-
thelial cell (BAEC) growth were investigated. The
BAECs used in the assays express both the FGFR and
heparan sulfate proteoglycans, and they release FGF-2.
This drives growth or survival, and potently suppresses
apoptotic cell death; evidence for this has been reported
previously; FGF-2 activity can be inhibited using a
neutralizing anti-FGF-2 antibody, which resulted in
increased apoptosis.17 The BAECs are thus suitable as
models for identifying compounds that may act by
inhibiting or promoting FGF-2 induced signaling
pathways or that may alter cell growth and survival
pathways by other mechanisms. The effect of heparin on
endothelial cell growth in culture is complex, with both
proliferative18 and anti-proliferative19 effects being ob-
served. The conditions that were used in this case, where
the cells were grown in the presence of serum, favoured
anti-proliferative actions of heparin. Thus heparin-
albumin (HA) reduced BAEC growth by 32% at 10 lg/
mL. A number of the novel saccharide conjugates were
found to mimic the effect of heparin as they inhibited
endothelial cell survival under identical conditions, with
thiophenes 20 and 31 (thioglucamide) the most active;
these two compounds had similar potency, respectively,
inhibiting the growth of endothelial cells by 26% at
33 lM and 24% at 35 lM. The MTT assay developed by
Mosmann was used to determine cell populations during
the first assays that were carried out. The effects of the
growth inhibitor 31 and heparin-albumin (HA) on
absolute cell number were subsequently studied using
cell counting experiments; more specifically the number
of cells present in BAEC cultures after 24–72 h exposure
to thioglucamide 31 or HA was established and the re-
sults are shown in Figure 1. Thioglucamide 31 initially
reduced the BAEC number to a greater extent than HA;
however, its actions were over more rapidly than were
HA’s. Thus, after 24 h, thioglucamide 31 reduced BAEC
number by 47% compared to a 30% reduction by HA.
By 72 h, HA still caused a 25% reduction in cell number
whereas the number of cells in the presence of 31 had
returned to control levels. The cell counting experiments
In addition glucose conjugates were prepared; for
example, 31 was prepared from the azide 45 (Scheme 2)
by reduction to the amine and subsequent coupling to
thiophene-2-carboxylic acid followed by saponification.
The related glucoside 36 (not shown in Scheme 2) was
obtained by reaction of 45 with benzoyl chloride and
PPh3 and subsequent deacetylation. The tertiary amide
34 was prepared via the reaction of
D-glucose with
methylamine in MeOH followed by reaction of the
glycosylamine product with thiophene-2-carbonyl chlo-
ride in MeOH in the presence of Na2CO3. The reaction
of azide 43 with DBU gave 46, which after coupling with
thiophene-2-carbonyl chloride using triphenylphosphine
and deprotection gave 32 as a mixture of anomers
(Scheme 3). The synthesis of the divalent glucuronic acid
conjugate 30 has been described previously.16 The syn-
theses of other divalent conjugates are shown in Scheme
4. Thus EDC–HOBt promoted reaction of 44 with
isophthalic acid gave 48, which is converted to 38 by
coupling with pentane-1,5-diamine followed by depro-
tection. The coupling of 44 with 1,6-dioxaoctanedioic
acid and removal of protecting groups gave 39.

1876
N. Pitt et al. / Carbohydrate Research 339 (2004) 1873–1887
O
O
O
HO2C
HO
HO
O
O
O
G
N
H
N
H
N
G =
O
O
N
OH
5
6
7
8
O
O
Cl
O
O
O
O
O
N
H
N
H
N
H
HN
HN
HN
N
O2N
11
12
14
10
9
13
O
O
O
O
O
O
Cl
O
H
N
N
H
N
N
O
N
H
H
N
H
N
H
19
H
18
17
CF3
15
Cl
16
O
O
O
H3C
N
S
N
N
H
H
N
H
H
O
O
24
21
23
20
CH3
Cl
N
H
22
Ph
O
O
O
MeO
MeO
F
O
O
N
H
N
H
N
H
O
N
F
H
OMe
25
27
28
29
HO
H
N
S
CO2H
O
H
OH
CO2H
HO
HO
HO2C
O
HO
HO
HO
HO
N
HO2C
O
N
O
S
H
H
H
O
N
N
O
OH
O
OH
OH
32
OH
OH
O
O
O
OH
33
31
30
H
N
HO
HO
HO
HO
N
HO
HO
HO
S
HO2C
O
H
N
H
O
HO
O
O
HO
N
N
O
OH
34
OH
OH
OH
O
O
O
OH
37
35
36
HO2C
HO
HO
HO2C
HO
HO
H
N
O
O
HO2C
NH
H
H
N
O
O
OH
HO
N
OH
O
HO
OH
O
O
O
HO2C
H
O
O
HO
NH
N
HO
O
OH
HO2C
HO
HO
O
O
HO2C
HO
HO
O
O
NH
O
NH
OH
OH
38
39
40
Chart 3. Glucuronic acid and other saccharide conjugates synthesized and evaluated in BAEC growth assays.
did indicate that 31 is more potent after 24 h than
indicated by the MTT assay; this could imply that other
compounds in the saccharide library may be more
potent than they appear from MTT assay. The results
are indicative that another cell survival pathway is
up-regulated to compensate for the inhibitory effects
displayed by 31.
The compounds listed in Table 1 were also evaluated
for their effects on growth of a mouse mammary epi-
thelial cell line (NMuMG) in order to establish whether
the inhibition of endothelial cell growth was merely a
nonspecific toxic action. Heparin-albumin was found to
reduce the number of viable epithelial cells by 12.2%
after 24 h whereas no saccharide conjugate reduced the

N. Pitt et al. / Carbohydrate Research 339 (2004) 1873–1887
1877
MeO C
HO C
2
2
BF .OEt
MeO C
AgCO , AgClO ,
3
2
2
3
4
O
O
HO
AcO
O
CH Cl , ROH
ROH, CH Cl
2
2
2
AcO
O
2
HO
AcO
R
AcO
OH
AcO
deprotection
CCl
AcO
O
3
deprotection
Br
5, 6
42
41
NH
Amide coupling
chemistry and
deprotection
Ph P, CH CN,
RCOCl
HO C
3
3
2
MeO C
2
H
N
MeO C
2
O
O
HO
AcO
R
O
N
3
AcO
HO
AcO
NH
2
AcO
OH
deprotection
OAc
43
O
OAc
7-29
44
Scheme 1. Synthesis of glucuronides 5–29.
OAc
OH
O
(i) Pd-C, EtOH, H2
S
O
H
AcO
HO
HO
N3
AcO
N
(ii) Thiophene-2-
carboxylicacid, DCC,
DMAP, CH2Cl2
OAc
OH
O
N
45
31
(iii) NaOMe, MeOH
OH
OH
O
OH
S
(i) MeNH2, MeOH
O
HO
HO
O
HO
HO
HO
N
O
S
OH
HO
(ii)
S
OH
OH
OH
O
Cl
34a (33%)
34b (66%)
O
Na2CO3, MeOH
Scheme 2. Synthesis of glucosides.
thelial cell growth were also inhibitors of heparin or HS
binding to FGF-2 (see Table 1); in a preliminary com-
munication, we provided some evidence that this may
have been the case.20 Heparin and HS were evaluated as
standards in the binding assay; heparin showed a max-
imum inhibition (Imax) of 95% with 47.5% inhibition
observed at 1.6 ng/mL whereas HS was less potent in the
assay had a lower Imax (70%) and displayed 35% inhibi-
tion at 136.5 ng/mL. The average weight of the oligo-
saccharide fraction on HS that binds one FGF-2
molecule is estimated to be ꢀ1100;21 this would imply
that the concentration of the oligosaccharide fraction
required to inhibit FGF-2 binding to HS by 35% is thus
125 nM. The thiophene derivatives 31 and 32 (Imax 62%
@ 346 lM and 65% @ 350 lM, respectively) and alkyl
derivative 14 (Imax 51% @ 353 lM) were amongst the
more potent compounds in the binding assay. Some
compounds showed an apparent stimulation of the
binding of heparin to FGF-2 (e.g., 6 and 8) as increased
amounts of FGF-2 were bound to the 96 well plates
coated with HA in their presence. Experiments suggest
that the increased amounts of FGF-2 binding to plates
in presence of compounds is due to promotion of the
interaction between the 1ꢁ antibody, used in the assay,
and FGF-2, rather than the compound producing an
enhancement of heparin/FGF-2 binding. The hypothesis
Cl
DBU,
S
MeO2C
O
N3
O
CH2Cl2
43
67%
OAc
CH3CN,
Ph3P, CH2Cl2
OAc
46
H
N
H
LiOH, MeOH
THF, H2O
MeO2C
O
HO2C
O
N
S
S
O
O
OAc
OH
OAc
47 59%
OH
32
α:β 64:36
Scheme 3. Synthesis of 32.
proliferation of epithelial cells, nor did any alter gross
cell morphology. Dimethylsulfoxide, a known cytotoxic
agent, inhibited cell growth by >95%. These results
support the proposal that the reduction in BAEC
growth by the saccharide conjugates is a result of inhi-
bition of cell proliferation or survival rather than being
an induction of cytotoxicity.
