Interaction of PhenoLic Cosolutes with Polyethylenimine

The binding of drugs by macmmolecules is of interest because of its physioiogical, phar- macological, or pharmaceutical significance. Phenol and its related derivatives are used as model compounds of drugs. In this connection, for example, the interaction of polyvinylpyr- d idone with phenolic coeolutes has been investigated.'l

In a previous article the binding of acid a m dyea carrying phenolic hydmxyl p u p s such as orange I, orange 11, chrome violet, Chydroxyazobenzene4'-sulfonate, and 2.rl-dihydroxy- azobenzenea'-sdfonate by polyethylenimine (PEI) was studied. The results show that the extent of binding of chrome violet and 2,4dihydroxyauobenzene4'-sulfonate, which involve two hydroxyl groups in their StNCtUre, is extraordimwily large compared with the other dyes. It was further shown that there is an effect of added metal ions on the complex formation between the dyes and PEI.4

In thia article the combination of phenola and OH-aubstituted benzoic acids with PEI was examined by equilibrium dialyeia and spectmscopic methods. The effects of the number and position of phenolic hydroxyl groups on the small molecules upon the extent of binding were investigated to gain an inaight into the nature of the forces governing afiinity between two binding entities.

The phenolic consolutes used in this experiment were phenol, resorcinoi, benmic acid, & hydroxybemic acid (salicylic acid), 3 -hybxybemic acid, Chydroxybenzoic acid, 2.3dihy- droxybemic acid, 2,edihydroxybenzoic acid, and 2,5dihydroxybenzoic acid, which were pur- s e d by vacuum distillation or repeated recrys-tion from water. PEI, a commercially available polyethylenimine of average molecular weight 40,00060,000, was furnished from Tokyo Kaeai Co. The extent of binding of each of the coeolutes studied was measured by an equilibrium dialysis technique in 0.1MTrbacetata bufYer, pH 7.0, at 25CS

The ability of PEI to bind phenol and reeorcinol is shown in Figure 1, in which the extent of binding is expressed in terms of a common unit of weight: 1P g of PEI. As is evident in Figure 1, resorcinol, which has two OH p u p s in ita structure, exhibits the greater extent of binding for PEI compared with phenol. Phenol haa the value of pK, of 9.99 (2~5'0.~ Also the values of pKI and pK* of resorcinol are 9.15 and 11.3 (W, respectively.6 Accordingly, at pH 7.0, in which the binding experimenta were carried out, both compounds are unionized in bulk aque6us solution. Thus, these cosolutea are likely to be bound to PEI in the undissociated state. The results of binding observed indicate that the binding is subject to dramatic OH group dependence. The second hydroxyl group present in resorcinol plays a dominant role in the enhancement of the binding for PEI. Both hydroxyl groups of reeorcinol are apparently capable of interacting simultaneousIy with the polymer. Figure 2 shows the extent of binding of benzoic acid and OH-substituted benzoic acids by

PEI. These benmic acids behake similarly to phenols. Evidently the binding aEnity of PEI for these small molecules increases in the following order: benzoic acid, monosubstituted benzoic acid, disubstituted benzoic acid. Pronouncad differences are evident among these de- rivatives. The position of OH group in monosubstituted benzaic acids exerts no influence on the extent of binding. However, PEI show increasing affinity toward disubstituted derivatives in the order, 2,3.dihydroxybenmic acid < 2,4dihydmxybenzoic acid < 2.5dihydroxybenzoic acid. At pH 7.0 the carboxyl group on the aromatic nucleus is diseociated and the hydroxyl groups are undiasociated as judged by the values of pK. of these sub~tances.~ Therefore, its carboxylate anion must interact with the partially cationic nitrogen atoms on PEI since benzoic acid exhibita greater binding affiity relative to phenol. It is shown that PEI is highly branched and ca. 25% of the nitrogens are primary amines, 50% secondary, and 25% tertiary.' Fur- thermore, the polymer has a net positive charge in the pH used. Thus the interaction with the anionic small molecule reflects electmtatic influences. Also the OH groups on the aromatic nucleus contribute greatly to the enhancement of combination of the substances to PEI. The increased affinity of phenolic cosolutes for PEI is mainly due to .the formation of strong

Journal of Polymer Science: Polymer Chemistry Edition, Voi. 23, 2073-2076 (19851 @ 1985 John Wiley & Sons. Inc. CCC 0360-3676/85/072073$04.00

2074 J. POLYM. SCI. POLYM. CHEM. ED., VOL. 23 (1985)

Fig. 1. pH 7.0, at

2 5

d

w a. 0, 20

5!

y 1 5

2 = 10 in.

Ln

-- : fi -I

0 f =I 0 m L R 5 W

$ 0 - 4.0 - 3.5 - 3.0 -2.5

LOG (FREE SMALL MOLECULE)

d

w a. 0, 20

5!

y 1 5

2 = 10 in.

