inhibitory effect of dna structure on the efficiency of reaction of guanosine moieties with a...

Inhibitory effect of DNA structure on the ef®ciency ofreaction of guanosine moieties with a nitrenium ion

Michael Novak* and Sonya A. Kennedy

Department of Chemistry, Miami University, Oxford, Ohio 45056, USA

Received 30 April 1997; revised 8 July 1997; accepted 11 July 1997

ABSTRACT: The N-acetyl-N-(2-fluorenyl)nitrenium ion (2a) reacts very efficiently with monomeric 2'-deoxyguanosine (d-G) to form a C-8 adduct,N-(2'-deoxyguanosin-8-yl)-2-acetylaminofluorene (6), in an aqueousenvironment, with a selectivity ratio,kd-G/ks, of 13.1� 103 Mÿ1 at 0°C and 4.8� 103 Mÿ1 at 30°C. The reactivity ofthe self-complementary oligomer d-ATGCAT with2a can be separated into components due to the single-stranded(SS) and double-stranded (DS) forms. Within the error limits of the measurementskSS/kd-G� 0.27 andkDS/kd-G� 0.Another measure of the reactivity of d-G moieties in the DNA double helix can be obtained from measurements withthe circular super-coiled plasmid pUC19. This plasmid provides an upper limit forkpUC19/kd-Gof 0.02, wherekpUC19isthe average trapping rate constant per d-G moiety in pUC19. The strong inhibition of the trapping reaction caused bythe tertiary structure of the DNA double helix may be responsible for the change in product distribution of2a–d-Gadducts found from reaction with d-G, and denatured DNA (exclusive C-8 adduct,6) and native DNA [5–20% N-2adduct,3-(2'-deoxyguanosin-N2-yl)-2-acetylaminofluorene,7]. 1998 John Wiley & Sons, Ltd.

KEYWORDS: guanosine; nitrenium; DNA structure

INTRODUCTION

The ‘azide clock’ method has been used to characterizethe lifetimes and relative reactivities of carbocations innucleophilic solvents for over 25 years,1 and was firstused to characterize the lifetime of a nitrenium ion about10 years ago.2 Using this method, we have recentlyshown that certain carcinogenic esters ofN-arylhydroxyl-amines andN-arylhydroxamic acids (1) decompose inaqueous solution into nitrenium ions (2) that react veryselectively with N3

ÿ.3,4 These ions also react efficientlywith 2'-deoxyguanosine (d-G).5 Direct measurements ofthe aqueous solution lifetimes of2a–c confirm that theions have lifetimes in the range 0.1–10ms, and that theyreact with N3

ÿ with rate constants ofca 5� 109 Mÿ1

sÿ1.6,7

Although it is known that2a–creact efficiently with d-G, and the C-8 adducts generated from reaction withmonomeric d-G are equivalent to the major adductsisolated from in vivo and in vitro experiments withDNA,5,8,9 the effect of DNA structure on the adduct-forming reaction is unknown. In particular, it is notknown if the tertiary structure of the DNA double helix

promotes or inhibits the formation of the d-G–nitreniumion adducts.

We have compared the selectivity of reactions of theN-acetyl-N-(2-fluorenyl)nitrenium ion, (2a) with mono-meric d-G, the self-complementary hexamer d-ATGCATand the circular super-coiled plasmid pUC19. Our resultsshow that single-stranded DNA retains significantreactivity toward2a (ca 27% of that of d-G), but thatdouble-stranded DNA has negligible reactivity towardthis nitrenium ion. The implications of these results withrespect to carcinogenesis are discussed.

JOURNAL OF PHYSICAL ORGANIC CHEMISTRY, VOL. 11, 71–76(1998)

*Correspondenceto: M. Novak, Departmentof Chemistry,MiamiUniversity,Oxford, Ohio 45056,USA.Email: [email protected]/grant sponsor: American Cancer Society; contract grantnumber:CN-23K.

