Sulfonation and anticoagulant activity of fungal exocellular β-(1→6)-d-glucan (lasiodiplodan)

92 (2013) 1908–1914

ContentslistsavailableatSciVerseScienceDirect

Carbohydrate

Polymers

j o ur na l h o me p ag e : w w w . e l s e v i e r . c o m / l o c a t e / c a r b p o l

Sulfonation

and

anticoagulant

activity

of

fungal

exocellular

␤

-(1

_→

6)-

d

-glucan

(lasiodiplodan)

Ana

Flora

D.

Vasconcelos

_,

_Robert

_F.H.

_Dekker

_,

_Aneli

_M.

_Barbosa

_,

_Elaine

_R.

_Carbonero

_,

Joana

L.M.

Silveira

_,

_Bianca

_Glauser

_,

_Mariana

_Sá

_Pereira

_,

_Maria

_de

_Lourdes

_Corradi

_da

_Silva

a_Depto_{deFísica,QuímicaeBiologia,UniversidadeEstadualPaulista–UNESP,CEP19060-900,PresidentePrudente,SãoPaulo,Brazil} b_{BiorefiningResearchInstitute,LakeheadUniversity,ThunderBay,Ontario,CanadaP7B5E1}

c_Depto_{deBioquímicaeBiologiaMolecular,UniversidadeFederaldoParaná,CEP81531-980,Curitiba,Paraná,Brazil}

d_{LaboratóriodeTecidoConjuntivo,HospitalUniversitárioClementinoFragaFilho–UniversidadeFederaldoRiodeJaneiro,CEP21941-590,RiodeJaneiro,RJ,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received11June2012

Receivedinrevisedform31August2012 Accepted11October2012

Available online 14 November 2012

Keywords: ␤-(1→6)-d-Glucan Lasiodiplodiatheobromae Lasiodiplodan Sulfonation Anticoagulantactivity

a

b

s

t

r

a

c

t

An exocellular␤-(1→6)-d_{-glucan(lasiodiplodan) producedbya strainof Lasiodiplodiatheobromae}

(MMLR)grownonsucrosewasderivatizedbysulfonationtopromoteanticoagulantactivity.The struc-turalfeaturesofthesulfonated␤-(1→6)-d_{-glucanwereinvestigatedbyUV–vis,FT-IRand}13_CNMR

spectroscopy,andtheanticoagulantactivitywasinvestigatedbytheclassicalcoagulationassaysAPTT, PTandTTusingheparinasstandard.Thecontentofsulfuranddegreeofsubstitutionofthesulfonated glucanwas11.73%and0.95,respectively.UVspectroscopyshowedabandat261nmduetothe unsat-uratedbondformedinthesulfonationreaction.ResultsofFT-IRand13_{CNMRindicatedthatsulfonyl} groupswereinsertedonthepolysaccharide.Thesulfonated␤-(1→6)-d-glucanpresentedanticoagulant

activityasdemonstratedbytheincreaseindosedependenceofAPTTandTT,andtheseactionsmost likelyoccurredbecauseoftheinsertedsulfonategroupsonthepolysaccharide.Thelasiodiplodandidnot inhibitthecoagulationtests.

© 2012 Elsevier Ltd.

1. Introduction

␤-Glucansarefoundmainlyinthecellwallofyeastsand fil-amentousfungi,asminorityconstituentsinthecytosol,andcan alsobesecretedasexo-biopolymerstotheenvironment(Williams, 1997).Theyhaveemergedasanimportantclassofbioactive prod-uctswithbiologicalresponsemodifying(BRM)activities(Bohn& BeMiller,1995).Besidesimmunoprotectiveactivity, exopolysac-charidesofthe␤-glucantypehavebeenexaminedinrelationto theantithrombotic,antioxidant,antiviral,anti-inflammatory, anti-coagulantactions,andantiproliferativeactivityonbreastcancer cells (Brandi etal., 2011;Cunha et al., 2012;Kato et al.,2010; Martinichen-Herrero,Carbonero,Gorin,&Iacomini,2005;Wang etal.,2010).

