Nanostructured styrenic co-polymers containing glucopyranosyl residues and their functionalization

Article abstract of DOI:10.1016/j.tet.2009.05.033

Sugar-based co-polymers with saccharidic units in stable cyclic form and nanometric morphologies stabilized through crosslinking, adaptable through specific functionalizations to biochemical interaction studies with copper-containing amine oxidases, were synthesized from appropriate monomers and macromonomers. The most promising nanospherical co-polymer obtained, containing β-d-glucopyranosidic units, was employed in functionalization reactions with the help of model molecules, achieving useful transformations mainly at the 6-position and to a minor extent at the 2-position of the saccharidic system.

Full text of DOI:10.1016/j.tet.2009.05.033

Tetrahedron 65 (2009) 5684–5692
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Nanostructured styrenic co-polymers containing glucopyranosyl
residues and their functionalization
*
Marco Pocci , Silvana Alfei, Francesco Lucchesini, Vincenzo Bertini, Barbara Idini
`
Dipartimento di Chimica e Tecnologie Farmaceutiche e Alimentari, Universita di Genova, Via Brigata Salerno 13, I-16147 Genova, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Sugar-based co-polymers with saccharidic units in stable cyclic form and nanometric morphologies
stabilized through crosslinking, adaptable through specific functionalizations to biochemical interaction
studies with copper-containing amine oxidases, were synthesized from appropriate monomers and
Received 2 January 2009
Received in revised form 29 April 2009
Accepted 14 May 2009
macromonomers. The most promising nanospherical co-polymer obtained, containing
b-D-glucopyr-
Available online 21 May 2009
anosidic units, was employed in functionalization reactions with the help of model molecules, achieving
useful transformations mainly at the 6-position and to a minor extent at the 2-position of the saccharidic
system.
Keywords:
Glycopolymers
Macromonomers
Ó 2009 Elsevier Ltd. All rights reserved.
Nanostructured co-polymers
Glucopyranoside model
1. Introduction
stabilizers or emulsifiers, has been successfully exploited for the
synthesis of molecularly imprinted microspheres,16,17a,b nano-
Nanostructured polymeric systems find increasing applications
in various biomedical fields1 such as biosensors, tissue engineering
and drug delivery systems. In particular, hydrophilic nano-
structured glycopolymers with saccharidic moieties at the particle
surface extending towards the aqueous medium are attractive for
their possible interactions with proteins2 or cells3 and utility in
drug delivery.3
As a part of our ongoing interest in copper amine oxidases
(CAOs),4–9 a diverse class of enzymes10 that control important
cellular processes such as cell proliferation and crosslinking of
elastin and collagen, we have recently reported the synthesis of
crosslinked nanostructured gluconamidic vinyl co-polymers and
their functionalization by O-alkylation with benzaldehydic resi-
dues,11 as models for more complex fuctionalizations aimed to lo-
calized studies of angiogenesis and cell proliferation.
particles17a,b and nanostructured gluconamidic vinyl co-
polymers.11
In this work we report the synthesis and functionalization of
new sugar-based co-polymers having nanostructured morphol-
ogies stabilized through crosslinking and bearing saccharidic units
in stable cyclic glucopyranosidic form (structures a and b, Chart 1).
This enables an alternative ordering of the hydroxyl groups influ-
encing adhesion of proteins without altering the effect of the added
bioactive functions, allowing the production of materials, which
can be exploited in biomedical research based on the interaction
with copper-containing amine oxidases.
We achieved the preparation of the target sugar-based co-
polymers in the form of nanostructured systems through the
OH
The free radical polymerization (FRP) of sugar-based vinyl
monomers, successfully employed in the synthesis of glycopoly-
mers12–15 for simplicity of operation, solvent compatibility and
tolerance to many monomer functionalities, offers an interesting
variety of experimental polymerization conditions such as in bulk,
in solution and in multiphase systems including suspension,
emulsion, dispersion and precipitation polymerizations. Pre-
cipitation polymerization, which avoids the use of additives such as
HO
O
OH
OH
H
N
a
O
O
O
N
H
OH
O
H
n
HO
OH
OH
S
H
b
N
O
O
* Corresponding author. Tel.: þ39 010 3532685; fax: þ39 010 3532684.
E-mail address: pocci@dictfa.unige.it (M. Pocci).
Chart 1.
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.05.033

M. Pocci et al. / Tetrahedron 65 (2009) 5684–5692
5685
radical precipitation polymerization of new glucopyranosidic
monomers and macromonomers, and examined their functionali-
zation with the help of model molecules.
The deacetylation of Mac5 and Mac7 with MeONa in MeOH/
dioxane was monitored by observing the disappearance of the
strong absorptions at 1755 and 1230 cmꢀ1 of the ester groups and
the appearance of very strong hydroxyl bands at 3410 cmꢀ1 in the
FTIR spectra of Mac6 and Mac8, which proved soluble only in water,
DMSO, DMF and partially soluble under heating in MeOH and EtOH.
2. Results and discussion
2.1. Monomers and macromonomers
The new monomers 5, 6, 7 and 8, with the polymerizable
function linked to C-1 of the sugar moiety in stable cyclic structure,
were synthesized from 118 (Scheme 1) through the known inter-
mediates 2,19a–c 319b,20 and 4.21 The peracetylated monomers 5 and
7 were obtained in the form of stable spectroscopically character-
ized crystalline solids. Monomer 6, obtained from the deprotection
of 5, was a low melting solid, difficult to manipulate due to its
hygroscopicity and tendency to polymerize, so it was not employed
in polymerization experiments. Monomer 8, obtained in high yield
by deacetylation of 7, was a stable, lightly hygroscopic, white solid.
