Rev. bras. farmacogn. vol.25 número6

w w w . s b f g n o s i a . o r g . b r / r e v i s t a

Original

Article

Intraspecific

variation

of

meroditerpenoids

in

the

brown

alga

Stypopodium

zonale

guiding

the

isolation

of

new

compounds

Angelica

R.

Soares

_,

_Heitor

_M.

_Duarte

_,

_Luzineide

_W.

_Tinnoco

_,

_Renato

_C.

_Pereira

_,

Valéria

L.

Teixeira

a_{ProgramadePós-graduac¸ãoemQuímicaOrgânica,InstitutodeQuímica,UniversidadeFederalFluminense,Niterói,RJ,Brazil}

b_{GrupodeProdutosNaturaisdeOrganismosAquáticos,NúcleoemEcologiaeDesenvolvimentoSócioambientaldeMacaé,UniversidadeFederaldoRiodeJaneiro,Macaé,RJ,Brazil} c_{InstitutodePesquisasdeProdutosNaturais,UniversidadeFederaldoRiodeJaneiro,RiodeJaneiro,RJ,Brazil}

d_{DepartamentodeBiologiaMarinha.InstitutodeBiologia,UniversidadeFederalFluminense,Niterói,RJ,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received1May2015 Accepted7September2015 Availableonline23October2015

Keywords:

Phaeophyceae Dictyotales Meroterpenoids PCA

Biodiversity Seaweed

a

b

s

t

r

a

c

t

IntraspecificvariationonmeroditerpenoidsproductionbythebrownmarinealgaStypopodiumzonale atfourdifferentpopulationsalongtheBraziliancoastwasanalyzedusingPrincipalComponent Anal-ysisoverhigh-performanceliquidchromatographyprofilesfromalgaeextracts.Theordinationofthe samplesbythe similaritiesof theirchromatographic traitsshowed theexistenceofthree chemo-types:(i)thepopulationsBúziosandAbrolhoswhichwerecharacterizedbythepresenceofatomaric acid(1),(ii)thepopulationAtoldasRocaswhichcontainedthecompoundstypoldione(2),and(iii) thepopulationMarataízeswhichwascharacterizedbyotherpeaksthatguidedtheisolationofthree newmeroditerpenoidsstypofuranlactone(3),10,18-dihydroxy-5′_{a-desmethyl-5}′_{-acetylatomaricacid}

(4),andthe10-keto-10-deisopropyliden-5′_{a-desmethyl-5}′_{-acetylatomaricacid(5)togetherwiththe}

knowncompoundthe10-keto-10-deisopropyliden-atomaricacid(6).Thestructuresandrelative stere-ochemistryof3,4and5wereelucidatedbyNMRandMStechniques.Theobservedchemicaldifferences amongpopulationsofS.zonalecanberelatedtoitsgeographicdistributionandcanopenanavenueto thediscoveryofnewcompoundsinalgae.

©2015SociedadeBrasileiradeFarmacognosia.PublishedbyElsevierEditoraLtda.Allrightsreserved.

Introduction

Thetermmeroterpenoidsisappliedtodescribenatural

prod-uctsofmixedbiosyntheticorigin,whicharepartiallyderivedfrom

terpenoids.Theprefixmero(fromGreekmerus)means“part,

par-tialorfragment”(GerisandSimpson,2009).Therefore,anumberof

secondarymetabolitescanbedescribedasmeroterpenoids,which

assemblesahugerangeofstructuraldiversity,oftenlinkedwith

variedfunctionalities,arisingfromintra-andintermolecularring

closuresand/orrearrangementsoftheterpenechains(Gerisand

Simpson,2009;Macíasetal.,2014).Thisclassofnaturalproductsis

widelydistributedamongmarineorganisms,buttheyare

particu-larlyabundantinmarinebrownalgae,microorganismsandsponge

(Vallimetal.,2005;Mennaetal.,2013).

Within the marine brown algae, order Fucales and the

genusStypopodium(Dictyotales)havebeenrecognizedasarich

sourceofstructurallyunique,biologicallyandecologicallyactive

∗ Correspondingauthor.

E-mail:angelica@iq.ufrj.br(A.R.Soares).

meroditerpenoids(Vallsetal.,1993;Mesguicheetal.,1997;Dorta

et al.,2003;Soares etal.,2007; Mendeset al.,2011)thathave

beennamedbysomeauthorsasstypols(Macíasetal.,2014).Some

stypols,suchasstypoldiol,stypoldioneandepitaondiol,havebeen

tested for theircell proliferation inhibitoryactivity in different

celllines(WhiteandJacobs,1983;Sabryetal.,2005;Pereiraetal.,

2011),thusmakingthempromisingcandidatesasanticancerdrugs.

