Synthesis of poly(aspartimide)-based bio-glycoconjugates

Article abstract of DOI:10.1016/j.carres.2009.08.034

The purpose of this programme was to synthesize and analyze new bioconjugates of interest for the potential inhibition of the influenza virus, using poly(aspartimide) as a polymer support. The macromolecular targets were obtained by attaching various sial

Full text of DOI:10.1016/j.carres.2009.08.034

Carbohydrate Research 345 (2010) 33–40
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of poly(aspartimide)-based bio-glycoconjugates
b
c
a
a
Irina Carlescu a, , Helen M. I. Osborn , Jacques Desbrieres , Dan Scutaru , Marcel Popa
*
a Department of Natural and Synthetic Polymers, Faculty of Chemical Engineering and Environmental Protection, ‘Gh. Asachi’ of Iasi, Technical University,
Bd. D. Mangeron 71A, 700050 Iasi, Romania
b The School of Pharmacy, University of Reading, Whiteknights, Reading RG6 6AD, UK
c Universite de Pau et des Pays de l’Adour, IPREM/EPCP (UMR CNRS 5254), Helioparc Pau Pyrénées, 2 Avenue President Angot, 64053 Pau Cedex 09, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 June 2009
Received in revised form 21 August 2009
Accepted 25 August 2009
Available online 2 September 2009
The purpose of this programme was to synthesize and analyze new bioconjugates of interest for the
potential inhibition of the influenza virus, using poly(aspartimide) as a polymer support. The macromo-
lecular targets were obtained by attaching various sialic acid-linker-amine compounds to poly(asparti-
mide). 1H and 13C NMR studies were then performed to analyze the degree of incorporation of the
sialic acid-linker-amine compounds within the poly(aspartimide). These studies illustrated that the
incorporation was dependent on the nature of the spacer between the sugar and the amine functionality.
Thus aliphatic spacers favoured the inclusion of sialic acid onto the polymer support whereas compounds
having only an aromatic moiety between the sialic acid and the amine could not be easily incorporated.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Glycoconjugates
Polyvalent inhibitor
Poly(aspartic acid)
Sialic acid
Antiviral drugs
1. Introduction
act as inhibitors of hemagglutinin. The poly(aspartimide) was se-
lected as the polyvalent support because it affords linear biode-
One of the most promising methods to prevent infection of hosts
by the influenza virus is to inhibit the interaction of the virus with
sialic acid residues on host cells.1 In this regard, the development
of polyvalent inhibitors for the influenza virus has attracted recent
attention.2–6 Polyvalent binding is characterized by the simulta-
neous interactions between multiple ligands on one entity and mul-
tiple receptors on the target biomolecule. In order to have an
enhanced inhibition activity, the synthetic inhibitors must have a
greater affinity towards the virus than the natural receptors. The
connection of sialic acid molecules to a polyvalent backbone poten-
tially increases the avidity of these materials for the virus, by taking
advantage of the multivalent effect. Two types of carriers have been
studied for the preparation of glycoconjugates bearing multiple sia-
lic acid residues: natural backbones, such as proteins7–9 or polysac-
charides10–12 and synthetic backbones, such as spherical
dendrimers,13–15 liposomes16–20 and linear polymers.21–29 Com-
pared to other scaffolds, polymeric supports offer several advanta-
ges such as their ease of synthesis and capacity to display multiple
sialic acid residues.30
gradable compounds, it presents immunological activity and,
more importantly, it affords water soluble compounds.
2. Results and discussion
In order to obtain polymeric glycoconjugates with antiviral
activity, we took into consideration the fact that infection with
influenza virus occurs through attachment to the cell surface of
hemagglutinin, the major component of the influenza virus.
Literature studies have shown that hemagglutinin (HA) recog-
nizes the carboxylic acid function and the alcoholic chain of the
sialic acid (Fig. 1) while neuraminidase (NA) recognizes, in partic-
ular, the hydroxyl group on carbon 4 (C-4) of sialic acid. We there-
fore chose to synthesize a series of compounds which contained
sialic acid linked to a spacer that contained a terminal amine func-
tionality. Within the derivatives, the nature of the aglyconic chain
was varied to both probe the ease with which the sialic acid-linker-
amine units could be incorporated within the polymer, and the
importance of these structural changes on the biological activities
of the materials. Also, to minimize the potential for hydrolysis of
the derivatives, via the action of neuraminidase, a sulfur atom
was introduced into the structure.
The purpose of this paper is to report the design and synthesis
of new polyvalent sialosides, obtained by functionalization of
poly(aspartimide) (PAI) with compounds containing sialic acid.
