Poly(n-acryl amino acids): A new class of biologically active polyanions

Article abstract of DOI:10.1021/jm000089j

Poly(N-acryl amino acids) bearing side groups with a lipophilic character or having charged functional groups (i.e. -NH₂, -COOH, -SH, -OH, and phenols) were synthesized from the radical polymerization of N-acryl amino acid monomers. Monomers were prepared from the reaction of acryloyl chloride and amino acid esters in dry solvents. Polymers of a broad molecular weight ranging from 3 000 to 60 000 Da were obtained. The polymers were optically active, and their structures were confirmed by ¹H NMR and IR spectra and elemental analysis. Hydroxyl-containing polymers were sulfated in high conversion yields by SO₃/pyridine complex. The newly synthesized linear homopolyanions were tested for heparin-like activities: (i) inhibition of heparanase enzyme, (ii) release of basic fibroblast growth factor (bFGF) from the extracellular matrix (ECM), and (iii) inhibition of smooth muscle cell (SMC) proliferation. Polymers based on tyrosine and leucine were highly active in all three tests (microgram level). Polymers based on phenylalanine, tert-leucine, and proline were active as heparanase inhibitors and FGF release, and polymers of trans-hydroxyproline, glycine, and serine were active only as heparanase inhibitors. The polymer of cis-hydroxyproline was inactive. It was found that a net anionic charge (i.e. carboxylic acid) is essential for biological activity. Thus, methyl ester derivatives of the active polymers, zwitterionic amino acid pendent groups (lysine, histidine), and decarboxylated amino acids (tyramine, ethanolamine) were inactive. The above active polymers did not exhibit anticoagulation activity which is considered the main limitation of heparin and heparinomimetics for clinical use. These synthetic poly(N-acryl amino acids) may have potential use in the inhibition of heparanase-mediated degradation of basement membranes associated with tumor metastasis, inflammation, and autoimmunity.

Full text of DOI:10.1021/jm000089j

J . Med. Chem. 2000, 43, 2591-2600
2591
P oly(N-a cr yl a m in o a cid s): A New Cla ss of Biologica lly Active P olya n ion s
Alfonso Bentolila,† Israel Vlodavsky,‡ Rivka Ishai-Michaeli,‡ Olga Kovalchuk,‡ Christine Haloun,† and
Abraham J . Domb*,†
Department of Medicinal Chemistry, School of Pharmacy, Faculty of Medicine, The Hebrew University of J erusalem,
J erusalem 91120, Israel, and Department of Oncology, Hadassah University Hospital, J erusalem 91120, Israel
Received February 25, 2000
Poly(N-acryl amino acids) bearing side groups with a lipophilic character or having charged
functional groups (i.e. -NH2, -COOH, -SH, -OH, and phenols) were synthesized from the
radical polymerization of N-acryl amino acid monomers. Monomers were prepared from the
reaction of acryloyl chloride and amino acid esters in dry solvents. Polymers of a broad molecular
weight ranging from 3 000 to 60 000 Da were obtained. The polymers were optically active,
and their structures were confirmed by 1H NMR and IR spectra and elemental analysis.
Hydroxyl-containing polymers were sulfated in high conversion yields by SO3/pyridine complex.
The newly synthesized linear homopolyanions were tested for heparin-like activities: (i)
inhibition of heparanase enzyme, (ii) release of basic fibroblast growth factor (bFGF) from the
extracellular matrix (ECM), and (iii) inhibition of smooth muscle cell (SMC) proliferation.
Polymers based on tyrosine and leucine were highly active in all three tests (microgram level).
Polymers based on phenylalanine, tert-leucine, and proline were active as heparanase inhibitors
and FGF release, and polymers of trans-hydroxyproline, glycine, and serine were active only
as heparanase inhibitors. The polymer of cis-hydroxyproline was inactive. It was found that a
net anionic charge (i.e. carboxylic acid) is essential for biological activity. Thus, methyl ester
derivatives of the active polymers, zwitterionic amino acid pendent groups (lysine, histidine),
and decarboxylated amino acids (tyramine, ethanolamine) were inactive. The above active
polymers did not exhibit anticoagulation activity which is considered the main limitation of
heparin and heparinomimetics for clinical use. These synthetic poly(N-acryl amino acids) may
have potential use in the inhibition of heparanase-mediated degradation of basement
membranes associated with tumor metastasis, inflammation, and autoimmunity.
In tr od u ction
cally modified.6 The second group includes synthetic
carboxylic acid polymers.7
Regulation of physiological processes is mediated by
specific interactions between macromolecules, most
often by polyelectrolites. Their size, tertiary structure,
and nature of charge provide them with the necessary
chemical information to execute specific biological in-
teractions. Moreover, natural polyanions such as DNA
and glycosaminoglycans (GAG) are capable of binding
different proteins, resulting in a wide range of biological
activities. Therefore, synthetic linear polyanions have
been studied since the early 1960s as potential biologi-
cally active molecules.1 They present a wide range of
biological activities such as antimicrobial,2 antiinflam-
matory, and antitumor agents,3 excitation of the reticu-
loendothelial system (RES),4 and stimulation of inter-
feron production.5 These polymers lack a high degree
of selectivity, since their activity is based primarily on
their scaffold, electrical charge, or charge density along
the polymeric backbone.
