Olefin Functionalized Bis-Imidazolium Gold(I)

12 Pages·2013·7.89 MB·English

N, N9-Olefin Functionalized Bis-Imidazolium Gold(I) Salt
Is an Efficient Candidate to Control Keratitis-Associated
Eye Infection
Tapastaru Samanta1, Gourisankar Roymahapatra1¤, William F. Porto3, Saikat Seth4, Sudipta Ghorai4,
Suman Saha5, Jayangshu Sengupta5, Octa´vio L. Franco3, Joydev Dinda1, Santi M. Mandal2*
1SchoolofAppliedSciences,HaldiaInstituteofTechnology,Haldia,WestBengal,India,2CentralResearchFacility,IndianInstituteofTechnologyKharagpur,Kharagpur,
West Bengal, India, 3Centro de Ana´lises Proteoˆmicas e Bioqu´ımicas, Po´s-Graduac¸a˜o em Cieˆncias Genoˆmicas e Biotecnologia UCB, Bras´ılia-DF, Brazil, 4Mugberia
GangadharCollege,EgraSaradaSashibhusanMahavidyalaya,WestBengal,India,5PriyamvadaBirlaAravindEyeHospital,Kolkata,WestBengal,India
Abstract
Keratitistreatmenthasbecomemorecomplicatedduetotheemergenceofbacterialorfungalpathogenswithenhanced
antibiotic resistance. The pharmaceutical applications of N-heterocyclic carbene complexes have received remarkable
attention due to their antimicrobial properties. In this paper, the new precursor, 3,39-(p-phenylenedimethylene) bis{1-(2-
methyl-allyl)imidazolium}bromide(1a)anditsanalogousPF salt(1b)weresynthesized.Furthermore,silver(I)andgold(I)-
6
N-heterocycliccarbene(NHC)complexes[Ag LBr /Au LBr ;2a/3a],[(Ag L )(PF ) /(Au L )(PF ) ;2b/3b]weredevelopedfrom
2 2 2 2 2 2 62 2 2 62
their corresponding ligands. All compounds were screened for their antimicrobial activities against multiple keratitis-
associated human eye pathogens, including bacteria and fungi. Complexes 2a and 3a showed highest activity, and the
effectivenessof3awasalsostudied,focusingeradicationofpathogenbiofilm.Furthermore,thestructuresof1a,2aand3b
weredeterminedusingsinglecrystalX-rayanalysis,2band3awereoptimizedtheoretically.Themechanismofactionof3a
wasevaluatedbyscanningelectronmicroscopyanddockingexperiments,suggestingthatitstargetisthecellmembrane.
In summary, 3a may be helpful in developing antimicrobial therapies in patients suffering from keratitis-associated eye
infectionscaused bymultidrug-resistant pathogens.
Citation:SamantaT,RoymahapatraG,PortoWF,SethS,GhoraiS,etal.(2013)N,N9-OlefinFunctionalizedBis-ImidazoliumGold(I)SaltIsanEfficientCandidateto
ControlKeratitis-AssociatedEyeInfection.PLoSONE8(3):e58346.doi:10.1371/journal.pone.0058346
Editor:MichaelHamblin,MGH,MMS,UnitedStatesofAmerica
ReceivedAugust26,2012;AcceptedFebruary3,2013;PublishedMarch15,2013
Copyright:(cid:2)2013Samantaetal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermits
unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
Funding:Theauthorshavenosupportorfundingtoreport.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
*E-mail:[email protected]
¤Currentaddress:GlobalInstituteofScienceandTechnology,Haldia,WestBengal,India
Introduction action [5]. Among them, gold (I) and silver (I)-NHCs seem to be
remarkable candidates for antibiotic development, due to their
Keratitisisacommoncornealinfectionintropicalareasofthe higheractivityandrelativelylowtoxicitytomammaliancellswhen
world.Thiskindofinfectionisquitedangerous,andinmanycases comparedtoothermetals,suchasRu(II),Ru(I),Cu(I)andPd(II)-
can lead to permanent blindness, specially if not diagnosed NHC complexes. Hindi et al. [6] summarized the antimicrobial
promptly and treated effectively. Keratitis can develope from activitiesofseveralNHCsshowingclearbenefits,butdemonstrat-
fungal(Candida,Aspergillus,Fusariumetc.)andbacterial(Staphylococcus ed the need for higher amounts in order to obtain effective
aureus,Pseudomonasaeruginosaetc.)infections,especiallythosecaused pathogen control. However, NHCs are in a versatile ligand class
bytheuseofcontactlensesorbyeyeinjuries[1,2].Theemergence whichhastheadvantageofeasymanipulation.Furthermore,they
of bacterial and fungal pathogens with enhanced antibiotic also have the ability to bind simply to metals and can be readily
resistance has arisen due to a number of reasons, such as functionalized [7]. Ag(I) and Au(I)2NHC complexes have also
mutations,genetransfer,biofilmformationandinappropriateuse been found in many medicinal applications including antimicro-
of traditional antibiotics [3,4]. The association of antibiotic- bials[8].Goldinvivobiochemistrystillremainsenigmatic,mainly
resistant pathogens with keratitis infection is a matter of great due to a scarcity of adequate models and an incomplete
concern, since many infections have had no effective treatment understanding of gold reactivity. Imidazolium salts react with
yet. silveroxideandproduceanNHCsilvercomplex,whichservesas
Since antibiotic resistance seems to be inevitable, strenuous a useful trans-metallating reagent [9] which has been widely
effortshavebeenmadetodevelopnewantimicrobialagents.The studied.Finally,aninterestingaspectofsilver(I)-NHCcomplexes
pharmaceuticalapplicationofN-heterocycliccarbene(NHC)and is the aggregation of silver (I) centers by argentophilic attraction
their metal complexes have gained enormous attention due to withAg–Agseparations[10]shorterthanthesumoftheirvander
their antimicrobial properties. Recently, pyrazine functionalized- Waals radii.
