Synthesis of polymethacrylamides having a sugar moiety with an aliphatic hydrocarbon spacer and their application to control adhesion of hepatocytes cancer cells on the materials

Article abstract of DOI:10.1002/pola.26818

Polymers having a sugar moiety in the side group have been utilized as artificial matrices for cell adhesion in tissue engineering. In this study, methacrylamide - based polymers having lactose and maltose derivative structures in the side group with various aliphatic hydrocarbon spacers were synthesized, and their cell adhesion properties were examined. Methacrylamide monomers were prepared by two step amidation of a spacer diamine, first with a sugar lactone and then with a methacrylic anhydride. These monomers were radically polymerized in aqueous media using 4,4′-azobis(4-cyanovaleric acid) (ACVA) as radical initiator to give the corresponding polymethacrylamide. Specific interaction between these polymers and animal cell was investigated by adhesion of proliferated human liver cancer cell (WRL) to the polymethacrylamides. WRL interacted with polymers having a lactose structure with a hexamethylene or 1,4-cyclohexylene spacer by a specific manner and was promoted typical spheroid formation, while it did not interacted with polymers having a maltose structure.

Full text of DOI:10.1002/pola.26818

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Synthesis of Polymethacrylamides Having a Sugar Moiety with an
Aliphatic Hydrocarbon Spacer and Their Application to Control
Adhesion of Hepatocytes Cancer Cells on the Materials
Masayuki Toyoshima,1 Kozo Matsumoto,1,2 Mitsuaki Goto,1 Takeshi Endo1
1Molecular Engineering Institute, Kinki University, Kayanomori, Iizuka, Fukuoka 820-8555, Japan
2Department of Biological and Environmental Chemistry, Kinki University, 11-6 Kayanomori, Iizuka,
Fukuoka Prefecture 820–8555, Japan
Correspondence to: T. Endo (E-mail: tendo@moleng.fuk.kindai.ac.jp)
Received 8 January 2013; accepted 8 June 2013; published online 17 July 2013
DOI: 10.1002/pola.26818
ABSTRACT: Polymers having a sugar moiety in the side group
have been utilized as artificial matrices for cell adhesion in tis-
sue engineering. In this study, methacrylamide - based poly-
mers having lactose and maltose derivative structures in the
side group with various aliphatic hydrocarbon spacers were
synthesized, and their cell adhesion properties were examined.
Methacrylamide monomers were prepared by two step amida-
tion of a spacer diamine, first with a sugar lactone and then
with a methacrylic anhydride. These monomers were radically
polymerized in aqueous media using 4,40-azobis(4-cyanovaleric
acid) (ACVA) as radical initiator to give the corresponding poly-
methacrylamide. Specific interaction between these polymers
and animal cell was investigated by adhesion of proliferated
human liver cancer cell (WRL) to the polymethacrylamides.
WRL interacted with polymers having a lactose structure with a
hexamethylene or 1,4-cyclohexylene spacer by a specific man-
ner and was promoted typical spheroid formation, while it did
not interacted with polymers having
a maltose structure.
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KEYWORDS: radical polymerization; surfaces; water-soluble
polymers
Akaike and Kobayashi, as pioneers of such styrene-based
glyco-polymers, synthesized fascinating polymers showing
cluster effect of carbohydrate, which are applicable to cell
culture materials.13 Their polymers such as poly(p-N-vinyl-
INTRODUCTION Saccharides–protein interactions on the cell
surfaces play important roles in cell–cell communication,
immune response and pathogen invasion.1 Although, mono-
valent saccharide–protein interactions are generally consid-
ered weak, the interaction can be amplified by the clustering
effect. Therefore, polymers having a sugar moiety in the
repeating unit2 are expected to be useful as functional bio-
materials,3 because of their strong clustering effect of the
sugar moieties.4,5 In addition, such synthetic polymers are
considered as potentially important materials to study fun-
damentals of sugar–protein interactions. Actually, such poly-
mers with a sugar moiety (glyco-polymer) have been utilized
for recognition of protein,6,7 toxin,8,9 application of immuno-
chromatography,10 and culture of cells.11,12
benzyl-D-lactonamide) (PVLA) [Fig. 1(a)], which have
a
monovalent sugar moiety in each unit, showed strong affinity
to cells specifically by the clustering effect. Hepatocytes
attached to a PVLA-coated surface through the specific inter-
action between galactose groups in PVLA and cell surface
receptors (ASGP-R), which recognize galactose.
As PVLA has unique amphiphilic structural units composed of
polystyrene backbone as a hydrophobic part and an oligosac-
charide residue in the side chain as a hydrophilic part,14 PVLA
is strongly adsorbed to hydrophobic materials such as polysty-
rene and polyurethane.15 Besides that, PLVA has high biocom-
patibility and bioresorbability, so it is supposed to be widely
employed as scaffolds in tissue engineering. However, PVLA
has two problems to be solved. One is toxicity of a vinylbenzyl-
amine intermediate used in the preparation, and the other is
Glyco-polymer have been applied to tissue engineering and
worked as extracellular matrices (ECMs). Cell attachment, cell
growth, and cell behavior to these sugar carrying polymers
are regulated by their sugar groups, and these cellular effects
are completely different from natural ECM interaction.
Additional Supporting Information may be found in the online version of this article.
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hydrocarbon spacer and polymerized them19 (Fig. 2) via rad-
ical polymerization [Fig. 1(b)].