Thebiologicalactivitiescanbepresentedbythemoleculesin natura,orthroughchemicalmodification.Thechemical derivati-zationofglycansoffersanopportunitytoenhancetheiraction,

and even develop activity in non-bioactive molecules, which

can be used as newpharmacological agents (Mantovani et al.,

2008; Vetvicka, Vetvickova, Frank, & Yvin,2008).Furthermore,

∗Correspondingauthor.Tel.:+551832295743;fax:+551832215682. E-mailaddress:corradi@fct.unesp.br(M.d.L.CorradidaSilva).

the inclusion of sulfonate groups onglycans enablesthe

gen-eration of water-soluble molecules, and this is important for

anticoagulant and other biological activities (Mendes et al.,

2009).

Glucans of the ␤-(1→3)- and ␤-(1→3;1→6)-types are the mostdescribed inscientific articlesand patents.Theyhave lin-ear,branchedorcyclic(Laroche&Michaud,2007)structures,and aretargetsforresearchfortheirpharmacologicaland immunolog-icaleffects(Vetvickaetal.,2008).␤-(1→6)-d-Glucansarewidely

knownaspustulanproducedbylichensofUmbilicariaceaespecies (Narui,Sawada,Culberson,Culberson,&Shibata, 1999), andare commonlypresentasconstituentsofthefungalandyeastcellwall (Klis,Mol,Hellingwerf,&Brul,2002).Asexocellularbiopolymers, ␤-(1→6)-d-glucansareuncommon,ifnotrare,andareknownto

beproducedbyonlysomefungi(Cunhaetal.,2012;Vasconcelos etal.,2008).

Thebiological activityofthe␤-glucansfromfungiincluding mushrooms thatcompriseboth ␤-(1→3)-and (1→6)-d_-glucans

areconsideredthemosteffectiveimmunestimulatoryagents(Rop, Mlcek&Jurikova,2009),andthepresenceofbranchesatC-6onthe ␤-(1→3)-glucanchainaswellasatriplehelixconformationare

importantstructuralfeaturesdeterminingBRMactivityofthese

polysaccharides(Bohn&BeMiller,1995;Leung,Liu,Koon,&Fung, 2006).

0144-8617© 2012 Elsevier Ltd.

http://dx.doi.org/10.1016/j.carbpol.2012.10.034

Open access under the Elsevier OA license.

92 (2013) 1908–1914 1909

Substanceswithanticoagulantpropertieshavebeenusedboth

in therapeutic processes and in vitro to treat medical

condi-tions,andnaturalheparinismostwidelyused(Wang,Li,Zheng, Normakhamatov, & Guo, 2007). Unfortunately, heparin shows

somecontra-indicationssuchasbleeding,andmoreover,because

heparinisextractedfromanimaltissues,ittendstocausearisk

ofcontaminationbyanimal-derivedpathogens.Developmentof

alternativestoheparinthereforeisanimportantfieldofresearch. Nomammaliansourceofheparinoritsderivativesareconsidered tobeidealchoices,andconsequently,polysaccharidesulfonates (naturalorchemicallyderivatized)areofspecialinterest(Glauser etal.,2009;Pomin,2012).

Anabundantsourceofanticoagulantpolysaccharidesismarine algaethatcontainavarietyofnaturalsulfatedgalactansand sul-fatedfucans(Melo,Pereira,Foguel,&Mourão,2004).Othersulfated

polysaccharideswithanticoagulantactivityarefoundin marine

invertebrates (Pomin,2012).Chemically sulfonated

polysaccha-rides, which present anticoagulant and antithrombotic activity,

have been obtainedfrom various polysaccharidetypes suchas

microbial ␤-glucans (Brandi et al., 2011; Mendes et al., 2009)

anddextrans,andplant-derivedgalactoglucomannansand

galac-tomannans(Martinichen-Herreroetal.,2005;Yang,Du,Wen,Li,& Hu,2003).

Inthisworkwedescribeforthefirsttimethesulfonationofan exocellular ␤-(1→6)-d-glucan(lasiodiplodan) fromLasiodiplodia

theobromaeMMLRgrownonsucroseascarbonsource(Vasconcelos

et al., 2008), and the effect of sulfonation on promoting the

solubilityofthispolysaccharideinaqueoussolutions,andthe phys-iologicalactivityasananticoagulantasalternativetoheparin.

