Synthesis of simple heparanase substrates

Article abstract of DOI:10.1039/c1ob05165b

Heparanase degrades heparan sulfate (HS) chains on proteoglycans; elevated levels of heparanase expression correlate with tumour cell metastatic potential and vascularity, and reduced post-operative survival of cancer patients. Consequently, heparanase ex

Full text of DOI:10.1039/c1ob05165b

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Organic &
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PAPER
Synthesis of simple heparanase substrates
Andrew G. Pearson,†a Milton J. Kiefel,a Vito Ferro‡b and Mark von Itzstein*a
Received 30th January 2011, Accepted 25th February 2011
DOI: 10.1039/c1ob05165b
Heparanase degrades heparan sulfate (HS) chains on proteoglycans; elevated levels of heparanase
expression correlate with tumour cell metastatic potential and vascularity, and reduced post-operative
survival of cancer patients. Consequently, heparanase expression is considered a biomarker for cancer
detection. Although several heparanase assays have been developed, most require the preparation of
heterogeneous, (radio)labelled HS substrates and rely on the separation of enzymatically-degraded
products on the basis of molecular size. In studies directed towards the development of a more direct
heparanase assay, a series of glucuronides and glycosyl glucuronides were synthesised as putative
heparanase substrates. These compounds were designed with various aryl aglycones that could be
measured spectrophotometrically upon hydrolysis of the glycosidic linkage by heparanase. It was found
that the N-sulfated 4-nitrophenyl glycosyl glucuronide 24 and the N-sulfated methylumbelliferyl
glycosyl glucuronide 26 were hydrolysed by recombinant human heparanase. These compounds
represent the simplest substrates of heparanase reported to date.
Cleavage of HS chains is critical for the modulation of the
Introduction
biological function of HS-binding proteins, and profoundly affects
cell and tissue function involving migration and response to
changes in the extracellular matrix. It is also essential in the degra-
dation of the extracellular matrix by invading cells, particularly
metastatic tumour cells and leukocytes entering inflammatory
sites.3
Heparanase is an endo-b-D-glucuronidase that degrades HS
chains. Elevated levels of heparanase expression correlate with
metastatic potential, tumour vascularity, and reduced post-
operative survival of cancer patients.1b,3–4 Heparanase inhibitors
reduce the incidence of tumour metastases and therefore hep-
aranase is a potential target for anti-cancer drug development.3,4b,5
Heparanase hydrolyses the glucuronide linkage in HS, but only at
a few sites (e.g., as indicated in Fig. 1), yielding HS fragments of
10–20 disaccharide units. This suggests that heparanase recognises
a particular HS structure.3
Heparan sulfate (HS) proteoglycans are ubiquitous macro-
molecules located in the extracellular matrices and basement
membranes; they present a physical barrier to the movement of
cells (e.g., tumour cells and leukocytes) into tissues, and play
an important role in a variety of biological processes including
inflammation, metastasis and angiogenesis.1 HS proteoglycans
consist of a protein core, to which are attached several linear
HS chains that confer most of the biological properties. These
HS chains are sulfated linear polysaccharides of up to 400 sugar
residues, composed of D-glucuronic acid (GlcA) 1→4 linked to
D-glucosamine (GlcN), with various structural modifications, as
shown in Fig. 1.2
There have been several attempts to define the substrate
recognition properties of heparanases from various sources.6
The approaches taken have included structural comparison be-
tween polysaccharides susceptible or resistant to an enzyme,
sequence analysis of fragments generated by enzymatic cleavage,
studies of inhibitory effects of heparin derivatives, a series of
structurally-defined oligosaccharides isolated from heparin/HS,
and synthetic polysaccharides prepared using HS biosynthetic
enzymes. The majority of these studies allude to the impor-
tance of sulfate groups, but have otherwise failed to pro-
vide a unified picture. Of particular note are the studies of
Okada et al.6b using defined oligosaccharides and the more
recent investigations by Peterson and Liu6c using synthetic
polysaccharides.
Fig. 1 Schematic of heparan sulfate repeating disaccharide unit (left)
showing the heparanase cleavage site (right).
aInstitute for Glycomics, Griffith University, Gold Coast Campus, Queensland
4222, Australia. E-mail: m.vonitzstein@grifffith.edu.au
bDrug Design Group, Progen Pharmaceuticals Ltd, P.O. Box 2403, Toowong,
Brisbane 4066, Australia
† Current address: School of Medical Science, Griffith University, Gold
Coast Campus, Queensland 4222, Australia.
‡ Current address: School of Chemistry and Molecular Biosciences, The
University of Queensland, Brisbane, Queensland 4072, Australia.
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The lack of a convenient, functional assay hampered early
progress into the investigation of heparanase, although several
assays for heparanase activity have now been published7 or
are commercially available.8 Most assays rely on either labelled
substrates or separation of enzymatically-degraded substrates on
the basis of molecular size. HS fragments produced in these assays
may inhibit heparanase, complicating the assays. Additionally, it
is difficult to compare data from different assays. The use of a
homogeneous, low molecular weight substrate with a single enzy-
matic cleavage point greatly simplifies measurement of heparanase
activity, as recently demonstrated with the synthetic pentasac-
charide fondaparinux.9 However, whilst this assay represents a
significant improvement over previous assays, fondaparinux is not
an ideal substrate10 and the assay is not suitable for use in biological
matrices.
The primary objective of this investigation was to develop a
simple colourimetric or fluorometric assay for heparanase activity.
The general concept is shown in Fig. 2, wherein the reducing end
of the HS chain is replaced with a chromogenic or fluorogenic tag;
heparanase cleavage of glycosyl glucuronides 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 potential inhibitors and the correlation of
heparanase activity with tumour progression.
Fig. 3 Synthesis of the glycosyl glucuronide series of putative heparanase
substrates.
trichloroacetimidate donor 2 has the hydroxyl groups protected as
acetates and the GlcN nitrogen protected as a trifluoroacetamide.
Results and discussion
Synthesis
Our approach towards the key trichloroacetimidate donor 2
started with the known diol 3.12 Compound 3 was readily
acetylated to furnish 4 and subsequent azide reduction and tri-
fluoroacetylation provided the corresponding trifluoroacetamide
derivative 5 (Scheme 1). Cleavage of the benzyl ethers from 5
afforded 6, followed by acetylation to give 7. The anomeric acetate
in 7 was removed by treatment with benzylamine in THF to afford
the hemiacetal 8, which upon exposure to trichloroacetonitrile
and DBU in CH2Cl2 gave the desired key trichloroacetimidate
donor 2.
Fig. 2 Heparanase cleavage of the chromogenic or fluorogenic tag from
1 generates a measurable response.
In our approach towards the synthesis of compounds like 1, we
gave due consideration to selection of an appropriate protecting
group strategy. Typically in HS/heparin oligosaccharide synthesis,
hydroxyl groups that are to remain as free hydroxyl groups are
protected as benzyl ethers and O-sulfation is performed globally
with an excess of sulfating reagent. The benzyl ethers are then
removed and nitrogens, masked until that stage as azides, are
reduced to amines whereby they can be N-acetylated or N-
sulfated.11 In our approach towards compounds like 1, it was
necessary to remove the benzyl ethers prior to the introduction
of the aromatic aglycones, as the stability of aromatic aglycones
such as para-nitrophenyl under hydrogenolysis conditions was
questionable.
An advanced synthetic intermediate was preferred that could
furnish the desired target compounds with minimal synthetic steps
from the branch point of the synthesis. Thus a route was devised
for the synthesis of the desired glycosyl glucuronides that involved
the use of the advanced synthetic intermediate 2 (Fig. 3). The
Scheme 1 Reagents and conditions: a) Ac2O, DMAP, pyridine, quantita-
tive; b) 1. PPh3, THF, H2O; 2. (CF3CO)2O, pyridine, 81% over two steps;
c) H2, Pd/C, MeOH, AcOH, quantitative; d) Ac2O, DMAP, pyridine,
quantitative; e) BnNH2, THF, 87%; f) NCCCl3, DBU, CH2Cl2, 88%.
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Having developed an efficient method for the synthesis of the
trichloroacetimidate donor 2, it was possible to synthesise the
protected glycosyl glucuronides 9–12 (Scheme 2).
Thus compound 14 was de-esterified, N-acetylated to afford
15, and selectively 6-O-sulfated (DMF, 2 equivalents of SO3·Py
at room temperature for 16 h) to furnish 16. Deprotection of 14,
followed by N-sulfation afforded 17. Unfortunately, selective 6-O-
sulfation of 17 to afford 18 was not successful.
With appropriate conditions in hand for the deprotec-
tion, N-acetylation, and N-sulfation of the model compound
14, attention was turned towards the protected glycosyl
glucuronides. Compounds 9–12 were deprotected to furnish
amines 19–22, then either N-acetylated or N-sulfated to afford
compounds 23–30.
˚
Scheme 2 Reagents and conditions: BF3·OEt2, CH2Cl2, 4 A molecular
sieves, -15 ◦C to room temperature.
These four glycosyl glucuronides were synthesised in moderate
to good yield. The four compounds prepared were chosen because
the aglycones display a range of charge and spectroscopic proper-
ties. Interestingly, formation of the glycosyl glucuronosyl fluoride
13 was detected in the glycosidations of trichloroacetimidate 2
with 4-nitrophenol (25% yield of 13) and the cinnamyl ester (16%
yield of 13). It is believed that the formation of 13 occurred via a
similar mechanism to that previously reported by our group.13
Heparanase cloning and expression
The cloning strategy to produce recombinant human heparanase
followed the work of McKenzie et al.19 in which an insect
cell-baculovirus system was used to express the 8 and 50 kDa
heparanase subunits each as a fusion protein with a signal peptide
targeting for the excretion from the insect cell. In the work
of McKenzie et al., heparanase cDNA was amplified from a
mammary gland cDNA library using undisclosed primers. In
this work, repeated attempts to PCR amplify heparanase cDNA
from the same library proved unsuccessful. Therefore, a plasmid
incorporating the full length heparanase cDNA was used. Using
this plasmid, heparanase was expressed and purified according to
the literature.19
Deprotection, N-acetylation, N- and O-sulfation
Upon completion of the synthesis of glycosyl glucuronides 9–
12, their deprotection, N-acetylation/N-sulfation and O-sulfation
was investigated. Whilst there is literature precedence for the
chemoselective sulfation of a N-acetylglucosamine primary hy-
droxyl group (using either SO3·Py or SO3·NMe3) in the presence
of one or more secondary hydroxyl groups,14 selective primary
hydroxyl group mono-sulfation is difficult in the presence of
secondary hydroxyl groups, with both di- and trisulfation products
observed.15 Simultaneous N- and O-sulfation of oligosaccharides
has been reported; however, both primary and secondary hydroxyl
groups were O-sulfated.16 Successful O-sulfation in the presence
of one or more N-sulfates has been achieved by enzyme-catalysed
reactions.6c,17
To determine the optimal deprotection, N-acetylation/N-
sulfation and O-sulfation conditions on the glycosyl glucuronides,
the GlcN trifluoroacetamide derivative 1418 was utilised. The
readily accessible 14 was thought to be a good model compound
for this chemistry as it should behave in a similar manner to
compounds such as glycosyl glucuronides 9–12 under the reaction
conditions.
Heparanase assays
The synthesised compounds were evaluated for their ability to act
as heparanase substrates. Significant heparanase specific activity
was observed with 26 (48 nmol h-1 mg-1) and 24 (17 nmol h-1 mg-1).
