Antimicrobial N-brominated hydantoin and uracil grafted polystyrene beads

Journal of Controlled Release 216 (2015) 18–29

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Antimicrobial N-brominated hydantoin and uracil grafted polystyrene beads Shady Farah a, Oren Aviv a, Natalia Laout a, Stanislav Ratner a, Abraham J. Domb a,b,⁎ a Institute of Drug Research, School of Pharmacy-Faculty of Medicine, Center for Nanoscience & Nanotechnology and The Alex Grass Center for Drug Design and Synthesis, The Hebrew University of Jerusalem, 91120, Jerusalem, Israel b Jerusalem College of Engineering (JCE), Jerusalem, Israel

a r t i c l e

i n f o

Article history: Received 8 June 2015 Received in revised form 9 July 2015 Accepted 11 July 2015 Available online 26 July 2015 Keywords: N-bromoamine Hydantoin N-halamine disinfectant Uracil Polystyrene beads Water disinfection

a b s t r a c t Hydantoin-N-halamine derivatives conjugated on polystyrene beads are promising disinfectants with broad antimicrobial activity affected by the gradual release of oxidizing halogen in water. The objective of this work was to identify and test of hydantoin-like molecules possessing urea moiety, which may provide N-haloamines releasing oxidizing halogens when exposed to water at different rates and release profiles for tailored antimicrobial agents. In this work, several hydantoin (five member ring) and for the first time reported, uracil (six member ring) derivatives have been conjugated to polystyrene beads and tested for their lasting antimicrobial activity. Four molecules of each series were conjugated onto polystyrene beads from the reaction of the N-potassium hydantoin or uracil derivatives onto chloromethylated polystyrene beads. A distinct difference in bromine loading capacity and release profiles was found for the different conjugated derivatives. All tested materials exhibit strong antimicrobial activity against Escherichia coli and bacteriophages MS2 of 7 and ~4 log reduction, respectively. These results highlight the antimicrobial potential of halogenated cyclic molecules containing urea groups as water disinfection agents. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Extensive efforts have been made to control emerging disease infections [1,2]. There is a need for new biocidal compounds, which do not possess the limitations inherent in the disinfectants currently employed, i.e., long contact time, bacterial resistance, toxicity and limited regeneration ability [3]. N-halamine structures are among the most promising candidates. An N-halamine can be defined as a compound containing one or more nitrogen–halogen covalent bonds [4]. Nhalamine moieties are excellent biocides inactivating Gram-negative and Gram-positive bacteria, viruses, and fungi [5–8]. The bactericidal action of N-halamines is apparently a manifestation of a chemical reaction involving the direct transfer of positive halogen from the N-halamine to appropriate receptors in the bacterial cells [4]. Several commercial polymers have been functionalized with N-halamine moieties, rendering them biocidal upon surface contact with pathogens. These include cellulose [4,9–11], nylon [12], PET [13,14], Kraton rubber [15], and various surface coatings [14–23]. To date, however, the most important N-halamine polymers developed are the N-halogenated poly-

styrenehydantoins [24–26], because of their potential for economical disinfection of potable water, thus improving world health [24,27–31]. While N-bromohydantoin grafted polystyrene beads have been reported, mainly by the group of Worely, little has been reported on the effect of the N-haloamine structure on the conjugation synthesis and lasting disinfection activities in water. No reports have been published on the synthesis of N-halo-uracil, a urea containing cyclic molecule, and its potential use as a disinfection agent. Moreover, little attention has been given to the different parameters affecting oxidative halogen releasing profiles from polymeric beads and surfaces, such as substitutions and steric hindrance onto hydantoin and uracil rings. Diverse halogen release profiles influence antimicrobial activity and subsequently the material usage for different applications. Hydantoin/uracil derivatives were conjugated to chloromethyl polystyrene beads in different solvents and examined for conjugation yield %, halogen loading capacity and hypobromous acid release profile, halogen rechargeability and antibacterial activity for hundreds of liters against Escherichia coli (E. Coli) and MS2. 2. Materials and methods

⁎ Corresponding author at: Institute of Drug Research, School of Pharmacy-Faculty of Medicine, Center for Nanoscience & Nanotechnology and The Alex Grass Center for Drug Design and Synthesis, The Hebrew University of Jerusalem, 91120 Jerusalem, Israel. E-mail addresses: [email protected], https://medicine.ekmd.huji.ac.il/en/publications/researchersPages/pages/avid.aspx (A.J. Domb).

http://dx.doi.org/10.1016/j.jconrel.2015.07.013 0168-3659/© 2015 Elsevier B.V. All rights reserved.

2.1. Materials All reagents and solvents were of analytical grade. Bromine 99.8% was a gift from Israel Chemical, LTD — The Israel Bromine Compounds Company. Na2S2O3 pentahydrate, diethylether and acetic acid glacial

S. Farah et al. / Journal of Controlled Release 216 (2015) 18–29

19

A) Step 1: Hydantoin Derivative Ethanol

HN NH

O

Step 2:

O

O

HN

Reflux 2 hr

N

K

O

O

Hydantoin Salt

N

1. DMF ( )

KOH

2.

O

NH

Hydantoin-Ps (HD-Ps)

Cleaned with Acetone

Cl

OR NaOH

3. 300 rpm ,13 hr NH O

N H

H N

Ethanol

O

O O N

Reflux 2 hr

Uracil Derivative

K

N O

N H Uracil-Ps (UR-Ps)

O

Uracil Salt ( ) DMSO , 2hr . (1:1.5 - Reactive site: DMH or HD)

B) Hydantoin Derivatives O

O

O

HN

HN

HN NH

H

HN NH

NH

H3C

H

O

O

H2N

H

H3C O

Hydantoin

NH

HN

O

O

5,5'-DimethylHydantoin

O

Allantoin

5,5'-PhenylHydantoin

Uracil Derivatives O

O

HN NH

O

Uracil

H2N

O

O

HN

HN

HN NH

HOOC

O

6-AminoUracil

Active Nitrogen Site for Beads Conjugate

NH

O

Orotic Acid

NH

HN

O

N H

O

Uric Acid

Active Nitrogen Site for Bromination

Scheme 1. A) Conjugation reaction of representative hydantoin/uracil derivative molecules to polymeric resin by nucleophilic substitution of hydantoin/uracil salt. B) Studied derivatives were conjugation sites and potential sites for bromination (marked with circles).