The first polysaccharide applied in medicine was
heparin. It is a natural polyanion composed of repeating
disaccharide units of glucosamine and uronic acid. The
amino groups of glucosamine are partially sulfated or
acetylated.8 Some of the hydroxy groups are also sulfate
esters.9 The degree of sulfation and the chain size of
the polymer determine the biological activity of hep-
arin.10 Although heparin has a range of desired activi-
ties including inhibition of tumor metastasis and inhi-
bition of restenosis, its clinical use is limited to treating
blood-clotting disorders11 as the anticoagulation activity
of heparin may endanger the patient due to the risk of
hemorrhage.
By a two-step oxidation, Claes et al.12 have modified
several polysaccharides and studied their effectiveness
as interferon inducers and potential antiviral agents.
Polymers produced in this way contain carboxylic
groups distributed along a biodegradable polyacetal
backbone. These compounds showed low toxicity and
significant antitumor and antiviral activities. Sulfation
of other polysaccharides such as laminarin13 yielded
potent anticoagulants.
Two major groups of polyanionic substances have
been studied during the past three decades. One is
polyanionic polysaccharides, both natural and chemi-
* To whom correspondence should be addressed at David Bloom
Center for Pharmacy, The Hebrew University, School of Pharmacy,
P.O. Box 12065, Hadassa Medical Center, Ein-Karem, J erusalem
91120, Israel. Phone: 972-2-6757573. Fax: 972-2-6757629. E-mail:
adomb@cc.huji.ac.il.
One of the most studied synthetic polycarboxylic acids
is pyran7 a copolymer prepared from divinyl ether and
maleic anhydride (1:2). Its biological activity spectrum
is very wide, including the induction of interferon,
antiviral, antibacterial, and antifungal activities, stimu-
† The Hebrew University of J erusalem.
‡ Hadassah University Hospital.
10.1021/jm000089j CCC: $19.00 © 2000 American Chemical Society
Published on Web 06/13/2000

2592 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 13
Bentolila et al.
Sch em e 1. Synthesis of Poly(N-acryl amino acids)a
lation of immune responses, anticoagulant activity,
adjuvant disease inhibition, and anticancer activity.14
The polymer conformation is also important in deter-
mining its biological activity. Muck et al.15 reported that
isotactic poly(acrylic acids) are more effective than
atactic poly(acrylic acids) in providing protection against
picornavirus infection in vivo. The optimal activity was
reached at molecular weights ranging from 6 to 15 kDa.
Another interesting synthetic polyanion is aurintri-
carboxylic acid (ATA),16 obtained from the acid polym-
erization of phenols with formaldehyde. This polymer
exhibited activities such as inhibiting the cytopatic effect
of HIV-1,17 preventing the binding of interferon to its
receptor,18 and binding to nucleotides and to polynu-
cleotide domains of glucocorticoid receptors.19 Regan et
al.20 synthesized linear structures of polyaromatic poly-
anions as analogues of ATA, polymerizing properly
chosen aromatic rings and avoiding the presence of
strong oxidizing reagents. Polymers so obtained were
shown to mimic the polyanionic framework of DNA, as
revealed by their binding affinity for an anti-DNA
monoclonal antibody. They were also tested as hep-
arinoids, showing a low anticoagulant effect. Among the
polymers tested, 4-hydroxyphenoxyacetic acid gave best
results. It reverts autocrine cell transformation caused
by basic fibroblast growth factor (bFGF),21 abrogates
bFGF receptor binding, dimerization and mitogenic
activity,22 and efficiently inhibits the proliferation of
vascular smooth muscle cells (SMC).23
a
Reagents and conditions: A ) dry HCl in MeOH or EtOH;
B ) isobutylene, dioxane, H2SO4; C ) acryloyl chloride, NaHCO3,
H2O; D ) acryloyl chloride, triethylamine, CH2Cl2; E ) ammonium
persulfate/Fe2+, H2O; F ) azobis(isobutyronitrile), MeCN; G )
NaOH, MeOH and/or H2O; H ) trifluoroacetic acid:CH2Cl2 (1:1).
Fibroblast growth factors (FGF) are a family of
structurally related polypeptides that show high affinity
to heparin.24 They are highly mitogenic for a variety of
cell types, and their release from the extracellular
matrix (ECM) may induce tissue repair and new blood
vessel formation.25 They are bound to heparan sulfate
proteoglycans in the ECM and cell surfaces, exhibiting
a high degree of stability toward proteolytic enzymes,
and can be released in an active form by heparinoids
and heparan sulfate-degrading enzymes.25,26
Heparanase is an endo-â-D-glucuronidase that de-
grades heparan sulfate side chains in basement mem-
branes and ECMs.27 This activity facilitates cell invasion
in normal and pathological situations.28-30 The human
heparanase gene has recently been cloned and ex-
pressed,31,32 and it has been shown to play a role in
tumor progression and metastatic spread.30,31,33 High
levels of heparanase were detected in highly metastatic
tumor cells and in cells of hematopoietic origin such as
neutrophils and inflammatory macrophages and lym-
phocytes that must invade the blood vessel wall to
express their physiological function.29,30 Polyanionic
molecules, sulfated polysaccharides, and some hep-
arinoids that inhibit the enzyme also inhibit pathologi-
cal processes involving cell migration such as tumor
metastasis,30,33 inflammation, and autoimmunity.29,34
cal activities. The use of pendent amino acids along an
acrylate polymer provides specific distribution of charge
and of polar and hydrophobic groups arranged in a
stereoselective configuration, determined by the chiral-
ity of the amino acid R-carbon. Desired hydrophobicity
or charged side groups such as -NH2, -COOH, -OH,
and phenols can be easily introduced by choosing
different amino acid residues. To test this hypothesis,
we have synthesized and characterized polymers of a
series of N-acryl L-amino acids and tested them for their
heparin-like biological activity. This paper describes the
synthesis and biological activity of these polymers as
heparanase inhibitors, bFGF-releasing molecules, an-
ticoagulants, and inhibitors of SMC proliferation.