The 1H, 13C NMR and DEPT-135 spectra of 8 confirmed its structure
2.2. Precipitation co-polymerizations
Hydroxyl-rich Mac6, Mac8 and 8, were co-polymerized with
styrene or crosslinkers by radical precipitation polymerization in
the presence of appropriate solvents. The monomer feed molar
composition for the macromonomers was calculated on the basis of
an average degree of oligomerization of three, corresponding to
a nominal molecular weight of 1039 for Mac6 and 1267 for Mac8.
Mac6 and Mac8 were co-polymerized at 60 ꢁC with styrene in
mixtures H2O/EtOH (1/1.5–1.6 v/v, depending on macromonomer
feed molar composition in the range 0.17–0.80). Mac6 was co-
polymerized with 1,4-divinylbenzene (DVB) as crosslinker at 70 ꢁC
(in H2O/EtOH¼1:1.9) to afford co-polymers, which under obser-
vation with Scanning Electron Microscopy (SEM) showed irregular
morphologies of little interest.
and the b-stereochemistry of the anomeric carbon. In particular the
1H NMR spectrum recorded in DMSO-d6 showed peaks for NH,
secondary OH groups at C-2, C-3 and C-4, and primary OH at C-6
and H-1 as well separated signals (Experimental).
The macromonomers Mac6 and Mac8 were obtained by parallel
syntheses from peracetylated monomers 5 and 7 through radical
oligomerization to form Olig5 and Olig7 in the presence of 2-
aminoethanethiol hydrochloride,22 functionalization with 4-vinyl-
benzoyl chloride23 to form Mac5 and Mac7 and final deacetylation.
Scheme 2 shows the conversion of 5 and 7 into Mac6 and Mac8,
respectively.
The number average molecular weight values (Mn) of Olig5 and
Olig7 determined by acidimetric titration of the amino end group
were 1312 and 1580, respectively, close to the theoretical values of
1450 and 1678 calculated for a degree of oligomerization of three.
The styrenic Mac5 and Mac7 discoloured Br2 in CCl4 and
aqueous KMnO4 and gave a negative ninhydrin test, thus proving
the presence of the double bond and the absence of the amino
group. They both proved insoluble in hexane, Et2O, water and sol-
uble in benzene, CHCl3, CH2Cl2, acetone, dioxane, pyridine, THF,
DMF, EtOH.
Mac8 afforded co-polymers with peculiar lamellar structures
when co-polymerized as reported in Table 1 with DVB (CM8-1), or
with DVB and styrene (CM8-2), or with other crosslinkers such as
ethylenebisacrylamide (EBA) (CM8-3), or ethyleneglycol dimetha-
crylate (EGDM) (CM8-4). Figure 1a and b shows two typical mor-
phologies of the prepared co-polymers. The investigation of these
lamellar structures was beyond the aim of this work and it was not
pursued. Nevertheless, it must be observed that the co-polymers
CM8 prepared from the macromonomer Mac8 are expected to have
‘comb-like’ structure and amphiphilic nature, two properties that in
other glycoconjugated polystyrenes24 give rise to ordered confor-
mations, stable in aqueous urea solution, where they form cylindrical
‘bottle-brush’ micelles.
Monomer 8, subjected to precipitation co-polymerization with
DVB in MeOH, afforded spherically shaped co-polymers (C8) (Table 1)
with average diameter of 589ꢂ89 nm (Fig. 2), which were chosen for
functionalization experiments.
OAc
OAc
OAc
AcO
O
OAc
OAc
AcO
O
OAc
OAc
AcO
AcO
OAc
OAc
OH
NAN3
H2 (3.5 atm)
Pd/C 10%
TsOH
Br
N3
Br
DMF
O
BF3 OEt2
O
O
100 °C
2 (57%)
3 (87%)
1
OAc
OAc
OH
HO
acryloyl chloride
Et3N, rt
MeONa
MeOH
AcO
O
OAc
OAc
AcO
OAc
OAc
OH
OH
TsOH
H2N
HN
O
HN
O
O
O
O
O
O
5 (67%)
4 (99%)
6 (67%)
4-vinylbenzoyl
chloride
Et3N, rt
OH
OAc
HO
OH
OH
AcO
O
OAc
OAc
MeONa
MeOH
HN
O
HN
O
O
O
O
7 (83%)
8 (90%)
Scheme 1.

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M. Pocci et al. / Tetrahedron 65 (2009) 5684–5692
OAc
O
OAc
O
AcO
O
OAc
OAc
AcO
O
OAc
OAc
H
N
H
N
O
O
HS(CH2)2NH3Cl
AIBN/DMF/60 °C
83%
HS(CH2)2NH3Cl
AIBN/DMF/60 °C
80%
5
7
OAc
AcO
H
n
.
OAc
AcO
H2N
HCl
OAc
OAc
.
OAc
OAc
HCl
H2N
H
S
S
S
H
N
n
H
N
Olig7
S
Olig5
O
O
O
O
O
O
O
O
4-Vinylbenzoyl
chloride
Et3N
4-Vinylbenzoyl
chloride
Et3N
86%
88%
O
H
n
N
OAc
OAc
H
AcO
O
OAc
OAc
AcO
O
OAc
OAc
O
HN
H
n
H
N
H
N
S
O
O
Mac7
Mac5
O
NaOMe
MeOH/dioxane
NaOMe
MeOH/dioxane
91%
59%
H
n
O
OH
N
H
OH
HO
O
OH
OH
O
HO
O
OH
OH
HN
H
n
H
N
H
N
S
O
O
Mac6
Mac8
O
Scheme 2.
2.3. Functionalization of co-polymers obtained from 8
groups were involved. Model compound 10 based on the molecular
structure of monomer 8 was synthesized according to Scheme 3
from the intermediate 9 obtained from 4 or 1, with the latter route
being preferred to the former.
