PERSPECTIVE www.rsc.org/dalton | Dalton Transactions

Promotion of rare nucleobase tautomers by metal binding

Bernhard Lippert*a and Deepali Gupta*b

Received 23rd December 2008, Accepted 2nd February 2009First published as an Advance Article on the web 23rd February 2009DOI: 10.1039/b823087k

Metal binding to ligands with the potential of existing in different tautomeric structures candramatically alter the tautomeric equilibrium by stabilizing a particular, frequently minor, tautomer.The assumption that metal complexation of a minor tautomer is chemically irrelevant because of itsvery low abundance is misleading and in many cases wrong. In fact, from available X-ray structuraldata on metal–nucleobase complexes it is evident that metal binding to rare, as opposed to preferredtautomers, is anything but an exception. This “promotion of rare tautomers” through metalcoordination is of particular biological relevance in the case of nucleobases because any deviation fromWatson–Crick base pairing is potentially mutagenic. In recent years models of “metal-stabilized rarenucleobase tautomers” have been characterized for all common DNA nucleobases, including by X-raycrystallography. Though metal binding causes relatively minor structural changes in the nucleobases,electronic changes as expressed by acid–base properties, for example, can be substantial. In thisperspective article the biological consequences of the occupation of nucleobase sites by a metal entityand the altered acid–base chemistry of the nucleobase with regard to base mismatch formation,prevention of base pairing, and acid–base catalysis in nucleic acids are examined. Although not relevantto biology, the behaviour of the unsubstituted parent nucleobases is illuminating in this respect andtherefore included.

1. Introduction

The phenomenon of prototropic tautomerism, hence the existenceof equilibria between two or more species differing in thelocation of a hydrogen atom, is of considerable importance inorganic chemistry and biochemistry.1,2 Its biological significance

aFakultat Chemie, Technische Universitat Dortmund, D-44221, Dortmund,Germany. E-mail: bernhard.lippert@tu-dortmund.debSchool of Chemistry, University of Southampton, SO17 1BJ, Southampton,UK. E-mail: deepali.gupta@uni-dortmund.de

Bernhard Lippert

Bernhard Lippert received hisPhD degree from the TechnischeUniversitat Munchen (TUM) in1974. Following a stay at Michi-gan State University with Bar-nett Rosenberg (1974–1976), hereturned to TUM, where he ob-tained his “habilitation” degree(1982). In 1985 he moved toUniversitat Freiburg (associateprofessor) and in 1988 to Dort-mund (full professor) where heis Chair of Bioinorganic Chem-istry. His research include metal–

nucleic acid interactions, metal–metal bond formation, supramolec-ular chemistry of metal ions and N-heterocyclic ligands, includingnucleobases. He has been editorial board member of a number ofinorganic chemistry journals and is presently European Editor ofInorg. Chim. Acta.

Deepali Gupta

Deepali Gupta received her PhDdegree from the Technische Uni-versitat Dortmund (2005) un-der the supervision of Prof. DrBernhard Lippert. Her disserta-tion focused on the preparationand structural characterizationof Pt(II) and Pd(II) complexesof selected nucleobases. Thesecomplexes were studied with re-gard to the effects of metallationon hydrogen bonding propertiesand tautomerism. In 2005 shebegan post doctoral studies with

Prof. Dr Willem H. Koppenol at ETH Zurich. She moved to theUniversity of Southampton as a post doctoral fellow in 2007. Herresearch interests lie in the processes governing the interplay betweenbiological systems and redox-active species.

is particularly obvious in the case of the heterocyclic purine andpyrimidine nucleobases of DNA (Scheme 1), as any migrationof a N–H proton to another ring-N atom or to a carbonylgroup fundamentally changes the donor and acceptor propertiesof the various nucleobase edges. As a consequence, it can causemispairing during replication and, if it escapes repair, eventuallymay lead to a point mutation.3,4

It is well established that tautomer equilibria can be influencedby numerous factors such as temperature, solvent polarity, envi-ronment, the pKa of the “mobile” proton, chemical modification

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Scheme 1 Preferred tautomeric structures of the common nucleobasesof DNA and atom numbering schemes. T = thymine, A = adenine, C =cytosine, G = guanine, R = H (parent nucleobases), alkyl group (modelnucleobase), sugar (nucleoside), or sugar phosphate ester (nucleotide).In the common Watson–Crick or Hoogsteen base pairing schemes thesetautomeric forms occur.

of the skeleton, etc.. Remarkable shifts in tautomeric equilibria ofligands have been reported particularly upon encapsulation (e.g.b-diketone � keto,enol;5 b-ketoester � enolester6) and duringreceptor chemistry7 (e.g. creatinine detection or ureidopyrimidonedimerization) resulting from multiple specific interactions withthe environment. Enzymes capable of tautomerizing naturalsubstrates (“tautomerases”) use the very same principles. Amongthe chemical modifications, alkylation and metal coordinationare particularly relevant to nucleobases, and phenomenologicalstudies have provided ample evidence for the mutagenic potentialof such nucleobase alterations.

