Polymers with pendent functional groups

Polymers with pendent functional groups

European Polymer Journal 37 (2001) 1901±1906 www.elsevier.nl/locate/europolj Polymers with pendent functional groups VII. Polysaccharide derivatives...

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European Polymer Journal 37 (2001) 1901±1906


Polymers with pendent functional groups VII. Polysaccharide derivatives containing viologen groups Ecaterina Avram a, Cristina L~ ac~ atus b, Georgeta Mocanu a,* a

Petru Poni Institute of Macromolecular Chemistry, Aleea Gr. Ghica Voda Nr. 41A, 6600 Iasßi, Romania b Gr. T. Popa University of Medicine and Pharmacy, 6600 Iasßi, Romania Received 17 February 2000; received in revised form 1 December 2000; accepted 21 February 2001

Abstract The paper presents the synthesis of new polysaccharide derivatives containing viologen groups, by reaction of chloroacetylated crosslinked dextran microparticles with dipyridyl compounds as: 4,40 dipyridyl, N-n-octyldipyridinium chloride and N-benzyldipyridinium chloride. The in¯uence of the reaction conditions on the substitution degree with dipyridyl groups was discussed and a reaction mechanism was proposed. Some preliminary studies concerning antimicrobial activity of the polymeric dipyridyl compounds were also reported. Ó 2001 Elsevier Science Ltd. All rights reserved. Keywords: Polysaccharides; Dipyridyl; Viologen compounds; Dextran derivatives

1. Introduction The products containing dipyridyl derivatives are interesting materials with applications in industry and medicine. Due to the speci®c properties of the dipyridinium groups [1], by their including in various compositions one can obtain electroconductor materials [2], redox catalysts [3,4], photochromic and electrochromic materials with photosensibilisator properties in the process of water photolysis; they can also present herbicide [5] and bactericide action [6]. Macromolecular compounds with pendent viologen groups were also reported [7,8]. In previously reported papers are studied the reactions between chloroacetylated crosslinked dextran (CCD) microparticles with heterocyclic bioactive compounds such as metronidazole [9], nicotinic acid [10], theophylline [11], nalidixic acid [12] which lead to the obtaining


Corresponding author. Fax: +40-32-211-299. E-mail address: [email protected] (G. Mocanu).

of macromolecular conjugates with controlled release of the drug. This paper studies the obtaining of new dipyridyldextran conjugates through the reaction of CCD microparticles with 4,40 -dipyridyl, N-n-octyldipyridinium chloride and N-benzyldipyridinium chloride; these products were physico-chemical characterized with the aim to appreciate their performances in further speci®c utilizations. Preliminary studies concerning the antimicrobial activity of these polymeric dipyridyl compounds were also performed; there was established that for some bacteria macromolecular dipyridyl conjugates have improved antimicrobial activity than the small molecular compound. 2. Experimental 2.1. Materials · 4,40 -dipyridyl (DPy) anh. (Merk) recrystalized from diethylether. · N-n-octyldipyridinium chloride (ODPy) and N-benzyldipyridinium chloride (BDPy) synthesized in laboratory [13]; the monoquaternized derivatives of

0014-3057/01/$ - see front matter Ó 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 0 1 4 - 3 0 5 7 ( 0 1 ) 0 0 0 3 0 - 1



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E. Avram et al. / European Polymer Journal 37 (2001) 1901±1906

4,40 -dipyridyl were puri®ed by multiple recrystalizations from diethylether until the complete removal of traces of 4,40 -dipyridyl. Polymeric support: CCD microparticles 0.1 mm in diameter (dry) obtained in laboratory [14] by reaction in organic basic media of chloroacetyl chloride with crosslinked dextran microparticles; the degree of substitution with chloroacetyl groups was 2.2 per glucopyranosic unit (GU). Solvent: N,N 0 -dimethylformamide (DMF) p.a. dist. (Merk). Staphylococcus aureus, Staphylococcus saprophyticus, Escherichia coli ATCC, Sarcina lutea ATCC, Bacillus cerus, Klebsiella ATTC. agarized plates.