The hydrolysis of the remaining glycosyl glucuronides was at
the lower limit of detection, indicating negligible hydrolysis by
heparanase. The N-sulfated glycosyl glucuronides 24 and 26 are
the smallest molecules reported to be hydrolysed by heparanase—
the smallest molecules previously reported to be heparanase
substrates were tetrasaccharides based on the structure shown in
Fig. 4.6b,20
In order to ensure that 26 was indeed being hydrolysed
by heparanase and not simply decomposing under the assay
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Fig. 4 HS tetrasaccharides previously reported to be the smallest
substrates hydrolysed by heparanase.6b,20
Conclusions
A series of glucuronides and glycosyl glucuronides were synthe-
sised and evaluated as heparanase substrates. Hydrolysis of the N-
sulfated 4-nitrophenyl glycosyl glucuronide 24 and the N-sulfated
4-methylumbelliferyl glycosyl glucuronide 26 by heparanase was
detected. These are the smallest molecules reported to be hydrol-
ysed by heparanase. Glycosyl glucuronide 26 should serve as a
useful template in the development of a rapid diagnostic test for
heparanase activity.
conditions, a solution of 26, HS, and BSA in sodium acetate buffer
at pH 5.0 was incubated for 3 days. Low resolution mass spectral
analysis of the assay mixture gave a peak at 592.0, consistent with
the parent ion of 26, (m/z): 592.0 [M - 2Na + H]-. There were
no peaks to indicate that the aglycone had been hydrolysed, or
that O-sulfation of 26 had occurred. This would indicate that the
compound was relatively stable at pH 5.0 for at least 3 days and
that no cross-sulfation from HS had occurred.
The recombinant human heparanase used in this study
was determined via an ultrafiltration assay21 to digest
170 nmol HS h-1 mg-1 at 37 ◦C. This compares favourably
with the heparanase activity observed with the N-sulfated
4-methylumbelliferyl glycosyl glucuronide 26, which was
48 nmol h-1 mg-1. Previously, heparanase purified from human
platelets was shown to have a specific activity towards HS of
7800 nmol h-1 mg-1 using the cHRG-Sepharose beads separation
method.22 Care should be taken in comparing specific activities
using different assays. Not only are the HS preparations in the two
assays from different sources, but the manner by which heparanase
activity is measured is different. In using a substrate such as 26
to determine heparanase activity, every heparanase catalytic event
is measured. It is expected that the products resulting from the
hydrolysis of 26 would not be able to bind the heparanase active
site. This is in contrast to assays using HS chains, as the heparanase
substrate may be hydrolysed several times, making it difficult
to calculate the number of catalytic events. For example, the
heparanase ultrafiltration assay uses 30 kDa HS as the substrate;
degradation products are typically 7–10 kDa.7d This would suggest
that each substrate HS chain is hydrolysed 3–4 times. Additionally,
the resulting shorter HS chains may bind the heparanase active
site, resulting in feedback inhibition.
The advantages of the assay used in this work include the
lack of radiolabelling, the ability to process multiple samples,
ease of handling, the assay measures one catalytic event per
molecule, and that hydrolysed products should have no affinity
for the active site of heparanase. The results presented herein
indicate that 4-methylumbelliferone is an adequate leaving group
for the measurement of heparanase activity. As O-sulfate groups
on the GlcN moiety have been shown to increase hydrolysis by
heparanase roughly two-fold,6b,6c it would be of great interest to
evaluate the hydrolysis of the glycosyl glucuronides 31 and 32.
It is expected that the O-sulfates should increase the affinity of
the substrate for the heparanase active site, leading to greater
turnover by heparanase. If this is the case, it would be possible
to measure heparanase activity with shorter incubation times,
allowing a rapid assay for heparanase activity. Strategies towards
the synthesis of compounds such as 31 and 32 are currently being
pursued.
Experimental
General
Reactions were monitored by TLC on aluminium plates coated
with Silica Gel 60 F254 (E. Merck) and visualised with H2SO4 (5%
in ethanol) with heating to 200 ◦C or ultraviolet light where appli-
cable. Compounds were purified by flash chromatography using
R
either E. Merck Silica Gel 60 (0.040–0.063 mm) or Sephadexꢀ
1
LH-20. H and 13C NMR spectra were recorded using a Bru¨ker
300 MHz spectrometer. Data acquisition and processing were
performed using XWINNMR software (version 3.1) running on
a Silicon Graphics O2 workstation. Data editing was performed
using XWINPLOT software. Chemical shifts are given in parts
per million (ppm) relative to the solvent used (CDCl3: 7.26 for
1H, 77.2 for 13C; D2O: 4.79 for 1H). Two-dimensional COSY and
HSQC experiments were run to support 1H and 13C assignments.
Multiplicities of resonances are denoted as s (singlet), d (doublet),
t (triplet), q (quartet), dd (doublet of doublets), m (multiplet); (b)
indicates a broad signal and (obs) indicates the signal is obscured.
Low resolution mass spectra (LRMS) utilising electrospray ion-
isation (ESI) were obtained using a Bru¨ker Esquire 3000. High-
resolution mass spectra (HRMS) were carried out at either the
University of Queensland’s mass spectrometry service within the
School of Chemistry and Molecular Biosciences, or at the Eskitis
Institute, Griffith University, Nathan campus. All HRMS were
recorded using an electrospray method. Elemental microanalyses
were carried out at the University of Queensland’s Microanalysis
Facility. All H2O used was either distilled H2O or Milli-Q H2O. All
commercial solvents (acetone, chloroform, dichloromethane, ethyl
acetate, hexanes, methanol and toluene) were bulk distilled prior
to use. Dried solvents were distilled under N2.
General procedure for acetylation (A)
To a solution of alcohol (1.0 equiv) in pyridine (1 mL per mmol
of alcohol) and 4-dimethylaminopyridine (0.3–0.5 equiv) at 0 ◦C
under N2 was added acetic anhydride (25–40 equiv). The mixture
was allowed to regain ambient temperature, was stirred for 20 h,
then concentrated. The residue was dissolved in ethyl acetate,
washed with 1 N HCl, H2O, sat. aq. NaHCO3, and brine. The
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organic phase was dried over Na2SO4, filtered, and the solvent
was removed in vacuo.
5 ¥ OAc); dC(75 MHz; CDCl3) 171.4, 170.7, 169.8, 169.7, 169.1
(5 ¥ OC(O)CH3); 168.0 (GlcA C-6); 160.7 (OC(NH)CCl3); 157.6
(q, 38 Hz, C(O)CF3); 115.3 (q, 286 Hz, C(O)CF3); 96.6 (GlcN
C-1); 92.5 (GlcA C-1); 90.4 (CCl3); 73.5 (GlcA C-4); 71.7 (GlcA
C-5); 71.0 (GlcA C-3); 69.8 (GlcN C-3); 69.4 (GlcA C-2); 68.9
(GlcN C-5); 67.1 (GlcN C-4); 61.0 (GlcN C-6); 53.1 (CO2CH3);
52.3 (GlcN C-2); 20.7, 20.6, 20.5, 20.4, 20.4 (5 ¥ OC(O)CH3);
m/z (ESI) 841.059998 ([M + Na]+. C27H32Cl3F3N2NaO17 requires
841.061086).
General procedure for glycosidation (B)
To a solution of trichloroacetimidate 2 (1.0 equiv) in CH2Cl2
(10 mL per mmol of 2) was added alcohol (2.0–5.0 equiv) and
˚
4 A sieves. The mixture was stirred for 60 min at rt, then cooled
to -15 ◦C (4-methylumbelliferone reactions were performed with
exclusion of light). BF3·OEt2 (0.3–0.5 equiv) in CH2Cl2 was added.
The reaction mixture was allowed to warm to rt and was stirred
for 16 h. The mixture was diluted with CH2Cl2, filtered through
6-O-Acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-a-D-glucopyra-
nosyl-(1→4)-methyl 3-O-benzyl-D-glucopyranosyluronate (3).
Prepared as previously reported.12 a anomer dH(300 MHz; CDCl3)
7.39–7.26 (m, 15 H, 3 ¥ Ph); 5.43 (d, 1 H, J1,2 2.4 Hz, GlcA H-1);
5.25 (d, 1 H, J1,2 3.7 Hz, GlcN H-1); 4.89 (s, 2 H, PhCH2); 4.86,
4.59 (AB q, 2 H, JAB 11.2 Hz, PhCH2); 4.74, 4.68 (AB q, 2 H,
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Celiteꢀ, washed with aqueous Na2CO3, dried over Na2SO4, and
the solvent was removed in vacuo.
General procedure for N-sulfation (C)
J
AB 11.2 Hz, PhCH2); 4.62–4.56 (obs, 1 H, GlcA H-5); 4.31 (dd, 1
To a solution of methyl 2-amino-2-deoxy-b-D-glucopyranoside,23
19, 20, 21, or 22 (1.0 equiv) in H2O (50 mL per mmol of amine)
was added SO3·Py complex (10 equiv) portionwise. The pH was
maintained at 9.5 by the addition of 1 N NaOH. After 3 h, the
H, J6a,6b 12.1, J6a,5 2.0 Hz, GlcN H-6a); 4.27–4.24 (m, 1 H, GlcA
H-4); 4.20 (dd, 1 H, J6b,6a 12.1, J6b,5 4.6 Hz, GlcN H-6b); 3.99–
3.90 (m, 2 H, GlcA H-3, GlcN H-3); 3.81–3.74 (m, 2 H, GlcA
H-2, GlcN H-5); 3.63 (s, 3 H, CO2Me); 3.57–3.49 (m, 2 H, GlcN
H-2, GlcN H-4); 2.05 (s, 3 H, OAc); dC(75 MHz; CDCl3) 170.9
(OC(O)CH3); 169.4 (C-6); 137.4, 137.3, 137.3 (3 ¥ ipsoPh); 128.6,
128.6, 128.5, 128.2, 128.2, 128.1, 128.0, 128.0, 127.6 (Ph); 96.9
(GlcN C-1); 90.5 (GlcA C-1); 80.6 (GlcN C-3); 77.5 (GlcN C-4);
76.3 (GlcA C-3); 75.8, 75.1, 73.5 (3 ¥ PhCH2); 73.2 (GlcA C-5);
72.7 (GlcA C-4); 70.2 (GlcN C-5); 69.9 (GlcA C-2); 63.5 (GlcN C-
2); 62.5 (GlcN C-6); 52.5 (CO2CH3); 20.8 (OC(O)CH3); b anomer
dC(75 MHz; CDCl3) 170.9 (OC(O)CH3); 169.2 (C-6); 138.2, 137.6,
137.5 (3 ¥ ipsoPh); 128.6–127.6 (Ph); 97.5, 97.0 (C-1, GlcN C-1);
82.9, 80.2, 77.4, 74.7, 74.4, 69.7, 64.4, 63.3 (GlcA C-2, GlcA C-3,
GlcA C-4, GlcA C-5, GlcN C-2, GlcN C-3, GlcN C-4, GlcN C-5);
77.6, 77.1, 76.1 (3 ¥ PhCH2); 62.3 (GlcN C-6); 52.9 (CO2CH3);
20.9 (OC(O)CH3); m/z (ESI) 729.9 ([M + Na]+. C36H41N3NaO12
requires 730.3).
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reaction mixture was concentrated and purified on Sephadexꢀ
LH-20, eluting with 4 : 1 methanol–H2O.
General procedure for de-esterification (D)
To a solution of either a ~1 : 1 mixture of 8 and 10 or pure
compounds 9, 11, or 12 (1.0 equiv) in methanol (25 mL per mmol
of substrate) at 0 ◦C was added H2O (5 mL per mmol of substrate)
and 0.1 N LiOH (10 equiv). The solution was allowed to regain
ambient temperature and was stirred for 20 h. The solution was
acidified to pH 6 with dilute acetic acid, and then adjusted to
pH 7.5 with dilute NaOH. The solution was concentrated and
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purified on Sephadexꢀ LH-20, eluting with 4 : 1 methanol–H2O.