1998JohnWiley & Sons,Ltd. CCC0894–3230/98/010071–06$17.50

RESULTS AND DISCUSSION

The carcinogenicester1a decomposesvia rate-limitingheterolyticN—O bond cleavageinto the nitrenium ion2a, that is efficiently trappedby N3

ÿ andd-G (Scheme1).3–5 The rate constantratios kaz/ks and kd-G/ks can bedeterminedby measurementof theproductyieldsof themajorhydrolysisproduct3 andtheazideadducts4 and5,or thed-G adduct,6, asa functionof [N3

ÿ] or [d-G].3,4,5

In particular,the fractionalyield of hydrolysisproducts,fs, is given by

fs � ks

ks� kx�X� �1�

whereX canbe N3ÿ, d-G or any othernucleophilethat

reactsirreversiblywith 2a. The inverseof equation(1):

1fs� 1� kx

ks�X� �2�

providesa convenientmeansto evaluatekx/ks by linearregressionmethods.

Figure 1 shows1/fs as a function of [N3ÿ] for the

hydrolysisof 1a in 1 mM Na2HPO4–NaH2PO4 buffer [pH7.5,m = 0.5 (NaClO4)] at 0 and 30°C. Values of kx/ks

measuredat threetemperaturesfor X = N3ÿ andd-G are

givenin Table1 andln(kx/ks) is plottedvs1/T in Figure2.The slopesof the linear correlationlines in Figure2 are(DHs

‡ÿ DHx‡)/R. The lines are parallel within experi-

mentalerror with DHs‡ÿ DHaz

‡ = 5.4� 1.0 kcal molÿ1

and DHs‡ÿ DHd-G

‡ = 5.2� 1.2 kcal molÿ1 (1 kcal=

4.184kJ). SinceDHaz‡ � DHd-G

‡, the most significantdifferencein thesetwo trappingreactionsis dueto DS‡.From the interceptsof the plots,DSd-G

‡ÿ DSaz‡ =ÿ3.8

eu.Thetrappingof 2a by N3

ÿ occursat, or very near,thediffusionlimit. Directly measuredkaz for 2ageneratedbylaserflash photolysisat 20°C undertheseconditionsis4.2� 109 M ÿ1 sÿ1.6 Themeasuredvaluesof kaz at 20°Cfor severalnitrenium ions, including 2a, with lifetimes(1/ks) of 0.2–30ms are in the range 4.0� 109–5.0� 109 Mÿ1 sÿ1.6,7 This is very similar to diffusion-limited second-orderrateconstantsfor trappingof carbo-cationsby N3

ÿ and other small, strongnucleophiles.10

Scheme 1

Figure 1. Plot of 1/fs vs [N3ÿ] for 1a at 0 and 30°C.

Regression lines were determined from a weighted least-squares ®t

1998JohnWiley & Sons,Ltd. JOURNAL OF PHYSICAL ORGANIC CHEMISTRY, VOL. 11, 71–76(1998)

72 M. NOVAK AND S. A. KENNEDY

Competitionexperimentsshowthat thediffusion-limitedrateconstantfor reactionof purinenucleosideswith thenitreniumions2b and2c at 20°C undertheseconditionsis ca 2.0� 109 Mÿ1 sÿ1.8 Since ks for 2a has beenmeasuredas7.7� 104 sÿ1 undertheseconditions,6 kd-G

for 2a at 20°C is ca 6.2� 108 Mÿ1 sÿ1. This is within afactorof threeof theapparentdiffusion limit. Sincebothkaz and kd-G are so close to their respectivediffusion-controlled limits, it is not surprising that the majordifferencesin theserateconstantsappearin DS‡.