We selected two kinds of sugar, lactose and maltose. Lactose,
having a b-galactose moiety, can specifically interact with
WRL. In contrast, Maltose, having no b-galactose or GlcNAc
moiety, can’t interact with WRL.
FIGURE 1 Chemical structures of (a) PVLA and (b)
We evaluated their interaction with cells by observing effec-
tive adhesions of WRL on the polystyrene dish coated with
these polymethacrylamides having a sugar derived moiety.
methacrylamide-based glycol-polymers.
the cumbersome synthetic process. They prepared the poly-
mers by radical polymerization of a carbohydrate modified sty-
rene monomer. According to their methodology, at least five
step reactions were required to obtain the polymer.
EXPERIMENTAL
Chemicals
The following reagents were used as received; 4,40-azobis(4-
cyanovaleric acid) (ACVA) (Wako Pure Chemical Industries,
Oosaka, Japan), 1,3-diaminopropane, 1,6-diaminohexane, 1,4-
cyclohexanediamine (TCI, Tokyo, Japan), dulbecco’s modified
eagle medium (DMEM), fetal bovine serum (FBS), metha-
crylic anhydride (Sigma-Aldrich, Louisiana, MO).
In the most case, however, synthetic methods of such glyco-
polymers require protecting group chemistry and multiple-
step reactions, and purifications. These chemical strategies
are both time consuming and costly.
We consider that novel glyco-polymers are needed which
can be simply synthesized without protection of the sugar
hydroxyl groups, and without using a toxic intermediate.
The sugar lactones, lactonolactone and maltonolactone, were
synthesized according to the literature. Details are described
in the Supporting Information.20,21
Because many amines can easily add to sugar-derived lac-
tones16,17 and methacrylic anhydride18 under mild condi-
tions, various methacrylamide-based monomers with both
sugar moiety and hydrocarbon spacer can be readily synthe-
sized, by using aliphatic diamines as linking compounds.
Characterization
1H (300 MHz) and 13C NMR (75 MHz) spectra were recorded
on a JEOL AL300 NMR spectrometer in D2O. Chemical shifts
were determined using tetramethylsilane or the residual pro-
tons as the internal standards. IR spectra were taken on a
Thermo Scientific Nicolet iS10 spectrometer.
We report the facile synthesis of polymethacrylamides hav-
ing a sugar moiety with an aliphatic hydrocarbon spacer, tri-
methylene, hexamethylene, or 1,4-cyclohexylene, without any
protections and without using any toxic intermediates.
Gel permeation chromatography (GPC) was carried out with
a JASCO LC-Net II high-performance liquid chromatography
with TOSOH TSKgel ALPHA-M and phosphate buffer saline
(PBS) as eluent. Molecular weights were evaluated by a PEO
standard.
In this study, we synthesized methacrylamides having a
lactose or maltose lactone derivative with an aliphatic
FIGURE 2 Chemical structures of methacrylamide monomers synthesized in this study.
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The stained cells were observed by a fluorescence micro-
scope on Olympus IX71.
Synthesis of 6-Lactobionamidohexyl Amine (LAHA)
A solution of 1, 6-diaminohexane (3.9 g, 28 mmol, 10 equiv.)
in DMF (5 mL) was stirred in ice bath. Lactonolactone (1.0 g,
2.8 mmol, 1 equiv.) in DMF (80 mL) was added dropwise to
the mixture over 30 min. The product was washed with
chloroform and evaporated to give LAHA (1.13 g, 25 mmol)
in 89.0% yield.
General Procedure for Synthesis of Methacrylamides
Having a Sugar Moiety with an Aliphatic Hydrocarbon
Spacer
A DMF solution of methylene diamine (2.8 mol/mL, 10 mL)
was stirred in ice bath. A DMF solution of sugar lactone 1
(5.6 3 1022 mol/mL, 50 mL) was added dropwise to the
diamine solution over 30 min. The mixture was allowed to
1H NMR (300 MHz, D2O) d 5 4.56 (d, 1H, J 5 7.5 Hz, H-10),
4.40 (d, 1H, J 5 3.0 Hz, H-2), 4.19 (t, 1H, J 5 3.0, H-4), 3.94
(m, 3H, H-5, 20, 50), 3.79 (m, 7H, H-3, 6, 30, 40, 60), 3.28 (t,
2H, J 5 6.6 Hz, ANHACH2A(CH2)4ACH2ANH2), 2.82 (t, 2H,
J 5 6.9 Hz, A(CH2)4ACH2ANH2), 1.59 (m, 4H, NHACH2
ACH2ACH2A), 1.38 (m, 4H, NHACH2ACH2ACH2A)
ꢀ
react for 5 h at 0 C and then at ambient temperature over-
night. The DMF was evaporated to concentrate and the resi-
due was poured into chloroform, and an insoluble part was
collected by filtration. The remaining solid was dissolved in
H2O and the solution was washed with chloroform. The
aqueous solution was concentrated by evaporation to give an
intermediate product (white powder) 2, which was used for
the following reactions without further purification. A DMF
Synthesis of 6-Lactobionamidohexyl Methacrylamide
(LAHMA)
A solution of LAHA (1.0 g, 2.2 mmol, 1 equiv.) in DMF (50
mL) was stirred in ice bath. Methacrylic anhydride (407 mg,
2.6 mmol, 1.2 equiv.) in DMF (10 mL) was added dropwise
to the mixture over 10 min. The product was purified by
reverse-phase column (H2O) to give LAHMA (1.08 g, 2.1
mmol) in 94.1% yield.
solution of this compound
2 (0.4 mol/mL, 100 mL)
was stirred in ice bath. Methacrylic anhydride in DMF (1.2
mol/mL, 50 mL) was added dropwise to the solution over
10 min. The mixture was allowed to react for 5 h at 0 ꢀC.
