Comprehensive Natural Products Chemistry || Covalent Modification of DNA by Natural Products

7.14Covalent Modification of DNA byNatural ProductsKENT S. GATESUniversity of Missouri–Columbia, MO, USA

6[03[0 INTRODUCTION 381

6[03[0[0 Why Are DNA!damaìn` Natural Products of Interest< 3816[03[0[1 Classi_cation of DNA!damaìn` Natural Products 3816[03[0[2 General Mechanisms of Covalent DNA Modi_cation 382

6[03[1 DNA!DAMAGING NATURAL PRODUCTS 384

6[03[1[0 Imines 3846[03[1[0[0 Pyrroloð0\3Łbenzodiazepines 3846[03[1[0[1 Saframycins\ renieramycins\ and safracins 3866[03[1[0[2 Oxazolidine!containin` natural products 3886[03[1[0[3 Barminomycin I 4996[03[1[0[4 Other antibiotics that alkylate DNA via imine formation 490

6[03[1[1 Carbonyl!containin` Natural Products 4916[03[1[1[0 Aldehydes 4916[03[1[1[1 Isochrysohermidine 4926[03[1[1[2 Lactones 492

6[03[1[2 Cyclopropanes 4936[03[1[2[0 CC!0954 and the duocarmycins 4936[03[1[2[1 Myrocin C 4956[03[1[2[2 Ptaquiloside and the illudins 496

6[03[1[3 Epoxides 4976[03[1[3[0 Pluramycins and pluramycinones 4976[03[1[3[1 Kapurimycin A2 and the clecarmycins 4096[03[1[3[2 Alkoxyl radicals from vinyl epoxides 4096[03[1[3[3 Psorospermin 4006[03[1[3[4 Metabolically activated mycotoxins] the a~atoxins 4006[03[1[3[5 Other epoxides 402

6[03[1[4 Aziridines 4026[03[1[4[0 Azinomycin B:carzinophilin 4026[03[1[4[1 Azicemicins 4036[03[1[4[2 Other aziridines 403

6[03[1[5 Pyrrole!derived Cross!linkin` Aènts 4036[03[1[5[0 The mitomycins 4036[03[1[5[1 FR55868 and FR899371 4076[03[1[5[2 Oxidatively activated pyrrolizidine alkaloids 407

6[03[1[6 Alkenylbenzenes 4086[03[1[7 N!Nitroso Compounds 419

6[03[1[7[0 Streptozocin 4196[03[1[7[1 Nitrosamines 410

6[03[1[8 Nitroaromatics 4106[03[1[8[0 Azomycin 4106[03[1[8[1 Aristolochic acid 411

6[03[1[09 0\1!Dithiolan!2!one 0!Oxides 4116[03[1[09[0 Leinamycin 4116[03[1[09[1 0\1!Dithiole!2!thiones 415

380

381 Covalent Modi_cation of DNA by Natural Products

6[03[1[09[2 Polysul_des 4166[03[1[00 Quinones 416

6[03[1[00[0 Redox cyclin` of quinones and quinoid compounds 4176[03[1[00[1 DNA alkylation by quinone methides 4186[03[1[00[2 Formaldehyde!mediated covalent attachment of anthracyclines to DNA 4296[03[1[00[3 DNA alkylation by quinones 4206[03[1[00[4 Other DNA damaè by quinones 421

6[03[1[01 Heterocyclic N!Oxides 4216[03[1[01[0 Phenazine N!oxides 4216[03[1[01[1 Quinoxaline N!oxides 422

6[03[1[02 Resorcinols 4246[03[1[03 Bleomycin and Other Metal!bindin` Antibiotics 4256[03[1[04 Various Aènts That Reduce Molecular Oxyèn to Superoxide 4396[03[1[05 Diazo and Diazonium Compounds 430

6[03[1[05[0 Kinamycin 4306[03[1[05[1 Diazoketones 4316[03[1[05[2 Benzenediazonium ions 432

6[03[1[06 Enediynes 4336[03[1[07 Photochemically Activated Aènts 433

6[03[1[07[0 Li`ht!dependent DNA damaè not involvin` covalent adducts 4336[03[1[07[1 Formation of covalent photoadducts 434

6[03[1[08 Restriction and Methylation Enzymes 435

6[03[2 REFERENCES 435

6[03[0 INTRODUCTION

6[03[0[0 Why Are DNA!damaging Natural Products of Interest<

This chapter is devoted to the consideration of secondary metabolite natural products that formcovalent adducts with DNA and:or engage in reactions that lead to the rupture of covalentbonds in DNA[ Agents that cleave DNA via topoisomerase!mediated mechanisms are consideredseparately\ in Chapter 6[05 of this volume[

The utility\ if any\ of secondary metabolites to the organisms that produce them remains a matterof debate^0\1 however\ it is commonly believed that natural products which display potent biologicalactivity are the result of natural selection and that biosynthesis of these agents confers a selectiveadvantage upon the producing organism[1 DNA!damaging natural products frequently possesspotent cytotoxic\ cytostatic\ or mutagenic properties and\ in nature\ may serve as either o}ensiveor defensive weapons in the struggle for survival[ Regardless of the reason for their existence\ naturalproducts constitute a vast library of organic compounds that can serve as a useful resource[

A practical reason for the longstanding interest in DNA!damaging natural products is the factthat the cytotoxic or cytostatic e}ects of these agents sometimes endow them with useful medicinalproperties\ especially as potential anticancer therapeutics[2Ð5 Several DNA!damaging natural prod!ucts are currently in use for the treatment of various cancers and others have served as leadcompounds in the development of therapeutic agents[2Ð5 Another signi_cant impetus for the study ofnatural DNA!damaging agents lies in the fact that the chemical reactions and molecular recognitionprocesses employed by these compounds are sometimes unprecedented and strikingly e.cient[ Thus\natural products provide elegant examples of chemical processes that can e}ectively\ and oftenselectively\ modify DNA in a manner that has signi_cant biological consequences[ Finally\ naturalproducts with extremely potent biological activities sometimes reveal unforeseen biological pathwaysand these compounds can become useful tools for elucidating the details of complex life processes[For example\ studies with CC!09546 and a~atoxin7 have led to insights regarding mechanisms ofcell death and carcinogenesis\ respectively[

6[03[0[1 Classi_cation of DNA!damaging Natural Products

There are a bewilderingly large number of structurally diverse natural products that damageDNA^ however\ it is possible to divide this large number of DNA!damaging natural products intoa relatively small number of categories if one considers the chemical reactions by which thesecompounds modify DNA[ Although it is not possible to be all!inclusive\ this chapter attempts a

382Covalent Modi_cation of DNA by Natural Products

comprehensive survey of natural products known to covalently modify DNA[ The natural productsdiscussed in this chapter are divided into categories based upon the functional `roups and chemicalreactions by which these agents damage DNA[

The range of naturally occurring functional groups that have su.cient stability to survive in thecellular environment\ yet su.cient reactivity to modify DNA covalently\ is quite broad[ Thechemistry by which these functional groups react with DNA is diverse^ some natural productscontain functionality that is inherently reactive with DNA\ while others require in vivo chemical orenzymatic activation^ some natural products react with DNA stoichiometrically and others produceDNA damage catalytically[ It is not uncommon to _nd multiple DNA!reactive functional groupsin a single natural product or multiple mechanisms of DNA damage mediated by a single functionalgroup[

The emphasis on covalent reaction chemistry in this chapter is not meant to diminish the profoundimportance that functionality not directly involved in covalent reactions with DNA often has inDNA binding\ cell uptake and transport\ or triggering of DNA reactions[

6[03[0[2 General Mechanisms of Covalent DNA Modi_cation

The majority of all reactions involving covalent modi_cation of DNA can be placed in one oftwo categories] "i# reaction of electrophiles with nucleophilic sites on DNA\ or "ii# reaction ofradicals with DNA[ A very brief overview of these reactions is presented here[ The chemicalreactivity of DNA has been discussed in detail in Chapter 6[00 of this volume[

Electrophiles can react at a variety of nucleophilic sites in DNA\ although\ depending on theirchemical structure\ electrophiles generally show selectivity for certain nucleophilic sites inDNA[8Ð03 Common sites for modi_cation of duplex DNA by electrophilic natural products includeN!6 of guanine\ N!2 of adenine\ N!6 of adenine\ the exocyclic N1 amino group of guanine\ and N!2 of guanine "Figure 0#[8\00\02\03

Figure 0 WatsonÐCrick base pairs and the sugarÐphosphate backbone of DNA[

Electrophilic modi_cation at sites such as N!6 or N!2 of purine residues results in labilization ofthe glycosidic bond\03Ð05 ultimately leading to the formation of an abasic site "0# that\ under neutralconditions\ slowly hydrolyzes to yield a DNA strand break "Scheme 0#[06\07 Abasic sites are rapidlyconverted to strand breaks under alkaline conditions[04\05\08 Reaction of electrophilic species withthe exocyclic nitrogens or carbonyl oxygens of the DNA bases\ or with phosphate oxygens on theDNA backbone\ generally a}ords relatively stable adducts that do not lead to spontaneous strandscission under conditions of moderate pH[19Ð11

Many radical species are capable of reacting with DNA at a variety of positions[ Abstraction ofhydrogen atoms from the deoxyribose backbone of DNA by radicals represents an important modeof DNA damage that has been the subject of intense investigation[12Ð14 Abstraction of hydrogenatoms from the deoxyribose backbone almost universally leads to cleavage of the deoxyribosebackbone of DNA through complex reaction cascades "for example\ Scheme 1#[13\14 In addition totheir reactions with the sugar!phosphate backbone\ many radicals react extensively with the DNA


bases[15Ð18 These reactions do not necessarily lead to cleavage of the deoxyribose backbone^ however\such modi_cation of the structure of DNA bases is biologically important[ In the absence ofsequence!speci_c binding by a radical or radical precursor\ cleavage of duplex DNA by radicalsgenerally occurs with little sequence or base speci_city[29 This stands in contrast to DNA cleavageresulting from alkylating agents\ which commonly shows marked selectivity for one "or more# of theDNA bases[ DNA damage by various oxygen!centered\12Ð14\20 carbon!centered\15\21Ð23 and hydrogenradicals14 has been reported[

Although the majority of chemical reactions between natural products and DNA involve radicalsor electrophiles\ species such as carbenes\24 nitrenes\24 singlet oxygen\24Ð28 strong nucleophiles\04\39

and photoexcited molecules24Ð26 also can damage DNA[ Some of these reactions are relevant tocertain DNA!damaging natural products and\ as such\ will be discussed in the appropriate sectionsbelow[

This chapter is focused primarily on the chemical reactions of natural products with DNA and\in many cases\ does not attempt to address the biological relevance of these reactions[ Often\ thebiological relevance of in vitro studies is di.cult to assess accurately\ as these experiments are notintended to examine issues such as the repair of damaged DNA\ cellular uptake of xenobiotics\ andxenobiotic metabolism[ Similarly\ it must be remembered that demonstration of in vitro DNAdamage by a biologically active natural product by no means indicates that DNA damage is solelyresponsible for the compound|s biological activity[ In general\ however\ damage to cellular DNAhas profound biological consequences[5\6\16\30Ð37

N

NHN

N

O

NH2 E+ORO

RO

N

NHN

N

O

NH2

E

ORO

RO

N

NHN

N

O

NH2

E

ORO

RO

+

+H2O

ORO

RO

OHOH

RO

RO

OOH

RO

ROH

O

(1)

Scheme 1

BO

RO

R'O

BO

RO

R'O

BO

RO

R'O

OHO

O

O

BRO

OR'

HO

O

O

BRO

O

•RRH•

O-

O

+

BRO

O

H•

O2

-OR'

Criegee-typerearrangement

+

(e.g., RSH)

R, R'=DNA backbone

Scheme 2


6[03[1 DNA!DAMAGING NATURAL PRODUCTS

6[03[1[0 Imines

6[03[1[0[0 Pyrroloð0\3Łbenzodiazepines

A number of natural products containing a common pyrroloð0\3Łbenzodiazepine "PBD# core havebeen isolated from various strains of Streptomyces[38 These antibiotics show promising anticanceractivity in a variety of assays[38\49 The cytotoxic properties of the PBDs stem from the formation ofcovalent adducts with DNA that inhibit its processing by various enzymes[40\41

StructureÐfunction analysis of PBDs shows that an imine\ carbinolamine\ or methanolaminefunctionality at N!09ÐC!00 is required for the formation of DNA adducts and for biological activity""1#\ "2#\ Figure 1#[38\42 The imine\ carbinolamine\ and methanolamine forms of PBDs exist inequilibria\ the concentration of each species depending upon conditions "Scheme 2#[38 The imineform of PBDs "3# is generally thought to be the electrophilic species involved in adduct formationwith DNA "Scheme 2#\ although other species such as the iminium ion "4# or ring!opened amino!aldehyde "5# are possible intermediates "Scheme 3#[ Signi_cant amounts of species other than theimine have not been observed in NMR experiments and\ in chemical model reactions utilizingthiophenol as a nucleophile\ the rate of reaction of various PBDs was found to correlate with therates of imine formation[38\43 The S!con_guration found at C!00a in all naturally occurring PBDsconfers a slight right!handed twist down the long axis of the molecule which is complementary tothat of the minor groove of B!DNA[ For certain PBDs\ it has been shown that the R isomer doesnot bind DNA and is not biologically active[44

Figure 1 Pyrroloð0\3Łbenzodiazepine antibiotics[

Early experiments with the PBDs suggested that covalent attachment involves formation of anaminal linkage "6# with the exocyclic N1 of guanine in the minor groove of duplex DNA "see Scheme2#[45\46 This conclusion was based upon several experimental observations] "i# no adducts are formedin DNA that does not contain GC base pairs^ "ii# the adducts do not lead to strand cleavage uponalkaline work!up\ as N6 and N2 attachments would^ "iii# adduct formation proceeds normally on T3DNA\ which contains glycosylated 4!hydroxylmethylcytosine residues that place a steric blockade inthe major groove of DNA^ "iv# 7!2H!guanine!containing DNA shows no loss of tritium upon adductformation\ indicating that attachment is not at C!7 of guanine^ and "v# polydI!dC containing DNAdoes not form adducts[ Deoxyinosine "dI\ 7# is structurally analogous to deoxyguanosine\ but lacksthe exocyclic N1!amino group[ Covalent binding of the PBDs to DNA was measured by observingthe irreversible association of radiolabeled antibiotics with the double helix[47 Although isolation ofPBD!base adducts "6# has not been possible due to their instability\ high _eld NMR\48 ~uorescence


N

HN

OMe

H

O

R

RN

NH

O

R

RN

HN

OH

H

O

R

R

N

HN N

N

O

DNAH2N

N

HN

HN

H

O

R

R

-MeOH

+MeOH

+H2O

-H2O

N

HN N

N

O

DNA

(4)

(7)

Scheme 3

N

HN

H

O

R

RN

NH

O

R

R+H+

+H2O

-H2O

-H++

(4)(5)

N

HN

OH

H

O

R

RN

NH2H

O

R

R

O

(6)

Scheme 4

N

NN

N

O

dR

H

(8)


spectroscopy\48 and\ more recently\ X!ray crystallography59 have con_rmed that adduct formationinvolves formation of an aminal linkage between N1 of guanine and C!09 of the PBDs[

Formation of aminal adducts between PBDs and DNA is acid catalyzed at physiological pH[50\51

The adducts decompose below pH 4\ with the natural product released from the DNA unchanged[50\51

PBDs do not form adducts with single!stranded DNA or RNA[50\51 Furthermore\ it appears thatPBDÐDNA adducts\ sequestered in the minor groove of duplex DNA\ are protected from hydrolysis\as denaturation of PBD!modi_ed DNA results in reversal of adduct formation[51

Despite their small size\ PBDs display marked sequence selectivity in their reactions with DNA[38

In general\ 2?!PuGPu sequences are favored sites of reaction for PBDs^ however\ sequence speci!_cities di}er somewhat for each PBD[ Flanking sequences beyond the two base pairs adjacent tothe adducted guanine a}ect the e.ciency of DNA alkylation by PBDs[ NMR spectroscopy indicatesthat the aminal adduct formed by anthramycin "1# has the S!con_guration at C!00\52 although thestereochemistry of PBD adducts\ in general\ depends upon the sequence context of the alkylatedguanine and may di}er for each agent[48 Molecular modeling has been used to rationalize thesequence preferences and adduct stereochemistries for a number of PBDs[53

Hurley and co!workers have suggested that the sequence speci_city of the PBDs arises from thefact that the natural twist of these compounds does not exactly match that of the minor groove^thus\ more ~exible sequences\ such as the favored 4?!PuGPu sequences\ that can easily deform toaccommodate the adduct are preferred reaction sites[41 In agreement with this hypothesis\ it hasbeen found that PBD adduct formation in duplex DNA induces a moderate DNA bend of about5Ð04>[ Also consistent with the notion that DNA ~exibility plays a role in PBD sequence speci_cityis the fact that the amount of bending induced by several PBDs in various sequence contextscorrelates with the rate of adduct formation\ with sequences that adopt a more bent structurea}ording higher reaction rates[54 An alternate hypothesis for the sequence speci_city of the PBDshas been put forward suggesting that the 4?!PuGPu binding preference stems from the fact that\ ingeneral\ purineÐpurine steps have smaller than average twist angles\ thus producing a minor grooveshape that closely matches the inherent twist of the PBDs\ especially anthramycin "1#[59