were purchased from Frutarom Ltd., Israel. N,N-dimethylformamide anhydrous 99.8%, dimethylsulfuxide anhydrous 99.8% and starch were purchased from Sigma-Aldrich, Rehovot, Israel. Ethanol 99.8% was purchased from J.T. Baker, Israel. Potassium hydroxide, sodium hydroxide, hydantoin, 5,5′-dimethylhydantoin, 5,5′-diphenylhydantoin, uracil, 6-aminouracil, orotic acid and uric acid ≥ 98% were purchased from Sigma Aldrich, Israel. 1% crosslinked chloromethyl polystyrene (CMPS) was purchased from Tianjin Nankai Hecheng, China, C% 73.88–H% 6.34–Cl% 20.65%, size range(μm) 300–600, density (g/ml) 0.658. Hypobromous acid solution was prepared by bromine addition to an aqueous hydroxide solution followed by acetic acid to reach pH 6.5. 2.2. Methods 2.2.1. Conjugation hydantoin/uracil derivative to polymeric beads (HD-Ps, UR-Ps) The conjugation of hydantoin (HD) or uracil (UR) derivatives to chloromethyl polystyrene beads (CMPS) included two steps: first hydantoin/uracil salt preparation following acid–base reaction, followed by nucleophilic substitution, type SN2, where Cl− is released as leaving group, (Scheme 1A and B). Prior to conjugation, the starting material of chloromethyl polystyrene beads was cleaned from organic impurities

by soaking in acetone (10 ml/g) for 2 h at 25 °C, then filtering and washing three times with acetone (5 ml/g), and drying at 50 °C by an evaporator. The potassium salts of hydantoin/uracil derivatives were prepared by reacting equivalent molar ratio potassium hydroxide:hydantoin/uracil derivative in a minimum amount of ethanol with stirring under reflux for 2 h. Ethanol was removed under high vacuum to obtain a hydantoin/uracil derivative salt. Then 400 ml dimethylformamide (DMF, anhydrous) or dimethylsulfuxide (DMSO, anhydrous) were added, and the mixture was heated to 100 °C or 120 °C respectively, until all of the salt dissolved. In DMSO the salt dissolves faster. Then the system was connected to a nitrogen balloon, and 50.0 g of the previously cleaned CMPS were added at a ratio 1:1.5 of leaving group (CMPS):hydantoin/uracil salt. A mechanical stir was equipped with a stirring rod at 300 rpm, and the mixture was heated for 13 h or 2 h respectively. Resultant conjugated beads (HD-Ps and UR-Ps) were separated by filtration and washed 5 times with boiling 200 ml DDW and then with acetone for removal of unreacted hydantoin/uracil salt. Beads were evaporated until dryness at 70 °C. In DMF almost 25–35% weight increase was found due to conjugation hydantoin derivatives, although there was only 8–28% for uracil derivatives. On the other hand conjugation in DMSO resulted in 30% weight increase for both derivative groups.

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S. Farah et al. / Journal of Controlled Release 216 (2015) 18–29

Step 1: 2NaOH + Br2

NaBr + NaOBr +H 2O H HOBr

Step 2: Brominated Hydantoin-Ps (Br-HD-Ps) O

O N O

N NH

Hydantoin-Ps (HD-Ps)

1. 1.5 eqv HOBr to Reactive sites

O

N

Br H 2O

2.350 rpm, 48 hr

HOBr

O

O

N O

N H Uracil-Ps (UR-Ps)

Slow Release of

N O

Brominated Uracil-Ps (Br-UR-Ps)

N Br

Scheme 2. Bromination reaction of hydantoin and uracil conjugated polystyrene, (HD-Ps) and (UR-Ps) respectively, with hypobromous acid prepared in situ from bromine.

2.2.2. Bromination of hydantoin/uracil conjugated polystyrene (Br-HD-Ps and Br-UR-Ps) Active bromination, hypobromide (HOBr) was prepared in situ from bromine to fit molar ratio 1.5:1 to available active sites (Scheme 1B) for bromination, (Scheme 2). For example, to brominate 20.0 g uracil conjugated beads (UR–Ps), 1 reactive site/molecule with 5.82 mmol theoretical reactive sites/g conjugated beads was as follows: to a 500 ml round bottom flask 3 necked equipped with magnetic stir and icebath, a 27.94 g of NaOH was carefully added to 222 ml DDW to form a 3.15 N solution. After cooling, when the inside reaction solution temperature reached 20 °C, bromine (8.94 ml) was carefully added drop wise to form a dark-yellowish colorful solution. Stirring at 350 rpm was applied for 30 min, followed by adding 4 N acetic acid solution drop wise over 30 min to reach a pH 6.5 (almost 130 ml is needed). A dark-red solution resulted. Stirring continued for 48 h, followed by isolation of beads by filtration and washing with deionized water to remove unbonded bromine. The beads were dried using an evaporator at 50 °C. A similar procedure was applied for the other conjugated beads. A 19–26% weight increase was found for hydantoin derivatives and 15–25% for uracil derivatives.

2.2.5. Scanning electron microscopy analysis Bead size, morphology, tunnel diameter and porosity of the chloromethyl polystyrene beads before and after conjugation reaction were analyzed using SEM visualization. Beads were first frozen with liquid nitrogen, then using a sharp scalpel, the beads were cut to allow the inner and outer surface to be visualized. Samples were sputter-coated with platinum/palladium (Pt/Pd) to a thickness of about 10 nm using a sputtering deposition machine (Polarone E5100). They were then visualized by scanning electron microscopy (SEM), FEI E-SEM Quanta 2000 at constant acceleration voltage of 5 KV. Average intervals were measured using the SEM-internal dedicated software.

2.2.3. Elemental analysis Elemental microanalysis of nitrogen (%N), carbon (%C), and hydrogen (%H) was performed using the Perkin-Elmer 2400 series II Analyzer. Chlorine and bromine (%Cl, %Br) were determined by using the oxygenflask combustion method and subsequent potentiometric titration by the 835 Titrando Metrohm Titroprocessor and by Ion chromatography analysis using a Dionex IC system. All the bead samples, starting material chloromethyl polystyrene, hydantoin and uracil conjugated polystyrene and after bromination were fully analyzed for C, N, H, Cl and Br content. The standard deviation of the results is in the range of: %C, %H, %N ± 0.3%, %Cl, %Br ± 0.5%.

2.2.6. Quantitative analysis of conjugated hydantoin/uracil — Ninhydrin assay Beads (1 g) were transferred into 10 ml of aqueous barium hydroxide (20% w/v) and refluxed for 1 week to hydrolyze hydantoin/uracil molecules, followed by releasing α-amino acids/amino acrylic acid respectively, leaving the polymeric resin with primary amino groups (Scheme 3). Following hydrolysis, the particles were filtrated out, and sodium sulfate was added to the solution. Barium was precipitated and separated from the α-amino acid/acrylic acid solution by centrifugation for 20 min at 4000 rpm followed by filtration. The separated aqueous solution was lyophilized to obtain the released α-amino acid/ acrylic acid. The residue was dissolved in 5 ml DDW and amino acid determined by using Ninhydrin assay as follows: 100 μl of the solution or 100 mg for the amino beads was transferred to a glassy tube, and volume was completed to 4 ml with DDW. Then 1 ml of ninhydrin solution (5 g/100 ethanol) was mixed for 10 s and incubated in a boiling water bath for 15 min. 1 ml of ethanol:DDW 50:50 was added and mixed for an extra 10 s, then immediately analyzed using a spectrophotometer at 570 nm. Calibration curve of alanine 2–40 μg/ml was prepared in a similar manner. Results of the conjugation were presented in mmol/g conjugated beads.