DEPT-135 and HETCOR experiments allowed the assignment of
all the carbon signals of the saccharidic portion of 10. In addition,
the 1H NMR spectrum of 10 in DMSO-d6 showed signals for sec-
ondary OH groups at C-4, C-3 and C-2, primary OH at C-6 and
amidic NH (Experimental). These were very useful for revealing the
position and multiplicity of the alkylation reaction.
The study of co-polymer functionalization through deprotona-
tion of the saccharidic hydroxyl groups with strong bases and re-
action with alkyl chlorides to form stable ether bridges required the
synthesis of suitable model molecules to ascertain which hydroxyl
Table 1
Typical co-polymerization data of Mac8 and 8 at 70 ꢁC
Monomer
mg (mmol) g (mmol)
Comonomer Crosslinker AIBN
Solvent mL Time (h) Polymer
mg (%)
Model compound 10 was deprotonated with NaH in less than
stoichiometric ratio (Table 2) to limit multisubstitution followed by
alkylation with model chlorides carrying readily detectable alde-
hydic groups like the very reactive allylic chloride 1111 or the pri-
mary alkyl chloride 12.11 This afforded, under different reaction
conditions, products of O-alkylation mono or disubstituted at the
positions 6, 2 and 4.
The reaction of 10 with NaH (0.9 equiv) then with stoichiometric
(or excess) allyl chloride 11 in DMF at 24 ꢁC (Table 2) gave mixtures
of products, which by preparative layer chromatography (PLC)
afforded some unreacted 10, unresolvable mixtures of 13 and 14 in
ratios ranging from 1.44/1 to 2.25/1, small quantities of 15 and
dialkylation products, and non-saccharidic by-products. PLCs of
pooled fractions from different runs of mono and dialkylated
mixtures allowed the isolation of very small amounts of 15 and 16,
however sufficient for their characterization.
mg (%)
mg (%)
Mac8
350
d
DVB
53.0
(12.1)
DVB
5.1
(5.0)
EBA
4.0
H2O/EtOH
1/1.7
(72)
H2O/EtOH
1/1.5
(17.5)
H2O/EtOH
1/1.14
(15)
H2O/EtOH
1/1.4
(17)
24
24
74
48
70
CM8-1
110.5
(27.4)
CM8-2
11.4
(10.6)
CM8-3
11.5
(11.1)
CM8-4
12.2
(11.7)
C8
20.0
(4.6)
(0.22)a
Mac8
100
STY
2.05
(19.7)
d
16.7
(5.1)
(0.08)a
Mac8
100
5.2
(0.08)a
Mac8
100
(4.0)
EGDM
4.0
(4.0)
DVB
8.5
(5.2)
d
d
5.2
(5.2)
(0.08)a
8
MeOH
213
(0.60)
4.3
56.9
(3.2)
(1.9)
(43)
(25.9)
DVB¼divinylbenzene; STY¼styrene; EBA¼ethylenebisacrylammide; EGDM¼
ethyleneglycol dimethacrylate.
a
Mono and double alkylation products were established by ob-
serving the ratios of integrals of the NH signals with respect to the
Calculated from the reported nominal molecular weight of 1267 corresponding
to a nominal loading of 0.789 mmol/g of saccharidic units.

M. Pocci et al. / Tetrahedron 65 (2009) 5684–5692
5687
Figure 1. SEM images of the co-polymers CM8-3 (a), CM8-4 (b).
Figure 2. SEM images of the co-polymer C8.
H
N
OAc
OAc
OAc
AcO
O
OAc
OAc
HO
AcO
O
OAc
OAc
AcO
AcO
OAc
OAc
PhCOCl
Et3N, rt
(77%)
O
HN
H2N
.
O
BF3O OEt2
(75%)
O
.
O
O
TsOH
9
1
4
MeONa
MeOH
OH
HO
OH
OH
HN
O
O
O
10 (94%)
Scheme 3.
aldehydic ones in the 1H NMR spectra (1:1 for monoalkylation and
1:2 for double alkylation, respectively).
The attributions of the structures 13–16 were achieved by
comparing the DEPT-13513C NMR spectra of the saccharidic portion
of model 10 and alkylation products, and by looking for the
expected jump of about 7–9 ppm associated with the conversion of
hydroxyl carbon to alkoxyl carbon (Fig. 3, Chart 2).
The addition of dibenzo-18-crown-6 to the reacting mixture for
the alkylation of 10 with 11 to avoid possible preferential intra-
molecular complexation25 and to promote the reactivity of naked
alkoxy ions eliminated double substitution and lowered the re-
action selectivity slightly, reducing the 13/14 ratio.
The GC–MS analysis of the non-saccharidic portion of the various
runs revealed, besides unreacted 11 (11–34%), the presence of 4-(4-
hydroxy-2-butenyloxy)benzaldehyde (3–5%), N-(2-hydroxyethyl)-
benzamide (about 11%) and largely variable percentages of
4-hydroxybenzaldehyde under different reaction conditions, evi-
dencing the formation of spurious products.
A reactivity test of 11 treated at 24 ꢁC for 240 h with MeONa
in MeOH gave only the expected methyl ether by chlorine
substitution.
The 13C NMR spectrum of 16 confirmed the hydroxyl groups at C-6
and C-2 in 10 asthe preferred reactive sites, with the disappearance of
the signals at 62.00 ppm (C-6) and 74.40 (C-2) and the appearance of
signals at 70.81 and 81.81 ppm, respectively being observed.
The alkylation of 10 with 11 carried out at 0 ꢁC gave rise to
suppression of double substitution and increase of the 13/14 ratio
up to 5.88.