NHC complexes with clear deleterious effects against multidrug- Researchinthisareaismadeeasierandmoreinterestingbythe
resistant pathogens were seen to show an unusual mechanism of fact that steric and electronic properties as well the reactivity of
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AllylicAu(I)-NHCSaltinEyeInfection
metal–NHC complexes can be studied by varying the N- was added to it in N medium and stirred for 1h, and
2
substituents of imidazole [11]. Many NHC complexes have been precipitation of AgCl was observed. It was then filtered and the
reportedtoincludeNHCswithsimpleN-alkylorarylsubstituents. filtrate was collected; after removal of the solvent, the colorless
Various functional groups [5] such as pyridyl, phosphinyl, solidgoldcomplexproduced3,39-(p-phenylenedimethylene)bis{1-
pyrazolyl and thiazole have also attracted considerable attention (2-methyl-allyl)imidazoline}gold(I) bromide(3a)salt.
due to their use in constructing N-functionalized NHCs. In this 3,39-(p-phenylenedimethylene) bis{1-(2- methyl-allyl)imidazoli-
paper, welookatthisreaction,andreportthesynthesisofa new ne}silver hexafluorophosphate (2b) was synthesized by the
typeofsilver-NHCcomplex.Thehemi-labilenatureoftheallylic reaction of complex (1b) (64 mg, 0.1mmol) with excess Ag O
2
[12]systemchangesthemetal-organiccoordinationmodeduring (27 mg)inacetonitrile(CH CN).Themixturewasstirredatroom
3
theformation ofthecomplex, whichencouraged us todesign Ag temperature for 8 h with exclusion of light and then filtered to
and Au-complexes oftheallylic ligand. remove a small amount of unreacted Ag O. The filtrate was
2
Data reported here show the antimicrobial activity of N, N9- collectedandsolventwas removedtoproduce a whitesolidmass
olefin functionalized bis-imidazolium gold (I) and silver (I) salts of complex (2b). 90mg (0.15 mmole) of solid silver hexafluor-
against several resistant human fungal and bacterial pathogens, ophosphatecomplex(2b)wasputintoaround-bottomedflaskand
whichcancausetheseriouseyeinfectionkeratitis.Theirsynthesis, dissolved in 10mL of acetonitrile solvent. Au(SMe2)Cl (45 mg,
crystal structures, characterization, and mode of action were 0.15 mmol)wasaddedto(2b)inN mediumandstirredfor1 h.
2
analyzed andex vivobioassayswere carried out. Precipitation of AgCl was observed, which was then filtered.
Filtrate was collected and, after removal of solvent, producedthe
Materials and Methods colorless solid 3,39-(p-phenylenedimethylene) bis{1-(2- methyl-
allyl)imidazoline}gold(I) hexafluorophosphate complex (3b). The
Reagents and Chemicals chemicalshift(1HNMRand13CNMR)andmass(m/z)dataofall
Allthereagents-imidazole,a,a´ -dibromop-xylene,3-chloro-2- synthesizedcompounds weredescribed inthesupplementary file.
methyl propene, Ag O and NH PF - were purchased from
2 4 6
Sigma-Aldrich, UK, and were used without further purification.
X-Ray Structure Determinations
Allmanipulationswerecarriedoutinanopenatmosphereunless
Crystalsof1a,2aand3bwerecoatedinparaffinoil,mounted
otherwise stated. Au(SMe )Cl was prepared according to the
2 on a kryo loop, and placed on a goniometer at a temperature of
reported procedure [5]. Solvents EtOH, MeOH and CHCl of
3 296Kinanopenatmosphere.X-raydatasetswerecollectedusing
analytical-grade type were distilled and dried over appropriate
aBrukerApexCCDdiffractometerwithgraphitemonochromated
drying agents and dinitrogen prior to use. All the selected
MoK\aradiation(l=0.71073 A˚ ).Theunitcelldetermination
antibiotics and antifungals tested in this study were purchased
wasachievedbyusingreflectionsfromthreedifferentorientations.
from HIMEDIA, India. 1HNMR spectra were recorded on a
An empirical absorption and crystal refinement corrections were
BrukerARX400in(CD ) SO asthesolventand13CNMRdata
32 2 performed by using multiscan SADABS. Structure solution,
wererecordedon75MHzinstrumentrespectivelyandreferenced
toresidualprotonsand13Csignalsofdeuteriumsolvents.MALDI refinement and modeling were accomplished by using the
SHELXL-97 package [13]. The structure was obtained by full
MSspectrawererecordedonaAppliedBiosystem,Voyagertime-
matrix least square refinement of F2 and selection of appropriate
of-flight massspectrometer (Text S4).
atoms from the generated difference map. Additional data
collection and refinement parameters are summarized in Table
Synthesis of Compounds
S1and S2.