Our aim is to make new bioconjugate drugs that can potentially
Four new polyvalent compounds have been obtained by cou-
pling of sialic acid attached to four different linkers to poly(aspar-
timide). The spacers connected to sialic acid were of varying
length and functionality (Schemes 1–3) but each spacer group
* Corresponding author. Tel.: +40 232 278683/2176; fax: +40 232 271311.
E-mail addresses: icarlescu@yahoo.com, icarlescu@ch.tuiasi.ro (I. Carlescu).
0008-6215/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2009.08.034

34
I. Carlescu et al. / Carbohydrate Research 345 (2010) 33–40
that the sulfur-linked derivatives may offer better profiles in vivo,
OH
COO-Na+
due to their enhanced stability to glycosidase enzymes compared
with the O-linked glycosides. Thus 6 was coupled to protected
valerianic acid 12 (Scheme 2) to afford amide 7. This was then
treated with TFA to effect removal of the N-Boc group and to af-
ford amine 8.
HO
O
HO
AcHN
For the synthesis of the final sialic acid-spacer-amine derivative
11, the methyl ester of acetochloroneuraminic acid 2 was glycosyl-
ated with 4-hydroxy benzaldehyde under phase transfer condi-
tions34 (Scheme 3). The aldehyde functionality within the
obtained compound was coupled to the free amine functionality
within 13 under reductive amination conditions to afford amine
10. The terminal Boc-protecting group was then removed by treat-
ment with TFA, to afford the final sialic acid-spacer-amine deriva-
tive 11.
The obtained amino sialic acid intermediates 5, 6, 8 and 11
were then coupled in turn to poly (aspartimide) (Scheme 4)
according to the procedure described by Thoma et al.35 The reac-
tions were carried out in DMF at 50 °C in the presence of Et3N for
5 days and this was followed by treatment with an excess of 3-
amino-propanol at rt for 1 day. The acetate and methyl esters
within the compounds were then removed by treatment of the
glycopolymers with 1 M NaOH and the reaction products were
then purified by dialysis against water. After lyophilization, the
structures of the glycopolymers 18–21 were confirmed by spec-
troscopic methods.
HO
Figure 1. Representation of the sialic acid linked to body cell.
contained an amine functional group at its terminus to allow
attachment to the polymer. The syntheses of the amino spacer
functionalized sialic acid derivatives are described in detail
below.
2.1. Glycoconjugate synthesis
In order to attach the linker unit to the sialic acid, it was ini-
tially necessary to protect and activate the sialic acid to allow for
its subsequent glycosylation. This was achieved by reaction of sia-
lic acid with methanol, under acidic conditions, to form the
methyl ester 1. The reaction took place with good yield (90%)
and the obtained compound was sufficiently pure to be used in
the subsequent reaction without further purification. This was
then treated with acetyl chloride to form the fully acetylated
chloroneuraminic acid methyl ester 2 which proved a key inter-
mediate for the synthesis of the amino spacer functionalized sialic
acid derivatives 5, 6, 8 and 11. For entry to 3 and 4, chloride 2
was reacted with p-nitrophenol or p-nitrobenzenethiol under
phase transfer conditions22,31–33 to afford 3 and 4, respectively.
The nitro functionality was then reduced by treatment with tin(II)
chloride to afford the amines 5 and 6, ready for coupling to
poly(aspartimide). Amine 6 was also used in the preparation a
further amine-spacer-sialic acid derivative 8, as it was anticipated
The integration of signals from the 1H NMR spectra correspond-
ing to the aromatic protons within the sialic acid-linker-amine
intermediates (7.42–7.28 ppm) and protons from polymer chain
(2.66 ppm) allowed the quantitative analysis of the product com-
position. The results for this analysis are indicated in Table 1 and
these illustrate that incorporation of sialic acid into the poly(aspar-
timide) backbone was of varying success for each sialic acid-
spacer-amine derivative. A satisfactory incorporation was obtained
for compounds 8 and 11 that contained an aliphatic spacer within
the structure. An insignificant degree of incorporation of the sialic
OH
OH
HO
COOH
O
HO
AcHN
HO
SA
MeOH, H+(resin), rt,
90%
OH
OAc
OH
Cl
HO
AcO
HX
NO2
AcCl
97%
COOCH3
COOCH3
O
O
HO
AcO
AcHN
TBAHS, EtOAc, Na2CO3 1M
56%, 76%
AcHN
HO
AcO
(1)
(2)
OAc
OAc
COOCH3
X
COOCH3
X
AcO
AcO
SnCl2 2H2O
NO2
NH2
O
O
AcO
AcHN
AcO
AcHN
EtOH
84%
AcO
AcO
(3), X=O
(4), X=S
(5), X=O
(6), X=S
Scheme 1. Synthesis of sialic acid-linker-amine intermediates 5 and 6.