2.2.4. Smart internal reflection (iTR) Bead samples were analyzed by a Smart iTR instrument, Nicolet iS10 (Thermo Scientific company, USA). Beads were placed directly on the diamond Nicolet and scanned in interval 500–4000 cm−1. The spectra were evaluated with OMNIC software for spectra similarity % calculations.

2.2.7. Iodometric titration The loaded bromine% in the polymeric samples was determined by the iodometric/thiosulfate titration method. Sodium thiosulfate solution (0.1 N, calibrated with potassium iodate) was used to titrate suspension of ~50 mg beads in: 50 ml of DDW with 1 g potassium iodide and 12.5 ml acetic acid (2 M) and 2 ml of 1% starch solution. The end point of the titration was the solution color change from blue to

S. Farah et al. / Journal of Controlled Release 216 (2015) 18–29

I. Hydantoin Derivatives Conjugated Beads Hydrolysis

Ba(OH)2 20% w/v Reflux, 1 week

O N NH

HO

II. Uracil Derivatives Conjugated Beads Hydrolysis

O

CO2

NH2

O

HO

NH2

N H UR-Ps

NH2

3-Amino-acrylic acid

HO HO

Ba(OH)2 20% w/v Reflux, 1 week

O

NH O DMH-Ps

CH3

O

HO

H

3,3-Diamino-acrylic acid NH2

N NH2 H AUR-Ps

HO

Ba(OH)2 20% w/v Reflux, 1 week

O CO2 NH2

NH2

NH

H

O

DPH-Ps

Ba(OH)2 20% w/v Reflux, 1 week

HO H

H

N NH

NH

O ALL-Ps

OH

2-Amino-but-2enedioic acid

O OA-Ps

O

O

NH2

OH

N H

O

CO2 C

O

2,2'-DiphenylGlycine

O

H2N

N N

CO2

NH2

N

NH2

O

H2N

O

2-Methyl Alanine

Ba(OH)2 20% w/v Reflux, 1 week

O

NH2

CO2

CH3

CH3

N

Ba(OH)2 20% w/v Reflux, 1 week

O

H3C

CO2

H

O

HD-Ps

O

H

N

H Glycine

NH2

Ba(OH)2 20% w/v Reflux, 1 week

O

H

H H

21

O

O NH2

CO2

O

HN NH2

N

O NH2

HO

Ba(OH)2 20% w/v Reflux, 1 week

H2N

O

Amino-ureido-acetic acid

NH2

H2N N H

H N

N N H H UA-Ps

O

O

CO2

O

NH2 5-Amino-2-oxo-2,3dihydro-1H-imidazole4-carboxylic acid

Scheme 3. Hydantoin/uracil ring hydrolysis from hydantoin/uracil derivative conjugated beads in barium hydroxide releasing α-amino acids or amino acrylic acid, respectively. Conjugated hydantoin derivatives: Hydantoin (HD-Ps), 5,5′-dimethylhydantoin (DMH-Ps), 5,5′-diphenylhydantoin (DPH-Ps) and allantoin (ALL-Ps). Conjugated uracil derivatives: Uracil (UR-Ps), 6-aminouracil (AUR-Ps), orotic acid (OA-Ps) and uric acid (UA-Ps).

colorless. Similar to Cl+ % calculation [7], the Br+ % was calculated from the following equation:   N  V  79:90 Brþ %¼  100% 2W ðWeightÞ

ð1Þ

where Br+ (%) N V W

Weight percent of oxidative bromine in the bead samples. Normality of the titrant sodium thiosulfate. Volume of the titrant sodium thiosulfate in milliliters. Weight of tested beads samples in milligrams.

2.2.8. Hypobromous acid release Upon contact with water brominated hydantoin/uracil beads release hypobromous acid (HOBr). The concentration in water was measured using DPD-1 tablets (diethyl-p-phenylene diamine), AQUALYTIC, AL250 and spectrophotometer at 528 nm wavelength. A HOBr release study from Br-HD-Ps and Br-UR-Ps derivatives was done with columns (28 mm wide) fitted with 16 g of dry brominated beads. Each column was washed with 25 l of water per day at a rate of 10 ± 1 min/l. Hypobromous acid concentration was measured every 10–15 l until

reaching the release of 0.01 ppm. Hypobromous acid partially dissociated in water to hypobromite. 2.2.9. Reloading/re-bormination cycles Beads were examined for reloading capability. After release, the remaining bromine was completely extracted by incubating the beads for one week in 5% Na2S2O3 aqueous solution followed by filtration and washing several times with DDW. Beads were re-brominated as detailed in the typical bromination method. Bead re-bromination and bromine extraction were conducted for at least 3 cycles. Samples were analyzed for bromine content % by elemental analysis. 2.2.10. Bacterial strains and growth conditions The E. coli (ATCC 8739) was provided by Hy Laboratories, Ltd., Rehovot, Israel. Bacteria for the test were prepared by overnight growth to obtain a stationary growth phase. A fresh stock of the test bacteria was prepared before the study started. Accordingly, the “prior to the challenge assay”, a Tryptic Soy Agar-TSA (Difco) plate, was inoculated and incubated overnight at 35 °C. Then, E. coli colonies were resuspended in a phosphate saline buffer (PBS) and thoroughly vortexed. The suspension obtained was additionally diluted in PBS to obtain initial concentration of 108CFU/ml that was used as a bacterial stock for a

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S. Farah et al. / Journal of Controlled Release 216 (2015) 18–29

Table 1 Chemical/physical characteristic parameters of flushing and challenge water. Test water

Flushing Challenge

Water basis

Acceptance criteria

RO + tap water (2:1) RO

Temperature (°C)

pH

Chlorine (ppm)

Turbidity (NTU)

TDS (ppm)

TOC (ppm)

20 ± 5 20 ± 5

6.5–8.5 6.5–8.5

Free (b0.1) Free (b0.05)

0.1–5 0.1–5

50–500 50–500

0.1–5 0.1–5

challenge test. Bacteriophage, type Coliphage MS2 (ATCC 15597-B1), was obtained from ATCC. It was grown and assayed in the bacterial host, E. coli (ATCC 15597), at 37 °C utilizing Tryptic Soy Broth-TSB (Difco). A high virus mixture was centrifuged at 6000 rpm for 15 min, and then a Coliphage-containing supernatant was removed and filtered through a 0.22 μ cellulose filter and diluted in PBS to 10 7 PFU/ml. The MS2 stock was refrigerated at 4 °C until used in the experiment. Host bacteria: A pure culture of the host bacteria E. coli, obtained from the ATCC collection, was rehydrated, cultured and stored according to the enclosed instruction. Fresh (4 h) stock of E. coli (ATCC 15597) host bacteria was prepared in TSB each time prior to the challenge test. It served as the inoculums for E. coli lawn on the top layer of agar used in the phage assay. Challenge preparation: For challenge preparation the resultant pellet of purified MS2 and E. coli (ATCC 8739) stocks was added to an appropriate volume of testing water to achieve an influent final concentration of 107 Colony Formation Units-CFU/100 ml and 107

