Effect of Phenyl Derivatization on the Stabilities of Antimicrobial N

Oct 7, 2010 - Nemanja Trišović , Tamara Timić , Jovana Divljaković , Jelena Rogan , Dejan Poleti , Miroslav M. Savić , Gordana Ušćumlić. Monatshefte f...
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Ind. Eng. Chem. Res. 2010, 49, 11188–11194

Effect of Phenyl Derivatization on the Stabilities of Antimicrobial N-Chlorohydantoin Derivatives Hasan B. Kocer,† S. D. Worley,*,‡ R. M. Broughton,† Orlando Acevedo,‡ and T. S. Huang§ Departments of Polymer and Fiber Engineering, Chemistry and Biochemistry, and Nutrition and Food Science, Auburn UniVersity, Auburn, Alabama 36849, United States of America

Phenyl substitution at position 5 on the hydantoin ring of a series of N-halamine derivatives, including an antimicrobial siloxane used for coating surfaces, has been employed to better understand the biocidal activities and stabilities of these compounds in the presence of water and exposure to ultraviolet irradiation. This phenyl derivatization of the hydantoin ring at its 5 position does, in fact, affect the antimicrobial efficacy of the siloxane against Staphylococcus aureus and Escherichia coli O157:H7, and its stability toward hydrolyses and ultraviolet light exposure. The phenyl substitution caused weakening of the N-Cl bond at the 1 position on the hydantoin ring, leading to an increase in biocidal activity but a decrease in stability. Model compounds were studied experimentally and theoretically at the (U)B3LYP/6-311++G(2d,p) level of theory in order to better explain the observations. It was found that the phenyl moiety at position 5 on the hydantoin ring can interact with the oxidative chlorine atom bonded to the amide nitrogen atom at position 1 which then results in a less stable N-Cl bond. Introduction Work in these laboratories for 3 decades has focused on a class of antimicrobial compounds termed N-halamines.1-3 These compounds can be used as disinfectants in aqueous solution,1,4 or they can be covalently bound to substrates via various tethering groups such as silanes.5,6 In general, N-halamine precursors are bound to the surfaces and then converted into N-halamine materials through a halogenation process, as shown in Figure 1.5,6 The N-halamine materials developed in these laboratories and elsewhere7 possess a number of advantages relative to other antimicrobial materials such as quaternary ammonium derivatives. These include relatively low cost, antimicrobial efficacies in brief contact times, substantial stability to loss of halogen through hydrolyses, and, possibly most important, the ability to be recharged upon loss of oxidative halogen by simple reexposure to dilute sources of household bleach or aqueous bromine. The mechanism of action for these N-halamine compounds is transfer of oxidative halogen to a microbe through direct contact, the rate of which depends upon the strength of the N-X bond, which in turn is related to the structure of the N-halamine molecule.8 Recent studies addressed this issue of N-X bond strength as affected by alkyl derivatization of the hydantoin moiety.9,10 The effect of extending the length of the alkyl groups at the 5 position of the hydantoin ring was minimal, so it was concluded that the dimethyl derivative, 3-triethoxysilylpropyl5,5-dimethylhydantoin, should be the silane coating material of choice because of its relatively low cost. However, the primary limitation of any N-halamine material is its tendency to lose its halogen upon exposure to ultraviolet light accompanied by partial decomposition of the compound. We undertook the present study in order to ascertain whether phenyl derivatization at the 5 position of the hydantoin ring would alter the stability of the stucture. * To whom correspondence should be addressed. Tel.: 1-334-8446980. Fax: 1-334-844-6959. E-mail: [email protected]. † Department of Polymer and Fiber Engineering. ‡ Department of Chemistry and Biochemistry. § Department of Nutrition and Food Science.

In this work several related model compounds have been prepared and characterized spectroscopically. The siloxanes of the dimethyl, methylphenyl, and diphenyl derivatives coated onto cellulose have been compared for antibacterial activity, stability to hydrolyses, and stability toward UV irradiation. This was done in order to address the issue of N-X bond lability. Also, DFT computations at the (U)B3LYP/6-311++G(2d,p) theory level have been performed to assist in the interpretation of the results. Experimental Section Materials. 5,5-Dimethylhydantoin and 5,5-diphenylhydantoin were purchased from Sigma Aldrich Co. (Milwaukee, WI, USA) and used without further purification. 5-Methyl-5-phenylhydantoin was prepared by the Bucherer-Berg reaction, the general procedure for which has been described.9 The triethoxysilylpropyl hydantoin derivatives were prepared by reacting the sodium salts of the three hydantoin derivatives with (3chloropropyl)triethoxysilane in anhydrous dimethylformamide (DMF) according to a general procedure which has been described.9 The structures of the three silanes were confirmed by 1H and 13C NMR; yields ranged from 92 to 97% by weight.

Figure 1. Preparation of antimicrobial coatings (X ) Cl, Br; R1, R2 ) Me or Ph).