I. Carlescu et al. / Carbohydrate Research 345 (2010) 33–40
35
HO
C
NH2
OAc
O
Boc2O
COOCH3
S
AcO
H
N
HO
O
NH2
+
O
C
O
AcO
AcHN
(12)
O
AcO
DCC, DMAP, CH2Cl2
62%
(6)
OAc
COOCH3
S
AcO
H
N
O
H
N
C
O
O
AcO
O
AcHN
(7)
AcO
CF3COOH/CH2Cl2
99%
OAc
COOCH3
AcO
NH2
H
S
N
C
O
O
AcO
AcHN
(8)
AcO
Scheme 2. Synthesis of sialic acid-linker-amine intermediate 8.
OAc
COOCH3
AcO
Cl
O
AcO
AcHN
AcO
(2)
NH2
HO
CHO
H2N
TBAHS, EtOAc, Na2CO3 1M, 64%
Boc-ON
OAc
COOCH3
AcO
NH
O
H2N
O
CHO
+
O
AcO
AcHN
(13)
O
NaCNBH3/MeOH
84%
AcO
(9)
OAc
COOCH3
AcO
H
N
O
NH
O
O
AcO
AcHN
AcO
(10)
O
TFA, 97%
OAc
COOCH3
O
AcO
NH
NH2
O
AcO
AcHN
AcO
(11)
Scheme 3. Synthesis of sialic acid-linker-amine intermediate 11.

36
I. Carlescu et al. / Carbohydrate Research 345 (2010) 33–40
SA
linker
NH
O
O
O
O
O
O
O
N
1. H2N-linker-SA
O
N
H
O
H2N
OH
OH
OH
OH
H2N
HN
2. H2N-(CH2)3-OH
3. NaOH, MeOH
4. Dialysis
x
NH
OH
O
b
a
PAI
5. Liophylisation
(18): SA-linker-NH2= 5
(19): SA-linker-NH2= 6
(20):
SA-linker-NH2= 8
(21): SA-linker-NH2= 11
Scheme 4. Glycopolymer synthesis.
acid derivative into the polymer structure was obtained for amine
derivatives 5 and 6, which correlates to glycopolymers 18 and 19.
The introduction of an aliphatic chain within the linker improves
the incorporation, as illustrated for 20 and 21 (Table 1). This may
be due to less steric hinderance for these compounds. By increasing
the chain flexibility, which is the case for compounds with termi-
nal aliphatic chains, the inclusion of sialic acid increased to 2%.
It is important to mention that all the glycopolymers are water
soluble, which makes these compounds suitable for in vivo studies.
The presence of poly(aspartimide) also adds other characteristics
to polyvalent glycoconjugates such as easy biodegradability and
immunological properties.36,37
The weight-average molar mass, radius of gyration and the
polydispersities of glycopolymers are presented in Table 2. The
polydispersity index values (MW/Mn) indicate relatively narrow
molecular weight distributions.
The degrees of polymerization obtained from size exclusion
chromatography are consistent with the data obtained for the ori-
ginal polymer from NMR experiments in dimethyl sulfoxide. From
these spectra it is possible to determine the number-average de-
gree of polymerization comparing the integral of signals assigned
to NH2 protons (at chain ends) at 8.5 ppm and the single proton
of cycle belonging to the polymer chain at 5.3 ppm (see Scheme 4).
It is equal to 220. As a conclusion because the degree of polymer-
ization of polymers 20 and 21 determined by SEC is 203 and 210,
respectively (Table 2), there is no polymer degradation during
the chemical modification.
3. Conclusions
In this work we have described the synthesis and character-
ization of two new glycopolymers with potential antiviral prop-
erties. In general, glycosylation of the N-acetylneuraminic acid
has been achieved in good yields, using phase transfer catalyzed
reactions. 1H NMR spectroscopic studies have shown that the
efficiency of incorporating the sialyl derivatives into the
poly(aspartimide) increases with the introduction of an aliphatic
chain into the structure. This has resulted in the preparation of
water soluble compounds of interest for the inhibition of the
influenza virus. The assessment of the properties of these com-
pounds to inhibit influenza virus is currently under investigation
in our laboratories.