In this perspective, the influence of metal coordination tonucleobases on the tautomeric structure of nucleobases and thepossible influence on base pairing patterns will be dealt with.Of course, other scenarios are feasible, by which metal ions maybecome the origin of a mutagenic event, which are unrelated totautomerism. Only some of these will be briefly discussed.

Theoretical calculations (ab initio, density functional theory(DFT) methods) are increasingly applied to this question. Al-though in many cases helpful for a deeper understanding offundamental aspects of metal-induced mutagenicity, they are notalways relevant to conditions in condensed phase. Here an attemptis made to review specifically experimental evidence pointing toalterations in tautomeric structures of nucleobases following metalbinding. This review is not intended to be exhaustive as far asexamples are concerned but rather wishes to highlight the generalsignificance of this topic.

2. Nucleobase tautomerism

Among the numerous types of prototropic tautomeric equilibriafeasible,1 keto � enol and amino � imino tautomerism arethe common ones with nucleobases. The former is relevant tothymine (T) and uracil (U), while the latter applies to adenine(A). With guanine (G) and cytosine (C), in principle both types oftautomerism are feasible.

It is widely accepted that of the four common, biologicallyrelevant bases, the N9-blocked purines and the N1-blockedpyrimidines, the keto (G, T (U)) and amino forms (A, C) exceed

the second most abundant tautomers by a factor of 104–105. Whilethis number may seem large, it has to be taken into considerationthat in terms of relative Gibbs free energies, the differences betweenthe preferred tautomer and the second most abundant tautomeris in the order of 23–29 kJ mol-1 only:

DG = -RT ln K t

with K t = preferred tautomer/rare tautomer = 104–105.Given these relatively modest differences, it becomes immedi-

ately evident that environmental effects (e.g. through hydrogenbond formation) and in particular any kind of chemical modifica-tion, will have a major influence on tautomer equilibria.

With regard to base pairing possibilities of the various tau-tomers, a further complication arises from the fact that theorientation of a single proton at an exocyclic group may bevariable. In other words, the various possible rotamers may interactdifferently with partners upon hydrogen bonding (Scheme 2).

It is obvious that the number of possible tautomers (andtheir respective rotamers) is substantially higher for the parentnucleobases as compared to the biologically relevant bases blockedat the N9-position (purine) or the N1-position (pyrimidine),respectively. At the same time it is evident that protonatednucleobases likewise display a larger number of possible tautomersthan the neutral nucleobases. Even then, however, solid statestructures usually reveal a preferred tautomeric structure. Withthe parent nucleobases of G and T (U), even monodeproto-nated, hence anionic bases, exist in an equilibrium of tautomers(Scheme 3).8

3. Nucleobase pairing

A central dogma in molecular biology is that Watson–Crickpairing between the neutral, predominant nucleobase tautomersis crucial in maintaining the integrity of the genome. If dis-regarded, mispair formation is feasible, leading eventually totransition or transversion mutations. Mispairs may involve non-complementary nucleobases, protonated or ionized bases, orrare nucleobase tautomers. Although true, the importance of“electronic complementarity” between the Watson–Crick partnersis possibly overestimated. Rather, additional features appear to beimportant in achieving proper Watson–Crick pairing, such as thegeometry of the base pair as a whole, for example. The virtual“systematic violation”9 of the Watson–Crick pairing schemes inRNA and the numerous alternative pairing patterns realized inRNAs,10 speak for themselves with regard to mispairs involvingrare nucleobase tautomers. There have been arguments in favourand against this hypothesis since it was first mentioned. A recentstudy provides strong evidence in support of the “rare tautomerhypothesis” by correlating the tautomer equilibrium constantof a nucleoside analogue with incorporation specificity of thetriphosphate by a DNA polymerase.11

4. Chemical modifications of nucleobases

Superficially, chemical modification of nucleobases, or of hetero-cycles in general, may be divided into two classes: in the first class,an atom has been substituted by another one. Examples includethe replacement of O in a carbonyl group by a sulfur atom, thesubstitution of an aromatic C–H proton by a halogen atom, or

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Scheme 2 Changes of donor (D) and acceptor (A) properties of keto � enol (top) and amino � imino tautomeric forms (bottom).