Cardiotoxicity observed for anthramycin "1# and sibiromycin "2# may be due to the formation ofo!quinoneimines that are capable of producing oxygen radicals through redox!cycling chemistry"see Section 6[03[1[00[0#[55 It is possible that redox cycling of anthramycin and sibiromycinÐDNAadducts could cause localized oxidative DNA damage[

Understanding of the covalent and noncovalent chemistry involved in adduct formation by thePBDs has allowed the successful design of novel DNA!cross!linking agents consisting of twocovalently tethered PBD units[ These cross!linking agents are more cytotoxic than the correspondingmonomeric natural products[56

6[03[1[0[1 Saframycins\ renieramycins\ and safracins

Some of the saframycin antibiotics "8\ 00# form electrophilic imine species that alkylateDNA[57Ð60 The early experiments implicating covalent attachment of saframycins to DNA involvedmeasuring the irreversible association of radiolabeled antibiotics with DNA[60 The structural simi!larities between the saframycins and the renieramycins "09# and safracins "00# suggests that thechemical reactivity of these agents with DNA will be similar^49 however\ the saframycins are theonly members of this group whose DNA!damaging chemistry has been studied in detail[ Thesaframycins and safracins are isolated from Streptomyces and the renieramycins are products ofmarine sponges[49

Lown and co!workers described three types of interactions between saframycins and DNA]58 "i#reversible noncovalent binding^ "ii# reversible\ acid!catalyzed formation of minor groove adductswith guanine^ and "iii# the predominant binding mode involving reversible formation of minorgroove adducts with guanine that is promoted by reducing agents\ such as dithiothreitol[

Signi_cantly\ only saframycins that bear a leaving group "i[e[\ cyanide or hydroxide# at C!10 arecapable of alkylating DNA[58 Adduct formation for these analogues is signi_cantly enhanced byreduction of the quinone moiety[57 On the basis of these observations\ it was proposed that\ foranalogues such as saframycin A "8a#\ reduction of the A!ring quinone to a hydroquinone facilitatesejection of the C!10 leaving group\ with corresponding formation of a DNA!alkylating iminium ion"01\ Scheme 4#[57


Although saframycinÐDNA adducts such as "02# have not been rigorously characterized\ experi!ments strongly suggest that reaction of the saframycin!derived iminium ion "01# with N1 of guanineyields aminal attachments similar to those observed for pyrroloð0\3Łbenzodiazepines "see Section6[03[1[0[0#[57 A requirement of GC base pairs is observed for the formation of the slowly reversible


covalent adducts and minor groove adduction is suggested by the fact that the DNA alkylationproceeds normally with major groove!glycosylated T3 DNA[ Activated saframycin can be trappedby added cyanide ions\ suggesting that the reductively activated form is not exclusively associatedwith DNA[

The quinone moiety of the saframycins\ in the presence of molecular oxygen and reducing agents\undergoes redox cycling "see Section 6[03[1[00[0# to produce the DNA!cleaving agent\ hydroxylradical[57 Such redox properties may explain the cytotoxicity of saframycin derivatives that do notform covalent attachments with DNA[ It is not clear whether saframycinÐDNA adducts can undergoredox cycling and\ thus\ e.ciently damage DNA via localized production of oxygen radicals[

Molecular modeling has been employed to predict the detailed binding mode of saframycin A"8a# in the minor groove of DNA[61 Footprinting and exonuclease stop assays with various saf!ramycins indicate that covalent attachment is favored at 4?!GGG sites[69

6[03[1[0[2 Oxazolidine!containing natural products

The family of natural products including naphthyridinomycin "03a#\ the cyanocyclines "03b\03d#\ the bioxalomycins "04#\ quinocarcin "05#\ and tetrazomine\ isolated from various strains ofStreptomyces\49 contain oxazolidine rings that may serve as DNA!damaging iminium ion precursors[Several members of this group of compounds display promising anticancer and antimicrobialproperties[49

Naphthyridinomycin "03a# forms covalent adducts with DNA[62 Experiments similar to thosedescribed above for the pyrroloð0\3Łbenzodiazepines and the saframycins suggest that these adductsinvolve attachment through N1 of guanine in the minor groove[ Formation of DNA adducts isfacilitated under reducing conditions "e[g[\ 0 mM dithiothreitol#[62 Thus\ it was suggested62 thatreduction of the quinone in "03a# stimulates the formation of iminium ion "06# by facilitatingelimination of the leaving group at C!6 "Scheme 5#[ This mechanism is analogous to that which waslater suggested to explain alkylation of DNA by saframycin A "see Scheme 4#[57 The same researchersalso suggested an alternative possibility in which reduction of the quinone moiety of "03a# promotesnoncovalent DNA association\ followed by an SN0CA reaction to yield the proposed aminal adductwith guanine[ Molecular modeling studies later indicated that both proposed mechanisms arereasonable[63

In addition to possible iminium ion formation at C!6 "06#\ the oxazolidine heterocycle in "03a#and the cyanocyclines may serve as a precursor for iminium ion formation at C!2a "07#[63 Oxazolidinering!opening to produce the reactive iminium ion at C!2a "see Scheme 5\ upper pathway# would befacilitated by reduction\ in a manner analogous to imine formation at C!6[ Interestingly\ treatmentof cyanocycline A "03b# with acid in the absence of reducing agents results in oxazolidine ringopening with concomitant iminium ion formation at C!2a[64 Whether DNA attachment by "03a#occurs at C!2a\ C!6\ or both sites remains unknown[

The possibility of successive formation of two electrophilic imines from "03a# and the cyanocyclinessuggest that DNA cross!links are formally possible^ however\ molecular modeling indicates thatsuch cross!links are unlikely\ because the initial covalent attachment at N1 of guanine positions thesecond electrophilic site outside the minor groove and away from nucleophilic sites on the doublehelix[63 ProteinÐDNA cross!links are a possibility with these agents[


Several natural products\ very similar in structure to "03a# and the cyanocyclines\ except con!taining two intact oxazolidine rings\ were isolated "from a di}erent strain of Streptomyces than thatwhich originally produced "03a## and were dubbed the bioxalomycins "04#[65 In the course ofcharacterizing the bioxalomycins\ researchers at Lederle attempted to isolate "03a# from the originalproducing strain[ Interestingly\ they were unable to isolate "03a# from these cultures\ but they didisolate bioxalomycin b1 "04b# from the original "03a#!producing strain[65 Thus\ although the pub!lished spectroscopic data for "03a# is clearly distinct from that of the bioxalomycins and wassupported by an X!ray crystal structure\ it is possible that "04b# may be the naturally producedform of "03a#[ It is possible that "03a# may have been transiently produced under certain fermentationconditions or may be an artifact of the original isolation process[

The NMR spectra of bioxalomycin a1 "04a# indicates that the compound exists in equilibriumwith the C!2a ring!opened iminium ion form[ Relative peak intensities indicate that 04) of thecompound exists in the ring!opened form[65 These NMR studies suggest that covalent attachmentof DNA at C!2a of the bioxalomycins is a strong possibility[

Two additional oxazolidine ring!containing antitumor antibiotics\ quinocarcin "05#66 and tetra!zomine67 have the potential to alkylate DNA via iminium ion intermediates[ Possible alkylation ofDNA by quinocarcin has been examined using molecular modeling\68 but has not been exper!imentally veri_ed[ Interestingly\ a second mechanism for DNA damage\ distinct from DNA alkyl!ation\ has been observed for quinocarcin and tetrazomine[79\70 These agents generate DNA!cleavingoxygen radicals through reduction of molecular oxygen to superoxide radical[ This chemistry isdescribed further in Section 6[03[1[04[

6[03[1[0[3 Barminomycin I

Barminomycin I "08#\ sometimes referred to as SN!96\ is a member of the anthracycline familyof antibiotics\ but is unique among the anthracyclines in that the anthraquinone core is substitutedwith an unusual carbinolamine!containing sugar moiety[ Experiments similar to those describedabove for the pyrroloð0\3Łbenzodiazepines\ suggest that "08# forms covalent adducts with the N1!amino group of guanine\ preferentially at 4?!GC sites[71 This reaction is mechanistically related tothe formaldehyde!mediated cross!linking of anthracyclines to DNA "see Section 6[03[1[00[2#[

N

N

N

O

OH

OH

MeO

R

H H

H

H

H

OH

N

N

N

OH

O

OH

MeO

R

H H

H

OH

N

N

N

O

OH

O

MeO

RH

H

H

OHN

N

N

O

OH

OH

MeO

RH

H

H

OH

N

N

N

OH

OH

OH

MeO

R

H H3a

78

H

1a OH

H+

OH

H+

OH

+

+

OH

-OH

(14a)

(17)

(18)

Scheme 6


6[03[1[0[4 Other antibiotics that alkylate DNA via imine formation

Alkylation of duplex DNA at N1 of guanines by the novel carbinolamine!containing antibioticecteinascidin 632 "19#\ isolated from a Caribbean tunicate\ has been reported[72

A number of pyrroloiminoquinones such as discorhabdin A "10#\ makaluvamine F "11#\ andepinardin D "12#\ isolated from marine sponges\ have the potential to form DNA!reactive imines[73\74

Imine formation resulting from elimination of the methoxy substituents or opening of the sulfur!containing rings of these natural products would be facilitated by reduction of the iminoquinonemoiety[ Inhibition of topoisomerase II by these agents has been studied^74 however\ the possibilityof covalent adduct formation has not been explored[

O

O

OH

OHOH

O

O

OH

O

HN

O

OH

O

Barminomycin I

OH

(19)

HN

N

HN

O

S

OBr

HN

N

HN

O

S

OH

Br

N

N

OO

OO

NHMeO

HO

O

OMe

HO

S OH

Me

(22) Makaluvamine F(21) Discorhabdin A(20) Ecteinascidin 743

HN

N

HN

OH

HO Br

O

OMe

(23) Epinardin D

O


6[03[1[1 Carbonyl!containing Natural Products

6[03[1[1[0 Aldehydes

A number of aldehyde!containing compounds occur naturally in plants and other organisms[75Ð78

Some of these aldehydes are not formally secondary metabolites^ rather\ they are products of naturaloxidative decomposition of lipids[ Some naturally occurring aldehydes can react with nucleophilicsites in DNA^ for example\ a\b!unsaturated aldehydes\ such as 1!hexenal\ have been isolated fromvarious plant sources\ are mutagenic\ and form covalent adducts with DNA[89

DNA adducts of simple unsaturated aldehydes\ such as acrolein and crotonaldehyde "13# havebeen well characterized80 and may be viewed as models for naturally occurring unsaturated alde!hydes[ Guanine is the most reactive base toward a\b!unsaturated aldehydes\ although adducts withall four of the DNA bases have been isolated "Figure 2#[ Many of the products obtained from thereaction of DNA bases with these compounds are the apparent result of Michael addition\ followedby attack on the aldehyde carbonyl to yield cyclic adducts "Scheme 6#[80

Figure 2 DNA adducts with a\b!unsaturated aldehydes[

Cyclic adducts attached at N!6 and C!7 of guanine undergo spontaneous depurination to give anabasic site on the DNA[ Other adducts\ such as those spanning N!0 and N!1 of guanine\ are notlabile and\ when formed in vivo\ may interfere with enzymatic processing of DNA[

Malondialdehyde is a naturally occurring carcinogen that is formed by endogenous lipid per!oxidation[81 Marnett and co!workers have extensively characterized the adducts of malondialdehydewith DNA bases\ examined the mutagenicity of these adducts\ and con_rmed the existence ofmalondialdehydeÐDNA adducts in human cells[81

Formaldehyde is ubiquitous in the environment and\ although it exists primarily "½87)# as thehydrated form in aqueous solution\ it reacts readily with DNA to form a variety of DNA mono!adducts as well as cross!links "14#[82\83 Hopkins and co!workers found that 4?!d"AT# sequences arepreferred targets for formaldehyde cross!linking\ possibly because cross!link formation at these sitesrequires minimal distortion of the DNA duplex[83 Formaldehyde also reacts readily with certainanthracyclineÐDNA complexes to yield drugÐDNA cross!links "see Section 6[03[1[00[2#[

O

HH

H HN

N N

N

O

H2NdR

HN

N N

N

O

NH dR

O

N

N N

N

O

NH dR

OH

Scheme 7

(24)

N

NN

N

HN

O

RO

RO

NH

N

N N

N

O

OR

OR

R=DNA backbone(25)


The mutagenic properties of the sesquiterpene lactone hymenovin "15# have been ascribed to itsbis!hemiacetal functional group[84 Although adducts of this natural product with DNA have notbeen reported\ hymenovin may be capable of cross!linking DNA\ its bis!hemiacetal moiety servingas a masked dialdehyde[ Similarly\ DNA damage reported for the fungal natural product patulin"16#85 may be due to the formation of a reactive aldehyde from the hemiacetal functional group[86

6[03[1[1[1 Isochrysohermidine

Isochrysohermidine "17# derives from an oxidative rearrangement of the secondary metabolitehermidin found in certain plant species[87 The symmetrical dimer "18# has been characterized by X!ray crystallography and occurs in the plant as a mixture of the D\L!racemate and the meso compound[The presence of two carbinolamide functional groups suggested to Boger and Baldino that thisnatural product might cross!link DNA[ It was shown that racemic and meso isochrysohermidine\at millimolar concentrations\ do\ in fact\ cross!link DNA[87 The slow cross!linking reaction ismoderately a}ected by the pH of the reaction mixture\ with cross!linking facilitated slightly byeither acidic or basic conditions[ The pH pro_le of this reaction suggests that DNA alkylation doesnot involve acyliminium ion formation\ a process that would be acid catalyzed[ Rather\ it isproposed87 that acidic or basic conditions catalyze a ring!opening reaction that yields an electrophilic0\1!dicarbonyl moiety "18# on each half of the symmetrical isochrysohermidine molecule "Scheme7#[

The vicinal dicarbonyl 1\2!butanedione "diacetyl# is produced by several microbial species anddisplays mutagenic77 and antibacterial activity[88 Bis!tricarbonyl species have been successfullyemployed as designed DNA!cross!linking agents[099

6[03[1[1[2 Lactones

There are reports that a\b!unsaturated lactones may react with DNA[86\090 It is noteworthy\however\ that studies with the natural a\b!unsaturated lactone helenalin show that\ while thiolnucleophiles readily engage in Michael reactions with the a\b!unsaturated lactone helenalin\ thisnatural product does not alkylate purine bases[091

OO

HO

OHO

(26) Hymenovin

O

O

O

OH

(27) Patulin

NN

OMe

MeO2CO O

OH

MeO

HO

CO2Me

Me Me

NHN

OMe

O O

OH

MeO

CO2Me

Me Me

O

O

O

Me

Electrophilic Dicarbonyl

DNAReactions

(28) (29)

Scheme 8


6[03[1[2 Cyclopropanes

6[03[1[2[0 CC!0954 and the duocarmycins

Appropriately substituted cyclopropanes are potent electrophiles[092 A number of cyclopropane!containing antibiotics that derive biological activity through DNA alkylation have been identi_ed[093

CC!0954 "29#\ isolated in 0863 from Streptomyces\ is extremely cytotoxic and shows activity againstsome cancer cell lines at picomolar concentrations[51\094Ð096

The cyclopropane ring of "29# is electrophilic[ Attack of nucleophiles on the strained ring isfavored by aromatization of the indole quinone system that results from the reaction "Scheme 8#[Early experiments showed that "29# forms stable covalent attachments with DNA\095\096 but that\under thermal treatment\ the covalent adducts decompose to yield strand breaks[097 Isolation andcharacterization of the adduct "24# released by thermal treatment of DNA alkylated by "29# clearly

N

N

O

NO

MeO2C

H O

H

OMe

OMe

OMe

H

N

N

O

N

O

MeO2C

H

H

OMe

OMe

OMe

OH

X

N

N

N

H

O

H

OH

OMe

N

O

N

N

H2NO

OH

OMe

H

O

HN

N

O

NO

MeO2C

H OH

H

OMe

OMe

OMe

H

(a) Duocarmycin B1: X=Br(b) Duocarmycin C1: X=Cl

(30) CC-1065 (31) Duocarmycin A

X

(a) Duocarmycin B2: X=Br(b) Duocarmycin C2: X=Cl

(33) (34)

N

N

O

N

MeO2C

H O

H

OMe

OMe

OMe

(32) Duocarmycin SA


demonstrated that covalent modi_cation involves nucleophilic attack of the N!2 nitrogen of adenineon the cyclopropane ring of the natural product "see Scheme 8#[097 DNA alkylation occurs almostexclusively at adenine^ however\ small amounts of an adduct resulting from the attack of N!2 ofguanine have been identi_ed[098\009 Alkylation of DNA by "29# is essentially irreversible in neutralaqueous solution^094 however\ DNA alkylation by some structurally related cyclopropane!containingantibiotics is reversible[094\000

The linked pyrroloindole moieties of "29# lend a right!handed curvature to this antibiotic\ remi!niscent of the well!known minor groove!binding agents distamycin and netropsin[ In accord withits structural similarity to netropsin\ early experiments demonstrated that "29# binds in the minorgroove of DNA at AT!rich sequences[095\096 It is shown that netropsin inhibits the binding of "29# toDNA\ that major groove glycosylation present in T3 DNA does not inhibit binding\ and that theantibiotic shows markedly better binding to homopolymeric AT DNA vs[ GC DNA[ Subsequent1D!NMR experiments have conclusively con_rmed minor groove binding by this antibiotic[001