The solution was poured into chloroform to give a white pre-
cipitate, which was collected by filtration. The resulting pow-
der was dissolved in H2O and the solution was washed with
chloroform. Finally, the product was purified by reverse-
phase column chromatography (LH-20 2 3 30 cm2 in H2O)
to yield the desired methacrylamide 3 as a white powder.
1H NMR (300 MHz, D2O) d 5 5.67 (d, 1H, J 5 0.9 Hz, CH2@C),
5.44 (d, 1H, J 5 0.9 Hz, CH2@C), 4.57 (d, 1H, J 5 7.5 Hz, H-
10), 4.40 (d, 1H, J 5 2.7 Hz, H-2), 4.19 (t, 1H, J 5 2.7 Hz, H-4),
3.94 (m, 3H, H-5, 20, 50), 3.75 (m, 7H, H-3, 6, 30, 40, 60), 3.59
(t, 2H, J 5 7.2 Hz, ANHACH2A(CH2)4ACH2ANHACOAC@C),
3.26 (t, 2H, 6.9 Hz, ACH2ANHACOAC@C), 1.94 (s, 3H,
C@CACH3), 1.55 (m, 4H, NHACH2ACH2ACH2A), 1.36 (m,
4H, NHACH2ACH2ACH2A)
Synthesis of 3-Lactobionamidopropyl Amine (LAPA)
A solution of 1,3-diaminopropane (2.65 g, 28 mmol, 10
equiv.) in DMF (5 mL) was stirred in ice bath. Lactonolac-
tone (1.0 g, 2.8 mmol, 1 equiv.) in DMF (80 mL) was added
dropwise to the mixture over 30 min. The product was
washed with chloroform and evaporated to give LAPA (905
mg, 22 mmol) in 78.3% yield.
Synthesis of 6-Lactobionamidocyclohexyl Amine (LACHA)
A solution of 1,4-cyclohexanediamine (3.18 g, 28 mmol, 10
equiv.) in DMF (10 mL) was stirred in ice bath. Lactonolac-
tone (1.0 g, 2.8 mmol, 1 equiv.) in DMF (80 mL) was added
dropwise to the mixture over 30 min. The product was
washed with chloroform and evaporated to give LACHA (998
mg, 22 mmol) in 78.7% yield.
1H NMR (300 MHz, D2O) d 5 4.57 (d, 1H, J 5 7.5 Hz, H-10),
4.40 (d, 1H, J 5 3.0 Hz, H-2), 4.19 (t, 1H, J 5 3.0, H-4), 3.93
(m, 3H, H-5, 20, 50), 3.75 (m, 7H, H-3, 6, 30, 40, 60), 3.32 (t, 2H,
J 5 6.9 Hz, ACH2ACH2ACH2ANH2), 2.67 (t, 2H, J 5 6.9 Hz,
ACH2ACH2ACH2ANH2), 1.65 (m, 2H, NHACH2ACH2ACH2A)
1H NMR (300 MHz, D2O) d 5 4.47 (d, 1H, J 5 7.2 Hz, H-10),
4.29 (d, 1H, J 5 7.5 Hz, H-2), 4.09 (t, 1H, J 5 2.4 Hz, H-4),
3.83 (m, 3H, H-5, 20, 50), 3.67 (m, 7H, H-3, 6, 30, 40, 60), 3.46
(m, 1H, ANHACHA(CH2ACH2)2ACHANH2), 2.53 (m, 1H,
ACHANH2), 1.50 (m, 8H, ANHACHA(CH2ACH2)2ACHANH2)
Synthesis of 3-Lactobionamidopropyl Methacrylamide
(LAPMA)
A solution of LAPA (850 mg, 2.1 mmol, 1 equiv.) in DMF (50
mL) was stirred in ice bath. Methacrylic anhydride (388 mg,
2.5 mmol, 1.2 equiv.) in DMF (10 mL) was added dropwise
to the mixture over 10 min. The product was purified by
reverse-phase column (H2O) to give LAPMA (775 mg, 1.6
mmol) in 76.0% yield.
Synthesis of 6-Lactobionamidocyclohexyl
Methacrylamide (LACHMA)
A solution of LACHA (980 mg, 2.2 mmol, 1 equiv.) in DMF
(50 mL) was stirred in ice bath. Methacrylic anhydride (400
mg, 2.6 mmol, 1.2 equiv.) in DMF (10 mL) was added drop-
wise to the mixture over 10 min. The product was purified
by reverse-phase column (H2O) to give LACHMA (915 mg,
1.8 mmol) in 81.2% yield.