Scheme 3 Tautomer equilibrium between thymine (R = CH3) and uracil(R = H) anions.

the replacement of an amino proton of an exocyclic NH2 groupby methoxy substituent or a methyl group. The effects on acid–base properties of the nucleobase, on its tautomer equilibrium, andits hydrogen bonding properties may be qualitatively different, butthey are in all cases measurable. For example, N6-methoxyadenine,the mutagenic adenine derivative formed upon reaction of A withhydroxylamine, adopts an imino tautomer structure and mispairswith cytosine.12

In the second class, a reactive group such as an alkyl group orsimply a proton, for example, has been added to the nucleobase(e.g. N7 of G or O6 of G) to produce a cationic species. Dependingon the acidifying effect of this modification the ligand may rapidlylose a proton to regain its neutral state (Scheme 4).

Metal binding to nucleobases can be seen accordingly, hence asbinding to an anionic nucleobase, with a N–H or a C–H protonsubstituted by the metal ion, or as coordination to a ring–N, andexocyclic O atom of the neutral nucleobases, or even p bonding toC=C double bonds.

Scheme 4 Alkylation reactions of a guanine nucleobase at N7 and O6and its different effects on the protonation state of the nucleobase.

5. Metal complexes of parent nucleobases

The parent nucleobases, because of their additional tautomerequilibria, are not necessarily good models for the naturalnucleosides and nucleotides. Nevertheless they are of interest inthe context of this review, because numerous computational andX-ray structural studies with such systems confirm an importantaspect: namely, that tautomeric structure and reactivity (here:metal coordination) are not necessarily interrelated. In otherwords, a particular tautomeric structure, even if predominant,does not automatically ensure that in a metal complex thistautomeric structure is maintained. After quite some debates andmisconceptions, this view is generally accepted today.1

5.1 Adenine

Of the various (14) possible tautomers of unsubstituted adenine,the 9H-amino tautomer is the most stable and hence predominant

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species in the gas phase,13,14 in water,15 and in the solid state (matrixisolation).16 The second most stable tautomer in the gas phase is the7H-amino form, and this is likewise the case in water. Accordingto calculations, the 3H-amino tautomer and the 1H,9H-iminotautomer follow (Scheme 5).17–19 All the other tautomers areconsiderably higher in energy and are not considered here.

Scheme 5 The four most stable tautomers of adenine. Relative stabilitiesin the gas phase are 0, �31 kJ mol-1, �35 kJ mol-1 and �52 kJ mol-1 forthe four tautomers shown and between 70 and 190 kJ mol-1 for the others(not shown). The term anti refers to the relative positions of the protonsat N1 and N6. This situation is occasionally also termed cis to indicate itspointing to the imidazole ring.

As the dipole moment of the 7H-amino tautomer (6.77 D) islarger than that of the 9H-amino tautomer (2.65 D),18 the formeris more stabilized in polar solvents or under conditions, in whichthe 7H-amino tautomer is efficiently stabilized by non-covalentintermolecular interactions. For example, cocrystallization ofadenine with a neutral Mn(II) oxalate shows the disfavoured gasphase 7H-amino tautomer to be present in the solid state, stabilizedby hydrogen bonds involving all three N–H protons as donorsand the three other ring N atoms as acceptors.20 It demonstratesthat the order of stability can be changed by relatively minorenvironmental effects.

Metal coordination can be expected to have a considerablylarger influence, but its effect can be modulated by the environ-ment. For example, Cu(II) binding to adenine may occur throughN3 with maintenance of the preferred 9H-amino tautomer struc-ture, because the co-ligand of the metal involves the proton at N9in both intra- and intermolecular fashion in bifurcated hydrogenbonds and consequently stabilizes this form.21 On the otherhand, metal coordination (e.g. of Zn(II)22 or Co(III)23) may takeplace through N9 of the 7H-amino tautomer, with reinforcementby an intramolecular hydrogen bond between a co-ligand (e.g.H2O or NH2R) at the metal and the N3 position of adenine.Computational studies of a series of +1 cations (Li+,24 Cu+,25

Ag+17,18) have provided the astonishing result that in the gas phasechelation of the metal ion through N6 and N7 to a minor iminotautomer (1H, 9H-imino (syn); ca. 80 kJ mol-1 less stable thanmajor tautomer) is the most favourable arrangement (Scheme 6,(i)–(iii)).