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M. Pocci et al. / Tetrahedron 65 (2009) 5684–5692
Table 2
Reaction data of the alkylation of 10 with 11 and 12 in DMF
Run
10 mg (mmol)
Chloride
Molar ratio 10/NaH/chloride
t
ꢁC
Time H
A mg (%)
16 mg (%)
C-6/C-2 alkylation ratio
1
2
3
4
5
6
7
8
89.4 (0.273)
95.8 (0.293)
101.2 (0.309)
96.4 (0.295)
100.7 (0.308)
87.2 (0.266)
91.9 (0.281)
96.9 (0.296)
11
11
11
11
11
11
12
12
1/0.9/1
1/0.9/1
1/0.9/3
1/0.9/1
1/0.9/1b
1/0.9/1b
1/0.9/1
1/0.9/1
24
24
24
0
24
24
24
80
24
72
24
144
8
24
99
4
21.5 (17.9)
24.0 (18.4)
25.0 (17.9)
23.3 (17.5)
30.8 (22.1)
24.7 (20.5)
7.9 (6.2)
4.8 (2.6)
4.7 (2.4)
7.8 (3.7)
d
d
d
d
d
2.25 (13/14)a
2.10 (13/14)a
1.44 (13/14)a
5.88 (13/14)a
1.50 (13/14)a
1.70 (13/14)a
5.08 (17/18)c
1.50 (17/18)c
12.1 (9.0)
A¼mixture of monoalkylated products.
a
From the –CH]CH– signals.
In the presence of 18-dibenzo crown ether (0.9 equiv).
From the anomeric CH signals.
b
c
A stability test of a mixture 13/14 treated with NaH in DMF at
Samples of the nanospherical co-polymer C8 were subjected to
treatment with NaH then with allyl chloride 11. It was concluded
that 11 was more reactive and attractive than 12 as a model for
appropriate bioactive reagents. The co-polymers underwent func-
tionalization parallelling those of the model molecule 10. After the
removal of low molecular weight aldehydes and spurious alkyl-
ation products by careful washing and centrifugation, the func-
tionalized co-polymers gave positive Schiff’s tests as shown by the
fuchsia colour of variable intensity developed on the surface of the
solid co-polymers, not removable by washing.
24 ꢁC for 48 h produced both N-(2-hydroxyethyl)benzamide and 4-
hydroxybenzaldehyde as shown by GC–MS.
Side reactions and the formation of by-products contributed to
make the overall alkylation yields rather modest, nevertheless the
obtained yields, if transferred to co-polymers, would correspond to
functionalization in the range 0.4–0.6 mmol/g, sufficiently good for
the planned bioactive co-polymers.
The reaction of 10 with NaH, then with 12, carried out in
a similar way as with 11, at 24 ꢁC gave only mono-substituted
products at positions 6 and 2 producing an unresolvable mixture of
17 and 18 (Table 2). This is in agreement with the lower reactivity of
the primary alkyl chloride, with lower yields for longer reaction
time being observed.
The increase of reaction temperature to 80 ꢁC never gave rise to
the formation of dialkylation products and promoted a modest
increase of yields of monoalkylation with the formation of an un-
determined small amount of 19 alkylated at C-4. Since the GC–MS
of the non-saccharidic portion of the mixture revealed the presence
of 4-(2-butenyloxy)benzaldehyde indicating alteration of 12, pro-
longed reaction times were not explored further.
3. Conclusions
With the view of attaining new research tools in the field of
copper-containing amine oxidases based on new saccharidic
nanostructured co-polymers functionalized to make them capable
of specific interaction with the enzyme active site, we synthesized
monomers 5, 6, 7 and 8.
Monomer 6 was discarded for its poor physical properties. Per-
acetylated monomers 5 and 7 were transformed into the macro-
monomers Mac6 and Mac8, respectively.
In the strongly basic conditions of the functionalization of 10
with 11 or 12, undesired reactions of aldehydic carbonyls with
hydroxyl groups were never observed.
Mac6, Mac8 and 8 were subjected to precipitation co-poly-
merization in the presence of crosslinkers affording polymeric
materials with different morphologies. Co-polymers from Mac6
Figure 3. DEPT-135 spectra of the saccharidic portion of 10 (a), mixture 13þ14 (b), 15 (c) and 16 (d).

M. Pocci et al. / Tetrahedron 65 (2009) 5684–5692
5689
O=HC
OH
CH=O
CH=O
O
HO
O
OH
H
N
O
O
O
Cl
O
Cl
O
13
12
11
O=HC
O=HC
O=HC
O=HC
OH
O
O
O
OH
OH
O
O
HO
O
O
O
OH
OH
O
OH
HN
OH
HN
HN
O
O
O
O
O
O
O
O
15
14
16
O=HC
O=HC
O=HC
O
O
OH
O
O
OH
OH
O
HO
O
OH
O
OH
OH
HO
O
H
N
O
HN
HN
OH
O
O
O
O
O
O
18
19
17
Chart 2.
were discarded because they were not satisfactory for further
functionalization. Co-polymers from Mac8 (CM8) were character-
ized by lamellar structures, not studied further in this context. Co-
polymers from 8 (C8) showed very attractive spherical shapes with
average diameter of 589 nm. These were chosen for a study of
functionalization through deprotonation and alkylation of the
saccharidic hydroxyl groups.
Model molecules 10 (for the saccharidic moiety) and 11 or 12
(for the electrophilic reagent) were synthesized and used to model
the alkylation process. The functionalization of 10, performed using
10% less than stoichiometric NaH in order to limit the introduction
of more than one aldehydic group per saccharidic unit, afforded
products of mono or dialkylation involving positions 6, 2 and to
a much lesser extent the position 4. Double alkylation was easily
avoided by lowering the reaction temperature. Monoalkylation
reactions, giving mixtures of 6 and 2-O-alkyl derivatives showed
a selectivity in favour of the 6-alkylated product, which increased
on lowering the reaction temperature.
procedures, with the exception of 4, which was carefully extracted
with dry Et2O to transform the white glassy foam into crystals (mp
150–152 ꢁC, purity 98% by HPLC).