Ch em istr y
P olya cr yla te Syn th esis. N-Acryl amino acid meth-
yl, ethyl, or tert-butyl esters were prepared from the
reaction between acryloyl chloride and the desired
amino acid ester in dry organic solvent in the presence
of an acid acceptor, i.e. triethylamine. The desired
acrylate derivatives were obtained quantitatively and
in high purity after a short workup of the reaction
mixture, which involved crystallization or precipitation
(Scheme 1). The structures and the analytical data of
synthesized N-acryl amino acids are given in Table 1.
Trytil and carbobenzoxy groups were used for protection
of the amine side chain functionality of histidine and
lysine, respectively. No protection group was used for
the hydroxyl functionality of hydroxyproline, serine, and
tyrosine. Short purification by chromatography was
needed for monomers containing cis-4-hydroxyproline,
tyrosine, and histidine in order to obtain pure com-
pounds. The ester derivatives were easily obtained by
Although these polyanions exhibited a range of de-
sired biological activities, they lack specificity and may
possess contradicting activities. Moreover, most of these
polymers exhibit strong anticoagulation activity which
may result in hemmohrage, thus limiting their potential
clinical use.
On the basis of the fact that polyanions usually mimic
heparin, we hypothesized that polyanions based on
N-acryl L-amino acids may express heparin-like biologi-

Poly(N-acryl amino acids)
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 13 2593
Sch em e 2. Synthesis of N-Substituted Acryl Amides
Ta ble 1. Structures and Analytical Data of N-Acryl Amino
Acid Monomers
Section). The IR spectra of the monomers and polymers
exhibited absorption peaks confirming the structure of
functional groups. For example, poly(N-acrylhydroxy-
proline methyl ester) showed peaks at: 3412 (hydroxyl,
narrow), 1742 (ester, sharp), and 1627 cm-1 (amide,
sharp), whereas the free carboxylic acid derivative
showed peaks at: 3504 (hydroxyl, broad), 1731 (car-
boxylic acid, sharp), and 1634 cm-1 (amide, sharp).
Almost all monomers were stable at room temperature.
Their 1H NMR remained unchanged after 1 year of
storage at room temperature. Monomers 2b (Scheme 1)
and 5c (Scheme 2) were unstable, polymerizing within
hours at 4 °C.
The polymerization was achieved in high yields
(>90% conversion) by radical initiation either by azobis-
(isobutyronitrile) in acetonitrile or by ammonium per-
sulfate/Fe(II) in water or water-methanol mixtures,
depending on monomer solubility (Scheme 1). Tables 2
and 3 summarize the structures and analytical data for
the poly(N-acryl amino acids) and the corresponding
decarboxylated polymers, respectively. The polymers
melted (Tmax, by DSC) at temperatures between 80 and
160 °C with a narrow heat absorption peak and heat of
fusion ranging between 60 and 140 J /g, indicating
relatively crystalline polymers. Most polymers had
negative optical rotation, except for the tyrosine (4g,
Table 2) and phenylalanine (4h ) based polymers. Mo-
lecular weights were determined by GPC analysis.
Purification by precipitation afforded polymers with
narrower polydispersity than the bulk. Methyl and ethyl
esters were cleaved by basic hydrolysis and tert-butyl
esters by hydrolysis with trifuoroacetic acid (TFA).
Sodium or potassium salts of all polymers described in
Tables 2-4 were water-soluble, while the free carboxylic
acid polymers were slightly soluble. The ester deriva-
tives were soluble in aliphatic alcohols, chlorinated
hydrocarbons, and acetonitrile. The methyl esters and
free acids of hydroxyproline (2a , 2b) and serine (2j)
polymers were water-soluble. Although the 1H NMR
spectra of the polymers showed poor resolution, it was
possible to monitor the polymerization process by fol-
lowing the disappearance of the signals representing the
vinyl protons.
* Codes are related to Scheme 1.
bubbling dry HCl through the methanolic or ethanolic
amino acid suspension or by acid-catalyzed reaction of
the amino acid with isobutene. Attempts to synthesize
N-acryl amino acid by direct amidation of acryloyl
chloride with amino acids in aqueous solutions afforded
satisfactory results only when glycine and proline were
used. The structures obtained were corroborated un-
1
ambiguously by H NMR and elemental analysis. The
1H NMR of all monomers synthesized showed the same
pattern for the vinyl moiety. Proton Hc is downfield
shifted because of its proximity to the carbonyl group.
Protons Ha and Hb are nonequivalent due to the planar
geometry of the amide bond. Hence, their coupling to
Hc produced split doublet signals. The integration of
acrylic protons is in good correlation with the ester ones.