2. Experimental

2.1. Materials

Sodium heparin (5000UI/mL) was purchased from Akzo

Organon (São Paulo, Brazil). Human plasma was obtained by

centrifugation (450×g/15min at25◦_{C)of citrated blood. Blood}

coagulationtimereagents:activatedpartialthromboplastintime

(APTT), thrombin time (TT) and prothrombin time (PT), were

acquired from In-Vitro Diagnóstica S/A (Itabira, MG, Brazil).

Thrombin and antithrombin were obtainedfrom Haematologic

Technologies,USA,andchromogenicsubstrateS-2238from

Chro-mogenix,Sweden. ChlorosulfonicacidwasobtainedfromVetec

(RiodeJaneiro,RJ,Brazil).

2.2. Productionandpreparationofˇ-(1→6)-d-glucan

(lasiodiplodan)

␤-(1→6)-d-Glucan(lasiodiplodan)wasproducedbyL.

theobro-maeMMLRandgrownonnutrientmediumcontainingsucroseas

previouslydescribed(Vasconcelosetal.,2008).Cell-freeculture fluidwasobtainedafterremovalofthemyceliumbycentrifugation (5500×g/20min)at4◦_{C.Thesupernatantwastreatedwith3}

vol-umesofabsoluteethanol,theprecipitatedmaterialrecoveredand dissolvedindistilledwater,followedbyextensivedialysisagainst frequentchangesofdistilledwaterover48h,andthenfreeze-dried.

2.3. Analyticaltechniques

Carbohydrate was determined by the phenol–sulfuric acid

method(Dubois,Gilles,Hamilton,&Rebers,1956)withd-glucose

as standard. Protein was measured by the Bradford method

(Bradford,1976)usingbovineserumalbuminasstandard.

2.4. Sulfonation

Sulfonationof␤-(1→6)-d-glucanwasperformedaccordingto O’Neill (1955) with some modifications: lasiodiplodan powder (50.0mg)wassolubilizedindryformamide(10.0mL)with vigor-ousstirringfor24hatroomtemperature.Then10.0mLofpyridine

wasaddedtothemixturefollowed bycontinuationofvigorous

stirringforanother30hatroomtemperature.Chlorosulfonicacid

(4.0mL)wasnextaddeddrop-wisetothemixtureinanice-bath

overanintervalof2h,andthenleftat4◦_{Cfor12h.Thereaction}

wasterminatedbyaddingice-water,andneutralizedbyaddinga

solutionof10%(w/v)NaHCO3untilallCO2evolutionceased.The

reactionmixturewasthendialyzedexhaustivelyagainstdistilled waterfor6dayswithseveralchangesofwater,andthedialysate concentratedunderreducedpressure(<39◦_{C)andlyophilized.The}

productobtainedwasreferredtoassulfonated␤-(1→6)-d-glucan.

Thesulfonationreactionwasrepeatedtwofurthertimesuntila

degreeofsubstitution(DS)ofgreaterthan0.80wasobtained.

2.5. Determinationofthedegreeofsubstitution(DS)

Samples of sulfonated ␤-(1→6)-d_{-glucan (1.0mg) were}

hydrolyzedusing1.0MHCl(1.0mL)for5hat100◦_{C.Todetermine}

the DS, 0.2mLof hydrolyzedthe sulfonated ␤-(1→6)-d-glucan

samplewasreactedwith3.8mLof3%(w/v)trichloroaceticacid

(TCA)inaglasstube,andthen1.0mLofprotectorsolution(6.0g NaCl,0.5mLc.HCl,2.5mLof0.1%(w/v)gelatinand47.0mLdistilled

water) and 0.03gBaCl2 wereadded. Thecontents were stirred

for 1minand left for 15min.The resulting BaSO4 formed was

measuredturbidimetricallyat360nm.TheDS,whichdesignates

the average number of sulfonyl groups oneach sugar-residue,

wasestablishedfromthesulfurcontentaccordingtoWhistlerand Spencer(1964),inwhichS=%sulfur:

S(%)= (BaSO4,␮g)×0.1374×100

Sample,␮g

DS= 162×S

3200−102×S

2.6. Homogeneityoftheˇ-(1→6)-d-glucanandsulfonated ˇ-(1→6)-d-glucan

Thehomogeneityofthe␤-(1→6)-d-glucans(naturaland

sul-fonated) was determined by gel permeation chromatography.