All the reagents including Schiff’s fuchsin-sulfite reagent and
solvents were purchased from Aldrich. The solvents were dried and
distilled according to standard procedures. Petroleum ether refers
to the fraction with boiling point 40–60 ꢁC. Styrene (STY), 1,4-
divinylbenzene (DVB, containing 80% of DVB isomers) and ethyl-
eneglycol dimethacrylate (EGDM) were distilled at reduced
pressure under N2 and stored at ꢀ20 ꢁC. 2,20-Azobis(2-methyl-
proprionitrile) (AIBN) was crystallized from MeOH.
4.2. Methods
Melting points, determined on a Leica Galen III hot stage ap-
paratus, and boiling points are uncorrected. FTIR spectra were
recorded as films or KBr pellets on a Perkin Elmer System 2000
spectrophotometer. 1H and 13C NMR spectra were acquired on
a Bruker Avance DPX 300 Spectrometer at 300 and 75.5 MHz, re-
spectively, using TMS as internal standard and assigned through
DEPT-135, COSY, HETCOR and decoupling experiments. GC–MS
analyses were obtained with an Ion Trap Varian Saturn 2000 in-
The functionalization of co-polymer C8 performed in analogy to
10 with NaH and the more suitable model 11 was confirmed by the
fuchsia colour of the Schiff’s test, developed on the surface of the
solid co-polymer, which was not removable by washing.
strument (CI mode, filament current 10
mA) equipped with a DB-
4. Experimental
4.1. Materials
5MS (J&W) 30 m, i.d. 0.32 mm, film 1 m capillary column.
m
HPLC analyses were performed at rt, constant flow rate (1 mL/
min) and UV detection (254 nm) using a Merck LiChrocart 125-4
HPLC cartridge, with a mixture acetonitrile/water¼60:40 as eluent.
Flash chromatography (FC) was performed on Merck Silica gel
(0.040–0.063 mm). TLCs were obtained on Merck F254 silica gel
aluminium sheets. The saccharidic compounds were evidenced by
spraying the TLC with a 0.3% solution of o-aminodiphenyl in H2SO4/
EtOH (5:95) followed by heating. Preparative layer chromatogra-
phies (PLCs) were performed on Merck F254 60 silica gel plates
(20ꢃ20 cm, 0.5 mm).
1,2,3,4,6-Penta-O-acetyl-b-D
-glucopyranose (1),18 2-bromoethyl
2,3,4,6-tetra-O-acetyl-
2,3,4,6-tetra-O-acetyl-
2,3,4,6-tetra-O-acetyl-
b-
b-
b-
D
D
D
-glucopyranoside (2),19a–c 2-azidoethyl
-glucopyranoside (3),19b,20 2-aminoethyl
-glucopyranoside toluene-4-sulfonic acid
salt (4),21 4-vinylbenzoyl chloride,23 4-vinylbenzoic acid,26 (E)-4-
(4-chloro-2-butenyloxy)benzaldehyde (11)11 and 4-(4-chlor-
obutoxy)benzaldehyde (12)11 were prepared according to known

5690
M. Pocci et al. / Tetrahedron 65 (2009) 5684–5692
Scanning Electron Microscopy (SEM) images were obtained
with a Leo Stereoscan 440 instrument (LEO Electron Microscopy
A sample of monomer 6 in the form of highly hygroscopic
amorphous solid, for characterization purposes was chromato-
graphed by PLC (CHCl3/MeOH 4:1 as eluent) and obtained as
a glassy foam.
Compound 6. Yield 67%. FTIR (KBr,
(amide), 1627 (C]C), 1077, 1036 (C–O).
`
Ltd) at Dipartimento di Chimica e Chimica Industriale (Universita di
Genova). The average diameter of co-polymers C8 was calculated by
measuring directly on the screen the diameters of 41 nanospheres
of the SEM image in Figure 2a.
n
, cmꢀ1) 3392 (OH), 1659
Microanalyses of solid compounds 5, 7, 8 and 9 were obtained
1H NMR (DMSO-d6) 2.95–3.38 (m, 10H), 4.15 (d, H-1, J¼7.4 Hz),
4.58 (br s, 1OH), 5.03 (br s, 3OH), 5.59 (dd, 1H, Jgem¼2.3 Hz,
Jcis¼10.1 Hz), 6.09 (dd, 1H, Jgem¼2.3 Hz, Jcis¼10.1 Hz), 6.28 (dd, 1H,
Jcis¼10.1 Hz, Jtrans¼17.1 Hz), 8.15 (br t, NH). 13C NMR 38.58, 60.97,
67.73, 69.96, 73.33, 76.46, 76.77, 103.05, 125.05, 131.61, 164.63.
The glassy monomer 8 was obtained as a white powder by
dissolution in MeOH and precipitation in dry Et2O.
`
from the Laboratorio di Microanalisi (Facolta di Farmacia, Uni-
`
versita di Pisa).
4.3. Monomers 5–8
Crystalline 4 (3.0 mmol) in dry CH2Cl2 (18 mL) was treated un-
der N2 at rt with dry Et3N (1.7 mL) then with acryloyl chloride or 4-
vinylbenzoyl chloride23 (3.0 mmol). After stirring for 4 h at rt the
mixture was hydrolyzed with water (18 mL, pH¼7–8), extracted
with CH2Cl2 (3ꢃ20 mL) and dried over anhydrous MgSO4. The
removal of the solvent at reduced pressure afforded the crude N-
Compound 8. Yield 90%. Mp 104–106 ꢁC, purity 98% by HPLC.