2.2. Methods 2.2.1. Synthesis The amination reaction was performed in a glass reactor provided with stirrer, condenser and thermostating bath. 0.3 g of crosslinked dextran microparticles were quantitatively introduced in the reactor; the solvent (DMF) was added (10 ml/g) and the microparticles were left to swell 12 h at room temperature. After that, 0.15 g 4,40 -dipyridyl was added and the reaction was performed under stirring and heating at a chosen temperature for the established period of time. Then, the obtained microparticles were ®ltered on a crucible ®lter, washed with a solvent (methanol) until in the washing e‚uents no reagent was identi®ed (checking by UV spectrophotometry) and ®nally dried at vacuum up to constant weight. All operations were performed carefully and the microparticles were weighted before and after reaction at an analytical balance. 2.2.2. Physico-chemical characterization · Ionic chlorine content (Cl , g%) with 0.02 N AgNO3 solution with a Titrator TTT1C Copenhagen. · IR spectroscopy (Specord M-80, Carl Zeiss Jena, Germany); 1 H NMR in DMSO-d6 spectroscopy (Jeol C 60 Hz); this spectrum was performed on a linear chloroacetylated dextran sample which was reacted with an excess amount of BDPy, in the same conditions. · Tests of chemical stability: 0.05 g samples were immersed in 50 ml testing solution, at room temperature; at established intervals of time aliquots were withdrawn and the amount of released heterocyclic compound was determined by UV spectrophotometry (Specord M42 Carl Zeiss Jena Spectrophotometer) at 240 nm wavelength. · Water uptake of the microparticles was determined after Pepper' s method [15].

The substitution degrees with dipyridyl groups were calculated by two ways: · From the Cl content, with the formula: DS ˆ

330:5  Cl % 35:5  100 MWPy  Cl %


where 330.5 is the molecular weight of glucopyranosic unit substituted with 2.2 chloroacetyl groups (neglecting the small contribution of crosslinking hydroxypropyl bridges); Cl %: the ionic chlorine content; MWPy : the molecular weight of the dipyridyl compound used. · From the yield: if all operations were e€ected quantitatively, carefully, after reactions occurs an increase of the weight of the crosslinked microparticles which corresponds with the amount of dipyridinium compound introduced on the macromolecular backbone; hence one can calculate the DS from the weight increase, with the formula: DS ˆ

DW  330:5 MWPy  Wi


where DW represents the weight increase after reaction and Wi represents the initial microparticle weight. 2.2.3. Testing of antimicrobial activity [16±21] Testing of bacterial susceptibility was performed using di€usion method, a Petri plates with Mueller± Hinton agar (0.2 ml suspension/100 ml agar) inoculated with 0.5 MacFarland standard bacterial suspension. We placed ®lter paper discs impregnated with polymer solutions of two concentrations 50 lg/ml and 25 lg/ml. We interpreted the results after 16±18 h incubation at 37°C. The minimum inhibitory concentrations (MICs) determination was performed using dilution method in Mueller±Hinton agar with an inoculum of approximately 104 CFU (formatting colonies units)/spot, following recommendations of the HCCLS (National Committee for Clinical Standards) for sensitivity testing. The results were read after overnight incubation at 37°C.

3. Results and discussion 3.1. Synthesis The amination reaction of the chloroacetyl groups with 4,40 -dipyridyl occurs according to the mechanism of the bimolecular nucleophilic substitution; taking into account that the dipyridyl compound is a bifunctional reagent, one can suppose that two kinds of products can be formed:

