Design and synthesis of biomimetic polyacrylamides

Reactive Polymers, 10 (1989) 211-217

211

Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

DESIGN AND SYNTHESIS OF BIOMIMETIC POLYACRYLAMIDES * D. ROIZARD, A. BREMBILLA and P. LOCHON

CNRS-UA 494, Chimie-Physique Macromol$culaire, ENSIC-INPL, 1 rue Grandville, BP 451, 54001 Nancy (France) (Received August 1, 1988; accepted in revised form November 5, 1988)

Water-soluble biomimetic (co)polyacrylamides have been synthesized. Their preparation requires radical (co)polymerizations of new cyclic monomers in dimethylformamide solution, followed by appropriate chemical modifications. The starting acrylamides are obtained with good yields from acryl chloride and aniline derivatives substituted in the 3, 4-positions by NO2 and N H 2 groups. The aromatic moiety is linked to the acryl group by means of a hydrophobic spacer arm [-(CH2) . - type]. For n = O, 4-amino-3-nitroaniline is used as the starting reagent," for n v~ O, symmetrical diaminoalkanes must first be monofunctionalized with the help of SNar reaction carried out on 4-nitro-3-aminochlorobenzene. Benzimidazole rings are then synthesized via reduction of the nitro group with Na2S204 and cyclization with glycolic acid. The first esterolytic tests of picolinate activated ester show enhanced hydrolysis rates, which means that the esterolytic activity of the 2-hydroxymethylbenzimidazole residues is totally retained in the macromolecular structures elaborated, and that the use of appropriate esters shouM result in increased hydrolysis rates induced by hydrophobic complexation.

INTRODUCTION

The study and comprehension of the action of enzymes have been a challenge for many years. Many researchers [1-3] have already contributed to the increasing knowledge in this field, showing evidence, for instance, of the catalytic cooperation mechanism of some particular chemical functions located in the * Paper presented at the 4th International Conference on Polymer Supported Reactions in Organic Chemistry, Barcelona, Spain, June 26-July 1, 1988. 0923-1137/89/$03.50

active site of the enzymes. However, these studies have also thrown light on the complexity of the overall fundamental action of enzymes, which makes them some of the most powerful reagents in nature, with regard to their extraordinary efficiency and selectivity. In our laboratory, we were first involved in the modelling of hydrolytic enzymes such as a-chymotrypsin [4-6] and, more recently, in the modelling of carboxypeptidase A [7]. This paper reports on how macromolecular models bearing active units were designed and synthesized.

© 1989 Elsevier Science Publishers B.V.

212

MODELLING O F H Y D R O L Y T I C ZYMES: BACKGROUND

EN-

The first simple models studied in our laboratory were of the papain type. These models combine within the same compound a benzimidazole ring as the basic site and a thiol function as a potential nucleophile (Scheme 1). The esterolytic properties of such compounds were examined through their action in the hydrolysis of the acetate of p-nitrophenol in aqueous medium. The rate of appearance of the p-nitrophenolate ion in the m e d i u m is used to determine the activity of the models (Scheme 2). Among the compounds studied, the particular benzimidazole bearing a mercaptomethyl group at the 2-position exhibits a large acceleration of the reaction rate. According to our kinetic results, this acceleration is interpreted in terms of a bifunctional action of both functional groups of the model.

/N~CH2

)n'OH

Scheme 3. Hydroxyalkylbenzimidazoles.

Similar studies were also carried out with 2-hydroxyalkylbenzimidazole derivatives, in order to model metallo-enzymes (Scheme 3). This type of enzyme, such as carboxypeptidase A, requires the formation of a cationic complex with Zn 2+ to be active. The hydrolysis rate of picolinate-activated ester is used to test the activity of these models (Scheme 4). UV spectrophotometry is also used to follow the kinetics of the esterolysis. The largest acceleration rate observed is due to the 2-hydroxymethylbenzimidazole derivative, which indicates the formation of a very active metal ion complex.

NH

POLYMER MODELS: AIM AND DESIGN

Scheme 1. Mercaptoalkylbenzimidazoles.

