Preparation and Investigation of Antibacterial

7/28/2019 Preparation and Investigation of Antibacterial

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Tanya Abel,' JaimeLee . Cohen,", JasmineEscalera,'Robert Engel,'b Maya Filshtinskaya,' RussellFincher,cAlice Melkonianc and Karin Melkonian*cPaceUniversity, QueensCollege ofC.U.N. Y., Long Island University, C.W. Post Campus

Surfaces bearing protein units (wool, silk) have been modified in a two stepprocess to incorporate at thefree side-chain hydroxyl groups unctionalities (lipophilic with polycationic units) that bear antibacterialactivity. The approach has involved tosylation of the hydroxyl groups followed by displacement with atertiary amine bearing cationic and lipophilic components.The effectiveness f thesemodified surfaces orantibacterial action against a series of Gram + and Gram- bacteria is reported Structural factorsmaximizing the activity against all species ested have been studied and appropriate surfaces have beengenerated Preparative procedures along with methods of investigation of the antimicrobial activity areincluded along with a discussionof modeof activity.Keywords: antibacterial, surfaces,antimicrobial

INTRODUCTIONIt has for some time been recognized hatcationic surfactants bear antibacterial activity .1-5Investigations of structure-activity relationshipshave demonstrated hat in addition to a cationicsite, a significant lipophilic component of thesurfactant is involved in optimization of activity.With simple alkylbenzyldimethylammoniumchlorides in their action against Pseudomonasaeruginosa, the optimal length of the alkyl chainhas been noted to be twelve carbon atoms.3Optimal activity toward a variety of bacterialspecies or numerous structural variations of thewater soluble cationic surfactants appears tooccur when an alkyl chain of between ten andfourteen carbon atoms s present.6-IOThe mechanismof action of such cationicsurfactants on bacteria s understood o be one of

electrostatic interaction and physical disruption,as opposed to interference with a metabolicpathway, as is commonly the situation withantibiotic species.I After the cationic site of theagent attached to a significant lipophiliccomponent binds to anionic sites of the cell wallsurface it is then able to diffuse through the cellArticle Designation: Refereed

AIMVolume3. Issue2, Fall 2003

Preparation and Investigation of AntibacterialPro'tein-basedSurfaces

ABSTRACT

wall and bind to the membrane. Acting as asurfactant, t is able to disrupt the membrane andpermit the release of electrolytes and nucleicmaterials, eading to cell death.While the construction of antibacterialagents that express their activity in such amanner has beenwell investigated, he remainingchallenge has been to impart such activity to asurface from which the active agent is notreleased and will be able to maintain activityindefinitely. The binding of quaternaryammonium sites to glass surfaces through theuse of silyl-ether linkages was found to impartantibacterial activity to such surfaces.12Severalpolymeric surfaces have also been investigated,including polystyrenelJ,14and poly(propylene

imine).15In this light, it appeared a reasonablepossibility that other types of surfaces could berenderedantibacterial by the covalent attachmentof polycationic units having lipophilic adjuncts.Prior efforts of our laboratory had demonstratedthe facility with which such polyammoniumunits having lipophilic adjuncts could beprepared,16,17s well as the manner in which

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/k-


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such materials could be attached o a variety ofprimary hydroxyl sites n complex structures.SFollowing from this we previously havereported on the preparation and nvestigation of a

variety of carbohydrate derived surfacematerialsthat were similarly functionalized.1s Thesefunctionalized surfaces have been noted to beantibacterial with regard to a broad series ofGram + and Gram - bacteria.Success ith suchcarbohydrate-derived surfaces, particularlycotton, has suggested hat other types of fabricsurfaces might also be rendered antibacterial.Using active cationic surfactant components aspreviously described, a similar approach andresultant activity might be anticipated forproteinaceous surfaces with a significantconcentration of serine residues. The primaryhydroxyl group of the amino acid appears o bean ideal site for incorporation of the activecomponents. In this way, both wool and silkfabrics might be rendered antimicrobial as theserine content of each is significant (minimumvalues are for wool -10%,19,20 nd for silk, 13-17%19). he current report is concernedwith the

R= -CH2(CH2)12 CH)-CH2 (CH2) 14CH)-CH2(CH2)16 CH)-CH2(CH2)18 CH)

Article Designation: Refereed

functionaJizationof wool and silk surfaces n thismanner and the investigation of their resultantantimicrobial activity.RESULTS AND DISCUSSION

For the present effort parentpolyammonium units having lipophilic adjunctswere prepared by a modification of previouslynoted techniquesl6,17sing the bis-tertiaryamine1,4-diazabicyclo[2.2.2]octane in reaction withappropriate haloalkanes n ethyl acetate solution(Equation I), The resultant monocationic salts (1- 4), which readily precipitated rom solution,were isolated by suction filtration and driedunder vacuum. All exhibited IH and 13CNMR inaccord with their proposed structures, as well aselemental analyses n accord with hydrated statesof their elemental formulae, which have beenreported previously.16,17,ZJ

Protein-based surfaces (100% silk cloth,commercial grade; 100% wool cloth, commercialgrade) were activated and functionalized (asnoted in Equation 2), followed by washing anddrying, to generate the modified surfaces.