E. Avram et al. / European Polymer Journal 37 (2001) 1901±1906


Hence, this amination reaction can lead either to the monoquaternized (MQ) or to the biquaternized product (BQ), as function of the reaction conditions. This biquaternized product causes a supplementary crosslinking of the dextran network previously crosslinked with hydroxypropyl bridges. If the reaction occurs with the formation of monosubstituted derivative, the DS calculated from the Cl content (formula 1) must correspond with that calculated from the weight rise (formula 2); for the bisubstituted dipyridyl compound, at a content of 2 Cl corresponds a rise with one pyridyl unit, hence the ratio between the two DS (calculated with the formulas 1 and 2) will be 2/1. In conclusion, by comparing the two DS above mentioned, one can appreciate the ratios between the monosubstituted and bisubstituted products. In the case of N-n-octyldipyridinium chloride and Nbenzyldipyridinium chloride, which have only one N atom capable to quaternize, the reactions occur with the formation of the compounds:

half corresponds to the initial Cl content of the reactant and one-half to the new formed quaternary group. The data presented in Table 1 sustain the above mentioned suppositions. By comparing the data presented in Table 1 one can see that with the bifunctional dipyridyl reagent, at a molar ratio reagent/GU of 1/1 was obtained almost only biquaternized derivative of DPy compound, while at 2/1 molar ratio was obtained preponderantly monoquaternized DPy derivative. With N-n-octyldipyridinium chloride and N-benzyldipyridinium chloride, which contain only one reactive site, the two DS are equivalents, as expected. As a result of these above presented data, one can conclude that the DS calculated on the DW =Wi basis represents better the content of dipyridyl units introduced on the macromolecular matrix; the DS calculated from the Cl content represents the amount of the chloroacetyl groups involved in quaternization reaction and may be 1 or 2 dipyridyl units.

In these cases, the two DS calculated with the formulas 1 and 2 must be the same, mentioning that from the Cl content determined in the reaction products, one

In Figs. 1 and 2 are presented the in¯uence of the temperature and duration reaction of dipyridyl with CCD microparticles on the DSMQ and DSBQ . As can be


E. Avram et al. / European Polymer Journal 37 (2001) 1901±1906

Table 1 In¯uence of the reaction conditions on the DS in amination reaction (solvent: DMF 10 ml/g support; duration: 24 h; temperature: 60°C) Sample


Molar ratio (reagent/GU)

DW =Wi

Cl content (g%)

DSMQ (with formula 2)

DSBQ (with formula 1)

Ratio (DSBQ =DSMQ ):2/1



1/1 2/1 1/1 1/1

0.5 0.47 0.33 0.73

11.40 8.99 6.43/2 9.96/2

1.0 1.04 0.388 0.796

2.02 1.39 0.40 0.81

2/2 1.33/1 1/1 1/1

seen, with the temperature and duration rise, both DSMQ and DSBQ increase, but their ratio is maintained about 2/1, which suggests that at 1/1 molar ratio (reagent/GU) the reaction occurs preponderantly with the formation of the biquaternized product. At elevated temperatures, the maximal DS is obtained in a shorter time than at lower temperatures. For 2/1 dipyridyl/GU molar ratio the curves representing the DSMQ and DSBQ are closer, which suggests the preponderance of obtaining of the monosubstituted dipyridyl derivative (Fig. 3). Fig. 4 represents the variation of DS in the reaction of CCD microparticles with N-n-octyldipyridinium chloride and N-benzyldipyridinium chloride. As can be seen, the N-octyl derivative of dipyridyl is more reactive

Fig. 3. Variation of DSMQ and DSBQ in the amination reaction with 2/1 molar ratio DPy/GU; temperature: 60°C.

Fig. 4. Variation of DS in the amination reaction with BDPy and ODPy; temperature: 60°C; BDPy (1) and ODPy (1): DS calculated with formula (1); BDPy (2) and ODPy (2): DS calculated with formula (2).

Fig. 1. Variation of DSMQ as temperature and duration function of the amination reaction with 1/1 molar ratio DPy/GU.

than N-benzyl derivative, due to the fact that in the last compound the nucleophilicity of the pyridinium nitrogen is decreased by the involving of the p-electrons of heterocycles in conjugation with those of the benzyl substituent.