10.1021/ie101258s  2010 American Chemical Society Published on Web 10/07/2010

Ind. Eng. Chem. Res., Vol. 49, No. 22, 2010

Model compounds containing a butyl group at the 3 position on the hydantoin ring were prepared by the following procedure. The potassium salts of the hydantoin derivatives prepared as described previously9 were dissolved in anhydrous DMF at 95 °C, and then an equimolar amount of 1-bromobutane (J. T. Baker Chemical Co., Phillipsburg, NJ, USA) was added to the solution. The resulting mixture was stirred for 3 h.10 The KBr produced in the reaction was removed by filtration, and DMF was removed at reduced pressure. The residual mixture was dissolved in chloroform, and then the solid impurities (salt) were removed by filtration; the chloroform was removed at reduced pressure. The structures of the 3-butyl-5-substituted hydantoin derivatives were confirmed by 1H and 13C NMR; yields ranged from 90 to 95% by weight. Spectral Data for the Synthesized Compounds. (i) 3-(Triethoxysilylpropyl)-5,5-dimethylhydantoin (MM). MM was obtained as a colorless viscous liquid. 1H NMR (CDCl3, 250 MHz): δ 0.58 (t, J ) 8.50, 2H), 1.19 (t, J ) 7.00, 9H), 1.40 (s, 6H), 1.70 (pent, J ) 7.94, 2H), 3.45 (t, J ) 7.38, 2H), 3.78 (q, J ) 7.00, 6H), 6.95 (s, 1H). 13C NMR (CDCl3, 63 MHz): δ 177.53, 156.98, 61.80, 58.43, 40.97, 24.91, 21.57, 18.18, 7.57. (ii) 3-(Triethoxysilylpropyl)-5-methyl-5-phenylhydantoin (MP). MP was obtained as a colorless viscous liquid. 1H NMR (CDCl3, 250 MHz): δ 0.54 (t, J ) 8.25, 2H), 1.15 (t, J ) 7.00, 9H), 1.67 (pent, J ) 8.25, 2H), 1.77 (s, 3H), 3.44 (t, J ) 7.25, 2H), 3.73 (q, J ) 7.00, 6H), 7.31 (m, 3H), 7.51 (d, J ) 6.75, 2H), 7.84 (s, 1H). 13C NMR (CDCl3, 63 MHz): δ 175.46, 157.40, 139.01, 128.73, 128.19, 125.22, 63.51, 58.33, 41.13, 25.68, 21.52, 18.21, 7.42. (iii) 3-(Triethoxysilylpropyl)-5,5-diphenylhydantoin (PP). PP was obtained as a slightly yellow liquid. 1H NMR (CDCl3, 250 MHz): δ 0.60 (t, J ) 8.50, 2H), 1.20 (t, J ) 7.00, 9H), 1.77 (pent, J ) 7.75, 2H), 3.57 (t, J ) 7.25, 2H), 3.78 (q, J ) 7.00, 6H), 7.38 (m, 10H), 7.94 (s, 1H). 13C NMR (CDCl3, 63 MHz): δ 173.43, 157.16, 139.36, 128.74, 128.40, 126.83, 69.98, 58.33, 41.46, 21.61, 18.33, 7.52. (iv) 3-Butyl-5,5-dimethylhydantoin (MMm). MMm was obtained as a colorless viscous liquid. 1H NMR (CDCl3, 250 MHz): δ 0.94 (t, J ) 7.25, 3H), 1.33 (sext, J ) 8.00, 2H), 1.44 (s, 6H), 1.60 (pent, J ) 7.25, 2H), 3.49 (t, J ) 7.25, 2H), 6.70 (s, 1H). 13C NMR (CDCl3, 63 MHz): δ 13.60, 19.82, 24.87, 30.09, 38.22, 58.56, 157.09, 177.73. (v) 3-Butyl-5-methyl-5-phenylhydantoin (MPm). MPm was obtained as a white powder: mp, 89.8 °C. 1H NMR (CDCl3, 250 MHz): δ 0.90 (t, J ) 7.25, 3H), 1.29 (sext, J ) 7.50, 2H), 1.58 (pent, J ) 7.25, 2H), 1.82 (s, 3H), 3.49 (t, J ) 7.25, 2H), 6.82 (s, 1H), 7.29-7.44 (m, 3H), 7.51 (d, J ) 6.50, 2H). 13C NMR (CDCl3, 63 MHz): δ 13.62, 19.86, 25.64, 30.07, 38.61, 63.51, 125.23, 128.41, 128.88, 138.79, 157.16, 175.39. (vi) 3-Butyl-5,5-diphenylhydantoin (PPm). PPm was obtained as a white powder: mp,129.1 °C. 1H NMR (CDCl3, 250 MHz): δ 0.90 (t, J ) 7.25, 3H), 1.31 (sext, J ) 8.00, 2H), 1.61 (pent, J ) 7.00, 2H), 3.55 (t, J ) 7.25, 2H), 7.24 (s, 1H), 7.29-7.53 (s, 10H). 13C NMR (CDCl3, 63 MHz): δ 13.62, 19.89, 30.08, 38.83, 70.01, 126.83, 128.50, 128.80, 139.29, 156.99, 173.38. General Procedure for the Synthesis of 3-Butyl-1-chloro5-substituted Hydantoin Derivatives. Each of the model compounds discussed above and trichloroisocyanuric acid (molar ratio, 1:3) were dissolved in acetone and stirred for 1 h at room temperature.10 The acetone was removed, and hexane was added to the mixture. The insoluble solids were removed by filtration, and then the hexane was removed by evaporation. The chlo-