Table 1
Results of the synthesis of poly(aspartic acid)–sialic acid conjugatesa
Glycopolymer
Sialic acid-linker-amine
intermediates
PAI:SA
ratio
Incorporation
of SA (mol %)
Yield
(%)
4. Experimental
18
19
20
21
5
6
8
10:1
10:1
10:1
10:1
ND
ND
2
44
35
55
55
4.1. General methods
All reagents, solvents and starting materials were purchased
from Aldrich and Fluka and were used without further purification
unless otherwise noted. Poly(aspartimide) was prepared according
to the procedure described by Neri et al.38
11
1
a
Conditions: DMF/NEt3/50 °C/5 days and then excess of 3-aminopropanol for
1 day. Incorporation of ligand units was estimated on the basis of the results of 1H
NMR spectra. ND: not determined due to small amount.
Table 2
The calculations of molar mass and radius of gyration were carried out according to the Zimm fit method
Glycopolymer
MW (g/mol)
Mn (g/mol)
MW/Mn
Rg (nm)
dn/dc
Ip
DP
20
21
36,910
38,020
20.853
18.367
1.77
2.07
8.2
8.2
0.1420
0.145
1.77
2.07
203
210

I. Carlescu et al. / Carbohydrate Research 345 (2010) 33–40
37
Thin-layer chromatography was performed on Silica Gel 60-
4.3. Methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
D-
F254 (Merck) and detection was realized by either of the following
methods: charring with a solution of KMnO4 (aq), ninhydrin re-
agent spray, 5% (v/v) H2SO4 in EtOH and subsequent heating or
UV detection. Column chromatography was performed on Silica
Gel (Merck, Kieselgel 60). Dialysis was performed using dialysis
membranes (MW cutoff of 12–14,000 Da). 1H NMR spectra were re-
corded at 250 MHz on a Bruker spectrometer at room temperature.
Chemical shifts are reported in ppm relative to the solvent: CHCl3
in CDCl3 at 7.24 ppm, CD2HOD in CD3OD at 3.30 ppm and HOD in
D2O at 4.8 ppm (internal Me4Si, d = 0), or D2O. For carbon spectra
the references are 77.0 ppm for CDCl3 and 49.9 ppm for CD3OD.
Infrared spectra were recorded on a Perkin–Elmer 1720-X series
Fourier Transform spectrometer as thin films, Nujol mulls or KBr
glycero-b- -galacto-2-nonulopyranosyl)onate chloride (2)
D
A suspension of 1 (1.5 g, 4.64 mmol) in freshly distilled acetyl
chloride (50 mL) was stirred in a securely stoppered flask at room
temperature for 72 h and then concentrated under reduced pres-
sure (azeotroped three times with toluene). Drying under high vac-
uum afforded 2 (2.29 g, 97%) as a foam and this was used directly
in the subsequent reaction without further purification. 1H NMR
(250 MHz, CDCl3) d 1.92 (3H, s, NAc), 2.06, 2.07, 2.09, 2.13 (12H,
4 ꢂ s, 4 ꢂ OAc), 2.29 (1H, dd, J3a,3e = 13.9 Hz, H-3a), 2.80 (1H, dd,
J3e,3a = 13.9 Hz, H-3e), 3.89 (3H, s, CO2Me), 4.07 (1H, dd,
J9 = 12.5 Hz, H-9), 4.24 (1H, t, J5 = 10.2 Hz, H-5), 4.36 (1H, dd,
J6 = 10.2 Hz, H-6), 4.43 (1H, dd, J9 = 12.5 Hz, H-90) 5.18 (1H, ddd,
0
discs, all absorptions are quoted in cmꢀ1
.
J8 = 7.2 Hz, H-8), 5.33 (1H, d, JNH = 10.2 Hz, NH), 5.41 (1H, ddd,
J4 = 11.2 Hz, H-4), 5.49 (1H, dd, J7 = 7.2 Hz, H-7).
Size Exclusion Chromatography (SEC) analysis was conducted
using multiple detectors. Waters Alliance 2690 (USA) chromato-
graph was equipped with two Shodex OHpak columns (SB-
804HQ and SB-802.5HQ) and two online detectors: a differential
refractometer (Waters 2410) and a Dawn Helios light scattering
detector (MALS) from Wyatt (USA) equipped with a K5 cell and
120 mW solid-state laser operating at 658 nm. A 0.1 M solution
of NaNO3 was used as the eluent at a flow rate of 0.5 mL/min.
The weight-average molar mass, radius of gyration and polymole-
cularity were obtained from data collected and analyzed using AS-
TRA SEC-software (version 5.3.4, Wyatt Technology Corp., USA).