Onemilligramofeachofthe2polysaccharidesampleswas

dis-solvedinwater(1.5mL)andappliedtoaSepharoseCL-4Bcolumn (1.5cm×30cm),andelutedwithdistilledwaterataflowrateof 0.5mL/min.Fractions(1.5mL)werecollectedandanalyzedfor

car-bohydrate(490nm).Thevoidvolume(19.5mL)wasdetermined

usingbluedextran.

2.7. Spectroscopyanalysis

Fourier-transform infra-red (FT-IR) spectra of the

exopolysaccharides samples (␤-(1→6)-d-glucan and sulfonated

␤-(1→6)-d_{-glucan, 1mg) were recorded using KBr pellets}

(250.0mg) on a Bruker Vector 22 Model spectrometer. The

ultraviolet–visible(UV–vis)absorptionspectrafordiluteaqueous solutions (1.0mg/mL) of ␤-(1→6)-d_{-glucan and sulfonated ␤}

-(1→6)-d-glucanweredeterminedusingaShimadzu1601UV-Vis

spectrophotometer. Nuclear magnetic resonance spectroscopy

ofcarbon thirteen(13_{CNMR)analysisof␤-(1→6)-d-glucanand}

sulfonated ␤-(1→6)-d-glucanwerecarriedoutusinga400MHz

Bruker model DRX Avance spectrometer incorporating Fourier

1910 92 (2013) 1908–1914

examinedat50or70◦_{C.Chemicalshiftsareexpressedinppm(ı)}

relativetoresonanceofMe2SO-d6at39.70forsamplesexamined

inthissolvent.

2.8. Bloodclottingassays

Theanticoagulantactivityofthesamples(␤-(1→6)-d-glucan

andsulfonated␤-(1→6)-d-glucan;0–200␮g/mL)wasdetermined

bymeasuringthe clottingtimes (inseconds)of humanplasma

using the reagents prothrombin time (PT), thrombin time (TT)

and activatedpartial thromboplastin time (APTT) according to

the manufacturer’s instructions. Each assay was performed at

37◦_{C.Heparin(0–30␮g/mL)wasusedasstandard.Normalhuman}

plasma (900␮L) wasmixed with 100␮L of exopolysaccharides

solution (␤-(1→6)-d_{-glucan and sulfonated ␤-(1→6)-}d_-glucan),

orheparindissolvedinisotonicsaline,withintheconcentration rangesindicated above.Isotonicsaline(100␮L)wasusedinthe controlgroup.Ineachofthebloodclottingassays,thereagents(PT, TTandAPTT)andallglasstubeswerepre-heatedat37◦_Cbeforeto

use.Theassayswereperformedintriplicateandtheresults repre-sentthemeans±SD.

2.9. Inhibitionofthrombinbyantithrombininthepresenceof

ˇ-(1→6)-d-glucan,sulfonatedˇ-(1_→6)-d-glucanandheparin

The inhibition of thrombin by antithrombin wasperformed

according to Melo et al. (2004) with the following

modifica-tions:incubationswereperformedin disposablemicrocuvettes.

Thereactionmixturecontained25␮Lofhumanplasmaorpurified

antithrombin (40nM), and 25␮L of exopolymer solution (␤

-(1→6)-d_{-glucanorsulfonated␤-(1→6)-}d_{-glucan;0–400␮g/mL),}

orheparin(0–1␮g/mL)inTS/PEGbuffer(0.02MTris/HCl,0.15M NaCl,and1.0mg/mLpolyethyleneglycol,pH7.4).Thrombin(10␮L, 20nM)wasnextaddedtothemixturetoinitiatethereaction,and after60sincubation(roomtemperature),500␮LofTS/PEGbuffer

containing25␮MchromogenicsubstrateS-2238forthrombinwas

added,andtheabsorbanceat405nmrecordedfor300s.No

inhi-bitionoccurredinthecontrolexperimentinwhichthrombinwas

incubatedwithantithrombinintheabsenceofsulfated

polysac-charides.

