Synthesis of a trisulfated heparan sulfate disaccharide analog and its use as a template for preliminary molecular imprinting studies

Article abstract of DOI:10.1016/j.carres.2007.12.020

A heparan sulfate disaccharide analog was synthesized by a multistep route. This synthesis was designed in such a way that one intermediate could be selectively deprotected to provide versatility during both synthesis and homologation of heparan sulfate related polysaccharides. Non-covalent imprinted polymers were prepared by using the synthesized disaccharide as a template and a primary amine functionalized acrylate as the key functional monomer suitable for specific sulfated sugar recognition. The binding of related sugars to the imprinted and non-imprinted polymers and the binding of template to the chemically modified polymers have been also investigated.

Full text of DOI:10.1016/j.carres.2007.12.020

Available online at www.sciencedirect.com
Carbohydrate Research 343 (2008) 587–595
Synthesis of a trisulfated heparan sulfate disaccharide analog and
its use as a template for preliminary molecular imprinting studies
Yasunori Ikeda,a Fernando Sineriz,a Laurent Bultel,b Eric Grand,b
˜
b,
a,
*
*
´
Jose Kovensky and Dulce Papy-Garcia
aLaboratoire CRRET-UMR CNRS 7149, Faculte´ des Sciences et Technologie, Universite´ Paris 12 Val de Marne, 61,
Av. du Ge´ne´ral de Gaulle, 94010 Cre´teil Cedex, France
bLaboratoire des Glucides, UMR CNRS 6219, Universite´ de Picardie-Jules Verne, 33, rue Saint Leu, F-80039 Amiens, France
Received 1 April 2007; received in revised form 19 December 2007; accepted 19 December 2007
Available online 28 December 2007
Abstract—A heparan sulfate disaccharide analog was synthesized by a multistep route. This synthesis was designed in such a way
that one intermediate could be selectively deprotected to provide versatility during both synthesis and homologation of heparan
sulfate related polysaccharides. Non-covalent imprinted polymers were prepared by using the synthesized disaccharide as a template
and a primary amine functionalized acrylate as the key functional monomer suitable for specific sulfated sugar recognition. The
binding of related sugars to the imprinted and non-imprinted polymers and the binding of template to the chemically modified poly-
mers have been also investigated.
Ó 2007 Elsevier Ltd. All rights reserved.
Keywords: Heparan sulfate; Disaccharide; Imprinting; Molecular recognition
1. Introduction
rides, is therefore essential to understand the relation-
ship between HS structures and their biological
functions. However, most of the information related to
the HS structure for protein specificity at the molecular
level remains obscure because of the HS structure hetero-
geneity.4 Although a number of antibodies that recog-
nize sulfated sugars as HS have already been developed,
only few is known about their specificity at the fine
structure level.5–7 Furthermore, sequencing, analytical,
and chemical methods in this area are extremely labo-
rious and complex and require approaches that should
include the development of new recognition entities
capable to detect, and even quantify, well characterized
fractions of HS in biological extracts. Advances in this
direction will result in a significant progress in the area
of glycomics.
Heparan sulfates (HS) are a class of naturally occurring
polysaccharides recognized to be crucial regulators of
several biological processes such as cell proliferation,
differentiation, and migration. It is now wholly accepted
that the biological activities of HS are largely related to
the sulfation pattern of their sugar chains.1,2 The basic
disaccharide unit of HS, composed of an uronic acid
(L-iduronic [IdoA] or D-glucuronic [GlcA]) (1?4)-
linked to a N-acetyl-D-glucosamine (GlcNAc), can be
differently sulfated to display specific sequences that
bind to particular proteins named heparin binding pro-
teins (HBP).3 These saccharide–protein interactions are
at the basis of a number of HS fundamental biological
roles.4 The knowledge of the fine structural features of
these polysaccharides, or of their derived oligosaccha-
We have recently reported the applicability of a new
strategy for the recognition of sulfated sugars based
on Molecular Imprinting Technologies by a non-cova-
lent imprinting process using glucose-6-sulfate [Glc(6S)]
as a template.8 Molecular Imprinting is a methodology
*
Corresponding authors. Tel.: +33 145177081; fax: +33 145171816
(D.P.-G.); e-mail addresses: jose.kovensky@u-picardie.fr; papy@
univ-paris12.fr
0008-6215/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2007.12.020

588
Y. Ikeda et al. / Carbohydrate Research 343 (2008) 587–595
that makes possible the introduction of recognition sites
into highly crosslinked polymers via templated-directed
assembly of functionalized monomers in a polymeric-
forming mixture. This enables the formation of discrete
cavities with a precise spatial arrangement of functional
groups to provide specific interactions with the template
when rebinding.9,10 This technology was recently used
for a wide range of applications including chromato-
graphic purification,11,12 solid-phase extraction,13,14
reaction catalysis,15 and some biological systems such
as enzyme mimetics16 or biomimetic sensors.17,18 In this
report, we show the chemical synthesis of a trisulfated
disaccharide analog of a HS disaccharide, and by using
this compound as a template, we describe our prelimin-
ary results on the synthesis of a series of molecularly im-
printed polymers (MIPs) and of their corresponding
NIPs (non-imprinted polymers) and examine their abil-
ities to bind the template and other related sugars.
sidered for the synthesis of the oligosaccharide binding
to antithrombin III and/or to blood coagulation factor
Xa without losing its biological activity.19
2.1. Synthesis of disaccharide 1
We prepared 4,6-di-O-p-methoxybenzylidene acetal 3
(75%) from the known allyl 3-O-benzyl-b-D-glucopyran-
oside20 2 (Scheme 1). After purification, 3 was selectively
reduced with LiAlH4–AlCl3 to afford 4-O-p-methoxy-
benzyl derivative 4 in 38% yield and 47% of recovered
3. Then, the selective oxidation of the primary hydroxyl
group of 4 with TEMPO furnished the expected acid
that was immediately benzylated to uronate 5 in 69%
yield (two steps). Afterwards, an acetylation of the free
2-OH was performed and the totally protected deriva-
tive 6 isolated. Deprotection of the anomeric position
has been accomplished in a classical two steps transfor-
mation. Allyl isomerization of 6 with (Ph3P)3RhCl–
DABCO followed by a reaction with HgCl2–HgO
smoothly led to compound 7 as a mixture of the a and
b anomers in 83% yield (two steps). Finally, the reaction
of trichloroacetonitrile–DBU with the previously ob-
tained hemiacetal 7 offered the glycosyl donor 8 in a very
good yield (93%). As a confirmation of compound 8
structure, we have attributed the 160.6 ppm signal of
the 13C NMR spectrum to the imidate group carbon
introduced at position 1 of the glucose moiety.