1H NMR (300 MHz, D2O) d 5 5.66 (d, 1H, J 5 0.9 Hz, CH2@C),
5.35 (d, 1H, J 5 0.9 Hz, CH2@C), 4.59 (d, 1H, J 5 7.5 Hz, H-10),
4.44 (d, 1H, J 5 2.7 Hz, H-2), 4.20 (t, 1H, J 5 2.7 Hz, H-4), 3.94
(m, 3H, H-5, 20, 50), 3.78 (m, 7H, H-3, 6, 30, 40, 60), J 5 3.13 (t,
2H, J 5 6.9 Hz, ACH2ACH2ACH2ANHACOAC@C), 3.04 (t, 2H,
6.9 Hz, ACH2ACH2ANHACOAC@C), 1.94 (s, 3H, C@CACH3),
1.80 (m, 2H, NHACH2ACH2ACH2A)
1HANMR (300 MHz, D2O) d 5 5.55 (d, 1H, J 5 1.2 Hz,
CH2@C), 5.24 (d, 1H, J 5 1.2 Hz, CH2@C), 4.46 (d, 1H, J 5 7.5
Hz, H-10), 4.33 (d, 1H, J 5 2.7 Hz, H-2), 4.10 (t, 1H, J 5 2.7 Hz,
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H-4), 3.80 (m, 3H, H-5, 20, 50), 3.62 (m, 7H, H-3, 6, 30, 40, 60),
3.45 (m, 1H, ANHACHA(CH2ACH2)2ACHANHACOAC@C),
3.23 (m, 1H, ACHANHACOAC@C), 1.72 (s, 3H, C@CACH3),
1.50 (m, 8H, ANHACHA(CH2ACH2)2ACHANHA)
reverse-phase column (H2O) to give MAHMA (977 mg, 1.6
mmol) in 85.3% yield.
1H NMR (300 MHz, D2O) d 55.45 (d, 1H, J 5 0.9 Hz, CH2@C),
5.20 (d, 1H, J 5 0.9 Hz, CH2@C), 4.32 (d, 1H, J 5 2.4 Hz, H-
10), 4.23 (d, 1H, J 5 3.6 Hz, H-2), 4.10 (t, 1H, J 5 2.4 Hz, H-4),
3.90 (m, 3H, H-5, 20, 50), 3.75 (m, 7H, H-3, 6, 30, 40, 60), 3.42
(t, 2H, J 5 7.2 Hz, ANHACH2A(CH2)4ACH2ANHACOAC@C),
3.26 (t, 2H, 6.9 Hz, ACH2ANHACOAC@C), 1.94 (s, 3H,
C@CACH3), 1.55 (m, 4H, NHACH2ACH2ACH2A), 1.36 (m,
4H, NHACH2ACH2ACH2A)
Synthesis of 3-Maltobionamidopropyl Amine (MAPA)
A solution of 1,3-diaminopropane (2.65 g, 28 mmol, 10
equiv.) in DMF (10 mL) was stirred in ice bath. Maltonolac-
tone (1.0 g, 2.8 mmol, 1 equiv.) in DMF (80 mL) was added
dropwise to the mixture over 30 min. The product was
washed with chloroform and evaporated to give MAPA (950
mg, 23 mmol) in 81.3% yield.
Synthesis of 6-Maltobionamidocyclohexyl Amine
(MACHA)
1H NMR (300 MHz, D2O) d 5 4.31 (d, 1H, J 5 2.4 Hz, H-10),
4.19 (d, 1H, J 5 3.6 Hz, H-2), 4.13 (t, 1H, J 5 3.0, H-4), 3.91
(m, 3H, H-5, 20, 50), 3.71 (m, 7H, H-3, 6, 30, 40, 60), 3.31 (t,
2H, J 5 6.9 Hz, ACH2ACH2ACH2ANH2), 2.70 (t, 2H, J 5 7.2
Hz, ACH2ACH2ACH2ANH2), 1.67 (m, 2H, ANHACH2ACH2
ACH2ANHA)
A solution of 1,4-cyclohexanediamine (3.18 g, 28 mmol, 10
equiv.) in DMF was stirred in ice bath. Maltonolactone (1.0 g,
2.8 mmol, 1 equiv.) in DMF (80 mL) was added dropwise to the
mixture over 30 min. The product was washed with chloroform
and evaporated to give MACHA (1.0 g, 22 mmol) in 79.5% yield.
1H NMR (300 MHz, D2O) d 5 4.30 (d, 1H, J 5 1.8 Hz, H-10),
4.12 (m, 2H, H-2, 4), 3.95 (m, 3H, H-5, 20, 50), 3.77 (m, 7H,
H-3, 6, 30, 40, 60), 3.46 (m, 1H, ANHACHA(CH2ACH2)2
ACHANH2), 2.24 (m, 1H, ACHANH2), 1.60 (m, 8H, ANHA
CHA(CH2ACH2)2ACHANH2)
Synthesis of 3-Maltobionamidopropyl Methacrylamide
(MAPMA)
A solution of MAPA (900 mg, 1.9 mmol, 1.0 equiv.) in DMF
(50 mL) was stirred in ice bath. Methacrylic anhydride (845
mg, 2.2 mmol, 1.2 equiv.) in DMF (10 mL) was added drop-
wise to the mixture over 10 min. The product was purified
by reverse-phase column (H2O) to give MAPMA (832 mg, 1.5
mmol) in 80.2% yield.
Synthesis of 6-Maltobionamidocyclohexyl
Methacrylamide (MACHMA)
A solution of MACHA (900 mg, 1.9 mmol, 1 equiv.) in DMF
(50 mL) was stirred in ice bath. Methacrylic anhydride (345
mg, 2.2 mmol, 1.2 equiv.) in DMF (5 mL) was added to the
mixture dropwise over 10 min. The product was purified by
reverse-phase column (H2O) to give MACHMA (898 mg, 1.8
mmol) in 82.2% yield.