A

C

Plague Formation Units-PFU/l (104 PFU/ml). Challenge water was thoroughly mixed, so that the organisms would maintain uniform suspension during the challenge. 2.2.11. Antimicrobial activity Antimicrobial evaluation was conducted according to NSF-231 protocol for testing microbiological water purifiers with minimal modifications. Tested beads including starting materials and hydantoin/uracil conjugated beads before and after bromination were placed inside the identical columns. The system used 25 l per day of flushing water. Antimicrobial efficacy of the tested materials was measured at different challenge points. For each challenging point 2.5 l of challenge water containing 107 CFU/100 mL of E. coli bacteria and 10 4 PFU/mL of MS2 bacteriophages were passed through the column. The first 250 mL of water was allowed passing through the system prior to sampling to assure that sufficient spike had been added to the system. The next 2 l of challenging water was collected and part was transferred in a sterile bottle containing

B

Outer Surface

D

Inner Core

Fig. 1. Representative scanning electron micrographs for chloromethyl polystyrene, 1% crosslinked, used in conjugation reactions: (A) Polymeric bead size 300–600 μm, magnification ×100, scale bare 500 μm. (B) Low porosity of the polymeric beads, magnification ×400, scale bare 200 μm. (C) Porosity/tunnels at the top of beads outer surface, magnification ×20,000, scale bare 2 μm. (D) Porosity inside the beads core, magnification ×300,000, scale bare 0.2 μm. No significant changes in particle size and porosity were found after conjugation reaction for the studied hydantoin/uracil derivatives.

S. Farah et al. / Journal of Controlled Release 216 (2015) 18–29

A

23

(A) Chloromethyl polystyrene (CMPS)

(B) HD-PS

(C) DMH-PS

(D) DPH-PS

(E) ALL-PS

B

(A) Chloromethyl polystyrene (CMPS)

(B) UR-PS

(C) AUR-PS

(D) OA-PS

(E) UA-PS

Fig. 2. (A). FTIR spectra of chloromethyl polystyrene conjugated to hydantoin derivatives. (A) Starting material chloromethyl polystyrene (CMPS), (B) hydantoin conjugated polystyrene (HD-Ps), (C) 5,5′-dimethyl hydantoin conjugated polystyrene (DMH-Ps), (D) 5,5′-diphenyl hydantoin conjugated polystyrene (DPH-Ps) and (E) allantoin conjugated polystyrene (ALLPs). The marked peak at spectrum A, 1264 cm−1, attributed to –CH2–Cl vibration in the starting material which disappeared while new carbonyl peaks were identified at 1630–1780 cm−1. For CMPS: _C–H Str (3030–3080 cm−1), −CH2–Cl (2921, 2849 cm−1), C_C Vib (1610, 1510, 1444 cm−1), C–Cl Vib (1264, 825 cm−1). For conjugated samples: N–H (3) Str 3336 cm−1, N–H (1) Str 3070–3100 cm−1, _C–H Str (3030–3080 cm−1), CH3(C–H) Str 2976 cm−1, C_C Vib(1513,1445,1415 cm−1), C_O Str(1770–1779,1661–1705 cm−1). (B). FTIR spectra of chloromethyl polystyrene conjugated to uracil derivatives. (A) Starting material chloromethyl polystyrene (CMPS), (B) uracil conjugated polystyrene (UR-Ps), (C) 6-aminouracil conjugated polystyrene (AUR-Ps), (D) orotic acid conjugated polystyrene (OA-Ps) and (E) uric acid conjugated polystyrene (UA-Ps). The marked peak at spectrum A, 1264 cm−1 attributed to –CH2–Cl vibration in starting material which disappeared while new carbonyl peaks were identified at 1630–1780 cm−1. For CMPS: _C–H Str (3030–3080 cm−1), −CH2–Cl (2921, 2849 cm−1), C_C Vib (1610,1510, 1444 cm−1), C–Cl Vib (1264,825 cm−1). For conjugated samples: N–H (3) Str 3336 cm−1, N–H (1) Str 3070–3100 cm−1, _C–H Str (3030–3080 cm−1), CH3(C–H) Str 2976 cm−1, C_C Vib(1513,1445,1415 cm−1), C_O Str(1770–1782,1651–1726 cm−1).

sodium thiosulfate (0.01% final concentration) to neutralize any residual bromine. In addition, residual bromine in effluent was measured at each microbiological sampling point. Test water: A bromine inactivation experiment was carried out in general (type 1) water. Chemical and physical characteristic parameters are presented in Table 1. The specific characteristic parameters were obtained by mixing reverse osmosis treated water (RO) and tap water

(flushing water) or by the adjustment of RO water (challenge water) as described in Guide Standard and Protocol for Testing Microbiological Water Purifiers [33,34]. Microbial assay techniques: The collected samples were tested for microbial analysis immediately. Aliquots of the sample (100 ml for E. coli bacteria and 1 ml for MS2 phages) were portioned for analysis. All samples were processed in duplicate.

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S. Farah et al. / Journal of Controlled Release 216 (2015) 18–29

Table 2 Elemental analysis of hydantoin/uracil conjugated polystyrene, before and after hydantoin/uracil hydrolysis.⁎ Descriptiona

Hydantoin/uracil hydrolysisb

C%

H%

N%

Cl%

CMPS HD-Ps (DMF) HD-Ps (DMF) HD-Ps (DMSO) HD-Ps (DMSO) DMH-Ps (DMF) DMH-Ps (DMF) DMH-Ps (DMSO) DMH-Ps (DMSO) DPH-Ps (DMF) DPH-Ps (DMF) All-Ps (DMF) All-Ps (DMF) All-Ps (DMSO) All-Ps (DMSO) UR-Ps (DMF) UR-Ps (DMF) AUR-Ps (DMF) AUR-Ps (DMF) AUR-Ps (DMSO) AUR-Ps (DMSO) OA-Ps (DMF) OA-Ps (DMF) UA-Ps (DMF) UA-Ps (DMF)

– Before After Before After Before After Before After Before After Before After Before After Before After Before After Before After Before After Before After

73.88 72.64 55.15 72.99 61.16 72.04 70.22 71.41 69.59 75.91 74.56 70.79 70.88 71.98 68.54 74.20 68.20 73.72 71.29 72.10 69.45 73.44 76.68 75.10 74.38

6.34 6.31 5.30 6.35 5.48 6.77 6.54 7.01 6.78 5.70 5.81 6.59 6.07 6.43 6.38 6.05 5.72 6.28 6.31 6.48 5.77 6.57 6.75 6.32 6.21