The calculations of molar mass (Fig. 2) and radius of gyration were
carried out according to the Zimm fit method (angles 5–16).
4.4. General method of glycosylation of acetochloroneuraminic
acid
To a solution of freshly prepared SA2 in ethyl acetate was added
a solution of the phenol compound and TBAHS in Na2CO3 (1 M
solution). The mixture was stirred vigorously for 3 h at room tem-
perature and next diluted with equal amounts of ethyl acetate and
NaHCO3 (saturated solution). The organic phase was separated and
washed twice with NaHCO3 (saturated solution), water and brine.
The organic phase was dried over MgSO4 and concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography using the mixture ethyl acetate/dichlorometh-
ane/triethylamine = 1:1:1%.
4.2. Methyl 5-acetamido-3,5-dideoxy-
nonulopyranosylonate (1)
D-glycero-b-D-galacto-2-
4.4.1. Methyl (4-nitrophenyl 5-acetamido-4,7,8,9-tetra-O-
To a stirred solution of Neu5Ac (3 g, 9.7 mmol) in dry methanol
(100 mL) at room temperature under N2 was added Amberlite IR-
120 (H+) (1.5 g). After stirring for 48 h, the resin was filtered off
and evaporated to a volume of ꢁ5 ml. Ether was added to turbidity
and 1 crystallized as a white solid on standing. It was recuperated
by filtration (2.82 g, 90%) and used in the subsequent reaction
without further purification; mp 191–193 °C, lit.39 179–180 °C.
1H NMR and 13C NMR spectral data matched those reported.
acetyl-3,5-dideoxy-2-oxy-a-D-glycero-D-galacto-2-
nonulopyranosid)onate (3)
By using the above-mentioned procedure, 2 (1 g, 1.96 mmol),
EtOAc (30 ml), p-nitrophenol (0.545, 3.92 mmol), Na2CO3 (1 M,
30 mL), TBAHS (0.798 g, 2.354 mmol) were combined to afford 3
(0.68 g, 56%) as a yellow foam mp 88–90 °C, lit.22 87–88 °C. 1H
NMR (250 MHz, CDCl3) d 1.94 (3H, s, NAc), 2.05, 2.06, 2.07, 2.12
(12H, 4s, 4 ꢂ OAc), 2.31 (1H, dd, J3a,3e = 12.5 Hz, H-3a), 2.79 (1H,
Figure 2. Fractograms of polymers 20 and 21, concentrations (line) and molar masses (dots).

38
I. Carlescu et al. / Carbohydrate Research 345 (2010) 33–40
dd, J3e,3a = 4.7 Hz, H-3e), 3.67 (3H, s, CO2Me), 4.07–5.37 (8H, m),
7.14 (2H, d, J (Ho,m) = 9.3 Hz, ArH-ortho), 8.21 (2H, d, ArH-meta).
13C NMR (CDCl3): 170.94–168.12 (6 ꢂ C@O), 159.03, 143.41,
125.70, 118.57, 99.52, 68.35, 68.20, 67.01, 62.14, 60.44 (OMe),
49.32, 38.76, 23.26–20.78 (5 ꢂ Ac).
(3H, s, NAc), 2.02–2.10 (12H, 4s, 4 ꢂ OAc), 2.14 (1H, m, H-3a),
2.72 (1H, dd, H-3e), 3.62 (3H, s, CO2Me), 3.81–5.32 (8H, m), 6.59
(2H, d, coupling constant as for 5, ArH), 7.25 (2H, d, ArH).
4.6. Boc-5-aminopentanoic acid (12)
4.4.2. Methyl (4-nitrophenyl 5-acetamido-4,7,8,9-tetra-O-
To 5-aminopentanoic acid (0.5 g, 4.268 mmol) dissolved in
water (12 mL) was added NaHCO3 (0.717 g, 8.54 mmol). The mix-
ture was stirred at 5 °C and added to Boc2O (1.397 g, 6.402 mmol)
in previously cooled dioxane (5 ml). The mixture was stirred at 0 °C
for 1 h and then allowed to heat to room temperature overnight.
