Antifungal and antibacterial activity of 3-alkylpyridinium polymeric analogs of marine toxins Ana Zovko a , Maja Vaukner Gabri c b , Kristina Sep ci c a , Franc Pohleven b , Domen Jakli c a , Nina Gunde-Cimerman a , Zhibao Lu c , RuAngelie Edrada-Ebel d , Wael E. Houssen c , Ines Mancini e , Andrea Defant e , Marcel Jaspars c , Tom Turk a, * a Department of Biology, Biotechnical Faculty, University of Ljubljana, Vecna pot 111, 1000 Ljubljana, Slovenia b Department of Wood Science and Technology, Biotechnical Faculty, University of Ljubljana, Rozna dolina, Cesta VIII/34, 1000 Ljubljana, Slovenia c Marine Biodiscovery Centre, Department of Chemistry, University of Aberdeen, Aberdeen AB24 3UE, Scotland, UK d Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, John Arbuthnott Building, 27 Taylor Street, Glasgow G4 0NR, UK e Laboratorio di Chimica Bioorganica, Università di Trento, via Sommarive 14, I-38123 PovoTrento, Italy article info Article history: Received 27 September 2011 Received in revised form 27 October 2011 Accepted 28 October 2011 Available online xxx Keywords: Synthetic analogs Alkylpyridinium compounds Microwave-assisted polymerization Antifungal activity Antibacterial activity Wood-decay fungi abstract Analogs of marine sponge-derived 3-alkylpyridinium compounds (3-APS) were synthesized and screened for possible antibacterial and antifungal activities. They were found to exhibit moderate anti- bacterial activity. Antifungal potential was tested on pathogenic fungus Candida albicans, baker’s yeast Saccharomyces cerevisiae and hypersaline species Wallemia sebi. S. cerevisiae was the most susceptible to the action of selected 3-APS. Inhibitory effects on fungal growth were also studied on two wood-rotting fungi, brown-rot fungus Gloeophyllum trabeum and a white-rot fungus Trametes versicolor . The former showed a higher susceptibility to the action of 3-APS. The highest antifungal potential was observed with the poly-1,3-dodecyl pyridinium chloride (APS12-3, 7), while a complete loss of activity was noticed with the poly-1,3-butyl pyridinium chloride (APS3, 1), suggesting that this activity may closely correlate to the length of their alkyl chains. Based on our results, synthetic APS12-3 is a good candidate to be used as biocide or wood preservative against wood-rotting fungi. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction Natural and synthetic monomeric, oligomeric, and polymeric alkylpyridinium compounds (3-APS), bearing both positive charges and hydrophobic alkyl moieties, are known to exert a wide range of biological effects (Turk et al., 2008). Among the most studied naturally occurring polymeric 3-APS, there are the 3- octylpyridinium salts (poly-APS) (Fig. 1). These polymers were isolated from the marine sponge Reniera (Haliclona) sarai (Sepcic et al., 1997a,b), and show plenty of biological activity, including inhibition of acetylcholinesterase, antimicrobial potential, hemo- lytic and cytotoxic properties, as well as stable transfection of mammalian cells (Turk et al., 2008). They were also found to effi- ciently inhibit the settlement of several micro and macroorganisms on submerged surfaces, and to exert a reversible, non-toxic anti- fouling activity against Balanus amphitrite cypris larvae (Faimali et al., 2003; Garaventa et al., 2003). 3-APS bear significant struc- tural resemblance to tertiary amine salts and quaternary ammo- nium compounds that have been used in wood preservation (Eaton and Hale, 1993). The latter have been shown to exert their anti- bacterial activity by disrupting the bacterial cell membrane as a consequence of a hydrophobic interaction between their long alkyl chains and the cell surface (Shirai et al., 2006). However, the antifungal potential of natural poly-APS against two wood-decay fungal species, Trametes versicolor and Coniophora putaena, was found negligible up to the concentrations of 5 mg ml 1 (Elersek et al., 2008). In view of the potential use of poly-APS-like compounds as new eco-friendly antifouling compounds, or as transfection vehicles in gene therapy, further efforts to chemically synthesize their analogs have been made. Mancini et al. (2004) successfully synthesized dimers and tetramers of linear 3-alkylpyridinium salts. Their anti- fouling potential against settlement of Balanus amphitrite larvae was tested (Faimali et al., 2005), and found to be of lower potency compared to naturally occurring poly-APS. Recently, three new large analogs of natural poly-APS, APS12, APS12-2, and APS8 of * Corresponding author. Tel.: þ3861 423 33 88; fax: þ3861 257 33 90. E-mail address: [email protected](T. Turk). Contents lists available at SciVerse ScienceDirect International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod 0964-8305/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2011.10.014 International Biodeterioration & Biodegradation 68 (2012) 71e77
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International Biodeterioration & Biodegradation 68 (2012) 71e77