Stypoldiolandstypoldionehaveremarkableantioxidantproperties

throughradical-scavengingtogetherwithstructurallyrelated

com-pounds,taondiolandisoepitaondiol(Nahasetal.,2007).Inaddition

tothevarietyofbiologicalactivity,theecologicalfunctionas

defen-sivechemicalsagainstherbivoryof thesemeroditerpenoidshas

beendocumented(GerwickandFenical,1981;Pereiraetal.,2004).

Interestingly,theanalysesofmeroditerpenoidscompositionand

concentrationinStypopodiumrevealedanintra-specificvariation

amonggeographicallocations(GerwickandFenical,1981;Gerwick

etal.,1985;Soaresetal.,2003;Pereiraetal.,2004).

Inter- and intrapopulational chemical differences in marine

organisms(orchemotypes)havebeendescribedinseveralstudies

(e.g.Machadoetal.,2014),andthesevariationsareexplainedby

combinationofgenetic(Wrightetal.,2004),developmental(Paul

http://dx.doi.org/10.1016/j.bjp.2015.09.006

andVanAlstyne,1988),andenvironmental(Sudattietal.,2011) factors.

Hereweinvestigatedthespatialvariabilityofthe

meroditer-penoids in Stypopodium zonale (Lamouroux) Papenfuss

(Dictyotales),theuniquespecieofStypopodiumgenusdescribed

toBrazil(GuiryandGuiry,2015).PrincipalComponentAnalysis

(PCA) of the chemical profiles was employed in this study to

accessthechemicalvariabilityamongthepopulationsandguided

theisolationofthreenewcompounds(3–5),inadditiontoother

threeknownmeroditerpenoids(1,2and6).Thestructureswere

elucidated based on spectroscopic and spectrometric analysis

including1_Hand13_CNMR,1_H–1_{HCOSY,HSQC,HMBC,ROESYand}

HREIMS.

Materialsandmethods

Generalexperimentalprocedures

OpticalrotationsweremeasuredonaPerkin-Elmermodel241

polarimeter(Perkin-Elmer,USA)usingaNalampat25◦_C.IR

spec-tra wereobtainedwitha Perkin-Elmer1650/FTIR spectrometer

in CHCl3 solutions. HREIMS spectrum was taken on a Waters

MicromassAutospecmassspectrometer(Waters,EUA).1_HNMR,

13_{CNMR,HSQC,HMBCand}1_H–1_{HCOSYspectraweremeasured}

employingaVarianUnity300instrumentoperatingat300MHzfor

wereacquiredwiththestandardBrukerDRXpulsesequence

oper-ating at 400MHz for 1_{H NMR. The 2D ROESY experiment was}

performedwithmixingtimeof300ms,with16scansat25◦_C.

Sil-icagel(Merck,70–230and230–400mesh)wasusedincolumn

chromatography.Thinlayerchromatographywascarriedoutwith

silicagel60GF254(Merck).Oncedeveloped,spotsontheplates

werevisualizedbysprayingwith2%cericsulphateinsulfuricacid,

followedbygentleheating.

Algalmaterial

SpecimensofStypopodiumzonale(Lamouroux)Papenfuss

(Dic-tyotales)werecollectedbysnorkelingandSCUBAdivingatfour

distinctareas of theBrazilian coast:Forno inlet,Búzios,Rio de

JaneiroState(22◦ ₄₅′_S,41◦ ₅₂′_{W),atthedepthsofthe3–6min}

April,2002;Marataízes,EspíritoSantoState(21◦₀₂′_S,40◦₄₉′_W),

atthedepthsofthe13–15minApril,2002;AtoldasRocas,Rio

GrandedoNortestate(03◦₅₁′_S,33◦₄₀′_{W),at6–10minJune2002}

andAbrolhos,Bahiastate(14◦₄₆′_S,39◦₀₁′_{W),at10–12minApril}

1995.Thealgaewerewashedinseawatertoeliminateassociated

organismsandair-dried. Voucherspecimens (Búzios:HRJ8643;

Marataízes:HRJ5543;Abrolhos:HRJ5593andAtoldasRocas:HRJ

8678)weredepositedattheHerbariumoftheRiodeJaneiroState

University,Brazil(HRJ).