The rate constantratios for trapping of 2a by theoligomerd-ATGCAT weredeterminedby monitoringthechangein concentrationof thehydrolysisproduct3 asafunctionof [d-ATGCAT]. Figure3 showsaplot of 1/fs asa function of [d-ATGCAT] at 0 and30°C. The derivedvaluesof kd-ATGCAT/ks are given in Table 1 and ln(kd-

ATGCAT/ks) is plotted in Figure 2. The trappingproductwasnot characterizedin this case,but it hasbeenshownpreviouslythat shortDNA oligomersreactwith 1a andother estersof N-arylhydroxamicacids and N-arylhy-droxylamines to generatethe C-8 guanosineadductanalogousto 6 asthemajorobservedproduct.9

In theabsenceof oligomerthehydrolysisrateconstant,k0, for 1a is 0.010� 0.002sÿ1 at 0°C. In thepresenceof0.9mM d-ATGCAT the rate constantis unchangedat0.010� 0.001sÿ1. Under theseconditionsthe yield ofthe hydrolysis product,3, is reducedby 40% from its

yield in the absenceof the oligomer. Theseresultsaresimilar to thosereportedpreviouslyfor thehydrolysisof1a andsimilar estersin thepresenceof N3

ÿ andd-G,3–5

andareconsistentwith trappingof the nitreniumion 2aafter rate-limiting N—O bondheterolysis.

The temperature dependenceof kd-ATGCAT/ks isdifferent from that observedfor N3

ÿ andd-G. For bothof these nucleophileskx/ks decreaseswith increasingtemperature,but the opposite is true for kd-ATGCAT/ks

(Figure 2). A melting curve for d-ATGCAT (Figure 4)showsthat in this temperaturerange the self-comple-mentaryoligomer changesfrom predominantlydouble-strandedat 0°C (72%double-stranded)to predominantlysingle-strandedat 30°C (5% double-stranded).Theseestimatesweremadein thestandardfashionasindicatedin Figure 4. We concludethat the temperaturedepen-denceof kd-ATGCAT/ks is causedby the melting of theoligomerdimerover this temperaturerange,andthat thesingle-strandedform of the oligomer is much morereactive toward the nitrenium ion than is the double-strandedform.

A quantitativeestimateof the relative reactivitiesofthe double- and single-strandedforms of the oligomertoward2a canbe obtainedasfollows. If x andy aretherelativereactivitiesof thed-Gresiduesin thedouble-andsingle-strandedforms, respectively, compared withmonomericd-G, we canwrite theequation

(DS)x� (SS)y� kd - ATGCAT=ks

kd - G=ks�3�

whereDS andSSarethe fraction of theoligomerin thedouble- and single-strandedforms, respectively,at agiventemperatureasdeterminedfrom themeltingcurve.The rate constantratios kd-ATGCAT/ks and kd-G/ks aremeasuredat the sametemperatureasDS andSS.If weassumethatx andy aretemperatureindependentoverthenarrowtemperaturerangeemployedhere,we cansolvesimultaneousequationsto obtainx andy. Theassumptionthatx andy aretemperatureindependentis equivalenttoassumingthattherateconstantsfor trappingof 2a by thedouble-andsingle-strandedforms,kDS andkSS, varywithtemperaturein the sameway that kd-G does. This isprobablynot true over a large temperaturerange,but if

Figure 2. Plot of ln (kx/ks) vs 1/T. X = (*) N3ÿ, (*) d-G and

(~) d-ATGCAT. Regression lines were determined from anunweighted least-squares ®t

Table 1. Measured values of kx/ks at different temperatures

kx/ks (Mÿ1)a

Temperature(°C) X = N3ÿb X = d-G X = d-ATGCAT

0 (14.0� 1.0)� 104 (13.1� 1.0)� 103 (7.5� 0.4)� 102

20 (6.2� 0.4)� 104 c (8.0� 0.6)� 103 d

30 (5.4� 0.4)� 104 (4.8� 0.4)� 103 (12.5� 1.0)� 102

a Conditions:1 mM Na2HPO4–NaH2PO4 aqueousbuffer, pH 7.5,m = 0.5 (NaClO4), unlessindicatedotherwise.b Solventwas5 vol.% CH3CN–H2O.c Ref. 1d Ref. 3.


REACTION OF GUANOSINEWITH A NITRENIUM ION 73

thedataaretakenovera fairly narrowtemperaturerangethis assumptionwill not leadto majorerrors.Thevaluesof x andy calculatedfrom our dataareÿ0.02� 0 and0.27, respectively. Within the error limits of ourexperimentweconcludethatkDS� 0 andkSS= 0.27kd-G.