A solution of (680mg, 10mmol) imidazole and (400mg,
10mmol) NaOH was prepared in THF, 3-Chloro-2-methyl
Structure Optimization
propene (905mg, 10mmol) was added to the solution and the
In order to understand the structure of synthesized complexes
mixturewasrefluxedfor15hat65uC.Thereactionmixturewas
2band3a,thestructuresweredrawnaccordingtotheNMRand
cooled at room temperature and the solvent was removed. After
mass data found, and also further generated the structures of
removal of the solvent, a brown, oily liquid was isolated and
complexes2aand3b.GeometriesattheB3LYP/LANL2DZlevel
characterized as 1-(2-methyl allyl) imidazole. Then 1-(2-methyl-
allyl)imidazole (244 mg, 2 mmol) and a,a´ - dibromo p-xylene of theory using the Gaussian G-03-E01 [14] program were
optimized. The number of imaginary frequencies of all the
(264 mg, 1 mmol) was added to the reaction mixture (neat
molecules turns out to be zero, implying that they correspond to
reaction), heated for 6 h producing a colorless solid. The solid
masswaswashedwithPet-etherandrecrystallizedinMeOH.3,39- minimum energystructures on thepotential energysurface.
(p-phenylenedimethylene) bis{1-(2- methyl-allyl)imidazolium}
hexafluorophosphate salt (1b) was synthesized using the aqueous Microbial Strains and Growth Conditions
solution of ammonium hexafluorphosphate (NH PF ) in complex The Aspergillus fumigatus JM3 (filamentous fungus), Candida
4 6
(1a) and produces the colorless, water-insoluble hexafluorpho- albicans SJ11 (unicellular fungus) Pseudomonas aeruginosa (Gram-
sphate salt(1b). negativebacteria),andStaphylococcusaureus(Gram-positivebacteria)
Furthermore, 3,39-(p-phenylenedimethylene) bis{1-(2- methyl- used in this study were obtained from Priyamvada Birla Aravind
allyl)imidazoline}silver bromide (2a) salt was synthesized after a EyeHospitalinKolkata,India.Thephenotypiccharacteristicsof
reaction of complex 1a (102 mg, 0.2mmol) and Ag O (47mg, clinically isolated fungal strains and antibiotic sensitivity profiling
2
0.2 mmol) in dichloromethane, and the mixture was stirred at of bacterial strains were described in the supplementary section.
roomtemperaturefor12hwithexclusionoflight,thenfilteredto Allthestrainswereisolatedfrompatientsassociatedwithkeratitis,
remove a small amount of unreacted Ag O. The filtrate was and fungal strains were previously characterized [2,15]. Fungal
2
collected and solvent was removed producing a white solid mass species were collected from a mature solid medium culture plate
(2a). Furthermore, 144mg (0.2mmole) of solid silver bromide (Sabouraud dextrose agar) and mixed with liquid RPMI 1640
complex(2a)wastakeninaround-bottomedflaskanddissolvedin (Himedia,India),andwereincubatedfor24hat30uCtogetthe
20mL dichloromethane solvent. Au(SMe )Cl (59 mg, 0.2mmol) relevant turbidity of 0.56104CFU/mL. Bacterial cultures were
2
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AllylicAu(I)-NHCSaltinEyeInfection
maintainedinMueller-HintonBroth(MHB)andatotalinoculum ofthecontactlenses,complexeswereaddedtothebiofilminserial
loadof ca.105cells per well-maintained. double-diluted concentrations (0.97mM to 1 mM), and the final
volume of 2 mL with RPMI medium, which was then incubated
Antimicrobial Assays further for 24h at 30uC. A series of complex-free wells and
Minimum inhibitory concentration (MIC) values of all the biofilm-free wells was also included to serve as positive and
synthesized compounds and antifungal/antibacterial antibiotics negative controls, respectively. After incubation, the remaining
were determined according to CLSI guidelines [16]. The biofilm metabolic activity was quantified by the XTT-reduction
concentration of each compound used for the assay ranged from assay as described earlier [13]. For imaging, the lenses were
0.97 mMto1mM.MICvaluesweredeterminedwherenovisible flipped, and stained biofilms were visualized by fluorescence
growthwasobserved.Thecharacteristicfeaturesofclinicalisolates microscopy to compare their gross morphologies. Live-dead
withtheirantimicrobialsensitivityweredescribedinTextS3.The analysiswasperformedwithBacLightLive/Deaddye(Invitrogen
culture conditions and bacterial growth were monitored as Inc. USA). The images were captured from the stained biofilms
described earlier by Roymahapatra et al. [5]. Likewise, fungal under fluorescence microscope (OLYMPUS IX 51, fitted with
growth and culture conditions were monitored as described Evolution VF CCDCamera).