0.00 8.61 5.36 5.61 4.13 9.44 8.74 7.85 7.15 7.43 5.85 9.12 3.13 6.12 4.64 6.81 5.83 6.09 3.89 5.37 2.82 1.92 1.40 1.37 1.18

20.65 0.50 – 0.00 1.30 – 1.50 – 0.00 – 2.56 – 0.43 – 0.55 – 0.59 – 4.06 – 4.11 – 9.20 –

⁎ C, H, N, and Cl content of hydantoin/uracil conjugated beads (%C, %H, %N ± 0.3%, %Cl ± 0.5%). a Abbreviation of conjugated polystyrene: (HD) — Hydantoin, (DMH) — 5,5′dimethylhydantoin, (DPH) — 5,5′-diphenylhydantoin, (ALL) — allantoin, (UR) — uracil, (AUR) — 6-aminouracil, (OA) — orotic acid and (UA) uric acid. DMF/ DMSO, solvent used in conjugation reaction (Scheme 1A, step 2). b Hydrolyzed in aqueous barium hydroxide (20% w/v), reflux 1 week.

Influent samples were assayed with multiple dilutions. E. coli were enumerated in all samples by membrane filter using m-FC agar, following the method described in Standard Methods [35]. MS2 was assayed by the double layer over-lay method of Adams [36]. The detection limit for E. coli bacteria is 1 CFU/100 ml, and for MS2 bacteriophages it is 1 PFU/1 ml.

3. Results and discussion 3.1. Conjugation reaction of hydantoin/uracil derivatives to chloromethyl polystyrene resin (CMPS) and characterization Conjugation reaction between hydantoin/uracil derivatives to chlorometyl polystyrene resin (CMPS) was conducted as described in (Scheme 1A and B). Hydantoin/uracil molecules were first reacted with potassium hydroxide or sodium hydroxide in ethanolic solution to produce hydantoin/uracil salt (–CO–NH–CO–), followed by solvent evaporation and conjugation reaction hydantoin/uracil saltchloromethyl polystyrene resin. For the best comparison among conjugated derivatives, the same batch of crosslinked chloromethyl polystyrene beads of the following characteristics were used: 300– 600 μm beads size, 1% crosslinking and low porosity density of 30 nm porous size (Fig. 1A). The polymeric beads were SEM visualized before and after conjugation reaction. No significant change in particle size and porosity was found for the studied conjugated hydantoin/uracil derivatives (Fig. 1B–D). Conjugation reaction was studied into two polar solvents — N,N-dimethylformamide (DMF) and dimethyl sulfoxide (DMSO). In DMF, 25–35% weight increase was found due to conjugation of hydantoin derivatives and 8–28% for uracil derivatives. Conjugation in DMSO resulted in almost 30% weight increase for both derivatives. The conjugation of N-halamine molecules via a nitrogen atom to chloromethyl polystyrene was traced using FTIR instrumentation and elemental analysis. FTIR spectra of the conjugated hydantoin/uracil polystyrene are given in (Fig. 2A and B), respectively. The peak at 1264 cm−1 attributed to –CH2–Cl vibration in starting material disappeared, while new carbonyl peaks were identified at 1630–1780 cm− 1. No difference in the FTIR spectra was found for beads conjugated in DMSO compared to DMF mediated conjugation. Elemental analysis indicated nitrogen content of 7.35–9.93% for hydantoin conjugated polystyrene (HD-Ps) and 1.37–8.79% for uracil conjugated polystyrene (UR-Ps) (Table 2). Conjugation at DMF resulted in an increase in nitrogen content %. A low conjugation yield was found for uracil groups, particularly orotic and uric acid as determined by N% as low as 1.92 and 1.37 respectively (Table 2).

Fig. 3. Representative FT-IR spectra for hydantoin/6-aminouracil conjugated polystyrene before and after hydrolysis in aqueous barium hydroxide. (A) Starting material chloromethyl polystyrene (CMPS). (B) and (C) hydantoin conjugated polystyrene (HD-Ps) before and after hydrolysis, respectively. (D) and (E) 6-aminouracil conjugated polystyrene (AUR-Ps) before and after hydrolysis, respectively.

S. Farah et al. / Journal of Controlled Release 216 (2015) 18–29

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Table 3 FTIR spectra comparison and similarity % calculation of bead samples before and after conjugated hydantoin/uracil derivatives hydrolysis. Conjugated beadsa

Beads after hydrolysisb

Carbonyl peaksc

Spectra similarity%d

HD-Ps DMH-Ps DPH-Ps All-Ps UR-Ps AUR-Ps OA-Ps UA-Ps

HD/NH2-Ps DMH/NH2-Ps DPH//NH2-Ps All/NH2-Ps UR/NH2-Ps AUR/NH2-Ps OA/NH2-Ps UA/NH2-Ps

Completely disappeared Decreased intensity⁎ Slight decreased intensity Completely disappeared Decreased intensity⁎ Completely disappeared Completely disappeared Completely disappeared

32.15 80.98 94.13 51.27 91.63 59.56 77.08 61.83

a Abbreviation of conjugated polystyrene: (HD) — hydantoin, (DMH) — 5,5′-dimethylhydantoin, (DPH) — 5,5′-diphenylhydantoin, (ALL) — allantoin, (UR) — uracil, (AUR) — 6-aminouracil, (OA) — orotic acid and (UA) uric acid. b Hydrolyzed in aqueous barium hydroxide (20% w/v) at reflux for 1 week. c Carbonyl peaks indicate the presence of conjugated hydantoin or uracil derivatives monitored by Smart iTR instrument, Nicolet iS10. d Spectra similarity % was conducted using OMNIC software by overlaying FTIR spectrums before and after hydrolysis. ⁎ Carbonyl peaks intensity decreased significantly, indicating incomplete hydrolysis/or partially hydrolyzed molecules still attached.

3.2. Quantitative analysis of conjugation and Ninhydrin assay When hydantoins are heated for relatively long periods of time with a large excess of barium hydroxide in aqueous solution, a profound breakdown takes place, and α-amino acids are obtained [32]. Using

this property of hydantoins, a quantitative method for determination of conjugation of hydantoin molecules to polymeric resin (mmol/g) was developed. This also applies to uracil conjugated beads that were conjugated hydantoins and uracil molecules hydrolyzed in aqueous barium hydroxide for released α-amino acids and amino acrylic acid

Fig. 4. Quantification of released α-amino acid/amino acrylic acid using Ninhydrin assay. (A). focus on color development resulted from the reaction between α-amino acid/amino acrylic acid and ninhydrin, monitored by spectrophotometer at 570 nm. Alanine standard samples, 2–40 μg/ml, were prepared according to experimental section (Quantitative analysis of conjugated hydantoin — Ninhydrin assay). (B). Calibration curve of alanine 2–40 μg/ml analyzed by the ninhydrin test. Samples were diluted and analyzed, and results were presented in mmol/g, Table 4.