This is an indication that no additional acrylic group
was attached to the unprotected side chain functionality
of compounds 2a , 2b, 2g, and 2j (Table 1). Acrylate
monomers lacking the carboxylic groups of tyramine,
ethanolamine, and 3-hydroxypyrrolidone which cor-
respond to tyrosine, serine, and hydroxyproline but
without the carboxylic acid were prepared by direct
amidation in water by the same procedure used to
synthesize the hydroxyproline methyl ester derivatives
and purified by chromatography (see Experimental
To increase the anionic nature of the polymers in
biological media, sulfation of hydroxyl side groups was
performed. Polymers based on trans-4-hydroxyproline
(3a ), tyrosine (3g), serine (3j), and 3-hydroxypyrrolidine
(6a ) were sulfated on the hydroxyl side group using SO3/
pyridine complex followed by known procedures de-
scribed for polysaccharides.12,35 Sulfate content was

2594 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 13
Ta ble 2. Structures and Analytical Data of Poly(N-acryl amino acids)
Bentolila et al.
* Codes are related to Scheme 1. Tmax was determined by DSC; optical activity was determined in water (40 mg/mL) at 25 °C at the
D-line of sodium. APTT (in s) was determined for 25 µg/mL polymer in pooled normal plasma; the data in parentheses are for polymer
concentration 250 µg/mL. Control: 32 s. No coagulation was observed with heparin (positive control).
Ta ble 3. Analogues of Poly(N-acryl amino acids) Lacking Carboxylic Acid Group*
* Codes are related to Scheme 2. Tmax was determined by DSC; molecular weight was determined by GPC.
determined by sulfur analysis. The data analysis for
these polymers is given in Table 4. Sulfation of between
40% and 74% of the hydroxyls was obtained. These
polymers were water-soluble.
prepared as previously described.29-31,36 Incubation of
partially purified human placental heparanase37 with
this ECM releases into the medium high- and low-
molecular-weight sulfate-labeled degradation products.
The high-molecular-weight material is composed pri-
marily of nearly intact heparan sulfate proteogly-
cans, serving as a readily accessible heparanase sub-
strate.29,30,38 In the absence of heparanase inhibitors,
low-molecular-weight sulfate-labeled degradation frag-
Biology
Hep a r a n a se In h ibition . The effect of the polymers
on heparanase activity was tested using [35S]O4
2-
labeled ECM produced by cultured endothelial cells and

Poly(N-acryl amino acids)
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 13 2595
Ta ble 4. Physical and Biological Data Obtained for Sulfated Poly(N-acryl amino acids)
* Codes are related to Scheme 3. APTT is expressed in seconds (s). Pooled normal plasma was used as control with 32 s. Polymers were
tested at 25 µg/mL.
F igu r e 1. Effect of heparin and polyanions on heparanase
activity. Sulfate-labeled ECM-coated 35-mm dishes were
incubated (37 °C, 48 h, pH 6.2) with human placental hepara-
nase in the absence (control) (0) and presence of 5 µg/mL
heparin ([), poly(N-acryl-trans-4-hydroxy-L-proline) (4), or
poly(N-acryl-cis-4-hydroxy-L-proline) (b). Sulfate-labeled mate-
rial released into the incubation medium was analyzed by gel
filtration over Sepharose 6B columns. Heparanase activity is
represented by the amount of radioactivity eluted in peak II
(fractions 23-35).
F igu r e 2. Inhibition of heparanase by poly(N-acryl amino
acids). Sulfate-labeled ECM-coated 35-mm dishes were incu-
bated (37 °C, 48 h, pH 6.2) with human placental heparanase
in the absence (control) and presence of 1 µg/mL (gray bars)
or 5 µg/mL (white bars) polyanionic compounds. Sulfate-
labeled material released into the incubation medium was
analyzed by gel filtration over Sepharose 6B columns. Hepara-
nase activity is expressed by the amount of radioactivity eluted
in peak II (sum of cpm in the highest nine fractions of peak
II) multiplied by the Kav of this peak.30
ments of heparan sulfate are released into the incuba-
tion medium29-31,38 (Figure 1, peak II). Heparin and
N-substituted nonanticoagulant species of low-molecu-
lar-weight heparin are inhibitors of heparanase, pre-
venting the degradation of heparan sulfate.28,30,31,38 All
polymers were assayed for heparanase inhibition activ-
ity (Figure 2). Poly(N-acrylates) of glycine (4d ), tyrosine
(4g), glutamic acid (4l), leucine (4e), tert-leucine (4f),
and sulfated hydroxyproline (7a ) yielded 70-80% inhi-
bition of heparanase activity already at a concentration
of 1 µg/mL, similar to heparin. The most effective
compound was poly(N-acrylleucine) (4e) (Figure 2).
Polymers containing trans-hydroxyproline (4a ) (Figure
1), proline (4c), and phenylalanine (4h ) inhibited the
enzyme only at 5 µg/mL. Polyanions bearing cis-hy-
droxyproline (4b) (Figure 1), histidine (4i), and lysine
(4k ) were inactive. The corresponding decarboxylated
poly(acryl amino acids) (series 6) were inactive even at
25 µg/mL (not shown).
induce release of bFGF from the ECM.25,26,39 The
released bFGF promotes, among other effects, prolifera-
tion of vascular endothelial and smooth muscle cells.
As shown in Figure 3, polymers containing proline (4c),
leucine (4e), tyrosine (4g), and sulfated hydroxyproline
(7a ) were highly active at 5 µg/mL, releasing 40-50%
of the ECM-bound bFGF, compared to about 85%
release of bFGF induced by heparin under the same
conditions. A significant bFGF release was also exerted
by polyanions bearing tert-leucine (4f), phenylalanine
(4h ), and glutamic acid (4l), releasing 30-45% and 55-
65% of the ECM-bound bFGF at 5 and 25 µg/mL,
respectively. Other poly(N-acryl amino acids) tested (4b,
4d , 4j, 4k ) were inactive and yielded percent release
similar to the spontaneous release of bFGF obtained in
Relea se of ECM-Bou n d bF GF . It has been dem-
onstrated that heparin and heparin-like molecules

2596 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 13
Bentolila et al.
