ORI GI
NALPAPERRutheniumversusplatinum:interactionsofanticancermetallodrugswithduplexoligonucleotidescharacterisedbyelectrosprayionisationmassspectrometryMichaelGroesslYuryO.TsybinChristianG.HartingerBernhardK.KepplerPaulJ.DysonReceived:5November2009
/ Accepted:3February2010 /
Publishedonline:7March2010SBIC2010Abstract
Thebindingoftheruthenium-basedanticancerdrug candidates KP1019,
NAMI-A and RAPTA-Ttowards different double-stranded
oligonucleotides wasprobedbyelectrosprayionisationmass
spectrometryandcompared with that of the widely used
platinum-basedchemotherapeuticscisplatin,carboplatinandoxaliplatin.Itwas
foundthat theextent of adduct formationdecreasedin the following
order: cisplatin[oxaliplatin[NAMI-A[RAPTA-T[carboplatin [KP1019. In
addition to thecharacterisation of the adducts formed with the
DNAmodels, thebindingsites of themetallodrugs
ontheoli-gonucleotides were elucidated employing top-down
tan-demmassspectrometryandwerefoundtobesimilar forall
themetallodrugsstudied, irrespectiveof thesequenceof the
oligonucleotide. Astrong preference for
guanineresidueswasestablished.Keywords Ruthenium Platinum
Anticancer Oligonucleotides MassspectrometryIntroductionFollowing
the discoverythat cisplatin inhibits cell
divisioninbacteriabyRosenberginthe1960s,
anditssubsequentidentication as a potent anticancer agent, it
quicklybecame, andstill remains, themost
widelyusedchemo-therapeutic drug for the treatment of a wide range
ofmalignancies [1, 2]. Although cisplatin is extremelyeffective
against several cancers, e.g. testicular and
ovariancancers,itsapplicationisalsoaccompaniedbyseveresideeffects,
such as nausea, vomiting and hearing loss, andmany tumours show
intrinsic or acquired resistance againstthedrug.
Thesefactsledtothedevelopment andclinicalapproval of platinum-based
derivatives, i.e. carboplatin
andoxaliplatin(Fig.1)[3],whichmainlydifferfromcisplatinintheir
rateof aquationandthereforetheir overall reac-tivity. More
recently, drugs based on metals other thanplatinum,
suchasruthenium, gallium, titaniumandgold,have been developed to
treat cisplatin-resistant cancers [4],and it is thought that these
drugs exhibit a different mode ofaction compared with
platinum-based drugs and
couldthereforewidentherangeofcancersthatcanbetreated.Indazolium
trans-[tetrachloridobis(1H-indazole)ruthe-nate(III)] (KP1019),
imidazoliumtrans-[tetrachlorido(1H-imidazole)(S-dimethylsulfoxide)ruthenate(III)](NAMI-A)and
[dichlorido(g6-toluene)(PTA)ruthenium(II)],
wherePTAis1,3,5-triaza-7-phosphaadamantane(RAPTA-T),allof
whichincorporate rutheniumas thecentral atom, areamong the most
promising candidates (Fig.1) [5]. KP1019and NAMI-A have
successfully completed phase 1 clinicaltrials;
NAMI-Aenteredphase2clinical
trialsin2008(incombinationwithacytotoxicdrug), andKP1019isaboutto
follow. NAMI-Aand RAPTA-T seemto be
highlyeffectiveagainstmetastases,whereasKP1019showsgoodactivity
towards primary tumours. For all of the
compoundsElectronicsupplementarymaterial
Theonlineversionofthisarticle(doi:10.1007/s00775-010-0635-0)containssupplementarymaterial,
whichisavailabletoauthorizedusers.M.Groessl(&) Y. O.Tsybin P.
J.DysonInstitutdesSciencesetIngenierieChimiques,EcolePolytechniqueFederaledeLausanne(EPFL),1015Lausanne,
Switzerlande-mail:[email protected]. G.Hartinger B.K.
KepplerInstituteofInorganicChemistry,UniversityofVienna,
WahringerStr.42,Vienna1090, Austria1
3JBiolInorgChem(2010)15:677688DOI10.1007/s00775-010-0635-0lowgeneral
toxicity combined with excellent clearancerateswasobserved.The
antiproliferative activity of platinum-based anti-cancer agents
relies on interactions with DNA,
bindingmainlytoadjacentguaninemoieties,whichleadstokinksin the
molecular structure and ultimately to inducingapoptosis[6].
Interactionswithproteins, especiallyinthecase of ruthenium, have
alsobeensuggestedtoplayanimportant role, although direct
interactions with
DNAcannotbeexcluded[7,8].Overtheyears,virtuallyalltheavailable
bioanalytical/biophysical techniques, includingspectroscopy,
chromatography, electrophoresis, X-raydif-fractionanalysis,
isotopiclabelingandmassspectrometry(MS), as well as hyphenated
techniques, have been appliedtothestudy
ofmetallodrug(model)DNAinteractions[9].Following the development of
electrospray ionisation(ESI) MSandmatrix-assistedlaser
desorption/ionisation(MALDI) MStechniques inthelate1980s,
theyquicklybecame invaluable for the analysis of nucleic acids
andoligonucleotides [10], and therefore also for the analysis
ofmetal complexDNA interactions [11, 12]. AlthoughESI-MS analysis
of duplex DNAdrug interactions
hasbeenreportedonseveraloccasions[1315],eventhemostrecent studies
focused primarily on the adducts formedwith single strands and the
mechanisms underlyingdissociation in the gas phase [16, 17].