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S. Farah et al. / Journal of Controlled Release 216 (2015) 18–29

Table 4 Conjugation analysis of hydantoin/uracil–polystyrene. Descriptiona

Conjugation-Method I (mmol/g)b

Conjugation-Method II (mmol/g)c

Conjugation-Method III (mmol/g)d

HD-Ps (DMF) HD-Ps (DMSO) DMH-Ps (DMF) DMH-Ps (DMSO) DPH-Ps (DMF) All-Ps (DMF) All-Ps (DMSO) UR-Ps (DMF) AUR-Ps (DMF) AUR-Ps (DMSO) OA-Ps (DMF) UA-Ps (DMF)

3.85 2.51 4.80 4.00 5.70 2.84 1.75 3.24 2.05 1.81 1.17 0.41

3.83 2.42 3.80 3.07 3.45 2.79 1.67 2.67 2.03 1.75 1.13 0.43

3.50 2.31 3.37 2.81 3.21 2.63 1.52 2.53 1.93 1.97 1.07 0.38

a b c d

Abbreviation of conjugated polystyrene — See Table 3. Method I: Calculated conjugation (mmol/g) using elemental analysis reported in Table 2. Method II: Calculated conjugation (mmol/g) using Ninhydrin assay for quantification released α-amino acid/amino acrylic acid (Scheme 3). Method III: Calculated conjugation (mmol/g) using Ninhydrin assay for quantification of resulted primary amino groups formed on-beads (Scheme 3).

respectively (Scheme 3). Barium was separated in the form of fast solid precipitate of barium sulfate after adding sodium sulfate. Conjugated molecule hydrolysis resulted in primary amino functionalized resin as confirmed by FTIR. Representative FTIR spectra are given in (Fig. 3). An overlay between spectra before and after hydrolysis was produced using OMNIC software as summarized in Table 3. All the conjugated molecules were found to hydrolyze. Hydantoin derivative conjugatedPs substituted a conjugated hydantoin ring to exhibit an increased chemical stability. The least ring hydrolysis was found for 5,5′-diphenylhydantoin conjugated molecule, with 94% spectra similarity%, while 80% and 32% for 5,5′-dimethylhydantoin and hydantoin conjugated, respectively (Table 3). Quantification of released α-amino acid/amino acrylic acid was determined using the Ninhydrin assay based on color development. The reaction between α-amino acid/amino acrylic acid and Ninhydrin was monitored by spectrophotometer at 570 nm (Fig. 4A). A calibration curve of alanine 2–40 μg/ml was prepared (Fig. 4B). For bead/release amino molecule solution, samples were diluted before being analyzed,

and the results of conjugation were presented in mmol/g of derivative conjugated beads. Using the previous calibration curve, conjugation loading for the different derivatives was tested and calculated, and compared to conjugation values calculated from elemental analysis (Table 4). The same assay was also used to quantify the resulted primary amino groups onto polymeric resin. Hydantoin derivatives conjugation was more successful than uracil derivatives. Moreover, quantification analysis of the resultant amino polymeric resin was less accurate and of lower values than freely released amino acid/amino acrylic acid (Table 4). These aminobeads were analyzed by elemental analysis for nitrogen content (Table 2). 3.3. Bromination of conjugated hydantoin/uracil polystyrene Conjugated bead bromination at basic condition was ineffective with less than 5% after 48 h. Therefore, all conjugated hydantoin/uracil polystyrene bromination was conducted as described in (Scheme 2). A

Table 5 Elemental analysis of brominated hydantoin/uracil conjugated polystyrene, before/after release study and loading/reloading capacity.⁎ Descriptiona

Bromine release

C%

H%

N%

Br%

Descriptiona

Bromine release

C%

H%

N%

Br%

Br-HD-Ps (DMF)b Br% (weight)c Br-HD-Ps (DMF)b Reloading after 3 cyclesb,d Br-HD-Ps (DMSO)b Br% (weight)c Br-HD-Ps (DMSO)b Reloading after 3 cyclesb,d Br-DMH-Ps (DMF)b Br% (weight)c Br-DMH-Ps (DMF)b Reloading after 3 cyclesb,d Br-DMH-Ps (DMSO)b Br% (weight)c Br-DMH-Ps (DMSO)b Reloading after 3 cyclesb,d Br-DPH-Ps (DMF)b Br% (weight)c Br-DPH-Ps (DMF)b Reloading after 3 cyclesb,d Br-All-Ps (DMF)b Br% (weight)c Br-All-Ps (DMF)b Reloading after 3 cyclesb,d

Before Before After 270 l

54.03

4.41

6.07

3.73

6.79

Before Before After 180 l

4.55

5.20

62.88

4.77

3.68

54.50

4.61

4.32

56.39

4.37

4.70

61.49

4.71

4.26

59.99

4.44

5.00

52.85

4.91

6.77

53.37

4.55

3.62

63.46

5.62

7.98

61.79

4.70

3.44

53.34

5.01

3.92

53.68

4.20

2.46

64.24

5.49

6.75

59.82

4.30

2.41

60.46

4.50

5.40

54.90

4.55

1.61

68.21

4.86

5.59

65.24

5.12

1.66

58.41

4.99

4.89

59.43

5.17

1.07

64.87

5.19

4.86

Br-All-Ps (DMSO)b Br% (weight)c Br-All-Ps (DMSO)b Reloading after 3 cyclesb,d Br-UR-Ps (DMF)b Br% (weight)c Br-UR-Ps (DMF)b Reloading after 3 cyclesb,d Br-AUR-Ps (DMF)b Br% (weight)c Br-AUR-Ps (DMF)b Reloading after 3 cyclesb,d Br-AUR-Ps (DMSO)b Br% (weight)c Br-AUR-Ps (DMSO)b Reloading after 3 cyclesb,d Br-OA-Ps (DMF)b Br% (weight)c Br-OA-Ps (DMF) b Reloading after 3 cyclesb,d Br-UA-Ps (DMF)b Br% (weight)c Br-UA-Ps (DMF) b Reloading after 3 cyclesb,d

58.21

63.03

25.27 24.13 13.82 25.43 23.07 22.59 18.18 23.67 24.12 21.32 13.69 24.32 25.26 22.42 15.19 25.11 19.28 16.43 12.14 19.45 21.51 19.23 14.13 22.07

66.50

5.11

0.89

19.90 17.78 14.76 19.88 21.49 19.98 18.82 21.72 25.27 23.47 19.25 25.43 25.36 23.03 20.60 25.67 24.90 22.28 16.80 25.12 16.64 15.12 13.39 16.88

Before Before After 110 l Before Before After 300 l Before Before After 110 l Before Before After 300 l Before Before After 180 l

⁎ C, H, N, and Br content of brominated hydantoin/uracil conjugated beads (%C, %H, %N ± 0.3%, %Br ± 0.5%). a Abbreviation of brominated-conjugated polystyrene: See Table 3. b Bromine content % determined by elemental analysis. c Found bromine % using iodometric/thiosulfate titration method and Eq. (1). d Bromine content % after 3 cycles of bromine extraction and reloading for brominated beads used for release study.