ELISA assays were developed for determination of
binding of the monosaccharide conjugates to FGF-2 in
competition with a heparin-albumin conjugate (HA) as
it was possible that the compounds that altered endo-

1878
N. Pitt et al. / Carbohydrate Research 339 (2004) 1873–1887
NH2
HO2C
CO2H
MeO2C
(i)
H2N
H
N
O
DCC, HOBt, DMAP,
AcO
CH2Cl2, THF
AcO
O
38
44
OAc
HOBt, EDC, DMF
(ii) NaOMe, MeOH then
LiOH, THF, H2O, MeOH
O
O
(i)
O
DCC, HOBt, DMAP,
CH2Cl2, THF
HO
HO
2
48
(ii) LiOH, THF, H2O, MeOH
39
Scheme 4. Synthesis of divalent glucuronic acid conjugates.
150
that there is generally a correlation between inhibition of
BAEC survival and inhibition of FGF-2 binding to HA
is not supported by the results obtained; 14 and 32 were
inactive and 31 was active in the endothelial cell assay;
each of these compounds would have been expected to
inhibit BAEC growth if their mechanism was due to the
inhibition of action of FGF-2. Furthermore the syn-
thesis of the disaccharide 3, which is the simplest
disaccharide component of heparin, has recently been
completed within our group; it is inactive in the BAEC
assay and did not inhibit FGF-2 binding to heparin in
the ELISA; this clearly shows that 20 and 31 are not
mimics of simple heparin disaccharides.22 These obser-
vations, based on evaluation of a wider range of com-
pounds than that reported in the preliminary
communication and on the biological evaluation of 3
suggest thus that although the saccharide conjugates
were designed as putative FGF-2 inhibitors and puta-
tive mimetics of 3, their ability to cause a reduction in
viable cell populations is not through their inhibition
of heparin/FGF-2 binding interactions; it may be
through the alteration of another step in the FGFR-
mediated signal transduction pathway, or through the
alteration of an entirely different signal transduction
pathway.
Galactose amide conjugates that are structurally
related to 31 and 36 are weak inhibitors of an a-galac-
tosidase (mM range). Indeed other compounds struc-
turally related to the aromatic-saccharide conjugates
described herein have been synthesized for evaluation as
glycosidase inhibitors.23 We considered the possibility
that a possible mode of action for 31 might include
glycosidase inhibition; however 1-deoxynojirimycin was
found to be inactive as an inhibitor of BAEC growth.24
It is known that glycosidase inhibitor castanospermine is
an angiogenesis inhibitor but neither it nor N-methyl-1-
deoxynojirimycin altered the proliferation of cultured
endothelial cells when evaluated previously.25 These
observations rule out the possibility that 20 and 31 are
acting as glycosidase inhibitors.
Thioglucamide 31
Heparin
% Control
100
50
0
24h
48h
72h
Figure 1. The effect of thioglucamide 31 (35 lM) and heparin-albumin
(10 lg/mL) on the growth of BAECs as determined by cell counting
experiments.
Table 1. Biological data for bioactive glycoconjugates and heparin/
heparan sulfatea
Compound
FGF-ELISA
Concn
BAEC survivalb
Imax
%
%
Concn
(lM)
(lM)
8
12
50c
45
51
24
40
20
50
35
25
23
62
65
97.5
34
Inactive
15
34
34
340
350
250
330
280
300
290
220
260
350
350
14
15
Inactive
Inactive
26
35
25
20
24
33
28
Inactive
8
25
27
30
29
Inactive
Inactive
15
28
29
22
26
31
32
24.5
Inactive
35
35
Heparin-
albumin
Heparin
(Fluka)
Heparan
sulfate
(Sigma)
0.1 mg/mL 32
0.1 mg/mL n.d.
0.1 mg/mL n.d.
10 lg/mL
95
70
––
––
aCompounds not included in the table were inactive.
bThe number of viable cells was estimated using the MTT assay.
cStimulates binding.

N. Pitt et al. / Carbohydrate Research 339 (2004) 1873–1887
1879
The order of activity in BAEC assay of 31 ¼ 20 > 32
may be suggesting that the compounds are inhibiting a
glucose or glucuronide receptor, although this would need
to be investigated in greater detail by evaluation of, for
example, the galactose and mannose conjugates. Also the
orientation of the thiophene with respect to the pyranose
appears to be important for biological activity. The for-
mal N-methylation of 31 to give 34 would alter aromatic
group presentation with respect to the pyranose: sec-
ondary amides such as 31 would be expected to adopt the
Z-anti structure (trans arrangement of the amide N–H
and C@O and H1–C1–N–H dihedral angle approaching
180ꢁ), whereas the N-methylated compound would be
expected to adopt preferentially E-anti structures; these
are based on comparisons with related benzamides.26 The
NMR spectra of 34 revealed, as expected, that there were
two signal sets;25 these correspond with the interconvert-
ing amide rotamers 34a (Z-anti, 33%) and 34b (E-anti,
66%), the relative proportions being quantified by inte-
gration.25 The lack of activity in the cell growth assay of
34 is thus explained by the E-anti isomer 34b adopting
preferentially the orthogonal arrangement of the thio-
phene and pyranose groups and the aromatic group of the
Z-anti isomer 34a being forced out of conjugation with
the carbonyl group due to the steric interaction between
the N-methyl group and aromatic protons.
Varian Inova 300 and 500, and Bruker DRX-600 MHz
spectrometers. Chemical shifts are reported relative to
internal Me4Si in CDCl3 (d 0.0) or HOD for D2O (d 4.80)
for 1H and (d 77.0) for 13C. 13C signals were assigned with
1
the aid of DEPT-135. H signals were assigned with the
aid of COSY. Coupling constants are reported in Hertz.
IR spectra were recorded with a Mattson Galaxy Series
FTIR 3000 using either thin film between NaCl plates or
KBr discs, as specified. Melting points were measured on
a Gallenkamp melting point apparatus. Elemental
analysis was performed on an Exeter Analytical CE440
elemental analyzer. Low and high-resolution mass
spectra were measured at the University of York, UK or
on a Micromass LCT KC420 or Micromass Quattro.
TLC was performed on aluminium sheets precoated with
Silica Gel 60 (HF254, E. Merck) and spots visualized by
UV and charring with 1:20 H2SO4–EtOH. Flash column
chromatography was carried out with Silica Gel 60
(0.040–0.630 mm, E. Merck) and employed a stepwise
solvent polarity gradient correlated with the TLC
mobility. Chromatography solvents used were EtOAc,
MeOH, acetone and CH2Cl2 (Riedel-de Haen), petro-
leum ether (bp 40–60 ꢁC, BDH laboratory supplies) and
toluene (Aldrich). Analytical HPLC separations were
performed using a Waters 600E pump and Waters 486
tuneable absorbance detector or Shimadzu LC10AT
pump and Shimadzu SPD10A tuneable absorbance
detector. All compounds were purified by semi-prep
HPLC using a Waters 600E pump before biological
evaluation (MeCN–water mixtures were used as eluant
with a flow rate 10 mL/min). The semi-preparative col-
umns used were YMC-Pack C-4 rev phase (S-10lm,
250 · 20 mm). Wavelength for both analytical and semi-
preparative HPLC was 220 nm. Reaction solvents were
dried and distilled where stated. The carboxylic acids
used for the library synthesis are sold as DiversiChemꢂ
Building Block Kits by NovaBiochem. Heparan sulfate
was obtained from Sigma (cat no. H9902, av MW
14,200) and heparin from Fluka (cat. no. 51536).
2.3. Summary
The strategy to synthesize a collection of structurally
diverse monosaccharide conjugates has provided novel
compounds that alter BAEC proliferation. Such bio-
logical activities for such simple glycoconjugates have
not been previously observed, to the best of our
knowledge. The active compounds have low molecular
weights and the synthesis of derivatives for further
biological evaluation would be straightforward. The
selectivity displayed by compounds in inhibiting endo-
thelial cell proliferation but not epithelial cell prolifer-
ation seems significant and worthy of further
investigation. The determination of the details of the
biological mode of action of the saccharide conjugates
could be important as it may yield new targets for
modulating signal transduction pathways. Such mecha-
nistic studies are underway as well as attempts to im-
prove the potency of the thiophene derivatives and the
results will be reported in due course.
3.2. Preparation of O-glycosides
3.2.1. A soln of bromide 4227 (1.0 equiv) in CH2Cl2 was
added to a stirred soln of the appropriate alcohol
(0.66 equiv), silver carbonate (2.25 equiv), silver per-
ꢀ
chlorate (0.1 equiv) in CH2Cl2 over activated 4 A
molecular sieves, under nitrogen. After 12 h the reaction
was diluted with CH2Cl2, filtered through Celite, washed
with water, dried (sodium sulfate anhyd) and the solvent
removed under diminished pressure. The residue was
purified by chromatography.