2. Results and discussion
The trisulfated disaccharide 1 (Chart 1), considered for
imprinting studies, is an analog of the rare HS disaccha-
ride unit GlcA(2S)b1,4Glc(NS,6S) where the N-sulfated
glucosamine residue Glc(NS,6S) was replaced by the
2-O-sulfated glucose moiety Glc(2S,6S). This particular
structural modification that reduces considerably the
number of required synthetic steps was previously con-
The glycosyl acceptor 9 (Scheme 2) was easily ob-
tained by selective 6-O-acetylation of known allyl-2-
O-acetyl-3-O-benzyl-b-D-glucopyranoside21 with acetyl
chloride in the presence of pyridine (98%). Thus reaction
of the glycosyl donor 8 with alcohol 9, using TMSOTf as
a promoter, led to the key disaccharide 10 in excellent
yield (96%). The b-anomeric selectivity of this reaction
has been confirmed by the 1H NMR spectrum of 10 that
NaO3SO
NaOOC
O
HO
HO
O
O
HO
O
NaO3SO
NaO3SO
1
Chart 1. Trisulfated disaccharide.
0
0
displays a coupling constant J1 ;2 of 8.0 Hz characteris-
PMP
HO
O
HO
O
O
O
i)
ii)
HO
PMBO
O
BnO
OAll
OAll
OAll
BnO
BnO
OAll
OH
OH
OH
2
4
3
7
iii)
BnOOC
BnOOC
BnOOC
PMBO
O
O
O
PMBO
BnO
PMBO
BnO
vi)
v)
OH
BnO
AcO
OAc
OR
OC(NH)CCl3
R = H
5
6
iv)
8
R = Ac
Scheme 1. Donor synthesis. Reagents and conditions: (i) p-MeOPhCH(OMe)2, PTSA, DMF, 75%; (ii) LiAlH4, AlCl3, CH2Cl2–Et2O, 38% and 47%
of recovered 3; (iii) TEMPO, NaClO2, NaOCl, CH3CN–phosphate buffer, then KHCO3, TBAI, BnBr, DMF, 69%; (iv) Ac2O, py, quant.; (v)
(Ph3P)3RhCl, DABCO, then HgCl2, HgO, acetone, 83%; (vi) Cl3CCN, DBU, 93%.

Y. Ikeda et al. / Carbohydrate Research 343 (2008) 587–595
589
BnOOC
AcO
HO
BnO
AcO
O
O
PMBO
BnO
i)
O
OAll
O
OAll
BnO
AcO
AcO
AcO
9
10
ii)
NaO SO
3
NaOOC
PMBO
HOOC
O
HO
O
O
iii)
PMBO
BnO
O
O
BnO
O
OAll
BnO
OAll
BnO
NaO SO
3
HO
NaO SO
3
HO
11
12
iv)
1
Å
Scheme 2. Disaccharide synthesis. Reagents and conditions: (i) 8, TMSOTf, toluene, ꢀ18 °C, 96%; (ii) KOH, THF–H2O, quant.; (iii) SO3 NMe3,
DMF, 66%; (iv) H2, Pd/C, 90%.
tic of 1,2-trans glycosidic linkages. This selectivity can
be explained by the presence of a neighboring acetate
protecting group at the C-2 position of the glycosyl
donor, which forms a disarmed donor known as a major
determinant of anomeric selectivity for the formation
of 2-trans glycosidic linkages. The key disaccharide pre-
cursor 10 yet to be synthesized can be homologated
from its reducing end, from its non-reducing end, or
both. Employment of orthogonal protecting groups
for the anomeric position (allyl) and for the C-4 position
(p-methoxybenzyl) makes compound 10 a very versatile
synthon for the preparation of tetra-, hexa-, or
octasaccharides.
Saponification of disaccharide 10 gave quantitatively
the partially protected uronate 11, which was directly
O-sulfonated to furnish the trisulfated disaccharide 12
(66%). Finally, deprotection of benzyl groups on prod-
uct 12 was accomplished on Pd/C. Disaccharide 1 was
thus isolated in very good yield (90%) after chromato-
graphy purification through a Sephadex G-10 column.
discriminating selectivity against its isomers and other
related molecules.8 In those studies hydrogen bonding
interactions between the primary amine group on 2-ami-
noethyl methacrylate (AEM) in the polymer side and the
sulfate group on the sugar were found suitable for tem-
plate recognition in Me2SO. Ethylene glycol dimethacry-
late (EGDMA) was used as cross-linker and the neutral
monomer methacrylamide (MAM) was introduced to
enhance template polymer interactions as it has been as-
sumed that using a combination of functional monomers
gives to MIPs the possibility to better interact with the
template leading to functional receptor sites.9
To study the applicability of this method to the recog-
nition of the heparin related disaccharide 1 (template),
we prepared a series of MIPs and their corresponding
NIPs by varying the amount of AEM (1, 3, and 4 equiv,
Table 1) related to the number of sulfate and carboxyl-
ate groups over the disaccharide. Once washed with
0.3 M NH4OAc pH 5 to eliminate the template from
the formed cavities, MIPs and NIPs were rinsed with
water and then with Me2SO and tested for their capac-
ities to bind disaccharide 1. Binding tests were per-
formed in Me2SO at equilibrium conditions (see
Section 3) in the presence of increasing amounts of the
corresponding polymer.
2.2. Molecular imprinting studies
In previous studies, we demonstrated that MIPs can
readily be prepared to specifically recognize Glc(6S) with
Table 1. Composition (mmol) of the reaction mixture components used to prepare polymers imprinted with disaccharide 1 and the percentage of
template binding to NIPs and MIPs
Polymer
Trisulfated disaccharide 1
AEM
MAM
EGDMAa
% Bindinga
MIP
NIP
NIP/MIP
P1
P2
P3
0.014 (1 equiv)
0.014 (1 equiv)
0.014 (1 equiv)
0.014 (1 equiv)
0.028 (2 equiv)
0.056 (4 equiv)
0.084 (6 equiv)
0.084 (6 equiv)
0.084 (6 equiv)
0.56 (40 equiv)
0.56 (40 equiv)
0.56 (40 equiv)
35.6
62.0
73.4
24.8
37.6
21.7
1.4
1.7
3.4
All reactions were carried out on Me2SO.
a The molar proportion of EGDMA regarding to all polymerizable components was 83%, 82%, and 80%, respectively, for P1, P2, and P3.