Cisplatin
formsmainlyDNAintrastrandcross-linkswithnucleobases[18],but the
formation of interstrand cross-links is also possible,and they
might contribute signicantly to the drugsactivity,
withenhancedrepair beingoneof thecausesofclinical
acquiredresistance[19, 20]. Althoughrutheniumdrugs have
alsobeenshowntobindtonucleotides andDNA[21], adduct
formationproceedsmoreslowlycom-pared with cisplatin, therefore
substantiating the hypothesisthat DNAmight not bethe(primary)target
forthiscom-poundclass.Inthis paper,
wedescribeaMS-basedtechniquefortheanalysis of adducts formed
between duplex DNAandanticancer metallodrugs. Using a multistage
mass-spec-trometric top-down approach, we have elucidated
thebindingsitesofthemetal-baseddrugs.MaterialsandmethodsESI-MSfor
characterisationof duplexDNAmetallodruginteractionswascarriedout
withanUltimaIIquadrupoletime of ight (TOF) mass spectrometer
(Waters,Manchester, UK) operated in positive and negative ionmode.
Theinstrumentwascalibrateddailyusinga0.01%PtNH3ClCl
NH3PtNH3NH3OOOOPtH2NNH2OOOORuClClClNSO
CH3CH3NHRuClClClNNClNHHNRuClClPNNNClB A CF E DFig.1
Platinum-basedpharmaceuticalscisplatin(a),carboplatin(b)andoxaliplatin(c)andtheruthenium-basedanticancerdrugcandidatesKP1019(d),
NAMI-A(e)andRAPTA-T (f). Counterions havebeenomittedforclarity678
JBiolInorgChem(2010)15:6776881
3phosphoricacidsolutionin50%acetonitrile. Determina-tionoftheextent
ofadduct formationwascarriedout bymonitoringthe5? and6?
chargestatesoftheduplexandthe corresponding adducts and integrating
their peak areas.Samples containing an effective complex to duplex
ratio of3:1were
freshlypreparedin20mMammoniumacetatebufferatpH7.4,andincubatedinEppendorftubesforupto120hat37
C(duplexconcentration25 lM).Aliquotsof20
lLweretakenafter1,3,6,24and120handstoredat -20 Cuntil analysis. The
samples were mixedwith100mMammoniumacetateinmethanol at
aratioof1:1immediatelyprior
toanalysistoenhancesprayformationandthestabilityofthe
duplexDNAinthegasphase. Dataacquisitionandanalysiswerecarriedout
usingtheMass-Lynxsoftwarebundle(Waters).Fragmentationexperimentsforbindingsiteelucidationwerecarriedout
withalineariontrapmassspectrometer(LTQXL, ThermoFisher Scientic,
Bremen, Germany),specically
innegativeionmodeowingtoitscapabilitytoperformmultiple-stageMS/MSexperiments.
For identi-cation of unmodied fragments the Web-based MongoOligo
Mass calculator version 2.06
(http://library.med.utah.edu/masspec/mongo.htm) was used [22].
Samples con-taininganeffectivecomplextoduplexratioof
1:1werepreparedinammoniumacetatebufferatpH7.4andincu-batedat 37
Cfor upto72h. For analysis,
thesamplesweredilutedtenfoldwithasolutioncontainingmethanol/water/n-propanol
in a ratio of 65:20:5 and placed into a
96-wellplateinanAdvionTriVersanano-ESIrobot(AdvionBiosciences,
Ithaca, NY, USA) equippedwitha 5.5-lmnozzlechip. TheESI robot
wascontrolledwithChipSoftversion7.2.0employingagaspressureof
0.45psi andaspray voltage of 1.7kV. The mass spectrometer
wasoperatedinenhancedresolutionmodeandforfragmenta-tionexperimentsanisolationwidthof3.0m/zandarela-tive
collision energy of 35%were used. The Xcalibursoftware bundle was
utilised for data acquisition
andanalysis(ThermoFisherScientic).MALDI-MS experiments were carried
out in linearmode usinganAximaCFRPlus (Kratos/ShimadzuBio-tech,
Kyoto, Japan) MALDI-TOF instrument. MALDImatrices(nicotinicacid,
anthranilicacid,
trihydroxyaceto-phenone,3-hydroxypicolinicacidandpicolinicacid)wereobtainedfromSigma-Aldrich(Buchs,Switzerland).High
performance liquid chromatography (HPLC)-puried synthetic
oligonucleotides were purchased fromMicrosynth(Balgach,
Switzerland)andDNATechnology(Risskov, Denmark). Duplex DNA was
prepared by mixingthe corresponding single strands (200 lM) in
100mMaqueous ammoniumacetate, heatingat 70Cfor
15minandthenannealingbyslowlycoolingto4C.
Annealingwasconrmedbygelelectrophoresis.
Ammoniumacetate(puritymorethan99.99%)
andcisplatinwerepurchasedfromSigma-Aldrich(Switzerland),
andHPLC-gradesol-vents(water, acetonitrile, n-propanol
andmethanol)werefromAcros (Geel, Belgium). Oxaliplatinwas
purchasedfrom Sequoia Research Products (Pangbourne, UK).KP1019,
carboplatin,
RAPTA-TandNAMI-Aweresyn-thesisedasdescribedelsewhere[2325].ResultsanddiscussionOptimisationofconditionsToestablishtheoptimumtechniqueandconditionsfortheanalysis
of the metallodrug-modied duplex DNA, bothESI-MSand MALDI-MSwere
evaluated for their suit-ability. The rst successful analysis of
intact double-stranded DNA by MALDI-MS was reported in 1995 [26]but
owingtotheconsiderabledissociationof
theduplexintothesinglestrandsforshortoligonucleotides(lessthan20-mer),
the approach has mainly been used to study longerDNAfragments[27].