From these results, obtained with simple compounds, we intend to prepare new esterolytic models of higher potential esterolytic properties. We believe that macromolecutar models could be interesting materials to deHzO

O= I C - O ~ - - ~ - N O z +

M O D E L S . . . ~ u v : SpectrophOtometer ]

CH 3 Release of p.nitrophenolate ion Scheme 2. Study of p-nitrophenyl acetate ester hydrolysis.

H2o z,,~÷

-NOz + MODELS---~-.~UVSpectrophotometer']

!,

Release of p.nitrophenolate ion Scheme 4. Study of p-nitrophenyl picolinate ester hydrolysis.

213 velop our research for the following reasons: enzymes themselves are macromolecules and, even if one does not plan to elaborate synthetic enzymes, one can assume that synthetic polymers offer better modelling possibilities; and from our point of view, the synthesis of polymer structures is also one of the methods which can help us to achieve our aim: successive and comprehensive macromolecular structures can be elaborated to give successive and complementary information on the way an enzyme works. The esterolytic properties of the polymer models should result from the presence of known active units, able to induce hydrolytic acceleration rates, and from the macromolecular structure itself, able to model other enzymatic properties such as complexation via hydrophobic bonds and selective hydrolysis towards esters of different natures. A synthetic approach was designed to lead to water-soluble polymers of variable physico-chemical properties. The methodology used has to solve the two following major problems: the introduction of active units in a variable polymer structure and the characterization of its esterolytic properties by means of the usual kinetic study, made on simpler models. We therefore designed a target macromolecule which could be described chemically as the association of a main polymer

chain, esterolytic units (Scheme 5).

and

SYNTHESIS OF ESTEROLYTIC POLYMERS General synthetic routes were easily found for the proposed target macromolecule; indeed, simpler starting structures can be proposed from the disconnections 1, 2 or 3. For our work, we focused on the structure coming from the disconnection 3; this case requires the synthesis and the copolymerization of appropriate monomers. This approach is the most general one because, on the one hand, a particular monomer could be polymerized with various comonomers to give different families of hydrolytic polymers, and on the other, variation of the spacer arm and of the esterolytic capacity could be made in a given family of polymers. These different synthetic possibilities should result in the overall modulation of the esterolytic properties of the macromolecular structures prepared.

Preparation of acrylic monomers The synthesis of acrylic monomers was chosen because of their known ability to give high-molecular-weight polymers with potential water-soluble properties and their easy access from acryl chloride (Scheme 6). The type of spacer arm was chosen for its predictable chemical inertness (methyl group) and its capacity to give hydrophobic bonds, in addition to leading to homogeneous polymer families [available (CH2) n residues]. The aromatic precursor unit was chosen for its potential ability to lead to the formation of

o

Scheme 5. Target macromoleculeand possible disconnections 1, 2, or 3.

spacer arms

Scheme 6. N-Substituted acrylamides.

214

benzimidazole rings, and for its possibility to be easily linked to the spacer arm or to the acryl group. The chemical linkage required to join spacer arms to aromatic residues was determined from the available starting products: for the spacer arms, either diamines, amino acids, amino alcohols or amino halogenated derivatives; and for the aromatic residues, either benzoic acids, chlorobenzenes or anilines. We chose a,~-diaminoalkanes as spacer arms because a large number of derivatives were available (n = 2-12), and also because it seemed to be the quickest synthetic route to N-substituted acrylamides, either from benzoic acids or from chlorobenzenes. Two general cases were considered and synthesized first: first, the case where the precursor unit is linked as closely as possible to the main polymer chain ( n - - 0 ) ; second, the case where a variable spacer is used (n +~ 0). The corresponding chemical routes studied were the acylation of substituted aniline by acryl chloride, and the functionalization of symmetric a,~-diaminoalkanes. The first route corresponds to the general Scheme 7. Starting anilines, appropriately ort h o - s u b s t i t u t e d in 3,4-positions (NO 2, N H 2 or C1 groups), can be used. Among these compounds we chose 3-nitro-4-aminoaniline; published results [8] indicate that 5(6)-aminobenzimidazole derivatives are not obtained from triaminobenzene; on the other hand, the use of 3-chloro-4-nitroaniline would certainly lead to a mixture of products given by the nucleophilic substitution reaction of chlorine by N H 3. The acylation of 3-nitro-4-aminoaniline leads to a mixture of mono- and diacryla-

o

x

S c h e m e 7. S y n t h e s i s o f s u b s t i t u t e d a c r y l a n i l i d e s .