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rinated derivatives were obtained as powders. The active chlorine content of the compounds was determined by a modified iodometric/thiosulfate titration. A derivative (about 0.05 g) was suspended in a solution of 90 mL of ethanol and 10 mL of 0.1 N acetic acid. After addition of 0.2 g of KI, the mixture was titrated with 0.0375 N sodium thiosulfate until the yellow color disappeared at the end point. The weight percent Cl+ on the samples was calculated by the following equation: %Cl+ ) (35.45NV)/(2W) × 100

(1)

where %Cl+ is the weight percent of oxidative chlorine on the samples, N and V are the normality (equiv/L) and volume (L) of the titrant sodium thiosulfate, respectively, and W is the weight of the sample (g). The purity of the chlorinated compounds was determined to be 97-99%. (i) 3-Butyl-1-chloro-5,5-dimethylhydantoin (MMm-Cl). MMm-Cl was obtained as white crystals: mp, 44.1 °C (lit.,10 45.4 °C). %Cl+ ) 16.0; theoretical, 16.2. 1H NMR (CDCl3, 250 MHz): δ 0.93 (t, J ) 7.50, 3H), 1.32 (sext, J ) 7.75, 2H), 1.46 (s, 6H), 1.61 (pent, J ) 7.50, 2H), 3.56 (t, J ) 7.25, 2H). 13 C NMR (CDCl3, 63 MHz): δ 13.57, 19.79, 22.20, 29.96, 39.47, 65.71, 154.71, 174.35. (ii) 3-Butyl-1-chloro-5-methyl-5-phenylhydantoin (MPmCl). MPm-Cl was obtained as a white powder: mp, 80.0 °C. %Cl+ ) 12.5; theoretical, 12.6. 1H NMR (CDCl3, 250 MHz): δ 0.92 (t, J ) 7.25, 3H), 1.31 (sext, J ) 7.75, 2H), 1.62 (pent, J ) 7.25, 2H), 1.90 (s, 3H), 3.59 (t, J ) 7.25, 2H), 7.32-7.55 (m, 5H). 13C NMR (CDCl3, 63 MHz): δ 13.57, 19.79, 21.48, 29.94, 39.82, 70.36, 125.96, 129.07, 129.09, 135.32, 155.07, 172.54. (iii) 3-Butyl-1-chloro-5,5-diphenylhydantoin (PPm-Cl). PPmCl was obtained as a white powder: mp, 83.1 °C. %Cl+ ) 10.0; theoretical, 10.3. 1H NMR (CDCl3, 250 MHz): δ 0.94 (t, J ) 7.50, 3H), 1.35 (sext, J ) 7.50, 2H), 1.69 (pent, J ) 7.00,2H), 3.68 (t, J ) 7.25, 2H), 7.26-7.37 (m, 4H), 7.37-7.56 (m, 6H). 13 C NMR (CDCl3, 63 MHz): δ 13.58, 19.89, 30.01, 40.21, 76.83, 128.34, 128.69, 129.33, 135.67, 154.64, 171.23. Characterization. The NMR spectra were obtained using a Bruker 250 MHz spectrometer; 1H and 13C spectra were recorded with 16 and 1024 scans, respectively. IR data were obtained with a Nicolet 6700 FT-IR spectrometer with ATR (attenuated total reflactance) accessory, recorded with 32 scans at 2 cm-1 resolution. Thermal data were obtained using a differential scanning calorimeter (DSC) Q2000 TA Instruments at a heating and cooling rate of 10 °C/min under N2 atmosphere. Coating Procedure. In this work the objective was to prepare coatings with as nearly as possible equal chlorine loadings to remove chlorine loading as a variable. Precursor silane monomers were first dissolved in an ethanol/water mixture (1:1 by weight) at concentrations ranging from 3 to 15 wt % so as to subsequently obtain chlorine loadings between 0.25 and 0.30%. The mixture was stirred for 15 min to produce a uniform solution. Cotton swatches (Style 400 bleached 100% cotton print cloth from Testfabrics, Inc., West Pittston, PA, USA) were soaked in the solution for 15 min and then cured at 95 °C for 1 h. Then the swatches were soaked in a 0.5% detergent solution for 15 min, rinsed several times with water, and dried at room temperature. The coating solution containing the dissolved silane derivatives weighed 50 g, and the cotton swatches were cut into areas of 320 cm2 each. Chlorination Procedure. The coated fabrics were chlorinated by soaking in 10% household bleach (0.6% sodium hypochlorite) at pH 7 (adjusted with 6 N HCl) for 1 h. After rinsing

