Synthesis and Characterization of N-Vinylpyrrolidone-tert-Butyl Methacrylate-Methacrylic Acid Terpolymers HavingAmino Sugar or Bioactive Amino Side Compounds

GERARDO MARTINEZ, MARTA FERNANDEZ-GARCIA, MANUEL SANCHEZ-CHAVES

Instituto de Ciencia y Tecnologıa de Polımeros (Consejo Superior de Investigaciones Cientıficas), Juan de la Cierva 3,28006 Madrid, Spain

Received 11 March 2004; accepted 11 June 2004DOI: 10.1002/pola.20347Published online in Wiley InterScience (www.interscience.wiley.com).

ABSTRACT: Free-radical copolymerization of N-vinylpyrrolidone (VP) with tert-butylmethacrylate (TBMA) was carried out in bulk at 50 °C, using 2,2�-azobisisobutyronitrileas initiator. From copolymer composition determined by 1H-NMR analysis and usingthe modified Kelen–Tudos equation, the monomer reactivity ratios were determined.After acidic hydrolysis, the modification reaction of some N-vinylpyrrolidone-tert-butylmethacrylate-methacrylic acid (VP-TBMA-MAA) terpolymers with several model bio-active amino compounds (benzocaine, phenethylamine, tyramine) and a model aminosugar (2-amino-2-deoxy-D-glucose (glucosamine)) was studied in the homogeneousphase. Interaction experiments between glucosamine-carrying VP-TBMA-MAA ter-polymers and Concanavalin A (Con A) in a phosphate buffer (pH � 7.2) at roomtemperature showed that VP-TBMA-MAA terpolymer interacts with Con A. © 2004Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 18–27, 2005Keywords: copolymerization; N-vinyl pyrrolidone-tert-butyl methacrylate copoly-mers; modification; bioactive amino compounds; pendent glucosamine; biopolymers;Concanavalin A

INTRODUCTION

In recent years, a great deal of attention has beenpaid to saccharide-containing synthetic polymers(so called glycopolymers) because of their poten-tials as cell-specific biomedical materials,1 aspharmacological substances,2 and as biologicalrecognition substances.3,4 The synthesis of glyco-polymers usually requires the preparation of po-lymerizable sugar derivatives,5,6 although theless frequently employed chemical modification ofpreformed polymers using saccharide-containingreagents offers an alternative synthetic route toglycopolymers.6 In both cases, the sugar is at-

tached to the polymer by amide, ester, ether, etc.,linkages.

On the other hand, the chemical linkage ofbioactive compounds to synthetic and naturalpolymers as a means to obtain delivery systems ofbioactive substances has attracted considerableinterest to optimize the duration of activity.7 Themacromolecular nature of the delivery system al-lows control of mobility and period of effective-ness of the biologically activated component.

Related to that before mentioned, amphiphilicN-vinylpyrrolidone-tert-butyl methacrylate (VP-TBMA) copolymers possess several propertiesthat make them feasible macromolecular bioac-tive carrier candidates. VP is a hydrophilic non-ionic monomer of interest for the preparation ofbioactive copolymers.8–16 TBMA, a hydrophobicmonomer, has been selected to prepare amphiphi-

In memoriam of Dr. Enrique Lopez Madruga.Correspondence to: G. Martınez (gmart@ictp.csic.es).

Journal of Polymer Science: Part A: Polymer Chemistry, Vol. 43, 18–27 (2005)© 2004 Wiley Periodicals, Inc.

18

lic graft and block copolymers17,18 since subse-quent hydrolysis of the tert-butyl groups leads toblock copolymers containing hydrophobic and hy-drophilic segments. As far as we are aware, stud-ies on VP-TBMA copolymers and subsequent hy-drolysis have not been reported in the literature.

The first part of this paper reports the statis-tical copolymerization of VP with TBMA in bulkand the subsequent acidic hydrolysis of some ofthe resulting VP-TBMA copolymers to achieveterpolymers with pendent reactive carboxylicacid groups, that is, N-vinylpyrrolidone-tert-butylmethacrylate-methacrylic acid (VP-TBMA-MAA).The second part deals with the applicability ofthese pendent carboxylic acid groups in the cou-pling of 2-amino-2-deoxy-D-glucose (glucosamine)to obtain a new type of sugar-carrying amphi-phile. In addition, a study of the coupling of somemodel bioactive amino compounds such as benzo-caine, phenethylamine, and tyramine to get poly-mer-bioactive compound conjugates is also made.Finally, the interaction of glucosamine-carryingpartially hydrolyzed VP-TBMA copolymers with alectin, Concanavalin A, is also investigated.