1H NMR (300 MHz, D2O) d 5 5.42 (d, 1H, J 5 0.9 Hz, CH2@C),
5.18 (d, 1H, J 5 0.9 Hz, CH2@C), 4.33 (d, 1H, J 5 2.4 Hz, H-
10), 4.22 (d, 1H, J 5 3.6 Hz, H-2), 4.14 (t, 1H, J 5 3.0, H-4),
3.94 (m, 3H, H-5, 20, 50), 3.74 (m, 7H, H-3, 6, 30, 40, 60), 3.43
(t, 2H, J 5 6.9 Hz, ACH2ACH2ACH2ANHACOAC@C), 3.31 (t,
2H, 6.9 Hz, ACH2ACH2ACH2ANHACOAC@C), 1.99 (s, 3H,
C@CACH3), 1.78 (m, 2H, ANHACH2ACH2ACH2ANHA)
1H NMR (300 MHz, D2O) d 5 5.55 (d, 1H, J 5 1.2 Hz, CH2@C),
5.24 (d, 1H, J 5 1.2 Hz, CH2@C), 4.46 (d, 1H, J 5 7.5 Hz, H-
10), 4.10 (m, 2H, H-2, 4), 3.90 (m, 3H, H-5, 20, 50), 3.70 (m,
7H, H-3, 6, 30, 40, 60), 3.45 (m, 1H, ANHACHA(CH2ACH2)2
ACHANHACOAC@C), 3.23 (m, 1H, ACHANHACOAC@C),
1.72 (s, 3H, C@CACH3), 1.50 (m, 8H, ANHACHA(CH2ACH2)2
ACHANHA)
Synthesis of 6-Maltobionamidohexyl Amine (MAHA)
A solution of 1,6-diaminohexane (3.9 g, 28 mmol, 10 equiv.)
in DMF was stirred in ice bath. Maltonolactone (1.0 g, 2.8
mmol, 1 equiv.) in DMF (80 mL) was added dropwise to the
mixture over 30 min. The product was washed with chloro-
form and evaporated to give MAHA (1.12 mg, 25 mmol) in
88.0% yield.
General Procedure for Synthesis of Polymethacrylamides
Having Sugar Moieties with Aliphatic Hydrocarbon
Spacers
A mixture of monomers 3, an initiator (ACVA), and water
was charged in a schlenk flask and degassed by three freeze-
pump-thaw cꢀycles. The flask was sealed under vacuum and
heated at 60 C with occasional agitation. The polymerization
was stopped by cooling the tube, and the products were pre-
cipitated in acetone and collected by centrifugal sedimenta-
tion (10,000 G for 30 min). The polymeric products were
purified by dialysis in cellulose tubes with a molecular
weight cutoff of 2000. Freeze drying of the solution by
FD510 gave the desired polymer as a white solid powder.
1H NMR (300 MHz, D2O) d 5 4.30 (d, 1H, J 5 2.4 Hz, H-10),
4.22 (d, 1H, J 5 3.6 Hz, H-2), 4.09 (t, 1H, J 5 2.4 Hz, H-4),
3.88 (m, 3H, H-5, 20, 50), 3.74 (m, 7H, H-3, 6, 30, 40, 60), 3.36
(m, 1H, ANHACH2A(CH2ACH2)2ACH2ANH2), 2.42 (m, 1H,
ACH2ANH2), 1.60 (m, 8H, ANHACH2A(CH2ACH2)2ACH2
ANH2)
Synthesis of 6-Maltobionamidohexyl Methacrylamide
(MAHMA)
A solution of MAHA (1.0 g, 1.9 mmol, 1 equiv.) in DMF (50
mL) was stirred in ice bath. Methacrylic anhydride (354 mg,
2.3 mmol, 1.2 equiv.) in DMF (5 mL) was added dropwise to
the mixture over 10 min. The product was purified by
Polymerization of LAPMA
LAPMA (100 mg, 207 mmol) was polymerized using 2.0 mol
% of initiator of ACVA (1.2 mg, 4.15 mmol) in water (830
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mL, 0.25 mol/L) at 60 ꢀC. The product was precipitated in
acetone, collected by centrifugal sedimentation, and purified
by dialysis in cellulose tubes with a molecular weight cutoff
of 2000 to give polyLAPMA (78 mg) in 78% yield.
in acetone, collected by centrifugal sedimentation, and puri-
fied by dialysis in cellulose tubes with a molecular weight
cutoff of 2000 to give polyMAHMA (70 mg) in 70% yield.
1H NMR (300 MHz, D2O) d 5 4.45–2.80 (maltose part), 2.35–
1.50 (spacer and linear), 1.40–0.70 (ACH2AC(CH3)A)
1H NMR (300 MHz, D2O) d 5 4.40–2.80 (lactose part), 2.35–
1.60 (spacer and linear), 1.55–0.70 (ACH2AC(CH3)A)
Polymerization of MACHMA
Polymerization of LAHMA
LACHMA (100 mg, 191 mmol) was polymerized using 2.0
mol % of initiator of ACVA (1.1 mg, 3.83 mmol) in water
LAHMA (100 mg, 191 mmol) was polymerized using 1.0 mol
% of ACVA (0.54 mg, 1.91 mmol) in water (770 mL, 0.25
mol/L) at 60 ꢀC. The product was precipitated in acetone,
collected by centrifugal sedimentation, and purified by dialy-
sis in cellulose tubes with a molecular weight cutoff of 2000
to give polyLAHMA (75 mg) in 75% yield.