Furthermore, MALDI-MShasbeenusedfor analysis of interactions
betweensingle-strandedoligonucleotideswithrutheniumdrugs[28].Forallthreedoublestrands(seeTable1forsequencesand
molecular masses), MALDI-MS experiments withcommonlyusedmatrices
containingeither nicotinicacidcombinedwithanthranilicacid[29],
6-aza-2-thiothymine[27, 30] and trihydroxyacetophenone [28] or a
combinationof 3-hydroxypicolinic with picolinic acid [31] were
carriedout. Independent of the matrix composition, the
ionisedspecies consisted mainly of the single strands in
bothpositiveandnegativeionisationmodes(seetheelectronicsupplementary
material). Also note that analysis could onlybecarriedout inlinear
modeastheadditional
ionighttimeinthereectronmodeinducedfragmentationof
theoligonucleotides. Consequently, no further experimentsprobing
the adducts formed between the duplexes andmetallodrugs were
carried out as dissociation of the
doublestrandshadtobeexpectedforthesesystemsaswell.Although it seems
logical to performthe analysis ofoligonucleotides in negative ion
mode owing to theirnegativelychargedphosphatebackbone, goodresults
forintact duplexDNAdruginteractioncanalsobeobtainedinthe positive
mode byESI-MS[14, 15]. Inthis case,dissociationof
thedoublestrandisprobablyhinderedbythepresenceof ammoniumions,
whichmaystabilisetheduplexduringiontransferfromsolutiontothegasphasein
the electrospray process. Still, the majority of publi-cations
dealing with ESI-MS of duplex DNA havereported the utilisation of
negative ion mode with highsalt concentrations in the spraying
solution [13], whichhas tobe consideredthe more widelyapplicable
instru-ment setting.JBiolInorgChem(2010)15:677688 6791
3Toestablishtheoptimalconditionsforourapplication,ESI-MSspectraof
theoligonucleotidesandmetallodrugswere recorded in positive and
negative ion mode
withvariousammoniumacetateconcentrationsbetween0and100mMandwithmethanol
contentsof upto50%. Sur-prisingly, the most satisfactory results
were obtained inpositive ion mode with a spraying solution
containing50mMammoniumacetate in50%methanol,
especiallywhenanalysingmetallodrugolignucleotideadducts(notethat
positive charges are introduced by the metallodrugfragments).
Althoughthesignal-to-noiseratioispoorerinpositiveionmode,owingtoadductformationbetweentheoligonucleotides
andammoniumions as well as sodiumand potassiumions leading to
additional peaks in thespectra, increased stabilisation of the
duplexes
wasobservedcomparedwiththenegativeionmode(seetheelectronicsupplementarymaterial),
especiallywhensam-ples containing the oligonucleotides and
metallodrugs wereanalysed. Therefore, this solvent system was
chosen for allfurther measurements which focused on the analysis
ofintact doublestrands. Negativeionmodewasselectedtoidentifythe
preferredstrandfor bindingof the metallo-drugs
incubatedwithduplexDNAinaccordancewithastudyreportedearlier[16].Interactionswithdoubled-strandedDNAPlatinumcomplexesFor
platinum-based compounds, the main focus of
attentionisontheirinteractionswithDNAsinceitisconsideredtobe the
ultimate target inside the cancer cell. A multitude
ofstudiesonthistopichasbeencarriedout over theyears,althoughenzymes
andproteins havealsobeenshowntoplaya role inthe inhibitionof
transcriptionandconse-quentlyintheinductionofapoptosis[32].
Thethreeplat-inum compounds were incubated with the
selectedoligonucleotidesfor upto120hunder simulatedphysio-logical
conditions. Following incubation, the samples
wereanalysedbyESI-MSandthespectraobtainedwerecom-paredwiththoseofthepureduplexes.For
samples containing the duplexes with multipleguanines (G; it is
known that binding occurs mainlythrough interaction with the
nucleophilic N7 of thisnucleotide),i.e.DS1andDS2,a
fastdecreaseofthe peakscorresponding to the unmodied
oligonucleotides wasobservedwhenincubationwascarriedout
withcisplatin.This was accompanied by an increase in the
relativeintensityof thepeaks
correspondingto[DS?Pt(NH3)2]and[DS?2Pt(NH3)2]species.
Ifthenumberofguaninesisreducedtooneineachstrand(DS3),
consequentlyalsorenderingintrastrandGGcross-linkingimpossible,
onlyabout40%oftheoligonucleotideismodiedafter24hofincubation,
whereasthisnumberismorethandoubledforDS1 and DS2 with more than one
guanine residue(Table2). Note that the extent of modication as
shown inFig. 3andTable2representsonlyanestimateasadductformation
with the metal complexes, which introduceadditionalpositivecharges,
mightleadtodifferentionisa-tioncharacteristics
comparedwiththeunmodiedoligo-nucleotides. However, an approximation
of the
relativereactivityofthedifferentcompoundscanbederivedfromthedata.Interestingly,
eveninthecaseofDS3(themost prom-inent peaks correspond to the 6?
and 5? species at 1,628.3and 1,953.8m/z, respectively; Fig. 2), the
major adduct is abifunctional oligo-[Pt(NH3)2] species
involvingcoordina-tion of the metal centre to the duplex as, for
example,evidenced by the peak at 1,999.3m/z corresponding
to[DS3?Pt(NH3)2]5?(Fig.2). This infers that
eitheradenosineservesasasecondbindingpartnertoformGAor GXA(XisC, T)
cross-links or interstrand
cross-linkingbetweenthetwoguanineresiduesoneachstrandtakesplace.Indeed,bothprocesseshavebeenshowntoberelevantinthebiologicalenvironment[33].