EXPERIMENTAL

Materials

VP (Aldrich) was deinhibited by distillation underreduced pressure, and the middle fraction storedby refrigeration overnight prior to polymeriza-tion. TBMA (Aldrich) was passed through a col-umn of activated basic aluminum oxide (Aldrich)and purged with high-purity nitrogen prior touse. Dimethyl sulfoxide (DMSO) and dioxanefrom Scharlau (Barcelona, Spain) were distilledunder nitrogen. N,N�-dimethylformamide (DMF;Scharlau) was purified by shaking with phospho-rus pentoxide for four days. Then, it was washedwith potassium hydroxide pellets and distilled at47 °C at 14 Torr. The middle fraction was used.Triethylamine and ethylchloroformate were pur-chased from Aldrich and purified by distillationand then dried for a few days with a Merck 4 Åmolecular sieve. 2,2�-azobisisobutyronitrile (AIBN)from Fluka was recrystallized twice from metha-nol and dried in vacuum (m.p. 104 °C). Benzo-caine (Fluka, �99%), D (�) glucosamine hydro-chloride (Fluka, �99%), tyramine (Aldrich, 99%),and phenethylamine (Fluka, �99%) as well asConcanavalin A (Con A) (Fluka) were used asreceived without further purification.

Copolymerization Reactions

VP-TBMA bulk copolymers were synthesized in200-mL glass ampoules with rubber septa. A free-radical initiator, AIBN, was used (0.4 wt % of themonomers). Dissolved oxygen was removed fromthe reaction solution by nitrogen purging for 60min prior to immersion in a water bath at 50 °C� 0.1 °C. The copolymerization system was homo-geneous in all cases investigated. After a specificlength of time, each ampoule was removed fromthe water bath, and the reaction was stopped with0.5 mL of a 10 wt % solution of hydroquinone intetrahydrofuran (THF). Conversion was alwayslower than 8%. A large excess of heptane wasused to precipitate the copolymers. All of thesewere reprecipitated from chloroform solution intoheptane, filtered, and dried at 40 °C under re-duced pressure in the presence of phosphoruspentoxide until constant weight was attained.Both the conversion and the monomer feed com-position were calculated gravimetrically.

Characterization of the Copolymers

1H-NMR spectra were recorded at 400 MHz on aVarian Inova 400 spectrometer with deuteratedchloroform as the solvent and locking agent at 45°C under standard conditions. The spectra wereobtained after accumulating 100 scans with asample concentration of 10 wt % solutions. Theproton solvent signal was used as chemical shiftmarker. The relative signal intensities of thespectra were measured from the integrated peakarea, calculated by means of an electronic inte-grator.

Acidic Hydrolysis

The reaction was performed in solutions of PT-BMA or VP-TBMA copolymers (0.5 wt %) in diox-ane/HCl (10/1 v/v) via refluxing for 3 days at 90°C. The solvent and HCl were then removed witha rotary evaporator. The products were furtherpurified from DMSO solutions into ethyl acetate(PTBMA) or acetone (VP-TBMA copolymers) andsubsequently dried to a constant weight at 40 °Cunder reduced pressure. The characterization ofthe resulting hydrolyzed products was made us-ing 1H-NMR spectroscopy and KBr pellets in aPerkin–Elmer FT-IR spectrometer.

VP-TBMA–METHACRYLIC ACID TERPOLYMERS 19

Reaction of Amino Compounds with VP-TBMA-MAA Terpolymers Activated with EthylChloroformate

Both fully hydrolyzed PTBMA and VP-TBMA-MAA terpolymers were dissolved in DMF and thesolution cooled to �8 °C. Calculated amounts oftriethylamine and ethyl chloroformate wereadded while stirring. After 1 h of stirring at �8°C, a cooled solution of the corresponding aminocompound in DMF was then added dropwise(Scheme 1). Stirring was continued at �8 °C foranother hour and then at room temperature for18 h. Triethylamine hydrochloride was removedby filtration and the filtrate containing the mod-ified polymer was precipitated into acetone. Allpolymers were purified by reprecipitation usingDMF as solvent and acetone as precipitant, thendried under vacuum in the presence of phospho-rous pentoxide. A schematic representation of theamine-carrying copolymer synthesis is presentedin Scheme 2.