F igu r e 4. Effect of heparin and polyanionic compounds on
proliferation of SMC. Twenty-four hours after seeding, the
SMC were arrested by 48 h incubation in medium containing
0.2% FCS, followed by addition of heparin or polymers (5 µg/
mL), 1 ng/mL bFGF, and [3H]thymidine, as described in the
Experimental Section. Cells were incubated for an additional
48 h and tested for thymidine incorporation into trichloroacetic
acid-insoluble materials. The background level of thymidine
incorporation by growth arrested SMC was 1000-1500 cpm
in different plates. Each data point represents the mean of
four wells, and the variation between different determinations
did not exceed (15%.
F igu r e 3. Release of ECM-bound bFGF. ECM-coated wells
(4-well plates) were incubated (16 h, 25 °C) with [125I]bFGF
(4 × 104 cpm/well). The ECM was washed (×3) to remove the
unbound bFGF and incubated (3 h, 37 °C) with 5 µg/mL (gray
bars) and 25 µg/mL (white bars) heparin and poly(N-acryl
amino acids). Released [125I]bFGF was counted in a γ-counter.
Released radioactivity is expressed as percent of total ECM-
bound [125I]bFGF (∼1 × 104 cpm/well).26,39 Each data point
represents the mean of four wells, and the variation between
wells did not exceed (10%.
Discu ssion
In the present study, polyanionic molecules based on
amino acids have been synthesized and tested for their
biological activity as heparin mimetics. Amino acids
were conjugated to polyacrylate via the amino group.
The polyanions obtained from the polymerization of
N-acryl amino acids or the corresponding decarbox-
ylated derivatives were tested for their ability to (i)
inhibit the heparanase enzyme, (ii) release ECM-bound
bFGF, and (iii) inhibit SMC proliferation. The polymers
of N-acrylates of tyramine, 3-hydroxypyrrolidine, and
ethanolamine were used as the decarboxylated ana-
logues of N-acryltyrosine, hydroxyproline, and serine,
respectively. Polymers obtained from these monomers
have the same pendent residues as the poly(acryl amino
acids) but without the carboxylic acid moiety. Sulfation
of polymers having hydroxyl side groups such as ty-
rosine, serine, and hydroxyproline yielded polymers with
enhanced anionic character that may confer additional
similarity to native heparin.
precense of PBS alone. Among the polymers lacking
carboxylic groups (series 6) only poly(acryltyramide) (6b)
showed activity at 25 µg/mL. The other polymers of this
series and those bearing zwitterionic residues (lysine
and histidine) were also inactive (Figure 3).
SMC P r olifer a tion . To test the antiproliferative
activity toward vascular SMC, the cells were first
arrested by incubation in medium containing 0.2% fetal
calf serum (FCS) followed by stimulation with 1 ng/mL
bFGF in the absence or presence of heparin or the
indicated poly(N-acryl amino acids). Among the syn-
thesized polymers, only substances that were found to
be active in the heparanase inhibition or bFGF release
tests [i.e. polymers based on trans-4-hydroxyproline
(4a ), glycine (4d ), leucine (4e), tyrosine (4g), serine (4j),
and glutamic acid (4l)] were selected for this assay. As
shown in Figure 4, polymers 4e and 4g effectively
inhibited the incorporation of [3H]thymidine into DNA
at 5 µg/mL, a concentration in which heparin was
inactive or slightly stimulatory.
None of the polymers tested in this study expressed
a significant anticoagulant activity, indicating that
these polymers will not suffer from the main limitation
of heparin, i.e. powerful anticoagulation effect.
An ticoa gu la n t Activity. The high anticoagulant
activity of heparin is attributed to its sulfate groups
since partially or fully desulfated heparin shows low or
no anticoagulant activity. The polymers were screened
for anticoagulant activity by the standard activated
partial thromboplastin time (APTT) method.40 All poly-
mers had a very low anticoagulant activity at a rela-
tively high concentration (25 µg/mL) (Table 2). In the
sulfated polymer series (Table 4), only sulfated hydroxy-
proline (7a ) exhibited significant anticoagulation activ-
ity.
The amino acid-containing polyacrylates showed some
correlation between the polymer structural features and
biological activity. These are summarized in Table 5.
In general, active polymers should display a net nega-
tive charge, hydrophobicity, and stereoregularity. Poly-
mers having at least one carboxylate group, providing
one negative charge per amino acid residue, were active
as heparanase inhibitors and bFGF-releasing agents.
Accordingly, methyl ester derivatives and zwitterionic
amino acid side groups such as poly(N-acryl-trans-
hydroxyproline methyl ester), poly(N-acryllysine), and

Poly(N-acryl amino acids)
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 13 2597
Ta ble 5. Structures of Polyelectrolytes versus Biological
Activity
bFGF, suggesting that these polymers interfere with the
mitogenic pathway of bFGF. Hence, poly(N-acrylty-
rosine or -leucine) may be potentially applied to inhibit
SMC proliferation and intimal thickening following
balloon deendothelialization and bypass coronary sur-
gery.