590
Y. Ikeda et al. / Carbohydrate Research 343 (2008) 587–595
HPLC analyses of supernatants showed that the bind-
60
50
40
30
20
10
0
ing abilities of MIPs were inferior to those of their cor-
responding NIPs. Moreover, increasing the amount of
AEM correlated with an increase of non-specific binding
(Table 1 and Fig. 1). Interestingly, binding experiments
in aqueous solutions (pH 3, 5, 7, 9, and 11) resulted in
null disaccharide 1 binding. This result is in agreement
with the Sineriz observation in where specific Glc(6S)
binding to AEM functionalized MIPs was avoided by
water.8 To investigate if the non-specificity is the result
of ionic interactions between template and protonated
AEM residues ðPolymer–NH3þÞ, we treated polymers
with 10% acetic acid or 0.5 N HCl solution followed
by washing with water and then with Me2SO. Under
these conditions disaccharide 1 rebinding þwas also high-
MIP-3
NIP-3
buond%
( )
Glc-3S
disacch
1
Glc
GlcA
þ
er on NIPs–NH3 than on MIPs–NH3 (results not
shown). This suggests that the charged amines are more
accessible in NIPs–NH3þ, possibly because they are
more abundant at the NIP than at the MIP surfaces.
In fact, if amines are present at the polymer surface,
ionic interactions with the template will block its access
Figure 2. Binding ability of polymers P3 (5 mg) to 1 mg/mL solutions
of disaccharide 1 (disacch1), glucose (Glc), glucose-3-sulfate [Glc(3S)],
and glucuronic acid (GlcA).
the absence of non-specific binding. However, GlcA
and GlcAꢀNa+ importantly bound to both, MIP–3
and NIP–3 (–NH2), while with the GlcA methyl ester
this non-specific binding was completely avoided
(Fig. 2). This result suggests that the non-specificity of
the interactions between disaccharide 1 and polymers
P3 is mainly due to the presence of carboxylic moieties
at the template side.
In an assay to avoid the non-specific binding at the
polymer surface and to improve the recognition proper-
ties of MIP–3, we attempted post-imprinting chemical
modifications.22–24 Polymers P3–NH2 were reacted with
common amines protecting groups including acetyl,
Troc, and Boc and the resulting polymers abilities to
bind disaccharide 1 were tested (Fig. 3). As expected, a
complete suppression of template binding was obtained
with acetylated polymers P3–Ac indicating that the
small acetyl group could enter and react inside the
polymer matrix. Importantly, MIP–3–Troc exhibited
þ
to the imprinted MIP–NH3 cavities. When binding
experiments were performed after washing polymers
with 0.1 M NaOH, or with 0.1 M Na2CO3 aqueous solu-
tion (pH 10), again, a high non-specific template binding
in Me2SO was observed in both, MIPs–NH2 and NIPs–
NH2 (results not shown). This is in disaccord with our
previous studies with similar polymers–NH2 where a
high specific binding was demonstrated with a sulfated
but non-carboxylated glucose derivative Glc(6S) as a
template.8 Thus, we investigated the influence of the car-
boxylate group in disaccharide 1 by performing binding
experiments of P3 (–NH2) polymers with glucose (Glc),
the sodium salt of glucose-3-sulfate [Glc(3S)], glucuronic
acid (GlcA), its sodium salt (GlcAꢀNa+), and its methyl
ester (GlcA methyl ester). It is important to remark that
disaccharide 1 presents sulfates at positions C-2 or C-6
but not at C-3 and consequently binding of Glc(3S) is
not expected. Thus, unsurprisingly, Glc and Glc(3S)
bound to polymers–NH2 in very limited extent indicate
90
80
MIP-1
NIP-1
MIP-2
NIP-2
MIP-3
NIP-3
100
80
60
40
20
0
70
60
50
MIP
MIP
-3
NIP
NIP
-3
40
30
20
10
0
Buond%
0
5
10
15
20
P3
P3-Ac
P3-Troc P3-Boc
polymer (mg/mL)
Figure 1. Binding isotherms of disaccharide 1 to polymers P1–P3.
Figure 3. Abilities of polymers, P3–Ac, P3–Troc, and P3–Boc (MIPs
and NIPs), to bind disaccharide 1 (1 mg/mL solution).

Y. Ikeda et al. / Carbohydrate Research 343 (2008) 587–595
591
disaccharide 1 specific binding, with improved binding
3.2. Allyl 3-O-benzyl-4,6-di-O-p-methoxybenzylidene-b-
D-glucopyranoside (3)
capabilities, when compared to MIP–3. This suggests
that, by blocking amines at the polymer surface, the
template can access to the polymer imprinted cavities.
Further studies to improve recognition specificity by
MIPs have to be considered.
To a soln of allyl 3-O-benzyl-b-D-glucopyranoside 220
(7.1 g, 22.9 mmol) in DMF (75 mL), p-anisaldehyde
dimethylacetal (7.4 mL, 43.4 mmol), and p-toluenesul-
fonic acid (0.3 g, 0.1 mmol) were added. After 2.5 h,
10% NaHCO3 (100 mL) and water (150 mL) were
added. The mixture was extracted with EtOAc
(2 ꢁ 250 mL). The organic layers were assembled,
washed with 10% NaHCO3 and brine, dried (Na2SO4),
filtered, and evaporated to syrup. Flash chromatogra-
phy on silica gel (4:1 to 3:1 hexanes–EtOAc) gave 3
In conclusion, the trisulfated disaccharide 1, which is
an analog of a rare repeating unit of heparin and HS,
was synthesized. The synthetic strategy makes use of
synthon 10 for the homologation of HS derivative poly-
saccharides. Molecularly imprinted polymers (MIPs)
and their respective NIPs were prepared through a
non-covalent approach by using disaccharide 1 as a tem-
plate and AEM as a functional monomer at the polymer
side. Results from binding experiments suggest that,
although low, specific binding of disaccharide 1 to MIPs
is possible by post-imprinting polymer chemical modifi-
cations. We demonstrate that the presence of the major
non-specific interactions between disaccharide 1 and the
polymers is the result of the presence of carboxylic
groups at the template side. These carboxylic groups
might interact with the amine functionalities mainly at
the polymers’ surfaces blocking in this way the access
of the template to the imprinted cavities. We do not
exclude the possibility by which a high polymer reticula-
tion degree also makes the disaccharide access highly
hindered. We are currently working on these and other
points to reach a convenient recognition of HS disaccha-
rides or even bigger HS fragments. Advance in this
direction will imply a great contribution to the field of
glycosciences.