Characterization of Amino Compounds-carryingVP-TBMA-MAA Terpolymers

The FTIR spectra were obtained using KBr discs.The content of amino compound was quantita-tively determined from 1H-NMR spectra regis-tered in DMSO-d6 at room temperature understandard conditions. For benzocaine, tyramine,and phenetylamine-based terpolymers, the com-position was calculated from the ratio of amidoproton absorption(� � 10.6; � � 8.2; � � 8.2,respectively) to the methylene protons (� � 1.7–1.8) in the main chain absorption. For glu-cosamine-carrying terpolymers, the composition

was determined by comparing the integrated in-tensities of the signals that appear in the interval� � 5.7–5.2 (corresponding to the � and � ano-meric protons) with the peaks at � � 1.8 and �� 1.0 (ascribed to the methylene and methyl pro-tons, respectively).

Interaction Between Glucosamine-Carrying VP-TBMA-MAA Terpolymers and Con A

Buffer solutions (0.01 M phosphate, pH 7.2, con-taining 0.5 M NaCl, 0.1 mM MnCl2, and 0.1 mM

Scheme 1

Scheme 2

20 MARTINEZ, FERNANDEZ-GARCIA, AND SANCHEZ-CHAVES

CaCl2) of glucosamine-carrying fully hydrolyzedPTBMA or glucosamine-carrying VP-TBMA-MAAterpolymers (0.549 and 0.316M fractions of glu-cosamine, respectively) and Con A were mixedand shaken at room temperature. At regular in-tervals, the turbidity of the solution was mea-sured at 360 nm on a Perkin–Elmer UV-VisLambda 16 spectrophotometer.

RESULTS AND DISCUSSION

Monomer Reactivity Ratios and Distribution of theMonomer Units

The free-radical copolymerization of VP-TBMA inbulk with AIBN as an initiator was carried out at50 °C.

The average molar fraction composition of thecopolymers was quantitatively determined fromthe corresponding 1H-NMR spectra of copolymersamples prepared with different monomer feeds.Figure 1 shows the 1H-NMR spectra of PVP, PT-BMA, and a couple of representative VP-TBMAcopolymer samples. The analysis was performedby comparing the integrated intensities of thesignals that appear at � � 1.4 (corresponding tothe protons in the tert-butyl ester group in themethacrylate units) with the peak at � � 3.2(ascribed to the methylene proton in the VP ring)(Fig. 1).

The molar fraction composition of monomerfeed (fVP), final conversion, and the experimentaland theoretical average molar fraction composi-tion of copolymer (FVP) for each conversion bulkcopolymerization are listed in Table 1. The mono-mer reactivity ratios were obtained from the co-polymer compositions with the modified Kelen–Tudos equation,19 in which the variation of themonomer feed composition with the conversion isconsidered. This concern has been taking intoaccount the inappropriate use of the differentialform of copolymer equation when the reactivityratios are quite different in magnitude. From theKelen–Tudos diagram shown in Figure 2a, theestimated values are rTBMA � 8.18 � 0.48 and rVP� 0.032 � 0.006. Additionally, in Figure 2b isrepresented the accuracy of the estimated datawhere the 95% joint confidence interval is drawn.

The copolymerization terminal model20 can beused for descriptive purposes21 although it is welldocumented that is only an approximate model.22

Therefore, to assess the quoted monomer reactiv-ity ratios, the cumulative copolymer compositionas a function of conversion for the different molar

fractions in the feed are compared to the theoret-ically calculated values.

The cumulative copolymer composition as afunction of conversion is shown in Figure 3. Thesolid lines are drawn according to the integratedcopolymer composition equation with the mono-mer reactivity ratios previously calculated. A rea-

Figure 1 1H-NMR spectra of poly (N-vinylpyrroli-done) (A), poly (N-vinylpyrrolidone-tert-butyl methac-rylate) FVP � 0.792 (B), poly (N-vinylpyrrolidone-tert-butyl methacrylate) FVP � 0.401(C), and poly (tert-butyl methacrylate) (D). The assignment of the labeledpeaks corresponds to the hydrogen marked on thestructural schematic.