Whenspectraare recorded in the negative mode, the majority of
thesignalsareattributable
tosingle-strandedoligonucleotides,indicating that the relative
amount of interstrand cross-links is below5%as covalent
bridgingviatheplatinummoiety should lead to a stable
double-stranded species.Also bisadducts detected in positive ion
mode([DS3?2Pt(NH3)2]5?at2,044.7m/z)indicatebindingofonemetallodrugmoietytobothofthestrands.Results
comparable with those for cisplatin were
obtainedforcarboplatinandoxaliplatin,
althoughadductformationproceeds more slowly compared with that for
cisplatin(Fig.3, Table2). This correlates tothe slower
aquationkinetics of carboplatinandoxaliplatinrelativetothat
ofcisplatin(theaquatedspeciesarebelievedtobeessentialintermediatesduringthereaction)
[33]. TheformationofTable1 Sequences and masses of the
complementary oligonucleotidesSequence Monoisotopicmass(Da)DS1
7,904.4SS1a 50-d(GTATTGGCACGTA)-303,987.7SS1b
50-d(TACGTGCCAATAC)-303,916.6DS2 7,905.4SS2a
50-d(TACGTGCCAATAC)-304,003.7SS2b 50-d(TACACACCGGTAC)-303,901.7DS3
9,753.7SS3a 50-d(ATATTATGCTTAATTA)-304,867.8SS3b
50-d(TAATTAAGCATAATAT)-304,885.9680 JBiolInorgChem(2010)15:6776881
3adducts occurs after exchange of the dicarboxylato and ox-alato
ligands, respectively, resultingin mainly bifunctionalcross-links,
as evidenced, for example, by the peakat 2,015.3m/z corresponding
to [DS3?Pt(DACH)]5?(DACHis1,2-diaminocyclohexane) for oxaliplatin
inFig. 2. Also, the formation of a bisadduct similar to the caseof
cisplatin can be observed ([DS3?2Pt(DACH)]5?at2,076.6m/z); for
carboplatin, the spectrumresembles that ofcisplatin (although with
lower intensities for the
metallodrugadductsrelativetothefreeunmodiedoligonucleotides),with a
major peak at 1,999.3m/z corresponding to[DS3?Pt(NH3)2]5?(not
shown). For reactionswith DS3,no signicant formation of interstrand
cross-links wasobserved, as evidenced by spectrarecordedin negative
ionmode. Alistofmetallodrugspeciesboundtotheoligonu-cleotides is
given in Table2.Eventhoughdissociationof thedoublestrands
inthenegative ion mode seems disadvantageous, it
providesinformation on the distribution of the metal
complexesbetween the single strands. For DS1 a slightpreference
forbindingtowardsSS1awasobserved,forDS2apreferenceforSS2aandforDS3adduct
formationwithbothstrandsproceeds at approximately the same rate.
These preferencescan be explained by the higher number of guanine
residuesintherespectivestrands,andadditionally,forexample,bythepresenceoftheadvantageoussequenceTGGCinSS1acomparedwithGTGinSS1b[34].RutheniumcomplexesThe
reactivityof rutheniumcompounds towards nucleo-tides has already
been demonstrated [35], although adifferent modeof actionas
comparedwiththeplatinumdrugsincludingproteininteractionshasbeenproposedbyseveralauthors[36].
Suchinteractionsmightexplaintheiractivitytowardscisplatin-resistant
cancer cellsaswell asthe antimetastatic properties of
NAMI-AandRAPTA-T[37,38].Analogoustotheexperimentswiththeplatinumcom-pounds,
all measurements were carried out in both positiveand negative ion
mode. When the spectra of the unmodiedoligonucleotides are compared
with those of the ruthenatedoligonucleotides, it can
bededucedfromthe differences inmass that the compounds have to
undergo ligand exchangebefore or on binding to the
oligonucleotides. For RAPTA-T,the major adducts were assigned to
[Ru(PTA)]- and[Ru(PTA)(T)]-containingspecies. For NAMI-A,
themostprominent adduct peaks correspond to the
oligonucleotideswiththe [Ru(Im)] moiety(Imis imidazole) is
attached,whereas [Ru(Ind)] and [Ru(Ind)2] adducts (Ind is
indazole)arefoundforsamplesincubatedwithKP1019.AsaseriesofaquatedspeciesaredetectedforNAMI-AandKP1019,whichresultsinahighernumberofpossibleadducts,dataanalysis
is complicated compared with that for the sampleswith
platinumcompounds. The apparently multidentatenature of the
bindingtoDNAseverelychanges its
sec-ondarystructure,leadingtodissociationoftheduplexintoits single
strands upon binding of the
ruthenium-baseddrugs.Consequently,almostnoadductformationbetweenintact
duplexDNAandthedrugs
couldbedetectedandnegativeionmodeprovidedinformationsimilartothatforpositive
ion mode, although at higher signal-to-noise
ratios(seetheelectronicsupplementarymaterial).