Ln

-- : fi -I

0 f =I 0 m L R 5 W

$ 0 - 4.0 - 3.5 - 3.0 -2.5

LOG (FREE SMALL MOLECULE) Extent of the binding of phenole by polyethylenimine in 0.1M lkbacetate 25%: (0) phenol, (0) resmcinol.

-4.0 - 3.5 - 3.0

buf€er,

LOG (FREE SMALL MOLECULE) Fig. 2. Extent of the binding of benzoic acid and OH-eubetituted benzoic acids by ply-

e t h y l e ~ e in O.lMTris9cetate buffer, pH 7.0, at 25C (0) benzoic acid, (8) Zhydroxybenzoic acid, (0) 3-hydmxybenzoic acid, (0) 4hydmxybenzaic acid, (8) 2,3dihydmxybenzoic acid, (a) 2,ktihydmxybenzoic acid, (cf) 2,5dihydmxybenzoic acid.

2075

300 4 00 500 6 00 WAVELENGTH (nm)

Fq. 3. Abeorption spectra of &&iihydroxybenzoic acid bound to polyethylenimine at var- ious valuw of r (moiea of bound small molecule per llts g of pfymer) in 0.1114 Tris-acetate buffer, pH 7.0, at 25C (1) 2.5dihydmxybenzoic acid alone, (2) r = 5.8, (3) r = 9.9, (4) r = 14.5; ( - - - ) 2,5Dihydmxybenzoic acid alone at pH 9.7 (its concentration was an arbitrary value, the pH w- adjusted with NaOH), ( - - - ) 2,5dihydroxybenzoic acid alone at pH 10.5.

hydrogen bond# to the amine residues of the polymer. It has been suggested that exceptionally strong hydrogen bonding occurs between phenolic hydroxyl group and amide bonds of poly- amide.8 However, there exists a possibility that the phenolic species bound by highly cationic PEX may be the phenolate form although the OH p u p a involved are undissociated at pH 7 in bulk environment.

Furthermore, additional forces aight be involved in the 2.5dihydroxybenzoic acid-PEI bind- ing syutem 811 reflected in Fm 3. in which spectra of bound 2,5dihydroxybenzoic acid on the macromolecule are ilIuetrate& The spectra were obtained by measuring the absorbance of a solution of the ~ m o l e c u l e / s ~ ~ l u ~ complex taken from within the dialysis bag vemw that of solution outside the bag. It is apparent in Figure 3 that a new absorption band ap- at a higher wavelength only when the compound binds to PEI. The visible abeorption band ia located at ea. 480 nm. This spectral observation implies that the formation of this complex ia very probably due to charge transfer. Only 2 ,~ydroxybenzoic acid among the phenolic small molecules examined in the present study showed the change in color when it combined with PEL The pronounced binding of the cosolute to PEI must take place by this additional force as shown in Figure 2

Them may be enother possibility that although the hydroxyl group of 2,5dihydroxybenzoic acid, with pK, near 9 (pK, means the dimxiation constant of its carboxyl group), wouid be largely uncharged at pH 7 in bulk water, the moleculea bound by highly cationic PEI are likely to be the phenolab form. The spectra in Figure 3 show a new band near 480 nm which may be that of phenolate form of compound. Thus, some spectra of the small molecule in aqueow solution in the abeence of the polymer at pH 9-11 were taken. "he results are illustrated in Figun, 3 also. As can be seen in Figure 3, the spectra do not exhibit any peak near 480 nm in the alkaline soiutions. Consequently, the absorption band near 480 nm detected in the presence of PEI might not be attributed to this situation.

References 1. P. Molyneux and H. P. Frank, d: Am &a Soc. 83,3169 (19611. 2. P. Bandyopaahyay and F. Rodiiguez, Polyrmr; 13, 119 (1972). 3. T. Takagiahi, K. Yoehikawa, H. Hamano, N. Kuroki, and H. Kozuka, J. Poiym Sci PoZym

Chem Ed, 23.37 (1985).

2076 J. POLYM. SCI. POLYM. CHEM. ED., VOL. 23 (1985)

4. T. Takagishi, K. Yo~hikawa, S. Okuda, N. Kuroki, and H. Kozuka, J , PoZym. Sci Polym

5. T. Takagishi and N. Kuroki, J, Polym Sci Pofym Chem Ed, 11, 1889 (1973). 6. G. Kortiim, W. Vogel, and K. Andrussow, Dissociation Constants of Organic Acids in

7. 1. M. Klotz, G. P. Royer, and A. R. Sloniewsky, Biochem~try, 8, 4752 (1969). 8. H. Endres and H. Homann, Angew. Chem. int.

Chem. Ed, 23,255 (1985).

Aqueous Solution, Butterworths, London, 1961.

EngL, 2,254 (1963).

TORU TAKAGISHI I(rY0AKI YOSHIKAWA N03UWXO KUROKI

Department of Applied Chemistry College of Engineering University of Osaka Prefecture Sakai, Osaka, 591, Japan

Hiroshima Bunkyo Women's Callege Asakitaku, Hiroshima, 73142, Japan

Received Orrober 10, 1984 Accepted January 18,1985