ꢀ
(830 mL, 0.25 mol/L) at 60 C. The product was precipitated
in acetone, collected by centrifugal sedimentation, and puri-
fied by dialysis in cellulose tubes with a molecular weight
cutoff of 2000 to give polyMACHMA (72 mg) in 72% yield.
1H NMR (300 MHz, D2O) d 5 4.45–2.80 (maltose part), 2.30–
1.45 (spacer and linear), 1.20–0.70 (ACH2AC(CH3)A)
1H NMR (300 MHz, D2O) d 5 4.50–2.80 (lactose part), 2.20–
1.50 (spacer and linear), 1.40–0.75 (ACH2AC(CH3)A)
Preparation of Polystyrene Substrates Coated with
Polymethacrylamides Having a Sugar Moiety with an
Aliphatic Hydrocarbon Spacer
Polymethacrylamides having a lactose and maltose derived
moiety with an aliphatic hydrocarbon spacer were diluted to
0.01% (w/v) concentration with by ultrapure water. One millili-
ter of the solution was put on a nontreated six-well cell culture
plate and the water was evaporated under atmospheric condi-
tions for overnight and then rinsed twice with PBS buffer.
Polymerization of LACHMA
LACHMA (100 mg, 191 mmol) was polymerized using 3.0
mol % of initiator of ACVA (1.6 mg, 5.74 mmol) in water
ꢀ
(830 mL, 0.25 mol/L) at 60 C. The product was precipitated
in acetone, collected by centrifugal sedimentation, and puri-
fied by dialysis in cellulose tubes with a molecular weight
cutoff of 2000 to give polyLACHMA (70 mg) in 70% yield.
1H NMR (300 MHz, D2O) d 5 4.50–2.90 (lactose part), 2.35–
1.45 (spacer and linear), 1.30–0.75 (ACH2AC(CH3)A)
Culture of Cells
1.5 mL of hepatic cancer cell line WRL cells (1.5 3 104 cell/
mL) in Dulbecco’s modified essential medium (DMEM) were
seeded for each polymer coated six wells plate and then cul-
tured in 5% CO2 at 37 ꢀC for 4 h. After 4 h incubation, the
medium was removed to separate nonadherent cells and 1.5
mL of 10% fetal bovine serum (FBS) containing DMEM was
added. The cell morphology was observed at 4, 24, 48, and
72 h after seeding the cells.
Polymerization of MAPMA
MAPMA (100 mg, 207 mmol) was polymerized using 2.0
mol % of initiator of ACVA (1.2 mg, 4.15 mmol) in water
ꢀ
(830 mL, 0.25 mol/L) at 60 C. The product was precipitated
in acetone, collected by centrifugal sedimentation, and puri-
fied by dialysis in cellulose tubes with a molecular weight
cutoff of 2000 to give polyMAPMA (80 mg) in 80% yield.
1H NMR (300 MHz, D2O) d 5 4.45–2.80 (maltose part), 2.35–
1.60 (spacer and linear), 1.55–0.70 (ACH2AC(CH3)A)
RESULTS AND DISCUSSION
Polymerization of MAHMA
MAHMA (100 mg, 191 mmol) was polymerized using 3.0
mol % of initiator of ACVA (1.6 mg, 5.74 mmol) in water
Syntheses of Polymethacrylamides Having a Sugar
Moiety with an Aliphatic Hydrocarbon Spacer
As shown in Scheme 1, methacrylamides having a sugar lac-
tone derivative of lactonolactone or maltonolactone with an
ꢀ
(770 mL, 0.25 mol/L) at 60 C. The product was precipitated
SCHEME 1 Synthesis of polymethacrylamides having a sugar moiety with an aliphatic hydrocarbon spacer.
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TABLE 2 Contact Angles of Polystyrene Surfaces Modified with
Polymethacrylamides Having a Sugar Moiety with an Aliphatic
Hydrocarbon Spacer
appropriate spacer were synthesized in good yields by an
amidation ofꢀthe corresponding sugar lactone with an amine
in DMF at 0 C, followed by a further amidation with metha-
crylic anhydride. These monomers were obtained as a white
powder over 60% yields. The chemical structures were iden-
Polymer
Contact Angle (ꢀ)
1
tified and analyzed by their H NMR and IR spectroscopy.
PLAPMA
PLAHMA
PLACHMA
PMAPMA
PMAHMA
PMACHMA
Control
78.6
27.1
30.3
81.6
39.9
71.5
87.7
We selected three kinds of aliphatic hydrocarbon spacers, tri-
methylene, hexamethylene, and 1,4-cyclohexylene. We made
a comparative review of polymer character, that trimethylene
and hexamethylene are difference in carbon number, and
hexamethylene and 1,4-cyclohexylene are difference in
structure.
Homopolymerizations of these monomers were conducted in
water using ACVA as an initiator via free radical polymeriza-
tion (Scheme 1). As shown in Table 1, the homopolymeriza-
tions smoothly proceeded with high conversions (over 90%)
in water. When the [initiator] / [monomer] ratio was
decreased from 3.0 to 1.0 mol %, the molecular weight of
the polymer increased. High molecular weight polymers
were obtained when DMF or DMAc was used as a solvent,
however the obtained polymers were insoluble in water.
modified with PLACHMA and PMACHMA are shown in Figure
3. The substrate modified with PLACHMA showed a main
emission peak at 530.0 eV, which can be assigned to O1s. It
also exhibited a peak at 397.0 eV, which can be assigned to
N1s. These observations suggest the presence of sugar moi-
ety on polystyrene surface. In contrast, PMACHMA modified
substrate didn’t show any peaks related to sugar moieties.