General procedure for N-acetylation (E)
6-O-Acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-a-D-glucopyra-
nosyl-(1→4)-methyl 1,2-di-O-acetyl-3-O-benzyl-D-glucopyrano-
syluronate (4). Prepared according to general procedure A from
3. Flash chromatography on silica gel (3 : 1 hexanes–ethyl acetate,
Rf 0.3) afforded 4 (100%) as a 2.5 : 1 a : b mixture (Found: C,
60.64; H, 5.79; N, 5.17. C40H45N3O14 requires C, 60.68; H, 5.73;
N, 5.31%); a anomer dH(300 MHz; CDCl3) 7.41–7.25 (m, 15 H,
3 ¥ Ph); 6.33 (d, 1 H, J1,2 3.6 Hz, GlcA H-1); 5.51 (d, 1 H, J1,2
3.9 Hz, GlcN H-1); 5.09 (dd, 1 H, J2,3 9.6, J2,1 3.6 Hz, GlcA H-
2); 4.94–4.78 (m, 4 H, 2 ¥ PhCH2); 4.86, 4.57 (AB q, 2 H, JAB
11.1 Hz, PhCH2); 4.36 (d, 1 H, J5,4 9.3 Hz, GlcA H-5); 4.30–4.16
(m, 3 H, GlcN H-6a, GlcN H-6b, GlcA H-4); 4.09 (dd, 1 H, J3,4
9.6, J3,2 9.6 Hz, GlcA H-3); 3.91 (dd, 1 H, J3,2 10.2, J3,4 8.7 Hz,
GlcN H-3); 3.77 (s, 3 H, CO2Me); 3.65–3.58 (m, 1 H, GlcN H-5);
3.52 (dd, 1 H, J4,5 10.2, J4,3 8.4 Hz, GlcN H-4); 3.36 (dd, 1 H, J2,3
10.2, J2,1 3.6 Hz, GlcN H-2); 2.20, 2.04, 1.95 (3 ¥ s, 9 H, 3 ¥ OAc);
dC(75 MHz; CDCl3) 170.7, 169.7, 168.8 (3 ¥ OC(O)CH3); 168.4
(GlcA C-6); 137.8, 137.5, 137.4 (3 ¥ ipsoPh); 128.8, 128.6, 128.5,
128.1, 128.1, 127.9, 127.8, 127.5, 127.2 (3 ¥ Ph); 97.9 (GlcN C-1);
89.2 (GlcA C-1); 80.2 (GlcN C-3); 79.3 (GlcA C-3); 77.4 (GlcN
C-4); 75.6, 75.2, 75.0 (3 ¥ PhCH2); 74.8 (GlcA C-4); 72.3 (GlcA
C-5); 71.4 (GlcA C-2); 69.8 (GlcN C-5); 63.3 (GlcN C-2); 62.2
(GlcN C-6); 53.0 (CO2CH3); 20.9, 20.9, 20.6 (3 ¥ OC(O)CH3); b
anomer dH(300 MHz; CDCl3) 7.40–7.24 (m, 15 H, 3 ¥ Ph); 5.73 (d,
To a solution of amine 19, 20, 21, or 22 (1.0 equiv) in methanol
(30 mL per mmol of amine) at 0 ◦C under N2 was added
triethylamine (2.0 equiv) and acetic anhydride (15 equiv). The
mixture was stirred for 90 min, H2O was added, and the solution
was concentrated. The product was purified by column chro-
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matography on Sephadexꢀ LH-20, eluting with 4 : 1 methanol–
H2O.
3,4,6-Tri-O-acetyl-2-deoxy-2-trifluoroacetamido-a-D-glucopy-
ranosyl-(1→4)-methyl 2,3-di-O-acetyl-a-D-glucopyranuronosyl
trichloroacetimidate (2). To a solution of 8 (532 mg, 788 mmol)
in CH2Cl2 (5 mL) and trichloroacetonitrile (790 mL, 7.88 mmol)
at 0 ◦C under N2 was added 1,8-diazabicyclo[5.4.0]undec-7-ene
(12 mL, 79 mmol). The mixture was stirred for 2 h and then
concentrated. Flash chromatography on silica gel (3 : 2 hexanes–
ethyl acetate, Rf 0.2) afforded 2 (571 mg, 88%); dH(300 MHz;
CDCl3) 8.74 (s, 1 H, NHC(O)CCl3); 6.56 (d, 1 H, J1,2 3.6 Hz,
GlcA H-1); 6.54 (bd, 1 H, NHC(O)CF3); 5.62 (dd, 1 H, J3,2 10.0,
J
3,4 9.3 Hz, GlcA H-3); 5.21 (d, 1 H, J1,2 3.7 Hz, GlcN H-1); 5.19–
5.08 (m, 2 H, GlcN H-3, GlcN H-4); 5.06 (dd, 1 H, J2,3 10.0, J2,1
3.6 Hz, GlcA H-2); 4.50 (d, 1 H, J5,4 9.8 Hz, GlcA H-5); 4.35 (dd,
1 H, J4,5 9.8, J4,3 9.3 Hz, GlcA H-4); 4.32–4.07 (m, 3 H, GlcN
H-2, GlcN H-6a, GlcN H-6b); 3.78 (s, 3 H, CO2Me); 3.72–3.66
(m, 1 H, GlcN H-5); 2.12, 2.03, 2.01, 2.01, 1.98 (5 ¥ s, 15 H,
4618 | Org. Biomol. Chem., 2011, 9, 4614–4625
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1 H, J1,2 6.9 Hz, GlcA H-1); 5.43 (d, 1 H, J1,2 3.6 Hz, GlcN H-1);
5.16 (dd, 1 H, J2,3 8.1, J2,1 6.9 Hz, GlcA H-2); 4.89–4.81 (m, 2 H,
PhCH2); 4.85, 4.72 (AB q, 2 H, JAB 11.1 Hz, PhCH2); 4.85, 4.56
(AB q, 2 H, JAB 10.8 Hz, PhCH2); 4.31–4.13 (m, 4 H, GlcN H-6a,
GlcN H-6b, GlcA H-4, GlcA H-5); 3.93–3.84 (m, 2 H, GlcN H-3,
GlcA H-3); 3.74 (s, 3 H, CO2Me); 3.66–3.57 (m, 1 H, GlcN H-5);
3.52 (dd, 1 H, J4,5 10.2, J4,3 8.7 Hz, GlcN H-4); 3.32 (dd, 1 H, J2,3
10.2, J2,1 3.6 Hz, GlcN H-2); 2.09, 2.05, 1.99 (3 ¥ s, 9 H, 3 ¥ OAc);
dC(75 MHz; CDCl3) 170.7, 170.5, 169.3 (3 ¥ OC(O)CH3); 168.2
(GlcA C-6); 137.5, 137.4, 137.4 (3 ¥ ipsoPh); 128.8–127.2 (3 ¥ Ph);
97.8 (GlcN C-1); 91.7 (GlcA C-1); 81.5 (GlcN C-3); 80.0 (GlcA
C-3); 77.1 (GlcN C-4); 75.6, 75.0, 74.7 (3 ¥ PhCH2); 74.6, 74.4
(GlcA C-4, GlcA C-5); 71.7 (GlcA C-2); 69.9 (GlcN C-5); 63.2
(GlcN C-2); 62.2 (GlcN C-6); 52.9 (CO2CH3); 20.9, 20.8, 20.86
(3 ¥ OC(O)CH3); m/z (ESI) 814.0 ([M + Na]+. C40H45N3NaO14
requires 814.3).
81.0, 78.6, 77.2, 74.6, 72.6, 71.9, 70.9 (GlcN C-3, GlcN C-4, GlcN
C-5, GlcA C-2, GlcA C-3, GlcA C-4, GlcA C-5); 75.1, 74.9, 74.2
(3 ¥ PhCH2); 62.0 (GlcN C-6); 53.0 (CO2CH3); 52.8 (GlcN C-2);
20.8, 20.8, 20.6 (3 ¥ OC(O)CH3); m/z (ESI): 884.272582 ([M +
Na]+. C42H46F3NNaO15 requires 884.271116).
6-O-Acetyl-2-deoxy-2-trifluoroacetamido-a-D-glucopyranosyl-
(1→4)-methyl 1,2-di-O-acetyl-D-glucopyranosyluronate (6).
A
solution of 5 (700 mg, 812 mmol) in methanol (20 mL) and acetic
acid (232 mL, 4.06 mmol) was stirred under an atmosphere of H2 in
the presence of Pd/C (10%) for 20 h at rt. After filtration through
R
a pad of Celiteꢀ, the solvent was removed in vacuo to afford 6
(480 mg, 100%) as a 2.3 : 1 a : b mixture; a anomer dH(300 MHz;
CDCl3) 8.28–8.20 (m, 1 H, GlcN NH); 6.30 (d, 1 H, J1,2 3.6 Hz,
GlcA H-1); 5.07 (d, 1 H, J1,2 3.0 Hz, GlcN H-1); 4.90 (dd, 1 H,
J2,3 9.6, J2,1 3.6 Hz, GlcA H-2); 4.45–3.93 (m, 8 H, GlcN H-2,
GlcN H-3, GlcN H-4, GlcN H-6a, GlcN H-6b, GlcA H-3, GlcA
H-4, GlcA H-5); 3.78 (s, 3 H, CO2Me); 3.54–3.44 (m, 1 H, GlcN
H-5); 2.17, 2.11, 2.07 (3 ¥ s, 9 H, 3 ¥ OAc); dC(75 MHz; CDCl3)
172.0, 170.7, 169.8 (3 ¥ OC(O)CH3); 167.7 (GlcA C-6); 158.7 (q,
37 Hz, C(O)CF3); 115.8 (q, 285 Hz, C(O)CF3); 99.9 (GlcN C-1);
89.1 (GlcA C-1); 80.4, 72.7, 72.0, 71.5, 71.5, 70.0, 69.9 (GlcN C-3,
GlcN C-4, GlcN C-5, GlcA C-2, GlcA C-3, GlcA C-4, GlcA C-5);
62.5 (GlcN C-6); 55.0 (GlcN C-2); 53.1 (CO2CH3); 20.8, 20.7, 20.5
(3 ¥ OC(O)CH3); b anomer dH(300 MHz; CDCl3) 8.28–8.20 (m,
1 H, GlcN NH); 5.67 (d, 1 H, J1,2 8.0 Hz, GlcA H-1); 5.04 (d, 1
H, J1,2 3.0 Hz, GlcN H-1); 4.90 (obs, 1 H, GlcA H-2); 4.45–3.93
(m, 8 H, GlcN H-2, GlcN H-3, GlcN H-4, GlcN H-6a, GlcN
H-6b, GlcA H-3, GlcA H-4, GlcA H-5); 3.82 (s, 3 H, CO2Me);
3.54–3.44 (m, 1 H, GlcN H-5); 2.12, 2.09, 2.04 (3 ¥ s, 9 H, 3 ¥
OAc); dC(75 MHz; CDCl3) 172.3, 171.2, 168.9 (3 ¥ OC(O)CH3);
167.3 (GlcA C-6); 158.7 (q, 37 Hz, C(O)CF3); 115.8 (q, 285 Hz,
C(O)CF3); 99.9 (GlcN C-1); 91.5 (GlcA C-1); 80.7, 75.3, 73.7,
73.1, 71.5, 71.2, 69.9 (GlcN C-3, GlcN C-4, GlcN C-5, GlcA C-2,
GlcA C-3, GlcA C-4, GlcA C-5); 62.6 (GlcN C-6); 54.9 (GlcN
C-2); 53.1 (CO2CH3); 20.7, 20.6, 20.5 (3 ¥ OC(O)CH3); m/z (ESI)
614.130081 ([M + Na]+. C21H28F3NNaO15 requires 614.130325).