Before Before After 110 l Before Before After 150 l Before Before After 110 l Before Before After 150 l Before Before After 150 l

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Table 6 Carbonyl peaks changes due to bromination of hydantoin/uracil conjugated polystyrene (Br-HD-Ps and Br-UR-Ps). Conjugated beadsa

Carbonyl peaks before bromination (cm−1)

Brominated beadsa

Carbonyl peaks after bromination (cm−1)

Spectra similarity%b

HD-Ps DMH-Ps DPH-Ps All-Ps UR-Ps AUR-Ps OA-Ps UA-Ps

1771.04, 1699.27 1771.79, 1704.50 1773.74, 1708.61, 1661.82 1779.28, 1698.70, 1603.39 1651.99 1698.99, 1622.09 1698.13, 1651.53,1604.50 1782.02, 1726.57, 1698.72

Br-HD-Ps Br-DMH-Ps Br-DPH-Ps Br-All-Ps Br-UR-Ps Br-AUR-Ps Br-OA-Ps Br-UA-Ps

1770.71, 1699.16 1768.64, 1697.96 1772.29, 1705.67 1783.20, 1697.76, 1603.38 1682.70, 1604.50 1697.47, 1602.91 1694.31, 1603.28 1779.16, 1724.57, 1698.16

85.35 83.75 92.54 85.67 65.06 49.59 78.76 80.72

a Abbreviation of conjugated/brominated-conjugated polystyrene: (HD) — hydantoin, (DMH) — 5,5′-dimethylhydantoin, (DPH) — 5,5′-diphenylhydantoin, (ALL) — allantoin, (UR) — uracil, (AUR) — 6-aminouracil, (OA) — orotic acid and (UA) uric acid. b Spectra similarity % was conducted using OMNIC software by overlaying FTIR spectra before and after bromination.

distinct difference in bromine loading capacity was found for the different conjugated derivatives (Table 5). The resultant bromine content% was found vary to 19–26% for conjugated hydantoin polystyrene while 16–25% for conjugated uracil polystyrene (Table 5). Due to amide bromination, nearby carbonyl groups are partially

affected and slightly decrease their vibrational values. Changes in vibration of carbonyl groups of conjugated hydantoin/uracil derivatives ring were followed by FTIR analysis. These changes in carbonyl group and spectra similarity % before and after bromination are summarized in Table 6.

Fig. 5. (A). Hypobromous acid release (ppm) from brominated hydantoin derivative conjugated polystyrene. Brominated beads were released till: Br-HD-Ps (DMSO) and Br-DMH-Ps (DMSO) 110 l, Br-ALL-Ps (DMF, DMSO) 180 l, Br-HD-Ps (DMF) 270 l, Br-DMH-Ps (DMF) and Br-DPH-Ps (DMF) 300 l. (B). Hypobromous acid release (ppm) from brominated uracil derivative conjugated polystyrene. Brominated beads were released until: Br-UR-Ps (DMF) and Br-AUR-Ps (DMSO) 110 l, Br-AUR-Ps (DMF) and Br-OA-Ps (DMF) and Br-UA-Ps (DMF) 150 l. Hypobromous acid was traced using kit DPD-1 and spectrophotometer at 528 nm wavelength. Release is per 16 g of brominated beads in columns of 28 mm in diameter. Each column was washed by passing 25 l of water per day at a rate of 10 ± 1 min/l. Hypobromous acid concentration was measured every 10–15 l until reaching the level of 0.01 ppm in the passing water.

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3.4. Hypobromous acid release from brominated hydantoin/uracil polystyrene derivatives (Br-HD-Ps and Br-UR-Ps) Hypobromous acid release from brominated hydantoin/uracil polystyrene derivatives (Br-HD-Ps and Br-UR-Ps) was studied in columns. Brominated 5,5′-dimethyl and diphenylhydantoin polystyrene beads exhibited extended release profile than brominated hydantoin polystyrene beads (Fig. 5A). This may be explained by the presence of two methyl/phenyl groups on the fifth carbon of the hydantoin ring. Another explanation may be electron donating groups and partially deactivatng the nearby N–Br bound by reducing the electronegativity difference, thus reducing reactivity of N–Br for oxidative Br release. It should be noted that the differences in the activity of the Nhaloamine may be explained by the effect of substitution groups on carbon 5 of the hydantoin ring. These substituents contribute to either, electron donation, steric hindrance, lyophilicity of the N-halamine moiety and intramolecular through-space interaction π-electrons of the aromatic ring and the hydrogen/halogen of the N1 amide, as reported by Kocer et al. [20,37]. Methyl/phenyl group presence above and below the hydantoin plane constitutes a steric hindrance, which limits water flow to reach the N–Br for hydrolysis. This is different from hydantoin, where the largest part of the released bromine is in the first few liters (b 25 l). An elemental analysis of bromine % after release study (Table 5) emphasizes the previously described issue where Br-HD-Ps was released (11.5%

for 270 l) and reached the lower limit of 0.01 ppm, while Br-DMHPs and Br-DPH-Ps were released 10.4% and 7.1%, respectively for 300 l. Nevertheless, hydantoin conjugated beads prepared in DMSO released faster than when prepared in DMF (Fig. 5A). For brominated uracilbeads, it was found that brominated 6-aminouracil, orotic acid and uric acid polystyrene beads exhibited a greater extended release profile than brominated uracil polystyrene (Br-UR-Ps) for 150 l (Fig. 5B). This may be explained by the presence of carboxylic/amino, electron withdrawing/donating group, respectively, on the carbon near the double bond of the uracil ring, they neutralize/minimize respectively. Br-OAPs released 8.1% of its bromine content, while Br-AUR-Ps released 6.0%, Br-UA-Ps — 3.3% and Br-UR-Ps — 2.7% (Table 5). Generally, the remaining bromine % after release study was too high with the possibility of extra extended release for hundreds of liters. Beads after release were extracted from the remaining bromine and examined for reloading ability and capacity for 3 cycles of re-bromination and extraction. Bromine loading % was examined and found identical to loading% before release study (Table 5). 3.5. Antimicrobial activity Evaluation of the bactericidal effectiveness of the above prepared brominated HD/UR-Ps resin was done in columns according to NSF 231 protocol with different testing points. A set of identical columns (diameter 28 mm) containing 16 g of the tested materials was prepared.