VP-TBMA–METHACRYLIC ACID TERPOLYMERS 21

sonably good agreement between experimentaland calculated data is found.

On the other hand, it is well known that neigh-bor groups play an important role in the modifi-cation reactions of polymer functional groups.23,24

Thus, the consideration of the monomer sequencedistribution along the copolymer chains is of in-

terest to study the effect in the reaction mecha-nism. The probability of formation of the differentTBMA-centered triads has been calculated ac-cording to the conditional probabilities, the mono-mer molar fraction in the feed, and the estimatedmonomer reactivity ratios. The changes in thetriad compositions as functions of the TBMA mo-lar fraction in the feed are illustrated in Figure 4,where TBMA and VP units are denoted as T andV, respectively. In this figure, the possible varia-tion of triads molar fraction with conversion hasnot been contemplated since the samples modifiedhave been performed at very low conversions. Fig-ure 4 reveals that the molar fraction of the TTT

Figure 3 Dependence on the cumulative copolymercomposition (FVP) with conversion for the copolymer-ization of the VP-TBMA system at 50 °C at various VPmolar fractions in the feed (fVP). The solid lines werecalculated according to the terminal model with esti-mated monomer reactivity ratios.

Figure 4 Molar fraction of TBMA-centered triads asa function of composition of the molar fraction of TBMAin the copolymer chain (FTBMA)

Table 1. Molar Feed Composition (fVP), Conversion,and Experimental and Theoretical CopolymerComposition (FVP) for the Free-RadicalCopolymerization of VP and TBMA at 50 °C in Bulk

fVP

Conversion(wt %) FVP FVP(theo)

0.30 4.5 0.050 0.050

0.50 6.3 0.104 0.106

0.60 6.4 0.150 0.147

0.70 6.9 0.211 0.205

0.80 7.8 0.274 0.293

0.90 8.0 0.401 0.447

0.96 7.4 0.613 0.649

0.98 3.2 0.792 0.731

Figure 2 (a) Kelen–Tudos diagram for the copoly-merization of VP and TMBA, and (b) The 95% jointconfidence interval of monomer reactivity ratios.

22 MARTINEZ, FERNANDEZ-GARCIA, AND SANCHEZ-CHAVES

triads increases monotonically, and the overallmolar fraction of TTT triads in the copolymerchain is higher than 0.5, for copolymers with aTBMA molar fraction in the copolymer chain,FTBMA, higher than 0.75, which corresponds tocopolymers performed with a fTBMA higher than0.25. Moreover, Figure 5 depicts the averagelengths of TBMA sequences (LTBMA), obtainedfrom the monomer sequence distribution of thecopolymers, as a function of TBMA molar fractionin the copolymer chain.

Acidic Hydrolysis

VP-TBMA-MAA terpolymers were prepared bythe hydrolysis of VP-TBMA copolymer precursorswith an adequate composition. The cleavage ofthe TBMA side-chain groups was achieved by areaction with concentrated HCl in dioxane heatedat reflux to afford the VP-TBMA-MAA terpoly-mers (Scheme 1). The average molar fraction ofterpolymers was quantitatively determined fromthe corresponding 1H-NMR spectra of terpolymersamples. Figure 6 shows the 1H-NMR spectra of aVP-TBMA copolymer and the corresponding ofthe same copolymer after hydrolysis. The analysiswas performed by comparing the integrated in-tensities of the signals that appear at � � 1.4(corresponding to the protons in tert-butyl estergroup in the methacrylic units) with the peak at �� 12.2 (ascribed to the OH protons in the carbox-ylic acid). The degree of hydrolysis was deter-mined from the ratio of carbonyl acid proton ab-sorption to the remaining tert-butyl protons.

Confirmation of the chemical transformationcan be seen in FTIR spectra such as those shownin Figure 7 and the analysis summarized in Table2. The predominant changes are the growth of anO-H stretch, which is extremely broad due tostrong hydrogen bonding in carboxylic acids from3500 to 2500 cm�1, broadening of the carbonylstretch because of overlapping ester and carbox-ylic acid bands, and attenuation of the doubletband at 1390 and 1370 cm�1 associated with theloss of the stretching vibration modes of the tert-butyl moiety. The TBMA content, extent of hydro-lysis, and final carboxylic acid group content arequoted in Table 2. Hydrolysis efficiency was vir-

Figure 5 Average lengths of TBMA sequences as afunction of the molar fraction of TBMA in the copoly-mer chain.