earlier, following Sengupta etal. [2]. Two wells, where no Biofilm formation by bacterial clinical isolates of P. aeruginosa
compounds were added, were used as positive controls, and and S. aureus on contact lenses were determined by using a
compounds with the absence of microorganisms were used as polystyrenecrystalvioletadherenceassay,asdescribedpreviously
negative controls in order to maintain the experimental sterility, by Croes etal. [18]. In brief, soft contact lenses were separately
and allindependent experiments were repeated four times. submerged in6wells offlat-bottomed polystyrene plates contain-
To examine the bacterial growth or killing kinetics in the ing 2 mL of Trypticase Soy Broth (TSB) with 0.5% glucose,
presenceofcompound3a,bacterialcellsweregrownin100mLof inoculated with the respective bacterial strain with an inoculum
Mueller-Hinton Broth (MHB) supplemented with different doses dose of 108CFU/mL and incubated for 48h at 37uC. The
ofcomplex(0.25to4.0mM),at37uCwithcontinuousagitationat biofilmthathadgrownovercontactlenseswasusedforminimum
180rpm. Growth or killing rates and bacterial concentrations biofilm eradication concentrations (MBEC) assay following
were determined by measuring OD at 600nm. The OD values Roymahapatra et al. [5] and complex concentrations were used
were converted into concentration of cells measured in CFU per inthesamewayasinthepreviousfungalassay.Thecrystalviolet
milliliter (1.0 OD corresponded to 2.166108CFU/mL). In vitro retainedwithinthebiofilmwasextractedbyadding100mlof70%
growth kinetics of fungal strains were determined following the (vol/vol) ethanol with 10% isopropyl alcohol (vol/vol) and
protocol described by Mitchell etal. [17]. In brief, fungal strains measured at 590nm (A590). All independent assays were
(103 CFU) were inoculated into 2 mL Sabouraud dextrose broth performed four times. Subsequently, the biofilm formations in
supplementedwithdifferentdosesofcomplex3a(0.25to4.0 mM), different concentrations were visualized under fluorescence
andincubatedat30uCwithcontinuousrockingat180rpm.Two microscope (OLYMPUS IX 51, fitted with Evolution VF CCD
millilitersofmock-inoculatedSabourauddextrosebrothservedas Camera) by staining with BacLight Live/Dead dye (Invitrogen
a negative control. The invitro growth rates were measured at Inc. USA).
OD andusingtheconversionfactorof36107CFU/mLper1
600
U OD [17]. All independent experiments were repeated four Cytoplasmic Material Release Study
600
times. Cytoplasmicmaterialreleasestudywascarriedoutaccordingto
Sahuetal.[19],withminormodifications.Tosummarize,1 mLof
Scanning Electron Microscopy (SEM) overnight bacterial (P. aeruginosa and S. aureus) and fungal (C.
Pseudomonas aeruginosa and Candida albicans cells were harvested albicans) culture (relevant turbidity of 0.5 6104CFU/mL) were
from the log phase of their respective growth medium. The cells centrifugedandthepelletwaswashedwithPBS(1X)buffer.Next,
were then washed three times with 1X PBS buffer and thepelletwassuspendedin1 mLofPBS(1X)bufferanddivided
resuspended in the same saline buffer. The cells were treated into five aliquots of 200mL each. Different aliquots of cell
withgold(I)complex(3a)withtheirindividualMICconcentration suspensions were treated with different concentration of complex
for30min.Cellswererepeatedlywashedwithsalinewaterand5– 3a(0.5to4 mM)andincubatedat30uCfor2 h.Thesampleswere
10ml of resuspended solution containing bacteria/fungus was filtered to remove bacteria, and OD values of supernatants were
placedonthelysine-coatedglasscoverslipasadrop-castemethod. recorded at 260nm. The percentage of cytoplasmic material
The fixed cells were dried and kept on desiccators until use. release was measured considering the OD values obtained with
Sampleswerethenfixedontoagraphitestubandkeptinanauto TrintonX-100 (0.1%) treatment with 100% release. The experi-
sputter coater (E5200, Bio-Rad) under low vacuum for gold ment was repeatedthree times.
coating up to 120 seconds. Surface morphology was studied by
using a scanning electron microscope (JEOL JSM5800) with an Molecular Docking
accelerated voltage between5–20kV. Two membrane models were built through the CHARMM-
GUI server [20] for simulating fungal and bacterial membranes,
Eradication of Biofilm on Contact Lenses both with 50A˚2. For fungal membrane, a proportion of
TheformationofAspergillusfumigatusandCandidaalbicansbiofilm 1:1:1:1:1:1 of POPA (palmitoyloleoylphosphatidylamine), POPS
oncontactlenseswascarriedoutaccordingtoSenguptaetal.[2]. (palmitoyloleoylphosphatidylserine), POPE (palmitoyloleoylpho-
Soft contact lenses were separately submerged in 6 wells of flat- sphatidylethanolamine), DOPC (dioleoylphosphatidylcholine),
bottomed polystyrene plates containing 2 mL of RPMI 1640 DPPC (dipalmitoylphosphatidylcholine) and cholesterol was used
medium, inoculated with the respective fungal strain with an according to Jo et al. [20]. For the bacterial membrane, a 3:1
inoculum dose of 3.56106 CFU/mL and incubated for 72h at proportionofofPOPEandPOPG(phosphatidylglycerol)wasused
30uC.After72h,thelenseswereremovedandtheplanktoniccells according toMurzynetal.[21].
were washed gently and repeatedly with 1X PBS buffer. To Molecular docking studies were performed by AutoDock Vina
determinetheeffectofgold(I)complex(3a)onbiofilmeradication and AutoDock Tools as previously described [5,22–24]. Grid
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AllylicAu(I)-NHCSaltinEyeInfection
boxes were set to the center of the membrane models and then spectra support the formulation of proligands 1a and 1b.