Water (10 ml) was added. The aqueous layers were extracted twice
with ethyl acetate. The organic layers were extracted twice with
NaHCO3 (saturated solution). The combined aqueous layers were
acidified to a pH 1 with 10% HCl. The aqueous layers were ex-
tracted again three times with ethyl acetate, dried over MgSO4
and concentrated under reduced pressure. The compound (0.85 g,
91.7%) was obtained as a white powder and used directly in subse-
quent reactions without further purification, mp 45–48 °C. 1H NMR
(250 MHz, CDCl3) d 1.45 (9H, s), 1.54 (4H, m), 2.32 (2H, t), 3.06 (2H,
t), 3.32 (1H, s). 13C NMR (CDCl3) 158.56, 117.36, 79.85, 40.93,
34.53, 30.42, 28.79, 23.26.
acetyl-3,5-dideoxy-2-thio-D-glycero-a-D-galacto-2-
nonulopyranosid)onate (4)
By using the above-mentioned procedure, 2 (0.5 g, 0.98 mmol),
EtOAc (15 mL), p-nitrobenzenethiol (0.304, 1.96 mmol), 15 mL
Na2CO3 1 M, TBAHS (0.332 g, 0.98 mmol) (0.41 g, 67%) mp 168–
170 °C, lit.22 170–171 °C. 1H NMR (250 MHz, CDCl3) d 1.89 (3H, s,
NAc), 2.06–2.04 (12H, 4s, 4 ꢂ OAc), 2.17 (1H, dd, coupling con-
stants as for 3, H-3a), 2.84 (1H, dd, H-3e), 3.61 (3H, s, CO2Me),
3.98–5.51 (8H, m), 7.64 (2H, d, ArH), 8.21 (2H, d, ArH). 13C NMR
(CDCl3): 171.23–167.78 (6 ꢂ C@O), 148.59, 138.22, 135.47,
124.23, 87.35, 74.92, 69.23, 68.89, 67.36, 62.15, 60.43, 53.10
(OMe), 49.17, 38.34, 23.16–20.82 (5 ꢂ Ac).
4.4.3. Methyl (4-formylphenyl 5-acetamido-4,7,8,9-tetra-O-acetyt-
3,5-dideoxy-D-glycero-a-D-galacto-2 nonulopyranosid)onate (9)
By using the above-mentioned procedure, 2 (0.5 g, 0.98 mmol),
EtOAc (15 mL), p-hydroxybenzaldehyde (0.24, 1.96 mmol), 15 ml
Na2CO3 1 M, TBAHS (0.399 g, 1.17 mmol) (0.37 g, 64%) mp 88–
90 °C, lit.34 90.4–91.6 °C. 1H NMR (250 MHz, CDCl3) d 1.93 (3H, s,
NAc), 2.05–2.20 (12H, 4s, 4 ꢂ OAc), 2. 31 (1H, dd, J3a,e = 13.1 Hz,
H-3a), 2.70 (1H, dd, J3e,a = 13.1 Hz, H-3e), 3.65 (3H, s, CO2Me),
4.08 (1H, dd, H-90), 4.11 (1H, ddd, H-5), 4.17 (1H, ddd,
J9 = 10.3 Hz, H-9), 4.58 (1H, dd, J6 = 10.8 Hz, H-6), 4.92 (1H, ddd,
J4 = 12.2 Hz, H-4), 5.27 (1H, d, JNH = 10.0 Hz, NH), 5.38 (1H, m,
J7 = 1.4 Hz, H-7), 7.15 (2H, d, J (Ho,m) = 8.64 Hz, ArH-ortho), 7.82
(2H, d, ArH-meta), 9.93 (1H, –CHO). 13C NMR (CDCl3): 191.48
(CHO), 171.36–168.69 (6 ꢂ C@O), 159.41, 132.34, 132.13, 119.22,
99.89, 74.03, 69.05, 68.77, 67.49, 62.43, 53.64 (OMe), 49.75,
39.07, 23.65–20.17 (5 ꢂ Ac).
4.7. N-Boc-1,4-diaminobutane (13)
1,4-Diaminobutane (0.25 g, 2.836 mmol) dissolved in 12 N HCl
(0.3 ml) was added to a solution of dioxane and water in equal pro-
portion (5 ml) and cooled down to 0 °C. To this mixture Boc-ON
(0.23 g, 0.945 mmol) was added and allowed to warm at room
temperature. The solution was suspended in H2O (2 ml), acidified
with 6 N HCl to pH 4 and washed with EtOAc (3 ꢂ 4 ml). The
remaining aqueous phase was adjusted to pH 11 with 10 N NaOH.
The organic phase was extracted with EtOAc, dried over MgSO4 and
concentrated under reduced pressure to give a pale yellow oil
(0.13 g, 23%). 1H NMR (250 MHz, CDCl3) d 1.44 (9H, s), 1.52 (4H,
m), 1.97 (2H, s), 2.72 (2H, m), 3.11 (2H, m), 4.29 (1H, s). 13C NMR
(CDCl3) 156.43, 77.40, 41.65, 40.27, 28.34, 27.38.