6-O-Acetyl-3,4-di-O-benzyl-2-deoxy-2-trifluoroacetamido-a-
D-glucopyranosyl-(1→4)-methyl 1,2-di-O-acetyl-3-O-benzyl-D-
glucopyranosyluronate (5). To a solution of 4 (1.16 g, 1.46 mmol)
in THF (10 mL) at 0 ◦C under N2 was added PPh3 (421 mg,
1.61 mmol). After 10 min, H2O (263 mL, 14.6 mmol) was added,
the solution was allowed to regain ambient temperature and was
stirred for 20 h. The solvents were removed in vacuo, the residue
was dissolved in pyridine (25 mL) at 0 ◦C under N2, to which
was added trifluoroacetic anhydride (2.03 mL, 14.6 mmol). The
solution was allowed to regain ambient temperature and was
stirred for 20 h. The mixture was then concentrated, the residue
was dissolved in ethyl acetate, washed with 1 N HCl, and H2O. The
organic phase was dried over Na2SO4, filtered, and the solvent
was removed in vacuo. Flash chromatography on silica gel (2 : 1
hexanes–ethyl acetate, Rf 0.3) afforded 5 (1.02 g, 81%) as a 3 : 1
a : b mixture; a anomer dH(300 MHz; CDCl3) d 7.38–7.17 (m, 15
H, 3 ¥ Ph); 6.74 (d, 1 H, JNH,2 9.5 Hz, NH); 6.29 (d, 1 H, J1,2
3.6 Hz, GlcA H-1); 5.23 (d, 1 H, J1,2 3.4 Hz, GlcN H-1); 5.05 (dd,
1 H, J2,3 9.7, J2,1 3.6 Hz, GlcA H-2); 4.84–4.76 (m, 2 H, PhCH2);
4.62 (s, 2 H, PhCH2); 4.60–4.54 (m, 2 H, PhCH2); 4.35–4.15 (m, 4
H, GlcN H-2, GlcN H-6a, GlcN H-6b, GlcA H-5); 4.14–4.08 (m,
1 H, GlcA H-4); 3.92 (dd, 1 H, J3,2 9.7, J3,4 9.2 Hz, GlcA H-3);
3.77 (s, 3 H, CO2Me); 3.74–3.57 (m, 3 H, GlcN H-3, GlcN H-4,
GlcN H-5); 2.18, 2.06, 1.77 (3 ¥ s, 9 H, 3 ¥ OAc); dC(75 MHz;
CDCl3) 170.7, 169.5, 168.6 (3 ¥ OC(O)CH3); 168.0 (GlcA C-6);
157.1 (q, 37 Hz, NC(O)CF3); 137.3, 137.3, 137.3 (3 ¥ ipsoPh); 128.7–
127.0 (3 ¥ Ph); 115.7 (q, 286 Hz, NC(O)CF3); 97.7 (GlcN C-1);
89.2 (GlcA C-1); 78.9 (GlcA C-3); 78.8 (GlcN C-3); 77.1 (GlcN
C-4); 75.6 (GlcA C-4); 75.1, 74.9, 74.9 (3 ¥ PhCH2); 72.6 (GlcA
C-5); 71.3 (GlcA C-2); 70.9 (GlcN C-5); 61.9 (GlcN C-6); 53.1
(CO2CH3); 52.9 (GlcN C-2); 20.9, 20.8, 20.3 (3 ¥ OC(O)CH3); b
anomer dH(300 MHz; CDCl3) 7.38–7.17 (m, 15 H, 3 ¥ Ph); 6.65 (d,
1 H, JNH,2 9.6 Hz, NH); 5.69 (d, 1 H, J1,2 6.7 Hz, GlcA H-1); 5.23
(d, 1 H, J1,2 3.4 Hz, GlcN H-1); 5.12 (dd, 1 H, J2,3 7.9, J2,1 6.7 Hz,
GlcA H-2); 4.84–4.76 (m, 2 H, PhCH2); 4.62 (s, 2 H, PhCH2);
4.60–4.54 (m, 2 H, PhCH2); 4.35–4.08 (m, 6 H, GlcN H-2, GlcN
H-6a, GlcN H-6b, GlcA H-3, GlcA H-4, GlcA H-5); 3.75 (s, 3 H,
CO2Me); 3.74–3.57 (m, 3 H, GlcN H-3, GlcN H-4, GlcN H-5);
2.08, 2.06, 1.86 (3 ¥ s, 9 H, 3 ¥ OAc); dC(75 MHz; CDCl3) 170.7,
169.1, 169.0 (3 ¥ OC(O)CH3); 168.1 (GlcA C-6); 157.1 (q, 37 Hz,
NC(O)CF3); 137.3, 137.2, 136.8 (3 ¥ ipsoPh); 128.7–127.0 (3 ¥ Ph);
115.7 (q, 286 Hz, NC(O)CF3); 97.2 (GlcN C-1); 91.8 (GlcA C-1);
3,4,6-Tri-O-acetyl-2-deoxy-2-trifluoroacetamido-a-D-glucopy-
ranosyl-(1→4)-methyl 1,2,3-tri-O-acetyl-D-glucopyranosyluronate
(7). Prepared according to general procedure A from 6. Flash
chromatography on silica gel (3 : 2 hexanes–ethyl acetate, Rf 0.3)
afforded 7 (100%) as a 2.3 : 1 a : b mixture (Found: C, 45.13; H,
4.86; N, 1.91. Microanalysis calculatedfor C27H34F3NO18: C, 45.19;
H, 4.78; N, 1.95%); a anomer dH(300 MHz; CDCl3) 6.57 (d, 1 H,
J
NH,2 9.3 Hz, NH); 6.30 (d, 1 H, J1,2 3.6 Hz, GlcA H-1); 5.49 (dd, 1
H, 10.1, 9.0 Hz, GlcA H-3); 5.21–4.98 (m, 4 H, GlcN H-1, GlcN
H-3, GlcN H-4, GlcA H-2); 4.42–4.06 (m, 5 H, GlcN H-2, GlcN
H-6a, GlcN H-6b, GlcA H-4, GlcA H-5); 3.77 (s, 3 H, CO2Me);
3.73–3.63 (m, 1 H, GlcN H-5); 2.23, 2.10, 2.02, 2.00, 1.98, 1.97 (6
¥ s, 18 H, 6 ¥ OAc); dC(75 MHz; CDCl3) 171.5, 170.7, 169.9, 169.6,
169.1, 168.7 (6 ¥ OC(O)CH3); 168.1 (GlcA C-6); 157.6 (q, 38 Hz,
C(O)CF3); 115.4 (q, 286 Hz, C(O)CF3); 96.6 (GlcN C-1); 88.7
(GlcA C-1); 73.4 (GlcA C-4); 71.6 (GlcA C-5); 71.2 (GlcA C-3);
69.7 (GlcN C-3); 68.9, 68.9 (GlcN C-5, GlcA C-2); 67.0 (GlcN C-
4); 61.0 (GlcN C-6); 53.0 (CO2CH3); 52.4 (GlcN C-2); 20.9, 20.7,
20.5, 20.5, 20.4, 20.3 (6 ¥ OC(O)CH3); b anomer dH(300 MHz;
CDCl3) 6.71–6.62 (bd, 1 H, NH); 5.77 (d, 1 H, J1,2 7.3 Hz, GlcA
H-1); 5.30 (dd, 1 H, J3,2 8.8, J3,4 8.8 Hz, GlcA H-3); 5.21–4.98 (m,
4 H, GlcN H-1, GlcN H-3, GlcN H-4, GlcA H-2); 4.42–4.06 (m, 5
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H, GlcN H-2, GlcN H-6a, GlcN H-6b, GlcA H-4, GlcA H-5); 3.77
(s, 3 H, CO2Me); 3.73–3.63 (m, 1 H, GlcN H-5); 2.10, 2.07, 2.01,
2.00, 1.99, 1.98 (6 ¥ s, 18 H, 6 ¥ OAc); dC(75 MHz; CDCl3) 171.4,
170.7, 169.8, 169.3, 169.1, 168.7 (6 ¥ OC(O)CH3); 167.8 (GlcA C-
6); 157.6 (q, 38 Hz, C(O)CF3); 115.4 (q, 286 Hz, C(O)CF3); 96.5
(GlcN C-1); 91.4 (GlcA C-1); 74.1, 73.7, 73.0 (GlcA C-3, GlcA
C-4, GlcA C-5); 70.2, 69.7, 68.9 (GlcN C-3, GlcN C-5, GlcA C-2);
67.0 (GlcN C-4); 61.1 (GlcN C-6); 53.0 (CO2CH3); 52.4 (GlcN
C-2); 20.9–20.3 (6 ¥ OC(O)CH3); m/z (ESI) 740.1 ([M + Na]+.
C27H34F3NNaO18 requires 740.2).
H-1); 5.19 (dd, 1 H, J2,3 8.7, J2,1 6.0 Hz, GlcA H-2); 5.21–5.09 (m,
2 H, GlcN H-3, GlcN H-4); 4.54 (dd, 1 H, J4,3 8.7, J4,5 8.4 Hz,
GlcA H-4); 4.33 (d, 1 H, J5,4 8.4 Hz, GlcA H-5); 4.32–4.11 (m, 3
H, GlcN H-2, GlcN H-6a, GlcN H-6b); 3.81–3.76 (m, 1 H, GlcN
H-5); 3.65 (s, 3 H, CO2Me); 2.10, 2.05, 2.04, 2.03, 2.01 (5 ¥ s,
15 H, 5 ¥ OAc); dC(75 MHz; CDCl3) 171.5, 170.7, 169.9, 169.3,
169.1 (5 ¥ OC(O)CH3); 167.6 (GlcA C-6); 160.7 (C-1¢); 157.5 (q,
38 Hz, C(O)CF3); 143.3 (C-4¢); 125.8 (C-3¢, C-5¢); 116.5 (C-2¢,
C-6¢); 115.4 (q, 286 Hz, C(O)CF3); 98.0 (GlcA C-1); 96.7 (GlcN
C-1); 74.0 (GlcA C-5); 73.1 (GlcA C-3); 72.8 (GlcA C-4); 71.3
(GlcA C-2); 69.8 (GlcN C-3); 69.0 (GlcN C-5); 67.1 (GlcN C-4);
61.2 (GlcN C-6); 53.0 (CO2CH3); 52.6 (GlcN C-2); 20.7, 20.5,
20.5, 20.5, 20.4 (5 ¥ OC(O)CH3); m/z (ESI) 819.1 ([M + Na]+.
C31H35F3N2O19 requires 819.2).
3,4,6-Tri-O-acetyl-2-deoxy-2-trifluoroacetamido-a-D-glucopy-
ranosyl-(1→4)-methyl 2,3-di-O-acetyl-D-glucopyranosyluronate
(8). ◦To a solution of 7 (2.69 g, 3.75 mmol) in THF (20 mL)
at 0 C under N2 was added benzylamine (820 mL, 7.50 mmol).
The mixture was allowed to regain ambient temperature and was
stirred for 16 h. The mixture was diluted with CHCl3 and washed
with H2O. The aqueous layer was back-extracted with CHCl3
and the combined organic phase was washed with 1 N HCl, sat.
aq. NaHCO3, brine, H2O, dried over Na2SO4, and the solvent
was removed in vacuo. Flash chromatography on silica gel (1 : 1
hexanes–ethyl acetate, Rf 0.3) afforded 8 (2.19 g, 87%) as a 5 : 1
a : b mixture; a anomer dH(300 MHz; CDCl3) 6.75 (d, 1 H, JNH,2
9.3 Hz, NH); 5.58 (dd, 1 H, J3,2 9.3, J3,4 9.3 Hz, GlcA H-3); 5.47
(d, 1 H, J1,2 3.4 Hz, GlcA H-1); 5.20 (d, 1 H, J1,2 3.7 Hz, GlcN
H-1); 5.18–5.07 (m, 2 H, GlcN H-3, GlcN H-4); 4.83 (dd, 1 H,
J2,3 9.3, J2,1 3.4 Hz, GlcA H-2); 4.58 (d, 1 H, J5,4 9.5 Hz, GlcA H-
5); 4.36–4.08 (m, 4 H, GlcN H-2, GlcN H-6a, GlcN H-6b, GlcA
H-4); 3.80 (s, 3 H, CO2Me); 3.78–3.72 (m, 1 H, GlcN H-5); 2.12,
2.05, 2.03, 2.00, 2.00 (5 ¥ s, 15 H, 5 ¥ OAc); dC(75 MHz; CDCl3)
171.2, 171.0, 170.4, 169.9, 169.4 (5 ¥ OC(O)CH3); 169.4 (GlcA
C-6); 157.8 (q, 38 Hz, C(O)CF3); 115.4 (q, 286 Hz, C(O)CF3);
96.4 (GlcN C-1); 90.2 (GlcA C-1); 73.8 (GlcA C-4); 71.3 (GlcA
C-3); 70.8 (GlcA C-2); 69.8 (GlcN C-3); 69.6 (GlcA C-5); 68.6
(GlcN C-5); 67.4 (GlcN C-4); 61.2 (GlcN C-6); 53.0 (CO2CH3);
52.2 (GlcN C-2); 20.7, 20.5, 20.5, 20.5, 20.3 (5 ¥ OC(O)CH3); b
anomer dH(300 MHz; CDCl3) 6.61 (d, 1 H, JNH,2 9.3 Hz, NH);
5.32 (dd, 1 H, J3,2 9.3, J3,4 9.3 Hz, GlcA H-3); 5.22–5.07 (m, 3 H,
GlcN H-1, GlcN H-3, GlcN H-4); 4.88–4.80 (m, 2 H, GlcA H-1,
GlcA H-2); 4.36–4.08 (m, 5 H, GlcN H-2, GlcN H-6a, GlcN H-6b,
GlcA H-4, GlcA H-5); 3.81 (s, 3 H, CO2Me); 3.78–3.72 (m, 1 H,
GlcN H-5); 2.12–2.00 (5 ¥ s, 15 H, 5 ¥ OAc); dC(75 MHz; CDCl3)
171.4, 171.0, 170.8, 170.1, 169.3 (5 ¥ OC(O)CH3); 168.8 (GlcA C-
6); 157.8 (q, 38 Hz, C(O)CF3); 115.4 (q, 286 Hz, C(O)CF3); 96.3
(GlcN C-1); 95.4 (GlcA C-1); 73.8, 73.7, 73.5, 72.7, 69.8, 68.7, 67.5
(GlcN C-3, GlcN C-4, GlcN C-5, GlcA C-2, GlcA C-3, GlcA C-4,
GlcA C-5); 61.1 (GlcN C-6); 53.1 (CO2CH3); 52.3 (GlcN C-2);
20.7–20.3 (5 ¥ OC(O)CH3); m/z (ESI) 698.150885 ([M + Na]+.