1
(7.4 g, 75%), as a syrup; [a]D ꢀ49 (c 0.045, CHCl3); H
NMR (CDCl3) d 7.40 (d, 2H, J 8.9 Hz, PMP), 7.30–
7.25 (m, 5H, Ar), 6.89 (d, 2H, J 8.9 Hz, PMP), 5.98–
5.83 (m, 1H, CH@), 5.50 (s, 1H, CHPh), 5.34–5.16 (m,
2H, @CH2), 4.95 (d, 1H, Jgem 11.7 Hz, CHPh), 4.77
(d, 1H, Jgem 11.7 Hz, CHPh), 4.43 (d, 1H, J1,2 7.4 Hz,
H-1), 4.40–4.28 (m, 2H, CHaCH@CH2, H-2), 4.12
(ddt, 1H, J 12.7, 6.3, and 1.2 Hz, CHbCH@CH2),
3.83–3.55 (m, 5H, H-3, H-4, H-6a, H-6b, –OH), 3.80
(s, 3H, OCH3), 3.44–3.32 (m, 1H, H-5); 13C NMR
(CDCl3) d 160.0, 138.17 (Ar), 133.5 (CH@), 129.6,
128.4–127.3 (Ar), 118.2 (@CH2), 113.6 (Ar), 102.2
(CH–PMP), 101.2 (C-1), 81.3, 80.2, 74.2, 66.1 (C-2, C-
3, C-4, C-5), 74.6, 70.5, 68.6 (C-6, CH2Ph,
CH2CH@CH2), 55.3 (OCH3); HRESIMS m/z: calcd
for [C24H28O7 + Na]+, 451.1733; found, 451.1716. Anal.
Calcd for C24H28O7: C, 67.28; H, 6.59. Found: C, 67.24;
H 6.45.
3.3. Allyl 3-O-benzyl-4-O-p-methoxybenzyl-b-D-gluco-
pyranoside (4)
3. Experimental
3.1. General methods
To a soln of allyl 3-O-benzyl-4,6-di-O-p-methoxybenzyl-
idene-b-D-glucopyranoside (3) (5.8 g, 13.7 mmol) in 1:1
CH2Cl2–ether (200 mL), LiAlH4 (2.3 g, 61.4 mmol)
was added at ꢀ10 °C under Ar. To this mixture, a soln
of AlCl3 (7.3 g, 54.6 mmol) in ether (50 mL) was slowly
added over 45 min. CH2Cl2 (250 mL) was added and the
mixture filtered through Celite. EtOAc (50 mL) and
water (20 mL) were added, and the organic layer was
separated, and concentrated to syrup. Flash chromatog-
raphy on silica gel (2:1 to 1:2 hexanes–EtOAc) gave 4
TLC was carried out on precoated aluminum plate
(0.1 mm) of Silica-Gel 60 F-254; detection was per-
formed by exposure to UV light and by spraying the
plate with 5% (v/v) H2SO4 in EtOH followed by heating.
1H and 13C NMR spectra were recorded at 300 and
75.5 MHz, respectively, chemical shifts are reported as
d values (ppm) relative to tetramethylsilane (TMS) or,
for compounds analyzed in D2O, to sodium 2,2-dimeth-
ylsilapentane-5-sulfonate (DSS). Mass spectrometry was
performed on a Q-TOF, Z-spray spectrometer. HPLC
analyses were performed isocratically at rt on a Waters
Symmetry Shield RP18 (5 lm, 4.6 ꢁ 250 mm) column
or a Dionex ProPac PA1 (4 ꢁ 250 mm) analytical col-
umn. Fifty microliters of samples was injected and
eluted with CH3CN (80%) for RP18 column or 0.1 M
aqueous ammonium acetate pH 5.0 for ProPac PA1 col-
umn, at a flow rate of 0.5 mL/min. The detection was
performed with an Evaporative Light Scattering System.
Water employed was milliQ quality.
20
25
(2.3 g, 38%) as a syrup, ½aꢂD ꢀ21 (c 0.2, CHCl3), ½aꢂD
(lit.25) ꢀ22 (c 3.3, CHCl3), along with recovered 3
(2.8 g, 47%). Anal. Calcd for C24H30O7: C, 66.96; H,
7.02. Found: C, 66.81; H 6.99.
3.4. Benzyl (allyl-3-O-benzyl-4-O-p-methoxybenzyl-b-D-
glucopyranosid)uronate (5)
A mixture of allyl 3-O-benzyl-4-O-p-methoxybenzyl-b-
D-glucopyranoside (4) (1.1 g, 2.6 mmol), TEMPO
(0.1 g, 0.6 mmol), NaClO2 (0.9 g, 10.6 mmol), and

592
Y. Ikeda et al. / Carbohydrate Research 343 (2008) 587–595
NaOCl (50 lL of a 2.1 M soln) in MeCN (12 mL) and
phosphate buffer pH 6.7 (10 mL) was stirred at 35 °C
for 4 days. 5 M NaOH (10 mL) and CH2Cl2 (40 mL)
were added, and the mixture was extracted with water
(2 ꢁ 50 mL). 0.5 M HCl (100 mL) was added and the
mixture extracted with CH2Cl2 (150 mL). The organic
layer was separated, washed with 10% Na2S2O3, dried
(Na2SO4), filtered, and evaporated to a syrup. Flash
chromatography on silica gel (1:1 to 1:1 hexanes–
EtOAc) gave the free acid (0.9 g, 76%). To a mixture
of the free acid (0.9 g, 2.0 mmol), KHCO3 (1.0 g,
10.1 mmol), and TBAI (0.1 g, 0.2 mmol) in DMF
(20 mL), benzyl bromide (1.0 g, 6.1 mmol) was added.
After 4 h at rt, water (80 mL) was added, and the mix-
ture extracted with EtOAc (2 ꢁ 70 mL). The organic
layers were assembled, washed with 10% NaHCO3 and
brine, dried (Na2SO4), filtered, and evaporated to syrup.