The polymer stereoregularity is also important for
determining its biological activity. We have found that
poly(N-acryl-cis-4-hydroxy-L-proline) (4b) was biologi-
cally inactive at the assays performed in this study, as
opposed to the trans homologue (4a ) which efficiently
inhibited heparanase activity (Figure 1). This finding
indicates that not only the anionic charge but also some
specific structural features have to be fulfilled to elicit
significant biological activity.
Altogether, some of the poly(N-acryl amino acids)
synthesized and characterized in this study may be of
clinical significance. For example, heparanase inhibitors
may have significant clinical applications in preventing
tumor metastasis and inflammatory/autoimmune pro-
cesses due to the involvement of this enzyme in the
extravasation of blood-borne tumor cells and activated
cells of the immune system.29-32 The fact that only one
species of heparanase was identified makes the hepara-
nase enzyme a promising target for potential antime-
tastatic drugs.41 Molecules that efficiently compete with
heparan sulfate and release active bFGF and other
heparin-binding growth factors from their storage in
ECMs and basement membranes may accelerate neovas-
cularization and tissue repair. Compounds which ef-
ficiently inhibit SMC proliferation may be applied,
systemically or when immobilized on intravascular
metal stents, to inhibit restenosis and accelerated
atherosclerosis.
poly(N-acrylhistidine) were inactive. Also, the corre-
sponding decarboxylated poly(acryl amino acid) deriva-
tives, poly(acryltyramide), poly(acryl-3-hydroxypyrro-
lidineamide), and poly(acrylethanolamide), were found
to be inactive, while their carboxylated analogues, poly-
(N-acryltyrosine), poly(N-acrylhydroxyproline), and poly-
(N-acrylserine), strongly inhibited heparanase activity.
Poly(N-acryltyrosine) also efficiently released bFGF
from ECM and inhibited SMC proliferation. Moreover,
polyacrylates with glutamic acid or O-sulfated hydroxy-
proline side groups exhibiting enhanced density of
negative charges per amino acid residue were potent
heparanase inhibitors and highly active in releasing
bFGF from ECM. In the case of the hydroxyproline
bearing polymer, the latter activity was attributed to
sulfation since the corresponding nonsulfated polymer
showed only heparanase inhibiting activity (Table 5).
Exp er im en ta l Section
Ma ter ia ls. Amino acids were purchased from Aldrich
(Milwaukee, WI), Fluka (Buch, Switzerland) and Sigma (St.
Louis, MO). Analytical grade solvents were purchased from
Frutarom (Haifa, Israel) and were used without further
purification. Triethylamine (Riedel-de Haen, Seelze, Germany)
was refluxed over KOH, distilled and stored over previously
desiccated molecular sieves (type 4A). Sepharose 6B was from
Pharmacia (Uppsala, Sweden). Sodium heparin from porcine
intestinal mucosa (PM-heparin, Mr 14 000, anti-FXa, 165 IU/
mg) was obtained from Hepar Industries (Franklin, OH).
Dulbecco’s modified Eagle’s medium (DMEM), fetal calf serum
(FCS), newborn calf serum (NBCS), penicillin, streptomycin,
L-glutamine and saline containing 0.05% trypsin, 0.01 M
sodium phosphate (pH 7.4) and 0.02% EDTA (STV) were
obtained from Biological Industries (Beit-Haemek, Israel).
Tissue culture dishes were obtained from Falcon Labware
Division, Becton Dickinson (Oxnard, CA). Four-well tissue
culture plates were obtained from Nunc (Roskilde, Denmark).
Na2[35S]O4 was obtained from Amersham (Buckinghamshire,
England). Recombinant human bFGF was kindly provided by
Takeda Chemical Industries (Osaka, J apan). Anticoagulation
tests were performed using IL Test APTT Liquid Silica kit
from Instrumentation Laboratory Co. (Lexington).
A second observation is related to the nature of the
amino acid side groups. Polymers bearing hydrophilic
residues showed only heparanase inhibition activity,
while polymers having more lipophilic residues also
induced bFGF release. These findings may indicate that
the inhibition of heparanase is caused primarily by
electrostatic interactions between the enzyme and the
polyanion. On the other hand, in the absence of the
strongly anionic sulfonamide groups (existing in hep-
arin), release of ECM-bound bFGF required both elec-
trostatic and some kind of lipophilic interactions.
In str u m en ta tion . Melting point of monomers was deter-
mined on an Electrothermal apparatus (Electrothermal, En-
gland). Tmax of polymers was determined on a Metler TA 4000-
DSC differential scanning calorimeter, calibrated with Zn and
In standards, at a heating rate of 10 °C/min. Molecular weights
of the polymers were estimated on a gel permeation chroma-
tography (GPC) system consisting of a Spectra Physics (Darm-
stadt, Germany) P1000 pump with RI detection (ERMA Inc.;
ERC-7510), a Rheodyne (Coatati, CA) injection valve with a
Only poly(N-acryltyrosine) and poly(N-acrylleucine)
efficiently inhibited SMC proliferation. These polymers
were 5-10-fold more active than heparin which, under
the experimental conditions applied in this study, failed
to inhibit and even stimulated thymidine incorporation
by SMCs at concentrations of 1 and 5 µg/mL (Figure 4).
This activity was more pronounced in the presence of

2598 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 13
Bentolila et al.
Sch em e 3. Sulfation of Hydroxyl-Containing
20-µL loop, and a Spectra Physics data jet integrator. Samples
were eluted with 0.05 M NaNO3 through a linear Shodex
OHpak KB-804 column at a flow rate of 1 mL/min. The
molecular weights were determined relative to pullulan stan-
dards (Showa Denko KK, J apan) with a molecular weight
range of 5 800-350 000 Da using a WINner/286 computer
program.