mide, even when stoichiometric proportions of reagents are used. However, with appropriate reaction conditions, monoacrylamide is the major product formed. We noticed that the nature of the solvent used [diethyl ether, benzene, dimethylformamide (DMF)] and the choice of the proton acceptor used (DMF, triethylamine) induce marked differences in the overall yield and in the relative amounts of both acrylamides formed. The use of a radical inhibitor does not avoid the formation of heavy by-products, which may suggest that these are due to nucleophilic addition on the double bond of the acryl structure. Careful purification of the monoacrylamide is needed prior to its polymerization. The second synthetic route requires the preparation of analogous acrylamides, N-substituted by a variable diamino spacer arm; their general formula is C H 2 = C H C O N R (CH2),Z , where Z is an aromatic residue. This target can be reached by two synthetic pathways (acylation, SNar) which lead to three different intermediates, N-aminoalkylacrylamides, N-aminoalkylbenzamides and N-aminoalkylanilines. Both of these routes involve the problem of the monofunctionalization of symmetrical diamines [9-11]. We found that here the best results were obtained from nucleophilic aromatic substitutions. The starting materials required are p-halogenonitrobenzenes. Amino-N-alkylanilines are easily obtained from 3-amino-4-nitrochlorobenzene; the yield of the reaction depends markedly on the diaminoalkane chain length (Scheme 8). Acylation of N-alkylanilines with n = 2 and 9 was carried out; these cases correspond to

n=2 n=3 n=4 n=5 n=9

% Yield / R 90 Me 30 H 45 H 36 H 75 H

NR.-(CH )-.NHR _.,J..,_ " 2n

NOz

I'Iz

S c h e m e 8. S y n t h e s i s o f N - s u b s t i t u t e d a c r y l a n i l i d e s .

215 the two most different cases of length of spacing arm; these monoacrylamides were obtained with good yield (ca. 85%).

Polymerization studies Radical homopolymerizations were carried out in solution for a temperature ranging from 55 to 120°C (solvent, DMF; initiator, azobisisobutyronitrile, azobisdimethylvaleronitrile or cumyl peroxide). These polymerizations are slow (20-70 h) compared with the usual acrylamide polymerizations; they give soluble polymers with conversion rates depending on the experimental parameters used. These results obviously show the occurrence of deactivating effects, certainly due to the aromatic substituents. Analogous copolymers were prepared with the use of acrylamide as comonomer because of its ability to give highly water-soluble polymers under the experimental conditions used. Most of the copolymer experiments were carried out for molar fractions of N-substituted acrylamides ranging from 10 to 50%. Copolymer formations were established from polymer solubility properties, 13C N M R spectroscopic data and gel permeation chromatographic observations; higher conversion rates were obtained for these copolymerizations. Their solubility in aprotic dipolar solvents and their hygroscopic characteristics vary with their composition. As expected, the average molecular weights obtained are higher.

amide groups of the polymers prepared. Model compounds such as 3-nitro-4-aminoisobutylanilide or o-phenylenediamine derivatives were first used to test different reagents required for the reduction or cyclization step. Two kind of reducing agents were efficient for the formation of the o-diamino ring: sodium dithionite (Na2S204) (yield--50%) and H 2 - P d / C (yield = 70%). In the cyclization step, the usual method of Phillips could not be used because the stability of the amide groups requires anhydrous conditions. Cyclization experiments were carried out with glycolic and mercaptoacetic acid, either in polyphosphoric or in bulk. The best results were obtained with glycolic acid. When applied to the copolyacrylamides, the H z - P d / C reducing agent was totally inefficient, even with the use of a high hydrogen pressure (80 bar), but the action of sodium dithionite gave the expected reduced copolymers with good yield. These water-soluble polymers were purified by dialysis. Successful cyclizations were finally obtained with a large excess of glycolic acid and confirmed by spectroscopic data (Scheme 9). The reaction used to test the esterolytic power of the modified polyacrylamides is the hydrolysis of p-nitrophenyl picolinate ester in a homogeneous aqueous medium. The metal ion complex formed with the ester and the benzimidazole moiety leads to the release of the p-nitrophenolate ion, the increasing concentration of which can be measured by UV spectrophotometry.