Figure 6 1H-NMR spectra of the VP-TBMA copoly-mer with FTBMA � 0.899 (A), after acidic hydrolysis (B),and after modification (0.472M fraction of glucosamine)(C). The assignment of the labeled peaks corresponds tothe hydrogen marked on the structural schematic. Theanomeric protons of the � and � isomers (see text) areshown as market.

VP-TBMA–METHACRYLIC ACID TERPOLYMERS 23

tually quantitative for PTBMA and increasingwith increasing TBMA content in the VP-TBMAcopolymer. As expected, extent of hydrolysis in-creases linearly as average lengths of TBMA se-quences does.

Coupling of Bioactive Amino Compounds

It is known that carboxylic acids can react withchloroformates in the presence of a basic catalystto give carbonic carboxylic anhydride. The amin-olysis of the anhydride groups formed gives theexpected amides with the formation of carbonicanhydride.25–27 The synthesis of amide deriva-tives of VP-TBMA-MAA terpolymers (R-COOH)was accomplished as shown in Scheme 2.

In one approach fully hydrolyzed PTBMA or VP-TBMA-MAA terpolymers were allowed to reactwith ethyl chloroformate followed by aminolysisof the formed anhydride groups with benzocaine,tyramine, phenethylamine, or glucosamine. Thestructure of the resulting polymers (Scheme 2)was confirmed by FTIR and 1H-NMR spectros-copy. All FTIR spectra showed marked changes inthe range of the carboxyl frequency compared tothe spectrum of the parent terpolymer. The re-sulting modified polymers show bands at 1690–1650 cm�1 due to the carbonyl in amido groups aswell as the characteristic -NH- deformation vibra-tion at 1550–1520 cm�1 (Fig. 7C). The 1H-NMRspectra of VP-TBMA-MAA modified by pendentbenzocaine, phenethylamine, tyramine (whichare not presented here), or glucosamine moietiesshow the characteristic bands of these groups.Benzocaine: � � 7.6 (-C6H4-), � � 10.6 (-NH-CO-);tyramine: � � 7.0 (-C6H4-), � � 8.2 (-NH-CO-);phenetylamine: � � 7.3 (-C6H5-), � � 8.2 (-NH-CO-).

As an example, the 1H-NMR spectrum of aVP-TBMA-MAA terpolymer (0.472M fraction ofglucosamine) shows the signals corresponding tothe grafted glucosamine groups (Fig. 6C). Themultiplet centered at � � 2.4–3.2 is due to thesugar protons.28 The bands at � � 5.7 and � � 5.2can be assigned to the anomeric protons of the �and � isomers,28 respectively. The content of thebioactive amino compounds in modified VP-TBMA-MAA terpolymers was determined by 1H-NMR spectroscopy, as mentioned in the experi-mental part.

Table 3 summarizes the results for the reactionbetween a fully hydrolyzed PTBMA or a VP-TBMA-MAA terpolymer (0.862M fraction of car-boxylic acid) and some model bioactive aminocompounds such as benzocaine, phenethylamine,and tyramine. It is seen from Table 3 that themodification extent of both the fully hydrolyzed

Table 2. Acidic Hydrolysis of PTBMA andVP-TBMA Copolymers

TBMAMolar Fraction LTBMA

Extent ofHydrolysis

(%)

FinalMolar Fraction

CarboxylicAcid Groups

1.000 — 96.5 0.96.5

0.89.9 9.2 95.9 0.86.2

0.72.2 2.9 83.7 0.60.4

0.61.1 2.0 74.3 0.45.4

Figure 7 FTIR transmission spectra of the VP-TBMAcopolymer with FTBMA � 0.722 (A), after acidic hydro-lysis (B), and after modification (0.316M fraction ofglucosamine) (C).

24 MARTINEZ, FERNANDEZ-GARCIA, AND SANCHEZ-CHAVES

PTBMA and the VP-TBMA-MAA terpolymer inthe reaction with benzocaine was notably lowerthan that of the reaction with phenethylamine ortyramine. The difference probably arises from thedifferent basicity of the amines, being higher inthe case of the aliphatic amines.