RBr G\+N~N-R'-=/ Br-(CH3)3COH(1)(2)(3)(4)

Equation 1

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R= -CUH2S-C14 H29

-C16 HJJ-c 18 HJ7

The structural componentspresumed or theprotein surfaces are deduced from thecorresponding results previously found forattachment of the cationic components tosubstrates bearing primary hydroxylgroups.J8.2J The reactivity of p-toluenesulfonyl chloride with alcohols iswell known!2 Primary hydroxyl sites can befunctionalized quite specifically relative tosecondary sites within the same molecule.Thus, tosylation (and subsequentdisplacement by the tertiary amine) of theavailable hydroxy s of the serine residuesof


Br-r:-\+N"'\;N-R'-=I(CH3)3COH

+ R+ $' 2 X-

~l;'J;Z' f0P-dabco-R

P-dabco-C12P-dabco-C14P-dabco-C.16P-dabco-C18

the proteins is presumed to occur withoutreaction also occurring at any availablesecondaryor aromatic hydroxyl sites. Verysignificantly, tosylation of thesesystemshasbeen accomplished without the use ofpyridine. Two alternative tosylationprocedures have been accomplished usingboth wool and silk, the first using solidsodium bicarbonate present as an acid sinkin acetonitrile medium. The second s mostpromising for applications, tosylation beingperformed in aqueousmedium with sodiumbicarbonate dissolved. This provides a

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homogeneous eaction medium (except forthe proteinaceous surface) minimizing theuse of noxious organic reagents. Theresultant modified proteinaceous materialsexhibit the same activity as those producedusing pyridine as a base in the tosylationstep. Four bacterial strains (two Gram- andtwo Gram +) were investigated, as notedbelow, with note of their American TypeCulture Collection referencenumber:

Escherichia coli(ATCC #14948)Proteus(ATCC#13315)Bacillus cereus(ATCC #14579)

Staphylococcus(ATCC #6538)Two types of experiments wereperformed to test the efficacy of thematerialsagainst hese bacteria.First, -105

Table 1Growth of bacteria on modified silk surfaces as apercentage of control

E. coli0

440

70.9

P-dabco-C12P-dabco-C14P-dabco-C16P-dabco-C18

E. coli


bacteria were added to a test sample of themodified surface having an area of 0.25 in2placed on a rich medium and incubated,along with a control surface on the sameplate. Control surfaces consist of the fabricof interest that have been washed with thesame solvent system as the experimentalsurfaces,but have not been reated with thelipophilic adjunct.Growth of bacteria off the edge ofthe surface was observedvisually, followingwhich the surfaces were placed in 4 mL ofliquid growth medium and incubated or 16hr. Growth of bacteria n these iquid mediawere measured turbidimetrically. (In allinstances, nvolving each of the two types ofprotein-based surfaces, the controlexperiments exhibited full growth of thebacteria in all growth studies.) Growth ofeach of the bacteria in the liquid mediaexperiments with modified silk is shown inTable 1. Corresponding esults for modifiedwool surfaces are shown in Table 2.

vulgaris

aureus

P- vulgaris0

4.60

24.6

S. aureus0000

B. cereus0000

P. vulgaris S. aureus. cereus

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Fundamentally, this workdemonstrateshat broad antibacterialactivitycan be imparted to silk surfaces hrough thecovalent attachment of cationic agentswithlipophilic adjuncts. Both Gram + and Gram- bacteriawere killed completely (no growthof bacteria in rich liquid medium afteraddition to treated surface on nutrient richplate) with two of the four attached agents(P-dabco-C12 and P-dabco-C16). Three ofthe cationic agents(P-dabco-C12, P-dabco-C14 and P-dabco-C16) exhibited complete killing of Gram +bacteria but not (the more resistant)Gram -bacteria.In a further type of experiment, 106bacteria were spread on agar platescontaining a rich medium, and modifiedsurfaceswere placed on top. Incubation was

Table 3Growth of bacteria on agar under modified silk surfacesas a percentage of control


Table 4Growth of bacteriaon agar under modifiedwool surfacesasa percentage f control