Additionally,thenatureoftheadductswasconrmedbymeasurementsTable2
Assignment of
adductsformedbetweenoligonucleotidesandthemetallodrugsstudiedaswellastheestimatedpercentageofoligonucleotidemodiedafteranincubationperiodof24hDACH1,2-diaminocyclohexane,PTA1,3,5-triaza-7-phosphaadamantane,
Indindazole,ImimidazoleDrug Adducts
Averagemass(Da)Relativeamountofmodiedduplexafter24h(%)DS1 DS2
DS3Cisplatin Pt(NH3)2229.1 89 84 40Pt(NH3)2Cl 264.6Carboplatin
Pt(NH3)2229.1 29 24 24Oxaliplatin Pt(DACH) 309.3 68 51 34RAPTA-T
Ru(PTA) 258.2 46 37 36Ru(PTA)(T) 350.4Ru(PTA)(T)Cl 385.8KP1019 Ru
101.9 13 18 15Ru(Ind) 219.2Ru(Ind)(H2O)
237.2Ru(Ind)(H2O)2255.2Ru(Ind)2337.3NAMI-A Ru 101.9 60 53 27Ru(Im)
169.1Ru(Im)(H2O)
187.1Ru(Im)(H2O)2205.1JBiolInorgChem(2010)15:677688 6811
3innegativeionmodeaslossofthechloridoligandscouldotherwisebeinterpretedasanartefactduringionisationinpositive
ion mode. Furthermore, data on the preferredstrand were obtained
owing to the dissociation of thedouble strand, showing a higher
extent of adduct formationfor singlestrandsSS1aandSS2asimilar
totheplatinumcompounds. Again, nodifferencewasobservedfor
SS3aandSS3b.ItwasexpectedthatRAPTA-Twouldformthegreatest number of
adductssincetherutheniumcentreinthiscompoundisinthemorereactive?IIoxidationstatecompared
with ?III in KP1019 and NAMI-A[7]. Sur-prisingly, thedoublestrands
aremodiedtothegreatestextent by NAMI-A, whereas KP1019 only shows
very littleinteraction with the oligonucleotides (Fig.3,
Table2).Theseobservationsmight beexplainedbythehydrolysiskineticsof
thecompounds; accordingtopreviousstudies[39, 40], NAMI-Ais
aquatedmorerapidlythanRAPTAcompounds.DS2time [min]rel. amount of
modified DNA0.00.20.40.60.81.0DS3time [min]rel. amount of modified
DNA0.00.20.40.60.81.0CisplatinOxaliplatin
CarboplatinRAPTA-TKP1019NAMI-ADS1time [min]0 200 400 600 800 1000
1200 14000 200 400 600 800 1000 1200 14000 200 400 600 800 1000
1200 1400rel. amount of modified DNA0.00.20.40.60.81.0Fig.3
Estimated extent of binding of the metallodrugs
studiedtowardsduplexDNA.TopDS1,middleDS2andbottomDS31703.92044.71999.31628.3DS6+1666.1[DS+
Pt(NH3)2]6+[DS+ 2Pt(NH3)2]6+1953.8DS5+[DS+ Pt(NH3)2]5+[DS+
2Pt(NH3)2]5+1628.3DS6+1953.8DS5+1628.3DS6+1953.8DS5+1740.92076.62015.31679.5[DS+
Pt(DACH)]6+[DS+ 2Pt(DACH)]6+[DS+ Pt(DACH)]5+[DS+ 2Pt(DACH)]5+1500
1700 1900 2100 m/zRelative Intensity(%)100100100000Fig.2
Electrosprayionisationtime
ofight(ESITOF)massspectrashowingtheinteractions
betweenDS3andplatinum-basedchemo-therapeutics. TopunmodiedDS3,
middleDS3?cisplatin(incuba-tion with carboplatin yields the same
adducts albeit with lowerrelative intensity) and bottom
DS3?oxaliplatin. DACH 1,2-diaminocyclohexane682
JBiolInorgChem(2010)15:6776881 3Asdepictedin Fig. 4
forDS2,themostintenseionsarefound for the -5 charge state, with the
monoisotopic peaksat 799.8and779.4m/zassignedtounmodiedSS2aandSS2b,
respectively, whentheinstrument was operatedinnegative ionmode.
Minor peaks stemmingfromadductformation with sodium, potassium and
ammonium ions arealsopresent inthespectrabut havenot
beenlabelledforclarity. For RAPTA-T, the two chlorido ligands are
lost onbinding, but during prolonged incubation also the
aromatictoluene ring is displaced, as noted previously for
otherRAPTAcompounds [41, 42], resulting in peaks at m/zvalues of
832.4and850.9for [SS2b?Ru(PTA)]5-and[SS2b?Ru(PTA)(T)]5-(Fig.4,
topspectrum).
Similarly,peakswithslightlyhigherrelativeintensitiesat852.9and871.3m/z
were assigned to [SS2a?Ru(PTA)]5-and[SS2a?Ru(PTA)(T)]5-,
respectively. Adducts
containingthe[Ru(PTA)(T)Cl]-moietyareonlypresentatverylowintensitiesforincubationtimeslongerthan6h.NAMI-A
undergoes a more complicated series ofhydrolysis reactions prior to
the formation of stable adductswithbiomolecules. Indeed,
inapreviousstudycharacter-ising the interactions of NAMI-A with
cytochrome c, it wasshown that the chlorido ligands have to be
substituted(partially by aqua ligands) to form a stable
adduct[43].Consequently, thepeaksinFig. 4(bottomspectrum)at 814.6,
818.2 and 821.8m/z have been assignedto [SS2b?Ru(Im)]5-,
[SS2b?Ru(Im)(H2O)]5-and[SS2b?Ru(Im)(H2O)2]5-, respectively, whereas
thepeaksat832.6, 836.2and839.8m/zcorrespondtosimilaradducts formed
with SS2a. Furthermore, the peaks at868.8, 872.4, 876.0 and
879.6m/z indicate binding of
eventwopartiallyaquatedRu(Im)containingmoietiestoSS2a,which in
general seemed to be the preferred binding
partnerowingtothehighernumberofguanosineresidues.AlthoughtherateofaquationofthechloridoligandsofKP1019iscomparabletothatofNAMI-A,
thereisasec-ondnitrogen-containingheterocyclecoordinatedtoruthe-nium
which is more strongly coordinating than thedimethyl
sulfoxideligandinNAMI-A. Asthesenitrogendonorligands arein a
transpositionto
eachother,bindingtotheoligonucleotidecouldbestericallyhindered,
there-foreexplainingthelowerextentofadductformation. Themajor peaks
in Fig. 4 for the KP1019DS2 interaction(middle spectrum) where
assigned as follows: 824.5m/z to[SS2b?Ru(Ind)]5-, 848.6m/z to
[SS2b?Ru(Ind)2]5-,845.0m/z to [SS2a?Ru(Ind)]5-and 868.5m/z
to[SS2a?Ru(Ind)2]5-. Alsonote that there is a series ofminor peaks
correspondingtotheaquatedspecies of
theRu(Ind)moiety.ForbothKP1019andNAMI-A,
evenadductswithonlytherutheniumcentre(lossof all original ligands)
coordi-nated to the oligonucleotides were detected after
incubationtimesof 5days, whereas the PTA ligand
stayscoordinatedtorutheniuminRAPTA-T.