Extractspreparation

Adultandwell-developedspecimensofS.zonalefromBúzios

(115galgadryweight,dw),Marataízes(12.8gdw),Abrolhos(173g

dw)and Atoldas Rocas(106gdw) were air-dried, milled and

extractedthreetimeswithCH2Cl2(using100mlofsolventtoeachg

ofdw)atroomtemperature.Thesolventwasremovedinvacuum

toyieldadarkgreentarinallcases(19g,16.5%dw;1.8g,14.2%

dw;31g,17.9%dw;12g,12.9%dw,respectively).Individual

spec-imens(n=3)fromeachpopulationwereindependentlyextracted

inanidenticalfashionandhadtheirchemicalprofilesanalyzedby

HPLC-DAD.

HPLC-DADchemicalprofiles

Thecrudeextractsandpurecompounds(1)and(2)were

ana-lyzedbyhigh-performanceliquidchromatography(HPLC)and

car-riedoutonaVP-ODSShim-packcolumn(5␮M,250mm×4.6mm)

usinga Shimadzu HPLCchromatographequipped witha Diode

Array Detector (DAD, Shimadzu, Japan) and a coupled system

consistingof a vacuumdegasser,pumpLC-20AT, and detectors

SPD-M20Aand CBM-20A.Abinarygradientelutionatflowrate

1.0ml/min wasemployedusingwater (pH3.0 withphosphoric

acid)assolventAandacetonitrileassolventB,asfollows:60–100%

Bat 0–25min.Theeluent remainedat100%acetonitrileduring

5min,thenchangeddirectlytotheinitialgradientconditions

dur-ing10minforthecolumnre-conditioningbetweentwosequential

injections.TheUV–Visabsorbancespectrawereregistered.Crude

extractswere dilutedin acetonitrileto a final concentrationof

10mg/mlandfilteredina0.45␮MPTFEsyringefilter(Millipore,

USA)prior injection.Analiquotof 20␮lof filtratesolutionwas

injectedforHPLCanalysis.

PrincipalComponentAnalysis(PCA)

ThefirststepofthedatapreparationforthePCAanalysiswas

theremovalofthefirst5minoftheretentiontime(tR)fromthe

two-dimensionalchromatogrammatricesfromeachextract

sam-plefromspecimensofS.zonaleobtainedbytheHPLC-DADsystem,

sinceitcontainsaverylowpeakresolutioncausedbythestrong

overlapping signal from polar compounds. The chromatograms

werethenbaselinealignedandcorrectedforthetimeshiftofpeaks

among them using the Correlation Warping Algorithm (COW),

asdescribed by Nielsenetal. (1998),usingthesoftware “COW

tool” (http://www2.biocentrum.dtu.dk/mycology/analysis/cow/).

Data fromwavelengthof 280nm andtR between5 and27min

wereextractedfromeachofthetwo-dimensionalchromatograms

andplacedinamatrixcontainingallsamples.Eachchromatogram

wasnormalizedbythehighestpeakfoundineachsampleinother

toenhancetheproportionalinformationamonghighpeaks.The

matrixwithallchromatogramswasthen meancenteredbefore

runningthePCAroutine,whichwasconductedusingtheRlanguage

and environment (http://www.R-project.org) withthe installed

package“ChemometricsWithR”(Wehrens,2011).

Isolationandidentificationofthemeroditerpenes

Toobtainthepurestandardsoftheknowncompounds1and

2previouslydescribedbySoaresetal.(2003),theextractsfrom

specimenscollectedatBúziosandAtoldasRocaswere

fraction-ated.FollowingthemethodusedbySoaresetal.(2003),partofthe

extractofthespecimenscollectedatBúzios(2.1g)wassubmittedto

anacidic–basicextraction.Theacidfraction(198mg)was

fraction-atedonsilicagelvacuumliquidchromatographytoyield8mgof1.

TheextractobtainedfromalgaecollectedatAtoldasRocas(2.5g)

waselutedina columnchromatographyover silicagelwithan

increasinggradientofhexane,dichloromethane(CH2Cl2)andethyl

acetate(EtOAc)toprovideaslightlyimpurefractionofcompound

2.Thisfractionwaspurifiedbyvacuumliquidchromatographyover

silicagelandelutedwithhexane:EtOAc(1:1,v/v)toyield2(6mg).

Theextract(1.8g)fromMarataízeswasfractionatedbysilicagel

vacuumliquidchromatographyusingastepwisegradientsolvent

systemofincreasingpolaritystartingfrom100%hexaneto100%

methanol(twelve fractions,A–M).The fractioneluted with15%

EtOAcinCH2Cl2(fractionG,247mg)wasfurtherpurifiedwithsilica

gelcolumnchromatographyusingastepwisegradientofmethanol

(MeOH)inCH2Cl2 toafford3(12.0mg).Thefractionelutedwith

50%EtOAcinCH2Cl2(fractionI,126mg)wasre-chromatographed

usingsilicagelcolumnchromatographywithEtOAc20%inCH2Cl2

toyield4mgofthecompound4.Finally,thefractionelutedwith

5%MeOHinEtOAc(91mg,fractionL)waspurifiedwithsilicagel

columnchromatographyelutedwithincreasingamountofEtOAc

inCH2Cl2togive38mgofmixtureof5and6.