Poly(N-acryl amino acids) and Poly(acryl amides)
1H NMR spectra were recorded on a Varian 300-MHz
spectrometer. CDCl3 (99.5% D) and D2O (99.75% D) were
purchased from Merck (Darmstadt, Germany). Scale position
was determined by chloroform or water signals, respectively,
and results are reported in ppm. Optical activity was measured
on an automatic polarimeter apparatus from Optical Activity
(England). InfraRed spectra were recorded on a Perkin-Elmer
FTIR 2000 (Columbia), and results are reported in cm-1
.
Samples were prepared as KBr pellets at a concentration of
1-2%.
N-Acr yl Am in o Acid s (2, R′ ) H). 10.0 mmol of amino
acid was dissolved in 10 mL of 2 M KOH (2.0 equiv); 1.0 mL
of acryloyl chloride (1.2 equiv) was added dropwise with
vigorous stirring to the chilled solution. The solution was
stirred at room temperature for 1 h and then washed with
ether (20 mL × 2). The water layer was then acidified with 5
M HCl to pH ∼3. The solution was extracted with chloroform
(20 mL × 3) and dried over MgSO4 and the solvent was
evaporated to an oily residue. Products usually crystallized
from chilled mixtures of ethyl acetate-ether. See Table 1 for
structures and physical data of monomers.
hexane or liquid chromatography (silica gel 60, 70-230 mesh).
See Scheme 1.
N-Acr yl-4-tr a n s-h yd r oxyp r olin e Meth yl Ester (2a ).
Acryloyl chloride (1.8 mL, 20 mmol) was added dropwise to a
vigorously stirred chilled solution of 4-trans-hydroxyproline
methyl ester hydrochloride (3.63 g, 20 mol) and NaHCO3 (3.36
g, 40 mol) in water. The stirring was continued at room
temperature for 30 min. Then, the reaction mixture was
acidified with 1 M NaHSO4 solution to pH∼3. The solution
was extracted three times with ethyl acetate. The washings
were dried over MgSO4 and filtrated and the solvent removed
by flash evaporation. The oily residue was recrystalized from
ethyl acetate and ether, yielding 1.83 g (48%).
P olym er iza tion in Aqu eou s Solu tion . 2.0 mmol of N-
acryl amino acid ester was dissolved in 5 mL of methanol and
1 mL of water was added. A stream of N2 was bubbled trough
the solution for 3 min; 1% ammonium persulfate solution (450
mL, 1% mol/mol monomer), 2% sodium methabisulfite (450
mL) and 2% ferrous ammonium sulfate (250 mL) were added.
Polymerization was carried out at room temperature for 10 h.
Solvents were removed by flash evaporation and the residue
was precipitated in ether from methanolic solution. Precipi-
tates were collected by vacuum filtration, washed with ether
and air dried.
P olym er iza tion in Or ga n ic Solven ts. 2.0 mmol of N-
acryl amino acid ester and 4 mg of azobis(isobutyronitrile) were
dissolved in about 3 mL of acetonitrile in a borosilicate ampule.
The solution was degassed by bubbling N2 for 2 min. The
ampule was sealed and heated overnight to 70 °C. Ampule was
smashed; the polymer was ground in a mortar, washed on a
sinter funnel with ether and dried by air stream.
N-Acr yl-4-cis-h yd r oxyp r olin e Meth yl Ester (2b). 3.63
g of 4-cis-hydroxyproline methyl ester hydrochloride (20 mmol)
was treated as described above for the trans derivative. The
product did not crystallize and the residue was purified by
chromatography (silica gel 60, 70-230 mesh, eluent: gradient
of 0 to 5% MeOH in dichloromethane): 2.5 g of pasty solid
was obtained (65%); low stability and polymerizes readily.
Meth yl Ester Hyd r olysis. Poly(N-acryl amino acid methyl
esters) were dissolved in methanol or acetonitrile and 1 M
sodium hydroxide (2 equiv per ester group) was added.
Solutions were stirred overnight at room temperature and
neutralized by 1 M sodium bisulfate. Organic solvent was
removed and the aqueous solutions were freeze-dried. The
solids were dissolved in methanol and salts were removed by
filtration. Pure polymers were obtained by evaporation of the
clear methanolic solutions or by precipitation with ether or
petroleum ether. Poly(N-acryl diethyl glutamate) was hydro-
lyzed by the same procedure.
ter t-Bu tyl Ester Hyd r olysis. Poly(N-acryl amino acid tert-
butyl esters) were dissolved in dichloromethane:trifluoroacetic
acid (1:1). Solutions were left at room temperature for 3-4 h.
Most of the solvent was removed by flash evaporation. Ether
or petroleum ether was added to precipitate the residue. The
bulk polymers were grounded and triturated with petroleum
ether, ether or hexane.
O-Su lfa ted P oly(N-a cr yl a m in o a cid s). 1.0 mmol of poly-
(N-acryl amino acid) was suspended in dry DMF and 0.5 mL
of dry pyridine (6 equiv). The suspension was heated to 80 °C
and 0.480 mg of SO3/pyridine complex (3 equiv) was added.
The mixture was refluxed for 2 h. Solvent was removed by
flash evaporation and the residue dissolved in 1 M KOH (about
10 mL). The solution was dialyzed against DDW for 3 days at
4 °C and freeze-dried. Extent of sulfation was measured by
elemental analysis (Scheme 3). The analytical and biological
data are compiled in Table 4.