4.5. General method of reduction of nitro compounds:
4-nitrophenyl derivatives
4.8. Methyl(tert-butyl-5-(4-aminophenyl-5-oxopentylcarbamate)-
5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-
a-D-
Compounds 3 and 4 were suspended in absolute ethanol to
which was added tin(II) chloride dihydrate. The reaction mixture
was stirred at 70 °C for 2 h and then cooled and poured onto ice-
water and the final pH was adjusted to 8 with NaHCO3. The result-
ing mixture was filtered and the clear filtrate extracted with EtOAc.
The organic layers were combined, washed successively with satu-
rated NaHCO3, H2O and saturated NaCl, dried over MgSO4 and con-
centrated under reduced pressure. The compounds 5 and 6 were
obtained as off yellow foams and were used directly in subsequent
reactions without further purification.
glycero- -galacto-2-nonulopyranosid)onate (7)
D
Compound 6 (0.31 g, 0.518 mmol), 12 (0.113 g, 0.518 mmol),
DCC (0.118 g, 0.57 mmol) and catalytic amount of DMAP in anhy-
drous CH2Cl2 were stirred at room temperature overnight. The dicy-
clohexylurea was filtered off and the solution was concentrated. The
foam residue was purified by silica gel column chromatography
using the mixture ethyl acetate: triethylamine in a ratio 1:1%. The
compound 7 was obtained as a pale yellow foam (0.26 g, 62%). 1H
NMR (250 MHz, CDCl3) d 1.44 (9H, s), 1.85 (3H, s, NAc), 2.07–2.00
(12H, 4s, 4 ꢂ OAc), 2.41 (2H, t), 2.56 (1H, s), 2.80 (1H, dd,
J3e = 17.27 Hz, H-3e), 3.17 (2H, m), 3.58 (3H, s, CO2Me), 3.93–5.31
(8H, m), 5.83 (1H, m), 7.44 (2H, d, J (Ho,m) = 8.47 Hz, ArH-ortho),
7.56 (2H, d, ArH-meta), 8.44 (1H, s). 13C NMR (CDCl3): 172.13–
168.34 (8 ꢂ C@O), 156.76, 149.05, 140.62, 138.69, 137.69, 123.09,
119.56, 114.93, 88.12, 75.08, 70.46, 68.13, 62.40, 60.83, 53.15
(OMe), 49.39, 38.36, 28.80, 23.47–21.22 (5 ꢂ Ac).
4.5.1. Methyl(4-aminophenyl5-acetamido-4,7,8,9-tetra-O-acetyl-
3,5-dideoxy-2-oxy-a-D-glycero-D-galacto-2-nonulopyranosid)-
onate (5)
Compound
3
(0.66 g, 1.077 mmol), SnCl2 ꢂ 2H2O (1.526 g,
6.767 mmol), ethanol (40 ml), (0.52 g, 83%). 1H NMR (250 MHz,
CDCl3) d 1.93 (3H, s, NAc), 2.05–2.12 (12H, 4s, 4 ꢂ OAc), 2.31 (1H,
dd, J3e,a = 12.9 Hz, H-3a), 2.7 (1H, dd, J3e,a = 5.1 Hz, H-3e), 3.66
(3H, s, CO2Me), 4.07–5.36 (8H, m), 6.62 (2H, d, J (Ho,m) = 8.48 Hz,
ArH), 6.89 (2H, d, ArH).
4.9. Methyl (5-amino-N-(4-phenyl)pentanamide-5-acetamido-
4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-a-D-glycero-D-galacto-
2-nonulopyranosid)onate (8)
4.5.2. Methyl (4-aminophenyl 5-acetamido-4,7,8,9-tetra-O-
acetyl-3,5-dideoxy-2-thio-
nonulopyranosid)onate (6)
Compound 4 (0.4 g, 0.637 mmol), SnCl2 ꢂ 2H2O (0.9 g, 4 mmol),
ethanol (30 ml), (0.32 g, 84%). 1H NMR (250 MHz, CDCl3) d 1.86
a
-
D
-glycero-
D
-galacto-2-
Compound 7 (0.33 g, 0.413 mmol) and TFA (6.20 g, 54.38 mmol)
were stirred in anhydrous CH2Cl2 (8 ml) at room temperature for
1.5 h then concentrated under reduced pressure. Drying under
high vacuum afforded 8 (0.285 g, 99%) as a foam mass and was

I. Carlescu et al. / Carbohydrate Research 345 (2010) 33–40
39
used in the subsequent reaction without further purification. 1H
4.12.1. Polymer (18)
NMR (250 MHz, MeOD) d 1.76 (4H, m), 1.84 (3H, s, NAc), 2.12–
1.98 (12H, 4s, 4 ꢂ OAc), 2.13 (1H, m), 2.48 (2H, m), 2.86 (1H, dd,
H-3e), 2.99 (2H, m), 3.60 (3H, s, CO2Me), 3.83–4.80 (8H, m), 7.47
(2H, d, J (Ho,m) = 8.49 Hz, ArH-ortho), 7.61 (2H, d, ArH-meta). 13C
NMR (CDCl3) 173.95–169.38 (7 ꢂ C@O), 141.85, 136.47, 124.39,
121.09, 88.83, 75.37, 71.25, 70.76, 68.97, 63.11, 53.36 (OMe),
40.44, 39.18, 37.01, 28.03, 23.28–20.87 (5 ꢂ Ac).