C25H32F3NNaO17 requires 698.151454.
4¢-Methylumbelliferyl (3,4,6-tri-O-acetyl-2-deoxy-2-trifluoro-
acetamido-a-D-glucopyranosyl)-(1→4)-methyl 2,3-di-O-acetyl-b-
D-glucopyranosiduronate (10). Prepared according to general
procedure
B
from
2
and 4-methylumbelliferone. Flash
chromatography on silica gel (3 : 2 hexanes–ethyl acetate, Rf
0.3) afforded 10 (~31%); dH(300 MHz; CDCl3) 7.52 (d, 1 H, J5¢,6¢
9.5 Hz, H-5¢); 6.94–6.91 (m, 2 H, H-6¢, H-8¢); 6.75 (d, 1 H, JNH,2
9.3 Hz, NH); 6.19 (q, 1 H, J3¢,Me¢ 1.2 Hz, H-3¢); 5.39–5.25 (m, 2
H, GlcA H-1, GlcA H-3); 5.22–5.05 (m, 4 H, GlcN H-1, GlcN
H-3, GlcN H-4, GlcA H-2); 4.49 (dd, 1 H, J4,3 8.7, J4,5 8.4 Hz,
GlcA H-4); 4.33–4.07 (m, 5 H, GlcN H-2, GlcN H-5, GlcN
H-6a, GlcN H-6b, GlcA H-5); 3.70 (s, 3 H, CO2Me); 2.40 (d,
JMe¢,3¢ 1.2 Hz, Me¢); 2.11–1.98 (5 ¥ s, 15 H, 5 ¥ OAc); dC(75 MHz;
CDCl3) 171.5–169.1 (5 ¥ OC(O)Me); 167.7 (GlcA C-6); 160.9
(C-2¢); 158.8 (C-7¢); 157.8 (q, 38 Hz, C(O)CF3); 154.7, 152.3 (C-4¢,
C-8a¢); 125.8 (C-5¢); 115.6 (C-4a¢); 115.4 (q, 286 Hz, C(O)CF3);
113.8 (C-6¢); 113.2 (C-3¢); 103.9 (C-8¢); 98.2 (GlcA C-1);96.5
(GlcN C-1); 74.0–72.9 (GlcA C-3, GlcA C-4, GlcA C-5); 71.3
(GlcA C-2); 69.9 (GlcN C-3); 69.0 (GlcN C-5); 67.2 (GlcN C-4);
61.1 (GlcN C-6); 52.9 (CO2CH3); 52.5 (GlcN C-2); 20.7–20.4 (5 ¥
OC(O)CH3); 18.7 (C-4¢ CH3); m/z (ESI) (m/z) 856.187146 ([M +
Na]+. C35H38F3NNaO19 requires 856.188241) and 6 (~31%), which
were not separated.
4¢-[3¢¢-Methyl prop-2¢¢(E)-enoate]phenyl (3,4,6-tri-O-acetyl-2-
deoxy-2-trifluoroacetamido-a-D-glucopyranosyl)-(1→4)-methyl 2,
3-di-O-acetyl-b-D-glucopyranosiduronate (11). Prepared accord-
ing to general procedure B from 2 and methyl 4-hydroxy-
cinnamate.24 Flash chromatography on silica gel (3 : 2 hexanes–
ethyl acetate) afforded 11 (59%, Rf 0.3); dH(300 MHz; CDCl3)
7.64 (d, 1 H, J3¢¢,2¢¢ 16.0 Hz, H-3¢¢); 7.48 (d, 2 H, J3¢,2¢ 8.7 Hz, H-
3¢); 6.97 (d, 2 H, J2¢,3¢ 8.7 Hz, H-2¢); 6.53 (d, 1 H, JNH,2 9.0 Hz,
NH); 6.34 (d, 1 H, J2¢¢,3¢¢ 16.0 Hz, H-2¢¢); 5.36 (dd, 1 H, J3,2 8.8,
J3,4 8.8 Hz, GlcA H-3); 5.29–5.26 (m, 2 H, GlcN H-1, GlcA H-
1); 5.20–5.12 (m, 3 H, GlcN H-3, GlcN H-4, GlcA H-2); 4.53
(dd, 1 H, J4,3 8.8 Hz, J4,5 8.8 Hz, GlcA H-4); 4.32–4.11 (m, 4 H,
GlcN H-2, GlcN H-6a, GlcN H-6b, GlcA H-5); 3.82–3.77 (m, 4
H, GlcN H-5, CO2Me); 3.64 (s, 3 H, CO2Me); 2.11, 2.05, 2.04,
2.03, 2.02 (5 ¥ s, 15 H, 5 ¥ OAc); dC(75 MHz; CDCl3) 171.4, 170.7,
170.0, 169.4, 169.1 (5 ¥ OC(O)CH3); 167.8, 167.5 (GlcA C-6, C-
1¢¢); 157.7 (C-1¢); 157.5 (q, 38 Hz, C(O)CF3); 143.8 (C-3¢¢); 129.6
(C-4¢); 129.6 (C-3¢, C-5¢); 116.8 (C-2¢, C-6¢); 116.8 (C-2¢¢); 115.4
(q, 286 Hz, C(O)CF3); 98.4 (GlcA C-1); 96.6 (GlcN C-1); 73.9
(GlcA C-5); 73.5 (GlcA C-3); 72.9 (GlcA C-4); 71.6 (GlcA C-2);
69.8 (GlcN C-3); 68.9 (GlcN C-5); 67.2 (GlcN C-4); 61.1 (GlcN
4¢-Nitrophenyl
(3,4,6-tri-O-acetyl-2-deoxy-2-trifluoroacet-
amido-a-D-glucopyranosyl)-(1→4)-methyl 2,3-di-O-acetyl-b-D-
glucopyranosiduronate (9). Prepared according to general
procedure B from 2 and 4-nitrophenol. Flash chromatography
on silica gel (2 : 1 hexanes–ethyl acetate, Rf 0.2) afforded 9 (80%);
(Found: C, 47.06; H, 4.56; N, 3.25. C31H35F3N2O19 requires C,
46.74; H, 4.43; N, 3.52%); dH(300 MHz; CDCl3) 8.26–8.19 (AB m,
2 H, H-3¢, H-5¢); 7.10–7.04 (AB m, 2 H, H-2¢, H-6¢); 6.57 (d, 1 H,
J
NH,2 9.1 Hz, NH); 5.36 (d, 1 H, J1,2 6.0 Hz, GlcA H-1); 5.36 (dd, 1
H, J3,2 8.7, J3,4 8.7 Hz, GlcA H-3); 5.27 (d, 1 H, J1,2 3.8 Hz, GlcN
4620 | Org. Biomol. Chem., 2011, 9, 4614–4625
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C-6); 52.9, 51.7 (2 ¥ CO2CH3); 52.5 (GlcN C-2); 20.7, 20.5, 20.5,
20.5, 20.4 (5 ¥ OC(O)CH3); m/z (ESI) 858.206055 ([M + Na]+.
C35H40F3NNaO19 requires 858.20443) and 3,4,6-tri-O-acetyl-2-
deoxy-2-trifluoroacetamido-a-D-glucopyranosyl-(1→4)-methyl
2,3-di-O-acetyl-a-D-glucopyranosyluronate fluoride (13) (16%, Rf
0.4); dH(300 MHz; CDCl3) 6.67 (d, 1 H, JNH,2 9.3 Hz, NH); 5.73
(dd, 1 H, J1,F 52.8 Hz, J1,2 2.7 Hz, GlcA H-1); 5.56 (dd, 1 H, J3,2
9.9 Hz, J3,4 9.3 Hz, GlcA H-3); 5.20 (d, 1 H, J1,2 3.9 Hz, GlcN
H-1); 5.18–5.10 (m, 2 H, GlcN H-3, GlcN H-4); 4.89 (ddd, 1
H, J2,F 24.0, J2,3 9.9, J2,1 2.7 Hz, GlcA H-2); 4.47 (d, 1 H, J5,4
9.6 Hz, GlcA H-5); 4.32 (dd, 1 H, J4,5 9.6, J4,3 9.3 Hz, GlcA H-4);
4.30–4.09 (m, 3 H, GlcN H-2, GlcN H-6a, GlcN H-6b); 3.82 (s,
3 H, CO2Me); 3.72–3.66 (m, 1 H, GlcN H-5); 2.12, 2.06, 2.03,
2.01, 2.01 (5 ¥ OAc); dC(75 MHz; CDCl3) 171.5, 170.8, 169.9,
169.7, 169.2 (5 ¥ OC(O)CH3); 167.9 (GlcA C-6); 157.6 (q, 38 Hz,
C(O)CF3); 115.4 (q, 286 Hz, C(O)CF3); 103.6 (d, J1,F 230 Hz,
GlcA C-1); 96.5 (GlcN C-1); 73.0 (GlcA C-4); 71.3 (d, J5,F 4 Hz,
GlcA C-5); 70.0 (GlcA C-3); 69.9 (d, J2,F 24 Hz, GlcA C-2); 69.7
(GlcN C-3); 69.0 (GlcN C-5); 67.0 (GlcN C-4); 61.0 (GlcN C-6);
53.2 (CO2CH3); 52.4 (GlcN C-2); 20.7, 20.5, 20.4, 20.4, 20.4 (5 ¥
OC(O)CH3); m/z (ESI) 700.14607 ([M + Na]+. C25H31F4NNaO16
requires 700.147111).
(C-2); 22.1 (NHC(O)CH3); m/z (ESI) 360.032980 ([M + Na]+.
C9H16NNa2O9S requires 360.033568).
Methyl 2-deoxy-2-sulfonamido-b-D-glucopyranoside sodium salt
(17). Prepared according to general procedure C from methyl
2-amino-2-deoxy-b-D-glucopyranoside23 to afford 17 (86%);
dH(300 MHz; D2O) 4.46 (d, 1 H, J1,2 8.4 Hz, H-1); 3.93 (dd, 1
H, J6a,6b 12.3, J6a,5 1.9 Hz, H-6a); 3.67 (AX m, 1 H, H-6b); 3.64
(AX m, 1 H, H-3); 3.55 (s, 3 H, OMe); 3.44–3.40 (m, 2 H, H-
4, H-5); 2.99 (dd, 1 H, J2,3 10.0, J2,1 8.4 Hz, H-2); dC(75 MHz;
D2O) 102.5 (C-1); 75.6 (C-4/5); 74.7 (C-3); 70.0 (C-4/5); 60.7 (C-
6); 59.9, 57.2 (C-2, OCH3); m/z (ESI) 318.023732 ([M + Na]+.
C7H14NNa2O8S requires 318.02355).