FTIR (KBr, n
, cmꢀ1) 3351 (OH), 1627 (amide), 1078, 1053 (C–O), 987
and 909 (vinyl). 1H NMR (DMSO-d6) 2.94–3.24 (m, 4H), 3.32–3.76
(m, 5H), 3.78–3.94 (m, 1H), 4.20 (d, H-1, J¼7.7 Hz), 4.54 (t, OH,
J¼5.8 Hz), 4.94 (d, OH, J¼4.8 Hz), 4.99 (d, OH, J¼4.5 Hz), 5.07 (d, OH,
J¼4.3 Hz), 5.37 (d, 1H, Jcis¼11.0 Hz), 5.96 (d, 1H, Jtrans¼17.6 Hz), 6.79
acryloyl-2-aminoethyl 2,3,4,6-tetra-O-acetyl-
(5) as a glassy foam or N-(4-vinylbenzoyl)-2-aminoethyl 2,3,4,6-
tetra-O-acetyl- -glucopyranoside (7) as a white solid.
b-D-glucopyranoside
(dd, 1H, Jcis¼11.0 Hz, Jtrans¼17.6 Hz), 7.70 (m, 4H), 8.44 (t, NH). 13
C
b
-D
NMR 39.96, 61.68, 68.20, 70.65, 74.08, 77.20, 77.53, 103.75, 116.77,
126.58, 128.18, 134.17, 136.53, 140.34, 166.61. Anal. Calcd for
C17H23NO7: C, 57.78; H, 6.56; N, 3.96. Found: C, 57.67; H, 6.58; N,
3.95.
Monomer 5 was purified by two cycles of dissolution in dry THF
and precipitation in pentane, dissolution in EtOAc, filtration
through a short column of silica gel (h¼10 cm,
tion of the solvent and crystallization from Et2O: yield 67%; mp
114–117 ꢁC, purity 98% by HPLC. FTIR (KBr, , cmꢀ1) 3351 (NH), 1759
4¼1 cm), evapora-
n
4.4. Oligomerizations of monomers 5 and 7
and 1744 (ester), 1667 (amide), 1634 (C]C), 1066 and 1040 (C–O),
989 (vinyl). 1H NMR (CDCl3) 2.01 (s, 3H), 2.03 (s, 3H), 2.04 (s, 3H),
2.09 (s, 3H), 3.55 (m, 2H), 3.75 (m, 2H), 3.88 (m, 1H), 4.17 (dd, 1H,
J¼2.5, 12.4 Hz), 4.25 (dd, 1H, J¼4.8, 12.4 Hz), 4.51 (d, 1H, J¼7.9 Hz),
5.00 (m, 2H), 5.21 (t, 1H, J¼9.5 Hz), 5.65 (dd, 1H, Jcis¼10.2 Hz,
Jgem¼1.6 Hz), 6.11 (dd, 1H, Jcis¼10.2 Hz, Jtrans¼17.0 Hz), 6.10 (br s,
NH), 6.29 (dd, 1H, Jtrans¼17.0 Hz, Jgem¼1.6 Hz). 13C NMR 20.58, 20.72,
39.25, 61.87, 68.33, 69.30, 71.40, 72.01, 72.61, 101.05, 126.59, 130.74,
165.52, 169.42, 169.55, 170.16, 170.62.
The peracetylated monomer (5 or 7) (4.0 mmol), 2-amino-
ethanethiol hydrochloride (0.8 mmol) and AIBN (4% by weight of
the monomer) were dissolved under N2 in MeOH (20 mL/mmol of
monomer 5) or dry DMF (13 mL/mmol of monomer 7) in a vial with
side arm, subjected to two freeze-pump-thaw cycles at ꢀ78 ꢁC,
siphoned into the polymerization flask and magnetically stirred at
60 ꢁC for 72–79 h. The mixture was cooled, concentrated under
reduced pressure and poured into a large excess of Et2O (400 mL/g
of oligomer). The oligomer was filtered and further purified
through two dissolution/precipitation cycles as follows: Olig5
[MeOH (solvent)/Et2O (non-solvent)]; Olig7 [THF (solvent)/Et2O
(non-solvent)] until the disappearance of the residual monomers (5
or 7) was observed by TLC using petroleum ether/acetone 1:1 as
eluent (Rf: 5¼0.49, 7¼0.73, Olig5 or Olig7¼0).
Anal. Calcd for C19H27NO11: C, 51.23; H, 6.11; N, 3.14. Found: C,
51.16; H, 6.09; N, 3.13.
Monomer 7 was purified by dissolution in EtOAc (100 mL), fil-
tration through a short column of silica gel (h¼13 cm,
evaporation of the solvent and crystallization in Et2O: yield 83%;
mp 156–157 ꢁC, purity 98% by HPLC. FTIR (KBr, , cmꢀ1) 3338 (NH),
4
¼2 cm),
n
1755 (ester), 1649 (amide), 1065 and 1048 (C–O), 992 and 905
(vinyl). 1H NMR (CDCl3) 1.92 (s, 3H), 2.00 (s, 3H), 2.03 (s, 6H), 3.53–
3.90 (m, 4H), 3.96 (m, 1H), 4.10 (dd, 1H, J¼2.4, 12.4 Hz), 4.24 (dd, 1H,
J¼5.0, 12.4 Hz), 4.54 (d, 1H, J¼7.9 Hz), 5.05 (m, 2H), 5.21 (t, 1H,
J¼9.5 Hz), 5.36 (dd, 1H, Jcis¼10.8 Hz, Jgem¼0.8 Hz), 5.83 (dd, 1H,
Jtrans¼17.6 Hz, Jgem¼0.8 Hz), 6.60 (br t, NH), 6.74 (dd, 1H,
Jcis¼10.8 Hz, Jtrans¼17.6 Hz), 7.61 (m, 4H). 13C NMR 20.58, 20.66,
39.65, 61.87, 68.34, 69.19, 71.39, 72.00, 72.62, 101.03, 116.00, 126.30,
127.29, 133.34, 135.94, 140.77, 167.02, 169.43, 169.54, 170.15, 170.58.