It may be noticed that VP-TBMA-MAA terpoly-mers modified by pendent benzocaine, phenethyl-amine, or tyramine moieties, which are water-insoluble polymers, can find potential applicationas drug delivery systems. The release of the activecompound from the terpolymer matrix can beachieved by hydrolytic cleavage of the linkingbond. Further investigation evaluating the result-ing conjugates is now in progress.

Table 4 shows satisfactory couplings in the re-action between glucosamine and a fully hydro-lyzed PTMBA or some VP-TBMA-MAA terpoly-mers with different carboxylic acid groups con-tent. Total completion was not attained for each ofthe four reactions that were carried out. However,it is interesting to point out that solubility in

water of the parent VP-TBMA-MAA terpolymerswas notably improved. Thus, glucosamine-carry-ing VP-TBMA-MAA terpolymers with a glu-cosamine molar fraction higher than 0.30 are per-fectly soluble in water.

Interaction Between Concanavalin A andGlucosamine-Carrying VP-TBMA-MAA Terpolymers

It is known that lectins are highly specific carbo-hydrate-binding proteins that agglutinate cellsand/or precipitate glycoconjugates.29,30 Con-canavalin A (Con A), the most extensively inves-tigated member of the lectin family, has beenwidely used in protein-carbohydrate interactionstudies.31–34 Con A is a tetramer with four carbo-hydrate-binding sites and it recognizes and binds,specifically �-D-glucopyranosyl, �-D-manopyrano-syl, or �-D-fructofuranosyl residues. All these car-bohydrate residues have the same configurationof the hydroxyl groups at the C-3, C-4, and C-6positions of pyranosyl or furanosyl rings.

Table 3. Reaction of Both a Fully Hydrolysed PTBMA Homopolymer and a VP-TBMA-MAA Terpolymer,Activated by Ethyl Chloroformate, using Triethylamine as Catalyst and DMF as Solventa,with Bioactive Amino Compounds

Molar Fraction ofCarboxylic Acid

GroupsbAmino

Compoundc

Conversion ofCarboxylic Acid

Groups (%)

Final Molar Fraction Composition

VP TBMA Acid Amide

Hydrolysed PTBMA 0.96.5 Benzocaine 27.9 0.00 0.03.5 0.69.6 0.26.9

0.96.5 Phenethylamine 46.2 0.00 0.03.5 0.51.9 0.44.6

0.96.5 Tyramine 50.6 0.00 0.03.5 0.47.7 0.48.8

VP-TBMA-MAA 0.86.2 Benzocaine 28.1 0.10.1 0.03.7 0.61.9 0.24.3

0.86.2 Phenethylamine 45.3 0.10.1 0.03.7 0.47.1 0.39.1

0.86.2 Tyramine 49.7 0.10.1 0.03.7 0.43.4 0.42.8

a [Ethyl chloroformate] � [Triethylamine] � 0.11 mol � L�1.b [Acid groups] � 0.10 mol � L�1.c [Amino compound] � 0.11 mol � L�1.

Table 4. Reaction of a Fully Hydrolysed PTBMA Homopolymer and Some VP-TBMA-MAA Terpolymers,Activated by Ethyl Chloroformate, using Triethylamine as Catalyst and DMF as Solventa, with Glucosaminec

Molar Fraction ofCarboxylic Acid

Groupsb

Conversion ofCarboxylic Acid

Groups (%)

Final Molar Fraction Composition

VP TBMA Acid Glucosamide

Hydrolysed PTBMA 0.96.5 56.9 0.00 0.03.5 0.41.7 0.54.9

VP-TBMA-MA 0.86.2 54.8 0.10.1 0.03.7 0.39.0 0.47.2

VP-TBMA-MAA 0.60.4 52.3 0.27.8 0.11.8 0.28.8 0.31.6

VP-TBMA-MAA 0.45.4 53.1 0.38.9 0.15.7 0.21.3 0.24.1

a [Ethyl chloroformate] � 0.11 mol � L�1; [Triethylamine] � 0.22 mol � L�1.b [Acid groups] � 0.10 mol � L�1.c [Glucosamine] � 0.11 mol � L�1.