Thedouble-strandedoligomerhasvery little reactivitytowardthenitreniumion,buttheactuallevelof reactivityis difficult to determinewith precisionbecauseof thepresenceof themuchmorereactivesingle-strandedform.The selectivity of the double-strandedsuper-coiledplasmid pUC19 provides another estimate of thereactivity of d-G residueswithin double-strandedDNA.At 0°C in thepresenceof sufficientpUC19to produceasolution 0.66mM in d-G residues,the yield of thehydrolysisproduct3 is reducedby 14.7� 0.5%,while k0

remainsconstantat ca 0.010sÿ1. This resultcorrespondsto a valueof kpUC19/ks of 260� 10Mÿ1, wherekpUC19 isthetrappingrateconstantperd-Gmoietyin pUC19.Thisis anupperlimit since2acanreactwith thesmallamountof nickedDNA presentin the sampleandcanalsobindnon-specifically to the phosphatebackbone of theDNA.11 Sincethe reactivity of nitrenium ions with themonomericpurineandpyrimidinebasesd-A, d-C andd-T is negligible, these probably do not contribute

significantly to the measuredreactivity.8 This resultshowsthat d-G residuesin double-strandedDNA trapnitrenium ions very inefficiently, on average.The d-Gresidueswithin pUC19 do not have identical environ-mentsandthey may showa rangeof reactivitiestoward2a,but it is clearfrom ourdatathatmostd-GmoietiesinpUC19showvery little reactivity toward2a.

Table2 summarizesthereactivitiesof d-G moietiesinvariousenvironmentswith the nitrenium ion 2a at 0°C.Monomeric d-G is most reactive. It traps 2a veryefficiently (>50% trapping)at [d-G] as low as0.1mM.Thereactivityof d-G residuesin single-strandedDNA isreduced,but is still substantial.Thetrappingefficiencyat0.1mM in d-Gresiduesis abouthalf of thatof monomericd-G. The d-G residuesin double-strandedDNA shownegligiblereactivitytoward2awith amaximumtrappingefficiencyat 0.10mM in d-G residuesof about2.5%.

Theseresultsshow that the tertiary structureof theDNA doublehelix significantlyinhibits the formationofthe C-8 adduct. The magnitudeof this inhibition issurprisingbecausethe C-8 adductis apparentlyformedby rearrangementof an initially formedN-7 adduct,andN-7 of d-G is accessiblein themajorgrooveof theDNAdoublehelix.8,12 The largebulk of 2a mayplay a role inlimiting the accessibilityof N-7 to this nitrenium ion.This inhibition of theformationof theC-8adductmaybethe reasonthat treatmentof native DNA with 1a or itsprecursorsleads to the formation of a minor (5–20%comparedwith theC-8 adduct)N-2 adduct,7.9a–e,13Thisadductis not detectedin studieswith d-G or denaturedDNA.5,9d,14It appearsthat this adductis only detectedifthe formation of the more abundantC-8 adduct isinhibited.Thishasimportantimplicationswith respecttocarcinogenesisbecausethe N-2 adductis moreresistantthan the more abundantC-8 adduct to excision andrepair.9,13 Ironically, theresistanceof thedoublehelix toformation of the C-8 adduct may make it moresusceptibleto the formation of the potentially moredangerousN-2 adduct.

Our datashow that, in vivo, duplex DNA shouldbefairly resistantto attackby 2a. Only in cells undergoingDNA replicationor transcriptionis is likely thatefficient

Figure 4. Melting curve for d-ATGCAT at pH 7.5 (5 mM

Na2HPO4±NaH2PO4 buffer), m = 0.5 (NaClO4). Absorbancemeasurements were made at 260 nm

Figure 3. Plot of 1/fs vs [d-ATGCAT] for 1a at 0 and 30°C.Regression lines were determined from a weighted least-squares ®t

