Synthesis and Characterization of Functionalized Polystyrene Beads

Polystyrene microspheres SX3 (35-75 pm in diameter, Biorad) were modified in three steps (Fig. 6). First, 10 mol of monochlorosulfonic acid (HSO3CI) (excess) was added to polystyrene in dichloromethane (CH2Cl2). The mixture was stirred at room temperature for 1 h, then the suspension was filtered and washed with CH2C12. This nucleophile substitution led to poly(parachlorosulfonyl)styr-ene—a reactive intermediate—immediately used in the second step of the func-tionalization process. Poly(parachlorosulfonyl)styrene was condensed with dimethyl ester of amino acids in CH2Cl2 at room temperature in the presence of triethylamine (Et3N). Resin was extensively and successively washed with etha-nol (100%) and aqueous solutions of sodium hydroxide (from 10-2 M to 2 M, and back to 10-2 M) in order to gently hydrolyze ester groups. Five amino acids (AAs) were substituted: arginine (R), aspartic acid (D), phenylalanine (F), asparagine (N), and serine (S). Functionalized polymers were characterized by elemental analysis and their chemical compositions are reported in Table 2. Results showed that 77% ± 10% of styrene units were substituted by sodium sulfonate groups (PS-SO3Na) and by amino acid sulfamide groups (PS-SO2AA); the substitution rate is directly related to the amount of introduced dimethyl ester amino acid in the reaction mixture. In addition to its amino acid substitution percentage, each polymer was characterized by R values with R = [COO-/ (COO- + SO- )]. This parameter gives the molar ratio of carboxylate upon the total anionic groups present along the macromolecular chains of the polymers. The different values of R reported in Table 2 allowed comparison of the different functionalized polymers.

In aqueous medium, functionalized polystyrene beads displayed swelling properties varying with their chemical composition. In order to evaluate and normalize the surface area really accessible to biological molecules such as protein and virus, human serum albumin (HSA) adsorption experiments were achieved on all the synthesized polystyrene derivatives. It is well known that HSA adsorbs most of the polymer surfaces in a nonspecific way [30-32]. Previous studies showed that the amount of adsorbed HSA at saturation on calibrated nonporous silica beads equaled approximately 0.12 pg/cm2 of available surface

Step 1 ; Reaction of monoch loro sulfonic acid

Step 1 ; Reaction of monoch loro sulfonic acid

Step 2: Condensation of dimethyl ester amino acid

I EtjN

Step 3: Hydrolysis of methyl ester group

-f CHJ-CHJ-1

j- + NaOH n

-

-iCHj-CH^ + CHjOH

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9

R

R

[30]. Therefore, adsorption experiments of radiolabeled HSA ( I-HSA) onto polystyrene derivatives were performed; the analysis of the adsorption isotherms showed that the adsorption followed a one-site Langmuir law and allowed determination of maximal adsorption capacity of the polymers (Bmax) and the apparent affinity constant Ka. The available surface area for portein or virus was determined for each polymer as SA (cm2/mg) = Bmax/0.12 (Table 2). Results showed that SA varied with the chemical composition of the polymer. Indeed, except for phenylalanine derivatives, SA values of the polymers decreased vs. the amino acid substitution rate to reach a plateau value, at which all the polymers displayed the same swelling behavior. Phenylalanine is a hydrophobic amino acid

FACING PAGE

FIG. 6 Functionalization of polystyrene beads SX3 (Biorad) by various amino acids. R = -CH2-CH2-CH2-NH-C-NH2 for arginine

0"

for aspartic acid

for serine

for asparagine

for phenylalanine that can induce the development of several interactions with the hydrophobic domains of albumin, this may explain the different behavior of this polymer as compared to the other polystyrene derivatives. Therefore, the calculated mean value of Ka was about 2 x 105 M-1 and corresponded to a nonspecific adsorption value. Taking this result into account, surface of the different polymers was precoated with BSA before the adsorption of viruses onto polymers in order to screen nonspecific sites. Thus, any observed variation in viruses adsorption will be due to the chemical composition of the polymer and not to its physical properties.

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