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with tap and distilled water, the swatches were then dried at 45 °C for 1 h to remove any occluded free chlorine from the material. FTIR Confirmation of Siloxane Derivative Bonding. FTIR spectra of the coated fabrics confirmed that the N-halamine precursors bonded to the cotton fabric, as a band at ca. 1770 cm-1 was detected, which can be assigned to the carbonyl band of the amide structure in the hydantoin moieties. After the treatment with bleach, this band shifted to ca. 1780 cm-1, indicating disruption of N-H · · · OdC hydrogen bonding as conversion of N-H to N-Cl occurred. Analytical Titration. The chlorine concentration loaded onto the samples was determined by the iodometric/thiosulfate titration method discussed above. The weight percent Cl+ was determined from eq 1. Biocidal Efficacy Testing. Gram-positive Staphylococcus aureus (S. aureus) and gram-negative Escherichia coli (E. coli) were selected as the test organisms for determining the effectiveness of disinfection by the chlorinated hydantoinyl siloxane derivatives. The chlorinated coated fabric swatches were first held at 45 °C for 1 h to remove all occluded chlorine from the cotton fibers. Coated fabric swatches were challenged with S. aureus (ATCC 6538) and E. coli O157:H7 (ATCC 43895) bacterial suspensions in pH 7 phosphate buffer solution (100 mM). Suspensions (25 µL) of the bacterial solution were added to the center of a 2.54 cm square fabric swatch, and a second identical swatch was placed on top of the first swatch. A sterile weight was used to ensure sufficient contact of the swatches with the inocula. The contact times for the bacteria with the swatches were 1, 5, 10, and 30 min. At those contact times the fabric swatches were quenched with 0.02 N sodium thiosulfate solution to remove any oxidative chlorine which could cause extended disinfection. Serial dilutions of the solutions contacting the surfaces were made, and these were plated on Trypticase agar and incubated for 24 h at 37 °C, and colony counts were made to determine the presence or absence of viable bacteria. Unchlorinated control samples were treated in the same manner. It should be noted that our method of evaluating antimicrobial coatings, termed “sandwich testing”, is very similar to the Japanese industrial standard method, JIS Z 2801santimicrobial surface test. It differs only in the bacteria sample size, the fact that only one swatch of cloth is used in the latter, and that the moisture content is controlled for long contact times to prevent dryness which would kill the bacteria. Our N-halamine contact times are generally less than 10 min for a complete inactivation so that the moisture content is not a factor. Washing Testing. The stability and rechargeability of chlorine on the samples were evaluated by using a standard washing test according to AATCC test method 61. The cotton samples were washed for the equivalents of 5, 10, 25, and 50 machine washes in a Launder-Ometer. The %Cl+ loadings on the samples after the washings, with or without prechlorination and after rechlorination, were determined by the titration procedure outlined above. UVA Light Stability Testing. UVA light stability of the bound chlorine and the derivatized hydantoinyl siloxane coatings on the cotton fabric were measured by using an Accelerated Weathering Tester (The Q-panel Co., Cleveland, OH, USA). The samples were placed in the UV (Type A, 315-400 nm) chamber for contact times in the range of 1-6 h. After a specific time of exposure to UVA irradiation, the samples were removed from the UV chamber and titrated, or rechlorinated and titrated.

The temperature was 37.6 °C, and the relative humidity was 17% during the UVA light irradiation. Theoretical Computations. Density functional theory (DFT) calculations at the restricted and unrestricted (U)B3LYP/6311++G(2d,p) theory level11,12 were used to characterize the transition structures and ground states for the unchlorinated and chlorinated hydatoinyl siloxanes and butyl-substituted model compounds in vacuum using Gaussian 09.13 The DFT calculations were used for geometry optimizations and computations of vibrational frequencies, which confirmed all stationary points as either minima or transition structures, and provided thermodynamic corrections. This level of theory has been shown to accurately reproduce hydantoin energies and geometries in previous computational studies.14 All calculations were performed on computers located at the Alabama Supercomputer Center. Results and Discussion Coating Procedure. The biocidal characteristics of a coated fabric primarily depend on the structure of the coating compound and the concentration of the biocidal sites.2 In this regard, an attempt was made to maintain the Cl+ active site concentration on the fabric at a constant level, about 0.30% of Cl+ loading, for all of the hydantoinyl siloxane-derivatized cotton fabrics. Past work in these laboratories has demonstrated that changes of 0.03% in chlorine loading do not materially affect the performance of a given N-halamine biocide,9 so any differences observed among the derivatives in this work should not be attributed to slightly different chlorine loadings on the swatches. The concentration of the coating solution was 8 wt % for the MM derivative, while it was 3 wt % for the MP and PP derivatives, because phenyl substitution reduces the water solubility of the compounds. Antimicrobial Efficacies. The treated cotton swatches were challenged with S. aureus and E. coli O157:H7 at concentrations between 107 and 109 CFU (colony-forming units), as summarized for E. coli in Figure 2 (the raw data for both pathogens are presented in the Supporting Information). The unchlorinated control samples provided only about 0.01 to 0.25 log reductions, due to the adhesion of bacteria to the cotton swatches, within 30 min contact time intervals. All of the chlorinated treated samples showed excellent antimicrobial activity. PP-Cl and MPCl provided a total inactivation of E. coli O157:H7 within 5 min, while MM-Cl required 10 min contact, in test I. The phenyl-substituted derivatives (PP-Cl and MP-Cl) exhibited the same trends, i.e., relatively higher inactivation rates in tests II and III. The same qualitative observations held for S. aureus (see the Supporting Information). We conclude that the results of this study indicate slight increases in the biocidal efficacies of the derivatives when phenyl derivatization is performed at position 5 of the hydantoin ring for the hydantoinylsiloxane coatings. This implies that the oxidative chlorine is donated to the bacterial cell more facilely for the phenyl derivatives, which further suggests that the presence of the phenyl group at the 5 position on the hydantoin ring causes a weakening of the N-Cl bond relative to that of the dimethyl derivative. We also conclude that all of the derivatives produced extremely effective biocidal coatings on cotton with 8-log reductions of E. coli O157:H7 and S. aureus within 10 min of contact. Stabilitiy toward Hydrolyses. The stabilities toward machine washing of coated fabric swatches are presented in Table 1. Three types of washing experiments were performed: prechlorinated coatings at the concentration levels indicated at 0