A number of antibiotics with structures similar to "29# have been reported[49\000 Duocarmycins A"20# and SA "21# possess a cyclopropane!containing core very similar to "29#[ Duocarmycins C0\ B0

"23#\ C1\ and B1 "22# display properties identical to duocarmycin A and are\ in fact\ prodrugs thatconvert to duocarmycin A in aqueous solution[ Prior to the discovery of "22b# and "23b# the samecompounds had been isolated independently from a di}erent strain of Streptomyces than theduocarmycins and named pyrindamycin B and A\ respectively[49\000 In general\ the duocarmycinsdisplay DNA binding\ DNA alkylating\ and potent biological properties that are similar to thoseof "29#[

The duocarmycins and "29# display marked sequence speci_city in their alkylation of DNA andthe molecular origin of this selectivity has been the subject of intense study[094\000 Preferred sites ofalkylation for "29# include sites such as 4?!PuNTTA and 4?!AAAAA[094 Both "29# and the duo!carmycin antibiotics preferentially bind to AT!rich sites\ although binding of these antibiotics toDNA does not necessarily lead to e.cient alkylation unless the binding site contains an appropriatelypositioned nucleophile "i[e[\ N!2 of adenine#[ For example\ the sequence 4?!GAATT is a strongnoncovalent binding site for "29#\002 but is not alkylated with the same e.ciency as the comparablebinding site 4?!AGTTA[003

Interestingly\ Hurley et al[ found that simple CC!0954 analogues\ such as "25#\ alkylate duplexDNA with sequence selectivity nearly identical to the intact natural product[004\005 This result suggeststhat sequence selectivity of DNA alkylation by "29# may arise in the chemical bonding steps\ possiblystemming from sequence!dependent properties of the DNA target site\ such as conformational~exibility or the presence of DNA functional groups at the binding site that catalyze the alkylationreaction[094\006 Other examples of sequence!dependent e}ects on the reactivity of DNA are known[007

Consistent with the notion of DNA!catalysis in the alkylation reaction\ NMR studies have identi_edwater molecules coordinated to an AT base pair and a phosphate residue of the target site thatmight catalyze the alkylation reaction[094 DNA a.nity provided by the pyrroloindole {{tail|| of "29#\while apparently not required for sequence speci_city\ clearly improves the e.ciency of the alkyl!ation reaction\ and relatively high concentrations of "25# are required to obtain alkylation levelssimilar to those achieved by the natural product under a given set of conditions[004\005

In results that appear in stark contrast to those described above for "29#\ Boger|s group hasreported that simple duocarmycin analogues\ such as "26# display markedly reduced sequence

N

N

H

O

O

N

NN

N

NH

H

DNAN

N

H

HO

O

N

N

N

N

N H

DNA

H+

(30) (35)

Scheme 9


selectivity relative to intact duocarmycins[000\008 In addition\ analogues of duocarmycin bearingnoncyclopropane electrophiles alkylate DNA with sequence selectivity identical to the naturalproduct[ These and other results have led Boger and co!workers to suggest that the sequence!selective alkylation of DNA by the duocarmycins derives primarily from noncovalent interactionsbetween DNA and the natural products[000 The precise roles of noncovalent binding and covalentbonding steps in the high sequence speci_city displayed by the duocarmycins and "29# remain atopic of debate and the subject of ongoing experimentation[

Boger|s group has proposed that a DNA binding!induced conformational change causes groundstate destabilization of "29# and the duocarmycins thereby activating the agents for DNAalkylation[019 They suggest that this activation is the result of a DNA binding!induced twist in theN!1 amide that links the cyclopropane!containing moiety to the pyrroloindole group on the {{right!hand|| portion of the molecule[

Analogues of the duocarmycins and "29# have been prepared in which the reactivity of thecyclopropane moiety is altered[ Examination of a wide range of natural and synthetic analogueshad led Boger|s group to the _nding that duocarmycin analogues whose cyclopropane groups aremore solvolytically stable are more e.cient DNA alkylators and are more potent cytotoxins "Figure3#[000\010 Increased cytotoxicity of the more stable analogues presumably re~ects\ at least in part\a decrease in the nonproductive hydrolytic destruction of the compounds or a decrease in thenonproductive alkylation of biomolecules[ The partitioning of compounds between DNA reactionand hydrolysis has been quantitatively examined in some cases[098

Figure 3 Relative stability of some duocarmycin and CC!0954 analogues[

The duocarmycins and "29# are exceedingly potent cytotoxins[ In the case of "29#\ as few as threecovalent lesions per 095 base pairs of DNA can lead to cell death[011 The extreme toxicity of theseagents even at low levels of DNA damage suggests that adducts of "29# with DNA are particularlylethal[ In e}orts to elucidate the molecular origin of the biological activity of "29#\ Hurley|s grouphas extensively characterized the e}ect that DNA lesions involving "29# have on the processing ofDNA by polymerases\ helicases\ ligases\ and other enzymes[094 In addition\ they have characterizedDNA bending\ winding\ and ~exibility of DNA adducted by "29#[094 Hurley and co!workers havesuggested that the potent toxicity of "29# may derive in part from its ability to act as a surrogatetranscriptional activator protein[6 DNA bending induced by formation of adducts of "29# with DNAmay mimic the bending of DNA by transcriptional activators such as Sp0[ Thus\ formation ofadducts with "29# at some sites in genomic DNA may irreversibly activate gene transcription\thereby leading to excessive and unregulated production of messenger RNA and proteins[

6[03[1[2[1 Myrocin C

Myrocin C "27#\ isolated from a soil fungus\ is a cyclopropane!containing antibiotic that displaysantitumor properties[012 Chu!Moyer and Danishefsky suggested a mechanism by which "27# might

N

N

H

O

O

(36)


alkylate biomolecules[013 In the proposed mechanism\ allylic displacement of the C!8 hydroxylactivates the cyclopropane moiety by bringing this strained ring into conjugation with the C!6carbonyl group[ It was further speculated that the electrophilicity of the cyclopropane moiety couldbe increased through ring!chain tautomerism to produce "28#[ Nucleophilic cyclopropane ringopening of "28# would result in favorable aromatization of the B!ring of "27# "Scheme 09#[

The viability of this proposal was supported by chemical model reactions[ Utilizing thiophenolas a nucleophile\ the expected bis!thiophenol adduct of "27# was obtained[013 If DNA is\ in fact\ abiological target for "27#\ the model reactions suggest that this antibiotic could either serve as across!linking agent or as a thiol!activated alkylating agent[ As yet\ there have been no reportsregarding reactions of DNA or other biomolecules with "27#[

6[03[1[2[2 Ptaquiloside and the illudins

Investigations seeking the chemical agents responsible for the carcinogenic properties of brackenfern uncovered the potent carcinogen ptaquiloside "39#\ containing a spiro!cyclopropane system[014

Under mildly basic conditions\ ptaquiloside eliminates D!glucose to yield the dienone "30#[014 Com!pound "30# may represent an activated form of ptaquiloside because nucleophilic attack on thecyclopropane ring of this aglycone is favored by the formation of an aromatic ring "Scheme 00#\similar to the reactions of CC!0954\ the duocarmycins\ and myrocin C with nucleophiles[

O

OH

O

O

HOH

O

OO

HOH

Nu

O

CO2H

H

Nu

O

(38) Myrocin C

9 9

7

6 6

O

CO2H

Nu

OH

OH

CO2H

Nu

OH

Nu

7

(39)

Scheme 10

Nu–

Nu–

OHO

OOH

O

OH

DNA

(43) Pterosin B

OHO

HOHO

OH

(40) Ptaquiloside

-glucose

ODNA-Nu

OH

H2O

(41) (42)

Scheme 11


The dienone "30# e.ciently cleaves DNA at micromolar concentrations\ while similar con!centrations of "39# produce only weak cleavage in a plasmid!nicking assay[015 Reactions of "30# withthe oligomer 4?!dGTAC\ followed by thermal workup\ yield adducts "31# attached to guanine N!6"0[1) yield# and adenine N!2 "9[4) yield#[ Pterosin B "32# is observed as a hydrolysis product[014\015

Studies with the adducted oligomer show that\ in TrisÐborate bu}er "pH 6[4#\ the adenine adductdepurinates at a markedly faster rate "t0:1�2[1 h# than the guanine N!6 adduct "t0:1�20 h#[ On thisbasis\ it is suggested that the relatively stable N!6!guanine adducts may be responsible for thecarcinogenic properties of ptaquiloside\ because the apurinic sites resulting from the N!2!adenineadducts would be repaired easily in vivo[015

Although the dienone "30# derived from ptaquiloside is a small molecule\ with little potential fornoncovalent DNA binding\ it is reported to alkylate DNA with some sequence selectivity[ Flankingsequences a}ect the e.ciency of adenine alkylation\ with 4?!dAAAT being the most favored alkyl!ation site[ The authors of this study note that sequence!dependent di}erences in the covalentreactivity of DNA may be responsible for the observed sequence selectivity[015

The illudins "33#\ isolated from poison mushrooms\ are cyclopropane!containing natural productssimilar in structure to ptaquiloside[016\017 The illudins are extremely toxic and are carcinogenic[Reaction of these compounds with DNA has been demonstrated018 and model reactions indicatethat\ at low pH\ these compounds can serve as alkylating agents[029

6[03[1[3 Epoxides

6[03[1[3[0 Pluramycins and pluramycinones

The pluramycins "e[g[\ "34# and "35## were _rst isolated from Streptomyces in the 0849s and foundto possess anticancer and antimicrobial activities[020 Over the years\ a large number of antibioticsfrom this family have been characterized[021 Similar to simple epoxides\022 the pluramycins formcovalent attachments with DNA that\ upon alkaline workup\ yield strand breakage at guanines[023\024

Isolation and characterization of the adduct "37# released by thermal workup of altromycin B!treated DNA showed conclusively that covalent attachment involves attack of the N!6 position ofguanine on the epoxide of the natural product[025 The chemistry of DNA attachment for thepluramycins is analogous to that for simple epoxides^ however\ unlike simple epoxides\ the plu!ramycins alkylate DNA very e.ciently and sequence speci_cally[

Work by Hurley|s group has revealed that the planar anthrapyrone!3\6\01!trione moiety of thepluramycins intercalates into DNA on the 4?!side of guanines that become adducted[021 Similar tosome anthraquinone antibiotics\ the pluramycins intercalate by a {{threading mechanism|| with theanthrapyrone system perpendicular to the long axis of the DNA base pair[ NMR experimentsindicate that the carbohydrate residues of the pluramycins contribute to sequence recognitionthrough hydrogen bonding and hydrophobic interactions with DNA[ The preferred sequences forDNA alkylation are 4?!AG for the altromycins and 4?!PyG for the {{classical|| pluramycins that donot contain sugar residues at the 4!position[021

The molecular basis for the sequence!speci_c alkylation of DNA by the pluramycins has beenstudied in detail[021 Neopluramycin\ a nonalkylating derivative of pluramycin A\ does not showstrong DNA binding that can be detected by Dnase I assays[ This suggests that sequence!selectivealkylation of DNA by pluramycin A does not simply re~ect strong sequence!speci_c binding at thepreferred alkylation sites[ Rather\ it appears that\ at favored pluramycin alkylation sites\ functional

O

HO

O

CH2R

Illudin S: R=OHIlludin M: R=H

(44)


groups of the DNA bases interact with the natural product to {{steer|| the electrophilic epoxide intothe appropriate position for reaction with N!6 of the target guanine[ Less e.cient alkylation ofguanines at nonpreferred alkylation sites may be the result\ not necessarily of weaker binding atthese sites\ but of inappropriate positioning of the epoxide for reaction with the N!6 position ofguanine[ The exact interactions involved in binding of the pluramycins and pluramycinones to theirpreferred target sites di}er somewhat for individual members of this class[ Flanking sequencesoutside the intercalation site also play a role in sequence speci_city[

Nucleophilic attack of DNA occurs at the C!07 position of hedamycin026\027 "34# and at C!05 ofaltromycin B "35#[025 The higher reactivity of "34# and the other diepoxide!containing pluramycinsmay be explained by the fact that the longer {{reach|| of the epoxide in these compounds allowsfavorable alignment of the DNA nucleophile for the stereoelectronically controlled epoxide ring!opening reaction\ with minimal distortion of the double helix[021

Studies examining the e}ects of DNA!binding proteins on modi_cation of DNA by the plu!ramycins indicate that these agents may serve as useful probes for protein!induced changes in DNAconformation[028 For example\ DNA alkylation by the pluramycins is enhanced at sites downstreamfrom TATA binding proteinÐDNA complexes[

In the presence of reducing agents and molecular oxygen\ the pluramycins and pluramycinonesmay be capable of producing oxygen radicals via redox cycling reactions involving their quinonemoieties "see Section 6[03[1[00[0#^ although\ to date\ DNA damage by this pathway has not beenreported for this class of natural products[

In theory\ proteinÐDNA cross!linking and interstrand DNA cross!linking022 could be mediatedby diepoxides such as hedamycin[

O

O

O

O

O

H

ON

OH

MeO

O

HOOMe

OH

OH

OOMe

O

O

O

O

O

O

O

NHO

OH

N

O

O

O

(47) Sapurimycin

O

O

O

O OH

O

OH

HO2C

O

O

O

(A Pluramycinone)

O

2

(45) Hedamycin

H

(46) Altromycin B

8

Sugar

(48) Altromycin B–guanine adduct

Sugar

OH

5

H

2

10

5

10

N

NHN

N

O

16

18

Pluramycins

NH2

8

(A "Classical" Pluramycin)


6[03[1[3[1 Kapurimycin A2 and the clecarmycins

The epoxide!containing antibiotic kapurimycin A2 "38# is similar in structure to the plu!ramycinones and has been found to alkylate guanines in duplex DNA[039 A covalent adduct of thisantibiotic with guanine has been isolated by thermal treatment of "38#!treated DNA[ Charac!terization of the product shows attachment of DNA to C!05 of the epoxide through N!6 ofguanine[030 Analysis of the DNA end products resulting from alkylation of a self!complementaryoligonucleotide by "38# is consistent with the products known to arise from DNA cleavage resultingfrom alkylation at N!6 of guanine[030 The sequence speci_city of DNA alkylation by "38# has notbeen examined[

The reported clecarmycins "49# and "40# are similar in structure to "38#\ but bear epoxides onboth ends of their aromatic core[031 If the clecarmycins bind to DNA via threading mechanismssimilar to the pluramycins\ it can be expected that an epoxide in the diepoxide fragment will alkylateguanine in the major groove of DNA[ In addition\ it can be imagined that\ with some distortion ofthe double helix\ the additional epoxide "on the end opposite the diepoxide moiety# of the cle!carmycins could alkylate nucleophilic positions in the minor groove of DNA\ thus leading to inter!or intrastrand cross!links[

6[03[1[3[2 Alkoxyl radicals from vinyl epoxides

Studies with synthetic vinyl epoxides032 "41# suggest a second mode of DNA damage\ in additionto alkylation of DNA\ that may be possible for vinyl epoxide!containing natural products such as"38#\ "36#\ pluramycin A\ and rubi~avin A[ Thiyl radicals react with the double bond of simple vinylepoxides\ leading to homolytic opening of the epoxide ring "Scheme 01#[ The resulting alkoxy radical

O

O

O

O

O

OH

O

O

O

O

HO

OO

OAc

OH

O

CO2H

O

(51) Clecarmycin C

(50) Clecarmycin A1(49) Kapurimycin A3

14

16

17

O

O

O

O

O

OH

O

O

O

HOOH


"42# is capable of abstracting hydrogen atoms from a deoxyribose mimic in model reactions[032 Therelevance of this model chemistry to actual DNA cleavage was shown by the _nding that a vinylepoxide covalently linked to a phenanthrolinium intercalator "43# mediates DNA cleavage in thepresence of thiyl radicals generated by a horseradish peroxidase:hydrogen peroxide:glutathionesystem[20 This radical chemistry of vinyl epoxides could be biologically relevant\ as thiyl radicalsresulting from thiol oxidation may be present in cells[

6[03[1[3[3 Psorospermin

The epoxide!containing phytotoxin psorospermin "44# displays potent anticancer properties and\similar to the other epoxides discussed in this section\ has been found to alkylate N!6 of guanine[033

Experiments utilizing two!dimensional proton NMR show that\ similar to the pluramycins\ "44#intercalates to the 4?!side of guanine residues that become alkylated[ Unlike the pluramycins\however\ in the "44#ÐDNA adduct\ the natural product is intercalated with its long axis parallel tothe long axis of the DNA base pairs[033 Attack of the N!6 position of guanine occurs at C!3? of "44#[The preferred alkylation site of "44# is 4?!GG\ a site that is one of the least favored sites for thepluramycins[033

6[03[1[3[4 Metabolically activated mycotoxins] the a~atoxins

The a~atoxins\ sterigmatocystin\ the versicolorins\ and the austocystins "45#Ð"48# are a group ofhighly carcinogenic\ fungal secondary metabolites that contain a common dihydrofurofurancore[33\034 These mycotoxins are not inherently reactive with DNA\ but are converted to reactive\highly carcinogenic epoxides by cytochrome P349 oxygenase systems in vivo "Scheme 02#[ The DNAreactions of a number of the dihydrofurofuran mycotoxins have been studied\ but a~atoxin B0 "45#is perhaps the best characterized member of this class[

O

R'

O

R'RS

R'RS

O

RS••

(52) (53)

Scheme 12

N

NO

-OTs+

(54)

