A double stranded DNA fragment shows a significant decrease in double-helix stability after binding of monofunctional platinum amine compounds

4123 (N CjH£)' 101 (N‘H)* 2mtorr (N»CjH5) 101 (N+Hl* 73 JJL

J. Am. Chem. Soc. 1989, ¡11, 4123-4125

3

Scheme 1, Figure 1, and below, the reaction islimitedto those molecules (1,2,6) that containa _{3-hydroxy functionality}inthe

A ring.

Acknowledgment. This work was _{supportedby the} National

ScienceFoundation_{(CHE-8721768, RGC;}CHE8717380,

HIK).

Professor J. _Cassadyisacknowledged forsamples of 1-6.

Figure1. The productdistributionsobtainedfortwo protonated, isomeric

diterpenoiddilactones upon collisionswith ethyl vinylether in the center quadrupoleofa_{triple quadrupole} mass _{spectrometer. The} base_peak

(101+) arises from ethylation of ethyl vinyl ether byprotonated ethyl vinyl ether(73+).5c·7

A

Double-Stranded

DNA

Fragment Showsa

_Significant

Decrease in

_{Double-Helix Stability After Binding}

of

Monofunctional Platinum

Amine Compounds

terium at a nonacidic site, as _{evidenced by the} fact that this

deuteriumcannotbeexchanged toa_{hydrogen atom upon collisions} withethylvinyl ether. The large difference in the heat of for-mation of simple neutral alkyl vinylethersandalcohols _(A(AHf)

= -22_{kcal/mol forethylvinylether and ethanol)}isthe most_likely

drivingforceforthisentropicallydisfavored reaction. Weestimate thereactionto beat least 4_{kcal/molexothermic for protonated}

4-hydroxy-2-butanoneand _{ethyl vinyl}ether.10 Note thatthe

proton affinitiesof,8-hydroxycarbonyl compoundsare comparable to _{the proton} affinities of alkyl vinyl ethers.10 _{This precludes}

efficient_{competition by the}reactionthat_{usually dominates}the

ion chemistry of alkyl vinyl ethers, i.e., deprotonation of the

reactant ions.

Themost _intriguingresultofthis studyisthe_discovery that

“vinylation” oforganicions_byalkylvinylethersis_highlyselective

towarddifferent_{oxygen-containing}functionalitiesandthat this

behaviorisnotlimitedto simple modelionsbutapplies to complex

polyfunctional ions as well. Under the same _{conditions, only}

dissociation and _{deprotonation reactions} were observed for a

number of protonatedalcohols, ethers, aldehydes, ketones, esters, lactones, and epoxides, including mono- and polyfunctional,

saturatedand _{unsaturated, cyclic}and _acylicaswell asaromatic

molecules. Totestthe_{selectivity of}the_{“vinylation”}reactionmore

rigorously,we decided toexamine the_reactivityof_{polyfunctional} molecules _{that only}differby the positionofthe hydroxy group

expected tobe necessaryforthe reaction. The_{isomeric diterpenoid}

dilactones2and3_(Figure 1)presenta_challengingtestsincethese

protonated moleculesare

difficult

to _distinguishon thebasisof

their_{dissociation product distributions. However,}due tosteric

constraints in the isomer3,only2 is_{expected toexchange}a_proton

to a_{vinyl groupupon}collisionswith_ethylvinylether. We found

that protonated 2does indeed _{undergo the reaction}ofinterest,

giving a _production with arelative abundanceof_upto 10%of

thebase_{peak (101+),}while3onlygivesa traceatthemass value

of interest (<1% of the base _{peak; Figure 1). Moreover,} the

reactionseems to be _independentofthestructure oftherest of the molecule. Forthesix_{diterpenoid dilactones(1-6)}shown in

(10)AH, ofprotonated4-hydroxy-2-butanonewas estimatedto be>62.9

kcal/mol byassuming that theprotonaffinity of4-hydroxy-2-butanoneis

equal toor less that ofacetylacetone (i.e., <207.8± 5_kcal/mol111),and estimating AH,for neutral 4-hydroxy-2-butanone (estimated to be-95.4 kcal/mol by the methodofBenson12and usingAH, of2-butanone: -57.5

kcal/mol11);AH, of -deprotonated 4-hydroxy-4-methylpyranwas estimated

to be+80.9 _{kcal/mol by} the methodof Benson12andusingAH, = ₊₁₂₉ kcal/molfor -deprotonatedpyran;11 AH, ofethyl vinyl etheris-34 _kcal/ mol;11 AH, ofethanolis_{-56.1 kcal/mol.11}

(11) Lias,S.G.; Bartmess,J.E.;Liebman,J, F.;Holmes,J.L.; Levin,R.