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Figure 3. Stability toward UVA light exposure of cotton coated with derivatized hydantoinyl siloxanes (%Cl+ remaining).

Figure 2. Biocidal tests: microorganism, E. coli O157:H7; total bacteria, 2.10 × 108 (8.32 logs), 1.56 × 108 (8.19 logs), and 2.30 × 108 (8.36 logs) for tests I-III, respectively. Chlorine loadings on the coated swatches (MMCl, MP-Cl, and PP-Cl) were as follows: (test I) 0.31, 0.32, and 0.29%, respectively; (test II) 0.28, 0.33, and 0.31%, respectively; (test III) 0.31, 0.33, and 0.29%, respectively. Table 1. Stability toward Washing of Cotton Coated with Derivatized Hydantoinyl Siloxanes (%Cl+ Remaining)a MM

MP

PP

machine washesc

X

Y

Z

X

Y

Z

X

Y

Z

0 5 10 25 50

0.39 0.21 0.16 0.11 0.08

0.39 0.24 0.21 0.13 0.09

0.07 0.05 0.03 0.03

0.38 0.12 0.06 0.03 0.01

0.38 0.17 0.12 0.08 0.05

0.10 0.07 0.05 0.04

0.41 0.12 0.09 0.06 0.01

0.41 0.19 0.18 0.14 0.07

0.17 0.16 0.14 0.06

b

b

b

a The error in the measured Cl+ weight percentage values was (0.01. X, Chlorinated before washing; Y, chlorinated before washing and rechlorinated after washing; Z, unchlorinated before washing but chlorinated after washing. c A washing cycle is equivalent to five machine washes in AATCC test method 61. b

machine washes in Table 1 (X), prechlorinated and rechlorinated after a given number of machine washes (Y), and unchlorinated

until after a given number of machine washes (Z). All coatings were partially lost upon successive washing, although loadings of 0.05-0.09 wt % Cl+ after 50 machine washes would still provide residual disinfection. Several observations can be made pertaining to the data in Table 1. First, the phenyl-substituted derivatives lost the bound chlorine to somewhat greater extent than did the methyl-substituted derivatives (Table 1 (X)). Moreover, this rate is not a result of the dissociation of tethering groups (siloxane) from cotton because rechlorination of the derivatives provided similar chlorine loadings for all derivatives (Table 1 (Y)). Second, unchlorinated phenyl derivatives (Z) are slightly more resistant toward washing cycles as compared to methyl-substituted derivatives, most likely because of the more hydrophobic character of the phenyl-derivatized coatings. Again, these washing tests, as for the biocidal results, imply that phenyl derivatization of the hydantoin ring at the 5 position weakens the N-Cl bond. Stability toward Ultraviolet Light Irradiation. The UV light stabilities of the N-Cl bonds of the synthesized Nhalamine siloxane derivatives were quite dependent upon the presence or absence of the phenyl group (Figure 3). Phenyl substitution in PP-Cl and MP-Cl caused a more dramatic loss in bound chlorine upon UVA exposure than did methyl substitution alone (MM-Cl). Clearly, there was some decomposition (around 40%) of all of the coatings over the 6 h exposure as evidenced by the lower Cl+ loadings after rechlorination. The mechanism of this decomposition is under study and will be discussed in future work. However, the primary decomposition process was the dissociation of N-Cl bonds, with again the presence of the phenyl group accentuating the process. The stability was reasonable for an N-halamine derivative given that a 6 h exposure in the UV chamber was equivalent to the same time in direct midday summer sunlight. However, one of the goals in performing this work was to determine if phenyl substitution on the hydantoin ring would stabilize the N-Cl bond toward UV degradation. Clearly the effect is destabilizing. Potential Factors Affecting Nitrogen-Halogen Bond Lability. There are several factors which could influence the nitrogen-halogen bond lability upon phenyl derivatization at position 5 of the hydantoin ring which, in turn, would influence the rate of disinfection of microbes and the stability toward hydrolyses and UV light irradiation. (1) Phenyl groups are superior electron donors to methyl groups, and thus phenyl substitution should strengthen the N-Cl bond by increasing its polarity via a through-bond interaction. (2) Phenyl groups at position 5 of the hydantoin ring could cause steric hindrance to the approach of microbial cells, thus reducing the rate of disinfection. However, the disinfection rate of the phenylsubstituted derivatives was higher implying that the N-Cl bond

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Figure 4. Structures of the synthesized model compounds.