dislocatedtocoveronelayerofmembranemodels,whileforother Complex 2a was prepared (Yield of 2a: 115mg, 0.15 mmol,
receptors the grid boxes were kept in the center of the receptor. 81%) by the complexation of proligand- 1a and Ag O in
2
Thegridboxsizesformembranemodelsweresetto20,20and30 dichloromethane with exclusion of light (Fig. 1) as reported in
for X, Y and Z axes, respectively. The 3a structure was set as a thegeneralprocedure[5].Complex3awaspreparedbythesilver
flexible ligand by using the default parameters of the AutoDock carbene transmetallation [5] method (Yield of 3a: 135mg, 0.17
Tools. Final models were analyzed on PyMol (The PyMOL mmole, 83%). Afterward, complex 2a and Au(SMe )Cl were
2
Molecular Graphics System,Version1.4.1, Schro¨dinger, LLC). combined in DCM under nitrogen atmosphere at room temper-
ature. Complex 2b was prepared by mixing proligand 1b with
MTT Assay Ag O in acetonitrile under stirring (4 h) at room temperature
2
Non-carcinomamouseembryofibroblastcell(3T3)andhuman (Yieldof2b:52mg,0.085mmol,86%).3bwasalsopreparedby
breast carcinoma cell (MDA-MB-231) lines were used in this a transmetallation method using 2b and Au(SMe2)Cl in acetoni-
study. The cells were cultured as monolayers in Dulbecco’s trile (Yield of 3b: 94mg, 0.14mmol, 92%). Formation of the
Modified Eagle Medium (DMEM) supplemented with 10% (v/v) complexes 2a, 2b, 3a and 3b was confirmed by the absence of
heat-inactivated fetal bovine serum and antibiotics. After over- imidazolium protons (C2H) and a downfield shift of aromatic
nightincubation,themediumwasaspiratedoff,washedwithPBS protons. In the case of 2a, the carbenic signal appeared at
(1X) buffer and 200mL of complex 2a, and 3a (0–100mM 178.2 ppm in the 13CNMR spectra, and a downfield shift of
concentration) medium were added separately in triplicates. The 38.9 ppm in comparison to free imidazolium salt was observed
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro- after complexation. A significant 37–39ppm shift of carbenic
mide] assay was performed following the method described by carbon in the 13CNMR spectrum confirmed the formation of
Mandal etal.[25]. complexes2b,3aand3b.Thiswasfurthersupportedbythemass
spectrometric data. The single crystal X-ray structures of 1a, 2a
Compatibility Test and 3b were determined (see supplementary file). 1HNMR,
13CNMR and MALDI mass spectra of all synthesized complexes
Ophthalmologists generally have a large repertoire of antibac-
were supplied in the supplementary file and their corresponding
terial and antifungal antibiotics in the form of eye drops,
structures were determined (Fig.S1 toS5,TextS1andS2).
ointments, tablets and parenteral applications to control eye
infections. Complex 3a was used in an eye drop and tested for
48h, six times a day on goat (Capra aegagrus hircus) eyes, and the Antimicrobial Activity
concentration was 5-fold of that used in invitro experiments. The The antibacterial and antifungal activities of all synthesized
goateyeswerecollectedfromabutcher’sshopatthelocalmarket. compoundsweretestedagainstbothGram-positiveand-negative
In general, enucleation of the entire globe of the eyes was done bacteriaaswellasagainstunicellularandfilamentousfungus.The
after death. The cornea was excised from globe under an aseptic antibacterial activities of complexes 2a and 3a were higher than
conditioninabiosafetycabinet.Next,theexcisedcorneawaskept 2b and 3b (Tables 1 and 2). Complex 3a was more active
in a McCarey-Kaufman medium for preservation without any against P. aeruginosa and A. fumigatus than complex 2a and other
antibiotics. Experiments were repeated with three eyes. Each eye tested antibiotics. The eradication of pathogen-formed biofilm is
waskeptat4uCinapetridishanddropswereappliedat4hour an important criterion for the successful development of new
intervals. After 48 hours of treatment, the blubber conjunctiva, generationantimicrobials.Allthepathogenicstrainswereusedto
retina and lens were evaluated for any damage, cracking or develop their biofilm over contact lenses and the biofilm
dilution byvisual observation. eradication ability of complex 2a and 3a was checked. The
minimum biofilm eradication concentrations of all the tested
Statistical Analysis antibiotics,complex2aand3aweredetermined(Table2).The
The results are presented as the mean 6 SD. The statistical N,N9-olefinfunctionalizedbis-imidazoliumgold(I),3asaltsare3
to10foldmoreactivethanthesilver(I)complexandcommercial
significance of the experimental results was determined by one-
antibiotics.Thefluorescencemicrographofcontrolbiofilmtreated
way Student’s t-test or one-way ANOVA followed by Dunnett’s
test. Values of p,0.05 were considered statistically significant. with complex 3a revealed the effectiveness of biofilm eradication
ability after 24h of treatment (Fig. 2). The killing activity of
Prism version5.0was usedfor allstatistical analyses.
complex3aforP.aeruginosawasdifferentfromS.aureus(Fig.S6).