Contents lists available
International Biodeterioration & Biodegradation
journal homepage: www.elsevier .com/locate/ ibiod
Antifungal and antibacterial activity of 3-alkylpyridinium polymeric analogsof marine toxins
Ana Zovko a, Maja Vaukner Gabri�c b, Kristina Sep�ci�c a, Franc Pohleven b, Domen Jakli�c a,Nina Gunde-Cimerman a, Zhibao Lu c, RuAngelie Edrada-Ebel d, Wael E. Houssen c, Ines Mancini e,Andrea Defant e, Marcel Jaspars c, Tom Turk a,*
aDepartment of Biology, Biotechnical Faculty, University of Ljubljana, Ve�cna pot 111, 1000 Ljubljana, SloveniabDepartment of Wood Science and Technology, Biotechnical Faculty, University of Ljubljana, Ro�zna dolina, Cesta VIII/34, 1000 Ljubljana, SloveniacMarine Biodiscovery Centre, Department of Chemistry, University of Aberdeen, Aberdeen AB24 3UE, Scotland, UKd Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, John Arbuthnott Building, 27 Taylor Street, Glasgow G4 0NR, UKe Laboratorio di Chimica Bioorganica, Università di Trento, via Sommarive 14, I-38123 Povo Trento, Italy
a r t i c l e i n f o
Article history:Received 27 September 2011Received in revised form27 October 2011Accepted 28 October 2011Available online xxx
0964-8305/$ e see front matter � 2012 Elsevier Ltd.doi:10.1016/j.ibiod.2011.10.014
a b s t r a c t
Analogs of marine sponge-derived 3-alkylpyridinium compounds (3-APS) were synthesized andscreened for possible antibacterial and antifungal activities. They were found to exhibit moderate anti-bacterial activity. Antifungal potential was tested on pathogenic fungus Candida albicans, baker’s yeastSaccharomyces cerevisiae and hypersaline species Wallemia sebi. S. cerevisiae was the most susceptible tothe action of selected 3-APS. Inhibitory effects on fungal growth were also studied on two wood-rottingfungi, brown-rot fungus Gloeophyllum trabeum and a white-rot fungus Trametes versicolor. The formershowed a higher susceptibility to the action of 3-APS. The highest antifungal potential was observed withthe poly-1,3-dodecyl pyridinium chloride (APS12-3, 7), while a complete loss of activity was noticed withthe poly-1,3-butyl pyridinium chloride (APS3, 1), suggesting that this activity may closely correlate to thelength of their alkyl chains. Based on our results, synthetic APS12-3 is a good candidate to be used asbiocide or wood preservative against wood-rotting fungi.
� 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Natural and synthetic monomeric, oligomeric, and polymericalkylpyridinium compounds (3-APS), bearing both positive chargesand hydrophobic alkyl moieties, are known to exert a wide range ofbiological effects (Turk et al., 2008). Among the most studiednaturally occurring polymeric 3-APS, there are the 3-octylpyridinium salts (poly-APS) (Fig. 1). These polymers wereisolated from the marine sponge Reniera (Haliclona) sarai (Sep�ci�cet al., 1997a,b), and show plenty of biological activity, includinginhibition of acetylcholinesterase, antimicrobial potential, hemo-lytic and cytotoxic properties, as well as stable transfection ofmammalian cells (Turk et al., 2008). They were also found to effi-ciently inhibit the settlement of several micro and macroorganismson submerged surfaces, and to exert a reversible, non-toxic anti-fouling activity against Balanus amphitrite cypris larvae (Faimali
: þ3861 257 33 90.
All rights reserved.
et al., 2003; Garaventa et al., 2003). 3-APS bear significant struc-tural resemblance to tertiary amine salts and quaternary ammo-nium compounds that have been used in wood preservation (Eatonand Hale, 1993). The latter have been shown to exert their anti-bacterial activity by disrupting the bacterial cell membrane asa consequence of a hydrophobic interaction between their longalkyl chains and the cell surface (Shirai et al., 2006). However, theantifungal potential of natural poly-APS against two wood-decayfungal species, Trametes versicolor and Coniophora putaena, wasfound negligible up to the concentrations of 5 mgml�1 (Eler�seket al., 2008).
In view of the potential use of poly-APS-like compounds as neweco-friendly antifouling compounds, or as transfection vehicles ingene therapy, further efforts to chemically synthesize their analogshave been made. Mancini et al. (2004) successfully synthesizeddimers and tetramers of linear 3-alkylpyridinium salts. Their anti-fouling potential against settlement of Balanus amphitrite larvaewas tested (Faimali et al., 2005), and found to be of lower potencycompared to naturally occurring poly-APS. Recently, three newlarge analogs of natural poly-APS, APS12, APS12-2, and APS8 of
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12.5, 14.7 and 11.9 kDa sizes, respectively, were synthesized bymeans of a microwave-assisted polymerization technique. Theywere assayed for their antibacterial, anti-acetylcholinesterase, andhemolytic activity, as well as for their potential to induce stabletransfection in mammalian cells (Houssen et al., 2010).