VP-TBMA–METHACRYLIC ACID TERPOLYMERS 25

The interaction of a water-soluble glu-cosamine-carrying VP-TBMA-MAA terpolymer(0.316M fraction of glucosamine) with Con A atroom temperature is reflected in Figure 8, wherethe dependence of the absorbance, when anamount of Con A is added to an aqueous buffer-solution (pH � 7.2) of the glucosamine-carryingterpolymer, is shown. The curve obtained shows aprecipitin type reaction with Con A. The turbidityof the solution gradually increased and reached a

maximum after about 25 min; then decreased anda precipitate was deposited. The precipitation canbe interpreted on the basis of crosslinkagesamong the tetrameric Con A molecules and mul-tivalent terpolymer molecule carrying glucoseresidues. In addition, when free glucose wasadded to the coagulated suspension mixture ofglucosamine-carrying terpolymer and Con A, theprecipitate was dissolved and a clear solution wasrestored. This result may be due to the exchangeof free glucose with glucosamine-carrying terpoly-mer and to the dissociation of the (glucosamine-carrying terpolymer) Con A complex. This impliesthat Con A associates more preferentially withfree glucose than with the pendent glucose inglucose-carrying terpolymer, which is reasonablein view of the greater steric freedom of free glu-cose. A similar phenomenon was observed whenmannose was added to the coagulated suspensionmixture of glucosamine-carrying terpolymer andCon A, which may be attributed to the fact thatmannose expels the glucose residues from thebinding sites, resulting in dissociation of the mul-tivalent interaction, according to the known factthat the ability of Con A to form a complex withmannose is greater than that with glucose.32

Table 5 presents the dependence of the maxi-mum absorbance value at 360 nm (�360) with theglucose-carrying terpolymer and Con A concen-trations. The results obtained with the glu-cosamine-carrying fully hydrolyzed PTBMA arealso included. From the values collected in Table5 it is clear, as could be expected, that the absor-bance values increase as both the amount of glu-cosamine-carrying polymer and the protein do.

All these results support the attribution thatthe observed turbidity changes are due to the

Figure 8 Turbidity as a function of time in the inter-action of a glucosamine-carrying terpolymer (0.316Mfraction of glucosamine [glucosamine-carrying terpoly-mer � 6 mg.mL�1]) with Con A (4 mg.mL�1) in anaqueous buffer solution (pH � 7.2) at room tempera-ture.

Table 5. Interaction of a Glucosamine-Carrying VP-TBMA-MAA Terpolymer(0.31.6 molar fraction of glucosamine) and a Glucosamine-Carrying Fully HydrolysedPTBMA (0.54.9 molar fraction of glucosamine) with Con Aa

[Glucosamine-CarryingPolymer] mg � mL�1

[Con A]mg � mL�1 �360

VP-TBMA-MAA 2 1 0.0163 1 0.0206 1 0.0276 2 0.0336 4 0.052

Hydrolysed PTBMA 6 1 1.3006 2 1.470

a Aqueous buffer solution (pH � 7.2) at room temperature.

26 MARTINEZ, FERNANDEZ-GARCIA, AND SANCHEZ-CHAVES

specific recognition of sugars by the tetramericprotein Concanavalin A.

CONCLUSION

The results presented here provide an easy way toincorporate bioactive amino compounds on copol-ymers based on TBMA with potential applicationas matrices for environmentally stimulated drugrelease. Perhaps the most interesting feature ofthis method is to synthesize glucosamine-contain-ing copolymers through chemical modification.The water solubility of these copolymers remark-ably improved with an increased number of glu-cosamine units. Glucosamine-carrying VP-TBMA-MAA terpolymers and Con A in phosphatebuffer (pH � 7.2) at room temperature wereproven to interact with each other.

The authors gratefully acknowledge financial supportfrom the Comision Interministerial de Ciencia y Tec-nologıa (CICYT Contract Grant No. MAT 2000-1008).

REFERENCES AND NOTES

1. Nakaya, T.; Nishio, K.; Memita, M.; Imoto, M. Mak-romol Chem Rapid Commun 1993, 14, 77.

2. Rozalski, A.; Brade, L.; Kunh, H. M.; Brade, J.;Kosma, P.; Appelmk, B. J; Kusumoto, S.; Paulsen,H. Carbohydr Res 1989, 193, 257.