The killing activity was concentration-dependent and no cell
Results
growth was seen even after 0.5 and 2.5mM concentration of
Synthesis of the Complexes complex 3a for P. aeruginosa and S. aureus, respectively. Similarly,
Theligand1awassynthesizedbytheadditionofimidazoleand nocellgrowthwasobservedafter2.5and0.5 mMconcentrationof
NaOHindryTHFunderstirringconditionsatroomtemperature complex3aforC.albicansandA.fumigatus,respectively(Fig.S7).
and a subsequent addition of 3-chloro-2-methylpropene. The
mixturewasrefluxedat65uCfor15handallowedtocoolatroom Insights into Mechanism of Action
temperature, and the solvent was removed. A brown oily liquid The interactions between microbial cell membrane and
form of 1-(2-methyl allyl)imidazole was obtained. Then 1-(2- complex 3a were observed by using a scanning electron
methyl-allyl)imidazole and a,a´ – dibromo- p-xylene were mixed microscope (SEM). SEM pictures clearly indicated a morpholog-
and the reaction mixture was heated for 6 hours to obtain a icalchangeafter1 hoftreatmentwithcomplex3a(Fig.3).The
colorlesssolid1a(Yield:490mg,0.96mmol,96%).Quantitative morphologicalchangeindicatesthatthetargetshouldbefoundon
conversion of PF salt (1b) of the proligand was achieved by the the microorganism surface such as lipid bilayer or cell walls. In
6
additionofaqueoussolutionofammoniumhexafluorphosphatein order to determine the morphological changes observed in SEM
solution1a.ThepresenceofNCHNprotonin1HNMRspectra imageswereduetothemembranedamageornot,theleakageof
confirmed the formation of ligands 1a (d=9.47 ppm) and 1b cytoplasmicmaterialswasmonitoredat260nmwavelength.The
(d=9.25ppm). Elemental analysis, 13CNMR and MALDI mass leakage of cytoplasmic materials is considered a characteristic
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AllylicAu(I)-NHCSaltinEyeInfection
Figure1.Schematicdiagramofthesynthesisprocedureofligands1aand1b,andtheirderivedcompounds2a,3aand2b,3b.
doi:10.1371/journal.pone.0058346.g001
indicationofdamagetothecytoplasmicmembrane[19].Different observed after increasing the complex concentration, and maxi-
concentrations of complex 3a were incubated with bacterial and mumleakagewasfoundinP.aeruginosaincomparisontoS.aureus
fungal cellsuspensions for2 h.Cytoplasmic material leakage was andC.albicans(Fig.4),whichindicatesthatthemaintargetmight
belipid bilayer.
Therefore, docking experiments were performed in order to
Table1. Minumuminhibitory concentrationofthe verify the binding affinities of 3a compound to microbial cell
synthesized compounds, antibacterialand antifungal membranes (Fig. 5). Docking data shows the complexes formed
antibiotics against keratitis-associated eyepathogens. between 3a compounds with bacterium- and fungus-like mem-
branes. The 3a molecule is clearly more buried into bacterium-
like membrane in comparison to fungus-like lipid bilayer.
Compounds C.albicans A.fumigatus P.aeruginosa S.aureus Moreover, docking results suggested that 3a affinity to bacteri-
um-likemembranesisstrongerthanfungus-likemembranessince
2a 3.90 3.90 3.90 3.90 the affinities were 27.4kcal.mol21 and 26.1kcal.mol21 respec-
2b 125 125 125 125 tively.Furthermorenonepolarinteractionswereobservedinboth
3a 1.95 0.97 0.97 1.95 cases,beingcomplexationmainlydrivenbyvanderWallsforces.
3b 125 125 125 125
Voriconazole 15.62 0.97 Nt nt Compatibility Test
Bothcomplexes2aand3ainhibitedcellproliferationofMDA-
AmphotericinB 1.95 0.97 Nt nt
MB-231 and 3T3 cells in a dose-dependent manner. Cell
Fluconazole 31.25 500 Nt nt
inhibition was prominent mainly at concentrations after 25 mM
Ceftazidime nt nt 31.25 31.25
for both the complexes, as confirmed by MTT assay (Fig. S8).
Vancomycin nt nt 7.81 15.62 The IC values of complex 3a against MDA-MB-231 and 3T3
50
Piperacillin nt nt 31.25 62.5 cancer cells were 29.2162.38 mM and 23.4462.01mM, and the
values of 2a against MDA-MB-231 and 3T3 cells were
DataarerepresentingasmMconcentration.ntmeans‘‘nottested’’.
32.9462.72mM and 26.2161.88mM, respectively. In addition,
doi:10.1371/journal.pone.0058346.t001
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AllylicAu(I)-NHCSaltinEyeInfection
Figure2.Imagesofbiofilmeradicationfromsoftcontactlensesbycomplex3a.Imageswerecapturedafter24htreatmentwith0.1%
DMSO (left panel) and with complex 3a (as MBEC values listed in Table 2) over 72h-grown biofilm (right panel). Bacterial pathogens were P.
aeruginosa(a&b);S.aureus(c&d)andfungalpathogenswereA.fumigetus(e&f);C.albicans(g&h).Complex3asignificantlyreducedlivecells
(greenfluorescence)andmostofthecellsweredead(redfluorescence)againstalltestedpathogensoncontactlenses.Imageswerecapturedata
magnificationof400X.
doi:10.1371/journal.pone.0058346.g002
complex 3a was used as an eye drop at five times the through theoretical optimization. 2band 3blooked like a ‘chair’
concentration observed in the invitro experiment and applied withtwoligandsbondingbymetalcenterfromtheoppositeside.