Anal. Calcd for C25H31NO11: C, 57.58; H, 5.99; N, 2.69. Found: C,
57.50; H, 6.01; N, 2.68.
The peracetylated monomer (5 or 7) (2.0 mmol) was dissolved
in dry dioxane (24 mL) and treated with 1.32 M MeONa in MeOH
(1.6 mL) at rt for 60 min (the appearance of deacetylated monomers
6 or 8 was observed by TLC using a mixture CHCl3/MeOH¼4:1 as
eluent) (Rf: 5¼0.79, 6¼0.16, 7¼0.90, 8¼0.40). The dioxane mixture
was diluted with MeOH (15 mL) then treated with Amberlite
IR120(plus) up to pH¼6, filtered and the resin washed with MeOH.
Washings and filtrate were combined and evaporated at reduced
The loading of amino groups in the oligomers in the form of
hydrochlorides was determined by dissolving the oligomer (50–
80 mg) in THF (1–2 mL), treating the solution with a known excess
of 0.1 N NaOH and titrating the unreacted NaOH with 0.1 N HCl.
FTIR spectra of Olig5 and Olig7 are characterized by strong
absorption bands at 1755 cmꢀ1 (ester), 1230 and 1039 cmꢀ1 (C–O).
4.5. Macromonomers Mac6 and Mac8
A solution of Olig5 or Olig7 (1.0 mmol, as determined by titra-
tion of NH2 group) in dry CHCl3 (55 mL for Olig5 or 44 mL for
Olig7), and Et3N (2.0 mmol) was cooled to 0 ꢁC, treated under N2
with 4-vinylbenzoyl chloride23 (1.0 mmol), allowed to reach rt and
magnetically stirred until the disappearance of the purple colour
developed on TLC after elution, with 0.2% solution of ninhydrin in
95% EtOH. The mixture was then hydrolyzed with water (10 mL),
extracted with CHCl3 (3ꢃ20 mL) and dried over anhydrous MgSO4.
After removal of the solvent under reduced pressure, the solid
macromonomer was purified by repeated dissolution in CHCl3 and
precipitation in Et2O, and brought to constant weight under re-
duced pressure: Mac5 (yield 86%); Mac7 (yield 88%).
pressure to afford the crude N-acryloyl-2-aminoethyl -gluco-
b
-D
pyranoside (6) or N-(4-vinylbenzoyl)-2-aminoethyl b-D-glucopyr-
anoside (8) as pink syrups. The syrups were dissolved in a mixture
of CHCl3/MeOH 1:1 for 6 or CHCl3/MeOH¼4:1 for 8 and passed
A solution of Mac5 or Mac7 (0.50 mmol of 4-vinylbenzamido
groups) in dry dioxane (35 mL for Mac5 and 20 mL for Mac7) was
treated with 1.12 M MeONa in MeOH for 90 min at rt until the
disappearance of the peracetylated macromonomer as shown both
through a short column of silica gel (h¼13 cm, ¼2 cm) using the
4
same mixture of solvents (90 mL) and vacuum evaporated.

M. Pocci et al. / Tetrahedron 65 (2009) 5684–5692
5691
by TLC (petroleum ether/acetone¼1:1 as eluent) and by the lack of
the ester band at 1755 cmꢀ1 in the FTIR spectrum. The milky sus-
pension was then treated with MeOH in large excess to favour
coagulation and filtered. The solid deacetylated macromonomer
was dried and brought to constant weight under reduced pressure:
Mac6 (yield 59%); Mac8 (yield 91%).
was concentrated under reduced pressure to afford 10 as an oil that
was passed through a short column of silica gel (h¼13 cm, ¼2 cm)
4
using a mixture CHCl3/MeOH¼4:1 as eluent. The evaporation of the
solvent gave a spongy solid that by treatment with dry Et2O
changed to a hygroscopic powder when stored over P2O5. Yield 94%.
Purity 98% by HPLC. FTIR (KBr, n
, cmꢀ1) 3392 (OH), 1640 (amide),
1077 and 1036 (C–O). 1H NMR (DMSO-d6) 2.93–3.21 (m, 4H), 3.37–
3.75 (m, 5H), 3.84 (m, 1H), 4.19 (d, H-1, J¼7.7 Hz), 4.54 (t, OH,
J¼5.7 Hz), 4.95 (d, OH, J¼4.7 Hz), 5.00 (d, OH, J¼4.3 Hz), 5.08 (d, OH,
J¼3.9 Hz), 7.49 (m, 3H), 7.84 (m, 2H), 8.43 (t, NH, J¼5.1 Hz). 13C NMR
39.62 (CH2N), 62.00 (C-6), 68.51, 70.98 (C-4), 74.40 (C-2), 77.53 (C-
3), 77.87 (C-5), 104.07 (C-1), 128.12, 129.21, 132.09, 135.36, 167.33.
4.6. Precipitation co-polymerizations of Mac8 and 8 with
crosslinkers
Monomer 8 or macromonomer Mac8 or mixture Mac8/styrene
1/20.5 w/w and AIBN (Table 1) was dissolved under N2 with the
proper solvent in a vial with side arm, siphoned into the poly-
merization flask equipped with silicone septum and a Teflon stir-
ring bar, then treated with the crosslinker through a microsyringe.
The mixture, after 30 min of N2 purging, was magnetically stirred at
70 ꢁC for 24–74 h. The milky suspension was centrifuged at
3000 rpm for 30 min to remove the liquid phase and subjected to
two cycles of washing/sonication/centrifugation (15 min each) first
with THF then Et2O. The co-polymer was brought to constant
weight under reduced pressure (yields 11–27%).