In this work, we report on synthesis of five further poly-APS-like3-alkylpyridinium synthetic analogs (3-APS), and their antibacte-rial and antifungal potential.
APS12 (5), APS12-2 (6), APS12-3 (7), and APS8-2 (8) (Table 1) weredissolved in deionized water to a final concentration of 10 mgml�1,and kept at 4 �C until use.
2.1.2. MicroorganismsThe Gram negative Escherichia coli, strain EXB-V1, and the Gram
positive Staphylococcus aureus, strain EXB-V54 were obtained fromthe local collection at the Department of Biology, University ofLjubljana.
The following wood-decay basidiomycetes were used: a brown-rot fungus Gloeophyllum trabeum, strain ZIM L 017, and a recentlyisolated white-rot fungus T. versicolor strain ZIM L*. Both fungalstrains were from the culture collection at the Department ofWoodScience and Technology, Biotechnical faculty, Ljubljana (Rasporet al., 1995).
Candida albicans EXF 525, Saccharomyces cerevisiae EXF 3362and Wallemia sebi EXF-958 strains were obtained from the localcollection at the Department of Biology, University of Ljubljana.
Table 1Molecular weights and antibacterial activity of 3-alkylpyridinim synthetic analogsagainst E. coli and S. aureus, expressed as the minimal inhibitory concentration(MIC).
2.1.3. Wood blocksAntifungal tests with the brown-rot fungus were carried out on
wood blocks made fromNorway spruce (Picea abies) and thewhite-rot fungus on blocks made of beech wood (Fagus sylvatica). Thedimensions of wood blocks in mini block test (MBT) were25�10� 5 mm, and the dimensions of wood blocks for the EN 113standard assay were 50� 25�15 mm. Orientation and quality ofthe wood as well as fungicidal tests met the requirements of thestandard EN 113 (European Committee for Standardization, 1996).
2.2. Methods
2.2.1. Synthesis e general methodsAll starting chemicals were purchased from Aldrich. The
microwave-assisted polymerizations were performed on BiotageInitiator Microwave Synthesizer (Uppsala, Sweden). NMR spectrawere recorded in CDCl3, CDCl3/CD3OD or D2O (as specified) ona Bruker AC-F 250 MHzNMR spectrometerwith Tecmag acquisitionsystem or on Varian Unity-INOVA 400 MHz system. Chemical shiftswere reported as d values relative to an internal standard, tetra-methylsilane (dH¼ 0.00 ppm) or 4,4-dimethyl-4-silapentane-1-sulfonic acid (DDS) in D2O. Coupling constants are given in Hz.Electrospray weight spectra of the synthesized compounds weremeasured on an LTQ/Orbitrap weight spectrometer equipped witha high resolution FT weight analyzer set up at 100,000 and exter-nally calibrated at 3 ppm. The samples were dissolved inMeOH:H2O (1:1 v/v) and were injected at a volume of 20 ml, runwith direct infusion of acetonitrile:H2O (90:10 v/v with 0.1% formicacid) at a flow rate of 200 ml min�1. Positive mode ionization wasaccomplished at a capillary temperature of 200 �C, a capillaryvoltage of 35.5 V and a source voltage of 4.0 kV. MS data was pro-cessed utilizing the centroid algorithm mode of the LTQ at a mergewidth of 5.0. High resolution MS data was processed using Xcalibur2.1 and the deconvolution software Proweight 1.2.8.
2.2.1.1. Compound 1 (APS3). Thionyl chloride (4.2 g, 36 mmol,1.2 equiv.) was added drop wise to a solution of 3-(3-hydroxypropyl)-pyridine (4.1 g, 30 mmol) in dichloromethane(25 mL) at room temperature over 30 min. The mixture was thenstirred for another 2 h and the solution was neutralized with 2 Mpotassium carbonate aqueous solution. The organic layer was driedwith magnesium sulfate, and concentrated. The residue was sub-jected to flash chromatography on silica gel using a mixture ofpetroleum ether and ethyl acetate (1:1 v/v) to give 3-(3-chloropropyl) pyridine (4.0 g, 85%). A solution of the later (4.0 g)in methanol (8.0 g) with trace of sodium iodide was subjected tomicrowave-assisted polymerization (130 �C, 8 bar, 30 w) for 60 h(monitored by 1H-NMR, CDCl3/CD3OD). The resulting mixture wasconcentrated and extracted with a mixture of petroleum ether anddichloromethane (1:1 v/v) to remove un-reacted monomer andsome oligomers. Final purificationwas carried out by size exclusionon Sephadex LH-20 column to give 1 (APS3) as brown oil (3.5 g).