3. Experimental section
3.1. General
3.2.2. The alcohol (1.0 equiv) and trichloroacetimidate
4128 (1.0 equiv) in anhyd CH2Cl2 over activated 4 A
molecular sieves was placed under a nitrogen atmo-
sphere and cooled to )15 ꢁC. Boron trifluoride diethyl
ꢀ
Optical rotations were determined with a Perkin–Elmer
241 model polarimeter at the sodium D line at 23 ꢁC.
NMR spectra were recorded with JEOL JNM-GX270,

1880
N. Pitt et al. / Carbohydrate Research 339 (2004) 1873–1887
etherate (0.5 equiv in CH2Cl2) was added and the mix-
ture stirred for 12 h at rt. The reaction mixture was
then diluted with CH2Cl2, washed with Na2CO3 (aq),
the organic phase was dried (sodium sulfate anhyd), the
solvent removed under diminished pressure and the
residue purified by chromatography.
matography and then semi-preparative C-4 RP HPLC
(flow rate: 10 mL/min) to give, after freeze drying, the
desired compound.
3.7. 1,4-Dioxaspirol[4,5]dec-2-yl-b-D-glucopyranosidu-
ronic acid (5)
3.3. Preparation of acid chlorides
(1,4-Dioxaspirol[4,5]dec-2-yl)-2,3,4-tri-O-acetyl-b-D-glu-
copyranosiduronic acid, methyl ester (235 mg, 41%) was
prepared as in Section 3.2.1. This intermediate (85 mg,
0.17mmol) was deprotected as described in Section 3.6 to
Thionyl chloride (2 equiv) was added to the carboxylic
acid (1 equiv) in anhyd toluene at 0 ꢁC. The reaction
mixture was then heated at 70 ꢁC for 3 h. The acid
chlorides were purified by distillation.
afford 5 as a gum (39 mg, 66%); ½aꢁ )22.7 (c 0.30, MeOH);
D
IR (KBr) cmꢂ1 3424, 2928, 1617, 1420, 1284, 1054, 663; 1H
NMR (D2O, 300 MHz): d 4.56–4.53 (m, 2H), 4.51–3.37
(m, 7H), 3.46–3.36 (m, 1H), 1.80 (m, 10H); 13C NMR
(D2O, 125 MHz): d 178.6, 114.0, 113.9 (each s), 105.1,
105.0, 78.8, 78.1, 77.0, 76.6, 75.7, 75.6, 74.5 (each d), 73.4,
72.8, 67.7, 38.1, 38.0, 36.4, 27.2 (each t); ESIHRMS: m=z
[M+Na]þ: calcd 371.1336, found 371.1318.
3.4. Synthesis of amides from azide 4329
To azide 4330 (1 equiv) and the acid chloride (2 equiv),
dissolved in anhyd MeCN (4 mL), was added dropwise a
soln of PPh3 (1.3 equiv) in anhyd CH2Cl2 (1 mL) at rt. The
reaction was allowed to stir for 12 h, diluted with CH2Cl2
(20 mL), washed with satd NaHCO3 (10 mL) and then
with water (10 mL). The organic phase was dried (sodium
sulfate anhyd), the solvent removed under diminished
pressure and the residue purified by chromatography.
3.8. (3-Benzoylphenyl)-b-D-glucopyranosiduronic acid (6)
The reaction of 3-benzoylphenol with 42 gave the pro-
tected precursor (269 mg, 52%); a portion (153 mg,
0.29 mmol) was deprotected to give 6 as an off-white solid
3.5. Synthesis of amides from amine 44
(50 mg, 46%); ½aꢁ )4.2 (c 0.26, MeOH); IR (KBr) cmꢂ1
D
3400, 2907, 1625, 1435, 1204, 1062; 1H NMR (D2O,
300 MHz): d 7.68–7.38 (m, 9H), 5.05 (br s, 1H), 3.79 (br s,
1H), 3.69 (m, 3H); 13C NMR (D2O, 125 MHz): d 202.6,
178.0 (each s, C@O), 159.0, 140.8, 139.1 (each s), 136.4,
133.0, 132.8, 131.3, 127.7, 124.5, 120.8 (each d), 102.5 (d,
C-1), 78.9, 78.1, 75.4, 74.5 (each d); ESIHRMS: m=z
[M+Na]þ: calcd 397.0899, found 397.0978.
Reaction of azide 4330 with Pd/C in anhyd THF at )5 ꢁC
under H2 gave amine 44 and its a-anomer in a 13:1 ratio
after filtration and removal of solvent; 1H NMR
(CDCl3, 300 MHz): d 5.30 (t, 1H, J 9.6), 5.16 (t, 1H, J
9.6), 4.86 (t, 1H, J 9.6), 4.23 (d, 1H, J 8.6), 4.04 (d, 1H, J
10.0), 3.75–3.72 (overlapping signals, 5H), 2.07, 2.03,
2.02 (each s, each 3H). This freshly prepared amine 44
(1 equiv) was added to a soln of the carboxylic acid
(1.2 equiv), HOBT (1.2 equiv), DMAP (1.2 equiv) and
DCC (1.2 equiv) in anhyd THF at 0 ꢁC. The reaction
mixture was then stirred at rt for 12 h, the precipitated
dicyclohexyurea was filtered off and the solvent removed
under diminished pressure. The residue was dissolved in
CH2Cl2, washed with NaHCO3 (aq), brine and dried
(sodium sulfate anhyd). The organic phase was then
concentrated under diminished pressure, and the residue
purified by chromatography.
3.9. 1-Deoxy-1-[pyrazinylcarbonylamino]-b-D-glucopyr-
anuronic acid (7)
The protected precursor was prepared (250 mg, 39%)
from amine 44 and a portion (70 mg, 0.16 mmol) de-
protected to give 7 as an off-white solid (43 mg, 90%);
½aꢁ )11.6 (c 0.25, water); IR (KBr) cmꢂ1 3453, 3253,
D
2917, 1790, 1659, 1562, 1368, 1239, 1065, 1065, 1041; 1H
NMR (D2O, 270 MHz): d 7.95–7.40 (m, 3H), 5.25 (d, 1H,
J 8.6), 3.88 (d, 1H, J 9.5), 3.66 (m, 3H); 13C NMR (D2O,
125 MHz): d 175.4, 169.2 (each s), 150.5, 147.1 (each d),
146.8 (s), 146.3, 82.3, 79.2, 78.8, 74.2, 74.0 (each d);
ESIHRMS: m=z [M)H]ꢂ: calcd 298.0687, found
298.0687.
3.6. Deprotection of glucuronic acid conjugates
A soln of 0.05 M LiOH in 2.5:1.0:0.5 MeOH–water–
THF (6 equiv) was added to the glycosylamide or O-
glycoside (1 equiv). The soln was stirred at 0 ꢁC until
complete deprotection (TLC, 3:1 EtOAc–MeOH). The
soln was then diluted with water and the pH adjusted to
3.0 with Amberlite IR120 (Hþ). The Amberlite was re-
moved by filtration and the MeOH and THF were re-
moved under diminished pressure. The resulting aq soln
was freeze dried and the residue purified by flash chro-
3.10. 1-(Benzoylamino)-1-deoxy-b-D-glucopyranuronic
acid (8)
The protected intermediate (270 mg, 62%) was prepared
from azide 43 and a portion deprotected (110 mg,
0.25 mmol) to give 8 as an off-white solid (54 mg, 72%);
20
D
½aꢁ +2.5 (c 0.62, water); IR (film) cmꢂ1 3399 (OH),

N. Pitt et al. / Carbohydrate Research 339 (2004) 1873–1887
1881
1
2900, 1649 (C@O), 1644 (C@O), 1258; H NMR (D2O,
270 MHz): d 8.00 (m, 5H), 5.18 (d, 1H, J 8.2), 4.01 (d,
1H, J 9.0), 3.50 (m, 3H); 13C NMR (D2O, 125 MHz): d
176.0, 174.6, 149.9 (each s), 135.6, 131.6, 130.3, 82.6,
79.6, 78.9, 74.2, 74.1 (each d); ESIHRMS: m=z
[M+Na]þ: calcd 320.0746, found 320.0757.
protected to give 12 as an off-white solid (118 mg, 65%);
½aꢁ )1.3 (c 0.6, water); IR (KBr) cmꢂ1 3401 (OH),
D
2960, 1889, 1634, 1551, 1418, 1300, 1087, 1027; 1H
NMR (D2O, 270 MHz): d 8.63 (s, 2H), 7.71 (d, 2H) 5.19
(d, 1H, J 8.2), 3.83 (d, 1H, J 9.1), 3.55 (m, 3H); 13C
NMR (D2O, 125 MHz): d 178.4, 172.2 (each s), 152.3,
152.3 (each d), 144.1 (s), 124.7, 82.3, 81.0, 79.0, 74.5,
74.4 (each d); ESIHRMS: m=z [M+H]þ: calcd 299.0879,
found 299.0861.