3.2. Characterization of dipyridinium products

Fig. 2. Variation of DSBQ as temperature and duration function in the amination reaction with 1/1 molar ratio DPy/GU.

The IR spectrum of dipyridyl-dextran conjugate (Fig. 5) presents the characteristic band for pyridinium group at 1640 cm 1 . The 1 H NMR spectrum in DMSO-d6 of a linear Nbenzyldipyridinium-dextran conjugate shows signals for polysaccharidic protons between 3±4 ppm and 5.4 ppm; for CH2 (acetyl) at 4.3 ppm; for CH2 (benzyl) at 6.0

E. Avram et al. / European Polymer Journal 37 (2001) 1901±1906

ppm; for phenyl protons at 7.6 ppm, for dipyridyl protons between 8.6 and 9.3 ppm. Chemical stability of conjugates was studied on two kinds of products: sample DCAB 2 and DCAB 30 (Table 1), which represent preponderantly bisubstituted dipyridinium-dextran conjugate (BQ), and respectively, monosubstituted dipyridinium-dextran conjugate (MQ). The elution curves in acidic medium (pH ˆ 1:2) and in solution tampon with pH 7.4 were presented in Fig. 6. In acidic solutions (pH ˆ 1:2) both conjugates release the dipyridyl slower than in solutions with weak basic pH (7.4); the preponderantly monosubstituted dipyridyl derivative releases the small molecular compound easier than the bisubstituted one. Water uptake of dipyridyl-dextran conjugates varies with the degree of substitution with heterocyclic compound. Initially, the CCD microparticles with 2.2 DS with chloroacetyl groups are hydrophobic; water uptake


Fig. 7. Variation of water uptake as function of DS (DPy: dipyridyl-dextran conjugate; BDPy: N-benzyldipyridyl-dextran conjugate; ODPy: N-n-octyldipyridyl-dextran conjugate).

of the substituted with dipyridyl microparticles increases with rise of DS, as one can see in Fig. 7. The more hydrophilic dextran derivative is the dipyridyl-benzyl substituted; the more hydrophobic is, as expected, the dipyridyl-octyl substituted. These hydrophilic±hydrophobic properties can in¯uence the behaviour of these conjugates in various applications. 3.3. Testing of antimicrobial activity

Fig. 5. The IR spectra of the dipyridyl-dextran conjugate (Ð) and of the chloroacetylated crosslinked dextran microparticles (- - -).

Fig. 6. Elution pro®le of DPy from conjugates preponderantly monosubstituted (DCAB 30) and bisubstituted (DCAB 2) derivatives.

Preliminary studies of sensitivity of bacteria towards dipyridyl itself and its polymeric conjugates were effected. In Table 2 is presented the diameter of the inhibition area of the bacteria using the new obtained products. As can be seen from the presented data, the area of inhibition have approximately the same values for DPy itself and its polymeric conjugates, but taking into account that they contain about 40 g% DPy, this means that in fact the polymeric products have a double area of inhibition. One can suppose in this case a synergistic e€ect when used DPy linked to the polymeric support. Only in the case of E. coli the e€ect of the tested products is the same; in the case of S. lutea ATCC, the inhibition area of the polymeric conjugates is much greater than for the small molecular product. MICs for DPy and its macromolecular conjugates were determined against the same bacteria. The results obtained as MICs (lg/ml) are reported in Table 3. MICs for the tested products varies with the bacteria used: polymeric conjugate of DPy (DCAB 30) have a smaller inhibition concentration than DPy itself, in the case of B. cerus, K. ATCC and E.coli ATCC (taking into account that the polymer contain only about 40% active substance); the polymeric conjugate DCAB 22, have smaller MICs for B. cereus and K. ATCC and approximately the same MICs for E. coli, as the small molecular product. Hence, one can arm that polymeric conjugates of dipyridyl compounds have improved antimicrobial activity than the small molecular product.