Thisresultisrathersurprisingaslossof
thenitrogen-containingligandsinKP1019andNAMI-A was not observed in
earlier protein bindingexperiments; inour study,
theligandexchangecouldbeinducedstericallyowingtotheexibilityof
theoligonu-cleotide.
AlistofmetallodrugspeciesfoundboundtotheoligonucleotidesisgiveninTable2.BindingsitedeterminationToestablishthebindingsiteof
themetallodrugs, i.e. themetalated nucleobases, MS/MSexperiments
using colli-sion-induceddissociation(CID) werecarriedout.
CIDofdouble-stranded DNA yields mainly the intact singleFig.4
Negative ion mode ESI TOF mass spectra showing
theinteractionsbetweenDS2andruthenium-basedcompoundsafter
anincubationperiodof 6h. Dissociationintothesinglestrands SS2a(open
diamonds) and SS2b (lled diamonds) (charge states -6 to
-4)takesplaceundertheselectedconditionsandthedifferent extent
ofadduct formation depending on the strand and the compound
isdemonstrated(topRAPTA-T, middleKP1019and
bottomNAMI-A).Onlythemostintenseadductpeaksforeachcompoundarelabelledforthe-5chargestate.
PTA1,3,5-triaza-7-phosphaadamantane,
Indindazole,ImimidazoleJBiolInorgChem(2010)15:677688 6831 3strands,
and their further fragmentation in positive
ionmoderesultsonlyinlargeoligonucleotidefragments(thephosphategroupsonthenucleotidesinducelowionisationefciencyfor
short sequences inpositiveionmode)
thatgivelittlestructuralinformation. Therefore,
fragmentationexperiments were carried out in negative ion mode with
aniontrapinstrumentcapableofMSnanalysis.Owingtothecharacteristicisotopicpattern,
inducedbycoordinationofthe metals to the oligonucleotides,
metal-free species couldeasily be distinguished from the modied
oligonucleotides.Thecommonlyappliednomenclaturefor oligonucleo-tide
fragments is summarisedinFig. 5[44]. For n-typefragments, Bn (B is
one of the nucleobases A, C, G or T) isusually lost by an
elimination reaction prior to strandbreaking, which is caused by a
second elimination reaction,leading toa furan ring system[44].
Internal fragments,resultingfromtwostrandbreaksatthea/wsite,
possessaphosphategroupat their 50terminus, whereas
the30ter-minuscarriesafuransystem.PlatinumcomplexesTheinteractionofcisplatinwithDS1andDS2waschar-acterised
by ESI-MS/MS earlier, revealing GG and GTG asthemajor
bindingsites[16]. Inthisstudy, wewantedtoverify that the preference
towards guanine residues ismaintained even if adenine and thymine
are present inlarge excess as in DS3, and, additionally, establish
thepreferredbindingsitesfortheplatinum-basedpharmaceu-ticals
oxaliplatin and carboplatin. Previous studies haveshownthat
bothoxaliplatinandcarboplatinformadductswith DNA comparable to
those of cisplatin, showing also astrong preference for guanosine
as the major bindingpartner[46,
47].Forthispurpose,theionsofthePt(NH3)2(forcisplatinand
carboplatin) and Pt(DACH) (for oxaliplatin) adducts ofthe
single-stranded oligonucleotides were isolated andsubjectedtoCID.
As expected, bindingtowards theoli-gonucleotides takes place mainly
at guanine-containingresidues, eveninthecaseofDS3.
Theformationofcom-plementary modied and unmodied (anBn),
wnandinternal (Bn:Bm) fragments
comparabletothosefoundinpeptidefragmentationexperimentsinproteomicsgivestheexactmetal
bindingsite. Asthecoordinationofthemetalmight result in
fragmentation mechanisms additional
tostandardsinglefragmentationpathways, i.e. a, b, c, dandw, x, y, z
fragments as well as internal a/w-type
fragments,notallthepeakscouldbeassigned.InFig.6,anexpandedsegmentofarepresentativeCIDspectrumforthebindingsitedeterminationonDS2incu-bated
with a metallodrug (in this case oxaliplatin) is depic-ted. In this
case, the peak corresponding to [SS2b?Pt(DACH)]3-at 1,402.2m/z was
isolated in the ion trap oftheinstrument
andsubjectedtofragmentation.
ThemajorpeaksintheresultingCIDspectrumcorrespondto[SS2b-C?Pt(DACH)]3-at1,365.0m/z,aswellasthenon-plat-inum-containing
fragments [a4A]-and [a5C]-at 1,003.1and 1,316.0m/z, respectively.
It can be seen that all (anBn)-type fragments from the 50terminus
up to the guanine resi-due at position 9 remain unmodied (as
evidenced, e.g., bythe non-platinumpeak at 1,247.6m/z, which was
assigned to[a9G]2-), whereas all (anBn) fragments fromthere
onexhibitanisotopicpatterncharacteristic for platinum. Thiscan be
attributed to the attachment of the Pt(DACH) moiety,which leads to
a mass increase of 307.1Da (neutral
species).Consequently,thepeakat1,566.0m/zcanbeidentiedas[a10G?Pt(DACH)]2-.