Substrates modified with PLAHMA and PMAHMA also
showed emission peaks of sugar moieties, while substrates
modified with PLAPMA or PMAPMA did not show them
(Supporting Information).
These polymethacrylamides synthesized here were subjected
to the examination of culture of cells.
Modification of Polystyrene Substrates with
Polymethacrylamides Having a Sugar Moiety with an
Aliphatic Hydrocarbon Spacer
The polymethacrylamides prepared in this study have both
hydrophilic saccharide moieties and hydrophobic spacer in
the side groups. The saccharide moieties increase polymer’s
solubility toward water in addition to the ability of cellular
recognition, while the hydrophobic spacer parts should be
strongly absorbed to the polystyrene substrate. Therefore,
the hydrophilic—hydrophobic balance, in sugar moiety22 and
spacer part of these polymers are quite important not only
for cellular recognition but also for efficient coating of the
substrates. In the case of PLACHMA, PLAHMA, and PMAHMA,
substrates modified with these polymers showed much
smaller contact angle than that of the control experiment,
although the polymers had hydrophobic hexamethylene or 1,
4-cyclohexylene spacers. We consider that these polymers
had enough hydrophobicity to be absorbed on polystyrenes
Polystyrene substrates were coated by an addition of 0.01
wt % aqueous solution of the polymethacrylamides with lac-
tose or maltose derived structure. Water contact angles of
some of the substrates thus prepared were significantly
reduced by the modification. As shown in Table 2, contact
angles of substrates modified with PLAHMA, PMAHMA, and
PLACHMA reduced less than 40ꢀ from the control contact
angle of 87.7ꢀ. Conversely, the contact angle of the substrates
were almost unchanged (>71.5ꢀ) by the modification with
PLAPMA, PMAPMA and PMACHMA.
The polymethacrylamides with lactose or maltose—derived
structure on polystyrene substrate were further investigated
by XPS analysis. Typical XPS wide scans of substrates
TABLE 1 Polymerization of Lactose or Maltose Derivative Monomers with Various Aliphatic Hydrocarbon Spacersa
Conv. (%)b
Yield (%)c
Mn (g/mol)d
Mw/Mn
d
Run No.
Monomer
[Initiator]/[Monomer]
1
2
3
4
5
6
LAPMA
2.0 mol %
1.0 mol %
3.0 mol %
2.0 mol %
3.0 mol %
2.0 mol %
94
97
96
95
97
92
78
75
70
80
70
72
38,000
120,000
82,000
14,000
20,000
14,000
4.9
3.3
2.7
3.0
8.1
4.5
LAHMA
LACHMA
MAPMA
MAHMA
MACHMA
a
Concentration: 0.25 mol/L, solvent: H2O, 60 ꢀC, reaction time: 15 h.
Determined by 1H NMR.
b
c
Acetone insoluble part was dialyzed (under cut 2,000).
Estimated by GPC (Eluent : PBS(-), poly (ethylene oxide) standards).
d
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TABLE 3 The Number of WRL Recognized on Polystyrene Sur-
face Modified with Polymethacrylamides Having a Sugar Moi-
ety with an Aliphatic Hydrocarbon Spacer
The Number of
Polymer
Cells (cells/nm2)
PLAPMA
PLAHMA
PLACHMA
PMAPMA
PMAHMA
PMACHMA
Control
21
70
68
20
5
FIGURE 3 Typical XPS spectra of substrates modified with (a)
PLACHMA and (b) PMACHMA.
22
24
substrates. As a result, the contact angles of the substrates
modified with these polymers may become smaller. Conversely,
in the case of PLAPMA, PMAPMA, and PMACHMA, the hydro-
phobic—hydrophilic balance might not good to be absorbed
on a polystyrene substrate.
same as the aggregates cultured on PVLA coated surface
(Supporting Information).23,24
Conversely, WRL did not interact with MAHMA, which has
glucose moieties at the end of the side groups. These results
suggested that PLAHMA and PLACHMA specifically inter-
acted with the WRL by b-galactose recognition, while in the
case of PLAPMA, PMAPMA, and PMACHMA, WRL interacted
onto the surface by a nonspecific way similar to the control
experiment.
Cell Culture
To investigate specific interactions between polymers having
sugar-derived structures with various aliphatic hydrogen car-
bon spacers and cell, the cell adhesion experiments were
carried out on a polystyrene dish coated with these poly-
mers. The human hepatic cancer cell line WRL specifically
recognized glycoproteins having terminal galactose/GalNAc
moieties. The WRL exclusively adhered onto the polystyrene
surface coated with 0.01% (w/v) solution of PLAHMA and
PLACHMA as shown in Figure 4. As expected, WRL inter-
acted with polymers having lactose derived moieties with ali-
phatic hydrocarbon spacers, PLAHMA and PLACHMA, which
possess terminal galactose moieties. On the PLAHMA and
PLACHMA, WRL cells became round shape at 4 h after seed-
ing the cell and then formed spheroids at 24 h after seeding,
which is well-known as a phenomenon for keeping high
hepatic function. When hepatocytes were cultured by these
polymers, adhesion and proliferation of the cells were almost
WRL adhesion with surface modified sugar polymer was
compared in term of number of cells at 4 h after seeding the
cell (Table 3). The numbers of adhesion cells for PLAHMA
and PLACHMA were 70 (cells/nm2) and 68 (cells/nm2),
respectively, whereas that for PMAHMA was 5 (cells/nm2),
and those for the other polymers ranged from 20 to 22
(cells/nm2).