4¢-Nitrophenyl (2-amino-2-deoxy-a-D-glucopyranosyl)-(1→4)-
sodium b-D-glucopyranosiduronate (19). Prepared according to
general procedure D from 9 to afford 19 (65%); dH(300 MHz;
D2O) 8.21 (AB m, 2 H, H-3¢, H-5¢); 7.18 (AB m, 2 H, H-2¢, H-6¢);
5.41 (d, 1 H, J1,2 4.0 Hz, GlcN H-1); 5.22 (d, 1 H, J1,2 7.8 Hz, GlcA
H-1); 3.98 (AX m, 1 H, GlcA H-5); 3.87–3.81 (m, 2 H, GlcA H-3,
GlcA H-4); 3.77–3.72 (m, 2 H, GlcN H-6a, GlcN H-6b); 3.70–3.60
(m, 2 H, GlcN H-5, GlcA H-2); 3.51 (dd, 1 H, J3,2 10.1, J3,4 9.6 Hz,
GlcN H-3); 3.35 (dd, 1 H, J4,3 9.6, J4,5 9.6 Hz, GlcN H-3); 2.67
(dd, 1 H, J2,3 10.1, J2,1 4.0 Hz, GlcN H-2); dC(75 MHz; D2O) 174.5
(GlcA C-6); 161.6 (C-1¢); 142.5 (C-4¢); 126.0 (C-3¢, C-5¢); 116.4
(C-2¢, C-6¢); 99.2 (GlcA C-1); 99.0 (GlcN C-1); 76.7 (GlcA C-5);
76.0, 75.9, 73.6, 72.6, 72.0 (GlcN C-3, GlcN C-5, GlcA C-2, GlcA
C-3, GlcA C-4); 69.4 (GlcN C-4); 60.0 (GlcN C-6); 55.1 (GlcN
C-2); m/z (ESI) 499.116688 ([M + Na]+. C18H24N2NaO13 requires
499.117061).
3¢,5¢-Dimethoxycarbonyl (3,4,6-tri-O-acetyl-2-deoxy-2-trifluo-
roacetamido-a-D-glucopyranosyl)-(1→4)-methyl 2,3-di-O-acetyl-
b-D-glucopyranosiduronate (12). Prepared according to general
procedure B from 2 and dimethyl 5-hydroxyisophthalate.25 Flash
chromatography on silica gel (3 : 2 hexanes–ethyl acetate, Rf 0.3)
afforded 12 (61%); dH(300 MHz; CDCl3) 8.40 (t, 1 H, J4¢,2¢ = J4¢,6¢
1.4 Hz, H-4¢); 7.83 (d, 2 H, J2¢,4¢ = J6¢,4¢ 1.4 Hz, H-2¢, H-6¢); 6.52
(d, 1 H, JNH,2 9.1 Hz, NH); 5.40–5.30 (m, 2 H, GlcA H-1, GlcA
H-3); 5.27 (d, 1 H, J1,2 3.8 Hz, GlcN H-1); 5.22–5.09 (m, 3 H,
GlcN H-3, GlcN H-4, GlcA H-2); 4.54 (dd, 1 H, J4,3 8.6, J4,5
8.4 Hz, GlcA H-4); 4.34 (d, 1 H, J5,4 8.4 Hz, GlcA H-5); 4.32–4.08
(m, 3 H, GlcN H-2, GlcN H-6a, GlcN H-6b); 3.95 (s, 6 H, 2 ¥
PhCO2Me); 3.84–3.77 (m, 1 H, GlcN H-5); 3.65 (s, 3 H, CO2Me);
2.11, 2.07, 2.05, 2.04, 2.02 (5 ¥ s, 15 H, 5 ¥ OAc); dC(75 MHz;
CDCl3) 171.4, 170.7, 169.9, 169.4, 169.1 (5 ¥ OC(O)CH3); 167.8
(GlcA C-6); 165.5 (2 ¥ PhCO2Me); 157.5 (q, 38 Hz, C(O)CF3);
156.3 (C-1¢); 132.1 (C-3¢, C-5¢); 125.5 (C-4¢); 121.9 (C-2¢, C-6¢);
115.4 (q, 286 Hz, C(O)CF3); 98.5 (GlcA C-1); 96.7 (GlcN C-1);
73.8 (GlcA C-5); 73.3 (GlcA C-3); 72.9 (GlcA C-4); 71.4 (GlcA
C-2); 69.8 (GlcN C-3); 69.0 (GlcN C-5); 67.1 (GlcN C-4); 61.2
(GlcN C-6); 52.8 (CO2CH3); 52.6 (2 ¥ CO2CH3); 52.5 (GlcN C-2);
20.7, 20.5, 20.5, 20.5, 20.4 (5 ¥ OC(O)CH3); m/z (ESI) 890.194956
([M + Na]+. C35H40F3NNaO21 requires 890.19426).
4¢-Methylumbelliferyl (2-amino-2-deoxy-a-D-glucopyranosyl)-
(1→4)-sodium b-D-glucopyranosiduronate acetate (20). Prepared
according to general procedure D from a ~1 : 1 mixture of 8 and
10 to afford 20 (100% from 10); dH(300 MHz; D2O) 7.34 (d, 1
H, J5¢,6¢ 8.9 Hz, H-5¢); 6.87 (dd, 1 H, J6¢,5¢ 8.9, J6¢,8¢ 2.3 Hz, H-6¢);
6.73 (d, 1 H, J8¢,6¢ 2.3 Hz, H-8¢); 5.95 (s, 1 H, H-3¢); 5.70 (d, 1
H, J1,2 3.7 Hz, GlcN H-1); 5.12 (d, 1 H, J1,2 7.8 Hz, GlcA H-1);
4.07–4.00 (AX m, 1 H, GlcA H-5); 3.98–3.88 (m, 2 H, GlcA H-
3, GlcA H-4); 3.88–3.80 (m, 3 H, GlcN H-3, GlcN H-6a, GlcN
H-6b); 3.80–3.73 (m, 1 H, GlcN H-5); 3.72–3.65 (AX m, 1 H,
GlcA H-2); 3.49 (dd, 1 H, J4,3 9.6, J4,5 9.6 Hz, GlcN H-4); 3.24
(dd, 1 H, J2,3 10.6 Hz, J2,1 3.7 Hz, GlcN H-2); 2.18 (s, 3 H, CH3¢);
dC(75 MHz; D2O) 176.2 (GlcA C-6); 166.0 (C-2¢); 161.2, 157.9,
155.2 (C-4¢, C-7¢, C-8a¢); 128.4 (C-5¢); 116.6 (C-4a¢); 115.8 (C-6¢);
112.9 (C-3¢); 105.1 (C-8¢); 101.2 (GlcA C-1); 97.8 (GlcN C-1);
78.4 (GlcA C-5); 77.9, 77.7 (GlcA C-3, GlcA C-4); 74.8 (GlcA
C-2); 74.2 (GlcN C-5); 72.0 (GlcN C-3); 71.1 (GlcN C-4); 61.7
(GlcN C-6); 56.3 (GlcN C-2); 19.8 (CH3¢); m/z (ESI) 512.143037
([M - Na]-. C22H26NO13 requires 512.140962) and a mixture (900
mg) of sodium acetate and 2-amino-2-deoxy-a-D-glucopyranosyl-
(1→4)-sodium b-D-glucopyranosyluronate acetate.27
Methyl 2-acetamido-2-deoxy-6-O-sulfo-b-D-glucopyranoside,
sodium salt (16). To a solution of 1526 (89 mg, 378 mmol) in
DMF (2 mL) was added SO3·Py complex (120 mg, 757 mmol).
◦
The mixture was stirred at rt for 16 h, and then cooled to 0 C,
methanol (1 mL) was added and the mixture was concentrated.
4¢-[3¢¢-(Sodium prop-2¢¢(E)-enoate)]phenyl (2-amino-2-deoxy-a-
D-glucopyranosyl)-(1→4)-sodium b-D-glucopyranosiduronate ac-
etate (21). Prepared according to general procedure D from 11
to afford 21 (77%); dH(300 MHz; D2O) 7.50 (d, 2 H, J3¢,2¢ 8.8 Hz,
H-3¢); 7.28 (d, 1 H, J3¢¢,2¢¢ 16.0 Hz, H-3¢¢); 7.04 (d, 2 H, J2¢,3¢ 8.8 Hz,
H-2¢); 6.35 (d, 1 H, J2¢¢,3¢¢ 16.0 Hz, H-2¢¢); 5.53 (d, 1 H, J1,2 3.7 Hz,
GlcN H-1); 5.07 (d, 1 H, J1,2 7.8 Hz, GlcA H-1); 3.95–3.90 (m, 1
H, GlcA H-5); 3.84–3.80 (m, 1 H, GlcA H-4); 3.77–3.54 (m, 6 H,
R
The product was purified on Sephadexꢀ LH-20, eluting with 4 : 1
methanol–H2O afforded 16 (100 mg, 78%); dH(300 MHz; D2O)
4.40 (d, 1 H, J1,2 8.4 Hz, H-1); 4.29 (dd, 1 H, J6a,6b 11.2, J6a,5
2.1 Hz, H-6a); 4.17 (dd, 1 H, J6b,6a 11.2, J6b,5 5.1 Hz,H-6b); 3.68–
3.58 (m, 2 H, H-2, H-5); 3.54–3.42 (m, 5 H, H-3, H-4, OMe); 1.98
(s, 3 H, NAc); dC(75 MHz; D2O) 174.6 (NHC(O)CH3); 102.0 (C-
1); 73.7, 73.6, 71.7 (C-3, C-4, C-5); 66.9 (C-6); 57.1 (OCH3); 55.3
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GlcN H-3, GlcN H-5, GlcN H-6a, GlcN H-6b, GlcA H-2, GlcA
H-3); 3.38 (dd, 1 H, J4,3 9.5 Hz, J4,5 9.5 Hz, GlcN H-4); 2.95 (dd,
1 H, J2,3 10.3 Hz, J2,1 3.7 Hz, GlcN H-2); dC(75 MHz; D2O) 181.5
(OC(O)CH3); 175.9, 174.5 (GlcA C-6, C-1¢¢); 157.4 (C-1¢); 140.2
(C-3¢¢); 130.6 (C-4¢); 129.3 (C-3¢, C-5¢); 122.7 (C-2¢¢); 116.6 (C-2¢,
C-6¢); 99.8 (GlcA C-1); 97.1 (GlcN C-1); 76.4 (GlcA C-5); 76.0,
75.8 (GlcA C-3, GlcA C-4); 72.8, 72.1, 71.5 (GlcN C-3, GlcN C-5,
GlcA C-2); 69.2 (GlcN C-4); 59.8 (GlcN C-6); 54.5 (GlcN C-2);
23.2 (OC(O)CH3).
4¢-Methylumbelliferyl (2-acetamido-2-deoxy-a-D-glucopyranos-
yl)-(1→4)-sodium b-D-glucopyranosiduronate (25). Prepared ac-
cording to general procedure E from 20 to afford 25 (100%);
dH(300 MHz; D2O) 7.41 (d, 1 H, J5¢,6¢ 8.9 Hz, H-5¢); 6.92 (dd, 1 H,
J
6¢,5¢ 8.9, J6¢,8¢ 2.4 Hz, H-6¢); 6.81 (d, 1 H, J8¢,6¢ 2.4 Hz, H-8¢); 6.01 (q,
1 H, J3¢,Me¢ 1.1 Hz, H-3¢); 5.45 (d, 1 H, J1,2 3.7 Hz, GlcN H-1); 5.15
(d, 1 H, J1,2 7.8 Hz, GlcA H-1); 4.04–3.96 (AX m, 1 H, GlcA H-5);
3.91 (dd, 1 H, J2,3 10.7, J2,1 3.7 Hz, GlcN H-2); 3.90–3.84 (m, 2 H,
GlcA H-3, GlcA H-4); 3.83–3.80 (m, 2 H, GlcN H-6a, GlcN H-
6b); 3.80–3.73 (m, 2 H, GlcN H-3, GlcN H-5); 3.69–3.62 (AX m, 1
H, GlcA H-2); 3.49 (dd, 1 H, J4,3 9.5, J4,5 9.5 Hz, GlcN H-4); 2.22
(d, 3 H, JMe¢,3¢ 1.1 Hz, CH3¢); 2.07 (s, 3 H, NAc); dC(75 MHz; D2O)
176.9 (GlcA C-6); 176.9 (NHC(O)CH3); 166.6 (C-2¢); 161.8, 158.5,
155.9 (C-4¢, C-7¢, C-8a¢); 129.1 (C-5¢); 117.3 (C-4a¢); 116.4 (C-6¢);
113.5 (C-3¢); 105.7 (C-8¢); 101.7 (GlcA C-1); 99.6 (GlcN C-1); 79.4
(GlcA C-5); 78.7, 78.3 (GlcA C-3, GlcA C-4); 75.6 (GlcA C-2);
74.4, 73.2 (GlcN C-3, GlcN C-5); 72.2 (GlcN C-4); 62.6 (GlcN C-
6); 56.2 (GlcN C-2); 24.5 (NHC(O)CH3); 20.4 (CH3¢); m/z (ESI)
554.151152 ([M - Na]-. C24H28NO14 requires 554.151525).