3. Resultsanddiscussion

3.1. Sulfonationandstructuralcharacterization

Theexocellular␤-(1→6)-d_{-glucanwasselectedforthiswork}

asitpresentsimportantcharacteristicsforbiologicalactivitywith regardstopurity,uniformity,homogeneity(Fig.1)andtriplehelix

conformational structure at previously described (Vasconcelos

etal.,2008).Ashydratedthebiopolymerformedaviscous solu-tion,andthederivatizationbysulfonationwasawaytoimprove itssolubilityinaqueoussolutions,andtopromoteanticoagulant activity.Itiswellknownthattheintroductionofchargedgroups

onaneutralpolysaccharidechainimproveswatersolubilityand

canenhanceitsbiologicalactivitiessuchasanticoagulation(Brandi etal.,2011;Jung,Bae,Lee,&Lee,2011).

Inthis work,sulfonation(threecycles) wasperformedusing

formamideassolvent,pyridineascatalyticreagentand chlorosul-fonicacidasthehydroxylgroupdonor.Theeffectivenessofeach

cycleof thesulfonationreactionwasmonitoredbyUV–vis and

FT-IRanalysis.Theintegrityofsulfonatedmaterialwasperformed

bygelpermeationchromatographyonSepharoseCL-4B(Fig.1),

andpresentedasinglecarbohydratepeak.Followingsulfonation,

UV–visspectroscopyshowedanewbandat261nm(Fig.2)that

canbe attributabletothen→␲* transition ofsulfonate orthe

Fig.1. Gelpermeationchromatographyprofileofthe␤-(1→6)-d-glucan(---)and

sulfonated␤-(1→6)-d-glucan(—)onacolumnofSepharoseCL-4B.Thecolumn (1.5cm×30cm)waselutedwithwaterataflowrateof0.5mL/min.

unsaturatedbondformedinthesulfonationprocess(Brandietal., 2011;Yangetal.,2003).

Thesuccessofthereactionwasaccompaniedbytheappearance oftwocharacteristicsabsorptionbandsonFT-IRspectraofthe sul-fonated␤-(1→6)-d-glucan(Fig.2):oneat1258cm−1describingan

asymmetricalS Ostretchingvibration(Yang,Du,Huang,Wan,&Li, 2002;Zhang,Zhang,Zhou,Chen,&Zeng,2000),whereastheband at810cm−1 _{representingasymmetricalC O Svibration}

associ-atedwithaC O SO3group(Brandietal.,2011;Nie,Shi,Ding,&

Tao,2006)demonstratedthatlasiodiplodanwassuccessfully sul-fonated.Additionally,anewbandat1631cm−1_{couldberelatedto}

theunsaturatedbondformedduetothesulfonationprocess(Yang etal.,2003).

Thesulfurcontentofthesulfonatedpolysaccharidewas

deter-mined by calculating the DS, and in this case was 0.95. In

studiesrelatedtoanticoagulantactivitiesofchemicallymodified␤ -glucans,theDSwasaround1.95(Martinichen-Herreroetal.,2005), 1.74(Nieetal.,2006)and1.54(Brandietal.,2011).Accordingto publishedreports,DSvaluesequalorhigherthan0.80arerequired foranticoagulantactivity(Han,Yao,Yang,Liu,&Gao,2005).The contentof sulfurobtainedfor thesulfonated␤-(1→6)-d-glucan

was11.73%,andwithintherangeofvaluesnecessaryfor antico-agulantactivity.

Thepositionsofthesulfonylgroupsinpolysaccharidescanbe determinedby13_{CNMR(Hanetal.,2005;Zhangetal.,2008),and}

theliteratureshowsthatthemainpositionfortheentryof sul-fonylgroupsin␤-(1→3)-glucansistheprimarycarbon,i.e.atC-6, followedbyC-2andC-4(Tellesetal.,2011;Zhangetal.,2000).