The dimer C16H19N22þ exhibited at m/z¼ 239.2, represented
a multiple-charged species of þ5 and þ16 for 1.2 and 3.8 kDa,respectively. Deconvoluted data of the high resolution weightspectral data of APS3 indicated a polymerization grade of m¼ 10and 32 at a ratio of 9:1 for the monomer C8H9Nþ (seesupplementary information, Fig. 1).
2.2.1.2. Compound 2 (APS7). Thionyl chloride (4.2 g, 36 mmol,1.2 equiv.) was added drop wise to a solution of 7-(3-pyridyl)heptanol (5.8 g, 30 mmol) in dichloromethane (25 ml) at room
A. Zovko et al. / International Biodeterioration & Biodegradation 68 (2012) 71e77 73
temperature over 30 min. The mixture was then stirred for another2 h. The solution was neutralized with 2 M potassium carbonateand the organic layer was dried with magnesium sulfate, andconcentrated. The residue was subjected to flash chromatographyon silica gel using a mixture of petroleum ether and ethyl acetate(1:1 v/v) followed by ethyl acetate 100% as eluent to give 3-(7-chloroheptyl) pyridine (4.0 g, 63%). The later (4.0 g) was dissolvedin methanol (8.0 g) and subjected to microwave-assisted poly-merization (130 �C, 8 bar, 30 w) for 60 h (monitored by 1H-NMR,CDCl3/CD3OD). The resulting mixture was concentrated andextracted with a mixture of petroleum ether and dichloromethane(1:1 v/v) to remove un-reacted monomer and some oligomers.Final purificationwas carried out by size exclusion on Sephadex LH-20 to give 2 (APS7) as brown oil (3.5 g).
For APS7, its monomer C12H18Nþ shown as a multiple-chargedion peak at m/z¼ 176.1 yielded a mixture of two grades of poly-merization for m¼ 8 and 24 at a ratio of 2:1, which were indicatedby deconvoluted weight peaks at 1.4 and 4.2, respectively (seesupplementary information, Fig. 2).
2.2.1.3. Compound 3 (APS7-2). A mixture of 7-(3-pyridyl) heptanol(3.7 g, 20 mmol) and hydrobromic acid (4.1 g) in toluene (20 g) washeated under reflux overnight. The resulting mixture was thensubjected to flash column chromatography on silica gel usingdichloromethane 100% followed by 5% methanol in dichloro-methane as eluent to give 3-(7-bromoheptyl) pyridine bromide asa brown oil (0.3 g) which can be neutralized to give 3-(7-bromoheptyl) pyridine. The later (0.42 g) was dissolved in meth-anol (2.0 g) and subjected to microwave-assisted polymerization(130 �C, 8 bar, 30 w) for 12 h (monitored by 1H-NMR, CDCl3/CD3OD). The resulting mixture was concentrated and extractedwith a mixture of petroleum ether and dichloromethane (1:1 v/v)to remove un-reacted monomer and some low molecular weightoligomers. Final purification was carried out by size exclusion onSephadex LH-20 to give 3 (APS7-2) as brown oil (0.3 g).
1H-NMR spectrum is practically superimposable to the one forcompound 2. A similar monomer, C12H18N, as in APS7 was utilizedfor APS7-2. APS7-2 is a mixture of three grades of polymerization atm¼ 12, 24, and 35 which were indicated by deconvoluted weightpeaks at 2.1, 4.2, and 6.1 kDa, respectively at a ratio of 3:2:1(supplementary information, Fig. 3).
2.2.1.4. Compounds 4e6 (APS8, APS12, APS12-2). The compoundswere synthesized as described earlier (Houssen et al., 2010).
2.2.1.5. Compound 7 (APS12-3). Thionyl chloride (2.3 g, 20 mmol,2.0 equiv.) was added drop wise to a solution of 12-(3-pyridyl)-1-dodecanol (14) (2.63 g, 10 mmol) in dichloromethane (25 ml), atroom temperature over 30 min. The mixture was then stirred foranother 2 h. After that, the solution was neutralized with 2 Mpotassium carbonate aqueous solution. The organic layer was driedwith magnesium sulfate, and concentrated. The residue was sub-jected to flash chromatography on silica gel using a mixture ofpetroleumether and ethyl acetate (1:1 v/v) followedbyethyl acetate100% as a mobile phase to give 3-(12-chlorododecyl) pyridine(1.97 g, 70%). The later (0.5 g) in methanol (4.0 g) was subjected tomicrowave-assisted polymerization (130 �C, 8 bar, 30 w) for 60 h(monitored by 1H-NMR, CDCl3/CD3OD). The resulting mixture wasconcentrated and extracted with a mixture of petroleum ether anddichloromethane (1:1 v/v) to remove un-reacted monomer andsome oligomers. Final purificationwas carried out by size exclusionon Sephadex LH-20 column togive 7 (APS12-3) as brownoil (0.40 g).