17.899.450.774.9

21.491.719.822.1

0000

000

19.5then perfonned overnight, after which themodified surfaceswere discarded. Scrapingsof agar from the region beneath the addedsurface were placed in 4 mL of liquidgrowth media and further incubatedovernight. Growth of surviving bacteria inthese liquid media was measuredturbidimetrically. (Control surfaces werealso included on each plate as noted before;all controls exhibited full growth ofbacteria.) Full data for these growthexperiments are shown in Table 3 for

modified silk and Table 4 for modified woolsurfaces. Again, with silk surfaces, P-dabco-C12 and P-dabco-C14 exhibited thegreatest activity against both Gram + andGram - bacteria, and P-dabco-C16exhibited significant activity against Gram +bacteria

E. coli00

32.742.4

P. vulgaris26.5

022.3NA

B. cereus000

]5.5

S. aureus0

NA0

NA

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An optimal chain length of thelipophilic unit for antibacterial activity isobserved or these protein-basedsurfacesaswas previously with carbohydrate-basedsurfaces.S Maximal antibacterialactivitycan be observed oward particular types ofbacteria using various chain lengths oflipophilic species. We are continuinginvestigation of variations in nature of thelipophilic chain in an effort to understandthis structure/activity relationship.The antibacterial activity may beunderstood as occurring in a stepwisemaJU1er. The lipophilic chains can besubsumedby the bacterial species o a stagewhere the cationic portion is brought intointimate contact with the cell surface,and issubsumedsufficiently ar that it is not easilyexpelled. Detergent-like action then resultsin cell surface disruption initiating celldestruction. A particular advantageof suchaction is the lack of consumption of theantibacterial agent; it is not changed n the

processand remainsattached o the surface.Clearly, modification ofproteinaceous surfaces (silk and wool) bythe covalent attachment oflipophilic/cationic adjuncts imbues thosesurfaces with an antibacterial character.However, the activity against both Gram +and Gram - bacteria s not as great as thatpreviously reportedusing similarly activatedcarbohydrate-derived surfaces. We ascribethis characteristic to the necessarilydecreased loading of the agent to theproteinaceoussurfaces.Only 10-20% of thesurface constituent units (serines) of theproteinaceous surfaces s capable of beingmodified, whereasvirtually all of the surfaceunits of a carbohydrate-basedurface can beso modified. It is unlikely that antibacterialaction results from a single activefunctionality interacting with the bacterial


E. coli31.728

82.593.9

P. vulgaris00

63.7NA

B. cereus0000

S. aureus000

NA

cell. Rather, with the large cell settling onthe surface, he greater he number of activefunctionalities in a relatively small regionthat can interact with the cell the greater helikelihood that a sizable fissure of the cellenvelope can result. This action issignificantly less likely for a surface that ispartially-serine than one that bears an activefunctionality throughout the exposedsurface. Nonetheless, significant activity isobserved for the modified proteinaceoussurfaces.Several other comparisonswith thepreviously reported carbohydrate surfaceresults may be noted. With proteinaceoussurfaces (compared to carbohydrate-basedsurfaces) the adjunct with a saturated 16-carbon chain is no longer most active.18Shorter chains, particularly the 12-carbonchain, exhibit greater activity. This is incloser correspondencewith prior work onother surfaces.3.6-10t would appear hat theactivity of such adjuncts s influenced by the

nature of the surface to which they areattached. Further, Gram + bacteria arereadily dispatchedby all except he saturated18-carbon chain adjuncts on proteinaceoussurfaces, whereas Gram - species remainheartier.We are continuing with efforts: I) tobroaden he range of surfaces hat can be somodified; 2) to seek optimal structure or theantibacterialagent; and 3) expand he libraryof microorganisms susceptible to suchactivity. It would also be of interest toidentify proteinaceous materials that aresignificantly higher in serine content thanthe silk and wool currently availablecommercially.

EXPERIMENTALGeneralprocedure or the preparation ofmodified urfaces:6 JTATMVolume3, Issue 2,Fall 2003


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A strip (2" x 10", 1.2 g, maximally 2.2mmol of free serine hydroxyl for silk and1.1 mmol for wool) of the raw surfacematerial was activated by addition to asolution containing an excess of p-toluenesulfonyl chloride (10 g, 52.5 mmol)and sodium bicarbonate 5.0 g, 59.5 mmol)in water (100 mL). After standing in thestirred solution for 4 hr, the strip wasremoved and washedwith water chilled withice. Attachment of the polycationic ligandwas then accomplished by placement in astirred I-butyl alcohol solution (25 mL)containing the monocation unit (1-4) (anexcess - e.g. with 4 to generate P-dabco-CU, 20 g, 44.7 mmol) and agitated for 24hours. After this time the modified surfacematerial was removed from the reactionmedium and washed epeatedly with water,followed by a brine wash, and finally driedin air without heating.General procedure for determination of theantibacterial characteristics of the modifiedsurfaces:Experiment 1:The rich medium used for bacterialgrowth was prepared from Bacto-tryptone,Bacto-agar, yeast extract and sodiumcWoride. Each modified surface sample wasinvestigated for its antibacterial effect witheach of the bacteria studied in a two-partexperiment. Specifically, on the sameplate,bearing the growth medium, were placedtwo separateexperimental uns, thosebeing:A - surface material that had been