ESTEROLYTIC POLYMER MODELS The principle of the formation of polymer models is usually simple from o-nitroamino aromatic rings; benzimidazole rings can result either from a single reductive cyclization, or from a two-step synthesis, with prior reduction to the cyclization step. Here the choice of the synthetic route and experimental conditions were determined by the stability of the

~

NF~ ~ NF~ HOC2HCOOH [N l~ INH N~ ~ OH Scheme 9. Synthesis of esterolytic polymers.

216 TABLE 1 Kinetic measurements on N-benzimidazole acrylanilide (mole fraction = 50%) Conditions: ester, 5 × 1 0 -5 M; p H = 7.14; T = 3 0 ° C Compound

Concentration (tool/l)

ZnNO 3 kobs a (mol/1 × 10 3) (s × 10 3)

Bim-OH 5 x 10 -4 0.75 PA-A1/ Bim-OH b 5.2>(10 - 4 1 Polyacrylamide > 10- 3 1

3.3 4.07 Not measurable

a Spontaneous picolinate hydrolysis: kobs = 0.1 × 1 0 -~ S-1. b Active complex concentration: 2.8 X 10-5 s-1.

Qualitative tests were carried out with the different types of copolymers prepared; they all revealed a marked acceleration of the esterolysis reaction compared with the spontaneous hydrolysis of the activated ester. First kinetic measurements were also made with a copolymer of N-benzimidazole and acrylanifide (molar fraction = 50%). They showed that the esterolytic activity of the benzimidazole units is totally retained (Table 1). This means that efficient metal ion complexes are formed along the copolymer and that their activity is governed by a catalytic mechanism. From these results we hope that further esterolytic experiments carried out with polymers possessing a hydrophobic spacer arm will show the appearance of hydrophobic complexation with appropriate activated esters.

EXPERIMENTAL

Synthesis of polyacrylamides Commercial-grade reagents were used as starting materials for the synthesis of acrylamide monomers and polymers. Monomers were synthesized easily from alkyl or aromatic amines [3,12] by acylation with stoichiometric

amounts of acryl chloride. Typically, 0.065 mol of amine is dissolved in 100 ml of distilled solvent (DMF or diethyl ether) in the presence of small amounts of hydroquinone under an inert atmosphere; after cooling of the reaction mixture to 0 ° C and adding 3 equivalents of proton acceptor, the acylating agent is slowly added. After 10 h, the reddish heterogeneous medium is filtered over Celite. The products are isolated by evaporation of the solvent under reduced pressure and recrystallization from benzene (yield 70-85%). IR and 1 H N M R spectra were in good agreement with the predicted absorption bands and chemical shifts; specifically, the acrylamide group introduced is very well characterized by its strong carbonyl IR absorption (v = 1650-1680 cm -1) and the N M R peaks of the three vinylic protons (8 p p m in DMSO-d6: H x = 5.8, H a = 6.2 and H b = 6.3). Polyacrylamides were synthesized by radical polymerization in solution. Typically, the monomer (0.01 mol), the solvent (10 ml, freshly purified) and the initiator (AIBN, 1-2 mol.%) are mixed in a glass vessel and degassed twice under reduced pressure. Then the reaction mixture is heated at 5 5 ° C in a thermostated bath for 20-70 h. Quantitative isolation of the polymer is effected by precipitation of the reaction mixture under vigorous stirring in the solvent used for the starting reagents. The IR spectra of the polymers showed characteristic absorption due to the formation of the polymeric chain (v = 2920-2960 cm -1) and the the disappearance of the C=C double bond ( v = 1625 cm-1). The nitro groups are quantitatively reduced in presence of a small excess of Na2S204 in aqueous solution to give o-phenylene rings responsible for strong IR absorptions at 3500 and 1610 cm -1. The final formation of benzimidazole rings was carried out as follows: under an inert atmosphere, the reduced polymer (0.5 g) is heated to l l 0 ° C with a large excess of glycolic acid for 1 h; after cooling, the polymer is isolated by dialysis (yield =