3¢,5¢-Disodium carbonyl (2-amino-2-deoxy-a-D-glucopyranosyl)-
(1→4)-sodium b-D-glucopyranosiduronate acetate (22). Prepared
according to general procedure D from 12 to afford 22 (97%);
dH(300 MHz; CDCl3) 7.93 (bt, 1 H, H-4¢); 7.58 (bd, 2 H, H-2¢, H-
6¢); 5.62 (d, 1 H, J1,2 3.7 Hz, GlcN H-1); 5.10 (d, 1 H, J1,2 7.8 Hz,
GlcA H-1); 4.03–3.97 (AX m, 1 H, GlcA H-5); 3.89–3.79 (m, 2
H, GlcA H-3, GlcA H-4); 3.79–3.71 (m, 3 H, GlcN H-3, GlcN
H-6a, GlcN H-6b); 3.71–3.64 (m, 1 H, GlcN H-5); 3.61 (dd, 1 H,
J2,3 8.2, J2.1 7.8 Hz, GlcA H-2); 3.42 (dd, 1 H, J3,2 9.5, J3,4 9.5 Hz,
GlcN H-4); 3.21 (dd, 1 H, J2,3 9.5, J2,1 3.7 Hz, GlcN H-2); 1.84 (s,
3 H, OAc); dC(75 MHz; CDCl3) 183.3 (OC(O)CH3); 176.4, 176.1
(GlcA C-6, 2 ¥ PhCO2Na); 158.2 (C-1¢); 139.9 (C-3¢, C-5¢); 125.7
(C-4¢); 121.2 (C-2¢, C-6¢); 102.1 (GlcA C-1); 97.4 (GlcN C-1); 78.1,
77.7, 77.6 (GlcA C-3, GlcA C-4, GlcA C-5); 74.8 (GlcA C-2); 74.1
(GlcN C-5); 71.6 (GlcN C-3); 70.9 (GlcN C-4); 61.6 (GlcN C-6);
56.0 (GlcN C-2); 25.2 (OC(O)CH3).
4¢-Methylumbelliferyl
(2-deoxy-2-sulfonamido-a-D-glucopy-
ranosyl)-(1→4)-sodium b-D-glucopyranosiduronate, sodium salt
(26). Prepared according to general procedure C from 20 to
afford 26 (67%); dH(300 MHz; D2O) 7.52 (d, 1 H, J5¢,6¢ 8.9 Hz,
H-5¢); 7.00 (dd, 1 H, J6¢,5¢ 8.9, J6¢,8¢ 2.4 Hz, H-6¢); 6.92 (d, 1 H,
J8¢,6¢ 2.4 Hz, H-8¢); 6.09 (q, 1 H, J3¢,Me¢ 1.1 Hz, H-3¢); 5.69 (d, 1
H, J1,2 3.7 Hz, GlcN H-1); 5.23 (d, 1 H, J1,2 7.9 Hz, GlcA H-1);
4.07–3.89 (m, 3 H, GlcA H-3, GlcA H-4, GlcA H-5); 3.85–3.80
(m, 2 H, GlcN H-6a, GlcN H-6b); 3.79–3.62 (m, 3 H, GlcN H-3,
GlcN H-5, GlcA H-2); 3.51 (dd, 1 H, J4,3 9.5, J4,5 9.5 Hz, GlcN
H-4); 3.27 (dd, 1 H, J2,3 10.3 Hz, J2,1 3.7 Hz, GlcN H-2); 2.31 (d,
3 H, JMe¢,3¢ 1.1 Hz, CH3¢); dC(75 MHz; D2O) 174.4 (GlcA C-6);
164.3 (C-2¢); 159.3, 156.1, 153.5 (C-4¢, C-7¢, C-8a¢); 126.6 (C-5¢);
114.9 (C-4a¢); 113.9 (C-6¢); 111.1 (C-3¢); 103.3 (C-8¢); 99.2 (GlcA
C-1); 97.4 (GlcN C-1); 76.8 (GlcA C-5); 76.2, 75.9 (GlcA C-3,
GlcA C-4); 72.4, 71.6, 71.2 (GlcN C-3, GlcN C-5, GlcA C-2);
69.6 (GlcN C-4); 60.0 (GlcN C-6); 58.0 (GlcN C-2); 17.9 (CH3¢);
m/z (ESI) 592.095721 ([M - 2 Na + H]-. C22H26NO16S requires
592.097775).
4¢-Nitrophenyl (2-acetamido-2-deoxy-a-D-glucopyranosyl)-(1→
4)-sodium b-D-glucopyranosiduronate (23). Prepared according
to general procedure E from 19 to afford 23 (48%); dH(300 MHz;
D2O) 8.29–8.24 (AB m, 2 H, H-3¢, H-5¢); 7.27–7.22 (m, 2 H, H-2¢,
H-6¢); 5.44 (d, 1 H, J1,2 4.0 Hz, GlcN H-1); 5.33 (d, 1 H, J1,2 7.8 Hz,
GlcA H-1); 4.22 (d, 1 H, J5,4 9.3 Hz, GlcA H-5); 3.96–3.63 (m, 8
H, GlcN H-2, GlcN H-3, GlcN H-5, GlcN H-6a, GlcN H-6b,
GlcA H-2, GlcA H-3, GlcA H-4); 3.54 (dd, 1 H, 10.1, 8.8 Hz,
GlcN H-4); 2.06 (s, 3 H, NAc); dC(75 MHz; D2O) 174.4 (GlcA
C-6); 172.0 (NHC(O)CH3); 161.4 (C-1¢); 142.7 (C-4¢); 126.0 (C-
3¢, C-5¢); 116.5 (C-2¢, C-6¢); 99.2 (GlcA C-1); 97.5 (GlcN C-1);
75.8, 75.4 (GlcA C-3, GlcA C-4); 74.4 (GlcA C-5); 72.8, 72.3, 70.5
(GlcN C-3, GlcN C-5, GlcA C-2); 69.3 (GlcN C-4); 59.8 (GlcN
C-6); 53.5 (GlcN C-2); 21.9 (NHC(O)CH3); m/z (ESI) 517.131970
([M - Na]-. C20H25N2O14 requires 517.131129).
4¢-[3¢¢-(Sodium prop-2¢¢(E)-enoate)]phenyl (2-acetamido-2-
deoxy-a-D-glucopyranosyl)-(1→4)-sodium b-D-glucopyranosi-
duronate (27). Prepared according to general procedure E from
21 to afford 27 (100%); dH(300 MHz; D2O) 7.51 (d, 2 H, J3¢,2¢
8.8 Hz, H-3¢); 7.37 (d, 1 H, J3¢¢,2¢¢ 16.0 Hz, H-3¢¢); 7.04 (d, 2 H,
J2¢,3¢ 8.8 Hz, H-2¢); 6.34 (d, 1 H, J2¢¢,3¢¢ 16.0 Hz, H-2¢¢); 5.37 (d, 1
H, J1,2 3.7 Hz, GlcN H-1); 5.08 (d, 1 H, J1,2 7.8 Hz, GlcA H-1);
3.91–3.64 (m, 8 H, GlcN H-2, GlcN H-3, GlcN H-5, GlcN H-6a,
GlcN H-6b, GlcA H-3, GlcA H-4, GlcA H-5); 3.54 (dd, 1 H, J2,3
8.7, J2,1 7.8 Hz, GlcA H-2); 3.42 (dd, 1 H, J4,3 9.5, J4,5 9.5 Hz,
GlcN H-4); 1.99 (s, 3 H, NAc); dC(75 MHz; D2O) 175.0, 174.5,
174.3 (GlcA C-6, C-1¢¢, NHC(O)CH3); 157.6 (C-1¢); 140.9 (C-3¢¢);
129.6 (C-4¢); 129.4 (C-3¢, C-5¢); 121.8 (C-2¢¢); 116.6 (C-2¢, C-6¢);
99.6 (GlcA C-1); 96.9 (GlcN C-1); 76.8 (GlcA C-5); 76.3, 75.6
(GlcA C-3, GlcA C-4); 73.1 (GlcA C-2); 71.8, 70.6 (GlcN C-3,
GlcN C-5); 69.6 (GlcN C-4); 60.0 (GlcN C-6); 53.6 (GlcN C-2);
21.8 (NHC(O)CH3); m/z (ESI) 542.151100 ([M - 2 Na + H]-.
C23H28NO14 requires 542.151525).
4¢-Nitrophenyl (2-deoxy-2-sulfonamido-a-D-glucopyranosyl)-
(1→4)-sodium b-D-glucopyranosiduronate, sodium salt (24). Pre-
pared according to general procedure C from 19 to afford 24 (32%);
dH(300 MHz; D2O) 8.26–8.23 (AB m, 2 H, H-3¢, H-5¢); 7.24–7.20
(m, 2 H, H-2¢, H-6¢); 5.68 (d, 1 H, J1,2 3.6 Hz, GlcN H-1); 5.30
(d, 1 H, J1,2 8.1 Hz, GlcA H-1); 4.05–3.90 (m, 3 H, GlcA H-3,
GlcA H-4, GlcA H-5); 3.80 (m, 2 H, GlcN H-6a, GlcN H-6b);
3.75–3.66 (m, 2 H, GlcN H-5, GlcA H-2); 3.63 (dd, 1 H, J3,2 10.2,
J3,4 9.6 Hz, GlcN H-3); 3.50 (dd, 1 H, J4,3 9.6, J4,5 9.6 Hz, GlcN
H-4); 3.26 (dd, 1 H, J2,3 10.2, J2,1 3.6 Hz, GlcN H-2); dC(75 MHz;
D2O) 174.4 (GlcA C-6); 162.0 (C-1¢); 142.9 (C-4¢); 126.5 (C-3¢,
C-5¢); 116.8 (C-2¢, C-6¢); 99.6 (GlcA C-1); 97.7 (GlcN C-1); 77.1,
76.4, 76.3 (GlcA C-3, GlcA C-4, GlcA C-5); 72.8, 72.0, 71.5 (GlcN
C-3, GlcN C-5, GlcA C-2); 69.9 (GlcN C-4); 60.4 (GlcN C-6); 58.4
(GlcN C-2); m/z (ESI) 577.059334 ([M - Na]-. C18H22N2NaO16S
requires 577.059315).