subjected to the solvent washingprocedures of reaction but withoutaddition of the reagent materials, towhich the bacteria beinginvestigated were added. In eachinstance ~~ of the stock dispersionof bacteria n log growth phasewereadded using an Oxford BenchmatePipetman, placing the entire load ofbacteria at the center of the testswatch of material (square,0.5 in toa side). For the strains investigated,this corresponded o addition of thefollowing number of bacteria:


Escherichia coli1.41 x 105Proteus vulgaris2.34 x 105Bacillus cereus1.54 x 105Staphylococcusaureus 1.47x 105B - modified surface material, to

which the bacteria beinginvestigated were added n the samemanner and amount as noted for Babove.The growth plate was incubated overnight at35C. Growth was noted visually in theregion around the material surface.Subsequently, the surface material wasremoved from the growth medium andplaced n 4 mL of fresh growth medium andincubated at 35C for 16 hr. Growth ofbacteria in this instance was measuredturbidimetrically using a Beckman Model 25UVNIS spectrophotometer t 60 nm.Experiment2:Approximately 106 bacteria (7S 1tL)were spread on an agar plate using a cellspreader. A test swatch of material (as inExperiment I) was added to the top of thebacteria. Plates were incubated overnight at3S"C. Material swatches were removed anddiscarded. Scrapings of agar located in theregion under the placement of the materialwere coIIected and were placed in 4 mL ofthe growth medium and incubated at 3S"Cfor 16 hr. Growth was measuredturbidimetrically as n Experiment I.

ACKNOWLEDGEMENTThe authors wish to acknowledge heassistance of Dr. Elizabeth Lowe in thedeterminationsof surfacecharacteristics.REFERENCES1. M.R.J. Salton, J Gen. Physiol.,

1968, 2,227S-252S.2. W.B. Hugo and M. Frier, M. Appl.Microbiol., 1969,17,118-127.3. E. Tomlinson,M.R. Brown andS.S.Davis, J Med. Chern., 1977, 20,1277-1282.7 JTATMVolume 3, Issue 2,Fall 2003


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S.P. Denyer, Int. Biodeterior.Biodegrad., 1995,36,227-245.G. McDonnell and A.D. Russell,Clin. Microbiol. Rev., 1999, 12,147-179.H. Nagamune, T. Maeda, K.Ohkura, K. Yamamoto, M.Nakajima, and H. Kourai,Toxicology in Vitro, 2000, 14, 139-147.C.R. Birnie, D. Malamud, and R.L.Schnaare,Antimicrobial Agents andChemotherapy, 2000, 44, 2514-2517.G. Viscardi, P. Quagliotto, C.Barolo, P. Savarino,E. Barni, and E.Fisciaro, J: argo Chem., 2000, 65,8197-8203.

J. Pernak, J. Kalewska, H.Ksycinska, and J. Cybulski, Eur. J:Med. Chem.,2001, 36,899-907.C. Campanac,L. Pineau, A. Payard,G. Baziard-Mouysset, and C.Roques, Antimicrobial Agents andChemotherapy, 2002, 46, 1469-1474.W. Hugo, and G. Snow,Biochemistry of AntibacterialAction, 1981, Chapman and Hall,Ltd., London.A.J. Isquith, E.A. Abbott, and P.A.Walters,Appl. Microbiol., 1972,24,859-863.A. Kanazawa, T. Ikeda, and T.Endo, J: Poly. Sci., Part A, 1993,31,1441-1447.

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Melkonian, Carbohydr. Res., 2002,337,2495-2499.H.L. Needles, Handbook of TextileFibers, Dyes and Finishes, 1981,Garland STPM Press,New York, pp87-91.K.L. Hatch, Textile Science, 1993,West Publishing, St. Paul, MN, p.145.J.I. Cohen, S. Castro, J.-a. Han, V.Behaj, and R. Engel, HeteroatomChem.,2000, 11,546-555.W.S. Johnson, J.C. Collins, R.Pappo, M.B. Rubin, P.J. Kropp,W.F. Johns, J.E. Pike, and W.Bartman, W. JAm. Chern. Soc.,1963, 85, 1409-1430.

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Preparation and Investigation of Antibacterial