O

OH

OMe

O

OH

O

(55) Psorospermin

C4'


Enzymatic oxygenation of "45# yields both the exo and endo isomers[034 Both epoxides are highlyreactive^ methanolysis of the exo isomer yields a mixture of cis and trans products\ suggesting anSN0 character to this reaction\ while the endo isomer produces only the trans methanolysis product[The exo epoxide "59# reacts readily with DNA\ while the endo epoxide is less reactive[ Accordingly\the exo isomer is highly mutagenic and the endo isomer is not[034

The major covalent adduct "50# resulting from treatment of DNA with activated a~atoxins hasbeen well characterized and clearly shows that a~atoxin epoxides alkylate guanine at the N!6position[035\036 In addition\ minor adducts resulting from reaction of activated a~atoxin B0 with N!6 of adenine037 and with cytosine038 have been identi_ed[

Due to their instability\ the epoxides of "45# have not been isolated from biological systems^however\ a~atoxin epoxides may be generated in situ by oxidation of "45# with agents such as 2!chloroperbenzoic acid049 or by conversion of "45# to the dichloride using chlorine gas\ followed bythe addition of this product to aqueous solution[034 The exo epoxide has been unambiguouslysynthesized by oxidation of "45# with dimethyldioxirane in acetone[040 The half!life of the exoepoxide in water is ½ 0 s^ nonetheless\ addition of this material to aqueous\ DNA!containingmixtures produces high yields of covalent adducts[041\042

OO

O

O

O O

OO

O

O

O O

O

H

H

OO

O

O

O O

H

H

HO

H

H

H

H

N

N N

N

O

i, DNAii, heat(depurination)

(56) Aflatoxin B1

H2N

H

cytochromeP450s

(60)

(61)

Scheme 13

OO

O

O OO

O

O

O

OH

O

OO

OH O

O

OH

OH

(56) Aflatoxin B1 (57) Sterigmatocystin (58) Versicolorin A

O

OO

OMe

O

O OMe

Cl

(59) Austocystin A


Proton NMR experiments using oligomeric DNA duplexes indicate that "45# intercalates intoduplex DNA\043 and that intercalation of the exo epoxide to the 4?!side of a guanine residuewould position the epoxide ideally for reaction with N!6 of this guanine[041 Consistent with thisintercalationÐalkylation picture\ modi_cation of single!stranded DNA\ A!form DNAÐRNAduplexes\ and Z!form DNA duplexes by "45# is ine.cient[044 Investigations of the sequence!speci_cityof DNA modi_cation by "45# indicate that 4?!GGG sequences are modi_ed\ on average\ 19 timesmore e.ciently than the least!favored sequence 4?!TGA[045 It is known that guanine residues thatare ~anked by guanines possess enhanced nucleophilicity^ however\ the precise origin of the sequencespeci_city of the a~atoxins remains a subject of investigation[ The reasons for the potent car!cinogenicity of the a~atoxins have been reviewed[7\046

6[03[1[3[5 Other epoxides

A number of environmental carcinogens are metabolically converted to DNA!reactive epoxidesin a manner similar to the a~atoxins[047\048 Azinomycin\ which contains a DNA!reactive epoxide\ isdiscussed in Section 6[03[1[4[0[ Neocarzinostatin contains an epoxide that is involved in the trig!gering of enediyne cycloaromatization^ however\ the epoxide of neocarzinostatin is not thought toreact directly with DNA[

6[03[1[4 Aziridines

Aziridine and aziridinium ion species have long been known as the active DNA!alkylatingspecies generated by nitrogen mustards[09\007 Although aziridine!containing natural products are notcommon\ several antibiotics bearing this functional group have been reported[

6[03[1[4[0 Azinomycin B:carzinophilin

Carzinophilin was isolated from Streptomyces in 0843\059 although the structure of this naturalproduct remained elusive for many years[ The structure of carzinophilin ultimately became clearwhen it was realized that the natural product was identical in structure to an antibiotic isolatedfrom a di}erent strain of Streptomyces in 0875\ azinomycin B "51#[050

Compound "51# contains two potentially electrophilic functional groups\ an epoxide and anaziridine[ Thus\ it is not surprising that early experiments showed anomalously facile renaturationof DNA treated with "51#\ thereby suggesting that this agent forms interstrand DNA cross!links[051

Work involving electrophoretic isolation of cross!linked oligonucleotides\ followed by piperidinetreatment and sequencing gel analysis of the resulting cleavage sites on each strand\ conclusivelydemonstrated that reaction of "51# with DNA yields base!labile cleavage sites and that DNA cross!links form at 4?!GNT and 4?!GNC sequences "Figure 4#[050 Cross!linking does not occur at targetsequences in oligonucleotide duplexes when the guanine residues normally involved in the cross!link are replaced with 6!deazaguanine "52#^ however\ cross!link formation still occurs at sites whereguanine is replaced with inosine "7#[050 Thus\ cross!link formation apparently involves covalentattachment of "51# to DNA through the N!6!positions of adenines and guanines in the major grooveof DNA[ Compound "51# alkylates guanines in single!stranded DNA\ but does not react with

MeO

O

O

O

N

O

H O

N

N

H

O

OH

H

H

AcO

HO

(62) Carzinophilin/Azinomycin B


adenines in single!stranded DNA[050 This suggests that GÐA cross!links may result from initialattachment of the natural product to guanine followed by reaction with adenine on the oppositestrand\ or that noncovalent binding of "51# to DNA is required to facilitate reaction at adenines[

Figure 4 Sequences cross!linked by "51#[

6[03[1[4[1 Azicemicins

An intriguing pair of aziridine!containing antimicrobial agents\ the azicemicins "53#\ have beendescribed[052 Although there have been no reports regarding the interaction of these agents withDNA\ the fact that they contain potentially electrophilic aziridine moieties\ along with their struc!tural similarity to the epoxide!containing\ DNA!alkylating agents kapurimycin A2 "38# and thepluramycins "e[g[\ "36##\ suggest that these agents may be capable of DNA alkylation[

6[03[1[4[2 Other aziridines

The mitomycins FR899371 and FR55868 are DNA!cross!linking agents that contain aziridinemoieties[ These compounds are discussed in Sections 6[03[1[5[0 and 6[03[1[5[1[

Research indicates that alkylation of phosphate residues in DNA by certain synthetic aziridinesmay yield substituted aminoethylphosphate triester lesions that lead to hydrolysis of the phos!phodiester backbone of DNA[053

6[03[1[5 Pyrrole!derived Cross!linking Agents

6[03[1[5[0 The mitomycins

The _rst mitomycins "Figure 5# were isolated in the 0849s[054 Mitomycin C "54a# has provenclinically useful in the treatment of certain cancers[055\056 Early experiments implicated DNA as thebiological target of these drugs056 and\ based upon unusually facile renaturation of mitomycin!treated DNA\ it was suggested that this agent covalently cross!links opposing strands of double!

N

NH

N

O

NH2

DNA

(63) 7-Deazaguanine

N

OH

O

HOMeO

OHOH

HO

MeO

O

R

Azicemicin A: R=MeAzicemicin B: R=H

(64)


helical DNA[057 Pioneering experiments of Iyer and Szybalski showed that cross!link formation wasdependent upon the presence of either chemical or enzymatic reducing agents[058 On this basis\ theseresearchers proposed that reduction of the quinone moiety of "54a# leads to elimination of methanol\thereby increasing the electrophilicity of the aziridine functional group in the natural product[058

Moore later re_ned this mechanistic proposal\ suggesting that alkylation of DNA by reduced "54a#involves SN0!type reactions with the extended quinone methide "57# formed by aziridine ring openingand with the a\b!unsaturated iminium ion "69# that results from expulsion of the carbamate groupfrom the monoadduct "58# "Scheme 03#[069 The early proposal of Iyer\ Szybalski\ and Moore hasserved as a starting point for many elegant experiments and\ over the years\ has proven largelycorrect[060Ð064

Figure 5 Mitomycin antibiotics[

Mitomycin C "54a# is remarkably stable prior to reduction of its quinone functional group[ Theelectron!withdrawing nature of the quinone group diminishes the basicity of the aziridine nitrogen\such that it is essentially unprotonated under physiological conditions "pKa of the aziridineR1NH¦�2[1#[056 In addition\ the electron!poor nature of the quinone group stabilizes the hem!iaminal group of "54a# by drawing electron density from the pyrrolo nitrogen and preventing theelimination of methanol[ Reduction of the quinone of "54a# to the hydroquinone "or semiquinone#form "55# increases the electron density on the pyrrolo nitrogen\ thereby facilitating elimination ofmethanol from the drug\ presumably by an E0!type reaction "Scheme 04# to yield the mitoseneform of the drug "56#[ Formation of the resulting C!80C!8a double bond in "56# increases theelectrophilicity of the aziridine ring by bringing this group into conjugation with the hydroquinone

N

OH

H2NCH2OCONH2

OMe

NH N

H2NCH2OCONH2

NH

N

O

OH

H2NCH2OCONH2

NH2

N

H2N

NH2

N

OH

H2NH2C

NH2

OH

N

OH OH

OH

OH

OHOH

H2NNu

NH2

OH

Nu-DNA

Nu

N

O

O

H2NCH2OCONH2

OMe

NH

Nu-DNA

H

+

ONH2

O

DNA

Nu

DNA

Nu

(65a) (66) (67)

(68) (69)

(70) (71)

Scheme 14


ring[ Model reactions\ as well as characterization of DNA adducts\ indicate that the _rst nucleophilicattack occurs at C!0ý of "57#[060Ð063 If the conditions are such that the quinone ring remains in thereduced form\ the _rst nucleophilic attack is followed by expulsion of the C!09ý carbamate groupto yield an electrophilic iminium ion "69# that can react with a second nucleophile[065 If the quinoneof "58# is oxidized subsequent to the _rst nucleophilic attack at C!0ý\ the pyrrolo nitrogen againbecomes vinylogously amidic "conjugated with the quinone carbonyl#\ is unable to facilitate expul!sion of the C!09!carbamate group\ and the DNA monoadduct "58# is obtained[

The quinone moiety of "54a# can be reduced chemically by agents such as sodium borohydrideand sodium dithionite056 or a number of enzymes\055 including DT!diaphorase\ NADPH!cytochromeC reductase\ and xanthine oxidase[066 The requirement for reductive activation of "54a# probablyplays a key role in its potent antitumor properties[ The oxygen sensitivity of the activation steprenders this agent selectively toxic to the oxygen!poor "hypoxic# cells found in solid tumors[055\067

Hypoxic conditions not only favor activation\ but further favor formation of DNA cross!links overmonoadducts[ DNA cross!links are thought to be particularly cytotoxic lesions[064 It appears likelythat either one or two electron reduction is su.cient to activate "54a# for DNA alkylation[056

Reductive activation of "54a# in the presence of DNA leads exclusively to covalent attachmentsat N1 of guanine[068 The exact nature of the adducts formed depends upon the activation conditions[Monofunctional alkylation of guanines occurs when the second alkylation step is inhibited byoxidation of the activated hydroquinone form of the drug back to the quinone form[ The oxidizingagent can be either molecular oxygen or excess\ unreduced "quinone form# "54a#[056 Treatment ofduplex DNA with "54a# under bifunctional alkylation conditions "for example\ anaerobic sodiumdithionite# leads to intrastrand079 and interstrand071 cross!link formation between exocyclic nitrogensof guanines that are proximal in the DNA double helix[ Mono! and bifunctional adducts formedboth in vitro and in vivo have been isolated and completely characterized[ In addition to interstrandand intrastrand DNA cross!linking\ "54a# can mediate proteinÐDNA cross!linking[070

Not surprisingly\ under highly acidic conditions "pH¾3[4#\ "54a# can alkylate DNA\ withoutreductive activation[ Interestingly\ under these conditions alkylation occurs primarily at N!6 ofguanine\ rather than at N1 of guanine[071 The shift in the site of adduct formation is rationalized bythe fact that\ under acidic conditions\ protonation of the aziridine leads to formation of a {{hard||alkylating species that preferentially reacts with N!6 of guanine[071

Noncovalent association of "54a# with DNA prior to covalent reaction is weak and is not highlysequence selective[056\064 The possible sequences that can be cross!linked by activated mitomycin Care dictated to a large extent by the 2[25 _ distance between the electrophilic C!0ý and C!09ý sitesin the activated form of the drug[ Due to the _xed distance between the electrophilic centers ofactivated "54a#\ and the preference for reaction at N1 of guanines\ only two possible sequences forinterstrand cross!link formation can reasonably be considered] 4?!GC and 4?!CG "Figure 6#[ It hasbeen unequivocally demonstrated that the preferred sequence for cross!linking is 4?!CG[072 In normalB!form DNA\ the distance between the guanine nitrogens in the preferred 4?!CG sequence is 2[51_ vs[ 3[0 _ in the less favored 4?!GC site[056 Thus\ the cross!link that induces minimal distortion ofthe double helix is favored^ however\ computations show that the two possible cross!linked structuresare approximately equal in energy\ suggesting that the preference for 4?!CG may be kinetic\ rather

N

OH

H2NCH2OCONH2

OMe

NH

OH

N

O

O

H2NCH2OCONH2

OMe

NHN

O-

O

H2NCH2OCONH2

OMe

NH

H

+

(65a) (66)

N

OH

H2N

CH2OCONH2

NH

OHH

N

OH

H2NCH2OCONH2

NH+

OH

(67)

Scheme 15


than thermodynamic[073 Molecular mechanics calculations on the possible monoadducts suggest thatthe 4?!GC preference could arise from the existence of unfavorable steric interactions encountered inthe _rst steps of the reaction leading to formation of the 4?!GC cross!link[072

Figure 6 Potential cross!linking sites for "54a#[

It appears that "54a# preferentially alkylates guanines in DNA sequences where cross!link for!mation is possible[ For example\ examination of various two!base combinations embedded in aparticular sequence showed that the favored sites of monoadduct formation by "54a# are as follows]4?!CG× 4?!GG× 4?!AG¼ 4?!TG[056\074 Several lines of evidence indicate that hydrogen bondingof the C!09!carbamate group to the exocyclic N1 amino group of the guanine that subsequentlybecomes adducted in the preferred 4?!CG sequence during cross!link formation plays a role in{{steering|| monoadduct formation to sites that can form cross!links "Figure 7#[064 This preference ismaintained in a mitomycin C analogue where an appropriate hydrogen bond acceptor "a hydroxylgroup# is present at this position[075 The 4?!CG preference for monoadduct formation is eliminatedwhen inosine "7# replaces guanine in the target sequence[064 Conversely\ a preferred monoalkylationsite presumably could be created by substitution of 1\5!diaminopurine "61# for the adenine oppositethe thymine in a 4?!TG sequence[ NMR studies on a monoadduct of "54a# with a DNA duplexprovide further evidence for hydrogen bonding between the C!09 substituent and the adjacentguanine N1 in a 4?!CG sequence[076

Only one intrastrand cross!link is possible for "54a#\ with attachment at two adjacent guanines ina 4?!GpG sequence[ Intrastrand cross!link formation induces an ½ 04> bend into duplex DNA[077

Figure 7 Possible noncovalent association of "54a# with DNA prior to adduct formation[

In addition to acting as a redox!sensitive trigger for the alkylation of DNA\ the quinone functionalgroup of "54a#\ in the presence of a reducing agent and molecular oxygen\ undergoes redox!cyclingreactions that lead to oxidative DNA damage "see 6[03[1[00[0#[078\089 Thus\ depending upon theconditions\ "54a# may damage DNA via two distinct mechanisms[ Covalent attachment of "54a# toDNA\ followed by redox cycling\ could result in localized regions of oxidative DNA damage[

N

NN

N

NH2

DNA

NH2

(72)


6[03[1[5[1 FR55868 and FR899371

Hopkins and co!workers showed that\ similar to mitomycin C\ FR55868 "62a# and FR899371"62b#\ when treated with a reducing agent such as sodium dithionite\ cross!link DNA preferentiallyat 4?!CG sequences[080\081 Substitution of deoxyinosine "7# for guanine at cross!linking sites eliminatescross!link formation\ suggesting that the cross!links\ similar to those obtained from "54a#\ involveattachments to DNA at N1 of guanine[080 GuanineÐguanine cross!links have been isolated bynuclease digestion of cross!linked DNA\ followed by reverse!phase HPLC puri_cation[ Completecharacterization of the isolated cross!links supports the structure "64#[081 This structure is consistentwith reduction!dependent formation of a mitosene!type reactive intermediate "63# from "62#\ asoriginally proposed by Fukuyama et al[082 "Scheme 05#\ and is not consistent with other possiblemechanisms that have been suggested083\084 for the alkylation of DNA by "62a# and "62b#[

6[03[1[5[2 Oxidatively activated pyrrolizidine alkaloids

In their early work with "54a#\ Iyer and Szybalski058 noted the structural similarities between thisagent and naturally occurring pyrrolizidine alkaloids\ such as retrorsine "65# and monocrotaline"66#[ These common plant!derived compounds are cytotoxic and carcinogenic[085\086 Pyrrolizidinealkaloids are relatively unreactive^ but in vivo oxidative metabolism by cytochrome P349 enzymesa}ords electrophilic pyrrole analogues "67# "Scheme 06#[085\086 It has been demonstrated that\ uponoxidative activation\ pyrrolizidine alkaloids\ such as "65# and "66#\ cross!link duplex DNA[087\088

Monoadducts of these natural products linked to DNA through N1 of guanine have been isolated[199