E. Avram et al. / European Polymer Journal 37 (2001) 1901±1906

Table 2 Diameter of inhibition area using DPy and its polymeric conjugates DCAB-22 and DCAB-30 (Table 1) Strains

S. aureus ATCC S. saprophyticus E. coli ATCC Sarcina lutea B. cerus K. ATCC a b

Diameter of inhibition area (mm) DCAB-22 (lg/ml)a

DCAB-30 (lg/ml)b

DPy (lg/ml)







10 11 12 30 17 15

8 7 11 25 11 10

10 10 11 25 15 11

7 8 10 15 14 10

15 14 24 40 17 16

10 11 21 34 15 15

Conjugate of N-benzyldipiridinium chloride. Conjugate of 4,40 -dipyridyl.

Table 3 MICs of DPy and its conjugates Strains S. aureus ATCC S. saprophyticus E. coli ATCC S. lutea ATCC B. cereus K. ATCC

MICs (lg/ml) DCAB-22



2 2 512 2 0.06 512

2 2 256 0.125 0.06 256

0.06 0.06 256 0.06 0.06 512

4. Conclusions New polymeric derivatives of dipyridyl were synthesized and characterized. Preliminary studies of the antimicrobial activity show, in some cases, larger inhibition area and smaller minimal inhibition concentration that the small molecular product. Improved properties obtained can be explained by a synergistic e€ect between the polymeric support and the biological active substance, linked covalently. References [1] Factor A, Heinson GE. Polym Lett 1971;9:289. [2] Kameyama Y, Nambu T, Endo T. J Polym Sci Polym Chem Ed 1992;30:1199. [3] Endo T, Takoda T, Kameyama A, Okawara M. J Polym Sci Polym Chem Ed 1991;29:135. [4] Sato T, Nambu Y, Endo T. J Polym Sci C: Polym Lett 1989;27:289.

[5] Kitazawa K, Kobayashi T, Shibamote T, Hirai K. Am Rev Respir Dis 1988;137:173. [6] Levery G, Rieger AL, Edwaed JO. J Org Chem 1991;46:1255. [7] Jing-Ji J, Haramoto Y, Nanasawa M. Makromol Rapid Commun 1999;20:135. [8] Drutßa I, Avram E, Cozan V. Eur Polym J 2000;36:221. [9] Mocanu G, Airinei A, Carpov A. J Bioactive Compatible Polym 1993;8:383. [10] Mocanu G, Airinei A, Carpov A. STP Pharma Sci 1994;4:287. [11] Mocanu G, Airinei A, Carpov A. J Controlled Release 1996;40:1. [12] Mihai D, Mocanu G, Carpov A. J Bioactive Compatible Polym 2000;15(3):245. [13] Kamogawa H, Mizuno H, Todo Y, Nanasawa M. J Polym Sci Chem Ed 1979;17:3149. [14] Mocanu G, Carpov A. Cellulose Chem Technol 1992; 26:675. [15] Pepper K, Reichenberg D, Hale DK. J Chem Soc 1952:3129. [16] Balsß M. Laboratorul Clinic ^õn Infectßii, Ed. Medical~a, Bucuresßti; 1982. [17] B^õlb^õe V, Pozsgi N. Bacteriologie Medical~a, Ed. Medical~a, Bucuresßti, vol. II; 1985. [18] Buiuc D. Microbiologie Medical~a, Univeristatea de Medicin~a ßs i Farmacie, Iasßi; 1992. [19] Buiuc D, Negutß M. Tratat de Microbiologie Clinic~a, Ed. Medical~a, Bucuresßti; 1999. [20] Iancu LS. Medical microbiology, vol. 144. Univeristatea de Medicin~a ßs i Farmacie, Iasßi; 1996. p. 154. [21] Thrupp LD. Susceptibility testing of antibiotics in liquid media. In: Larian V, editor. Antibiotics in laboratory medicine. Baltimore, MD: Williams and Wilkins; 1986.