4622 | Org. Biomol. Chem., 2011, 9, 4614–4625
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4¢-[3¢¢-(Sodium prop-2¢¢(E)-enoate)]phenyl (2-deoxy-2-sulfon-
amido-a-D-glucopyranosyl)-(1→4)-sodium b-D-glucopyrano-
(CIP) were purchased from New England Biolabs (Ipswich,
R
R
MA, USA). Bactoꢀ-tryptone, Bactoꢀ-yeast extract, and agarose
siduronate, sodium salt (28). Prepared according to general
procedure C from 21 to afford 28 (76%); dH(300 MHz; D2O)
7.57–7.51 (AX m, 2 H, H-3¢); 7.31 (d, 1 H, J3¢¢,2¢¢ 16.0 Hz, H-3¢¢);
7.10–7.06 (AX m, 2 H, H-2¢); 6.38 (d, 1 H, J2¢¢,3¢¢ 16.0 Hz, H-2¢¢);
5.63 (d, 1 H, J1,2 3.7 Hz, GlcN H-1); 5.15 (d, 1 H, J1,2 8.0 Hz,
GlcA H-1); 3.98–3.81 (m, 3 H, GlcA H-3, GlcA H-4, GlcA H-5);
3.78–3.53 (m, 5 H, GlcN H-3, GlcN H-5, GlcN H-6a, GlcN H-6b,
GlcA H-2); 3.45 (dd, 1 H, 9.7, 9.3 Hz, GlcN H-4); 3.22 (dd, 1 H,
J2,3 10.3, J2,1 3.7 Hz, GlcN H-2); dC(75 MHz; D2O) 175.9, 174.6
(GlcA C-6, C-1¢¢); 157.4 (C-1¢); 140.2 (C-3¢¢); 130.1 (C-4¢); 129.3
(C-3¢, C-5¢); 122.8 (C-2¢¢); 116.7 (C-2¢, C-6¢); 99.8 (GlcA C-1); 97.3
(GlcN C-1); 76.7, 76.1, 76.0 (GlcA C-3, GlcA C-4, GlcA C-5);
72.5, 71.6, 71.2 (GlcN C-3, GlcN C-5, GlcA C-2); 69.6 (GlcN
C-4); 60.0 (GlcN C-6); 58.0 (GlcN C-2); m/z (ESI) 580.099737
([M - 3 Na + 2 H]-. C21H26NO16S requires 580.097774).
were purchased from Oxoid (Basingstoke, Hampshire, UK). The
R
R
QIAprepꢀ spin miniprep kit, QIAquickꢀ gel extraction kit, and
the QIAGEN plasmid midi kit were supplied by QIAGEN Pty
Ltd (Doncaster, VIC, Australia). All custom made primers were
supplied by Bio-strategy Ltd (Paddington, QLD, Australia) and
GeneWorks Pty Ltd (Thebarton, South Australia). All sequencing
was carried out by the Australian Genome Research Facility
R
(AGRF, QLD, Australia). pCR-Blunt and the Zero Bluntꢀ PCR
cloning kit were purchased from Invitrogen (Carlsbad, CA, USA).
The pAcGP67-A baculovirus transfer vector and the pAcUW51
dual expression vector were purchased from BD Biosciences
(Franklin Lakes, NJ, USA). Microcon YM-10 ultrafiltration
devices for centrifugation were obtained from Millipore (Bedford,
MA, USA).
[3H]-HS was prepared by Progen Pharmaceuticals Ltd, by de-
N-acetylation of HS with hydrazine sulfate and re-N-acetylation
with [3H]-acetic anhydride (16 mmol, 8 mCi).7a The concentrations
of the [3H]-HS preparations were accurately determined using the
dimethylmethylene blue assay for GAGs.28 Unlabelled HS from
the same source was used as standard.
3¢,5¢-Disodium
carbonyl
(2-acetamido-2-deoxy-a-D-
glucopyranosyl)-(1→4)-sodium b-D-glucopyranosiduronate (29).
Prepared according to general procedure E from 22 to afford 29
(61%); dH(300 MHz; CDCl3) 7.98 (bt, 1 H, H-4¢); 7.63 (bd, 2
H, H-2¢, H-6¢); 5.43 (d, 1 H, J1,2 3.7 Hz, GlcN H-1); 5.14 (d, 1
H, J1,2 7.9 Hz, GlcA H-1); 4.03–3.97 (AX m, 1 H, GlcA H-5);
3.89 (dd, 1 H, J2,3 10.7, J2,1 3.7 Hz, GlcN H-2); 3.87–3.82 (m, 2
H, GlcA H-3, GlcA H-4); 3.82–3.78 (m, 2 H, GlcN H-6a, GlcN
H-6b); 3.78–3.69 (m, 2 H, GlcN H-3, GlcN H-5); 3.61 (dd, 1
H, J2,3 8.4, J2.1 7.9 Hz, GlcA H-2); 3.47 (dd, 1 H, J3,2 9.4, J3,4
9.4 Hz, GlcN H-4); 2.04 (s, 3 H, NAc); dC(75 MHz; CDCl3)
176.7, 174.4, 174.3 (GlcA C-6, NHC(O)CH3, 2 ¥ PhCO2Na);
156.4 (C-1¢); 138.1 (C-3¢, C-5¢); 123.7 (C-4¢); 119.3 (C-2¢, C-6¢);
100.4 (GlcA C-1); 96.9 (GlcN C-1); 76.8 (GlcA C-5); 76.2, 75.7
(GlcA C-3, GlcA C-4); 73.2 (GlcA C-2); 71.8, 70.6 (GlcN C-3,
GlcN C-5); 69.6 (GlcN C-4); 60.0 (GlcN C-6); 53.6 (GlcN C-2);
21.9 (NHC(O)CH3); m/z (ESI) 560.128451 ([M - 3 Na + 2 H]-.
C22H26NO16 requires 560.125706).
Cloning and expression of recombinant human heparanase
Heparanase cDNA was provided by Dr Mark Hulett and Prof.
Chris Parish, (ANU, Canberra, Australia). The strategy of
McKenzie et al. was then followed and proceeded essentially
as described.19 Insect cell expression was carried out at the
Protein Expression Facility, Institute for Molecular Biosciences,
University of Queensland. The expression protocol published by
McKenzie et al.19 was followed. The resultant crude supernatants
containing heparanase were kept at 4 ◦C until purification, which
was carried out at Progen Pharmaceuticals Ltd, Darra, QLD. Me-
dia was clarified by centrifuging at 17 700 g for 30 min at 4 ◦C and
the pH was adjusted by the addition of 10 mM sodium phosphate,
pH 7.0. The material was applied to a 145 mL SP Sepharose FF
(GE Healthcare) column. The column was then eluted with a 1.3 L
gradient of 0–0.75 M NaCl in 10 mM sodium phosphate, pH 7.0
buffer. Heparanase fractions were detected by electrophoresis and
pure heparanase was pooled and exchanged into 10 mM sodium
phosphate, pH 7.0 buffer. The amount of heparanase purified from
1 L of insect cell culture media was 0.4 mg.
3¢,5¢-Disodium carbonyl (2-deoxy-2-sodium sulfonamido-a-D-
glucopyranosyl)-(1→4)-sodium b-D-glucopyranosiduronate, sodium
salt (30). Prepared according to general procedure C from 22 to
afford 30 (39 mg, 65%); dH(300 MHz; CDCl3) 8.03 (bs, 1 H, H-4¢);
7.68 (bd, 2 H, H-2¢, H-6¢); 5.66 (d, 1 H, J1,2 3.7 Hz, GlcN H-1);
5.20 (d, 1 H, J1,2 7.9 Hz, GlcA H-1); 4.07–3.85 (m, 3 H, GlcA
H-3, GlcA H-4, GlcA H-5); 3.83–3.78 (m, 2 H, GlcN H-6a, GlcN
H-6b); 3.76–3.59 (m, 3 H, GlcN H-3, GlcN H-5, GlcA H-2); 3.48
(dd, 1 H, J4,3 9.5, J4,5 9.5 Hz, GlcN H-4); 3.25 (dd, 1 H, J2,3 10.3,
J2.1 3.7 Hz, GlcN H-2); dC(75 MHz; CDCl3) 174.6, 174.3 (GlcA
C-6, 2 ¥ PhCO2Na); 156.3 (C-1¢); 138.0 (C-3¢, C-5¢); 123.7 (C-4¢);
119.2 (C-2¢, C-6¢); 100.2 (GlcA C-1); 97.3 (GlcN C-1); 76.6, 76.1,
75.9 (GlcA C-3, GlcA C-4, GlcA C-5); 72.5 (GlcA C-2); 71.5,
71.1 (GlcN C-3, GlcN C-5); 69.5 (GlcN C-4); 59.9 (GlcN C-6);
57.9 (GlcN C-2); m/z (ESI) 598.074728 ([M - 4 Na+ + 3 H]-.
C20H24NO18S requires 598.071956).
Heparanase activity assay
The putative heparanase substrates were assayed against recom-
binant human heparanase as follows; reaction mixtures (50 mL)
were prepared in 96-well plates containing putative substrate (5.0
mM), BSA (0.1 mg mL-1), and heparanase (1.2 mg mL-1) in 60 mM
sodium acetate buffer, pH 5.0. The samples were incubated at 37 ◦C
with shaking at 400 rpm for 4 h–3 d. Reactions were stopped
by the addition of 200 mL of 0.2 M glycine buffer, pH 10.7.
Fluorescence was measured using a VICTOR3 Multilabel Plate
Reader at emission and excitation wavelengths of 460 and 355 nm,
respectively. Absorbance was measured using a VICTOR3 Multi-
label Plate Reader at 405 nm for 4-nitrophenol, and at 355 nm for
3-nitrophenol, 4-hydroxycinnamic acid, and 5-hydroxyisophthalic
acid. The assays were performed at least in duplicate and were
Materials
Ampicillin was purchased from Astral Scientific (Sydney, NSW,
R
Australia). One Shotꢀ TOP10 Competent Cells were purchased
from Invitrogen (Carlsbad, CA, USA). Restriction enzymes and
buffers, DNA ladders, and Calf Intestinal Alkaline Phosphatase
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corrected for background fluorescence/absorbance (reactions
were performed in the absence of heparanase).
8 (a) TaKaRa Bio, Heparan degrading enzyme assay kit. Available from:
<http://takaramirusbio.com>; (b) C. Tokuda, L. Jacquemart, M.
Pre´audat, Development of a homogeneous heparanase assay using
HTRF technology, Cisbio international technical document. Available
from: <http://www.htrf-assays.com>.
9 E. Hammond, C. P. Li and V. Ferro, Development of a colorimetric
assay for heparanase activity suitable for kinetic analysis and inhibitor
screening, Anal. Biochem., 2010, 396, 112–116.
10 A. Bisio, A. Mantegazza, E. Urso, A. Naggi, G. Torri, C. Viskov and B.
Casu, High-performance liquid chromatographic/mass spectrometric
studies on the susceptibility of heparin species to cleavage by hep-
aranase, Semin. Thromb. Hemost., 2007, 33, 488–495.
Stability assay
A 50 mL reaction mixture was prepared in a 96-well plate that
contained 26 (5 mM), HS (4 mM), and BSA (0.1 mg mL-1) in
60 mM sodium acetate buffer, pH 5.0. The plate was incubated at
37 ◦C with shaking at 400 rpm for 3 d and then analysed by low
resolution mass spectrometry.
11 For example see: (a) A. Adibekian, P. Bindscha¨dler, M. S. M. Timmer,
C. Noti, N. Schu¨tzenmeister and P. H. Seeberger, De novo synthesis of
uronic acid building blocks for assembly of heparin oligosaccharides,
Chem.–Eur. J., 2007, 13, 4510–4522; (b) L. Poletti and L. Lay, Chemical
contributions to understanding heparin activity: synthesis of related
sulfated oligosaccharides, Eur. J. Org. Chem., 2003, 16, 2999–3024;
(c) B. K. S. Yeung, P. Y. C. Chong and P. A. Petillo, Synthesis of
glycosaminoglycans, J. Carbohydr. Chem., 2002, 21, 799–865.
12 H. A. Orgueira, A. Bartolozzi, P. Schell, R. Litjens, E. R. Palmacci
and P. H. Seeberger, Modular synthesis of heparin oligosaccharides,
Chem.–Eur. J., 2003, 9, 140–169.
Acknowledgements
We wish to thank Dr Mark Hulett and Prof. Chris Parish,
(ANU, Canberra, Australia) for the donation of full-length
human heparanase cDNA, Dr Edward Hammond (Progen) for
the preparation of [3H]-HS and technical assistance, and the
Australian Research Council for an APA(I) Linkage grant and
associated PhD scholarship to AP.
13 A. G. Pearson, M. J. Kiefel, V. Ferro and M. von Itzstein, Towards
the synthesis of aryl glucuronides as potential heparanase probes. An
interesting outcome in the glycosidation of glucuronic acid with 4-
hydroxycinnamic acid, Carbohydr. Res., 2005, 340, 2077–2085.
14 For example see: (a) T. Sawada, S. Fujii, H. Nakano, S. Ohtake, K.
Kimata and O. Habuchi, Synthesis of sulfated phenyl 2-acetamido-
2-deoxy-D-galactopyranosides. 4-O-Sulfated phenyl 2-acetamido-2-
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