Flash chromatography on silica gel (4:1 to 2:1 hexanes–
EtOAc) gave 5 (1.0 g, 90%, 69% from 4), as a syrup; [a]D
ꢀ44 (c 0.27, CHCl3); 1H NMR (CDCl3) d 7.40–7.20 (m,
10H, Ar), 7.00] (d, 2H, Jo 8.5 Hz, PMB), 6.75 (d, 2H, Jo
8.5 Hz, PMB), 5.98–5.82 (m, 1H, CH@), 5.30–5.12 (m,
4H, CH2Ph, @CH2), 4.84 (d, 1H, Jgem 11.2 Hz, CHPh),
4.80 (d, 1H, Jgem 11.2 Hz, CHPh), 4.65 (d, 1H, Jgem
10.2 Hz, CHPh), 4.45–4.35 (m, 3H, H-1, CHPh,
CHaCH@CH2), 4.12–4.05 (m, 1H, CHbCH@CH2),
3.92 (d, 1H, J4,5 9.7 Hz, H-5), 3.85–3.52 (m, 3H, H-2,
H-3, H-4), 3.75 (s, 3H, OCH3); 13C NMR (CDCl3) d
168.33 (C-6), 159.20, 138.35, 138.01 (Ar), 133.41
(CH@), 129.9, 129.5–127.7 (Ar), 118.2 (@CH2), 113.7
(Ar), 102.0 (C-1), 83.5, 78.7, 74.8, 74.1 (C-2, C-3, C-4,
C-5), 75.1, 74.6, 70.4, 67.3 (CH2Ph, CH2CH@CH2),
55.2 (OCH3); HRESIMS m/z: calcd for [C31H34O8+
Na]+, 557.2151; found, 557.2165.
C-4, C-5), 74.6, 74.3, 69.5, 67.0 (2ꢁCH2Ph,
CH2CH@CH2), 54.9 (OCH3), 20.6 (CH3CO); HRE-
SIMS m/z: calcd for [C33H36O9+Na]+, 599.2257; found,
599.2247.
3.6. Benzyl (2-O-acetyl-3-O-benzyl-4-O-p-methoxy-
benzyl-a-D-glucopyranosid)uronate (7)
To a soln of 6 (0.9 g, 1.6 mmol) in 7:3:1 EtOH–toluene–
water (55 mL), (Ph3P)3RhCl (146.2 mg, 0.2 mmol) and
DABCO (45.4 mg, 0.4 mmol) were added. The mixture
was refluxed for 2 h and then filtered through Celite
and the solvent evaporated. The crude product was dis-
solved in 9:1 acetone–water (20 mL). HgCl2 (1.2 g,
4.6 mmol) and HgO (34.6 mg, 0.2 mmol) were added.
After 3 h, the mixture was filtered through silica gel,
then brine (40 mL) was added and the soln was
extracted with EtOAc (2 ꢁ 40 mL). The organic layers
were assembled, washed with brine, dried (Na2SO4), fil-
tered, and evaporated to syrup. Flash chromatography
on silica gel (2:1 hexanes–EtOAc) gave 7 (0.7 g, 83%),
as a syrup; [a]D +22 (c 0.11, MeOH); major isomer
1
(a): H NMR (CDCl3) d 7.35–7.20 (m, 10H, Ar), 7.00
(d, 2H, Jo 8.6 Hz, PMB), 6.75 (d, 2H, Jo 8.6 Hz,
PMB), 5.40 (dd, 1H, J1,2 3.3 Hz, J1,OH 3.2 Hz, H-1),
5.17–5.09 (m, 2H, CH2Ph), 4.85 (dd, 1H, J1,2 3.3 Hz,
J2,3 9.6 Hz, H-2), 4.77 (d, 1H, Jgem 11.5 Hz, CHPh),
4.68 (d, 1H, Jgem 11.5 Hz, CHPh), 4.60 (d, 1H, Jgem
10.4 Hz, CHPh), 4.48 (d, 1H, J4,5 9.6 Hz, H-5), 4.41
(d, 1H, Jgem 10.4 Hz, CHPh), 4.02 (dd, 1H, J3,4
9.0 Hz, J4,5 9.6 Hz, H-4), 3.81 (dd, 1H, J2,3 9.6 Hz, J3,4
9.0 Hz, H-3), 3.75 (s, 3H, OCH3), 2.00 (s, 3H, CH3CO);
13C NMR (CDCl3) d 170.3 (C-6), 169.2 (COCH3), 159.2,
138.2, 135.0, 129.3–127.3, 113.4 (Ar), 90.7 (C-1), 78.9,
78.7, 72.9, 70.6 (C-2, C-3, C-4, C-5), 75.4, 74.6, 67.3,
(CH2Ph), 55.2 (OCH3), 20.8 (CH3CO); HRESIMS
m/z: calcd for [C30H32O9+Na]+, 559.1944; found,
559.1964. Anal. Calcd for C30H32O9: C, 67.15; H, 6.01.
Found: C, 66.90; H 6.24.