217

70%). The most significant difference in the IR spectra of the cyclized polymers is the large decrease in absorption in the 3500-3300 c m - ] range, due to the disappearance of free N H 2 groups; indeed, the benzimidazole IR bands are weak (3200-2650 cm-1) and mostly masked by the large absorption of the amide groups. Kinetic m e a s u r e m e n t s

These were recorded with a Hitachi 320 spectrophotometer at 400 nm. Kinetic runs were carried out in water at 30 ° C, buffered to p H 7.14 by Nl-ethanesulphonic acid-N 2hydroxyethylpiperazine (HEPES) at an ionic strength of 0.1 mol 1-1 (NaNO3). Typically, a 3-ml standard quartz cuvette of 1-cm optical path length contains 5.2 X 10 - 4 M 2hydroxymethylbenzimidazole moieties, 10- 3 M ZnNO3, 0.05 M HEPES, 2.5 X 10 -3 M CTABr and 5 x 10 -s M p-nitrophenyl picolinate ester. The calculated concentration of the active metal-ligand complex is 2.8 x 10 -5 M. The rate constants (Table 1) are determined from the release of p-nitrophenolate ion according to the equation Log

(oo oo0) "DO~ - ~

= k°bst

CONCLUSION The general approach used to design esterolytic macromolecule models leads to the synthesis of various copolyacrylamides, Nsubstituted by 2-hydroxymethylbenzimidazole units (carboxypeptidase A models). The formation of an active metal ion complex with Zn 2÷ on lateral side-chains of a copolyacrylamide has been established, in addition to catalytic nature of the mechanism involved. Further studies should take advantage of the spacer arm present in the

macromolecular structure to point out hydrophobic effects and of the synthetic protocol used to prepare analogous models of hydrolytic cysteine enzymes. REFERENCES 1 G.R. Schonbaum and M.L. Bender, The hydrolysis of p-nitrophenyl acetate catalysed by o-mercaptobenzoic acid, J. Amer. Chem. Soc., 82 (1960) 1900. 2 H. Morawertz and W.R. Song, The interaction of chain molecules carrying reactive and catalytic chain substituents, J. Amer. Chem. Soc., 84 (1966) 5714-5718. 3 G. Wulff and A. Sharan, Chemical approaches to understanding enzyme catalysis, in: Proceedings of the 26th OHOLO Conference, Zichron Yaacow, Israel, 22-25 March 1981, pp. 106-118. 4 P. Lochon and J. Schoenleber, Comparaison de l'action catalytique de thiols monofonctionnels et de certains thiols bifonctionnels sur l'hydrolyse de l'acrtate de p-nitrophrnyle, Tetrahedron, 32 (1976) 3023-3030. 5 A. Brembilla, Etude de nouveaux sites catalytiques immobilisables sur un support macromolrculaire, Doctorat d'Etat, Nancy, 1982. 6 D. Roizard, Voie de synth+se de macromolecules modrles d'enzymes hydrolytiques, Doctorat d'Etat, Nancy, 1988. 7 A. Brembilla and P. Lochon, Un modrle de l'activit6 catalytique de m&alloenzymes: le systSme hydroxym&hyl-2 benzimidazole-Zn ++, J. Chim. Phys. Chim. Biol., 85 (1988) 309-314. 8 Van der Want, Uber 4(7)Amino-benzimidazol, Recl. Trav. Chim. Pays-Bas, 67 (1945) 45. 9 K. Kojima, N. Yoda and C.S. Marvel, base-catalysed polymerization of mateimide and some derivatives and related unsaturated carbonamides, J. Polym. Sci., Part A-l, 4 (1966) 1121-1134. 10 G.L. Stahl, R. Walter and C.W. Smith, General procedure for the synthesis of mono-N-acylated 1,6-diaminohexanes, J. Org. Chem., 43 (1978) 2285-2286. 11 F. Schwartz and A. Shanzer, Synthesis with silicon derivatives: Non-symmetrical derivatization of symmetrical diamines, Tetrahedron. Lett., 23 (1982) 978-982. 12 M.A.F.H. Lobry de Bruyn, Recherche quantitative sur la nitration des chloroacrtanilides avec de l'acide nitrique et des trois chloro-anilines en solution sulfurique, Recl. Trav. Chim. Pays-Bas, 36 (1916) 126-166.