Thestructureof allthecompoundswaselucidatedbasedon

thespectral data (opticalrotation,IR, EIMS and/orHREIMS,1D

and2DNMR).Theknowncompoundswereidentifiedcomparing

thesespectroscopicdatawiththeliterature(MoriandKoga,1992;

Wesselsetal.,1999;Dortaetal.,2003)and, whenavailable,by

directcomparisonwiththeoriginalpurifiedstandards.

Resultsanddiscussion

Marine algae of the Stypopodium genus are a rich source

of meroditerpenoids, which very often display potent

biologi-cal activities in addition to their ecological roles (Valls et al.,

1993; Mesguiche et al., 1997; Dorta et al., 2003; Soares et al.,

2007;Mendesetal.,2011).Thesecompoundshavebeenusedas

chemotaxonomicmarkerstodistinguishtherepresentativesofthe

genuswithintheDictyotales(Vallimetal.,2005).Thepresenceof

thesecompoundsinStypopodiumcanbeusefulfordereplication

purposeshelping onthedetectionof newcompounds amonga

plethoraofknowncompounds.

Here we compared four populations of S. zonale collected

alongofBraziliancoastlineaccordingtotheirHPLC-DADdata.The

ordinationofthepopulationsscoresbythefirsttwocomponents

PCA scores

CP2 (35.0%)

CP1 (62.6%)

Atol das Rocas Abrolhos

Búzios

Marataízes

5

4

3

2

1

0

–1

–2

–3

–4 –2 0 2 4 6

Fig.1.OrdinationofthefirsttwoPCAscores(CP1andCP2)obtainedfromthe analysisofchemicalprofilesfromfourdifferentpopulationsofStypopodiumzonale.

Atol das Rocas Abrolhos

A

B

C

D

E

Búzios

Marataízes

PCA loadings

CP2 (35%)

CP1 (62.6%)

Relative abundance

Retention time (tR)

5 10 15 20

2 1

1

25 –0.1 0.0 0.1 –0.1

0 5 10 0 5 10 0 5 10 0 5 10

–0.1 0.0

Fig.2. Meanchemicalprofiles(n=3)offourdifferentpopulationsofStypopodium zonaleandthePCAloadingsplottedagainsttheretentiontime.Eachchemical pro-filewasnormalizedbyitsmaximalpeakintensityinothertoenhancetherelative differenceamongpeaks.(1)and(2)markthepeaksofatomaricacidandstypoldione respectively.

variation, revealed that thesamples from Abrolhos and Búzios

sitesweregroupedcloselytogetherat thenegativeside ofCP1

(Fig.1).Thisisaresultoftheirsimilarchromatograms(Fig.2Aand

B),differentonlybypresenceofonepeak(retentiontime,tR=22.5)

intheextractsfromAbrolhos specimensofS.zonale.Thesetwo

populationsdifferfromtheotherpopulationsmostlybythe

pres-enceofapeakattR=18.9min,whichhadthestrongestloadingon

samplevariation(CP1,Fig.2E).Incontrast,thesamplesfromAtol

dasRocas,placedatthepositivesideofCP1(Fig.1),donothave

thispeakandweremostlycharacterizedbythreepeaks(tR’s=9.5,

12.9and20.4min)accordingtothePCAloading(Fig.2CandE).

Theextractof samplesfromMarataízeshad a weakcorrelation

withtheotherthreeanditwascompletelyseparatedfromthem

atCP2(Fig.1),withthestrongestpeakloadingsattR’s=8,9and

11min(Fig.2DandE),notobservedinotherpopulations.

Standardsofthemeroditerpenesatomaricacid(Soaresetal.,

2003)(1)andstypoldione(Soaresetal.,2003)(2)wereanalyzed

byHPLCinthesamewayastheextracts.Themajor

chromato-graphicpeakinFig.2AandB(tR=18.9min)wasidentifiedasthe

atomaricacid(1)in Búziosand Abrolhos extracts.In the

chro-matogramofAtoldasRocas(Fig.2C),thepeakattR=20.4minwas

identifiedasstypoldione(2).Tounambiguouslyassignthesepeaks

totheatomaric acid(1)and stypoldione (2), theextractsfrom

BúziosandAtoldasRocaswerefractionatedandthecompounds

wereidentifiedbythecomparisonoftheirspectroscopicdatawith

theliterature(Moriand Koga,1992; Wesselset al.,1999).