Figure 5. FTIR spectra of the model compounds in the N-H and C-H stretching region.

became weaker after phenyl substitution. Additionally, UVAlight stability and washing tests also suggested weaker N-Cl bonds in the phenyl derivatives as in the biocidal tests. Thus, neither through-bond electronic effects nor steric hindrance are the primary factors influencing the N-Cl bond dissociation. (3) An intramolecular through-space interaction could occur between the π-electrons of the aromatic ring and the hydrogen of the N1 amide,15,16 and this nonbonded interaction would be expected to be stronger after replacing the hydrogen atom with a chlorine atom, the latter having anisotropic electron density distribution. A less stable molecular conformation could occur due to nonbonded interaction between the aromatic ring and N-Cl moiety.17 It has been reported that the interaction between the chlorine atom and the π-cloud of an aromatic ring has an important role in determining the conformation of the overall molecule.17 To attempt to further address the topic of N-Cl bond stability for the derivatized hydantoin siloxanes, model compounds for the three siloxane derivatives were synthesized. Butyl groups were attached to the imide nitrogen (N3), simulating the siloxane groups (Figure 4).

The FTIR spectra of the model compounds (the N-H and C-H stretching mode regions) are presented in Figure 5; complete spectra are given in the Supporting Information. The N-H stretching band occurred at 3292 cm-1 for MMm but at relatively lower wavenumbers, 3254 and 3167 cm-1 for MPm and PPm, respectively. These bands disappear upon chlorination. Thus, the increasing red shift shift by about 38 cm-1 for MPm and 125 cm-1 for PPm suggests the existence of an interaction between the free N-H moiety and the aromatic rings.18,19 The second band at 3173 cm-1 appearing in the MPm spectrum corresponds to N-H...π hydrogen-bonded oscillator modes for the aromatic rings;19 this band is more pronounced in the PPm spectrum at 3094 cm-1, providing further evidence for an N-H · · · π interaction.19 Thus, there is definitely through-space interaction occurring between the phenyl groups and the amide H for the model compounds. Unfortunately spectral band overlap precludes a similar analysis for the N-chloro model compounds. The DSC thermograms of the chlorinated model compounds (see Supporting Information) each exhibited an exothermic peak representing the dissociation of the N-Cl bond. The peak maximum was at 221 and 199 °C for MMm-Cl and PPm-Cl, respectively, characteristic of N-Cl bond dissociation for N-halamine compounds,10 suggesting that the N-Cl bond becomes weaker due to phenyl substitution at the 5 position of the hydantoin ring for the model compounds, as was observed for the coated siloxanes discussed earlier. The corresponding peak for MPm-Cl was too broad to measure accurately. The 1H NMR data in the aromatic region for the unchlorinated and chlorinated model compounds, as shown in Figure 6, are relevant to the discussion above; the complete 1H NMR spectra are presented in the Supporting Information. The broad signal near 7.3 ppm for model compound MMm, which vanishes upon chlorination to form MMm-Cl, can be assigned to the amide proton resonance. The signals for the amide proton resonance for model compounds MPm and PPm, which disappear upon chlorination, are shifted to lower field at 7.84 and 8.43 ppm, respectively. This is a clear indication of a through-space interaction of the amide proton with the aromatic rings; a dominant through-bond interaction would have induced a shift to higher field.20,21 It is evident from the 1H NMR spectra for the model compounds (Figure 6) that chlorination drastically alters the signals for the aromatic protons. For the MPm derivative, the doublet at low field at 7.58 ppm had an integrated area of twofifths of the total signals in the 7.3-7.6 ppm region and can thus be assigned to resonance of the ortho protons on the phenyl ring. The complex multiplet near 7.4 ppm integrating to three-

Figure 6. 1H NMR spectra of the model compounds before and after chlorination (the aromatic region. The solvent was acetone-d6).