D.; Mallard,W.G,J. Phys. Chem.Ref. Data,Suppl.1 1988, 17. (12)Benson,S._{W. Thermochemical Kinetics', Wiley: New}York, 1976.

Carla J. van Garderen, Hansvan den Elst,

Jacques H.van Boom,and Jan _Reedijk*

Department

of

Chemistry, GorlaeusLaboratories

Leiden University, P.O. Box 9502, 2300 RA Leiden

TheNetherlands

Leo P.A. van Houte

Department

of

Biophysics, PhysicsLaboratory

Free University, DeBoelelaan 1081

1081 HV Amsterdam, TheNetherlands

ReceivedJanuary 17, 1989

The_{antitumor drug ci's-diamminedichloroplatinum(II)}(cDDP1)

preferentiallybinds to two _{neighboringguanine}basesofDNA.23"4 Ithasbeen suggestedthatthis chelation inducesaseriousdistortion

ofthe_{DNA,resulting}inadenaturation_{up to severalbase pairs.5·6}

Recently, NMR studies and molecular mechanics of

oligo-nucleotides containing eightor more base_pairsshowedthatthe

distortion is_{small; all} base _pairs, even those ofthe _platinated

guanines,are observed.7·8 Nevertheless, themelting temperature

(Tm) appeared to be loweredby 10-20 °C. These phenomena have been attributed to a “kinked” cDDP-DNA structure.9,10

For a _{detailed understanding} of_{the working mechanism} of

cDDPnot _onlytheultimate structuralchangebut alsothe

dis-tortion _resulting from the first binding step is _important.

(1) Abbreviations: cDDP,ci._{-PtCl2(NH3)2;tDDP,}ircms-PtCl2(NH3)2·,

dien,diethylenetriamine; dsNONA,d(TCTCGTCTC)-d(GAGACGAGA); Pt(dien)-dsNONA, Pt(dien)[d(Tt :TCGTCTC)-N7(5)]-d(GAGACGAGA); Pt(NH3)3-dsNONA, Pt(NH3)3[o'TCTCGTCTC)-N7(5)]-d(GAGACGA-GA);7"m, melting temperature.

(2) Fichtinger-Schepman, A. M. L;van der Veer,J. L.;denHartog,J.H.

J.;Lohman,P. . M.; Reedijk,J._biochemistry 1985, 24,707-713. (3) Inagaki, K.; Kidani, Y. ¡norg. Chim. Acta 1985, 106, 187-191. (4) Pinto, A. L.; Lippard, S. J. _{Biochim. Biophys. Acta} 1985, 780,

167-180.

(5) Munchausen, L. L.; Rahn,R. O._{Biochim. Biophys. Acta} 1975,414,

242-252.

(6) Macquet, J.-P.; Butour, J.-L. Biochimie 1978, 60,901-914. (7) denHartog,J.H.J.;Altona,C.;van Boom,J.H.;van derMarel,G.

A.;Haasnoot, C.A.G.;Reedijk,J.J.Am.Chem. Soc.1984,106, 1528-1530. (8)van _Hemelryck,B.;Guittet,E.;Chottard,G.;Girault,J.-P.; Huynh-Dinh, T.; Lallemand, J.-Y.; Igolen,J.; Chottard, J.-C.J.Am. Chem. Soc. 1984, 106,3037-3039.

(9)denHartog,J.H.J.;Altona,C.;van Boom,J.H.;van derMarel,G.

A.; Haasnoot, C. A. G.; Reedijk, J. J. Biomol. Struct. Dyn. 1985, 2, 1137-1185.