N-Acr yl-3-h yd r oxyp yr r olid in e (5a ): 1H NMR (CDCl3)
6.32 (2H,m), 5.66 (1H,d), 4.45 (1H,d), 4.10 (1H, broad), 3.60
(4H,m), 2.01 (2H,m); mp ) 48 °C. Anal. (C7H11NO2) Calcd: C,
59.56; H, 7.85; N, 9.92. Found: C, 59.51; H, 7.93; N, 9.73.
N-Acr yltyr a m id e (5b): 1H NMR (CDCl3) 7.05 (2h,d), 6.78
(2H,d), 6.25 (1H,d), 6.05 (1H,m), 5.60 (1H,d), 5.58 (1H,broad),
3.59 (2H,q), 2.80 (2H,t); mp ) 101 °C. Anal. (C11H13NO2)
Calcd: C, 69.09; H, 6.85; N, 7.33. Found: C, 68.98; H, 6.95;
N, 7.23.
N-Acr yleth a n ola m id e (5c): 1H NMR (D2O: 6.75 (1H,
broad), 6.20 (2H,m), 5.65 (1H,d), 3.74 (2H,t), 3.25 (2H,q); syrup;
low stability and polymerizes readily. Anal. (C5H9NO2) Calcd:
C, 52.16; H, 7.88; N, 12.17. Found: C, 53.27; H, 7.54; N, 11.79.
N-Acr yl Am in o Acid ter t-Bu tyl (2, R′ ) Bu t) or Meth yl
(2, R′ ) Me) Ester s. 20 mmol of amino acid ester was
dissolved in 100 mL of dichloromethane; 3.3 mL of triethyl-
amine (24 mmol, 1.2 equiv) was added. The solution was
chilled and acryloyl chloride (1.80 mL, 1.1 equiv) in 10 mL of
dichloromethane was added dropwise over 1 h. The cooling
bath was removed and the mixture was stirred overnight. The
solvents were removed in vacuum and the residues dissolved
in ethyl acetate (about 200 mL). The crystals (triethylamine
hydrochloride) were filtered out and the solution was washed
three times with 1 M NaHSO4 and 5% NaHCO3 and once with
brine (about 20 mL each wash). The organic layer was dried
over MgSO4, filtered and concentrated by flash evaporation.
Crudes were purified by crystallization, triturating with

Poly(N-acryl amino acids)
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 13 2599
P r ep a r a tion of Dish es Coa ted w ith ECM. Cultures of
bovine corneal endothelial cells were established from steer
eyes as previously described.36 Stock cultures were maintained
in DMEM (1 g of glucose/L) supplemented with 10% newborn
calf serum, 5% FCS, 50 U/mL penicillin, and 50 µg/mL
streptomycin at 37 °C in 10% CO2 humidified incubators.
Partially purified brain-derived bFGF (100 ng/mL) was added
every other day during the phase of active cell growth.36 For
the preparation of sulfate-labeled ECM coated dishes, bovine
corneal endothelial cells were dissociated from stock cultures
(second to fifth passage) with STV and plated into 35-well
plates at an initial density of 2 × 105 cells/mL. Cells were
maintained as described above except that 5% dextran T-40
was included in the growth medium and the cells were
maintained without addition of bFGF for 12 days. Na2[32S]O4
(540-590 mCi/mmol) was added (40 µCi/mL) 2 and 5 days
after seeding and the cultures were incubated with the label
without medium change.36 The subendothelial ECM was
exposed by dissolving (5 min, room temperature) the cell layer
with PBS containing 0.5% Triton X-100 and 20 mM NH4OH,
followed by four washes in PBS. The ECM remained intact,
free of cellular debris and firmly attached to the entire area
of the tissue culture dish.36
Hep a r a n a se Activity. Sulfate-labeled ECM was incubated
(24 h, 37 °C, 10% CO2 incubator) with 1 µg/mL partially
purified human placental heparanase37 at pH 6.2. To evaluate
the occurrence of proteoglycan degradation, the incubation
medium was collected and applied for gel filtration on Sepharose
6B columns (0.9 × 30 cm). Fractions (0.2 mL) were eluted with
PBS at a flow rate of 5 mL/h and counted for radioactivity
using Bio-fluor scintillation fluid. The excluded volume (Vo)
was marked by blue dextran and the total included volume
(Vt) by phenol red. The latter was shown to comigrate with
free sulfate.29-31,38 Degradation fragments of HS side chains
were eluted from Sepharose 6B at 0.5 < Kav < 0.8 (Figure 1,
peak II).29-31,38 A nearly intact HSPG released from ECM by
trypsin or plasmin was eluted next to Vo (Kav < 0.2, Figure 1,
peak I). Recoveries of labeled material applied on the columns
ranged from 85 to 95% in different experiments. Each experi-
ment was performed at least three times and the variation in
elution positions (Kav values) did not exceed (15%.
ing the radioactivity incorporated into trichloroacetic acid
(TCA) insoluble material.23
Ack n ow led gm en t. This work was supported by
grants from the German-Israeli Cooperation in Cancer
Research sponsored by the Deutsches Krebforschungs-
zentrum (DKFZ) and the Israeli Ministry of Science, the
Israeli Academy of Science.
Su p p or tin g In for m a tion Ava ila ble: Typical 1H NMR
spectrum of an N-acryl amino acid ester. This material is
available free of charge via the Internet at http://pubs.acs.org.
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