Complementarily, all wn-typefragments fromthe 30terminus up to
w4(1,252.1m/z)remain unmodied, whereas all further wnfragments
(whichinclude the G9 residue) are modied by Pt(DACH). This
isevidenced by, for example, the singly charged peak at1,888.1m/z,
with an isotopic pattern characteristic forplatinum and which can
be assigned to [w5?Pt(DACH)]-.By combining the information gained
from the modied andunmodied fragments from both the 30and
50terminus, wecan unambiguously identify the G9 residue as the
nucleotidemainly involved in the platinumbinding. Note that notall
detectedfragment ionsarecontainedintheexpandedFig.5
Nomenclatureofoligonucleotidefragmentsobservedby
MS/MS.adfragmentscorrespondto
fragmentswithanintact50terminusandwz-typefragmentshaveanintact30terminus.
Internalfragmentsresultingfromdoublefragmentationusuallyoccuratthea/wsite684
JBiolInorgChem(2010)15:6776881 3segmentsofthespectrum(Fig.6).
Calculatedandexperi-mental m/z values for the peaks in Fig. 6 are
listed
inTable3.Fragmentationexperimentswiththeotheroligonucleo-tidesandplatinumcomplexes
werecarried out ina
similarfashionandrevealedguanineasthemajorbindingpartnerfor the
other platinumcomplexes and all other singlestrands. For SS1a, the
neighbouring GG residues
atpositions6and7wereidentied.ForSS1b,G6appearstobe preferred over
G4. For SS2a, which contains veguanine residues, again
adjacentGGresiduesatpositions6and7showstrongerinteractionthanGTGsequences.ForSS3aandSS3b,despitethefactthatthereisonlyoneguanine
residue present, no binding to adenine and thymineresidues
wasdetected.In allcases, mainly (anBn),wn
andinternal(Bn:Bm)fragmentswereobserved.RutheniumcomplexesNMRandgel
electrophoresisstudiessuggest that NAMI-A,
RAPTAandKP1019exhibitsimilarreactivitytowardsadenineandguanineinplasmidDNA[35,
48], andcapil-lary electrophoresis inductively coupled plasma MS
studieshave shown that RAPTA compounds and KP1019 can
reactwiththeDNAmodel compound50-dGMP[39]. InNMRstudies with
different rutheniumarene complexes withsingle nucleotides, thymine
residues were also suggested
asbindingpartnersinadditiontoguanosine.However,underthese
conditions, steric effects resulting fromthe three-dimensional
structure of oligonucleotides, which makes theN3of thymine
virtuallyinaccessible, are not takenintoaccount,
whichisthemajordisadvantageofsinglenucle-otide DNA models [49].
Subsequent two-dimensionalNMR studies employing 14-mer
oligonucleotides con-rmed adduct formation with guanines and
suggestedintercalationandstackinginteractionsoftheareneligandwith
adjacent thymine residues [21]. Although DNA mightnot be the main
target of ruthenium compounds in terms
oftheiranticanceractivity,reactionswithsinglenucleotides,oligonucleotidesandRNAmoleculespresent
inthecyto-plasm, cannotberuledout.Asanexample,
expandedsegmentsofaCIDspectrumcorrespondingtoa[SS3a?Ru(PTA)]4-parent
ion(con-taining only one guanine residue) fragmentation
aredepictedinFig. 7. Assignment of thefragment ions wasconducted in
a fashion similar to that for the platinumcompounds, and the
majority of CID product ions could
beassignedtown-and(anBn)-typestrandbreaksaswell asinternal (Bn:Bm)
fragments. Again, some product ionscouldnotbeassigned,
mostprobablyowingtoadifferentfragmentationmechanisminducedbytheboundmetal.After
assigningthepeaks totheir
correspondingfrag-mentationpathwaysandconrmingcharacteristicisotopicTable3
Calculatedandexperimental m/zvaluesfor fragment
ionsfoundinFig.6(monoisotopicpeaks)Ion Calculatedm/z
Experimentalm/z[a4A]-1,003.2 1,003.1[a5C]-1,316.2
1,316.1[a6A]-1,605.3 1,605.0[a9G]2-1,247.7
1,247.6[a10?Pt(DACH)]2-1,565.8 1,566.0[a12?Pt(DACH)]2-1,882.3
1,882.1w4-1,252.2 1,252.1[w5?Pt(DACH)]-1,888.4
1,888.1[w6?Pt(DACH)]2-1,088.2 1,088.5[w7?Pt(DACH)]2-1,232.7
1,232.6[w9?Pt(DACH)]2-1,533.8 1,533.6[w10?Pt(DACH)]2-1,690.3
1,690.0[w11?Pt(DACH)]2-1,834.8 1,835.0[SS2b-C?Pt(DACH)]3-1,365.3
1,365.1Fig.6 An expanded segment
ofanLTQcollisioninduceddissociation(CID)massspectrumofSS2bmodiedbyoxaliplatinintherangefrom950to1,900m/zthatcontainsthemajorityofplatinum-containingfragments.UnidentiedfragmentsexhibitinganisotopicdistributioncharacteristicforplatinumarelabelledXJBiolInorgChem(2010)15:677688
6851 3patterns for modied residues, we identied the majorbinding
partner. Again starting fromthe 50terminus, itbecomes clear that
upto[a8G]2-(1,125.6m/z) mainlyunmodied residues are detected,
whereas from there on all(anBn)fragments,
whichincorporatetheguanineresidue,containtheRu(PTA) moiety(mass
increaseof 256.0Dafor the neutral species), for example, evidenced
by[a9C?Ru(PTA)]2-at 1,418.5m/z. Complementary infor-mationwas
obtainedbyassigningthefragments startingfrom the 30terminus: up to
w7, the peaks can be assigned
tounmodiedfragments,whereasw72-wasdetectedinboththe unmodied
(1,085.6m/z) and [w7?Ru(PTA)]2-(1,213.6m/z) forms. All further
wnfragmentswithn [7werefoundwiththeRu(PTA)moietyattached.