3.5. Benzyl (allyl 2-O-acetyl-3-O-benzyl-4-O-p-methoxy-
benzyl-b-D-glucopyranosid)uronate (6)
A soln of 5 (0.9 g, 1.7 mmol) in 1:1 pyridine-Ac2O
(1 mL) was stirred at rt for 20 h. The solvent was co-
evaporated with toluene (2 ꢁ 10 mL). Flash chromato-
graphy on silica gel (3:1 hexanes–EtOAc) gave 6 (0.9 g,
quantitative), as a syrup; [a]D ꢀ34 (c 0.11, CH3OH);
1H NMR (CDCl3) d 7.35–7.20 (m, 10H, Ar), 7.05 (d,
2H, Jo 8.6 Hz, PMB), 6.75 (d, 2H, Jo 8.6 Hz, PMB),
5.85–5.75 (m, 1H, CH@), 5.30–5.12 (m, 4H, CH2Ph,
@CH2), 5.08 (dd, 1H, J1,2 7.6 Hz, J2,3 9.1 Hz, H-2),
4.79 (d, 1H, Jgem 11.6 Hz, CHPh), 4.67 (d, 1H, Jgem
11.6 Hz, CHPh), 4.62 (d, 1H, Jgem 10.4 Hz, CHPh),
4.50 (d, 1H, J1,2 7.6 Hz, H-1), 4.43 (d, 1H, Jgem
10.4 Hz, CHPh), 4.35–4.27 (m, 1H, CHaCH@CH2),
4.15–3.90 (m, 4H, H-3, H-4, H-5, CHbCH@CH2), 3.75
(s, 3H, OCH3), 1.99 (s, 3H, CH3CO); 13C NMR
(CDCl3) d 169.1 (C-6), 167.9, 159.1, 137.8, 134.8 (Ar),
133.2 (CH@), 129.5, 129.4–127.5 (Ar), 117.0 (@CH2),
113.5 (Ar), 99.9 (C-1), 81.7, 78.7, 74.4, 72.5 (C-2, C-3,
3.7. Benzyl (2-O-acetyl-3-O-benzyl-4-O-p-methoxybenz-
yl-a-D-glucopyranosyl trichloroacetimidate)uronate (8)
˚
To a mixture of 7 (0.4 g, 0.7 mmol) and 4 A molecular
sieves (0.2 g) in CH2Cl2 (5 mL), trichloroacetonitrile
(1.2 mL, 0.7 mmol), and DBU (17.0 lL, 0.1 mmol) were
added. After 1 h, the mixture was filtered through Celite
and concentrated to syrup. Flash chromatography on
silica gel (5:1 hexanes–EtOAc containing 0.5% NEt3)
gave 8 (471.0 mg, 93%), as a white foam; [a]D +37 (c
1
0.12, CH3OH); H NMR (CDCl3) d 8.63 (s, 1H, NH),
7.40–7.22 (m, 10H, Ar), 7.01 (d, 2H, Jo 8.5 Hz, PMB),
6.75 (d, 2H, Jo 8.5 Hz, PMB), 6.51 (d, 1H, J1,2 3.5 Hz,
H-1), 5.22 (d, 1H, Jgem 12.3 Hz, CHPh), 5.13 (d, 1H,
Jgem 12.3 Hz, CHPh), 5.08 (dd, 1H, J1,2 3.6 Hz, J2,3
9.9 Hz, H-2), 4.85 (d, 1H, Jgem 11.5 Hz, CHPh), 4.63

Y. Ikeda et al. / Carbohydrate Research 343 (2008) 587–595
593
(d, 1H, Jgem 10.2 Hz, CHPh), 4.38 (d, 1H, Jgem 10.2 Hz,
CHPh), 4.45 (d, 1H, J4,5 10.0 Hz, H-5), 4.10 (dd, 1H, J3,4
9.7 Hz, J4,5 10.0 Hz, H-4), 3.85 (dd, 1H, J2,3 9.9 Hz, J3,4
9.7 Hz, H-3), 3.75 (s, 3H, OCH3), 1.98 (s, 3H, CH3CO);
13C NMR (CDCl3) d 169.8 (C-6), 168.0 (COCH3), 160.6
(C@NH), 159.3, 137.9, 134.8, 129.7–127.8, 113.7 (Ar),
93.5 (C-1), 78.6, 72.9, 71.6 (C-2, C-3, C-4, C-5), 75.4,
75.0, 67.4, (CH2Ph), 55.1 (OCH3), 20.4 (CH3CO); HRE-
SIMS m/z: calcd for [C32H32NO9Cl3+Na]+, 702.1040;
found, 702.1044.
4.56–4.50 (m, 3H, H-10, CH2Ph), 4.45–4.31 (m, 3H, H-
1, H-6a, CHPh), 4.29–4.20 (m, 1H, CHaCH@CH2),
4.09 (dd, 1H, J5,6b 4.9 Hz, J6a,6b 11.9 Hz, H-6b), 4.04–
3.96 (m, 1H, CHbCH@CH2), 3.90–3.72 (m, 3H, H-30,
H-40, H-50), 3.74 (s, 3H, OCH3), 3.63–3.53 (m, 2H, H-
3, H-4), 3.49–3.43 (m, 1H, H-5), 2.05, 1.98, 1.80 (3s,
9H, 3 ꢁ CH3CO); 13C NMR (CDCl3): d 170.5, 169.4,
169.6 (C-60, COCH3), 159.3, 138.5, 137.8, 134.8 (Ar),
133.5 (CH@), 129.6–127.3 (Ar), 117.26 (@CH2), 113.7
(Ar), 101.2 (C-10), 99.4 (C-1), 81.9, 80.6, 79.0, 78.2,
74.4, 72.9, 72.5, 72.3 (C-2, C-3, C-4, C-5, C-20, C-30,
C-40, C-50), 75.1, 74.6, 69.6, 67.3 (3 ꢁ CH2Ph,
CH2CH@CH2), 62.2 (C-6), 55.2 (OCH3), 20.7
(CH3CO); HRESIMS m/z: calcd for [C50H56O16+Na]+,
935.3466; found, 935.3454.
3.8. Allyl 2,6-di-O-acetyl 3-O-benzyl-b-D-glucopyran-
oside (9)
To a soln of allyl 2-O-acetyl-3-O-benzyl-b-D-glucopy-
ranoside21 (2.1 g, 6.0 mmol) in CH2Cl2 (15 mL), pyri-
dine (1 mL), and acetyl chloride (0.5 mL) were added
at 0 °C. After 1.5 h, MeOH (5 mL) was added, and the
solvent was co-evaporated with toluene. Flash chroma-
tography on silica gel (3:2 to 1:1 hexanes–EtOAc) gave
3.10. Allyl 3-O-benzyl-4-O-(3-O-benzyl-4-O-p-methoxy-
benzyl-b-D-glucopyranosiduronic acid)-b-D-glucopyran-
oside (11)
20
25
D
9 as a syrup; ½aꢂD ꢀ20 (c 1.0, CHCl3), ½aꢂ (lit25) ꢀ19
To a soln of 10 (74.6 mg, 0.1 mmol) in THF (2.5 mL), a
soln of KOH (28 mg) in water (0.5 mL) was added at
0 °C. After 22 h, additional KOH (25 mg) was added
and the mixture stirred until no more starting material
was detected by TLC (46 h). The mixture was extracted
with CH2Cl2 (2 ꢁ 3 mL), and then stirred with Amber-
lite IR-120 B to adjust the pH to 6. Solvents were co-
evaporated with toluene. Flash chromatography on sil-
ica gel (10:0 to 9:1 EtOAc–MeOH) gave 11 (57.0 mg,
1
(c 3.4, CHCl3); H NMR (CDCl3) d 7.40–7.20 (m, 5H,
Ar), 5.85–5.72 (m, 1H, CH@), 5.25–5.11 (m, 2H,
@CH2), 4.92 (dd, 1H, J1,2 8.0 Hz, J2,3 9.0 Hz, H-2),
4.75–4.63 (m, 2H, CH2Ph), 4.38 (d, 1H, J1,2 8.0 Hz, H-
1), 4.37–4.24 (m, 3H, CHaCH@CH2, H-6a, H-6b),
4.05–3.95 (m, 1H, CHbCH@CH2), 3.60–3.33 (m, 3H,
H-3, H-4, H-5), 3.19 (d, 1H, J4, OH 3.7 Hz, OH), 2.01,
2.98 (2s, 6H, 2 ꢁ CH3CO); 13C NMR (CDCl3) d
171.6, 169.5 (COCH3), 138.0 (Ar), 133.5 (CH@), 128.4,
127.7 (Ar), 117.1 (@CH2), 99.8 (C-1), 82.0, 73.7, 72.6,
69.9 (C-2, C-3, C-4, C-5), 74.5, 69.6 (CH2Ph,
CH2CH@CH2), 63.0 (C-6), 20.8, 20.7 (CH3CO); Anal.