3.11. 1-Deoxy-1-[[(tetrahydro-2-furanyl)carbonyl]amino]-
b-D-glucopyranuronic acid (9)
The protected precursor (224 mg, 35%) was prepared
from azide 44 and a portion (52 mg, 0.12 mmol) depro-
3.15. 1-Deoxy-1-[(2-methyl-1-oxopentyl)amino]-b-D-
glucopyranuronic acid (14)
tected to give 9 as an off-white solid (23 mg, 68%); ½aꢁ
D
)18.7 (c 0.08, MeOH); IR (KBr) cmꢂ1 3429, 2921, 2343,
1536, 1424, 1092, 668; 1H NMR (D2O, 300 MHz): d 4.95
(d, 1H, J 8.0), 4.42 (d, 1H, J 8.3), 3.95 (dd, 1H, J 6.0, 14.0),
3.85 (dd, 1H, J 6.5, 14.0), 3.75 (d, 1H, J 9.0), 3.50 (m, 3H),
2.28 (m, 1H), 2.02–1.84 (m, 3H); 13C NMR (D2O,
125 MHz): d 180.3, 178.5 (each s), 81.7, 80.5, 79.0, 74.5,
74.4, 72.7 (each d), 72.7, 33.0, 27.8 (each t); ESIHRMS:
m=z [M+Na]þ: calcd 314.0852, found 314.0886.
The precursor (54 mg, 42%) was prepared from azide 44
(mixture of diastereoisomers, 1:1.2) and a portion
(100 mg, 0.23 mmol) deprotected to give 14 as an off-
white solid (56 mg, 85%); ½aꢁ )16.6 (c 0.04, MeOH); IR
D
(KBr) cmꢂ1 3605, 1555, 1418, 1077, 1021, 639; 1H NMR
(D2O, 270 MHz): d 4.79 (m, 1H), 3.62 (m, 1H), 3.42–
3.19 (m, 3H), 2.28–2.03 (m, 5H), 0.94 (m, 3H), 0.70 (m,
3H); 13C NMR (D2O, 125 MHz): d 181.8, 181.7, 181.2,
175.5 (each s), 79.0, 78.9, 77.6, 76.2, 76.2, 71.6, 71.5,
40.5, 40.4 (each d), 35.5, 35.4, 19.7 (each t), 16.8, 16.7,
13.1, 13.1 (each q); ESIHRMS: m=z [M+Na]þ: calcd
314.1225, found 314.1216.
3.12. 1-[Cyclopropylcarbonylamino]-1-deoxy-b-D-gluco-
pyranuronic acid (10)
The protected precursor (910 mg, 48%) was prepared
from azide 44 and a portion (95 mg, 0.23 mmol) depro-
3.16. 1-Deoxy-1-[[(2,4-dichlorophenoxy)acetyl]amino]-b-
D-glucopyranuronic acid (15)
tected to give 10 as an off-white solid (52 mg, 87%); ½aꢁ
D
)20.4 (c 0.054, MeOH); IR (KBr) cmꢂ1 3374, 1569,
1415, 1069, 946, 649; 1H NMR (D2O, 270 MHz): d 4.82–
4.79 (d, 1H, J 9.0), 3.60 (d, 1H, J 9.5), 3.32–3.24 (m,
3H), 1.65–1.47 (m, 1H), 0.74 (d, 4H); 13C NMR (D2O,
125 MHz): d 181.2, 178.5 (each s), 79.0, 77.6, 76.1, 71.6
(2s) (each d), 17.0 (t), 10.6, 10.3; ESIHRMS: m=z
[M+Na]þ: calcd 284.0724, found 284.0746.
The protected precursor (250 mg, 12%) was prepared
from 44 and a portion (160 mg, 0.3 mmol) deprotected
to give 15 as an off-white solid (98 mg, 83%); ½aꢁ )7.3
D
(c 0.06, MeOH); IR (KBr) cmꢂ1 3452 (OH), 2386, 2569,
1417, 1238, 1078, 1019, 604; 1H NMR (D2O, 270
MHz): d 7.38 (s, 1H), 7.15 (d, 1H, J 9.0), 6.86 (d, 1H, J
9.0), 4.91 (d, 1H, J 9.2), 3.65 (d, 1H, J 9.5), 3.41–3.32 (m,
3H); 13C NMR (D2O, 125 MHz): d 181.9, 175.4, 151.7
(each s), 129.8, 128.0 (each d), 126.7, 123.1 (each s), 115.5,
78.7, 77.8, 76.1, 71.6, 71.5 (each d), 67.8 (t); ESIHRMS:
m=z [M)H]ꢂ: calcd 394.0096, found 394.0102.
3.13. 1-Deoxy-1-[(3-methyl-1-oxo-2-butenyl)amino]-b-D-
glucopyranuronic acid (11)
The protected precursor (273 mg, 33%) was prepared
from azide 44 and a portion (100 mg, 0.24 mmol) de-
protected to give 11 as an off-white solid (62 mg, 93%);
3.17. 1-Deoxy-1-[(1H-indol-2-ylcarbonyl)amino]-b-D-
glucopyranuronic acid (16)
½aꢁ )45.6 (c 1.1, MeOH); IR (KBr) cmꢂ1 3423, 2922,
D
1627, 1419, 1266, 1683, 945, 655; 1H NMR (D2O,
300 MHz): d 5.82 (s, 1H), 5.05 (d, 1H, J 9.0, 1H), 4.03
(d, 1H, J 9.0), 3.68–3.42 (m, 3H), 1.96 (s, 6H); 13C NMR
(D2O, 125 MHz): d 178.5, 173.4 (each s, C@O), 158.6 (s,
C@CH), 119.4 (d, C@CH), 81.8, 80.6, 79.0, 74.4, 74.4
(each d), 29.2, 22.4 (each q); ESIHRMS: m=z [M+Na]þ:
calcd 298.0903, found 298.0903.
The protected precursor was prepared from amine 44
(385 mg, 54%) and a portion (100 mg, 0.21 mmol) de-
protected to give 16 as an off-white solid (5.6 mg, 10%).
IR (KBr) cmꢂ1 3421, 2936, 2359, 1975, 1623, 1559,
1
1410, 1224, 1072, 882; ½aꢁ +5.0 (c 0.026, MeOH); H
D
NMR (D2O, 300 MHz): d 7.73 (m, 1H), 7.55 (m, 1H),
7.35 (m, 1H), 7.26 (s, 1H), 5.20 (d, 1H, J 8.9), 3.83 (d,
1H, J 9.3), 3.60 (m, 3H); 13C NMR (D2O, 125 MHz): d
178.5, 167.2, 139.8, 132.6, 129.8 (each s), 128.0, 125.2,
123.6 (each d), 115.2 (s), 108.3, 82.3, 80.9, 79.1, 74.6,
74.5 (each d); ESIHRMS: m=z [M+Na]þ: calcd
359.0889, found 359.0855.
3.14. 1-Deoxy-1-[(4-pyridinylcarbonyl)amino]-b-D-gluco-
pyranuronic acid (12)
The protected precursor was prepared from amine 44
(310 mg, 52%) and a portion (268 mg, 0.61 mmol) de-

1882
N. Pitt et al. / Carbohydrate Research 339 (2004) 1873–1887
3.18. 1-Deoxy-1-[[3-(trifluoromethyl)benzoyl]amino]-b-
glucopyranuronic acid (17)
D
-
NMR (D2O, 270 MHz): d 7.79–7.15 (m, 3H), 5.15 (d, 1H,
J 8.4), 3.82 (d, 1H, J 9.0), 3.56 (m, 3H); 13C NMR (D2O,
125 MHz): d 178.4, 168.0, 139.1 (each s), 135.4, 133.5,
131.0 (each d), 82.4 (d), 80.9, 78.9, 74.5, 74.4 (each d);
ESIHRMS: m=z [M+H]þ: calcd 304.0491, found
304.0529.
The protected precursor was prepared from amine 44
(115 mg, 38%) and a portion (60 mg, 0.12 mmol) de-
protected to give 17 as an off-white solid (30 mg, 83%);
½aꢁ )12.3 (c 0.39, water); IR (KBr) cmꢂ1 3403, 2903,
D
1723, 1661, 1551, 1330, 1128, 1074; 1H NMR (D2O,
270 MHz): d 8.13–7.55 (m, 4H), 5.19 (d, 1H, J 8.4), 3.83
(d, 1H, J 9.2), 3.53 (m, 3H); 13C NMR (D2O, 125 MHz):
d 173.0, 170.5, 133.8, 131.4 (each s), 129.9, 129.5, 124.8,
124.7, 80.1, 76.6, 76.4, 71.7, 71.5 (each d); ESIHRMS:
m=z [M+Na]þ: calcd 388.0620, found 388.1421.