The monomer, C17H28Nþ, was employed for APS12-3 whichyielded two grades of polymerization atm¼ 23 and 35 which wereindicated by the weight ion peak at m/z¼ 247.2 representing themultiple-charged species for 5.6 and 8.6 kDa, respectively at a ratioof 3:2 (see supplementary information, Fig. 4).
2.2.1.6. Compound 8 (APS8-2). The monomer used to producecompound 8 was synthesized as described in Gil et al. (1995). Themicrowave oligomerization was carried out in the same fashion asspecified for APS8.
1H-NMR (D2O): d 9.03 (br. s, 1H, H-2), 8.85 (br. s, 1H, H-6), 8.54(br. s 1H, H-4), 8.10 (m,1H, H-5), 4.63 (m, H-7 and H-14), 3.56 (m, H-9 and H-11), 2.06 and 1.64 (two m).
For APS8-2, the monomer, C12H18NOþ indicated by the weightion peak at m/z¼ 192.1, yielded 100% polymerization of m¼ 18resulting to a molecular weight of 3.4 kDa (see supplementaryinformation, Fig. 5). However, additional unexplainable weightion peaks were observed at m/z¼ 215.1 and 238.2 deconvolutingfor 5.3 and 1.6 kDa, respectively.
2.2.2. Determination of antibacterial activityAntibacterial activity was determined using the standard agar
diffusion test, as described by Mancini et al. (2004) in order toreveal the minimal inhibitory concentrations (MIC), e.g., the lowestconcentrations of the compounds that inhibit the growth of thetested microorganism. Briefly, bacteria were allowed to growovernight and their concentrations were then determined. Eachbacterial culture was incorporated in Luria Broth nutrient agarpreviously cooled to 42 �C. The final concentration of bacteria wasapproximately 5�105 colony forming unitsml�1. Twenty ml ofinoculated medium were poured into petri dishes and kept at 4 �Cuntil use. Rounded parts of agar (V¼ 1 cm) were cut out from thecooled medium. For estimating MIC, the compounds wereprogressively diluted in deionized water and 100 ml of each dilutionwas added to the wells in the inoculated agar plate. The plates werekept at 37 �C for 24 h. Finally, the diameters of inhibition zoneswere measured.
2.2.3. Antifungal activity on wood-decay fungiFor rapid determination of fungicidal activity, screening tests on
slope potato dextrose agar medium (PDA, Difco) were performedusing a broad concentration range of synthetic analogs (0, 0.001,0.005, 0.01, 0.05, and 0.1 mgml�1). Each concentrationwas assayedin 5 parallel trials. Each of 3-APS concentration (0.1 ml) has beenincorporated into agar and fungi were inoculated. After 7 days, themycelial growth of fungi was estimated by visual inspection.Fungicidal activity was estimated by fungal growth retardation,using the following visually determined marks:
0¼mycelium growth same like control,1¼ normal growth, insignificant retardation (area of colony�90% of area of controls),2¼ successful growth, visible signs of retardation (colony <90%and �60% of controls),3¼moderate growth, pronounced retardation (colony <60%and �25% of controls),4¼ unsuccessful growth, very marked retardation (colony<25%of controls),5¼ no growth
Further studies of fungicidal properties of 3-APS were per-formed according to EN 113 standard. Wood blocks were coated
Fig. 2. Synthesis of poly-(1, 3-alkylpyridinium) salts. Reagents and conditions: forR¼ alkyl chain: (i) HBr, toluene, reflux overnight followed by neutralization to yieldproducts with X¼ Br; thionyl chloride, dichloromethane, room temperature to yieldproducts with X¼Cl; (ii) reflux in acetonitrile or methanol (in the presence of a smallamount of KCl for monomeric chloride), followed by microwave irradiation at 130 �Cfor the time length stated for each compound under the experimental section.Numbering of compounds 1e8 is for convenience in the description of NMR data: APS3(1), APS7 (2), APS7-2 (3), APS8 (4), APS12 (5), APS12-2 (6), APS12-3 (7) and APS8-2 (8).
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with synthetic analogs with a concentration of 0.05 mgml�1. Jarswith PDA medium were inoculated with fungal mycelium. Beechwood specimens were exposed to white-rot fungi, and spruce oneswere exposed to brown-rot fungi. Sterilized and air-dried woodsamples were placed on a sterilized plastic grid on mycelia-overgrown PDA media and exposed to fungal decay in the growthchamber (25 �C, 75% relative humidity). In each inoculated jar, onetreated and one untreated (control) wood block were placed. Theresults were determined after 16 weeks. Weight losses of exposedblocks were determined gravimetrically on five parallel specimens(European Committee for Standardization, 1996). The treatmentresulted in a preservative uptake of about 100 kgm�3 for sprucewood and 90 kgm�3 for beech wood.