Calcd for C20H26O8: C, 60.90; H, 6.64. Found: C,
60.68; H 6.63.
1
quantitative), as a foam; [a]D ꢀ30 (c 0.15, MeOH); H
NMR (CD3OD) d 7.45–7.20 (m, 15H, Ar), 7.17 (d,
2H, Jo 8.6 Hz, PMB), 6.82 (d, 2H, Jo 8.6 Hz, PMB),
6.04–5.90 (m, 1H, CH@), 5.37–5.13 (m, 2H, @CH2),
5.02 (d, 1H, Jgem 11.0 Hz, CHPh), 4.92 (d, 1H,
Jgem 11.0 Hz, CHPh), 4.85–4.75 (m, 2H, CH2Ph),
4.69–4.60 (m, 3H, H-10, CH2Ph), 4.41–4.32 (m, 1H,
CHaCH@CH2), 4.35 (d, 1H, J1,2 7.8 Hz, H-1), 4.20–
4.12 (m, 1H, CHbCH@CH2), 4.00–3.70 (m, 5H, H-40,
H-5, H-50, H-6a, H-6b), 3.78 (s, 3H, OCH3), 3.60–3.35
(m, 5H, H-2, H-20, H-3, H-30, H-4); 13C NMR (CD3OD)
d 171.0 (C-60), 159.2, 139.3 (Ar), 134.7 (CH@), 130.5–
127.4 (Ar), 116.4 (@CH2), 113.6 (Ar), 103.4 (C-10),
102.5 (C-1), 84.5, 83.9, 79.6, 77.5, 75.8, 75.0, 74.8, 74.2
(C-2, C-3, C-4, C-5, C-20, C-30, C-40, C-50), 75.3, 74.6,
70.1 (2ꢁCH2Ph, CH2CH@CH2), 60.9 (C-6), 54.7
(OCH3); HRESIMS m/z: calcd for [C37H44O13Na]+,
719.2680; found, 719.2670.
3.9. Allyl 2,6-di-O-acetyl-3-O-benzyl-4-O-(benzyl 2-O-
acetyl-3-O-benzyl-4-O-p-methoxybenzyl-b-D-gluco-
pyranosiduronate)-b-D-glucopyranoside (10)
To a mixture of 8 (0.6 g, 0.8 mmol), and 9 (0.9 g,
˚
2.4 mmol), and 4 A molecular sieves (0.5 g) in toluene
(20 mL),
trimethylsilyl
trifluoromethanesulfonate
(8.0 lL, 44.0 lmol) in 80 lL of toluene was added at
ꢀ18 °C. After 40 min, diisopropylethylamine (0.1 mL)
was added and the mixture filtered through Celite and
concentrated to a syrup. Flash chromatography on silica
gel (10:1 to 3:1 toluene–EtOAc) gave 10 (0.7 g, 96%), as
1
a colorless foam; [a]D ꢀ7 (c 0.2, acetone); H NMR
3.11. Allyl 3-O-benzyl-4-O-(3-O-benzyl-4-O-p-methoxy-
benzyl-2-O-sulfonato-b-D-glucopyranosiduronate)-2,6-di-
O-sulfonato-b-D-glucopyranoside, tetrasodium salt (12)
(CDCl3) d 7.40–7.20 (m, 15H, Ar), 6.98 (d, 2H, Jo
8.6 Hz, PMB), 6.75 (d, 2H, Jo 8.6 Hz, PMB), 5.83–
5.72 (m, 1H, CH@), 5.25–5.12 (m, 2H, @CH2), 5.03
(dd, 1H, J1 ;2 8:0 Hz, J2 ;3 9:2 Hz, H-20), 5.01–4.93 (m,
2H, CH2Ph), 4.94 (dd, 1H, J1,2 8.0 Hz, J2,3 9.0 Hz, H-
2), 4.88 (d, 1H, Jgem 12.1 Hz, CHPh), 4.76 (d, 1H, Jgem
11.5 Hz, CHPh), 4.60 (d, 1H, Jgem 11.5 Hz, CHPh),
To a soln of (11) (330 mg, 0.47 mmol) in DMF (20 mL),
sulfur trioxide–trimethylamine complex (1.34 mg,
9.6 mmol) was added and the mixture stirred at 55 °C
for 16.5 h. The reaction was stopped by pouring the
0
0
0
0

594
Y. Ikeda et al. / Carbohydrate Research 343 (2008) 587–595
reaction mixture in to 10 mL of satd aq NaHCO3 aq
soln cooled in an ice-water bath. Chromatography on
Sephadex LH-20 (1:1 CH2Cl2–EtOH) gave 12 (322 mg,
66%), as a colorless foam; [a]D ꢀ42 (c 0.14, MeOH);
1H NMR (CD3OD) d 7.40–7.20 (m, 15H, Ar), 7.15 (d,
2H, Jo 8.7 Hz, PMB), 6.85 (d, 2H, Jo 8.7 Hz, PMB),
6.04–5.92 (m, 1H, CH@), 5.43–5.32 (m, 1H, @CHa),
5.19–5.07 (m, 1H, @CHb), 5.10 (d, 1H, J1,2 7.62 Hz,
H-1), 4.71–4.64 (m, 4H, CH2Ph, H-6a, H-10), 4.55
(d, 1H, Jgem 10.3 Hz, CHPh), 4.45–3.84 (m, 12H,
CHaCH@CH2, CHbCH@CH2, H-2, H-3, H-4, H-5, H-
6a, H-6b, H-20, H-30, H-40, H-50), 3.79 (s, 3H, OCH3);
13C NMR (CD3OD) d 171.6 (C-60), 159.3, 139.2, 139.0
(Ar), 134.8 (CH@), 129.6–127.5 (Ar), 116.5 (@CH2),
113.6 (Ar), 101.1 (C-10), 100.3 (C-1), 82.9, 81.6, 80.8,
79.5, 79.3, 76.1, 75.5, 74.4 (C-2, C-3, C-4, C-5, C-20,
C-30, C-40, C-50), 74.9, 74.3, 73.5 (2ꢁCH2Ph,
CH2CH@CH2), 68.3 (C-6), 54.7 (OCH3); HRESIMS
m/z: calcd for [C37H43O22S3Na2]+, 981.1204; found,
981.1209.