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Table 2. Dihedral Angles (deg) for PPm-Cl Isomers for Phenyl Substituent Twists N1-C5-CA-CB N1-C5-CA-CC N1-C5-CA′-CB′ N1-C5-CA′-CC′ a

Figure 7. 5,5-Diphenyl-3-butylhydantoin with Cl bonded at the N1 position optimized at the B3LYP/6-311+G(2d,p) level of theory.

fifths of the total signals in the 7.3-7.6 region can be assigned to the overlapping meta and para proton resonances. The ortho phenyl proton resonances shifted to higher field while the meta and para proton resonances shifted to lower field after chlorination to form MPm-Cl, such that a broad single signal at about 7.47 ppm resulted. A similar effect occurred for PPm upon chlorination to form PPm-Cl. For the PPm derivative the aromatic protons exhibited a single complex multiplet between 7.35 and 7.50 ppm. Upon chlorination, signals were resolved at 7.36 ppm (integrated area four-tenths that of the 7.3-7.6 ppm region) and 7.50 ppm (integrated area six-tenths that of the 7.3-7.6 ppm region), corresponding to resonances of the ortho and the meta/para protons, respectively. The dramatic effect of chlorination at the amide nitrogen on the NMR spectra of the phenyl derivatives is clearly indicative of substantial through-space interaction between chlorine and the phenyl rings. Similar effects have been reported previously in related aromatic systems.22,23 Computational Studies. The DFT methods described in the Experimental Section were employed to compute the geometries of PPm and PPm-Cl in order to assist in the interpretation of the observed NMR and FTIR data and to predict the N-Cl bond dissociation energies for MMm-Cl and PPm-Cl. To obtain the best comparison of the geometries of PPm with those of PPmCl, chlorinated isomers of PPm-Cl with the Cl placed at the C7 and C8 atoms on the butyl chain (Figure 4) were used to model PPm; this was done to minimize any anomalies introduced in the energy computations, under the assumption that Cl placed at C7 or C8 would have little or no effect on the geometries of the phenyl rings. The computed geometries for the hydantoin structures are in very good agreement with reported bond distances, bond angles, and torsional angles from X-ray crystallographic studies24,25 and semiempirical calculations;26 complete geometries for all of the computed structures are given in the Supporting Information. For example, the calculated N1-C5-CA and N1-C5-CA′ angles were ca. 112° for the PPm-Cl models of PPm, which were in excellent accord with experimental values of 111.5-112.5°.24 An illustration of the optimized PPm-Cl structure in which Cl is bonded to N1 is given in Figure 7. Of particular interest are the twists associated with the phenyl rings relative to the N1 position in the PPm-Cl models. Figure 7 and Table 2 provide the dihedral angles of the phenyl rings relative to the N1 position and predict the phenyl rings to be highly orthogonal when the Cl is bound to N1. In contrast, optimizations of the PPm-Cl models with an H bonded at N1 and the Cl at the C7 or C8 positions on the butyl chain find the twist of the phenyl’s to be less orthogonal (Table 2). The calculations are consistent with

N1-Cl

C7-Cl

C8-Cl

2.5 179.9 -85.5 91.1

6.2 -175.6 -102.7 73.9

5.5 -176.4 -104.2 72.7

Numbering scheme given in Figure 7.

experimental solid-state structures which determined the phenyl substituents to be fairly twisted with respect to the normals to the respective phenyl-hydantoin plane’s, yielding angles of 114 and 113° for 5,5-diphenylhydantoin24 and 104 and 105° for 5-phydroxyphenyl-5-phenylhydantoin.25 Chemical shifts in the 1H NMR spectrum for the aromatic protons of 5,5-diphenylhydantoin have been reported, and the resultant broad signal was attributed to the twist in the rings; ROSEY experiments estimate the N1-H · · · H(ortho)-phenyl distance to be 2.38 Å in solution.26 The present PPm-Cl gasphase calculations with Cl at the C7 or C8 position find the N1-H · · · H distances for the two closest ortho hydrogens on the aromatic ring to be ca. 2.76 Å; however, the distances are dramatically different, 2.95 and 3.35 Å, for N1-Cl · · · H(ortho)phenyl. The calculations indicate that the chlorine atom at the N1 position forces the phenyl rings to become more twisted and to cause the ortho protons to lie further in their shielding regions, as previously suggested.21,27 This anisotropic effect appears consistent with the current 1H NMR spectrum that finds the single complex multiplet in the PPm structure resolved into the two meta/para and ortho proton resonances for PPm-Cl with the ortho protons more shielded. In the current experimental work, phenyl substitution caused a more dramatic loss in the bound chlorine upon UVA exposure than did methyl substitution. To quantify this homolytic dissociation, the UB3LYP/6-311++G(2d,p) level of theory was used to compute the heats of formation (∆fH°298) for the Cl radical and PPm and MMm radical species, and their corresponding bond dissociation energies (BDE) were obtained (48.7 and 50.5 kcal/mol, respectively) using eq 2. Thus the calculations were consistent with the experimental findings predicting the BDE for the PPm-Cl system to be 1.8 kcal/mol lower in energy than for the MMm-Cl structure. BDE298 ) ∆fH◦298(hydantoin•) + ∆fH◦298(Cl•) ∆fH◦298(hydantoin-Cl) (2) Similar to UVA-light exposure, the destabilizing effect of the phenyl substitution relative to methyl groups was also found to occur in the antimicrobial and washing analyses. Further insight into the enhanced rate of Cl decomposition in water can be derived from our recent computational work elucidating the mechanism of the halogenation of the hydantoin ring moiety.14 In that work, aqueous-phase calculations were carried out using the B3LYP/6-311+G(2d,p) level of theory, and solvent was treated explicitly using the OPLS-AA force field28 and sampled with Monte Carlo statistical mechanics. The calculations predicted a ∆Gq of 20.3 kcal/mol for the base-induced dechlorination of 1-chloro-5,5-dimethylhydantoin to yield the amide anion and Cl+ (HOCl).14 Accordingly, the energy required to break the N-Cl bond in this heterolytic cleavage was significantly lower than the currently computed homolytic BDEs. Conclusions A series of (3-(triethoxysilyl)propyl)hydantoin derivatives and 3-butyl derivatives as model compounds with variation of methyl