(10) Kozelka,J.;Archer,S.; Petsko, G.A.; Lippard,S.J.;Quigley,G.J. Biopolymers 1987, 26, 1245-1271.

0002-7863/89/1511-4123$01.50/0 &copy; 1989American Chemical Society

Downloaded via LEIDEN UNIV on March 9, 2021 at 13:41:07 (UTC).

4124 J. Am. Chem. Soc., Vol.

Ill,

No. 11, 1989 Communicationsto theEditor

Table I. Melting Temperatures of Three Oligonucleotides Modified with cDDPandOneNonanucleotide Platinated with [Pt(dien)]2+or [Pt(NH3)3]2+7

oligonucleotide duplex addedsalt

Tm(-Pt)

(°C)

rm(+Pt)

(°C) (°C) ref

d(GATCCGGC)-d(GCCGGATCGC) (Pt-)A“ 1 M NaCl 55 28 27 8

d(GCCGGATCGC)-d(GCGATCCGGC) (Pt-)B6 1 M NaCl 58 49 9 21

d(TCTCGGTCTC)-d(GAGACCGAGA) (Pt-)Ca none 29 14 15 7

d(TCTCGGTCTC)-d(GAGACCGAGA) (Pt-ic4 0.1 M Mg2+ 52 30 22 22

d(TCTCGTCTC)-d(GAGACGAGA) e 0.5 M NaCl 42 26 16 this work

“A, 23

µ ;

Pt-A,8

µ .

b1

µ .

c5

µ .

46.4

_{µ .}

£4-5µ . ^The platinatedsequencesare underlined.

Figure1. _{Melting profiles of}dsNONA

_(---),

Pt(dien)-dsNONA (—). Pt(NH3)3-dsNONA

(--)

at 260nm, 4-5

µ

nonamer strand, 0.5 M

NaCl,pH7.temperatureprogression 1 °C/mm,%H = %hyperchroism.

Therefore, we have _investigated thedouble-stranded nonamer

d(TCTCGTCTC)-d(GAGACGAGA) modified withthe

mono-dentate_{complexes [PtCl(dien)]Cl} and _{[PtCl(NH3)3]Cl,} which

are bound to the_{central guanine} base.

Incontrast with the resultsofothers,1112our _{experiments show}

thatthe Tmofthestudiednonamer issignificantlydecreased upon

monofunctional platinum binding. This is indicative fora

de-stabilization ofthe doublehelix.

Thenonamers _d(TCTCGTCTC) andd(GAGACGAGA) were synthesized via an _{improved phosphotriester} method.13

Pt-(dien)[d(TCTCGTCTC)-N7(5)]

and

Pt(NH3)3[d-(TCTCGTCTC)-N7(5)]

were obtainedafteran _equimolar

re-actionofthe_{platinumcompound}with thenonamer. The reaction

productswere identified _by NMR. Afteraddition ofthe

com-plementary stranda_duplexis _formed; 1H NMR shows that all

imino protonsare _present,even the_{proton of}the_platinated G-C

base _{pair (data} not shown).

The melting behaviorofthe two _{Pt-containing fragments}and

theunmodifiedcompoundwere studied. The experimentalsetup

has been described _{previously.14}

Underlowsalt conditions,above2 _°C,the_{melting profiles of} the Pt nonamers couldnot beobserved_{completely. Therefore,} NaClwas addedto stabilize _{the duplexes. Figure} 1 shows the

melting profiles ofthe_{oligonucleotides under comparable}

con-ditions. The Tmoftheunmodified nonamer is42°C. Platination

ofthe_fragment w'ith _[Pt(dien)]2+or _[Pt(NH3)3]2+ reduces the

melting temperatureto 26 °C.15

Althoughseveralauthors predictedadestabilizationofthe DNA

duplexdue to fixationofmonodentate_{platinum,16 such}adrastic

(11) Brabec, V.;Vrána,O.;Kleinwáchter, V.;Kiss,F.Stud.Biophys. 1984, 101. 135-139.