The13_{CNMRspectraofnative␤-(1→6)-d-glucanandits}

sul-fonatedderivativearepresentedinFig.3.Thechemical shiftat 103.0ppmof␤-(1→6)-d-glucanwasattributedtotheanomeric

carbonandthatat69.0ppmtosubstitutedC-6.Thesignalsat75.9,

75.0,73.2and69.9ppmwereattributedtoC-3,C-5,C-2and

C-4,respectively (Naruiet al.,1999;Sassakiet al.,2002).The13_C

NMRspectrumofsulfonatedglucanswasmorecomplicatedwith

broader signals, resulting fromthe sulfonation of thehydroxyl

groups (Brandi et al., 2011). Usuallyfollowing sulfonation, the

spectrabecame morecomplicated becausethecarbons directly

attachedtotheelectronegativesulfonatedestergroupsshiftdown field,whilethecarbonsindirectlyattached(neighborhood)tothe sulfonylgroupshifttoanupfieldposition(Perlin&Casu,1982; Tellesetal.,2011).

Thechemicalshiftsinthe13_{CNMRspectrumofthesulfonated}

␤-(1→6)-d-glucanpresentedasmallvariation(+0.3ppm),in

92 (2013) 1908–1914 1911

Fig.2.FT-IRandUV–vis(inset)spectraof␤-(1→6)-d-glucanbefore(---)andaftersulfonation(—).

attributedtoC-1asthesignalofC-1splitswhenan OHgroupon C-2issubstitutedwithasulfonylgroup.Inaddition,thechemical

shiftat73.2ppmassignedasC-2alsosplitandmoveddownfield

(73.6ppm),suggestingthatpartofC-2wassulfonated.Theintense

signalat70.2ppmcorrespondedprobablytothesubstitutedC-6

andfreeC-4,whilethatat71.9ppmcouldbeassignedtoC-4 sub-stitutedbysulfonylgroups(␣shift).Thesmallsignalat68.6ppm (␤shift)canberelatedtocarbonindirectlyattachedtosulfonyl

groups.Probablypartof C-3wasalsosubstituted,however,the

attributionisdifficultbecausethechemicalshiftsmaycorrespond tomorethanonecarbon.Fromtheresultsweconcludedthat non-selectivesulfonationof␤-(1→6)-d-glucanhasoccurred,asC-2and

C-4weresplitindicatingpartialsubstitution.

Afterthesulfonationreaction,thesolutions ofsulfonated ␤

-(1→6)-d_{-glucan became less viscous and more water soluble,}

whichfacilitatesthebioassaystodetermineanticoagulant activ-itythroughtraditionaltestsofbloodcoagulationwithheparinas thereference.

3.2. Anticoagulantactivity

3.2.1. APTT,TTandPTclottingtimes

Toinvestigatetheanticoagulantpropertyof␤-(1→6)-d-glucan

andsulfonated ␤-(1→6)-d_{-glucan,APTT,PTandTTassays were}

conductedusingnormalhumanplasmaandtheeffectsofthe␤

-glucansontheclottingtimesmeasured.Theresultswerecompared withthoseforheparinasthereferencestandard.

TheAPTTtestisrelatedtothephaseofintrinsiccoagulationin plasmaandmeasuresthefunctionofbloodcoagulationfactorsXII, XI,IXandVIII.PTisrelatedtotheextrinsicphase,whichdepends

uponthetissuefactoroftheactivationprocessandmeasuresthe integrityofthecommonphaseofcoagulation.TheTTassay evalu-atestheconversionofplasmaticfibrinogentofibrininthepresence ofexogenousthrombin.Thecoagulationtimeinthelaststageof thecoagulationcascadeofeventsistheconversionoffibrinogento fibrinbythrombin(Beutler,Coller,Lichtman,&Kipps,2001;Melo etal.,2004;Mendesetal.,2009).

TheresultsfortheAPPT,TTandPTassaysareshowninTable1.

ThesulfonatedpolysaccharidewasabletoprolongtheAPPTand

TTtimes ina concentration-dependentmanner.Withregard to

theAPTTandTTresults,theanticoagulanteffectofsulfonated␤ -(1→6)-d-glucanat 30and 40_␮g/mLconcentrationwas_∼5and ∼7 times greater, respectively, than the control. These results demonstratedanimportantinvitroanticoagulantactivityforthe sulfonated ␤-(1→6)-d_{-glucan asdemonstrated by theincreases}

inthedose-dependenceofAPTTandTT,andthisactioncouldbe

attributabletothedegreeofsulfonation(DS0.95).