Compound 5 (0.22 g, 0.37 mmol), (PAI) (0.36 g, 3.71 mmol),
NEt3 (0.06 g, 0.56 mmol), DMF (4 ml), 3-amino-propanol (0.836 g,
11.14 mmol), methanol (10 ml), NaOH (2 ml), (0.21 g, 44%).
4.12.2. Polymer (19)
Compound 6 (0.36 g, 0.60 mmol), (PAI) (0.58 g, 5.98 mmol),
NEt3 (0.09 g, 0.9 mmol), DMF (10 ml), 3-amino-propanol (1.35 g,
17.98 mmol), methanol (15 ml), NaOH (3 ml), (0.26 g, 35%).
4.10. Methyl (tert-butyl-4-benzylamino)butylcarbamate-5-
acetamido-4,7,8,9-tetra-O-acetyt-3,5-dideoxy-2-oxy-
glycero- -galacto-2 nonulopyranosid)onate (10)
a
-
D
-
4.12.3. Polymer (20)
D
Compound 8 (0.37 g, 0.53 mmol), (PAI) (0.51 g, 5.29 mmol),
NEt3 (0.08 g, 0.79 mmol), DMF (7 ml), 3-amino-propanol (1.19 g,
15.95 mmol), methanol (16 ml), NaOH (3 ml), (0.43 g, 55%).
To 9 (0.37 g, 0.62 mmol) dissolved in methanol (20 ml), 13
(0.28 g, 1.49 mmol) was added. After 1 h of stirring at room tem-
perature NaCNBH3 (0.187 g, 2.976 mmol) and AcOH (0.178 g,
2.976 mmol) were added. After 24 h of stirring at room tempera-
ture, the reaction mixture was diluted with EtOAc (30 ml) and
Na2CO3 saturated solution (30 ml). After the filtration of salts, the
organic phase was separated, washed with Na2CO3 saturated solu-
tion (30 ml), dried over MgSO4 and concentrated under reduced
pressure. The compound 10 (0.4 g, 84%) was used directly in subse-
quent reaction without further purification. 1H NMR (250 MHz,
MeOD) d 1.38 (s, 9H), 1.45 (4H, m), 1.82 (3H, s, NAc), 1.95–2.08
(12H, 4s, 4 ꢂ OAc), 2.56 (2H, m), 2.64 (1H, s), 2.71 (1H, dd,
J3e = 13.34 Hz, H-3e), 2.99 (2H, t), 3.59 (2H, s), 3.67 (3H, s, CO2Me),
3.93–4.83 (8H, m), 5.32 (2H, m), 7.23 (2H, d, J (Ho,m) = 8.63 Hz, ArH-
ortho), 6.98 (2H, d, ArH-meta). 13C NMR (MeOD): 176.81–172.38
(7 ꢂ C@O), 161.43, 157.76, 138.54, 133.74, 126.28, 123.96,
104.39, 82.81, 77.30, 72.90, 66.20, 56.44, 52.76 (OMe), 50.09,
44.10, 31.87, 24.19–23.66 (5 ꢂ Ac).
4.12.4. Polymer (21)
Compound 11 (0.54 g, 0.81 mmol), (PAI) (0.79 g, 8.09 mmol),
NEt3 (0.123 g, 1.216 mmol), DMF (16 ml), 3-amino-propanol
(1.83 g, 24.36 mmol), methanol (21 ml), NaOH (4 ml), (0.56 g, 55%).
Acknowledgements
M.P. and I.C. gratefully acknowledge Ministry of Education of
Romania for financial support (Grant CEEX 5921/2006).
B. Grassl is acknowledged for his help in Size Exclusion chroma-
tography experiments.
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