3.22. 1-Deoxy-1-[(3,5-dimethylbenzoyl)amino]-b-D-gluco-
pyranuronic acid (21)
The precursor was prepared from azide 44 (560 mg, 56%)
and a portion (276 mg, 0.59 mmol) deprotected to give 21
as an off-white solid (162 mg, 85%); ½aꢁ )10.5 (c 0.2,
D
water); IR (KBr) cmꢂ1 3431 (OH), 2940, 1723, 1661,
1535, 1312, 1245; 1H NMR (D2O, 270 MHz): d 7.41–7.21
(m, 3H), 5.13 (d, 1H, J 8.6), 3.81 (d, 1H, J 9.1), 3.52 (m,
3H), 1.86 (s, 6H); 13C NMR (D2O, 125 MHz): d 178.5,
174.5 (each s), 142.0, 136.4, 135.5, 128.9, 82.5 (d, C-1),
80.6, 79.0, 74.4 and 74.2 (each d), 33.3 (q); ESIHRMS:
m=z [M+Na]þ: calcd 326.1240, found 326.1359.
3.19. 1-Deoxy-1-[[3-(2-furanyl)-1-oxo-2-propenyl]amino]-
b-D-glucopyranuronic acid (18)
The protected precursor (82 mg, 40%) was prepared
from azide 44 and a portion (150 mg, 0.33 mmol) de-
protected to give 18 as an off-white solid (73 mg, 71%);
½aꢁ )7.5 (c 6.9, MeOH); IR (KBr) cmꢂ1 3422, 2925,
D
1
1618, 1566, 1419, 1283, 1073, 883, 749; H NMR (D2O,
300 MHz): d 7.88 (br s, 1H), 7.68 (d, 1H, J 15.0), 7.25 (d,
1H, J 3.0), 6.83 (t, 1H, J 2.0), 6.70 (d, 1H, J 15.0), 5.28
3.23. 1-[([1,10-Biphenyl]-4-ylacetyl)amino]-1-deoxy-b-
D-
glucopyranuronic acid (22)
(d, 1H, J 9.0), 4.01 (d, 1H, J 9.0), 3.86–3.65 (m, 3H); 13
C
The protected precursor (0.59 g, 32%) was prepared from
azide 44 and a portion (52 mg, 0.1 mmol) deprotected to
NMR (D2O, 125 MHz): d 178.5, 173.4, 172.3, 153.3,
152.8 (each s), 148.2, 147.3, 132.7, 129.0, 119.8, 119.1,
118.5, 118.0, 115.4, 115.0, 82.1, 81.9, 80.7, 79.0, 74.6,
74.5, 74.0 (each d); ESIHRMS: m=z [M+H]þ: calcd
314.0876, found 314.0867.
give 22 as an off-white solid (35 mg, 91%); ½aꢁ )76.3 (c
D
0.08, MeOH); IR (KBr) cmꢂ1 3365, 2912, 1849, 1658,
1
1449, 1603, 1449, 1024; H NMR (D2O, 300 MHz): d
7.71–7.40 (m, 9H), 5.0 (d, 1H, J 9.0), 3.85 (d, 1H, J 9.0),
3.73 (s, 2H), 3.73–3.51 (m, 2H), 3.44 (t, 1H, J 9.0); 13C
NMR (D2O, 125 MHz): d 178.2, 177.2, 143.0, 142.3,
136.6 (each s), 132.6, 131.9, 130.4, 130.0, 129.6, 82.2,
80.0, 79.1, 74.5, 74.4 (each d), 44.7 (t); ESIHRMS: m=z
[M+Na]þ: calcd 410.1196, found 410.1216.
3.20. 1-Deoxy-1-[[3-(1H-indol-3-yl)-1-oxopropyl]amino]-
b-D-glucopyranuronic acid (19)
The protected precursor (286 mg, 37%) was prepared
from amine 45 and a portion (50 mg, 0.1 mmol) depro-
tected to give 19 as an off-white solid (29 mg, 81%); ½aꢁ
3.24. 1-Deoxy-1-[(2-naphthalenylcarbonyl)amino]-b-D-
glucopyranuronic acid (23)
D
)10.26 (c 1.49, water); IR (KBr) cmꢂ1 3420, 2920, 2358,
1578, 1417, 1076, 893, 512; 1H NMR (D2O, 300 MHz): d
7.74 (d, 1H, J 7.3), 7.55 (d, 1H, J 9.2), 7.48–7.06 (m,
3H), 5.0 (d, 1H, J 9.0), 3.71 (d, 1H, J 9.0), 3.68–3.49 (m,
2H), 3.43 (t, 1H, J 9.0), 3.15 (t, 2H, J 7.3), 2.75 (t, 2H, J
7.5); 13C NMR (D2O, 125 MHz): d 181.7, 177.8, 136.4,
126.8 (each s), 123.3, 122.1, 119.3, 118.8, 113.5 (each d),
112.2 (s), 79.3, 78.1, 76.5, 72.0, 71.9 (each d), 36.9, 20.8
(each t); ESIHRMS: m=z [M+Na]þ: calcd 387.1168,
found 387.1188.
The protected precursor (208 mg, 59%) was prepared
from amine 44 and a portion (60 mg, 0.12 mmol) de-
protected to give 23 as an off-white solid (31 mg, 76%);
½aꢁ 47.5 (c 0.18, water); IR (KBr) cmꢂ1 3447, 1658,
D
1
1596, 1422, 1077, 1024; H NMR (D2O, 300 MHz): d
8.37 (s, 1H), 7.97 (m, 3H), 7.81 (m, 1H), 7.60 (m, 2H),
5.20 (d, 1H, J 8.8), 3.83 (d, 1H, J 9.5), 3.54 (m, 3H); 13
C
NMR (D2O, 75 MHz): d 178.0, 174.5, 137.7, 135.0,
133.0, 131.9, 131.4, 131.4, 131.3, 130.6, 130.1, 126.5,
82.7, 80.6, 79.1, 74.5 (2s); ESIHRMS: m=z [M+H]þ:
calcd 348.1083, found 348.1063.
3.21. 1-Deoxy-1-[(2-thienylcarbonyl)amino]-b-D-glucopy-
ranuronic acid (20)
The protected precursor was prepared from azide 44
(260 mg, 53%) and a portion (159 mg, 0.36 mmol) de-
protected to give 20 as an off-white solid (90 mg, 83%);
3.25. 1-[(1,4-Dioxo-4-phenylbutyl)amino]1-deoxy-b-D-
glucopyranuronic acid (24)
½aꢁ )21.1 (c 0.21, water); IR (KBr) cmꢂ1 3400, 2928,
The protected precursor (200 mg, 35%) was prepared
from amine 44 and a portion (60 mg, 0.12 mmol) depro-
D
1913, 1729, 1655, 1614, 1545, 1419, 1296, 1089, 1024; 1H

N. Pitt et al. / Carbohydrate Research 339 (2004) 1873–1887
1883
tected to give 24 as an off-white solid (25 mg, 76%); ½aꢁ
124.6, 120.2, 118.0 (each s), 82.5, 79.3, 79.0, 74.6, 74.0
(each d), 13.8 (q); ESIHRMS: m=z [M)H]ꢂ: calcd
454.1138, found 454.1116.
D
)18.0 (c 0.05, water); IR (KBr) cmꢂ1 3441, 2910, 2343,
1647, 1448, 1211, 1038, 595; 1H NMR (D2O, 300 MHz): d
8.06 (d, 2H), 7.75 (t, 2H, J 7.5), 7.62 (t, 2H, J 7.5), 5.06 (d,
1H, J 9.0), 3.89 (d, J 9.0), 3.68 (m, 4H), 2.81 (t, 2H, J 6.7);
13C NMR (D2O, 125 MHz): d 203.5, 176.7, 175.9, 136.3
(each s), 134.3, 129.6, 129.1, 128.4, 128.4, 79.4, 78.0, 76.5,
72.0, 72.0 (each d), 33.8, 30.0 (each t, CH2); ESIHRMS:
m=z [M+Na]þ calcd 376.1008, found 376.0999.
3.29. 1-Deoxy-1-[(3,4,5-trimethoxybenzoyl)amino]-b-D-
glucopyranuronic acid (29)
The protected precursor (620 mg, 96%) was prepared
from azide 44 and a portion of this material (264 mg,
0.50 mmol) deprotected to give 29 as an off-white solid
(118 mg, 62%); ½aꢁ )48.8 (c 0.29, water); IR (KBr) cmꢂ1
3.26. 1-Deoxy-1-[(3,4-difluorobenzoyl)amino]-b-
pyranuronic acid (25)
D
-gluco-
D
3409, 1524, 1585, 1416, 1238, 1128, 1077; 1H NMR
(D2O, 270 MHz): d 7.03 (s, 2H), 5.17 (d, 1H, J 8.6), 3.80
(d, 1H, J 10.4), 3.56 (m, 3H), 3.29 (s, 9H); 13C NMR
(D2O, 125 MHz): d 175.9, 170.6 (each s, each C@O),
140.5, 129.1 (each s), 106.9, 105.8, 80.1, 78.2, 76.6, 72.1,
71.9 (each d), 61.2, 56.5 (each q); ESIHRMS: m=z
[M+Na]þ calcd 410.1063, found 410.1149.