To date "0886#\ no cross!links derived from natural pyrrolizidine alkaloids have been isolated^however\ cross!links involving simple synthetic derivatives of "67# have been isolated andcharacterized[

OH

R NO NCH3

OH

CH2OCONH2 OH

R NH

NH

O

OCONH2

(e.g. Na2S2O4)

Reduction

(a) FR900482: R=CHO(b) FR66979: R=CH2OH

(73)

NR

CH2OCONH2

NH

OH

NRNH2

OH

NH-DNADNA

NH-DNA

Cross-linked DNA

(74) (75)

Scheme 16

N

O

ROR'

O O

N

O

ROR'

O O

N

O

RO

N

O

RO

Nu-DNA

N

Nu-DNA

+

[O]

Nu-DNA

N

Nu-DNADNA-Nu

DNA-Nu

+

(78)

Scheme 17

N

O

O

O O

HO OH

(76) Retrorsine

N

O

O

O O

OH OH

(77) Monocrotaline


Hopkin|s group has shown that dehydroretrorsine and dehydromonocrotaline display a 09 ] 0preference for 4?!CG sites over 4?!GC sites[190\191 Although they are not as site selective as mitomycin\work with these pyrrolizidine alkaloids suggests that the common 4?!CG target site may result\ inpart\ simply from the fact that the distance between the electrophilic carbons involved in DNAmodi_cation is essentially the same for all pyrrole!derived bifunctional electrophiles "Figure 8#[

Figure 8 Common core structure for pyrrolizidine cross!linking agents[

6[03[1[6 Alkenylbenzenes

A number of substituted alkenylbenzenes occur naturally in plants\ including some plant speciesthat are used as herbs and spices[192 For example\ safrole "68# is a major component of oil ofsassafras and estragole "79# is found in the oils of tarragon and sweet basil[ Both "68# and "79# arecarcinogenic to rats and mice and have been found to modify DNA covalently[192\193

Investigations into the carcinogenic action of "68# and "79# led to the _nding that the 0?!hydroxy!lated metabolites "70# of these compounds are more potent carcinogens than the parent alk!enylbenzenes[ In vivo\ these allylic alcohols are enzymatically converted to sulfate derivatives "71#that are the ultimate carcinogens derived from "68# and "79#[194 DNA alkylation apparently results

N

O

ROR'

O O

N

O

ROR'

O O

N

O

RO

N

O

RO

Nu-DNA

N

Nu-DNA

+

[O]

Nu-DNA

N

Nu-DNADNA-Nu

DNA-Nu

+

(78)

Scheme 17


from an SN0? reaction in which departure of the sulfate leaving group yields a resonance!stabilizedcarbo!cation "72# that reacts with nucleophilic sites on DNA "Scheme 07#[192 Consistent with anSN0? mechanism\ nucleophilic attack occurs at both the 0? and 2? positions of the alkenylbenzenenucleus[

Acetylated derivatives of 0?!hydroxysafrole and 0?!hydroxyestragole serve as useful\ easily pre!pared models for the carcinogenic forms of these molecules[195 Reaction of these 0?!acetoxy modelcompounds with DNA in vitro yields a number of covalent DNA adducts[ For example\ adducts of"68# attached to C!7\ N1\ and N!6 of guanine and N5 of adenine have been reported[ Many of the sameDNA adducts have been isolated from in vivo experiments utilizing the 0?!hydroxyalkenylbenzenes orthe parent alkenylbenzenes[192

6[03[1[7 N!Nitroso Compounds

6[03[1[7[0 Streptozocin

Streptozocin "73# is an N!methyl!N!nitrosourea derivative of 1!deoxyglucose that possesses cyto!toxic and anticancer properties[196 Similar to the well!known methylating agent N!methyl!N!nitroso!urea "MNU#\ "73# decomposes in aqueous solution with release of dinitrogen\ presumably viamethyldiazoic acid "74# "Scheme 08#[49\197 Treatment of DNA by "73# yields methylation at sitesincluding N!6 and C!5 of guanine and N!0\ N!2\ and N!6 of adenine[197 In in vitro reactions\ theproducts resulting from alkylation of DNA by "73# are identical to those obtained with MNU^however\ in biological experiments "73# is less mutagenic than MNU[49\197

OO

OO

3'

(82)

(79) (81)

enzymaticoxidation

enzymaticsulfation

HO

OO

OHO3S

OO

+1'

2'

DNA DNAAdducts

(83)

Scheme 18

O

HO

HOHO

HNOH

N

NOO

(84) Streptozocin

H2O

N N OHH3C

O

HO

HOHO

HNOH

OOH

+

H3C N N+

DNAalkylation

(85)

Scheme 19


6[03[1[7[1 Nitrosamines

Nitrosamines are ubiquitous carcinogens that are formed during fermentation of various foodsand tobacco and by the in vivo reaction of nitrite preservatives with amines under the acidicconditions found in the gastrointestinal tract[198 For example\ N!methyl!N!nitrosamines are well!known carcinogens and DNA!methylating agents[ DNA alkylation by this class of compoundsinvolves metabolic oxidative activation\ followed by decomposition to yield alkylating species"Scheme 19#[198\109 The natural product dephostatin "75#\ isolated from Streptomyces\ is a tyrosinephosphatase inhibitor that contains the N!methyl!N!nitrosamino functional group[100 Inhibition oftyrosine kinases by "75# has been proposed to result from nitroso group!transfer to a cysteine thiolof these enzymes[101 If "75# can serve as a substrate for appropriate oxygenases\ alkylation of DNAand other biomolecules by this natural product is a possibility[ In addition\ the semiquinone formof "75# may decompose with loss of nitric acid which\ in the presence of molecular oxygen\ cancause DNA damage[

6[03[1[8 Nitroaromatics

6[03[1[8[0 Azomycin

Azomycin "76# is an antimicrobial agent _rst isolated in 0842 and structurally characterized in0844[102 Studies with "76#\ as well as with substituted 1!nitroimidazoles\ strongly suggest that "76#may be capable of covalently modifying DNA by several di}erent pathways[

Four!electron enzymatic reduction of 1!nitroimidazoles converts the nitro group to a hydroxy!amine "77#[103 These hydroxyamine derivatives of imidazoles are electrophilic and undergo hydrolysisto produce the known DNA!damaging agent glyoxal "78# "Scheme 10# "see Section 6[03[1[1[0#[103

Reductive activation of 1!nitroimidazoles\ including "76#\ in the presence of DNA results in theformation of glyoxal adducts with guanine[104\105 It is possible that electrophilic 1!hydroxy!aminoimidazoles may directly alkylate DNA\ although this has not been demonstrated[

1!Nitroimidazoles also can cause DNA damage by enzyme!driven redox cycling of the nitrofunctional group[106\107 In this process\ single!electron enzymatic reduction of the nitro group yieldsa radical anion that can be oxidized by molecular oxygen to generate superoxide radicals andregenerate the starting nitro compound "Scheme 11#[ Although enzyme systems protect cells fromits deleterious e}ects\ superoxide radical can lead to DNA damage through a cascade of reactionsinvolving dismutation to hydrogen peroxide followed by generation of hydroxyl radical via theFenton reaction "see Section 6[03[1[04#[

H3CN NO

H3C

enzymaticoxidation H3C

N NOH2C

OH

H3C N N OH

HCHO

+DNAalkylation

(85)

Scheme 20

OH

OH

N

CH3

NO

(86) Dephostatin

N NH

NO2

(87) Azomycin


6[03[1[8[1 Aristolochic acid

Aristolochic acids "e[g[\ "89## are carcinogens present in the leaves and roots of Aristolochia plantspecies that have been used medicinally for hundreds of years[108 Enzymatic reductive activation ofaristolochic acids in the presence of DNA leads to the formation of covalent adducts\ such as "82#and "83#\ attached through N!5 of adenine and N1 of guanine "Scheme 12#[086 DNA adducts havebeen prepared in vitro using a xanthine oxidase enzymatic reduction system119 and also have beenidenti_ed by in vivo experiments[110

Analysis of the DNA adducts formed by aristolochic acid led to the proposal that adductformation proceeds through reduction of the compound|s nitro group to a hydroxylamine "80#\followed by formation of a cyclic hydroxamide "81#[ It has been suggested that the ultimate alkylatingspecies is a nitrenium ion generated by spontaneous loss of hydroxide from the hydroxamide\086

although direct reaction of DNA with the hydroxamide is also reasonable[ N!Chloroaristolactamhas been used as a model for the presumed hydroxamide species and a}ords the same DNA adductsobtained by reductive activation of the natural product[119 Cyclic hydroxamide formation appearsto be crucial for the generation of a DNA!reactive electrophile in these systems^ 0\7!nitronaphthoicacid\ similar to the aristolochic acids\ is a reductively activated mutagen\ but the 1\2!substitutedanalogue is not[111 Reduction!dependent DNA adduction by aristolochic acid is similar\ in someregards\ to that by the well!known carcinogens 3!nitroquinoline N!oxide and 1!amino~uorene[112\113

6[03[1[09 0\1!Dithiolan!2!one 0!Oxides

6[03[1[09[0 Leinamycin

Leinamycin "84# was isolated from a strain of Streptomyces in 0878114 and its structure elucidatedby NMR spectroscopy\ chemical analysis\ X!ray crystallography\115 and total chemical synthesis[116

Compound "84# contains an unusual 0\1!dithiolan!2!one 0!oxide heterocycle "85# that has not beenfound in any other natural product[ The potent antitumor and antibacterial activity of this sulfur!containing natural product appears to stem from DNA damage[117

HN NR

NH

H2N NHR

NH2

HO OH

HH

O

O

+

+

DNAdamage

(89)

N NR

NO2

N NR

NH

N NR

NHHO

OH

+4e-

-OH

(enzymatic)

+H2O

(88)

Scheme 21

R-NO2

+1e-

R-NO2•-

O2O2•-

Scheme 22


Experiments have revealed that "84# cleaves DNA in vitro and that DNA cleavage requires thepresence of thiols in the reaction mixture[117 Other reducing agents such as NADH and ascorbatedo not trigger DNA cleavage by "84#[ It was also shown that S!deoxyleinamycin "86#\ prepared bycatalytic hydrogenation of the natural product\ displays greatly diminished biological activity anddoes not e.ciently cleave DNA in vitro[117 Thus\ the unusual 0\1!dithiolan!2!one 0!oxide heterocyclewas implicated in the cleavage of DNA by "84# and the data suggested that nucleophilic attack ofthiols on this sulfur heterocycle triggers subsequent DNA chemistry[

When "84# was discovered\ little was known about the reactivity of the 0\1!dithiolan!2!one 0!oxide heterocycle[ Therefore\ chemical model studies involving the reactions of simple 0\1!dithiolan!2!one 0!oxides "87# and "88# with thiols were able to provide insights regarding the chemistryunderlying the thiol!triggered cleavage of DNA by "84#[118 The dithiocarboxylic acid "099# andpolysul_des "090# were identi_ed as major products of the reaction of thiols with 0\1!dithiolan!2!one 0!oxides "Scheme 13#[118 It was suggested118 that these products result from an initial attack ofthe thiol on the central\ sulfenyl sulfur of the heterocycle\ followed by cyclization of the resultingsulfenic acid to yield an electrophilic oxathiolanone "091# and a hydrodisul_de "092#[ The observed_nal products of the reaction derive from the attack of excess thiol on the oxathiolanone intermediateand from decomposition of the unstable hydrodisul_de species[ Importantly\ it was shown118 that2H!0\1!benzodithiol!2!one 0!oxide "87# and the alicyclic dithiolanone oxide "88# react similarly with

O

O

CO2H

NO2

OMe

enzymaticreduction

(90) Aristolochic Acid I

O

O

CO2H

NHOH

OMe

O

O

OMe

N

O

OHi, DNA

ii, Isolation of DNA adducts

(91) (92)

MeO

N

N

NHN

NO

OH

N

NHN

NO

OH

O

NH

MeO

+HO

HN

O

HO

NH

O

OO

O

O

(94)

(93)

Scheme 23

SS

OH O

O

NH

H

H OH

O

O

S

N

SS

OH O

SS

R

RR

R O

O

(95) Leinamycin (97) S-Deoxyleinamycin(96)


thiols\ indicating that\ in these reactions\ "87# serves as a reasonable model for the oxygenated sulfurheterocycle of "84#[ It was noted that the electrophilic oxathiolanone or unstable hydrodisul_deintermediates might be involved in thiol!triggered DNA damage by "84#[118

Subsequent studies revealed that the reaction of "84# with DNA in the presence of thiols resultsin the formation of covalent DNA adducts[129 Thermal workup of DNA treated with "84# in thepresence of a thiol results in release of the adduct[ Characterization of the adduct "095# shows thatthe natural product has undergone a deep!seated rearrangement\ while forming a covalent bondwith N!6 of a guanine residue "Scheme 14#[129 It was proposed that\ analogous to the reaction ofthiols with simple 0\1!dithiolan!2!one 0!oxides\118 the reaction of a thiol with "84# leads to theformation of an electrophilic oxathiolanone[ The electrophilic oxathiolanone intermediate of thenatural product can be trapped in an intramolecular reaction with the C!50C!6 alkene of the 07!membered macrocycle resulting in generation of an episulfonium alkylating species "094#[129 Thisalkene!trapping reaction is precedented in the literature120 and is analogous to the well!knownreaction of alkenes with sulfenyl chlorides[ In chemistry similar to that observed for simple sulfurmustards\121 the thiol!generated episulfonium ion intermediate of "84# alkylates DNA at N!6 ofguanine[ The sequence speci_city of DNA alkylation by "84# has not been reported and it remainsunknown whether the 07!membered macrocycle of the natural product serves primarily as a sca}oldto position the C!50C!6 alkene properly for trapping of the thiol!generated oxathiolanone inter!mediate or whether this macrocycle is also involved in noncovalent association with duplex DNA[

Similar to the natural product\ simple analogues of the 0\1!dithiolan!2!one 0!oxide heterocycle in"84#\ "87#\ "88#\ and "097# are thiol!dependent DNA!cleaving agents[122 Obviously\ these leinamycinmimics lack alkene substituents that could be involved in the formation of episulfonium speciesanalogous to "094#[ Accordingly\ the mechanism of DNA cleavage by these 0\1!dithiolan!2!one 0!oxides is clearly distinct from that of the natural product and does not involve DNA alkylation[DNA cleavage by "87#\ "88#\ and "097# is inhibited by radical scavengers\ the hydrogen peroxide!destroying enzyme catalase\ and chelators of adventitious trace metals[122 These and other resultsindicate that simple 0\1!dithiolan!2!one 0!oxides\ in concert with thiols\ mediate the conversion ofmolecular oxygen to hydrogen peroxide which causes DNA cleavage via a trace metal!dependentFenton reaction[

These simple 0\1!dithiolan!2!one 0!oxides react rapidly and completely with thiols in aqueoussolution and the resulting products mediate thiol!dependent conversion of molecular oxygen toDNA!cleaving oxygen radicals[ Examination of each product in the mixture resulting from thereaction of thiols with "87# revealed that polysul_des are potent thiol!dependent DNA!cleavingagents[123 Mechanistic studies of polysul_des derived from 1!mercaptoethanol and other thiols

SO

O

SHS

SOH

S

O

S

SO

O

SS

SHO

OH

SS

S

HSS

S

SS

S

+

+TEA

(98)(102)

(100)

(103)

(101)

Scheme 24

SS

O

O

(99)


indicated that these compounds are thiol!dependent DNA!cleaving agents that mediate the con!version of molecular oxygen to DNA!cleaving oxygen radicals via a trace metal!dependent Fentonreaction[123 The detailed mechanism by which polysul_des mediate thiol!dependent generation ofoxygen radicals remains under investigation\ but may involve facile reduction of molecular oxygento superoxide by thiol!generated hydropolysul_de anions "e[g[\ 098# "Scheme 15#[123 It is likely thata number of processes not explicitly shown in Scheme 15\ including dimerization of species such as"098# and reactions involving hydrogen sul_de and thiol\ can lead to the production of superoxideradicals without net destruction of polysul_des[

S

N

O

O

S

HOH

H

NH

H

CO2H

OH

N

N NH

N

O

NH2

S+

S

N

O

O

OHH

H

OH

CO2H

H

NH

HH

HO

O

O

N

S

NH

OOH

OS

C6C7

no DNA

S

N

O

O

S

HOH

H

NH

H

CO2H

OH

R'

i, DNAii, thermal workup (depurination)

(a): R'=SSR(b): R'=OH

(104) (105) (107)

(106)

SS

OH O

O

NH

H

H OH

O

O

N

S

RSH

(95)

Scheme 25

S

S

O

O

HO

(108)


Consistent with Scheme 15\ at low concentrations of trisul_de and thiol "0Ð099 mM#\ where theratio of dissolved molecular oxygen "½199 mM# to hydrodisul_de "098# is relatively high\ thepolysul_de appears to serve as a catalyst for thiol oxidation "and presumably corresponding super!oxide production#\ with one equivalent of trisul_de leading to the production of approximately nineequivalents of disul_de[ The hydrogen sul_de by!product derived from the oxygen!independentreaction of thiols with polysul_des was identi_ed by bubbling the reaction headspace through a leadacetate solution\ followed by characterization of the resulting lead sul_de precipitate[ Superoxideradicals generated by polysul_de:thiol mixtures lead to DNA damage through a cascade of reactionsinvolving dismutation to hydrogen peroxide followed by generation of hydroxyl radicals via theFenton reaction "see Section 6[03[1[04#[