TheAPTTprolongationtimesuggestsinhibitionoftheintrinsic

coagulationpathway,whereasprolongationofTTtimeindicates

inhibition of thrombin-mediated fibrin formation (Wanget al.,

2007).NoprolongationofPTdemonstratedtherewasnoinhibition oftheextrinsicpathwayofcoagulation(Maoetal.,2009).Sincethe anticoagulanteffectofheparinisnotmediatedbymodulationof theextrinsicsystem,thesulfonated␤-(1→6)-d-glucanisapoor

inhibitoroftheextrinsicpathway.TheAPTTandTTvalueswere

compared withheparin,and a concentration∼10times greater

ofthesulfonated␤-(1→6)-d-glucanwasnecessarytoachievethe

sameeffectasexhibitedbyheparin.Thesulfonated␤-(1→3;1→

6)-d-glucan (named botryosphaeran) from Botryosphaeria rhodina

1912 92 (2013) 1908–1914

Fig.3.13_{CNMRspectraofexocellular␤-(1}

→6)-d-glucanandsulfonated␤-(1_→6)-d-glucan.

(Brandi et al., 2011), showed similar results as anticoagulants. AccordingtoMartinichen-Herreroetal.(2005),sulfated

polysac-charideswithaloweranticoagulantactivitythanheparin could

exhibit a potent antithrombotic effect with less hemorrhagic

risk.

Aswithheparin, theweakest effect wasobservedin thePT

assayfor thesulfonated ␤-(1→6)-d_{-glucan. Therelative lackof}

effectofsulfonated␤-(1→6)-d-glucanonthePTisconsistentwith

theobservationthatthistestisalsonotsensitivetoheparin,and severalothersulfatedpolysaccharides(Martinichen-Herreroetal.,

Table1

Anticoagulantactivityofnormalhumanplasmainthepresenceof␤-(1→6)-d-glucan,sulfonated␤-(1→6)-d-glucanandheparinasmeasuredbytheactivatedpartial

thromboplastintime(APTT),thrombintime(TT)andprothrombintime(PT).

Polysaccharide Amount(␮g/mL) Clottingtimes(s)

APTT TT PT

␤-(1→6)-d-Glucan Control(0) 46.65±0.8 19.45±0.4 12.87±0.8

1 39.45±0.2 15.92±0.4 16.04±0.1

5 39.94±0.6 16.56±0.5 14.87±0.1

10 42.30±0.1 14.24±0.3 15.77±0.1

15 41.07±0.7 14.53±0.2 14.27±0.2

20 41,09±0.2 14.43±0.4 16.87±0.1

Sulfonated␤-(1→6)-d-glucan Control(0) 46.65±0.8 19.45±0.4 12.87±0.8

1 46.79±0.2 15.67±0.3 15.12±0.0

5 58.27±0.1 26.75±1.6 17.56±0.1

10 70.55±3.6 50.87±1.1 17.52±0.4

15 96.97±1.3 70.06±0.6 18.65±0.2

20 105.69±0.1 187.54±1.5 18.61±0.4

30 228.49±3.7 227.06±2.2 17.45±0.4

40 298.13±1.0 249.45±0.6 17.17±0.7

Heparina _{Control(0) 46.65±0.8 19.45±0.4 12.87±0.8}

1 55.60±0.6 95.00±0.7 16.56±0.2

2 76.19±1.0 275.98±1.3 17.93±0.6

3 106.08±0.3 592.70±2.9 19.20±0.2

92 (2013) 1908–1914 1913

Fig.4.Concentrationdependenceof␤-(1→6)-d-glucan,sulfonated␤-(1→6)-d -glucanandheparinontheinactivationofthrombinby(a)antithrombinofhuman plasmaand(b)apurifiedantithrombinpreparation.

2005).The␤-(1→6)-d-glucan(lasiodiplodan)didnotinhibitthe

APTT,TTandPTassays,anddemonstratedthatthepresenceof sul-fonylgroupswasanessentialrequirementfortheseanticoagulant activities.