The protected precursor (323 mg, 62%) was prepared
from amine 44 and portion (162 mg, 0.34 mmol) depro-
tected to give 25 as an off-white solid (99 mg, 87%); ½aꢁ
D
)17.7 (c 0.62, MeOH); IR (KBr) cmꢂ1 3412, 2940, 1759,
1662, 1597, 1512, 1431, 1378, 1228, 1074; 1H NMR
(D2O, 270 MHz): d 7.76 (m, 1H), 7.65 (m, 1H), 7.34 (m,
1H), 5.16 (d, 1H, J 8.0), 3.82 (d, 1H, J 9.3), 3.56 (m, 3H);
13C NMR (D2O, 300 MHz): d 177.6, 172.3, 156.6, 153.6,
132.9, 127.6, 120.7, 120.1, 82.1, 80.4, 79.1, 74.4, 74.4;
ESIHRMS: m=z [M)H]ꢂ: calcd 332.0582, found
332.0576.
3.30. N-(b-D-Glucopyranosyl)-2-thiophenecarboxamide
(31)
Thiophene-2-carboxylic acid (0.07 g, 0.58 mmol), DCC
(0.14 g, 0.7 mmol) and DMAP (catalytic) were sus-
pended in anhyd CH2Cl2 (20 mL) and the reaction
mixture was allowed to stir at rt for 1 h. 2,3,4,6-Tetra-O-
3.27. 1-[[(4-Chlorophenyl)acetyl]amino]-1-deoxy-b-D-
glucopyranuronic acid (27)
acetyl-b-D
-glucopyranosylamine31 (0.2 g, 0.58 mmol)
obtained from azide 4532 was then added and the
reaction mixture was allowed to stir at rt; TLC analysis
(EtOAc) showed that the reaction was complete after
24 h. The solvent was removed and the residue purified
by chromatography (1:1 EtOAc–petroleum ether) to
give the protected precursor as a white solid (0.1 g,
41%); ESIHRMS: m=z [M+H]þ: calcd 458.1119, found
458.1121. This amide (0.06 g, 0.13 mmol) was suspended
in MeOH (5 mL) and NaOMe (0.1 mL of a 0.25 M soln)
was added followed by another 0.1 mL portion of the
NaOMe soln after 1.5 h. After 2 h Amberlite was added,
the reaction mixture was filtered and the solvent re-
moved to give 31 as white solid (20 mg, 50%), which was
The protected precursor (99 mg, 51%) was prepared
from azide 43 and a portion (92 mg, 0.19 mmol) depro-
tected to give 27 as an off-white solid (53 mg, 82%); ½aꢁ
D
)19.6 (c 0.12, MeOH); IR (KBr) cmꢂ1 3141, 1650, 1567,
1494, 1418, 1302, 736; 1H NMR (D2O, 300 MHz): d 7.54
(d, 2H, J 8.5), 7.25 (d, 2H, J 8.5), 5.18 (d, 1H, J 9.0),
3.99 (d, 1H, J 9.0), 3.91 (br s, 2H, CH2), 3.78 (m, 2H),
3.68 (t, 1H, J 9.0); 13C NMR (D2O, 125 MHz): d 175.8,
174.7 (each s, C@O), 134.0, 132.7 (each s), 131.5, 129.0,
79.7, 77.9, 76.7, 72.1, 72.1 (each d), 41.9 (t); ESIHRMS:
m=z [M+Na]þ: calcd 346.0694, found 346.0700.
1
purified by HPLC. H NMR (D2O, 300 MHz): d 7.89
3.28. 1-Deoxy-1-[[(3-methyl-4-oxo-2-phenyl-4H-1-benzo-
pyran-8-yl)carbonyl]amino]-b-D-glucopyranuronic acid
(28)
(dd, 1H, J 1.1, J 3.9), 7.86 (dd, 1H, J 1.2, J 5.1), 7.29
(dd, 1H, J 3.8, J 5.1), 5.24 (d, 1H, J 8.5), 3.97 (dd, 1H, J
2.27, J 12.4), 3.83 (dd, 1H, J 5.0, J 12.4), 3.52–3.71 (m,
4H); 13C NMR (D2O, 125 MHz): d 168.2, 139.2 (each s),
135.6, 133.7, 131.2, 82.8, 80.5, 79.4, 74.5, 72.1 (each d),
63.4 (t); FABHRMS m=z [M+Na]þ: calcd 318.0518,
found 318.0519.
The protected precursor (466 mg, 49%) was prepared
from amine 44 and a portion (80 mg, 0.13 mmol) de-
protected to give 28 as an off-white solid (47 mg, 84%);
½aꢁ )7.6 (c 0.05, MeOH); IR (KBr) cmꢂ1 3435, 2921,
D
1
2490, 1727, 1619, 1443, 1216, 1033, 836, 720; H NMR
3.31. 1-Azido-2,3-di-O-acetyl-1,4-dideoxy-a-
hex-4-enopyranuronic acid, methyl ester (46)
L
-threo-
(D2O, 300 MHz): d 8.24 (d, 1H, J 6.5), 8.07 (d, 1H),
7.82–7.56 (m, 2H), 7.56–7.51 (m, 4H), 5.16 (d, 1H, J
9.0), 3.79 (d, 1H, J 9.5), 3.81–3.47 (m, 2H), 3.36 (t, 1H, J
9.0), 2.09 (s, 3H); 13C NMR (D2O, 125 MHz): d 183.1,
170.5, 165.3, 155.5 (each s), 137.2 (d), 134.8 (s), 133.9,
131.9, 131.8, 131.4, 131.3, 131.3, 128.5 (each d), 126.8,
Azide 43 (2.00 g, 5.5 mmol) was dissolved in CH2Cl2
(20 mL), cooled to 0 ꢁC and DBU (0.9 mL, 6.1 mmol)
added. The reaction mixture was stirred for 40 min at
0 ꢁC and then applied directly to a silica-gel column,

1884
N. Pitt et al. / Carbohydrate Research 339 (2004) 1873–1887
which was eluted with an 1:3 EtOAc–petroleum ether
and gave 46 as a colourless oil (1.10 g, 67%); ½aꢁ )69.3
5.73 (d, 1H, J 3.1), 5.69 (d, 1H, J 8.2), 5.62 (d, 1H, J
1.3), 4.41 (m, 1H), 4.15 (m, 1H), 3.90 (m, 1H), 3.84 (m,
1H), 2.31 (s, 6H); ESIHRMS: m=z [M)H]ꢂ: calcd
278.0665, found 278.0666.
D
1
(c 2.1, CH2Cl2); H NMR (CDCl3, 300 MHz): d 6.24
(m, 1H), 5.58 (m, 1H), 5.26 (m, 1H), 5.04 (m, 1H), 3.86
(s, 1H), 2.12 and 2.11 (2·s, each 3H); 13C NMR
(CDCl3, 75 MHz): d 170.1, 169.5, 161.7, 143.0 (each s),
107.7, 85.3, 68.2 and 64.3 (each d), 53.0, 21.0 and 20.9
(each q); CIHRMS: m=z [M+NH4]þ: calcd 317.1097,
found 317.1098.
3.35. N-(b- -Glucopyranosyl)-N-methyl-2-thiophene-
D
carboxamide (34)
To a soln of methylamine in MeOH (5.4 mL, 11.2 mmol)
was added D-glucose (2.0 g, 11.2 mmol). The reaction
mixture was heated to 60–65 ꢁC for 30 min and then
stirred at rt for 2 h. The solvent was removed under
diminished pressure and the residue recrystallized from
3.32. 2,3-Di-O-acetyl-1,4-dideoxy-1-[2-thienylcarbonyl-
amino]-a- -threo-hex-4-enopyranuronic acid, methyl
L
ester (47)
EtOH to yield N-methyl-b-D-glucopyranosylamine (off-
Triphenylphosphine (0.37 g, 1.4 mmol) was added to a
soln of 46 (0.34 g, 1.1 mmol) and 2-thiophenoyl chloride
(0.32 g, 2.2 mmol) in anhyd MeCN (12 mL) and stirred
at rt for 16 h under a nitrogen atmosphere. The reaction
was diluted with CH2Cl2 (ꢀ10 mL) and washed with
satd NaHCO3 soln (2 · 25 mL) and water (2 · 25 mL).