ontothenormaleyethathadbeenexcisedfromagoat.After48h Thesynthesizedcomplexes2a,2b,3aand3b wereevaluated
of treatment with complex 3a, which was applied 6 times daily, for their antimicrobial properties against human pathogens
thecomplexwasrevealedtohavelowtoxicitytotheeye(Fig.6). causing keratitis infection. Among them, complexes 2a and 3a
Thepigmentandlenswereneitherdilutednorfracturedafter48h showed the strongest antimicrobial activities against resistant
oftreatmentwithcomplex3a,whichsuggestsitslowtoxicityand pathogenic strains. The bacterial strains were resistant to the
future application inkeratitis control. commonly used antibiotics ceftazidime, vancomycin, and piper-
acillin. The fungal strains were also resistant to amphotericin B,
Discussion fluconazole, andvoriconazole. Several resistant mechanismssuch
asenzymaticinactivation,chemicalmodification,alterationofthe
N,N9- olefin functionalized allylic ligands, 3,39-(p-phenylenedi- antibiotictargetsiteandeffluxpumpsarewellreported[27].The
methylene) bis{1-(2- methyl-allyl)imidazolium}bromide(1a) and mostpromisingfeatureofthisresearchwasthatallresistantstrains
3,39-(p-phenylenedimethylene)bis{1-(2-methyl-allyl)imidazolium} evaluated here were highly susceptible to complexes 2a and 3a.
hexafluorophosphate(1b) were used to synthesize four bis- Thisleadsustosuggestthatcomplexes2aand3afollowdifferent
imidazolium silver (I) and gold (I) salts as well as 2a, 2b and mechanismsin ordertoinhibit microorganismgrowth.
3a, 3b. Syntheses of these compounds were confirmed by using Inhibitionofbiofilmformationoreradicationofgrownbiofilm
NMR (1H and 13C) and MS analysis. Single-crystal X-ray isthemostimportantfeatureofthesecomplexes.Complex3awas
diffraction data were collected for compounds 1a, 2a and 3b. abletoeradicatethebiofilmformedbybacterialandfungalstrains
The molecules (2a, 2b and 3a, 3b) possessed N-Ccarbene bond bothsignificantlyandsuccessfully.Biofilmisacomplexmatrixof
distances N-Ccarbene-N angles in immidazole moity were compa- exopolymericsubstancesoftheorganismsthatpreventdrugsfrom
rable with other imidazolium systems. The carbene-Ag-carbene direct penetration. Hence, it is also believed that the ability of
bond angle in 2a was 170.83(15)u and C-Ag-Br bond angle was complex 3a to penetrate the biofilm matrix at very low doses
about170.6–171.6ui.e.linear,whereasAg-Ag-Brbondanglewas might be related to the presence of a dinuclear [Au2(1a-H)2]2+
nearly90u,whichindicatestheAg-Agbondwasperpendicularto olifinic motif. Indeed, gold complexes showed good and selective
C-Ag-Br in a ‘T’ shape. It revealed that the complex was of the activityagainstbothGram-positiveand-negativebacteria,aswell
bis-carbenetypewithitsolifinicsystemconfirmedbyitsdinuclear toward fungi. It is apparent that the functionalization of the
crystallinity [Ag2(1a-H)2]2+ motif. The same structural geometry nitrogenatomsoftheNHCligandsandthecomplexationwithAu
wasfoundintheoptimizedstructureofcomplex3a.For2a,Ag- (I) at the C2 site might be the cause of higher antimicrobial
Ag shortest bond distance was found to be 3.084(3)Au (Ag4-Ag1) activity. The difference in activities depends on several factors
[Fig. S2] and that of Au-Au for 3a was 3.08415Au(Au110– since these complexes are known to interact freely with cellular
Au112) [Fig. S3]. These metal-metal bond distances are shorter components when they are in a flexible co-planar [28] configu-
thanthereportedolefinsystemsandevenshorterthanthesumof rationandhaverotationalfreedom[29].Therelativeenergyasa
their van der Waals radii [26]. The single crystal structure of function of the torsional angle [5] is also a relative descriptor for
complex 3b was determined by XRD study and that of 2b the co-planarity and free rotation which endorse easy binding of
themetalcenterofthecomplexwiththeactivesite.Inthepresent
study,amongthefourcomplexes,themetalcentersof2aand3a
Table2. Minimumbiofilmeradication concentrationofthe
are bonded with a single ligand (other side attached to Br atom),
synthesized compounds, antibacterialand antifungal
having scope for free rotation. This free rotation promotes a co-
antibiotics against keratitis-associated eyepathogens.
planar configuration within the reaction medium for the easy
formation of complexes with cellular components. On the other
hand, ligand dissociation is prohibited in the case of cationic
Compounds C.albicans A.fumigatus P.aeruginosa S.aureus
tetrahedral Au (1) phosphine complexes as they gain stability
2a 15.62 7.81 7.81 7.81 through chelating. This is due to the high stability of the metal-
phosphorus bond [30], metal-F bond and probably formation of
2b 250 125 125 125
H-bondwith‘F’ofPF62,asisthecasefor2band3b.Sincemetal
3a 3.90 1.95 1.95 3.90
centersareconnectedwithtwoligands,freerotationisprohibited
3b 125 125 125 125 andtheyaremorestabilizedthroughchelatingwithPF 2,sotheir
6
Voriconazole 125 125 nt nt reactivity is lower than 2a and 3a. Our results are also in
AmphotericinB 31.25 15.62 nt nt agreement with an earlier report by O¨zdemir etal. [31] that
Fluconazole 500 500 nt nt imidazolium salt bearing Cl and Br salts is very active; but the
same derivative completely lost its activity when the anion was
Ceftazidime nt nt 62.5 62.5
changed toPF or BF .