Table 7 Antimicrobial activity of brominated hydantoin/uracil-beads against E. coli and MS2 bacteriophage. Challenge pointa

Brominated hydantoin-Ps (Br-HD-Ps) Output Br+ (ppm)

Flow rate (min/l)b

0l 75 l 150 l 300 l

20 1.04 0.03 0.01

10 10 10 10

Challenge pointa

Brominated diphenylhydantoin-Ps (Br-DPH-Ps) Flow rate (min/L)b

0L 75 l 150 l 300 l

16.3 1.73 0.75 0.33

10 10 10 10

Challenge pointa

Brominated uracil-Ps (Br-UR-Ps)

c

N7.4 N7.4 6.0 4.5

MS2

d

N3.9 N3.9 NTe NTe

Output Br+ (ppm)

Flow rate (min/l)b

40 1.20 0.48 0.11

10 10 10 10

Log reduction E. colic

MS2d

N7.4 N7.4 N7.3 N7.3

N3.9 N3.9 NTe NTe

Brominated allantoin-Ps (Br-HD-Ps) Log reduction E. coli

c

N7.4 N7.4 N7.3 N7.3

MS2

d

N3.9 N3.9 NTe NTe

Output Br+ (ppm)

Flow rate (min/L)b

9.5 0.22 0.01 b0.01

10 10 10 10

Log reduction E. colic

MS2d

N7.4 N7.4 6.2 b1.0

N3.9 N3.9 NTe NTe

Brominated aminouracil-Ps (Br-AUR-Ps)

Output Br+ (ppm)

Flow rate (min/l)b

0l 75 l 150 l

52.7 0.01 b0.01

10 10 10

Challenge pointa

Brominated orotic acid-Ps (Br-OA-Ps)

a

Log reduction E. coli

Output Br+ (ppm)

0l 75 l 150 l

Brominated dimethylhydantoin-Ps (Br-DMH-Ps)

Output Br+ (ppm)

Flow rate (min/l)b

12.3 0.45 0.20

10 10 10

Log reduction E. colic

MS2d

N7.4 4.5 b1.0

N3.9 N3.9 NTe

Output Br+ (ppm)

Flow rate (min/l)b

28.6 0.44 0.11

10 10 10

Log reduction E. colic

MS2d

N7.4 N7.4 N7.3

N3.9 N3.9 NTe

Brominated uric Acid-Ps (Br-UA-Ps) Log reduction E. coli N7.4 N7.4 N7.3

c

MS2 N3.9 N3.9 NTe

d

Output Br+ (ppm) 3.9 0.28 0.21

Flow rate (min/l)b

10 10 10

Log reduction E. colic

MS2d

N7.4 N7.4 N7.3

N3.9 N3.9 NTe

Bacterial Log Inlet (E. coli, MS2) were: 0 l (7.4, 3.9), 75 l (7.4, 3.9), 150 l (7.3, 3.9), 300 l (7.3, NTe). HOBr Release study from Br-HD-Ps and Br-UR-Ps derivatives were done in columns (28 mm wide) were fitted with 16 g of dry brominated beads, and each column was washed with 25 l of water per day at a rate of 10 ± 1 min/l. Hypobromous acid concentration was measured every 10–15 l until reaching 0.01 ppm. c E. coli: log (CFU/100 ml). d MS2: log (PFU/ml). e NT – not tested. b

S. Farah et al. / Journal of Controlled Release 216 (2015) 18–29

The microbiological quality of the water from the tested purifiers was monitored for 20 days. The ability of tested materials to inactivate the E. coli bacteria and MS2 bacteriophages in contaminated water was tested. The detection limit for E. coli bacteria is 1 CFU/100 ml, and for MS2 bacteriophages it is 1 PFU/1 ml. The average log reduction of E. coli bacteria was calculated at each challenge point. The concentration of residual bromine in effluent was also monitored at each challenge point (Table 7). All tested materials exhibit excellent antimicrobial properties, while Br-DMH-Ps (DMF) and Br-DPH-Ps (DMF) maintained 7 log reduction of E. coli for all tested points during 300 l. Brominated uracil derivatives were less active than brominated hydantoins, however Br-AUR-Ps (DMF), Br-OA-Ps (DMF) and BrUA-PS (DMF) maintained 7 log reduction of E. coli for all tested points during 150 l (Table 7). Both tested groups were tested against bacteriophages MS2 in the first 75 l, average log reduction of MS2 coliphages, and the concentration of residual bromine in effluent was traced. All the materials exhibited good activity of ~ 4 log reduction against MS2 (Table 7). Conjugated beads in DMSO after bromination exhibited moderated antimicrobial activity against E. coli and MS2. 4. Conclusions In summary, polystyrene beads with conjugated hydantoin or uracil derivatives were successfully synthesized in high yields from chloromethyl polystyrene beads in DMF or DMSO. A distinct difference between the loading capacity of bromine % for the different conjugated beads and also different release profiles was found. When comparing hydantoin derivatives (5 member ring) and uracil derivatives (6 member ring) the former showed extended release, while electron withdrawing groups substituted onto uracil increased the release of bromine to a sufficient level for antimicrobial activity. All tested materials exhibited strong antimicrobial activity against E. coli with 7 log reduction, while Br-DMH-Ps and Br-DPH-Ps maintained their activity for filtered 300 l. Brominated uracil derivatives maintained their antimicrobial activity for 150 l, which make them candidates for disinfection agents. All of the materials exhibit 4 log reduction against bacteriophages MS2 for 75 l. These results spotlight the antimicrobial potential of halogenated cyclic molecules containing urea groups.

OA-Ps UA-Ps

Abbreviation of polymeric resin and molecules CMPS chloromethyl polystyrene Ps polystyrene HD hydantoin DMH 5,5′-dimethylhydantoin DPH 5,5′-diphenylhydantoin ALL allantoin UR uracil AUR 6-aminouracil OA orotic acid UA uric acid Abbreviation of conjugated polystyrene Ps polystyrene HD-Ps hydantoin conjugated polystyrene DMH-Ps 5,5′-dimethylhydantoin conjugated polystyrene DPH-Ps 5,5′-diphenylhydantoin conjugated polystyrene ALL-Ps allantoin conjugated polystyrene UR-Ps uracil conjugated polystyrene AUR-Ps 6-aminouracil conjugated polystyrene

orotic acid conjugated polystyrene uric acid conjugated polystyrene

Abbreviation of brominated-conjugated polystyrene Br-HD-Ps brominated hydantoin conjugated polystyrene Br-DMH-Ps brominated 5,5′-dimethylhydantoin conjugated polystyrene Br-DPH-Ps brominated 5,5′-diphenylhydantoin conjugated polystyrene Br-ALL-Ps brominated allantoin conjugated polystyrene Br-UR-Ps brominated uracil conjugated polystyrene Br-AUR-Ps brominated 6-aminouracil conjugated polystyrene Br-OA-Ps brominated orotic acid conjugated polystyrene Br-UA-Ps brominated uric acid conjugated polystyrene

References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21]

Abbreviations DMF N,N-dimethylformamide DMSO dimethyl sulfoxide

29

[22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34]

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