TABLE 2. Relative reactivities of pUC19, d-ATGCAT and d-Gtoward the nitrenium ion 2a at 0°C

kx/kd-G

Trappingat 0.10mMin d-G residues(%) a

d-G 1 57� 3d-ATGCATb 0.27 26� 3(d-ATGCAT)2

c � 0 � 0pUC19 <0.02 <2.6� 0.2

a Calculatedfrom % trapping� kx=ks�X��100%�1� kx=ks�X� :

b Thesingle-strandedform.c Thedouble-strandedform.



reaction with 2a can occur. It is a well knownphenomenonthat actively growing cells, suchas tumorcells and stem cells in their mitotic phase,are moresusceptibleto anti-cancerdrugs that attack DNA.15 Itappearsthat the same generalizationholds true forsusceptibilityto nitreniumion-basedcarcinogens.

EXPERIMENTAL

The synthesisof 1a has been describedpreviously.16

Purification of solvents and preparation of buffersolutionshave beendescribed.3–5 NaN3 and d-G wereobtainedcommercially and were used without furtherpurification. The DNA hexamerd-ATGCAT was pur-chasedfrom National Biosciences.In all casesthe pHwasmaintainedat 7.5 with low concentration(1–5mM)Na2HPO4–NaH2PO4 (9:1) buffer.Theionic strengthwasmaintainedat 0.5 with NaClO4.

pUC19 was preparedby a generalprocedure.17 TheDH5a E.coli strainthat expressespUC19wasamplifiedin a rich medium(5 glÿ1 yeastextract,10 glÿ1 tryptone,10 glÿ1 NaCl,pH 7.5,autoclavedfor 30 min) containingampicillin and chloramphenicol.The cells were har-vestedand lysed with sodium dodecyl sulfate.pUC19was purified by equilibrium centrifugation in CsCl–ethidium bromidegradients.Concentrationswere mon-itored by UV methodsand purity was determinedby adensitometricscanof an imageof a UV illumination ofan ethidium bromide-stained gel. The purity wasdeterminedto be98%.

Product studies. The hydrolysis product 3, the azideadducts4 and5 andtheC-8 adductformedwith d-G, 6,have been characterized previously.3,4,14 Authenticsamplesof all compoundswereavailable.Productyieldsweredeterminedby HPLCmethodswith UV detectionat278or 280nm.

All productyieldsweredeterminedby 20ml injectionson to a C-8 Ultrasphereoctyl column using a MeOH–H2O eluent (6:4 or 6.5:3.5) buffered with 0.050M

NaOAc–HOAc(1:1) at a flow rateof 1 mlmin.ÿ1

Productstudieswereinitiatedby a 15ml injectionof a2.0mM stock solution of 1a in DMF into 3.0ml of theaqueoussolutioncontainingNaN3, d-G, d-ATGCAT orpUC19 to produce an initial concentrationof 1a of

1.0� 10-5 M. The concentrationsof N3, d-G and d-ATGCAT were kept in the range0.1–1.0mM. pUC19was usedat a concentrationthat resultedin a 0.66mM

concentrationof d-Gresidues.All reactionmixtureswereincubatedat 0, 20 or 30°C.

A melting curve for d-ATGCAT was obtained bymonitoringtheUV absorbanceat260nmfor asolutionoftheoligomerin thetemperaturerangeÿ12to 70°C undersolvent conditions identical with those used in theproductstudies.At temperaturesbelow aboutÿ5°C thesolutionis super-cooledandwill freezespontaneouslyifdisturbed. UV measurementscan be made on thissolution if care is taken to minimize dust particles inthesolutionandanyshockto thesolution.

Kinetics. Kinetic measurementsin NaN3 and d-G solu-tionshavebeendescribed.3–5 The rateof decompositionof 1a in 0.9mM d-ATGCAT at 0°C was monitoredbyUV methodsat 300nm. The concentrationsandsolventconditionswereidenticalwith thoseusedin the productstudies.

Acknowledgments

This work wassupportedby a grantfrom the AmericanCancer Society (CN-23K). The authors thank DrMichaelW. Crowder of this Departmentfor his assis-tancein preparingandpurifying pUC19.

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inhibitory effect of dna structure on the efficiency of reaction of guanosine moieties with a...

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