The reaction of thiols with "84#\ similar to the reaction of thiols with "87#\ results in the formationof polysul_des[ Consistent with the generation of polysul_des in the reaction of thiols with "84#\ ithas been demonstrated that the natural product "84#\ in the presence of physiologically relevantconcentrations of thiol\ mediates oxidative damage in addition to the DNA alkylation describedabove[123 The notion that polysul_des formed by reaction of thiols with "84# are derived from ahydrosul_de species "e[g[\ 098# is provided by the fact that\ when treated with thiol in the absenceof DNA\ the disul_de adduct "096a#\ resulting from attack of a hydrodisul_de species on theepisulfonium ion "094#\ is obtained in 10) yield\ along with the expected hydrolysis product "096b#"30) yield#[129 As it is known that hydrodisul_des decompose to polysul_des in aqueous solution\the thiol!dependent release of hydrodisul_de from "84# explains the formation of DNA!cleavingpolysul_des that results from treatment of the natural product with thiol[ Thus\ "84# is capable ofdamaging DNA by two distinct mechanisms involving either alkylation or the production ofpolysul_des that generate oxygen radicals in the presence of excess thiol[ The relative importanceof the two possible pathways will depend upon the conditions under which the thiol!triggeringreaction occurs[

0\1!Dithiolan!2!one 0!oxides\ such as "87#\ react with anilines and amine compounds under mildconditions\ suggesting covalent adducts could result from direct reaction of these sulfur heterocycleswith nucleophilic nitrogens in DNA[124

Not surprisingly\ leinamycin and simple 0\1!dithiolan!2!one 0!oxides are capable of inactivatingcysteine!dependent enzymes[125 The inactivation of cysteine!dependent enzymes clearly can haveimportant biological consequences[

6[03[1[09[1 0\1!Dithiole!2!thiones

It has been suggested that 0\1!dithiole!2!thiones occur naturally in plants such as broccoli[126 0\1!Dithiole!2!thiones\ similar to 0\1!dithiolan!2!one 0!oxides\ mediate thiol!dependent conversion ofmolecular oxygen to oxygen radicals and are capable of cleaving DNA in the presence of thiols[127

Research suggests127 that production of oxygen radicals might play a role in the induction of cancer!preventive phase II metabolic enzymes by these compounds\128 perhaps through reaction of theoxidizing radicals with protein transcription factors[ The chemical mechanism of oxygen!radicalgeneration by this class of compounds remains under investigation\ but appears similar\ in manyregards\ to that of the structurally related dithiolanone oxides "e[g[\ "87# and "88## discussedabove[

RSH RSSSR+ [RSSH] RSSR+

O2, RSH

O2•-

RSHRSSR + H2S

(109)(101)

DNAcleavage H2O2

Scheme 26


6[03[1[09[2 Polysul_des

The _nding that polysul_des are thiol!dependent DNA!damaging agents may be relevant to thebiological activity of several polysul_de!containing natural product antibiotics\ including varacin"009a#\139 lissoclinotoxin A "009b#\130 the leptosins "000#\131 and the sirodesmins "001#[132

Studies show that the varacin analogue\ 6!methylbenzopentathiapin\ is a potent thiol!dependentDNA!cleaving agent[131 The reaction of thiols with this pentathiapin results in the formation of acomplex mixture of polysul_des[ Mechanistic experiments show that DNA cleavage in this system\identical to the polysul_des described above\ involves the reduction of molecular oxygen to hydrogenperoxide "via superoxide#\ followed by a trace metal!dependent Fenton reaction[133 Interestingly\biological experiments have suggested that DNA damage might play a role in the antitumorproperties of the pentathiapin antibiotic varacin[139

In addition to their ability to produce oxygen radicals\ polysul_des may derive biological activitythrough reactions with thiol groups on proteins125 or through chemical reactions that lead todepletion of cellular thiols "Scheme 15#[

6[03[1[00 Quinones

A large number of quinone!containing natural products have been isolated and characterized[134\135

Anthraquinone!based natural products\ such as daunomycin "daunorubicin# "002a# and adriamycin"doxorubicin# "002b#\ have proven useful in the treatment of cancer and\ therefore\ have been thesubject of much investigation[136\137 In many natural products\ including the anthraquinones\ thequinone functional group occurs as part of a planar aromatic system that is involved in intercalativebinding to DNA[138 Noncovalent intercalation of these compounds into DNA often has importantbiological consequences[136\137 In addition\ there are a number of mechanisms by which quinone!containing compounds can covalently modify DNA[

S S

S

SS

NH2

NH

N N

O

O

H

OH

Sx

HN

OR

MeO

NN Sx

O

O

OH

OH

OHOAc

O

(a) Varacin: R=CH 3(b) Lissoclinotoxin A: R=H

(a) Leptosin E: X=3(b) Leptosin F: X=4

Sirodesmin C: X=3Sirodesmin B: X=4

(110) (111) (112)


6[03[1[00[0 Redox cycling of quinones and quinoid compounds

In the presence of molecular oxygen and appropriate reducing agents\ quinones can mediate thetransfer of electrons from the reducing agent to molecular oxygen\ resulting in the production ofsuperoxide radical anion[149Ð142 In this process\ reduction of the quinone yields a hydroquinone orsemiquinone radical "003#\ either of which can be oxidized by molecular oxygen to produce super!oxide and the quinone starting material "Scheme 16#[ Importantly\ the quinone is not destroyed inthis reaction cycle[ Superoxide radical leads to DNA damage through a cascade of reactionsinvolving dismutation to hydrogen peroxide followed by generation of hydroxyl radical via theFenton reaction "see Section 6[03[1[04#[

Quinones facilitate the reduction of molecular oxygen by a variety of reducing agents[ Forexample\ the rate of molecular oxygen reduction by the enzyme NADPH ] ferredoxin reductase isincreased markedly in the presence of quinones[143 Similarly\ quinones catalyze the reduction ofmolecular oxygen by thiols[144

Quinones can be reduced either chemically or enzymatically[142 For example\ thiols145 and NADHcan serve as chemical reducing agents in redox cycles involving quinones[ A variety of enzymes\including NADPH ] cytochrome P!349 reductase\ xanthine oxidase\ NADPH ] ferredoxin reductase\and NADPH ] quinone acceptor oxidoreductase\ have been found to reduce quinones[149 It remainsa matter of debate as to whether intercalated quinones can undergo e.cient redox cycling[145Ð148

For the anthraquinones\ nonintercalative DNA!binding modes may exist\ in which the quinonemoiety is capable of engaging in redox chemistry[148

Quinone antibiotics\ such as streptonigrin "004# and adriamycin "002b#\ chelate transition metalions that may serve to increase the e.ciency of oxygen!radical generation[148\159 These boundtransition metals may serve as intramolecular catalysts for various steps leading to the productionof hydroxyl radical\ thereby leading to e.cient\ highly localized generation of oxidizing species[

Q+1e-

Q•-

O2O2•-

Q = quinone

Scheme 27

O

O

O

O

O

HO

HOO

HO

MeO

O

H2N

O

(117) Elsamicin

NN

O

O

HO2C

H2N

H2N

CO2H

OH

OMe

OMe

O

N

C

O

NH2

CO O NH

Thr-L

HN

Thr-LO

L-MeVal

SarPro-L

Val-DD-Val

L-Pro

Sar

ValMe-L

O

(115) Streptonigrin (116) Actinomycin D


Redox!dependent DNA damage has been suggested or demonstrated for a variety of quinoneand quinoid natural products\ including daunomycin "002a#\150 adriamycin "002b#\147 streptonigrin"004#\151 saframycins A "8a# and C "8c#\57 mitomycin C "54a#\078\089 actinomycin D "005#\152\153

elsamycin "006#\154 and many others[55\74\155Ð158 Although for many quinone!containing natural products redox chemistry has not been explicitly examined\ in many cases toxic or mutagenic propertiesmay result from the production of oxygen radicals[

6[03[1[00[1 DNA alkylation by quinone methides

Under anaerobic conditions\ reduced anthracycline antibiotics eliminate the glycosyl or methoxylsubstituents at C!6 to yield reactive quinone methides "007# "Scheme 17#[140 It remains unclearwhether elimination occurs from the semiquinone or hydroquinone state[ Quinone methides areelectrophilic and have been shown to react with nucleophilic sites in DNA bases[169 Irreversibletautomerization to the 6!deoxyaglycone "008# often competes with the reaction of the quinonemethide and nucleophiles[160 The biological relevance of DNA alkylation by quinone methide speciesremains unclear^ however\ there is evidence that anthracyclines do\ in fact\ form covalent adductswith DNA in vivo[161\162 Koch has noted that the importance of such an attachment could beprofound\ as it tethers the quinone moiety\ a catalyst for the generation of oxygen radicals\ in closeproximity to DNA[169

In chemical model reactions\ quinone methides have been trapped by nucleophiles includingglutathione\ imidazole\ sul_te\ and the exocyclic N1!amino of guanine[169\160 Quinone methides ofmenogaril "019# and daunomycin have been observed by UVÐvis spectroscopy163 and the quinone

OOMe O

O OH

OH

OH

O

O

HONH2

OOMe OH

OH OH

OH

OH

O

O

HONH2

OH

O

HONH2

(113a)

2e-, 2H+

7

Scheme 28

OMe O

OH OH

OH

OH

O

OMe OH

OH OH

OH

OH

O

OMe O

O OH

OH

OH

O

Nu

Nu-H

(118)

(119)


methide of 00!deoxydaunomycin has been observed by NMR[164 Interestingly\ quinone methidesare also nucleophilic in character and can react with electrophiles such as protons and aldehydes[143

Simple\ synthetic compounds that yield DNA!damaging quinone methides upon reductive acti!vation have been designed and characterized[165Ð167

6[03[1[00[2 Formaldehyde!mediated covalent attachment of anthracyclines to DNA

The presence of formaldehyde as an impurity in a crystallization solvent led to the serendipitousdiscovery that this aldehyde e.ciently mediates covalent attachment of a daunomycin analogue toDNA[168 Subsequent experiments\ including a 0[4 _ resolution crystal structure\ demonstrated thatthe natural product daunomycin "002a# forms similar formaldehyde!mediated covalent links "010#with DNA "Scheme 18#[179 DNAÐdrug cross!link formation is apparently facilitated by the factthat\ in the noncovalent daunomycinÐDNA intercalation complex\ the N!2?!amino group of thedaunosamine glycoside and the exocyclic N1 of guanine in the minor groove are appropriatelypositioned for bridging by formaldehyde^ thus\ the resulting methylene bridge between the twonucleophilic sites does not lead to signi_cant distortion of the daunomycinÐDNA complex[179

Formation of the covalent attachment is absolutely dependent upon the N!2? on the anthracyclineantibiotic and N1 of guanine on DNA\ as shown by the fact that antibiotic analogues or modi_edDNA substrates lacking these functional groups do not become covalently associated in the presenceof formaldehyde[170

Under conditions that allow redox cycling of the quinone moiety\ oxidative decomposition ofadriamycin "002b# leads to generation of formaldehyde that can mediate subsequent covalentattachment of an intact molecule of "002b# to DNA "Scheme 29#[171 Oxidative degradation of "002b#yields the by!product "011#\ and it is proposed that the C!8 substituent is converted to formaldehyde[The formaldehyde by!product of this oxidation was detected in the reaction mixture[ Electrospraymass spectroscopic analysis of the adriamycinÐDNA adducts obtained by incubating adriamycin

OH O

O H

OH

OO

OH

HO

(Me)2N

OH

OMe

(120) Menogaril

OOMe O

O OH

OH

OH

O

O

HO NH2

OOMe O

O OH

OH

OH

O

O

HO NH

(113a)

3'

7 Double-stranded DNA

HCHO 3'

NN

N

NODNA

H

HN

7

(121)

Scheme 29


with dithiothreitol in phosphate bu}er is consistent with the adduct structure "010# "see Scheme 18#[Daunomycin "002a# does not undergo a similar oxidative self!degradation to produceformaldehyde[171

Interestingly\ incubation of either daunomycin or adriamycin with DNA and a reducing agent\such as dithiothreitol\ in tris"hydroxymethyl#aminomethane "Tris# bu}er leads to e.cient formationof the covalent adduct "010#[ Thus\ it appears that oxygen radical!mediated degradation of Trisbu}er produces formaldehyde[171 In addition\ it was observed that the anthracyclineÐthiol systemcan degrade the biological polyamine\ spermine\ to formaldehyde[171 This _nding\ coupled with theobservation that formaldehyde!mediated anthracyclineÐDNA cross!linking occurs e.ciently at lowformaldehyde concentrations\ suggests that adduct "010# could form under biological conditions[

The formaldehyde!mediated covalent attachment of anthracyclines to DNA is mechanisticallysimilar to the proposed reaction of barminomycin I with DNA "see Section 6[03[1[0[3#[

6[03[1[00[3 DNA alkylation by quinones

Quinones are electrophilic and\ in the case of simple quinones such as benzoquinone\ covalentadducts with DNA\ such as "012# and "013#\ have been isolated and characterized[172 Similar to a\b!unsaturated aldehydes "see Section 6[03[1[1[0#\ simple quinones yield cyclic adducts that are theapparent result of Michael addition and subsequent intramolecular attack of the DNA base on thequinone carbonyl\ followed by dehydration[

OOMe O

O OH

OH

OH

O

O

HO NH2

OH

OOMe O

O OH

OH

OH

O

O

HO NH2

OH

(113b)

3'

7

DTT(redoxcycling)

+ H2O2

(113b)

OOMe O

O OH

OH

OH

O

OH

O

HO NH2

HCHO+adriamycin

DNA

adriamycin–DNAcross-links

(122)

(121)

Scheme 30

ON

HO

HO

N

O

N

HO

(123)

ON

HO

HO

N

N

N

N

OH

(124)


Naturally occurring estrogens\ such as "014#\ undergo oxidative metabolism to catechol estrogens"015#[173 Oxidation of these catechols yields electrophilic o!quinone estrogens "016#[ These o!quinoneestrogens form a variety of DNA adducts\ such as "017#\ in vitro "Scheme 20#[173 In addition\ covalentadducts of the quinone estrogen "016# attached at C!7 of adenine have been obtained by in vitroreaction of the o!semiquinone radical with adenine[174

Electrophilic iminoquinones derived from oxidative metabolism of N1!methyl!8!hydroxy!ellipticinium also form covalent adducts with DNA[175\176

6[03[1[00[4 Other DNA damage by quinones

Photochemical DNA damage by anthraquinones has been reported and is discussed in Section6[03[1[07[

6[03[1[01 Heterocyclic N!Oxides

Several naturally occurring heterocyclic N!oxides have been isolated and found to possess cyto!toxic or mutagenic properties[177 Although no detailed studies of covalent DNA modi_cation bynaturally occurring N!oxides have been reported\ investigations of structurally related synthetic N!oxides provide insight into possible mechanisms of DNA damage by the natural products[

6[03[1[01[0 Phenazine N!oxides

Two naturally occurring phenazine N!oxides\ iodonin "018#178 and myxin "029#\189 have beenreported[ In vitro experiments demonstrated that the broad spectrum antimicrobial agent178 "018#inhibits RNA synthesis from a DNA template\ probably due to intercalative binding with duplexDNA[180 In addition\ when E[ coli cells were exposed to "018#\ single!strand DNA breaks wereobserved[181 The DNA!cleavage chemistry of "018# may be analogous to that of a synthetic phen!azinedi!N!oxide reported by Hecht and co!workers[182\183

O

HO

O

HO

O

O

O

HO

N

NN

HN

O

H2N

P-450peroxidasesor P-450

(in vitro)

dG

HOAc/H2O (1:1)

OHO

OH

(125) (126)

(127) (128)

Scheme 31


The phenazine N!oxide "020# studied by Hecht|s group mediates oxidative cleavage of DNA inconjunction with reducing agents such as dithiothreitol and NADPH "Scheme 21#[182 Under aerobicconditions\ DNA cleavage appears to involve a redox cycle in which the one electron!reducedphenazinedi!N!oxide "021# is reoxidized by molecular oxygen to product superoxide radical[182

Superoxide radical decomposes to yield the DNA!cleaving agent hydroxyl radical "see Section6[03[1[04#[ Compound "020# also cleaves DNA e}ectively under anaerobic conditions\ in the presenceof reducing agents[ Under anaerobic conditions\ "020# undergoes deoxygenation and it is proposedthat the one!electron reduced intermediate "021# fragments with the release of hydroxyl radical[182

Deoxygenated products "022# and "023# were detected only under anaerobic conditions[

Investigation of phenazinedi!N!oxideÐoligonucleotide conjugates revealed an interesting DNA!damage pathway distinct from the redox!activated chemistry described above[ Hybridization ofphenazinedi!N!oxide!modi_ed oligonucleotides to complementary deoxyoligonucleotides resultedin the production of heat and alkaline!labile lesions at guanine residues on the complementarystrand[183 All evidence is consistent with DNA cleavage resulting from a mechanism involvingalkylation at N!6 of guanine\ although alkylation at N!2 of guanine is also a possibility "Figure 09#[

Figure 09 Potential sites for nucleophilic attack on phenazinedi!N!oxides[

6[03[1[01[1 Quinoxaline N!oxides

1!Carboxyquinoxalinedi!N!oxide "024# is a fungal metabolite with mutagenic and antibacterialproperties[184 Results obtained with structurally related\ synthetic quinoxaline and triazine N!oxidessuggest that this N!oxide!containing natural product may be capable of damaging DNA[