Nei-therofthesecompoundswasidentifiedinthechromatogramof

theMarataízessamples,whichwerequitedifferentfromtheother

populationsasshownbyPCAanalysis.1_{HNMRdataofthisextract}

indicatedthepresenceofdifferentmeroditerpenes.Then,inorder

toevaluatetheextractofMarataízesforthepresenceofnew

sec-ondarymetabolites,itwasfractionatedandthreenewcompounds

(3–5)in additionof theknowncompound 6wereisolated and

identified.

Compound 3 wasobtained as an opticallyactive yellow oil

([␣]D26+6.36◦,c0.04,CH2Cl2).The1Hand13CNMRdata(Table1)

combinedwiththe[M]+_{peakatm/z484.2713(calc.forC}

29H40O6,

484.2814) in the HREIMS suggested a molecular formula of

C29H40O6,indicatingthatthiscompoundpossesses10degreesof

unsaturation.TheIRspectrumdisplayedstrongabsorptionbands

forahydroxylgroupat3435cm−1_{andtwocarbonylgroupat1749}

and1740cm−1_.The13_{CNMRspectrumshowedsignalsforall29}

carbons:sevenmethylgroups,sixmethylenegroups(allsp3_),six

methinegroups(4sp3_and2sp2_{)andtenquaternarycarbons(4}

sp3_and6sp2_{).Amongthequaternarycarbons,twoofthemwere}

resonatingatıCcarbonylgroupsofesterorlactonefunctionalities

atıC172.3andıC169.9.The1HNMRspectrumshowedsignalsfor

twometa-coupledaromaticprotonsatıH6.75(d,2.7)andıH6.72

(d,2.7)thattogetherwiththecharacteristicaromaticcarbon

sig-nalsindicatedthepresenceofatetrasubstitutedphenylring.Inthe

up-fieldregionsignalsappearforfivemethylgroupsatıH1.46(s),

ıH1.43(s),ıH1.18(d,6.9),ıH0.98(s)andıH0.80(s).Thesesignals,

inadditiontothebenzylicmethylenegroupatıH3.18(d,13.5)and

ıH2.12(d,13.5),suggestedaditerpenemoietyofthecompound3.

Additionally,theanalysisofthe1_{HNMRspectrumalsoindicated}

anaromaticmethylgroupatıH2.19(s),anacetylgroupatıH2.26

(s)andadeshieldedmethineresonanceatıH4.80(dd,1.2and4.2)

that,byHSQC,correspondedtoanoxygen-bondedmethine.

Theinspectionofthe1_H–1_{HCOSYspectrumallowedusto}

con-necttheprotonatıH4.80(H-13)tothemethylenegroupatıH1.48

(H-12)andthistothemethinegroupatıH1.93(H-11).HMBC

cor-relationoftheprotonatıH4.80(H-13)withthecarbonatıC81.3

(C-10)establishedanoxygenbridgebetweenthesecarbons(atıC

81.3and79.9).TheprotonatıH 4.80(H-13)alsoshowedHMBC

crosscorrelationtocarbonatıH41.4(C-11),whichcombinedwith

further1_H–1_{HCOSYcorrelationsestablishedthefive-membered}

ringfragmentcontainingaheteroatom.Finally,theHMBC

correla-tionsofıH1.43(H-19)tothequaternarycarbonsatıC81.3(C-10),

89.1(C-15),andthemethylgroupatıC26.4(C-20)inadditionto

thesecondcarbonylgroupatıC172.3(C-14)allowedtoestablish

theunusualstructureof3.Interpretationof1_H–1_{HCOSY,HSQCand}

HMBCexperimentsassignedallof1_Hand13_{CNMRsignals(Table1)}

Table1

NMR spectroscopic data for stypofuranlactone (3), 10,18-dihydroxy-5′_{a-desmethyl-5}′_{-acetylatomaric acid (4) and 10-keto-10-deisopropyliden-5}′_{a-desmethyl-5}′ -acetylatomaricacid(5)inCDCl3(300.0MHz).