The fungicidal potential of the most active compound APS12-3was further assayed with a mini block test. Wood blocks wereimmersed in 3-APS solution with three different concentrations(0.05, 0.1, and 0.2 mgml�1). Since the uptake of solution was quitelow, the mini block test was further modified and the wood spec-imens were vacuum-impregnated in order to achieve better uptakeof the compound. Moreover, higher concentrations of 3-APS (0.5, 1,and 3 mgml�1) were used. In both experiments wood blocks weresterilized and air-dried. After being immersed, wood blocks wereplaced on a sterilized plastic grid onmycelia-overgrown PDAmediain petri dishes, and vacuum-impregnated blocks were placed intojars. In each inoculated petri dish or jar, one untreated control blockand three blocks treated with various concentrations of a certaincompound were placed. The specimens were conditioned for eightweeks at 25 �C and 75% relative humidity. Weight losses of exposedblocks were quantified gravimetrically on five parallel trials(European Committee for Standardization, 1996). The treatment ofimmersed blocks resulted in an uptake of solution of 0.63 kgm�3,while the uptake for vacuum-impregnated samples was1.21 kgm�3. Due to different modes of impregnation and fungalgrowth, uptake of the compound was not proportional to thecompound concentration and consequently not considered repre-sentative. Therefore, results are presented as a content of activecompound in each block, calculated from the concentration of thedissolved compound and the average volume of impregnatedsolution. In addition, percent of weight loss that was induced byAPS12-3 is presented relative to control.
2.2.4. Antifungal activity on other fungiCultures of C. albicans and S. cerevisiae were cultured in malt
extract agar (MEA) medium. Culture tubes containing 5 ml of MEAliquid mediumwere inoculated with fungal cells. Final cell count ineach test tube was approximately 104 cellsml�1. Tests were per-formed with a broad range of concentrations of synthetic analogsAPS8, APS12, APS12-2 and APS12-3 (from 0.005 mgml�1 to0.1 mgml�1). Each concentration was assayed in 3 parallel trials.After 48 h of incubation at 30 �C, from each test tube 100 ml weretransferred and spread over MEA agar plates. After 48 h incubationat 30 �C the MIC was determined. The MIC of an antifungal agent isdefined as the lowest concentration which inhibits the visiblefungal growth.
Cultures of W. sebi were cultured in liquid and on solid YeastNitrogen Base (YNB) medium with 5% NaCl as described by KraljKun�ci�c et al. (2010). Pre-culturing in the liquid YNB media wasused to adapt the fungi to the media and growth conditions. Onepercent of the pre-culture in the exponential phase of growth wasinoculated in sterile liquid YNB medium. After the culture hadreached exponential growth phase it was inoculated onto a solidYNB medium. Circles of agar (V¼ 1 cm) were cut out from theinoculated solid medium. For estimating MIC, the compounds werediluted in deionized water and 100 ml of each dilutionwas added tothe wells on agar plates. Each concentration was assayed in 3
parallel trials. Plates had been incubated at room temperature andafter W. sebi overgrew the plate, MIC was determined.
3. Results and discussion
Compounds structurally similar to those used in this study,bearing both positive charge and hydrophobic alkyl moieties, areknown to be disinfection agents exerting a broad range of antimi-crobial activity against bacteria, viruses, fungi, and algae (Kawabataand Nishiguchi, 1988, Kourai et al., 1995, Wainwright and Crossley,2004, De Muynck et al., 2009). In fact, the naturally occurring 3-alkylpyridinium polymers (poly-APS) can successfully inhibit thegrowth of marine and freshwater algae and bacteria in theconcentrations of a few tens of micrograms ml�1 (Chelossi et al.,2006; Eler�sek et al., 2008). They may act as unspecific antibioticsaffecting micro and macroorganisms, or as natural repelling agents.However, it should be mentioned that, up to the concentration of5 mgml�1 they did not exert fungicidal properties against thebrown-rot fungus C. putaena, and the white-rot fungus T. versicolor(Eler�sek et al., 2008).
The selected biological activities of synthetic 3-APS, withemphasis on their antifungal potential, are represented in thecontinuation of the text, and the activity in correlation to theirstructural features is discussed.