N+

N+

O -

- O

OH

HO

(129) Iodinin (130) Myxin

N+

N+

O -

- O

OMe

HO

N+

N+

H2N(CH2)3NH

O -

O -

N+

N+

H2N(CH2)3NH

O -

O -

N

N

H2N(CH2)3NH N+

N

H2N(CH2)3NH

O -

anaerobic

anaerobic

+1e-

(RSH)

+1e-

(RSH)

O2O2•-

•

+ HO•+ HO•

(131) (132)

(133)

(134)

Scheme 32


In the 0869s\ biological experiments with the antibacterial and mutagenic agent quinoxalinedi!N!oxide suggested that this compound may cause degradation of DNA in vivo and further showedthat this N!oxide is selectively cytotoxic to oxygen!poor "hypoxic# cells[185\186 Substituted quin!oxaline187 and triazinedi!N!oxides\188 such as "025# and "026# have been extensively investigated ashypoxia!selective\ redox!activated cytotoxins for the possible treatment of solid tumors[ In vitrocleavage of DNA by quinoxaline!N!oxides in a well!de_ned system has not been reported "0886#^however\ the chemistry of DNA!cleavage by triazinedi!N!oxides has been investigated in somedetail[188\299

The compound 2!amino!0\1\3!benzotriazine 0\3!dioxide "027# shows selective toxicity toward thehypoxic cells in tumors and has reached phase II and III clinical trials as an antitumor agent[188

Early experiments suggested that this compound derives its biological activity from the cleavage ofcellular DNA and that strand scission may result from a species "028# generated by enzymatic one!electron reduction of the heterocycle[188 This theory was supported by several observations[ In theabsence of reducing systems\ "027# alone does not damage DNA[ In mammalian cells underanaerobic conditions\ "027# is ultimately reduced to "039#\ which is not highly cytotoxic\ and therates of reduction parallel cytotoxicity in several di}erent cell lines[188 A radical species resultingfrom the incubation of "027# with rat liver microsomes was observed by ESR^188 however\ no relationbetween this radical species and DNA cleavage was established[ The speci_c toxicity of "027# towardhypoxic cells may result from the fact that the {{activated|| radical form of the drug "028# is destroyedby reaction with molecular oxygen[188 This back!oxidation reaction would produce superoxide\whose in vivo cytotoxicity is mitigated by cellular enzymes such as superoxide dismutase andglutathione peroxidase[

Chemical experiments utilizing a xanthineÐxanthine oxidase enzyme system as a one!electronreductant show that "027# e.ciently cleaves DNA under hypoxic conditions[299\290 The two!electron!reduced metabolite "039# does not cleave DNA either alone or in the presence of the xanthineoxidase reduction system[299 Redox!activated DNA cleavage by "027# is e}ectively inhibited byoxygen!radical scavengers\ and strand scission occurs with little or no sequence speci_city[299 Inaddition\ "027#\ in concert with the xanthineÐxanthine oxidase enzyme system\ converts dimethylsulfoxide to methanesul_nic acid\ a reaction considered characteristic of hydroxyl radical[299 Takentogether these experiments suggest that one!electron reduction of "027# leads to a fragmentationreaction that produces hydroxyl radical and the deoxygenated metabolite "039#[ Thus\ hydroxylradical "rather than the radical form of the heterocycle "028## may be the major DNA!cleavingspecies generated by one!electron reduction of triazinedi!N!oxides\ under anaerobic conditions "seeScheme 22#[ This mechanism is analogous to that suggested by Hecht and co!workers to explain

N+

N+ CO2H

N+

N+ CN

NH2

R

R

O -O -

O - O -

N+

N+N

NH2

R

R

O -

O -

(135) (136) (137)

+ HO•

DNAdamage

.1e-, H+

.

N

N+N

+N

N+N

NH2

+N

NH2

N

N

OH

NH2

O _

O _

O _ O _

+N

O _

N

OH

N

NH2

O2O2•-

(138) (139) (140)

Scheme 33


redox!activated DNA cleavage by phenazine N!oxides\ such as "020#[182 It has been noted299 thatthe formation of the high!energy hydroxyl radical from these heterocyclic N!oxides may be thermo!dynamically driven by re!aromatization of the heterocyclic ring systems and by the entropicallyfavorable nature of the fragmentation reaction[

Triazene and quinoxaline di!N!oxides also produce photoactivated DNA damage[291

6[03[1[02 Resorcinols

A variety of 0\2!dihydroxybenzene compounds "resorcinols# have been isolated from plantsources[292 These natural products display a wide array of biological activities[ Hecht|s groupreported the isolation and characterization of a group of 4!alk"en#yl resorcinols\ "030#Ð"033#\ thate.ciently mediate DNA cleavage under aerobic conditions\ in the presence of copper"II#[292Ð295

These researchers noted that the DNA!cleaving ability of the freshly puri_ed resorcinols increasedupon incubation with copper"II# in basic\ aerobic solution[293 It was realized that the conditionswhich enhance the DNA!cleaving ability of these resorcinols are similar to reaction conditionsknown to cause hydroxylation of phenolic compounds[ Thus\ it was proposed that\ in basic solutioncontaining copper"II# and molecular oxygen\ the resorcinol ring system undergoes hydroxylationto yield 0\1\3!trihydroxybenzenes that are the active DNA!cleaving agents[293 The correspondingtrihydroxybenzene compounds were prepared and\ consistent with the proposed mechanism\ werefound to be 49Ð099 times better DNA!cleaving agents than the analogous resorcinols[295

The proposed in situ conversion of resorcinols to trihydroxybenzenes requires basic conditions\copper ion\ and molecular oxygen[ Studies with the trihydroxybenzene compounds demonstratedthat the subsequent DNA!cleavage process depends on copper ion and molecular oxygen\ but notbasic conditions[295 Experiments designed to elucidate the mechanism of DNA cleavage by thetrihydroxybenzenes showed that neocuproine\ a speci_c ligand for copper"I#\ inhibits cleavage andthat oxidation of these trihydroxybenzenes to their quinone form by 1\2!dichloro!4\5!dicyano!0\3!benzoquinone prior to the addition of DNA also prevents strand scission[ In addition\ the oxygenradical scavenger DMSO and the hydrogen peroxide!destroying enzyme catalase inhibit DNAcleavage by this system[295


Together\ these experiments suggest that DNA cleavage by 4!alkyl resorcinols "030#Ð"033#\ in thepresence of copper"II#\ base\ and molecular oxygen\ proceeds by initial conversion to the cor!responding trihydroxybenzene "034#\ followed by oxidation of the trihydroxybenzene by molecularoxygen to yield the corresponding quinone "035# and superoxide radical "Scheme 23#[295 Superoxideradical leads to DNA damage through a cascade of reactions involving dismutation to hydrogenperoxide followed by generation of hydroxyl radical via the metal!dependent Fenton reaction "seeSection 6[03[1[04#[ End!product analysis of resorcinol!cleaved DNA restriction fragments and thedetection of DNA free bases as products of these cleavage reactions shows that DNA cleavage byresorcinols is\ in this regard\ mechanistically identical to that by ironÐMPE methidiumpropyl!EDTA\ consistent with the notion that hydroxyl radical is the ultimate cleaving agent generated bythe resorcinolÐcopperÐoxygen system[295 It is possible that similar chemistry is involved in copper!dependent DNA damage that has been reported for the natural products tannic acid296 andurushiol[297

In the cleavage of DNA by 4!alkylresorcinols\ copper ion may play multiple roles\ converting theresorcinol system to a trihydroxybenzene\ possibly catalyzing the oxidation of the trihydroxybenzeneto quinone and also possibly catalyzing the decomposition of hydrogen peroxide to hydroxyl radical[This is similar in some regards to the role that has been proposed for transition metal ions chelatedby quinone antibiotics such as streptonigrin "004# and adriamycin "002b# "see Section 6[03[1[00[0#[

An interesting aspect of the work with alkylresorcinols\ such as "030#Ð"033#\ is the observationthat the e.ciency of DNA cleavage in this group of compounds is proportional to the length of thealk"en#yl substituent\ with analogues bearing longer alk"en#yl side chains producing more e.cientDNA cleavage[295 These resorcinol natural products provide a unique demonstration that simplealk"en#yl substituents can provide signi_cant DNA a.nity\ presumably through hydrophobic associ!ation with the grooves of duplex DNA[

6[03[1[03 Bleomycin and Other Metal!binding Antibiotics

The bleomycins "036# "BLM# are clinically useful antitumor agents that are known to damageDNA[298 Bleomycin A1 "036a# is a major component of the clinically used BLM mixtures and isprobably the most thoroughly studied of the BLMs[298Ð200 A subset of the functional groups in theBLMs converge to create a strong metal binding site "Figure 00# that\ in the presence of molecularoxygen\ reductants "such as thiols or ascorbic acid#\ and iron catalyzes single and\ to a lesserextent\ double!stranded cleavage of DNA[298Ð200 Although double!stranded cleavage is a relativelyinfrequent event for the BLMs\ these lesions are not easily repaired in vivo and are thus probablymore cytotoxic than single!strand cleavage[

OH

RHO

OH

O

RO

OH

CuH2O O

O

RHOO2 O2•-

H2O2

OH2

(145) (146)

Scheme 34

HO R

OH

basicconditions

O2 ,Cu2+

(141–144)


Figure 00 Metal binding site of bleomycin[

The exact nature of the reactive species involved in the cleavage of DNA by ironÐBLM remainsa subject of ongoing investigation^ however\ research over the last several decades has led to a fairlydetailed understanding of the events leading to strand scission "Scheme 24#[209\201 The iron"II#ÐBLMchelate readily forms a complex "037# with molecular oxygen[ One!electron reduction of this complexyields an iron"III#ÐBLM hydroperoxide "038# "activated ironÐBLM# that either directly cleavesDNA or undergoes heterolytic cleavage of the O0O bond to yield a reactive iron oxene species"049# that cleaves DNA[ In many regards\ the metal binding domain of ironÐBLM is analogous tothe iron!heme found at the active site of cytochrome P!349[ Similar to cytochrome P!349\ activatedironÐBLM can mediate the oxidation and oxygenation of many organic substrates[209 Under variousconditions\ complexes of BLM with iron\ cobalt\ copper\ manganese\ vanadium\ and nickel cancause DNA cleavage[209

The exact structure of the iron"III# intermediates in the DNA!cleavage reaction of BLM

N N

NH2O

HN NH2

O

H NH2

H2NO

HN

O

NH

N

NH

HOHN

ONH

O

N

S

S

NX

O

OHO

OH

OH

O

OOH

OH

OHO

O NH2

H

O

H

H

HO

H

H

H

H

H

NH

HN NH2

NH

NH

HN

HN

NHHN NH2

NH

NH

HN

HN

NHHN NH

NH HN NH2

NH

NH S+(Me)2X-

NHHNNH2

(147a) Bleomycin A2: X=

Bleomycin B2: X=

Bleomycin A5: X=

Bleomycin B4: X=

Bleomycin B6: X=


remains unclear due to experimental di.culties in analyzing this system^ however\ zinc"II#\ iron"II#\and cobalt"III# complexes of BLM have been characterized[209\201 The arrangement of ligands incobalt"III#ÐBLM has been deduced from 1D!NMR data "see Figure 00#[

Extensive studies involving product analysis and examination of isotope e}ects have revealedthat DNA cleavage by ironÐBLM involves exclusive abstraction of the hydrogen from C!3? ofdeoxyribose[209\201\202 The resulting C!3? deoxyribose radical "040# partitions between two distinctstrand scission pathways "Scheme 25#[ The relative predominance of each pathway depends uponthe reaction conditions "e[g[\ oxygen concentrations# and the surrounding environment "sequencecontext# of the radical[

BLM binds to DNA with an association constant of ½ 0×094 M−0[203\204 Investigations involvingcompetition binding experiments with agents such as distamycin\ suggested that BLM binds in theminor groove of DNA[205 In addition\ BLM binds e.ciently to T3 DNA\ which is glycosylated inthe major groove[206 Exclusive abstraction of the C!3? hydrogen of DNA\ which protrudes into theminor groove of duplex DNA\ is also consistent with minor groove binding by BLM[

BLM cleaves DNA primarily at 4?!dGPy sites\ with cleavage occurring between the guanine andadjacent pyrimidine base[209\201 Experiments with synthetic bithiazoles show that the bithiazole {{tail||of BLM does not bind to DNA with any sequence preference^ however\ this fragment of BLM doesprovide signi_cant DNA a.nity[207 Certain features of the binding of BLM to DNA are charac!teristic of intercalation\ including unwinding of supercoiled DNA and elongation of the doublehelix[204\208 Thus\ for many years it has been suspected that the bithiazole moiety of BLM mightassociate with DNA via intercalative!type binding[

A number of experiments suggested that the metal!binding domain of BLM contains the deter!minants for sequence!speci_c DNA cleavage[ For example\ deglyco!BLM cleaves DNA with speci!_city essentially identical to the natural product\ indicating that the sugar residues are not involved insequence!speci_c interactions[209 Furthermore\ investigations of synthetic deglyco!BLM derivativesrevealed that changes in the linker between the bithiazole moiety and the metal binding domainhave no e}ect on the sequence speci_city of the cleavage reaction[209 This data implicates the metal!binding domain in sequence!speci_c DNA binding\ because if the bithiazole {{tail|| controlledspeci_city\ it would be expected that\ as linker length between the tail and the cleaving domainincreased\ sites of DNA cleavage would be displaced relative to the binding site\ a phenomenonthat is not observed[

Fe2+BLM O2

O2-Fe3+BLM +1e-, +H+

HOO-Fe3+BLM [O-Fe3+BLM ]?

DNAcleaved DNA

Fe3+BLM

+1e-

(activatedbleomycin)

(148) (149) (150)

Scheme 35

OBRO

OR'

OBRO

OR'

B

O•O

HO- Strandcleavage

BLM-Fe3+O

OBRO

OR'

•

+

R'O-

HO

H2O, BLM-Fe 4+OH

BLM-Fe3++

BLM-Fe4+OH

HRO

'RO

O O

OBRO

OR'OHO

ROO-

O

B

CHO

+

O2

Strand cleavage

(151)

Scheme 36


Evidence that sequence speci_city involves interaction of BLM with the N1!amino group of theguanine adjacent to the DNA cleavage site was provided by the observation that replacement ofguanine with inosine "7# drastically diminishes DNA cleavage at these sites[219 Conversely\ sub!stitution of diaminopurine "61# for adenine converts weak 4?!APy cleavage sites into strong BLMcleavage sites[210

A published 1D!NMR structure of cobalt"III#!BLM complexed to a 4?!dCCAGGGCCTGGduplex provides a detailed view of a BLMÐDNA complex that is consistent with much of the existingexperimental data[201\211 This structure shows the bithiazole moiety intercalated on the 2?!side of the4?!GC target site[ This NMR structure further suggests that the observed 4?!dGPy sequence pref!erence for BLM cleavage derives from hydrogen!bonding interactions between the pyrimidinefunctional group of BLM|s metal!binding domain and the guanine residue at the 4?!GC cleavagesite[ Thus\ a unique\ minor groove base triplet "041# is formed by hydrogen bonding of N!2 and theamino group of BLM|s pyrimidine heterocycle with the exocyclic N1!amino group and N!2 ofguanine in the GÐC base pair at the target site[ The NMR structure places the hydroperoxide protonof cobalt"III#!BLM 1[4 _ from the C!3? hydrogen that is abstracted in the cleavage reaction of ironÐBLM[

The NMR structure reported by Wu et al[219 nicely accounts for double!stranded cleavage\supporting a previous proposal200 that cleavage of both strands at certain target sites involvesdissociation of the metal!binding domain of BLM from the DNA\ while the bithiazole remainsbound[ A 079> rotation of the metal!binding domain\ followed by reassociation with the DNA\would a}ord a BLMÐDNA complex that is able to e}ect the second cleavage event[ Consistent withthis picture\ palindromic sequences that provide the preferred 4?!dGPy target on each strand arehot spots for double!strand cleavage "Figure 01#[212

Figure 01 Preferred sequence for double!strand DNA cleavage by bleomycin[

Proton NMR investigations of ZnÐBLM complexes have yielded structures distinct from thoseobtained with the cobalt"III#ÐBLM complexes\ perhaps indicating that multiple binding modes arepossible for BLM[213

Over the last several years the Hecht group has extensively investigated BLM!mediated cleavageof biologically relevant substrates other than double!stranded DNA\ including RNA and DNAÐRNA hybrids[214 Their _ndings clearly indicate that BLM is able to cleave these substrates\ therebysuggesting that damage to nucleic acid targets other than double!stranded DNA could contributeto BLM|s biological activity[ In addition\ Hecht|s group has shown that metal!free BLM A1 mediatessequence!speci_c hydrolysis of RNA[215

A number of metal!binding antibiotics are structurally analogous to BLM\ including tallysomycin\zorbamycin\ the zorbonamycins\ victomycin\ the platomycins\ antibiotic YA!45\ cleomycin\ andphleomycin[298 These antibiotics are presumed to possess properties similar to BLM^ however\ mosthave not been thoroughly investigated[


6[03[1[04 Various Agents That Reduce Molecular Oxygen to Superoxide

A wide variety of naturally occurring organic compounds are capable of reducing molecularoxygen to superoxide radical[216 Some of these systems\ such as quinones "see Section 6[03[1[00[0#\are catalysts that mediate the transfer of electrons from reducing agents to molecular oxygen andothers stoichiometrically reduce molecular oxygen while being oxidized in the process[