degassed with nitrogen for 10 min and the tube sealed
and heated at 50 °C for 24 h. After polymerization, the
bulk polymer was wet-crushed in water with a mortar
and pestle. The milled polymer was washed widely on
a G4-glass filter funnel in deionized water. The template
was extracted with 0.3 M aq NH4OAc until it could no
longer be detected in the washing soln by HPLC. There-
after, the fine polymer particles were dried at 40 °C
under vacuum. Control non-imprinted polymer
(NIP–3) was prepared in the same way without the
addition of the template. 125.1 mg of MIP–3 (96%)
and 126.4 mg of NIP–3 (97%) were obtained.
3.14. General procedure for the preparation of P3–Ac,
P3–Troc, and P3–Boc. Capping of P3
The chemical modification of 1.0 g (1.1 lmol equiv of –
NH2) of template-free polymers P3 (MIP or NIP) with
10 equiv of the corresponding protective reagent,
Ac2O, TrocCl or Boc2O, was carried out in 0.2 mL of
Me2SO in the presence of 50 lL of Et3N. The reactions
were kept at rt for 2 h, followed by water quenching.
After MeOH and Me2SO washings the polymer particles
were dried at 40 °C under vacuum.
3.12. Propyl 4-O-(2-O-sulfonato-b-D-glucopyranosiduro-
nate)-2,6-di-O-sulfonato-b-D-glucopyranoside, tetra-
sodium salt (1)
To a soln of 12 (0.2 g, 0.2 mmol) in 4:3 EtOH–H2O
(7 mL), 5% Pd/C (155.0 mg) was added and the mixture
was stirred at 4 °C under hydrogen for 9 days; addition
of supplementary catalyst help to drive the reaction to
completion. The mixture was filtered through a Celite
pad, concentrated and chromatographed through
Sephadex G-10 (6 mL), using water as eluent. The
3.15. Binding experiments
Saturation studies of the polymer particles were carried
out to estimate their binding capacity. Increasing
amounts of polymer (5, 10, 15, 20 mg) were incubated
on a rocking table with 250 lL of 1 mg/mL soln of the
template or other analyte in Me2SO and allowed to
reach equilibrium. After 24 h, the particles were sedi-
mented by centrifugation and the supernatants were
analyzed by HPLC. Sample (50 lL) was injected and
eluted with CH3CN (80%) for separation on the RP18
column or with 0.1 M aqueous ammonium acetate pH
5.0 for separation on the ProPac PA1 column. The flow
rate was 0.5 mL/min in both the cases. The amount of
analyte bound to the polymer was obtained by subtract-
ing the peak area of the unbound analyte to the peak
area of the standard soln.
appropriate fractions were lyophilized to give
1
(0.13 g, 90%), as a white foam; [a]D ꢀ99 (c 0.1, water);
1H NMR (D2O) d 4.69 (d, 1H, J1,2 7.9 Hz, H-1), 4.55
(d, 1H, J1,2 7.9 Hz, H-10), 4.45 (dd, 1H, J5,6a 2.5 Hz,
J6a,6b 11.2 Hz, H-6a), 4.20 (dd, 1H, J5,6b 1.6 Hz, J6a,6b
11.2 Hz, H-6b), 4.05–3.95 (m, 3H, H-2, H-20, H-5),
3.87–3.50 (m, 7H, H-3, H-4, H-30, H-40, H-50, OCH2),
1.55 (m, 2H, CH2), 1.55 (t, 3H, J 7.4 Hz, CH3); 13C
NMR (D2O) d 174.4, (C-60), 101.0 (C-10), 100.2 (C-1),
80.4, 80.2, 77.4, 75.7, 74.6, 73.2, 72.6, 71.9 (C-2, C-3,
C-4, C-5, C-20, C-30, C-40, C-50), 72.8 (OCH2), 66.0 (C-
6), 22.6 (CH2), 10.0 (CH3); HRESIMS m/z: calcd for
[C15H24O21S3ꢀH+]ꢀ, 637.0050; found, 637.0066.
3.13. Synthesis of polymers. Typical procedure: prepara-
tion of P3
Acknowledgments
10.2 mg (0.014 mmol) of propyl 4-O-(2-O-sulfonato-b-
D-glucopyranosiduronate)-2,6-di-O-sulfonato-b-D-
glucopyranoside, tetrasodium salt (1), 3.2 mg of MAM
(0.084 mmol), 10.3 mg (0.056 mmol) of AEM (hydro-
chloric, 90%), 0.1 mL of EGDMA (0.56 mmol), and
1.2 mg (7.3 lmol) of AIBN were placed in a glass tube
and dissolved in 0.15 mL of Me2SO. The soln was then
Special acknowledgments are addressed to Professors
Karsten Haupt and Emmanuel Petit for their valuable
discussions concerning the Molecular Imprinting Tech-
nology and the analytical parts of this work. This work
was supported by the ‘French Centre National de la
Recherche Scientifique’ (CNRS) as a part of an ATIP/
Structure–Function Program.

Y. Ikeda et al. / Carbohydrate Research 343 (2008) 587–595
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1H and 13C NMR spectra of all reported compounds as
well as HPLC chromatogram of compound 1 are in-
cluded. Supplementary data associated with this article
can be found, in the online version, at doi:10.1016/
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