3.1. Synthesis and structural assignments of synthetic polymers
Poly-(alkylpyridinium) salts 1e8 were synthesized using themicrowave-assisted polymerization and following the scheme inFig. 2. The pyridyl-substituted alcohol substrates were prepared bycoupling of 3-picoline with either dibromoalkane or silyl-protectedbromoalcohol as previously reported (Houssen et al., 2010).Brominated monomer units were then prepared by refluxing of thealcohol substrates with hydrobromic acid followed by neutraliza-tion while chlorinated monomers were prepared by reaction of thesubstrates with thionyl chloride at room temperature. Monomer
Fig. 3. Effect of 3-alkylpyridinium synthetic analogs on mycelial growth of Gloeo-phyllum trabeum(A) and Trametes versicolor(B). The fungicidal activity of all thecompounds was assessed on mycelium-inoculated potato dextrose agar medium, asdescribed in the Materials and Methods section.
Table 2Effect of 3-alkylpyridinium synthetic analogs (0.05 mg/mL) on mass loss of woodsamples exposed for 16 weeks to the wood-decaying fungi Gloeophyllum trabeumand Trametes versicolor according to the EN 113 standard assay.
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solutions in acetonitrile or methanol were initially refluxed in thepresence of KI to generate small oligomers (Gil et al., 1995) thatwere concentrated and subjected tomicrowave irradiation. The sizeof the polymers was controlled by altering the irradiation time.
The synthetic materials were analyzed by electrospray ioniza-tion mass spectrometry (ESI-MS) with an LTQ/Orbitrap FT weightanalyzer, a method which was successfully used to identify thepreviously reported polymers 4e6 (Houssen et al., 2010). The dataindicate the low degree of poly-dispersity and the high degree ofpolymerization of the obtained products. APS3 (1) mixturecomprises polymers of 10 and 32monomer units, APS7 (2) containspolymers of 8 and 24 units; APS7-2 (3) was a mixture of polymerswith 12, 24, and 35monomer units while APS12-3 (7) has polymerswith 23 and 35 monomer units. Deconvolution with charge stateanalysis for each compoundwas consistent with its accurateweightanalysis and gave the same degree of polymerization. The cyclicnature of the final products was assumed based on the facts that thecharge state equaled the number of monomers in each moleculeand the terminal halogen was absent.
3.2. Determination of antibacterial activity
In accordance with previously published data on biologicalactivities of shorter 3-APS (Mancini et al., 2004), the activity of allthe tested compounds was the highest against a Gram positivebacterium S. aureus (MIC values from 0.03 to 1 mgml�1). Thehighest antibacterial potential was observed with APS12-3, fol-lowed by APS7-2, APS8B, and APS7. These activities were slightlyhigher than those obtained with APS12 and APS12-2 (Houssenet al., 2010; this work). APS3 and APS8-2 exhibited the lowestantibacterial potential (Table 1), with respectiveMIC values of 1 and10 mgml�1 against S. aureus and E. coli.
3.3. Antifungal activity on wood-decay fungi
The results of the screening tests on slope potato dextrose agarmedium (Fig. 3) showed the same trends of activity of different 3-APS, with APS12-3, APS12-2, and APS12 being the most active andcompletely inhibiting the growth of wood-decay fungi at theconcentration of 0.1 mgml�1. These are closely followed by APS7-2,APS8, and APS7. No activity up to the concentration of 0.1 mgml�1
was observed for APS3 and APS8-2. The brown-rot fungusG. trabeumwas slightly more susceptible to the action of 3-APS. Itsgrowth is already slightly inhibited at the concentration of fewmicrograms per milliliter, while comparable effects on the white-rot fungus T. versicolor were observed with approximately 10times higher concentrations.
The fungicidal potentialwas further surveyedwith the EN113 test,assaying the same concentration (0.05 mgml�1) of six different 3-APScompounds. The test revealed a series of active compounds from themost to the leastactiveone (APS12-3,APS12-2,APS8,APS12,APS7, andAPS7-2, respectively). The resultsof theaverageweight loss,presentedin Table 2, are not very significant, however they confirm the trendobserved in the previous test on mycelial growth inhibition, showing(i) the higher susceptibility of G. trabeum to the action of selected 3-APS, and (ii) the highest activity of APS12-3 (approximately 13%wood weight loss in the test with G. trabeum), followed by APS12-2,APS12, APS8, and APS7-2, each one induced a weight loss of approx-imately 5%. According to Unger et al. (2001) white-rot fungi havehigher resistance in comparison to brown-rot ones. The former aresupposed to be able to biotransformorganic biocideswhich can resultin unaltered growth or even in an increase in fungal growth, at least atthe lowest biocide concentrations.
Since the effects of 3-APS on fungal growth inhibition obtainedwith the EN 113 test were not so obvious, the test was repeated
with a mini block assay. The test was performed only with the mostactive compound APS12-3. Higher concentrations of APS12-3 (0.05,0.1, and 0.2 mgml�1) were used. As G. trabeum showed a highersusceptibility to the action of 3-APS only this fungal species wastested. APS12-3 showed to be a good inhibitor of wood-decayfungal growth. However, the uptake of 3-APS solution was quitelow since samples contained about 0.78 mg of aqueous solution ofAPS12-3. In order to achieve adequate retentions of compound,mini blocks were impregnated with different aqueous solution ofAPS12-3 (0.5, 1, and 3 mgml�1, respectively) by means of vacuumsuction. This method proved to be more efficient than withoutvacuum, since on average mini blocks retained 1.5 mg of aqueoussolution.