The organic layer was dried (MgSO4) and the soln was
concentrated under diminished pressure. The residue
was purified by chromatography (1:3 EtOAc–petroleum
white solid). Sodium carbonate (0.42 g, 4.03 mmol) was
added to a soln of the amine (0.30 g, 1.55 mmol) in
MeOH (15 mL) and this was cooled to 0 ꢁC and thio-
phene-2-carbonyl chloride (0.45 mL, 3.10 mmol) was
added dropwise over 5 min. The ice bath was then re-
moved and the reaction mixture was allowed to stir at rt
for 30 min. The solvent was removed and the residue
purified by chromatography (1:4 MeOH–EtOAc) to give
the title compound as a white solid (0.29 g, 63%); ½aꢁ
D
ether) to give 47 as a clear oil (0.25 g, 59%); ½aꢁ +21.1 (c
1
D
)0.78 (c 0.9, MeOH); H NMR (D2O, 300 MHz; Z/E,
0.33, CHCl3); IR (film) cmꢂ1 3395, 2924, 1742, 1660,
33:66): d 7.62, 7.44, 7.07 (m, 6H), 5.40 (br s, 1H, H-1, Z-
isomer), 5.01 (br s, 1H, H-1, E-isomer), 3.83–3.00 (m,
18H); 13C NMR (D2O): d 171.6 (2·s, C@O, E- and Z-
isomers), 137.5 (s), 133.8, 130.5 130.4 (each d), 90.4 (d,
C-1, E-isomer), 86.0 (d, C-1, Z-isomer), 80.3, 79.9, 79.0,
78.9, 78.8, 72.2, 71.8, 71.4 (each d), 63.0 (t), 34.0 (q, Z-
isomer), 31.3 (q, E-isomer); ESIHRMS: m=z [M)H]ꢂ:
calcd 302.0698, found 302.0690.
1
1543, 1239 and 1112; H NMR (CDCl3, 300 MHz): d
7.55 (m, 2H), 7.25 (m, 1H), 7.10 (m, 1H), 6.24 (m, 2H),
5.60 and 5.20 (each m, each 1H), 3.80 (s, 3H), 2.20 and
2.40 (each s, each 3H); 13C NMR (CDCl3, 125 MHz): d
171.0, 169.0, 162.0 and 161.0 (each s), 144.5 (s), 137.5
(s), 132.0, 129.9, 127.8, 106.0, 79.0, 69.0 (each d), 53.0
(q), 21.0 (2s) (q); CIHRMS: m=z [M+NH4]þ: calcd
401.1019, found 401.1022.
3.36. N-(b-D-Glucopyranosyl)-pyridine-4-carboxamide
(35)
3.33. 1,4-Dideoxy-1-[2-thienylcarbonylamino]-a/b-
threo-hex-4-enopyranuronic acid (32)
L-
Isonicotinic acid (0.13 g, 1.09 mmol), EDC (0.21 g,
1.09 mmol) and DMAP (0.13 g, 1.09 mmol) were sus-
pended in anhyd THF (10 mL) and the reaction mixture
Ester 47 (41.0 mg, 0.11 mmol) was treated with 0.05 M
LiOH as described above to afford the title compound
32 as an off-white solid [24.5 mg, 79%, a (64%) and b
was stirred at 0 ꢁC for 5 min. 2,3,4,6-Tetra-O-acetyl-b-
D-
1
(36%) isomeric mixture]; ½aꢁ )39.3 (c 0.08, MeOH); H
glucopyranosylamine33 (0.38 g, 1.09 mmol) was then
added and the reaction mixture stirred at rt. TLC
analysis (EtOAc) showed that the reaction was complete
after 12 h. The soln was diluted with EtOAc (20 mL) and
washed with water (2 · 20 mL), brine and dried (sodium
sulfate). The solvent was removed and the residue
purified by chromatography (1:1 EtOAc–petroleum
ether) to give the target compound as a white solid
(0.21 g, 43%). This intermediate (70 mg, 0.15 mmol) was
suspended in MeOH (5 mL) and NaOMe (0.1 mL of a
0.25 M soln) was added. Analysis by TLC (1:4 MeOH–
EtOAc) showed that the reaction was complete after 2 h.
The mixture was then filtered and the solvent removed
to give 35 as a clear oil (61%). CIHRMS: m=z [M+Na]þ:
calcd 307.0906, found 307.0898.
D
NMR (CD3OD, 300 MHz): d 7.87, 7.75 and 7.17 (m,
6H), 6.11 (d, 1H, J 4.8), 6.02 (d, 1H, J 3.4), 5.78 (m,
2H), 4.32 (m, 1H), 4.10 (dd, 1H, J 4.9, J 2.5), 3.84 (m,
1H), 3.80 (m, 1H); 13C NMR (CD3OD, 125 MHz): d
164.2, 139.5, 132.9, 132.8, 130.6, 129.1, 128.9, 111.7,
108.6, 80.6, 77.3, 71.1, 70.9, 69.5, 66.7; ESIHRMS: m=z
[M+Na]þ: calcd 308.0205, found 308.0294.
3.34. 1-[Benzoylamino]-1,4-dideoxy-a/b- -threo-hex-4-
L
enopyranuronic acid (33)
Reaction of benzoyl chloride with azide 46 and depro-
1
tection as described above gave 33; H NMR (CDCl3,
300 MHz): d 7.45, 7.43 and 7.30 (3s, 5H), 5.86 (m, 1H),

N. Pitt et al. / Carbohydrate Research 339 (2004) 1873–1887
1885
3.37. N-(b-
D
-Glucopyranosyl)benzamide (36)
of a 1.0 M soln in CH2Cl2) and DMAP (catalytic) were
then added and the reaction mixture was allowed to stir
at rt. TLC analysis (EtOAc) showed that the reaction
was complete after 19 h. Excess solvent was removed
and the residue extracted with CH2Cl2, washed with
NaHCO3 (3 · 50 mL) and water (3 · 50 mL), dried
(MgSO4), and the solvent removed. The residue was
purified by chromatography (EtOAc) to give the pro-
tected precursor as an off-white foam [0.03 g, 28%, Rf
0.58 (1:1 EtOAc–petroleum ether)]. This intermediate
was suspended in MeOH (10 mL), NaOMe (0.1 mL of a
0.25 M soln) was added and the mixture stirred at rt.
After 40 min another 0.1 mL of NaOMe was added. To
this soln was then added LiOH (1.6 mL of a 0.1 M soln
in THF–water–MeOH). Analysis by TLC showed that
the reaction was complete after a total of 3.5 h. The
mixture was diluted with water (5 mL) and neutralized
with Amberlite (Hþ), filtered and the solvent removed to
give the title compound as an orange oil (80 mg, quan-
titative); IR (film) cmꢂ1 3176, 3065, 2930, 2332, 1755,
1665, 1439, 1374, 1260, 1225, 1029, 732; 1H NMR (D2O,
300 MHz): d 7.55–8.04 (m, 4H), 5.36 (br s, 1H), 3.60–
4.31 (m, 4H), 3.50 (t, 2H, J 6.3), 1.49–1.78 (m, 4H); 13C
NMR (D2O): d 172.9, 172.3, 137.1, 135.7 (each s), 133.7,
133.3, 132.0, 128.9, 82.7, 79.0, 74.3, 74.1, 65.4 (each d),
42.2, 30.5, 25.6 (each t).
The title compound (0.04 g, 100%) was obtained when
benzoyl chloride was reacted with 2,3,4,6-tetra-O-acetyl-
b-D
-glucopyranosyl azide34 and PPh3 and subsequent
removal of the protecting groups by reaction with cat-
alytic NaOMe in MeOH as described above for related
compounds; mp 218–220 ꢁC [lit.36 mp 230–232 ꢁC]; Rf
0.18 (1:4 MeOH–EtOAc); ½aꢁ )45.0 (c 0.04, water)
D
[lit.35 ½aꢁ )11.6 (c 0.6, water)]; FABHRMS: m=z
[M+Na]þD: calcd 306.0952, found 306.0954.
3.38. 1,4-Dideoxy-1-[(3,5-dimethyl)benzoylamino]-a/b-
threo-hex-4-enopyranuronic acid (37)
L-
The protected precursor (50 mg, 0.1 mmol), prepared as
described for 47, was reacted with 0.05 M LiOH as de-
scribed above to give 37 as an off-white solid (10 mg,
33%; mixture of a (66%) and b (33%) anomers); 1H
NMR (CDCl3, 300 MHz): d 7.45, 7.43 and 7.30 (3s, 3H),
5.86 (m, 1H), 5.73 (d, 1H, J 3.1), 5.69 (d, 1H, J 8.2), 5.62
(d, 1H, J 1.3), 4.41 (m, 1H), 4.15 (m, 1H), 3.90 (m, 1H),
3.84 (m, 1H), 2.31 (s, 6H) ; ESIHRMS: m=z [M)H]ꢂ:
calcd 306.0978, found 306.0990.
3.39. N-(Methyl 2,3,4-tri-O-acetyl-b-D-glucopyranuro-
nosyl)amide-isophthalamic acid (48)
3.41. 3,6-Dioxaoctanedioic acid bis-N-(b-D-glucopyra-
nuronosyl)-amide (39)
To a vigorously stirred soln of amine 44 (0.5 g,
1.5 mmol) in DMF (10 mL) at rt, was added isophthalic
acid (0.5 g, 3.0 mmol), HOBt (0.2 g, 1.5 mmol), and EDC
(0.3 g, 1.5 mmol) successively. Triethylamine (8 drops)
was added and the mixture stirred and TLC analysis
(EtOAc) showed the reaction was complete after 24 h.