(12) Butour.J. _{L.; Macquet,} J. P. Biochim. Biophys. Acta 1981, 653.

305-315.

(13)van der Marel,G.A.;van Boeckel, C.A. A.; Wille,G.;van Boom,

J. H.Nucleic AcidsRes. 1982, 10. 2337-2351.

(14)van _Houte, L. P. A.; Westra, J. G.; Retel, J.; van Grondelle, R.

Carcinogenesis 1988, 9, 1017-1027.

(15) Ina_{temperaturedependencestudy}oftheiminoprotons thedifference in _{melting temperature} between the_platinated and the unbound nonamer appearsto bethesame as observedintheUV melting profiles.

decrease in Tm has not yet been reported. Hermann et al.l6a

explained the stabilization of the _{poly(l)-poly(C)} duplex after

[Pt(dien)]2+ binding with a _{triple-sandwich structure,} i.c., the

hypoxanthine-N7atoms above and below theone to which

Pt-(dien) is bound _{form hydrogen} bonds with protons ofthe two

amino groupsofthe _{Pt(dien) moiety. The}formation ofsucha

triple-sandwichstructure is_{neighbordependent and}isnot _likely tooccur inour nonamer _sequence. Theseauthorsalso_investigated

themonofunctional interactionof_{í/"a/zí-PtCl2(NH3)2}(tDDP)with poly(l)-poly(C).17,18 In agreement w-ithour results, a

destabi-lizationofthe_duplexwas found;18the7mwas lowered_{significantly}

aftertDDP _{binding. The observation that [PtCl(dien)]Cl}

faci-litatestheB— Z transition _in

poly(dG-dC)-poly(dG-dC)19,20is

anotherindication thatthe DNA structure can besignificantly

affected by monodentate platination.

For_{oligonucleotides} (8-10base_{pairs) modified with cDDP}a

reduction in the _{melting temperature}of9-27 °C has been

re-ported7,8,21,22(Table 1). Nevertheless,alliminoprotonsofthese duplexes are observedwith 1H NMR.

The melting temperature appears to be _dependent on the

numberofbase_{pairs involved induplex}formationandthe location

ofthe _platinationsite(compareTmand ATmof duplex A and B,

Table I). Thebase_sequenceofthe fragmentisalso important.

Thisisdemonstrated _byduplexBand C, whicharc both decanters

withthe_platinationsite inthemiddle ofthe sequence. However,

thereductionofthe _Tmofthesedecantersis_very different, namely 9 and 15 _{°C, respectively.}

In _comparison to _duplex Cour nonamer lacksa

guanine-cy-tosine base _pair in thecenter. _{For this fragment} we measured a Tmlow-cringof 16 °Cduetomonofunctional platinum binding,

which compares quite well to a reduction of 15-22 °C due to

bifunctional platination oftherelated C _duplex.

Although the resultscollectedinTable1 were obtained under

different conditionsitisobvious thatthereductionofthe Tmdue

to both bifunctionaland monofunctional platinum bindingis in

the same order of magnitude. This result is unexpected in the

light of earlierobservations.23-25 _{Binding of [PtCl(dicn)]Cl}to

randomsequenceDNA hardlyinfluences theCD spectrum, and

the changeoftheCD induced by bifunctional_{binding of cDDP} is_mainlydueto thechelation_step.23,24 Moreover, [PtCl(dien)]+

does not _disrupt the duplex ofcalfthymus DNA incontrast to

the effects ofcDDP and tDDP.25

In conclusion,_{binding of}amonofunctional platinum_compound

distortsthe DNA structure _{significantly.} Thedecrease in _melting

(16) (a) Hermann.D.;Fazakerley.G.V.; Guschlbauer, W. Biopolymers

1984, 23. 973-983. (b) Johnson, N. P.; Macquet, J.-P.; Wiebcrs.J L;

Monsarrat, B.Nucleic AcidsRes. 1982, 10,5255-5271.

(17) Fazakerley.G. V.; Hermann,D.;Guschlbauer. W.; Hawkes.G. E. Biopolymers 1984, 23,961-972.