Weight loss of unimpregnated control blocks was between 32and 53% which indicates that fungi were fully active and experi-mental proceeding was satisfactory (European Committee forStandardization, 1996).
Table 3Influence of different contents of 3-alkylpyridinium synthetic analog APS12-3 insamples on weight loss of mini wood blocks exposed for eight weeks to the wood-decaying fungus Gloeophyllum trabeum.
Table 4Antifungal activity of 3-alkylpyridinim synthetic analogs against C. albicans,S. cerevisiae and W. sebi, expressed as the minimal inhibitory concentration (MIC).
A. Zovko et al. / International Biodeterioration & Biodegradation 68 (2012) 71e7776
The results of average weight loss, presented in Table 3, showedthat APS12-3 is able to significantly decrease theweight loss, whichis only 9.5% relative to the control at concentration of 3 mgml�1.That corresponds to 5% actual weight loss (data not shown). Weightlosses lower than 3% are considered insignificant according to theEN 113 protocol. Due to an insufficient amount of compound wewere not able to determine which concentration of the compoundcompletely inhibited the growth of G. trabeum. Therefore, inter-polation techniques were required to obtain the most likelyconcentration that induced no weight loss in the wood samples.According to the interpolation graph (Fig. 4), for the total inhibitionof fungal growth a mini block should be impregnated with approx.7.6 mg of active compound. If we consider that average retentionusing vacuum impregnation was about 1.5 mg of aqueous solutionper mini block, than the APS12-3 concentration that entirelyinhibited the growth of G. trabeum should be approximately5 mgml�1. Concentration of 0.5% (w/v) (5 mgml�1) is acceptablefor commercial application. Based on these results, synthetic 3-APSAPS12-3 can be recommended as biocide in wood preservativesagainst G. trabeum. Its antifungal potential against other species ofwhite and brown wood-rotting fungi remains to be furtherelucidated.
3.4. Antifungal activity on other fungi
The antifungal potential of 3-APS was also checked againsta pathogenic fungus C. albicans, baker’s yeast S. cerevisiae, and thehypersaline species W. sebi. S. cerevisiae is a laboratory modelmicroorganism, C. albicans was chosen because of its opportunisticpathogenic characteristics, and W. sebi is a pathogenic
Fig. 4. Determination of concentration of APS12-3 required for total inhibition of thewood-decay fungus G. trabeum, obtained by interpolation technique. The dashed lineindicates the interpolation of weight loss values.
deuteromycete fungus. C. albicans EXF 525 strain was isolated froma human and these preliminary results should direct furtherinvestigations toward susceptibility tests against other pathogenicyeasts, black yeasts, and dermatophytic fungi which presenta serious problem in modern medicine. S. cerevisiae was slightlymore susceptible to the action of selected 3-APS than C. albicanswhileW. sebiwas the least susceptible. APS12-3 was again themostactive compound, with MIC values of 0.005, 0.01, and 0.02 mgml�1
against S. cerevisiae, W. sebi and C. albicans, respectively. The otherthree tested compounds (APS8, APS12, and APS12-2) showedcomparable potential against the tested fungi, being 1.25e12 foldless active than APS12-3 (Table 4).
4. Conclusions
Studies by different authors showed that the membrane-disrupting and antimicrobial potential of amphiphilic compoundscorrelates with their alkyl chain length, and increase in the positivecharge. We concluded that the fungicidal activity of 3-APS alsoclosely correlated to the length of their alkyl chains. In that regard,APS12-3 seemed to be the best candidate for the potential produc-tion of a protective fungicidal agent. Introduction of an oxygen atomto the alkyl chain, as in the case of APS8-2, dramatically reduces thebiological activity of the compound. The same trend could beobservedwith lowering the length of the alkyl chain, resulting in thecomplete loss of antifungal potential when applying 3-APS with theshortest (C3) linking chains. Considering the high stability of naturalpoly-APS, the environmental faith of 3-APS, as well as their putativetoxic effect on other micro and macroogranisms, should be consid-ered and will be studied in the future.
Acknowledgments
The authors gratefully acknowledge the Slovenian ResearchAgency for the financial support. This work was supported in partby a grant fund from NESTech. WEH is the recipient of a SULSApostdoctoral fellowship.
Appendix. Supplementary material
Supplementary data related to this article can be found online atdoi:10.1016/j.ibiod.2011.10.014.
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