Thissug-gests that the T10 residue might serve as an
additionalbindingpartner for therutheniumdrugtoformamulitd-entate
adduct. Additionally, modied internal fragmentssuch as
[T7:T11?Ru(PTA)]2-(969.5m/z), [A3:T11?Ru(PTA)]2-(1,598.5m/z) and
[T4:G8?Ru(PTA)]-(1,987.0m/z), all of whichcontaintheG8residue,
weredetected. Althoughsmall peaksattributableto, for exam-ple,
[w4?Ru(PTA)]-(1,508.0m/z) and
[a6A?Ru(PTA)]-(1,891.9m/z)indicatethatadductformationisalsopossiblewithadenineorthymineresidues(astheydonot
contain G8), the majority of the modied fragment
ionspointtoG8asthepreferredbindingpartner.Interestingly,no loss of
the PTA ligand was observed during CID.
Someofthepeakscorrespondtomorethanonespecies, as, forexample,
internal [T2:T5] and[T4:T7] fragments exhibitthesamemass(peaksat
700.6and1,402.0m/zforsinglyanddoublychargedspecies, respectively).
InTable4,
thecalculatedandobservedm/zvaluesforthemodiedfrag-mentionsfromFig.
7arelisted.ForRAPTA-T,
adductsofRu(PTA)withtheothersin-glestrandswere
furtheranalysed,andRu(Im)andRu(Ind)adductsweresubjectedtoCIDforNAMI-AandKP1019,respectively.
Surprisingly, evenfor DS3, whichcontainsmainlyadenine andthymine
residues, a behaviour com-parable tothat of the
platinum-basedchemotherapeutics(i.e.
preferenceforguanine)wasobserved.
Similarobser-vationsweremadeforKP1019andNAMI-A.Also for the other
strands, the results did not differstrongly fromthose obtained with
the platinum-basedcomplexes: in SS1a, G6 and G7 are preferred
bindingpartners, G6isthepreferredbindingpartnerinSS1b, G6and G7 are
preferred binding partners in SS2a and G9 is theFig.7
ExpandedsegmentsofanLTQCIDmassspectrumof[SS3a?Ru(PTA)]4-.Topm/zrangefrom650to1,275andbottomm/zrangefrom1,275to2,000.UnidentiedfragmentsdisplayinganisotopicpatterncharacteristicforrutheniumarelabelledX.Notethatnotalldetectedfragmentionsarecontainedintheexpandedsegments686
JBiolInorgChem(2010)15:6776881 3preferredbindingpartnerinSS2b.
Inaddition, low-inten-sitypeaks attributabletointeractions
withother residuesweredetected.DiscussionIn this paper, we have
presented a comparative study of
theadductformationbetweenduplexDNAandplatinum-andruthenium-basedanticancerdrugsbyMS.Notethatthisistherst
exampleofatop-downMS/MSapproachfortheelucidationofbindingsitesonDNAfragmentsusingCIDof
selectedadductsfor ruthenium-basedpharmaceuticals,as well as for
oxaliplatin and carboplatin. Interestingly,
MSperformedinthepositiveionmodewasmoreinformativefor
thecharacterisationof thedoublestrandmetallodrugadducts
thannegative ionmode MS. Positivelychargedadducts appear to be more
stable in the gas phase under theexperimental conditions employed
for this application.Time course experiments conrmed cisplatin,
with itsmonodentate chlorido ligands, to be the most
efcientcompound in terms of adduct formation over the
otherplatinumcomplexes, withtheir
bidentatebiscarboxylates,whereasNAMI-Awasmorereactivetowardsthedouble-strandedoligonucleotidesthanRAPTA-TandKP1019forruthenium-based
compounds. However, to conrm theapplicabilityof
theproposedmethodfor
accuratequanti-tativekinetics,validationwithacomplementaryanalyticaltechniquesuchasspectroscopy,
chromatographyor
(cap-illary)electrophoresiswouldhavetobecarriedout.Thetop-downapproachusedtodeterminethebindingpartner
ontheoligonucleotideshasseveral
advantagesincomparisonwithenzymaticdigestion: primarily,
exonuc-leases normally used for the removal of terminal
nucleotidesdo not recognise nucleotides modied by
metallodrugs,thereforeinhibitingthedigestionreactionandyieldinganincompletedataset.Thecombinationofenzymessimulta-neouslyattackingfromthe30and50endspartiallycircum-vents
this problem, but in the case of multiple binding, againonly
incomplete data are obtained. Moreover, the
top-downapproachismuchmorerapidthantheenzymaticdigestionprocedure,
which might also require change of pHandaddition of catalytically
active cations, which could interferewiththeinteractionof
themetallodrugwiththeoligonu-cleotide.Severalfactorshavetobeconsideredinthemul-tistage
mass-spectrometric approach: the adducts formedmust
besufcientlystabletonot becleavedduringCIDexperiments,
thereforerequiringcovalent bondingtotheoligonucleotide;
furthermore, positive charges are intro-duced by the metals, making
it difcult to characterise shortmodied oligonucleotides (consisting
of less than threebases) in negative ion mode, usually employed for
this kindof experiment. Nevertheless, as CID yields
complementaryfragments, i.e. ions from the 30and 50ends of the
biomole-cule, complete sequence coverage can be achieved
andinternalfragmentscansubstantiateassumptionsofspecicbindingpartners.
The results describedhereinshowthatguanine
isthepreferredbindingpartnerforbothplatinum-andruthenium-basedmetallodrugs,evenifotherbasesarepresentinlargeexcess,althoughminorpeaksindicatethatadenine
or thymine could also serve as a binding partner
fortherutheniumcompounds.Thisisingoodagreementwithdataobtainedbyother
techniques, includingtwo-dimen-sional NMRand biochemical assays,
which also showed thatadduct formation between DNAand both
platinumandrutheniumcompoundsoccursmainlyviaadductformationwith
guanosine [21].Acknowledgments Theauthors thank LaureMenin
forguidance inoperatingthequadrupoleTOFinstrument. M.G.
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