Position 3 4 5

ıC.m ıH(JinHz) HMBCa _{ıC,m ıH(JinHz) HMBC}a _{ıC,m ıH(JinHz) HMBC}a

1 38.4,CH2 2.12,d(13.5)

3.18,d(13.5)

17,1′_,2′_,6′ _{35.7,CH2 2.31,d(13.8)} 2.86,d(13.8)

2,3,17,1′_,2′_,3′ _{35.8,CH2 2.31,d(12.9)} 3.03,d(12.9)

6′

2 39.8,C 40.0,C 40.6,C

3 37.1,CH 1.75,m 35.5,CH 1.73,m 35.5,CH 1.75,m

4 24.8,CH2 1.31,m

1.82,m

16 25.3,CH2 1.75,m 15 25.4,CH2 1.42,m

1.82,m

5 31.2,CH2 1.37,d(6.6) 35.2,CH2 1.34,m

1.65,m

31.8,CH2 1.44,m 1.64,m

6 35.1,C 38.9,C 42.8,C

7 45.9,CH 2.46,m 46.8,CH 1.31,m 47.4,CH 1.99,d(10.2) 16,17

8 18.0,CH2 1.54,d(4.8)

2.03,m

19.1,CH2 1.38,m 24.1,CH2 1.62,m

2.03,m

9 29.8,CH2 1.85,d(9.3)

2.50,d(9.3)

28.7,CH2 2.47,m 2.54,m

42.3,CH2 2.36,m 2.40,m

10 81.3,C 87.5,C 212.5,C

11 41.4,C 1.93,ddd(1.2,

4.2,13.2)

45.0,C 1.60,m 63.4,CH 2.24,m

12 24.9,CH2 1.48,d(4.2) 17.4,CH2 1.89,d(12.3)

2.25,m

17.2,CH2 1.62,m 1.93,m

13 79.9,CH 4.80,dd(1.2,

4.2)

10,11 35.1,CH2 1.34,m 32.5,CH2 2.44,m

2.49,m

14 172.3,C 175.0,C 179.2,C

15 89.1,C 20.2,CH3 0.92,s 2,3,7 20.1,CH3 0.91,s 2,3

16 20.8,CH3 0.98,s 16.3,CH3 1.08,d(6.9) 16.4,CH3 1.15,d(6.9)

17 15.9,CH3 1.18,d(6.9) 19.5,CH3 1.07,s 6,11 16.9,CH3 0.78,s 5,11

18 16.2,CH3 0.80,s 5,6,7,11 76.9,C

19 22.7,CH3 1.43,s 10,15,20 25.5,CH3 1.29,s 10,18

20 26.4,CH3 1.46,s 27.2,CH3 1.27,s 10,18,19

1′ _{130.0,C 126.8,C 126.7,C}

2′ _{150.2,C 150.3,C 150.5,C}

3′ _{126.7,C 123.7,C 123.7,C}

4′ _{120.9,CH 6.72,d(2.7) 3}′_,6′_,7′ _{121.0,CH 6.73,d(2.7) 2}′_,5′_,7′ _{121.0,CH 6.71,d(2.4) 2}′_,5′_,7′

5′ _{143.6,C 143.1,C 143.0(C)}

6′ _{121.3,CH 6.75,d(2.7) 3}′_,4′_,6′ _{121.4,CH 6.81,d(2.7) 2}′_,5′ _{121.4,CH 6.82,d(2.4) 2}′_,4′_,5′

7′ _{16.6,CH3 2.19,s 2}′_,3′ _{16.3,CH3 2.23,s 2}′ _{15.9,CH3 2.21,s 2}′_,3′

8′ _{169.9,C 169.8,C 170.0,C}

9′ _{21.0,CH3 2.26,s 8}′ _{20.9,CH3 2.26,s 8}′ _{21.0,CH3 2.26,s 8}′

OH 4.58,s 4.56,s

a_{HMBCcorrelationsarefromproton(s)statedtotheindicatedcarbon.}

Therelativestereochemistryof3wasachievedbyROESY

exper-iment(Fig.3)alongwithbiogeneticconsiderations(Gonzálezetal.,

1982).IntheROESYspectrum,correlationsof17-Me(ıH1.18)with

H-7(ıH2.46),H-11(ıH1.93)andH-13(ıH 4.80),inadditionto

thecorrelationofthehydrogenH-13(ıH4.80)and20-Me(ıH1.46)

indicatedthatthesehydrogenatomsarespatiallyclose,and

accord-ingtoDortaetal.(2003),thesewereproposedina␣-configuration.

Ontheotherhand,ROESYcorrelationsofH-3(ıH1.75)and16-Me

(ıH0.98)and18-Me(ıH0.80),andalsothecorrelationof18-Me

(ıH0.80)and16-Me(ıH0.98)and19-Me(ıH1.43)suggestedthat

allofthemwere␤-oriented.