Excess solvent was removed and the residue was washed
with water (3 · 50 mL), extracted with CH2Cl2
(3 · 50 mL), dried (MgSO4), filtered and the solvent re-
moved. Chromatography (1:2 EtOAc–petroleum ether)
The protected precursor (0.1 g, 0.12 mmol) was prepared
from amine 44 and then suspended in LiOH soln
(2.87 mL, 1.44 mmol of a 0.1 M soln in 2.5:1.0:0.5
MeOH–water–THF) and the mixture was stirred at rt
and TLC analysis (EtOAc) showed that the reaction to
be complete after 1 h. The mixture was diluted with
water (10 mL), neutralized with Amberlite (Hþ), filtered
and the solvent removed. The residue was purified by
chromatography (MeOH) to yield the title compound 39
gave 48 as an off-white solid (0.29 g, 41%); ½aꢁ +40.0 (c
D
0.04, CHCl3); IR (film) cmꢂ1 3070, 2988, 2331, 1757,
1675, 1439, 1376, 1265, 1223, 1039, 732; 1H NMR
(CDCl3, 300 MHz): d 7.98 (d, 1H, J 9.5), 7.33–7.75 (m),
5.59 (t, 1H, J 9.5), 5.42 (t, 1H, J 9.5), 5.25 (t, 1H, J 9.5),
5.13 (t, 1H, J 9.5), 4.25 (d, 1H, J 9.5), 3.64 (s, 3H), 2.01,
1.97, 1.96 (each s, each 3H); 13C NMR (CDCl3): d 171.5,
170.2, 169.8, 169.6, 167.6, 167.0 (each s, each C@O),
133.5, 130.2 (each s), 133.9, 133.1, 129.3, 129.0, 78.8,
74.2, 72.4, 70.9, 69.9 (each d), 53.1 (q), 20.9, 20.8, 20.7
(each q); ESIHRMS: m=z [M)H]: calcd 480.1142, found
480.1142.
as an off-white solid (0.03 g, 52%); ½aꢁ )14.2 (c 0.04,
D
water); IR (KBr) cmꢂ1 3433, 2924, 2853, 1691, 1606,
1
1420, 1275, 1072; H NMR (D2O, 300 MHz): d 5.12 (d,
2H, J1;2 8.6), 4.28 (s, 4H), 3.90 (d, 1H, J 8.9), 3.89 (s,
2H), 3.56–3.69 (m, 3H); 13C NMR (D2O): d 175.9, 174.0
(each s, each C@O), 79.0, 78.2, 76.5, 71.9 (2s) (each d),
70.6, 69.7 (each t); ESIHRMS: m=z [M)H]ꢂ: calcd
527.1361, found 527.1384.
3.42. Octanedioic acid bis-(b-D-glucopyranuronosyl)-
amide (40)
3.40. Preparation of 38
The protected precursor (0.1 g, 0.12 mmol) was prepared
from amine 44 and then suspended in LiOH soln
(2.87 mL, of a 0.1 M soln in 2.5:1.0:0.5 MeOH–water–
THF, 1.44 mmol) and the mixture stirred at rt; TLC
analysis (EtOAc) showed that the reaction was complete
1,5-Diaminopentane (0.1 mL, 0.11 mmol), HOBt (0.03 g,
0.21 mmol) and 48 (0.1 g, 0.21 mmol) were stirred in
anhyd THF (15 mL) at 0 ꢁC. DCC (0.21 mL, 0.21 mmol,

1886
N. Pitt et al. / Carbohydrate Research 339 (2004) 1873–1887
after 1.5 h. The mixture was then diluted with water
(10 mL), neutralized with Amberlite (Hþ), filtered and
the solvent removed. The residue was purified by chro-
matography (MeOH) to give 40 as a white solid (60 mg,
seeded at the appropriate density. The methylthiazol
tetrazolium (MTT) assay, adapted from that described
in the literature,36 was used to assess cell viability.
Confluent monolayers of BAEC, grown in 24 well tissue
culture plates were treated with test compounds (10 lg/
mL) at the indicated concentration for 24 h at 37 ꢁC.
Following aspiration and washing with PBS, each well
was incubated with MTT (0.45 mg/mL) in RPMI 1640
for 3 h at 37 ꢁC. The overlying soln was then aspirated
and the cells solubilized by the addition of 1 mL Me2SO.
Absorbance was measured at 590 nm and viability ex-
pressed as percentage of control (untreated) wells. Sta-
tistical significance of differences between group means
was determined by ANOVA followed by a post-ANO-
VA Dunnett’s test.
92%); ½aꢁ )78.8 (c 0.04, water); IR (KBr) cmꢂ1 3428,
D
3174, 2926, 1619, 1439, 1070, 1022; Rf 0.39 (EtOAc); 1H
NMR (D2O, 300 MHz): d 5.04 (d, 1H, J 9.1), 3.86 (d,
1H, J 9.4), 3.43–3.67 (m, 3H), 2.40 (t, 2H, J 7.4), 1.70 (t,
2H, J 7.0), 1.20 (br s, 2H); 13C NMR (D2O): d 181.5 (s,
CO2H), 178.8, 176.0 (each s), 96.1, 92.4, 79.3, 76.5, 71.9
(2s) (each d), 35.9, 27.9, 25.0 (each t); ESIHRMS: m=z
[M)H]ꢂ: calcd 523.1775, found 523.1782.
3.43. Synthesis of heparin-albumin
Heparin [456 mg, 37.5 lmol; Fluka (cat. no. 51536)] and
BSA [17 mg, 0.25 lmol; Fluka (cat. no. 05470)] were
dissolved in 2.5 mL of 0.2 M potassium phosphate
buffer, pH 8.0. Sodium cyanoborohydride (12.5 mg,
198.9 lmol) was then added and the mixture was incu-
bated for 2 days at 37 ꢁC. The mixture was dialyzed at rt
against three changes of deionized water and freeze-
dried to yield the heparin-albumin complex as a white
solid (91 mg).
3.46. Epithelial cell assay
NmuMG cells were maintained in Dulbeccos modified
Eagle medium (DMEM) supplemented with 10% foetal
bovine serum, 4.5 g/L glucose and 10 lg/mL insulin.
Cells were grown to confluency in 75 cm2 tissue culture
flasks and maintained at 37 ꢁC in a humidified atmo-
sphere containing 95% O2 and 5% CO2. Subcultures
were created by passaging using a trypsin/EDTA
(0.125%/0.05%) mixture in phosphate buffered saline
(PBS), harvested by centrifugation (5 min at 1000 rpm)
and seeded at the appropriate density. The methyl-
thiazol tetrazolium (MTT) assay, was carried out as
described above except that after incubation with MTT,
cells were solubilized by the addition of 2.5 mL Me2SO.
Absorbance was measured at 590 nm and viability ex-
pressed as percentage of control (untreated) wells. Sta-
tistical significance of differences between group means
was determined by ANOVA followed by a post-ANO-
VA Dunnett’s test.
3.44. FGF-2 binding assay
A stock soln of heparin-albumin (5.0 mg/mL) was made
up in distilled water and diluted to a final working
concentration in a buffer containing 0.1 M Na2CO3 and
0.1 M NaHCO3 and coated onto 96-well assay
plates. Novel compounds, heparin-albumin and FGF-2
(100 ng/mL) were added to the wells in a 100 lL volume
of distilled water and incubated for 4 h at 37 ꢁC. Wells
were then washed sequentially with PBS/0.05% T20 to
remove any unbound protein and blot dried after each
wash. Goat polyclonal IgG antibody was added 100 lL/
well and incubated overnight at 37 ꢁC. Wells were wa-
shed as before. The amount of bound protein retained in
the wells was determined by ELISA using an alkaline
phosphatase-conjugated rabbit anti-goat IgG heavy and
light chain antibody. The ELISA absorbance readings
were read at 405 nm. Results were analyzed using a
nonlinear curve fitting programme (GraphPad PRISM).
Acknowledgements
The authors are grateful to Enterprise Ireland for Basic
Research Awards (SC/97/547 and SC/00/182), Health
Research Board (Irl.)/Wellcome Trust for an equipment
grant, BioResearch Ireland, IRCSET for a scholarship
to R.M.D., Science Foundation Ireland for a Pro-
gramme Investigator Award (P.V.M.) and Professor
Paul Cahill, Dublin City University (Ireland) for the gift
of BAEC cultures.
3.45. Endothelial cell assay
BAEC were maintained in RPMI 1640 medium sup-
plemented with 10% heat inactivated FCS, 25 mM glu-
tamine, 75 U/mL penicillin and 75 lg/mL streptomycin.
Cells were grown to confluency in 75 cm2 tissue culture
flasks and maintained at 37 ꢁC in a humidified atmo-
sphere containing 95% O2 and 5% CO2. Subcultures
were created by passaging using a trypsin/EDTA
(0.125%/0.05%) mixture in phosphate buffered saline
(PBS), harvested by centrifugation (4 min at 210g) and
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