Superoxide exists primarily as the radical anion "042# under physiological conditions[217\218 Only9[5) exists as the protonated hydroperoxyl radical "043# "pKa�3[7# at pH 6 "Equation "0##[Superoxide radical anion "042# is not believed to react directly with DNA^ however its conjugateacid\ hydroperoxyl radical "043# has been shown to produce sequence!speci_c DNA damage[217

Disproportionation "dismutation# of superoxide is of primary importance in the context of DNAdamage by this reduced form of molecular oxygen "Equation "1##[142\217Ð229 The disproportionationreaction involving a molecule of superoxide radical "042# with a molecule of hydroperoxyl radical"043# is rapid and\ although the concentration of "043# is low\ this reaction predominates over thedismutation reaction involving two molecules of "042#\ which is relatively slow "Equation "2##[Hydrogen peroxide generated by the disproportionation of superoxide decomposes via a tracemetal!catalyzed Fenton reaction to yield a potent DNA!cleaving agent that either is hydroxyl radicalor behaves very much like hydroxyl radical "Equation "3##[220Ð223 When substoichiometric quantitiesof transition metal ions are present\ e.cient generation of hydroxyl radical from hydrogen peroxiderequires the presence of a reducing agent that can recycle metal ions back to their active\ reducedform[ Superoxide radical can serve as such a reducing agent "Equation "4##[142\224 Interaction ofhydrogen peroxide with transition metals also leads to the formation of singlet oxygen\ which iscapable of reacting with DNA "see Section 6[03[1[07#[27

Several mechanistic tests are commonly used to implicate DNA cleavage by the cascade ofreactions shown in Equations "0#Ð"4#\ involving superoxide radical\ hydrogen peroxide\ trace metals\and hydroxyl radical[229 Addition of the hydrogen peroxide!destroying enzyme catalase to such aDNA!cleavage system results in a signi_cant decrease in strand scission[ Chelators of adventitioustrace metal ions\ such as desferrioxamine B "desferal# or diethylenetriaminepentaacetic acid\ willdecrease DNA cleavage by sequestering metal ions in a nonredox!active form\ thereby inhibitingthe Fenton reaction "Equation "4##[ The enzyme superoxide dismutase\ which catalyzes the dis!proportionation of superoxide radical "Equation "2## is also used as a mechanistic tool[ In caseswhere the enzyme superoxide dismutase diminishes DNA cleavage\ it is presumably due to the factthat\ in these systems\ superoxide radical is necessary as a reducing agent to return trace metals tothe active form needed in the Fenton reaction "Equation "4##[ In systems where other reducingagents\ such as thiols or ascorbate\ that can serve this function are present\ addition of superoxidedismutase may have no e}ect on\ or may even increase\ DNA!cleave e.ciency[ Agents such asethanol\ methanol\ mannitol\ and dimethyl sulfoxide are commonly employed as {{radical scav!enging|| agents and are sometimes considered diagnostic for the presence of hydroxyl radicals in aDNA!cleaving reaction[ While inhibition of DNA cleavage by these agents is consistent with theinvolvement of hydroxyl radical\ caution is necessary in interpreting the results obtained uponaddition of {{hydroxyl radical scavengers||\ as they also can react with other radical and nonradicalspecies[229

DNA damage involving reduction of molecular oxygen to superoxide has been reported for alarge number of organic compounds including thiols\141\225\226 hydroquinone and hydroquinoidcompounds "see Section 6[03[1[00[0#\ polysul_de:thiol mixtures "see Section 6[03[1[09[0#\ 0\1!dithiole!2!thiones:thiol mixtures "see Section 6[03[1[09[1#\ resorcinol:copper mixtures "see Section


6[03[1[02#\ enzyme:nitroaromatic systems "see Section 6[03[1[8[0#\ reductant:N!oxide systems "seeSection 6[03[1[01#\ ascorbate\227 3!hydroxy!4!methyl!2!"1H#!furanones\228 and hydrazines andhydroxylamines[239 In addition\ enzymes such as xanthine oxidase can cause oxidative DNA damagethrough the reduction of molecular oxygen to superoxide and hydrogen peroxide[230 There isevidence that neocarzinostatin\ under basic conditions\ either with or without added thiol\ generatessuperoxide radical\ although it appears that this chemistry is of minor importance relative to thiol!dependent\ biradical!mediated cleavage of DNA by this natural product[231

An interesting example of superoxide production by natural molecules is provided by the oxazo!lidine!containing antibiotics quinocarcin "05# and tetrazomine[79\70 Tetrazomine and "05# areexpected to be DNA!alkylating agents "see Section 6[03[1[0[2#^ however\ in addition to the expectedalkylation chemistry\ it has been found that these compounds mediate oxidative DNA damage[Identi_cation of the products resulting from the incubation of "05# and tetrazomine in aqueoussolution\ along with the study of synthetic analogues\ led to the proposal that these agents generatesuperoxide radical during the course of disproportionation reactions "Scheme 26#[

The cytotoxicity of superoxide and hydrogen peroxide is mitigated by the cellular enzymessuperoxide dismutase\232 catalase\ and glutathione peroxidase\233 and by ubiquitous radical scav!enging agents\ such as glutathione[ However\ despite these protective systems\ it is clear that anumber of antibiotics derive their biological e}ects\ at least in part\ through production of superoxideradical[

6[03[1[05 Diazo and Diazonium Compounds

6[03[1[05[0 Kinamycin

A reinvestigation of the kinamycin antibiotics "044# revealed that these natural products\ originallythought to be cyanocarbazoles\ actually contain diazo~uorene moieties[234\235 The biological target"s#of the kinamycins are not known^ however\ the DNA!damaging potential of these agents has beeninvestigated[236 Initially\ three possible mechanisms by which the kinamycins might damage DNAwere considered "Scheme 27#] "i# protonation of the diazo group would yield a diazonium ion "046#that could act as an alkylating agent^ "ii# reduction of the diazo group would lead to loss of nitrogen

N

N

OMeO

MeH

H

CO2H

H

N

N

OHMeO

MeH

H

CO2H

N

N

OMeO

MeH

H

CO2H

N

N

OMeO

MeH

H

CO2H

N

N

OHMeO

MeH

H

CO2H

N

N

OHMeO

MeH

H

CO2H

O

N

N

OHMeO

MeH

H

CO2H

O O•

N

N

OHMeO

MeH

H

CO2H

+

(16)

•

-1e-, -H+

+

+

+

•

O2O2•-DNA

cleavage

H2O

+1e-, +H+

Scheme 37


gas and formation of a reactive radical anion "047#^ or "iii# oxidation of the diazo group could leadto loss of dinitrogen with formation of a resonance!stabilized\ carbon!centered radical "048#[

Subjecting 8!amino~uorene to diazotization conditions\ followed by addition of the products toa DNA!containing assay mixture\ did not lead to DNA cleavage\ even in the presence of CuCl\which is known to trigger DNA damage by diazonium ions "see Section 6[03[1[05[2 below#[ Thefate of any diazonium ion "046# formed in this experiment remains unclear[ Experiments with simplemodels of the kinamycin diazo functionality demonstrate that DNA cleavage by the diazo compound"045# is triggered by copper"II# acetate\ and that cleavage is not triggered by CuCl[236 Thus\ it issuggested that DNA cleavage by the kinamycin model compound involves metal!mediated\ one!electron oxidation of the diazo functional group\ followed by loss of dinitrogen and concomitantformation of a carbon!centered radical "048#[ In earlier synthetic studies\ it was found that treatmentof "045# with copper"II# acetate a}ords reasonable yields of ~uorenone pinacol diacetate\ presumablyresulting from the dimerization of radical "048#[237 It was suggested that DNA cleavage in thissystem could actually stem from peroxy radicals formed by the reaction of molecular oxygen withthe resonance!stabilized carbon radical "048#[ In addition\ it was noted that an internal redoxreaction between the diazo and quinone functional groups of the kinamycins could trigger DNAdamage by the natural products[236

6[03[1[05[1 Diazoketones

A number of a!diazoketones with cytotoxic and anticancer properties have been isolated fromnatural sources "Figure 02#[238 DNA has not been suggested as a primary target for these agents^however\ one of these a!diazoketones\ 5!diazo!4!oxo!L!norleucine "059#\ has been found to cleave

H N2

N2AcO

H

+H+ -H+

-N2

reduction

-N2

oxidation

e.g. withCu(OAc)2-N2

(156)

•

+

+

+

DNAcleavage

•

_

_

(157)

(158)

(159)

Scheme 38

OH O

O

N2

OR2

OR3

OR4

R1O

Kinamycin A: R1=H, R2=R3=R4=AcKinamycin B: R1=R2=R4=H, R3=AcKinamycin C: R3=H, R1=R2=R4=AcKinamycin D: R1=R3=H, R2=R4=Ac

(155)


DNA\ though at relatively high concentrations "29Ð099 mM#\ in a plasmid!nicking assay[249 Involve!ment of carbon!centered radicals in DNA cleavage by "059# was suggested\ although a detailedmechanism for their formation was not proposed[

Figure 02 a!Diazoketone!containing antibiotics[

Reactions similar to those proposed for the diazo!containing antibiotic kinamycin can be imaginedfor a!diazoketone natural products[ In addition\ it is known that photochemical processes involvinga!diazoketones may yield carbenes and electrophilic ketenes capable of damaging DNA[240

6[03[1[05[2 Benzenediazonium ions

In certain species of mushrooms\ enzymatic metabolism of naturally occurring hydrazines isthought to produce aromatic diazonium ions[241 These diazonium ion metabolites are carcinogenic[242

Both DNA cleavage243 and DNA adduct244 formation have been reported for the mushroomdiazonium species 3!"hydroxymethyl#benzenediazonium ion "050#[ In vitro reactions of "050# withnucleosides have yielded adducts "051# and "052#\ in which the diazonium substituent has beenreplaced by C!7 of adenine or guanine\ respectively[244

Based upon radical scavenging data\ ESR experiments and other mechanistic experiments\ it hasbeen suggested that DNA cleavage by "050# results from the formation of carbon!centered phenylradicals[243 Previous work with synthetic benzenediazonium ions demonstrated that e.cient DNAcleavage by these compounds requires the presence of an agent\ such as CuCl\ that can serve as aone!electron reducing agent[245\246 It is believed that one!electron reduction of the diazonium ionfollowed by loss of dinitrogen a}ords a phenyl radical that can cleave DNA "Scheme 28#[ Inter!estingly\ DNA cleavage reported for "050# does not appear to require added reducing agents[243 Theauthors suggest that the formation of phenyl radicals from "050# is the result of a GombergÐBachman reaction^247\248 however\ the reason for the di}erences in conditions required for DNAcleavage by "050# and those for other reported diazonium ions is unclear[

Photolysis of benzenediazonium salts also leads to DNA cleavage^ under these conditions it ispostulated that DNA damage is due to the formation of arene cations[259

N NHOCH2

N

NN

N

NH2

OHO

HO

HOCH2

N

NHN

N

O

OHO

HO

HOCH2

+

(161) (162)

NH2

(163)

+N N

+1e-

-N2

• DNAcleavage

Scheme 39


6[03[1[06 Enediynes

Enediyne!containing antibiotics have captured the attention of many researchers250Ð252 because oftheir potent biological activity\ the synthetic challenge that they pose\ and also\ perhaps\ because ofthe unexpected and intriguing chemical reactions involved in the cleavage of DNA by these naturalproducts[ Enediynes undergo cycloaromatization reactions to yield carbon!centered radicals thatcause DNA strand cleavage via abstraction of hydrogen atoms from the deoxyribose backbone"Scheme 39#[ The enediyne antibiotics are discussed in detail in Chapter 6[04 of this volume[

6[03[1[07 Photochemically Activated Agents

6[03[1[07[0 Light!dependent DNA damage not involving covalent adducts

There are a number of mechanisms\ not involving the formation of stable covalent attachments\by which photoexcited states of organic molecules can damage DNA[24Ð26 Energy transfer from aphotoexcited state of an organic molecule to ground state molecular oxygen leads to the formationof singlet oxygen which can react with DNA\27\28\253 selectively causing damage to guanines[254Ð256

Some of the damage caused by singlet oxygen leads to spontaneous strand cleavage\28 although thedetailed chemistry of these processes remains to be elucidated[ Some photoexcited states of organicmolecules are potent oxidizing agents and can damage DNA by direct hydrogen atom abstractionor by electron transfer oxidation of the heterocyclic bases[24Ð26 Guanine is the most easily oxidizedbase and is probably the most common target for oxidations involving electron transfer[257Ð269

Finally\ the photoexcited states of some organic molecules can reduce molecular oxygen to super!oxide radical\24Ð26 which ultimately causes DNA damage through the formation of hydroxyl radical"see Section 6[03[1[04#[

A variety of natural products have been found to cause photosensitized DNA damage[ Forexample\ photolysis of daunomycin "002a#24 and gilvocarcin V "053#260 leads to the production ofsuperoxide radical[ Photolysis of simple anthraquinones leads to direct abstraction of hydrogenatoms from DNA and to oxidation of guanines through electron transfer reactions[261 Tetracyclines24

and gilvocarcin V260 act as singlet oxygen photosensitizers[ Upon irradiation with 259 nm light\camptothecin can directly abstract hydrogen atoms from DNA or produce superoxide radical\depending upon the reaction conditions[262 Certain metal complexes of bleomycin "036# catalyzelight!dependent DNA damage[209

O

SS

MeS

O-Sugar

HN COMe

HO

OO

S O-Sugar

HN COMe

HO

O

O

S O-Sugar

HN COMe

HO

O

Nu

hydrogen atomabstraction from DNA DNA

cleavage

Calicheamicin

Scheme 40


6[03[1[07[1 Formation of covalent photoadducts

A number of natural products engage in photochemically triggered cycloaddition reactionswith DNA that result in the formation of stable covalent adducts[ Perhaps the best characterizedof these natural products are the psoralens "054#\ a group of furocoumarin!containing plantmetabolites[263Ð265 Intercalation of psoralens at appropriate sites in double!helical DNA positionsthe molecule for 1¦1 cycloaddition reactions with pyrimidine bases[ Upon absorption of a 299Ð399 nm photon\ psoralens undergo a cycloaddition reaction involving either the 2\3!double bond ofthe pyrone moiety or the 3?\4?!double bond of the psoralen|s furan ring with the 4\5!double bondof an adjacent pyrimidine[

Photoreaction of psoralens with DNA yields monoadducts and also interstrand cross!links thatare the result of two sequential photoreactions[ Cross!links are possible only when the _rst photo!reaction involves the furan double bond of the psoralen\ such that the initial DNA adduct stillcontains an intact coumarin system that is capable of absorbing light between 299 and 399 nm[ Inaddition\ cross!link formation requires the presence of a second pyrimidine on the strand oppositethe initial adduct[ Thus\ monoadducts formed at 4?!PyPu or 4?!PuPy sequences can undergo asecond photoreaction with the adjacent pyrimidine on the opposite strand to a}ord a DNA cross!link[ Analysis of the yields of various monoadducts and cross!links shows that thymine is morereactive than cytosine in these photoaddition reactions and that 4?!TA sequences are preferred sitesof cross!link formation for the psoralens[ Monoadducts and cross!links\ e[g[\ "055#\ of psoralenswith DNA have been isolated and fully characterized[263Ð265

Another example of DNA photoadduction is provided by the naturally occurring C!glycoside!containing gilvocarcin antibiotics[ Structurally similar members of this class of antibiotics includechrysomycin A and ravidomycin[266 Notably\ analogues in this class of antibiotics that contain vinylside chains are more potent cytotoxins than those that do not[ In the case of gilvocarcin V "053#\intercalation of the benzonaphthopyranone moiety into DNA results in complexes that\ uponirradiation with UV light "×299 nm#\ yield covalent adducts with DNA[ Digestion of this modi_edDNA with 9[0N HCl at 099 >C results in release of 4\5!adducted pyrimidine bases[ Isolation andcharacterization of the product "056# shows that adduct formation is the result of a 1¦1 cyclo!

O

OH OMe

O

OMe

O

OH

OH

OH

(164)

O O O

3

454'

8 1

5'

(165)

O O O

OMe O

N

NH

O

NH

N

HH

H

H

H

OMe

DNA

O

O

O

DNA

OO

(166)

DNA/hν


addition between the vinyl group of gilvocarcin V and the 4\5!double bond of thymine[267 The acidicworkup conditions lead to isomerization of the furanose substituent of gilvocarcin V[

6[03[1[08 Restriction and Methylation Enzymes

Restriction enzymes catalyze the sequence!speci_c hydrolysis of double!stranded DNA[ Restric!tion enzymes occur in microorganisms as part of restrictionÐmethylation systems consisting ofDNA!cleaving enzyme:DNA!methylating enzyme pairs that recognize a common sequence[Sequence!speci_c methylation of its own DNA protects the host against DNA degradation by itsown restriction enzymes[ RestrictionÐmodi_cation systems appear to serve as a prokaryotic immunesystem that protects these organisms against foreign DNA that might enter the cell[268 The enzymes ofrestrictionÐmethylation systems in microorganisms formally can be considered secondary metabolitenatural products[ The chemistry and enzymology of restrictionÐmethylation enzyme systems hasbeen reviewed[268\279

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O

OH OMe

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OMe

NH

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O

OHOHO

HOH

(167)


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