(18) Hermann. D.; Fazakerley, G. V.; Houssier, C.; Guschlbauer, W. Biopolymers1984, 23.945-960.

(19) Malfov. B.; Hartmann, B.; Leng, M.Nucleic Acids Res. 1981, 9. 5659-5669.

(20) Ushav. . M.; Santella, R. M.: Caradonna, J. P.:Grunberger. D.;

Lippard,S.J.Nucleic AcidsRes. 1982, 10.3573-3588,

(21) van _Hemelryck,B.;Guittet,E.;Chottard,G.;Girault,J.-P.;Hermann, F.; Huynh-Dinh,T.: Lallemand. J.-Y.; Igolen,J.:Chottard, J.-C. Biochem. Biophys. Res.Commun. 1986, ¡38. 758-763.

(22) Marzilli. L.G. 1988. personalcommunication.

J. Am. Chem. Soc. 1989, 111, 4125-4126 4125

temperature appears to bealmost thesame for mono- and

bi-functional Pt compounds. Alreadyin the _{first platination step}

theduplexisdestabilized, whereafter chelationcan takeplace to

forma“kinked”structure, apparently withoutfurther_{major helix}

destabilization. The detailsofthehelixdistortion studiedwith

CDand NMR is _{the subject}of future _{investigations.}

Acknowledgment. We acknowledge EECsupport(GrantNo.

ST2J-0462-C), allowing regular scientificexchangewiththe group

of Prof. Dr J.-C. Chottard, and Johnson and Matthey Ltd.

(Reading,

UK)

for theirloanofK2PtCl4.

Elaboration

of

Fusedgem

-Dimethylcyclopropane

Systems via Cyclopropene

Cycloaddition. A

Stereocomplementary Approach James H. _Rigby* and Paul Ch. Kierkus

Department

of

Chemistry, WayneState University

Detroit, Michigan 48202

Received February 10, 1989

A gem-dimethylcyclopropane unit fused to a six-membered

carbocycleis a_{commonly displayed}architecturalfeature

char-acteristicofnatural products as _structurallydiverseas the

tu-mor-promoting diterpene, phorbol (l),1 and the _{sesquiterpene,}

aristolone (2).2

2

Todate,access intothe_{bicyclo[4.1.0]heptane carbon}skeleton

hasmost _{often employed dihalocarbene insertion-organocopper}

substitution technology, which often provides the target in only

modestoverallyields.3,4 Wewish toreporta_highlystereoselective,

general protocolbasedon _{cyclopropenecycloadditionchemistry}

as an alternative method for the elaboration offused

gem-di-methylcyclopropanespecies. Anoteworthyaspectofthis

meth-odology is the _{capability of}assembling systems with

comple-mentarystereoselection byminor reactionsequencemodification.

Whileseveralsubstituted cyclopropenespecieshavedisplayed

dienophilicbehavior in simplesystems,5gem-dimethylcyclopropene

itselfis a_{notoriouslypoorparticipant}intheDiels-Alderreaction.6

We were, however, intrigued with thenotion ofexploiting the

cycloaddition chemistry of the carbonyl-activated

dimethyl-cyclopropeneseries as an attractive entry into theCD _ringsof

phorbol (1) and _{related diterpenes.}

(1) For syntheticstudies on the phorbolsystem, see: _Wender, P. A.;

Keenan, R.M.;Lee, . Y.J.Am.Chem. Soc.1987,109,4390 and references

cited therein.

(2) Forrecent_syntheticstudiesonaristolone,see: Prasad, C. V. C.;Chan,

T. H.J. Org. Chem. 1987, 52, 120 and referencescited therein. (3) (a) Taylor, M. D.; Minaskanian,G.;Winzenberg, K. N.;Santone, P.;

Smith, A.B. J. Org. Chem. 1982, 47, 3960. (b)Marshall,J.A.; Ruth,J.A.

Ibid. 1974, 39, 1971.

(4) Higher ordercupratescan providesome improvement inthealkylation

step: Harayama, T.; Fukushi, H.;Ogawa,K.;Yoneda, I. Chem.Pharm.Bull. 1985, 33, 3564.