4.9. Alkylations of 10
4.9.1. General procedure
NaH (60% suspension inwhite oil) was washed under N2 with dry
pentane (2ꢃ2 mL), evaporated, suspended in dry DMF (2 mL),
treated with a solution of 10 in dry DMF (2 mL) and magnetically
stirred at rt until to get a clear solution (30 min). The clear solution
was treated with 11 or 12 under stirring at the desired temperature
until no further modifications of the composition of the mixture was
evidenced by TLC (CHCl3/MeOH¼7:1 as eluent). The mixture was
treated with MeOH (2 mL) and evaporated at 60 ꢁC under reduced
pressure to give a syrup that was chromatographed by PLC using the
same eluent as above (Table 2).
4.7. N-Benzoyl-2-aminoethyl 2,3,4,6-tetra-O-acetyl-
b-
D-glucopyranoside (9)
Compound 4 (2.4258 g, 4.30 mmol) was dissolved under N2 in
dry CH2Cl2 (20 mL) and added with dry Et3N (2.4 mL). After cooling
to 0 ꢁC the solution was treated with benzoyl chloride (0.6045 g,
4.30 mmol), stirred at rt for 4 h (until the disappearance of 4, TLC,
petroleum ether/EtOAc¼1:2 as eluent), hydrolyzed with water
(15 mL, pH¼10) and extracted with CH2Cl2 (3ꢃ15 mL). The extracts
were combined, dried over anhydrous MgSO4, concentrated under
reduced pressure to afford a dark oil that was passed through
4.10. Functionalization of the nanospherical co-polymer C8
NaH (60% suspension in white oil, 8.0 mg, 0.20 mmol) was
washed under N2 with dry pentane (2ꢃ1 mL), vacuum dried, sus-
pended in dry DMF (2.5 mL), added with solid C8 (50.0 mg, loading
of the saccharidic unit 2.83 mmol/g) and magnetically stirred at rt
for 30 min. The mixture was treated with 11 (42.0 mg, 0.20 mmol)
under stirring at 24 ꢁC for 72 h, added with water (1 mL) and THF
(1 mL), centrifuged at 3000 rpm (15 min) to remove the liquid
phase and subjected to washing/centrifugation cycles first with THF
then Et2O up to elimination of aldehydic compounds as evidenced
by TLC and the negative Schiff’s test in the last washings. The solid
was brought to constant weight under reduced pressure (13.0 mg)
and gave a positive Schiff’s test showing an intense fuchsia colour,
which after centrifugation of the sample remained on the solid,
leaving a colourless liquid phase.
a short column of silica gel (h¼13 cm, ¼2 cm). The obtained white
4
solid (1.65 g) was crystallized from Et2O to give 9 (1.39 g, yield 65%).
For characterization purposes a sample of 9 was chromatographed
by PLC (CHCl3/MeOH¼4:1 as eluent, Rf¼0.86). Mp 122–123 ꢁC;
purity 94% by HPLC. FTIR (KBr, n
, cmꢀ1) 3353 (NH), 1752 (ester),
1648 (amide), 1085 and 1044 (C–O). 1H NMR (CDCl3) 1.91 (s, 3H),
2.00 (s, 3H), 2.03 (s, 6H), 3.54–3.90 (m, 4H), 3.98 (m, 1H), 4.10 (dd,
1H, J¼2.3, 12.3 Hz), 4.24 (dd, 1H, J¼5.0, 12.3 Hz), 4.55 (d, H-1,
J¼7.9 Hz), 4.98–5.11 (m, 2H), 5.22 (t, 1H, J¼9.6 Hz), 6.63 (br s, NH),
7.40–7.56 (m, 3H), 7.78–7.81 (m, 2H). 13C NMR 20.54, 20.58, 20.66,
39.64 (CH2N), 61.86 (C-6), 68.31 (C-4), 69.18, 71.36 (C-2), 71.97 (C-
5), 72.62 (C-3), 101.01 (C-1), 126.95, 128.56, 131.60, 134.32, 167.41,
169.42, 169.51, 170.15, 170.57. Anal. Calcd for C23H29NO11: C, 55.75;
H, 5.90; N, 2.83. Found: C, 55.78; H, 5.91; N, 2.81.
Acknowledgements
Compound 9 was also prepared as follows: a mixture of 1
(0.6961 g, 1.8 mmol), N-(hydroxyethyl)benzamide (0.3527 g,
2.1 mmol) and CH2Cl2 (7 mL) was cooled to 0 ꢁC and treated in the
dark with BF3$OEt2 (1.30 g, 9.0 mmol) under stirring for 40 min.
The mixture was left at rt for 24 h, then hydrolyzed with water
(7 mL). The organic phase was washed in turn with satd NaHCO3,
satd NaCl, and dried over MgSO4. After evaporation of the solvent
under reduced pressure, the oily residue was crystallized from
a mixture Et2O/diisopropyl ether (1:1) to give 9 (0.6581 g, yield
75%) having FTIR, 1H and 13C NMR spectra coincident with those
reported above.
This work was financially supported by MIUR (Programmi di
Ricerca di Rilevante Interesse Nazionale 2006) and University
Funds.
Supplementary data
Supplementary data concerning the preparation of Olig5, Olig7,
co-polymers of Mac6 and Mac8 with styrene, and N-(hydroxy-
ethyl)benzamide, 1H, 13C NMR spectra of compounds 5–19 can be
found in the online version. Supplementary associated with this
article can be found in the online version, at doi:10.1016/j.tet.
2009.05.033.
4.8. N-Benzoyl-2-aminoethyl b-D-glucopyranoside (10)
A solution of 9 (0.8099 g, 1.6 mmol) in dry dioxane (20 mL) was
treated with 1.32 M MeONa in MeOH (1.2 mL) for 1 h at rt until
disappearance of 9 as shown by TLC (CHCl3/MeOH¼4:1; Rf: 9¼0.86,
10¼0.29). The solution was diluted with MeOH (5 mL) and treated
with Amberlite FTIR120(plus) to neutrality and filtered. The filtrate
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