Thus, the structure of 3 was determined and named as

stypofuranlactone,aderivativecompoundofthe5′

a-desmethyl-5′_{-acetylatomaricacid,isolatedoftheS.zonalefromPacific,north}

Atlanticand Caribbean area(Gerwicket al., 1985;Dorta et al.,

2003).Although moleculeswiththisfuran-lactonemoiety have

beenobtainedfromplantsandfungi(Macíasetal.,2014),thisis

thefirsttimethatcompoundswiththismoietyhavebeenobtained

frommarinebrownalgae(MarinLit,2015).

Compound4wasisolatedasapaleyellowoil([␣]D26+4.55◦,

c0.02,CH2Cl2)withthemolecularformula C29H44O7,as

deter-minedbyitsHREIMSatm/z504.3087 [M]+ _(calc.forC

29H44O7,

504.3081),20massunitsmorethancompound3.TheIRshowed

theabsorptionat3434cm−1_{forhydroxylgroupandat1698cm}−1

forcarbonylgroup.The1_Hand13_{CNMRdatafor4(Table1)were}

quitesimilartothoseof3,exceptbyabsenceoftheoxymethine

group,thedifferenceobservedinthechemicalshiftstothetwo

oxygenatedquaternarycarbonsatıC87.5(C-10)andıC76.9

(C-18),and tothegeminaldimethylgroups atıH 1.27(H-20)and

ıH1.29(H-19).IntheHMBCspectrum,thesetwomethylgroups

showedcross-peakswithbothoxygenatedquaternarycarbonsat

ıC87.5(C-10)andıC76.9(C-18).Thesedataindicateda

hydrox-yisopropylmoietyadjacenttothesecondhydroxygroupbonded

tothemainbackboneof4.Thecompleteanalysisofthe2DNMR

experimentsincluding1_H–1_{HCOSY,HSQCandHMBCallowedthe}

determinationoftheplanarstructureofthecompound4,which

wasnamedas10,18-dihydroxy-5′_{a-desmethyl-5}′_{-acetylatomaric}

acid.Therelativestereochemistryofallchiralcentersofthe

com-pound4,exceptforC-10(ıC87.5),wasdeducedbycomparisonof

theROESYcorrelations(Fig.3)withthoseofthecompound3.

The 10-keto-10-deisopropyliden-5′_{a-desmethyl-5}′

-acetyl-atomaric acid(5)and the10-keto-10-deisopropyliden-atomaric

acid (6) were isolated as a homogeneous mixture by TLC.The

compound5wasobtainedasthemajorcomponentin the

mix-turewith6.Themolecularformulaofthemajorcompoundwas

determinedtobeC26H36O6 byHREIMS(m/z[M]+444.2511)and

NMRdata.Comparisonofspectroscopicdataof5(Table1)with

the literature (Gerwick et al., 1985) suggested that compound

5 was closely related to 5′_{a-desmethyl-5}′_{-acetylatomaric acid,}

exceptbyreplacementofisopropylene groupof5′

a-desmethyl-5′_{-acetylatomaric acid by a ketone group at ıC 212.5 (C-10).}

Table2

IsolatedandidentifiedmeroditerpenoidsfoundinfourpopulationsofStypopodiumzonalealongtheBraziliancoast.

Meroditerpenoids Populations

Abrolhos Búzios AtoldasRocas Marataízes

Atomaricacid(1) X X

Stypoldione(2) X

Stypofuranlactone(3) Xa

10,18-Dihydroxy-5′_{a-desmethyl-5}′_{-acetylatomaricacid(4) X}a

10-Keto-10-deisopropyliden-5′_{a-desmethyl-5}′_{-acetylatomaricacid(5) X}a

10-Keto-10-deisopropyliden-atomaricacid(6) X

a_{Newcompounds.}

Fig.3.KeyROESYcorrelationsofthemetabolites3–5.

assigningallof1_Hand13_{CNMRsignals(Table1)tothecompound}

5.Theminorcompoundinthemixturewasidentifiedasthe

10-keto-10-deisopropyliden-atomaricacid(6)byanalysesofthe1_H

NMRand13_{CNMRdataandcomparisonthemwiththeliterature}

(Dorta et al., 2003). By comparison of the ROESY correlations

(Fig.3)ofthe5and6compoundswiththoseofthecompound3,

wecoulddeducethattheyhavethesamerelativestereochemistry

of3.