(5) Forexamplesofother gem-disubstitutedcyclopropenesasdienophiles, see: _{(a) Apeloig, Y.; Arad,D.; Kapon,M.; Wallerstein,}M.Tetrahedron Lett.

1987, 28, 5917. (b) Boger,D.L.;Brotherton,C. E. Tetrahedron1986, 42, 2777. _{For previous}studieson the_{cycloaddition}ofcarbonyl activated

gem-dimethylcyclopropenes and pyrazoles, see: (c) Dietrich-Buchecker, C.; Martina,D.;Franck-Neumann, M.J.Chem. Res.(S)1978, 78; J. Chem. Res.

(M) 1978, 1014. (d) Huisgen, R.; Reissig, H.-U. J. Chem. Soc., Chem.

Commun.1979, 568. (e)Huisgen,R.; Reissig,H.-U.Angew. Chem.,Int. Ed.

Engl. 1979, 18, 330.

(6)Closs, G. L.;Closs,L.E.;Boell, W. A.J.Am.Chem. Soc. 1963, 85, 3796.

Table I. Cycloadditions of Cyclopropaneand Pyrazole Addends with

Dienes

entry diene _dienophile pressure_(kbar) exo:endo

% _{yield of} cyclo-propane0 1 OMe 3a 10 1:22 92i,c 2 OTMS 3a 10 1:5 8 96,0 3 OMe 3b 10 1:19

1

4

O

3b 10 1:50 92iv 5 OMe 4 10 50:1 81c 6 ks. OTMS 4 10 50:1 £

"All new _{compoundsreported} herein_{exhibited satisfactoryspectral}

(IR, ‘H NMR, 13C _{NMR), analytical, and/or} high resolution mass

spectralcharacteristics. '’Overall yield for cycloadditionand

quantita-tive photochemical nitrogen extrusion. c_{Yield of}

isolated, purified

products.

The requisite addends 3a,b and 4 were _{readily prepared by}

cycloaddition of2-diazopropane to theappropriately functionalized

acetylenesfollowed, inthecase of4,byphotochemicallyinduced

nitrogenextrusion.7 Typically,thermal cycloaddition reactions

(CH2C12,50°C,96 h)ofthese_{reagentsrequired}massiveexcesses ofdiene to assure _{adequate yields} ofadducts. However,

per-formingthe additions at highpressure (CH2C12, 8-10kbar, 18

h) provided excellentyieldsofproducts employingessentially 1:1

diene-dienophile stoichiometry. Theresultsofthereactionof3a and 4 with (E)-1 -acetoxy-1,3-butadiene are illustrative.

3a, X.C02Me 3b, x,COMe ß C (1>3a (2) hv(93%) 1 4 (78%) 50 50 4

Exposureofthis diene to cyclopropene 4 (CH2C12, 10kbar, 18

h) provided adducts5(exo) and 6(endo) in 78%yieldwith an

exoiendoratioof50:18,9_parallelingthe establishedproclivityfor

exo addition exhibitedbyhinderedcyclopropenes.5a In marked

contrast, pyrazole3a gave,after cycloaddition(CH2Cl2, 10kbar,

18h)andquantitativephotochemical nitrogen extrusion (3500

Á,2.5 h)fromthe_{bicyclic pyrazoline intermediate,}a93%overall

yield

of

amixture

of

5and6inwhichthe endodiastereomer6

prevailedina ratio

_of

50:1.10 _{Thus, effective complementary}

(7)Padwa,A. 1,3-Dipolar Cycloaddition Chemistry:John_Wiley&Sons:

NewYork, 1987;Vol. 1, pp393-558. The photochemistryof3bwas not well-behaved,and_{the corresponding cyclopropene}was notreadily available for cycloaddition studies: Dietrich-Buchecker, C; Franck-Neumann, M. Tetrahedron1977, 33, 751.

(8) The exo/endo designationisrelativetothe_{gem-dimethylcyclopropane}

moiety.

(9) The corresponding Z-dienesdo not react to anyappreciable extent

under theseconditions as evidenced bythe totalabsence _{of reactivity of} (Z)-1-acetoxy-1,3-butadiene toward3aand 4.