3. Lee, Y. C.; Lee, R. T., Eds. Neoglycoconjugates:Preparation and Applications; Academic Press: SanDiego, CA, 1994.

4. Rathi, R. C.; Kopeckova, P.; Kopecek, J. MacromolChem Phys 1997, 198, 1165.

5. Wulf, G.; Schmid, J; Venhoff, T. Macromol ChemPhys 1996, 197, 259.

6. Okada, M. Prog Polym Sci 2001, 26, 67.7. Kenawy, E. R.; Sherrington, D.C.; Akelah, A. Eur

Polym Mater 1992, 28 841.8. Faragalla, M. M.; Hill, D. J. T.; Whittaker, A. K.

Polym Bull 2002, 47, 421.9. Bork, J. F.; Coleman, L. E. J Polym Sci 1960, 43,

413.10. Reddy, B. S. R.; Arshady, R.; George, M. H. Eur

Polym Mater 1985, 21, 511.11. Al-Issa, M. A.; Davis, T. P.; Huglin, M. B.; Yip,

D. C. F. Polymer 1985, 26, 1869.

12. Soundararajan, S.; Reddy, B. S. R. J Appl PolymSci 1991, 43, 251.

13. When, S.; Xiaonan, Y.; Stevenson, W. Polym Int1992, 27, 81.

14. Zaldıvar, D.; Peniche, C.; Bulay, A.; San Roman, J.Polymer 1992, 33, 4625.

15. Gallardo, A.; Lemus, A. R.; San Roman, J.; Cifu-entes, A.; Dıez-Masa, J. C. Macromolecules 1999,32, 610.

16. Sanchez-Chaves, M.; Martınez, G.; Madruga, E. L.;Fernandez-Monreal, C. J Polym Sci Part A: PolymChem 2002, 40, 1192.

17. Prochazka, K.; Kiserow, D.; Ramireddy, C.; Tuzar,Z.; Munk, P.; Webber, S. E. Macromolecules 1992,25, 454.

18. Smith, C. K.; Liu, G. Macromolecules 2060 1996,29.

19. Tudos, F.; Kelen, T.; Folds-Berezsnich, T.; Turcsa-nyi, B. J Macromol Sci Chem 1976, A10. 1513.

20. Mayo, F. R.; Lewis, F. M. J Am Chem Soc 1944, 66,1594.

21. Fukuda, T.; Goto, A.; Kwak, Y.; Yoshikawa, C.; Ma,Y. D. Macromol Symp 2002, 182, 53.

22. Coote, M.; Davis, T. P. Prog Polym Sci 1999, 24,1217.

23. Plate, N. A.; Noa, O. V. Polym Sci USSR 1979, 21,509.

24. Marechal, E. In Comprehensive Polymer Science;Eastmond, G. C.; Lewith, A.; Russo, S.; Sigwalt, P.,Eds.; Pergamon: Oxford, 1989; Vol. 6; p 1–47.

25. Boissonnas, R. A. Helv Chim Acta 1951, 34, 874.26. Hartmann, M.; Bauer, H. J.; Wermann, K. Polym

Bull 1985, 13, 195.27. Hartmann, M.; Wermann, K.; Clausen, F.;

Schwachula, G.; Feisel, L.; Kraus, D. Angew Mak-romol Chem 1988, 161, 185.

28. Kadokawa, J.; Kato, M.; Kobayashi, N.; Karasu,M.; Togaya, H.; Chiba, K. Macromol Rapid Com-mun 1994, 15, 971.

29. Kobayashi, K.; Sumitomo, H.; Ina, Y. Polym J 1985,17, 567.

30. Golstein, I. J.; Hayes, C. E. Adv Carbohydr ChemBiochem 1987, 35, 127.

31. Kobayashi, K.; Sumitomo, H.; Kobayashi, A.;Akaike, T. J Macromol Sci Chem 1988, 25, 655.

32. Nakamae, K.; Miyata, T.; Jikihara, A.; Hoffman,A. S. J Biomater Sci Polym Chem 1994, 6, 79.

33. Miyata, T.; Jikihara, A.; Nakamae, K.; Hoffman,A. S. Macromol Chem Phys 1996, 197, 1135.

34. Yoshizumi, N.; Kanayama, Y.; Maehara, M.; Ide,H. Langmuir 1999, 15, 482.

VP-TBMA–METHACRYLIC ACID TERPOLYMERS 27