Table2showsthemeroditerpenoidsidentifiedinthefour

stud-iedpopulations,inwhichwecoulddistinguishthreechemotypes

constitutedby algaefrom(i) Abrolhos andBúzios,(ii)Atol das

Rocasand(iii) Marataízes.Inthelast one,thenewcompounds

(3),(4)and(5)werefound.Chemicaldiversityamongpopulations

ofS.zonalewasreportedinsmallandlargespatialscale.Gerwick

etal.(1985)isolateddifferentcompoundsofthisspecieoccurring

atSouthAtlantic(Palao)andattheCaribbeanSea(Belize).Within

thecollectionsiteofBelize,theseauthorshavealsofoundchemical

diversitydifferencesbetweenpopulationsfromdeepandshallow

waters.DifferentandnovelcompoundsofS.zonalewerealsofound

inotherstudiescomparingpopulationsfromdistantgeographic

locations(Dortaetal.,2002,2003).Additionally,previousstudies

fromourresearchgrouphave demonstratedintraspecific

varia-tioninS.zonalefromBrazilianpopulations.Collectioncampaigns

in1998/1999showedthepresenceofatomaricacid(1)relatedto

otherpopulationsfromArchipelagoofAbrolhosandBúzios,Riode

JaneiroState,aswellasthepresenceofstypodione(2)in

popula-tionsfromAtoldasRocasandArchipelagoofFernandodeNoronha

(Soaresetal.,2003;Pereiraetal.,2004).Thesefindingsare

congru-entwiththechemotypesfoundinthiswork,wherethepopulations

fromAbrolhosandBúziosstillshowedthepresenceofatomaric

acidandAtoldasRocas,thestypodione.Thispatternwas

indepen-dentofthecollectiontime,indicatingatemporalconservationof

thechemotypesfound.

ThedifferencesobservedusingPCAonHPLCprofilesamongthe

samplesclearlyshowedthatthepopulationsofS.zonalealongthe

Braziliancoastcouldbeuniqueinrelationtosecondary

metabo-litecomposition.Thechemicaldiversityinalgaecouldarrivefrom

bioticandabioticfactors,andalsobytheinteractionbetweenthem.

CrossingexperimentswithspeciesofLaurenciagenusrevealedthat

geneticvariationwasresponsibleforthediversityofhalogenated

metabolitesinnaturalpopulationsofL.nipponica(Masudaetal.,

1997).Nevertheless, the intraspecific variations of halogenated

compoundsarerelatedtoalgalsexandlifehistoryphasesinthe

redalgaAsparagopsisarmata,whichresultsinaselectiveherbivory

byseahare(Vergésetal.,2008),andstresstheimportance

intraspe-cific variation on ecological and evolutionary consequences of

plant–herbivoreinteractions.Furthermore,abioticfactorssuchas

temperature(Sudattietal.,2011),salinity(Kuwanoetal.,1998),

lightexposure(Yuanetal.,2009),inductionofchemicaldefenses

againstherbivoresand spacecompetitionpressure (Amade and

Lemée,1998)arealsorelatedtointraspecificdifferencesin

chemi-calcompositionofmarineorganisms.Althoughthespecificsource

ofintraspecificvariationwasnotconsideredhere,thehigh

chem-icaldiversityofStypopodiumzonaleisoncemoresuggestedtobe

relatedtogeographicdistribution.

Moreover,theapplicationofanalyticalmethodstogetherwith

multifactorialstatisticshasproventobeausefultolltoordinate

samplesbythesimilaritiesoftheircomplexchromatographictraits,

andguidetheprioritization,whichhelpsonisolationand

identifi-cationofnewmeroditerpenesinS.zonale.

Author’scontribution

ARS collectedpart of thebiological material, performedthe

experiments,analyzedthedataandwrotethepaper.HMDwas

responsibleforthePCAandwrotethepaper.LTandVLTcontributed

workanddidcriticalreadingofthemanuscript.Alltheauthorshave

readthefinalmanuscriptandapprovedthesubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgements

ThisresearchwasfinanciallysupportedbyCNPqandFAPERJ.

ARS,RCPandVLTthankCNPqforprovidingResearchGrants.We

arealsogratefultoFAPERJforthe“Nota10”doctoralfellowship

toARS.Dr.RobertoC.Villac¸a(UFF)identifiedthespecimensfrom

allpopulationsandalsokindlycollectedthealgaeatMarataízes

site.The authorsthankLorenaSigilianofor technicalassistance

withtheHPLCanalysis.WealsothanktheNMRLaboratory

(Insti-tutode Química.UFF), Thomson MassSpectroscopy Laboratory

(InstitutodeQuímica,UNICAMP)and CNRMN(Institutode

Bio-química Médica, UFRJ) for analyses.We greatly appreciate the

helpfulcommentsofHosanaDebonsi(FCFRP-USP)thatimproved

themanuscript.

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