3.2.2. Inhibitionofthrombinbyantithrombininthepresenceof

ˇ-(1→6)-d-glucan,sulfonatedˇ-(1_→6)-d-glucanandheparin

Thebiologicalmechanismofsulfonatedpolysaccharidesoccurs bythepotentiationofplasmaticcofactors,whichare physiologi-calinhibitorsofthecoagulationcascadeasantithrombinthatacts byinhibitingthrombinandfactorsXa,XIIa,XIaandIX,andmay haveitsactionstrengthenedbythepresenceofheparin(Meloetal., 2004; Mendes etal., 2009).Toelucidate the inhibitory mecha-nismoftheanticoagulantactivityofsulfonated␤-(1→6)-d-glucan,

theeffectsonthrombinactivitywerestudiedusingchromogenic

substratesinthepresenceofplasmaasasourceofphysiological inhibitors(antithrombinandheparincofactorII),andalsopurified antithrombin.

Fig.4(a)and(b)showsthatthesulfonated␤-(1→6)-d_-glucan

wasabletopotentiatethrombininhibitioninamannersimilarto thatofheparin.However,comparingthevaluesofIC50

(concentra-tionofthesulfonated␤-(1→6)-d_{-glucannecessarytoobtain50%}

inhibitionofthrombinactivity)obtainedforbothsetsofassays, theactivityofthesulfonated␤-(1→6)-d-glucanwasapproximately

180-foldlowerthanthatofheparininexperimentsusingthe

puri-fiedantithrombin,and∼38-foldlowerwithhumanplasma.These

resultssuggestthatbesidestheactivationofantithrombin,the sul-fonated␤-(1→6)-d-glucancouldpossiblycontributetoanincrease

intheactionofanotherphysiologicalinhibitorofthrombin (hep-arincofactorII)absentintheassayswithpurifiedantithrombin. HeparincofactorIIisaninhibitorofserineproteaseandthrombin.

Antithrombininhibitsallintrinsicpathwaycoagulationenzymes

(Maoetal.,2009).

Therefore,theresultsoftheanticoagulanttestsdescribedinthis workdemonstratedthatthesulfonated␤-(1→6)-d-glucan

exhib-itedanticoagulantactivity,andwasmostlikelyinvolvedwiththe

intrinsic pathway. The lasiodiplodan didnot present inhibiting

activityatanyoftheconcentrationsexamined,demonstratingthat thepresenceofsulfonylgroupsinthis␤-(1→6)-d-glucanwasan

importantcharacteristicofanticoagulantaction.

4. Conclusions

Anexocellular␤-(1-6)-d-glucan(lasiodiplodan)wassulfonated

(three cycles)using formamide assolvent,pyridine ascatalytic reagentandchlorosulfonicacidasthehydroxylgroupdonor.The

effectiveness of each sulfonation reaction cyclewas monitored

byUV–visandFT-IRanalysis,andonlythesulfonated␤-(1-6)-d

-glucanshowedsulfonateortheunsaturatedbondformedinthe

sulfonation process. The content of sulfur and DS obtainedfor

the sulfonated ␤-(1→6)-d-glucanwas 11.73% and0.95,

respec-tively,whichindicatedthattherewasapproximatelyonesulfonyl groupperresidueofglucose.Thepositionsofthesulfonylgroups introducedinlasiodiplodanweredeterminedby13_CNMR,andfrom

theresultsitwasconcludedthatnon-selectivesulfonationofthe ␤-(1→6)-d-glucanhadoccurred,withC-2andC-4beingpartially

substituted.ItispossiblethatC-3alsoreceivedasulfonylgroup.The prolongationofAPTTinthepresenceofthesulfonated␤-(1→6)-d

-glucansuggestedinhibitionoftheintrinsicpathwayofcoagulation, whileanextensionoftheTTtimeprobablyindicatedinhibitionof thereactionresultingintheconversionoffibrinogenintofibrin. Thus,thisworkdemonstratedthatthechemicalderivatizationof exocellular␤-(1→6)-d-glucanbysulfonationproducedamodified

polysaccharideresultinginanticoagulationactivity.

Acknowledgements

AFDVasconcelosthanksCAPES(Brazil)foradoctoral scholar-ship.TheauthorsaregratefultoAnaMariaTovarandPauloA.de SouzaMourão(Lab.Tec.Conjuntivo,H.U.ClementinoFraga Filho-UniversidadeFederaldoRiodeJaneiro)forscientificassistanceand discussions.

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