It is also worth noting that the hydrophobicity of the spacer was
important for successful coating of the polystyrene dish with
the polymers with sugar moieties and good cell recognition.
FIGURE 4 Morphology of WRL cultured on polystyrene dishes with various polymethacrylamides having a sugar moiety. WRL
were cultured for 4 h (a) PLAHMA, (b) PLACHMA, (c) PMAHMA, (d) control PS dish, and cultured for 24 h (e) PLAHMA, (f)
PLACHMA, (g) PMAHMA, (h) control PS dish.
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5 J. Rao, L. Yan, J. Lahiri, G. M. Whitesides, R. M. Weis, H. S.
Warren, Chem. Biol. 1999, 6, 353–359.
In the case of polymers with a trimethylene spacer, coating
efficiency was not enough to attach the WRL cells onto the
corresponding polymers.
6 M. Toyoshima, Y. Miura, J. Polym. Sci. Part A: Polym. Chem.
2009, 47, 1412–1421.
7 A. Kobayashi, M. Goto, K. Kobayashi and T. Akaike, J. Bio-
mater. Sci. Polym. Ed. 1994, 6, 325–342.
CONCLUSIONS
In this study, we synthesized methacrylamides having a
sugar moiety with an aliphatic hydrocarbon spacer by two
steps starting from sugar lactones. These monomers were
easily polymerized via radical polymerization using ACVA as
an initiator.
8 M. Toyoshima, T. Oura, T. Fukuda, E. Matsumoto, Y. Miura,
Polym. J. 2010, 42, 172–178.
9 Y. Miura, Y. Sasao, H. Dohi, Y. Nishida, K. Kobayashi, Anal.
Biochem. 2002, 310, 27–35.
10 J. Ishii, M. Toyoshima, M. Chikae, Y. Takamura, Y. Miura,
Bull. Chem. Soc. Jpn. 2010, 84, 466–470.
Polymethacrylamides having strong hydrophobic part
(LAHMA, LACHMA and MAHMA) are well absorbed onto
hydrophobic surfaces such as the polystyrenes dish, exposing
the hydrophilic carbohydrate moiety in an aqueous phase.
Conversely, other polymers having weak hydrophobic part
could not be absorbed onto hydrophobic surfaces. The
exposed carbohydrates worked well as matrix ligands for
WRL recognition.
11 M. Oubihi, K. Oshima, N. Aoki, K. Kobayashi, K. Kitajima,
T. Matsuda, Biosci. Biotechnol. Biochem. 2000, 64, 785–792.
12 N. Ohtake, K. Niikura, T. Suzuki, K. Nagakawa, H. Sawa,
K. Ijiro, Bioconjug. Chem. 2008, 19, 507–515.
13 A. Kobayashi, T. Akaike, K. Kobayashi, H. Sumitomo, Makro-
mol. Chem. Rapid. Commun. 1986, 10, 645–650.
14 I. Wataoka, H. Urakawa, K. Kobayashi, T. Akaike, M.
Schmidt, K. Kajiwara, Macromolecules 1999, 32, 1816–1821.
15 K. Kobayashi, A. Kobayashi, T. Akaike, Methods Enzymol.
1994 247, 409–418.
We demonstrated that a specific function could be achieved
by culturing hepatocytes cancer cells in the PLAHMA and
PLACHMA. These polymethacrylamides may offer various
possibilities to be used in cell biological studies of liver func-
tions on polystyrene substrates.
16 R. Narain, S. P. Armes, Chem. Commun. 2002, 23, 2776–
2777.
17 R. Narain, S. P. Armes, Biomacromolecules, 2003 4, 1746–
1758.
18 B.-K. Chen, S.-H. Lo, S.-F. Lee, Chin. J. Polym. Sci. 2010, 28,
607–613.
19 A. Housni, H. Cai, S. Liu, S. H. Pun, R. Narain, Langmuir
2007, 23, 5056–5061.
ACKNOWLEDGMENT
20 K.Kobayashi, H. Sumitomo, Y. Ina, Polym. J. 1985, 17, 567–
575.
This work was financially supported by JSR Corporation.
21 Y.-I. Jeong, S.-J. Seo, I.-K. Park, H.-C. Lee, I.-C. Kangd,
T. Akaike, C.-S. Chob, Int. J. Pharm. 2005, 296, 151–161.
REFERENCES AND NOTES
22 C. A. Brown, In A Handbook of Sugar Analysis; Wiley: New
York, 1912; Chapter XVII–XX, pp 525–730.
1 R. Jelinek, S. Kolusheva, Chem. Rev. 2004, 104, 5987–6016.
2 Y. C. Lee, Carbohydr. Res. 1978, 67, 509–514.
23 A. Kobayashi, M. Goto, K. Kobayashi, T. Akaike, J. Biomater.
Sci. Polym. Ed. 1994, 6, 325–342.
3 R. Rene, Trends. Glycosci. Glyc. 1996, 8, 79–99.
24 Q. Yang, M. Strathmann, A. Rumpf, G. Schaule, M. Ulbricht,
ACS Appl. Mater. Interfaces, 2010, 2, 3555–3562.
4 J. Rao, J. Lahiri, L. Isaacs, R. M. Weisand G. M. Whitesides,
Science 1998, 280, 708–711.
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