Vancomycin nt nt 31.25 31.25 6 4
Theprobablemechanismofactionconsistsinthebacterialand
Piperacillin nt nt 250 250
fungalmembranesdestabilization,reducingthestructuralintegrity
and leading to clear microorganism shape modification. The
DataarerepresentingasmMconcentration.ntmeans‘‘nottested’’.
doi:10.1371/journal.pone.0058346.t002 membrane’s destabilizing effect is probably given by insertions of
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AllylicAu(I)-NHCSaltinEyeInfection
Figure 3. SEM micrographs of bacteria and fungi treated with compound 3a. Cells were treated with compound 3a (2mg.mL21) and
controlgroupwastreatedwith0.1%DMSO.ControlP.aeruginosacells(a)andtreatedwith3a(b);ControlC.albicanscells(c)andtreatedwith3a(d).
doi:10.1371/journal.pone.0058346.g003
Figure4.Effectofcomplex3aonthereleaseofcytoplasmicmaterialat260nm.P.aeruginosa(blueline),S.aureus(redline)andC.albicans
(green line) were incubated with different concentrations (0.5 to 5.0mM) of complex 3a at 30uC for 2h. Release of cytoplasmic material was
monitoredat260nmwavelengthandpercentvalueswerecalculatedinrelationtotritontreatment(positivecontrol).ThetreatmentwithtritonX-
100(0.1%)wasconsideredas100%ofcytoplasmicmaterialrelease.Datapointsrepresentmeanandstandarddeviationofthreeexperiments.
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AllylicAu(I)-NHCSaltinEyeInfection
Figure5.Theoreticaldockingstudiesofcomplex3awithbacteriumandfunguslikemembrane.Imagesarerepresentingthedocking
resultsofcomplex3awithbacterium-likemembrane(A)andfungus-likemembrane(B).Themembraneisrepresentedinsticks,carbonatomsarein
green,nitrogenatomsinblue,phosphorusatomsinorangeandoxygenatomsinred.3aisrepresentedinspheres:thegoldatomsareingold,
carbonatomsinwhite,nitrogenatomsinblueandbromineatomsinpink.Theinteractionsbetweenthemetalcomplexesandlipidsaredetailedat
thebottom.
doi:10.1371/journal.pone.0058346.g005
multiple molecules of 3a, intercalating the lipids and leading to However, a question arises: why was 3a more active than 2a?
membrane disruption (Figs. 2 and 3). However, 3a was more Themostcriticaldifferencebetweenthetwocompoundsisintheir
activeagainstbacteriathanfungi.Thisdifferencecouldberelated stability.ThecovalentbondbetweengoldandNHCismorestable
to two elements, the cell walls thickness and the membrane than the covalent bond constructed between silver and NHC.
affinities.Accordingtothedockingaffinities,thebindingof3ais Silver seems to be worse than gold for NHC r metal p-back
more favorable to bacterium-like membranes than fungus-like donation [3]. This fact makes 2a more reactive to free
ones. For bacterium-like membranes, a binding affinity of compounds, yielding more interactions with other molecules in
27.4kcal.mol21 was observed whereas the affinity for fungus- the environment than 3a complexes. Therefore, to compensate
like ones was 26.1kcal.mol21. In addition, the 3a activity is these interactions with non-target molecules, a larger number of
influencedbythecompositionofthecellwall,thecompoundmust 2acomplexesareneeded,while3acomplexesseemtogostraight
crossthecellwallbeforeactinginthelipidbilayer.Thedifferences tothemembranes.
betweentheamountsofleakagematerialsareduetothevariations Finally, the results obtained from the MTT assay are found to
of their cell wall compositions. P. aeruginosa, is a Gram-negative be in agreement with the low toxicity at concentrations of their
bacterium with a thinner peptidoglycan layer which makes MICandMBECvaluesbetween0.5to5.0 mM,andpreliminary
cytoplasmic leakage more likely compared to the thicker ex vivo toxicity studies demonstrated very low toxicity on the eye
peptidoglycan layer of Gram-positive bacterium, S. aureus. whenappliedexternally.Thus,itmayproveusefulasanexternal
Furthermore, in C. albicans, the main cell wall composition is eyedroptherapyinpatientssufferingwithkeratitis.Similarstudies
chitin,whichmakesarigidlayersurroundingthemembrane.The are in progress with some determining the molecular level of
cytoplasmicmaterialleakageismoreprominentandmorerapidin interactionwithbindingmodestobacterialandfungalmembrane
P. aeruginosa followed by S. aureus. The leakage was nominal in C. lipids while others are exploring the synthesis of other gold
albicans (Fig. 4). In addition, a small mammalian cell toxicity complexes with different halide groups to enable higher water
degree was observed in their corresponding MIC values between solubilityandlowertoxicity.Nevertheless,datareportedhereare
0.5 to5.0mMthrough the MTTassay. extremely promising, and the reported salts could probably be
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AllylicAu(I)-NHCSaltinEyeInfection
Figure6.Compatibilitytestofcomplex3aongoat(Capraaegagrushircus)eyes,asaneyedrop.Eyejustexcisedfromgoat(a);after48h
treatedwithcompound3a(b),conjunctivaisnotdamagedorcrackedafter48htreatmentwithcomplex3aapplied6timesdaily(c);andlensis
neitherdilutednorfracturedafter48htreatment(d).Imageswerecapturedatamagnificationof5X.
doi:10.1371/journal.pone.0058346.g006
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