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and phenyl substitution at the 5 position of the hydantoin moiety have been synthesized. The silane derivatives were coated onto cotton fabric and then treated with bleach to provide an antimicrobial property. The antibacterial activities, stabilities to hydrolyses, and UVA-light resistance of the silane derivatives have been compared. All of the resulting data were consistent with the conclusion that phenyl substitution at the 5 position of the hydantoin ring leads to weakening of the N-Cl bond in the compounds. NMR and FTIR data for the model compounds were suggestive of a through-space interaction between the Cl and the phenyl rings which influenced the N-X bond lability. DFT computational results obtained for the model compounds were consistent with the experimental analysis. Acknowledgment This work was supported by the U.S. Air Force through Grant FA8650-07-1-5908. Supporting Information Available: Additional information about chlorine loadings of synthesized compounds at different concentrations, biocidal test results of cotton coated with derivatized hydantoinyl siloxanes, NMR and FTIR spectra of the synthesized model compounds, DSC thermograms, and complete computational geometries for the model compounds. This information is available free of charge via the Internet at http://pubs.acs.org/. Literature Cited (1) Worley, S. D.; Williams, D. E. Halamine Water Disinfectants. CRC Crit. ReV. EnViron. Control 1998, 18, 133–175. (2) Worley, S. D.; Sun, G. Biocidal Polymers. Trends Polym. Sci. 1996, 4, 364–370. (3) Kenawy, E. R.; Worley, S. D.; Broughton, R. The Chemistry and Applications of Antimicrobial Polymers: A State of the Art Review. Biomacromolecules 2007, 8, 1359–1384. (4) Williams, D. E.; Elder, E. D.; Worley, S. D. Is Free Halogen Necessary for Disinfection? Appl. EnViron. Microbiol. 1988, 54, 2583– 2585. (5) Worley, S. D.; Chen, Y.; Wang, J. W.; Wu, R.; Cho, U.; Broughton, R. M.; Kim, J.; Wei, C.I.; Williams, J.F.; Chen, J.; Li, Y. Novel N-halamine Siloxane Monomers and Polymers for Preparing Biocidal Coatings. Surf. Coat. Int., Part B 2005, 88, 93–100. (6) For example, see: (a) Liang, J.; Barnes, K.; Akdag, A.; Worley, S. D.; Lee, J.; Broughton, R. M.; Huang, T. S. Improved Microbial Siloxane. Ind. Eng. Chem. Res. 2007, 46, 1861–1866. (b) Liang, J.; Chen, Y.; Ren, X.; Wu, R.; Barnes, K.; Worley, S. D.; Broughton, R. M.; Cho, U.; Kocer, H. B.; Huang, T. S. Fabric Treated with Antimicrobial N-Halamine Epoxides. Ind. Eng. Chem. Res. 2007, 46, 6425–6429. (c) Kou, L.; Liang, J.; Ren, X.; Kocer, H. B.; Worley, S. D.; Tzou, Y.-M.; Huang, T. S. Synthesis of a Water-Soluble Siloxane Copolymer and Its Application for Antimicrobial Coatings. Ind. Eng. Chem. Res. 2009, 48, 6521–6526. (7) For example, see: (a) Sun, Y. Y.; Chen, T.; Worley, S. D.; Sun, G. Novel Refreshable N-Halamine Polymeric Biocides Containing Imidazolidin-4-one Derivatives. J. Polym. Sci., Part A: Polym. Chem. 2001, 39, 3073–3084. (b) Makal, U.; Wood, L.; Ohman, D. E.; Wynne, K. J. Polyurethane Biocidal Polymeric Surface Modifiers. Biomaterials 2006, 27, 1316–1326. (8) Akdag, A.; Okur, S.; McKee, M. L.; Worley, S.D. The Stabilities of N-Cl Bonds in Biocidal Materials. J. Chem. Theory Comput. 2006, 2, 879–884. (9) Kocer, H. B.; Akdag, A.; Ren, X.; Broughton, R. M.; Worley, S. D.; Huang, T. S. Effect of Alkyl Derivatization on Several Properties of N-Halamine Antimicrobial Siloxane Coatings. Ind. Eng. Chem. Res. 2008, 47, 7558–7563.

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ReceiVed for reView June 9, 2010 ReVised manuscript receiVed September 13, 2010 Accepted September 14, 2010 IE101258S