Any attempts to predict metal binding patterns on the basisof relative stabilities of individual tautomers fail, if multiplemetal coordination is considered. Of the various possibilitiestwo crystallographically established cases are given in Scheme 6

Scheme 6 Examples of metal complexes of neutral adenine with metalbinding to the major tautomer (i)21 and two minor tautomers (ii)22,23 and(iii).17,18,24,25 (iv)26 and (v)27 represent scenarios with twofold metal bindingto rare amino tautomers.

((iv) and (v)), in which metals are bonded to N7 and N926 aswell as N3 and N9, respectively.27 In both the instances minortautomers are present rather than the most stable 9H-aminoform.

Adeninium cations (monoprotonated form) likewise come indifferent tautomeric forms. On the basis of experimental dataand theoretical calculations,28 1H,9H-adeninium is the most stableone, with the 3H,7H-form slightly less stable (1.9 kJ mol-1) andthe 3H,9H-form still somewhat less stable (8.2 kJ mol-1). Allthe other tautomers of the adeninium cation have considerablyhigher energies (35–215 kJ mol-1) and are not further consid-ered (Scheme 7). Not surprisingly, because of the small energydifference between the two most stable tautomers, the 3H,7H-adeninium cation can be crystallized with suitable counter ions.29

As far as metal complexation of adeninium cations is concerned,there are examples with the metal entity (ZnCl3

-)30 bonded to thepreferred tautomer (Scheme 7(iv)), but also cases with one (PtII)31

or two (Cd2+)32 metal ions bonded to a rare tautomer (1H,7Hform) of the adeninium (Scheme 7, (v) and (vi)). The latter is only5th in sequence of the most stable tautomer, and 45.5 kJ mol-1

higher in energy than the preferred 1H,9H-adeninium tautomer.

Scheme 7 Three most stable adeninium cations ((i)–(iii)) and selectedexamples of metal complexes ((iv)–(vi)).

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Deprotonation of adenine reduces the number of possibletautomers. In fact, di- and (formally) trianions no longer representN–H tautomeric systems, and the respective di-, tri- and eventetranuclear metal complexes (see, e.g. ref. 33) may form differentlinkage isomers. Of the adenine monoanion, with the acidic protonremoved from an endocyclic N atom and the exocyclic aminogroup retained, the metal may reside at N923 or more metalentities may be bonded to the available ring N atoms, e.g. N3,N9,34

N7,N9,35 or N3,N7,N9.35,36 In all these cases the amino tautomerstructure of anionic A is present.

Finally, a study deserves mentioning, in which a kineticallyrobust [CoIIICl(en)2]2+ entity is bonded to N9 of adenine, and inwhich, depending on the adenine protonation state, the nucleobasecan be present as its monoanion (amino form), its neutral form(rare 7H-amino tautomer form), or its protonated form (rare1H,7H-tautomer) (Scheme 8).23 The combination of X-ray dataand pKa values not only reveal the sequence of protonation whenstarting from the adenine monoanion ligand, but at the sametime show the dramatic shifts in acid–base equilibria upon Co(III)binding to N9. After all, pKa values for the free adeninium is 4.15,and deprotonation of neutral adenine occurs with a pKa of 9.8.37

Scheme 8 Top: Acid–base equilibria of (a) cis- and (b) trans-[CoCl(H2A-N9)(en)2]3+ (H2A = adeninium cation). According to ref. 23.Bottom: Comparison with pKa value of free forms of adenine.

5.2 Guanine

Of all four common nucleobases, guanine has the highest numberof possible tautomers, namely 20. Although there has beenagreement that in the solid state guanine adopts preferentiallya keto tautomer structure (with the N1 site carrying a proton),location of the second proton at one of the three other availableendocyclic N atoms was less certain. X-Ray crystallographyeventually solved this question for the solid state, proving that itis the N9 position, hence the 1H,9H-keto form is present.38 As weknow today from theoretical calculations, the considerably largerdipole moment of the 1H,9H-tautomer—in fact the largest oneamong all canonical bases—as compared to the 1H,7H-tautomer,is consistent with this finding. In solution an equilibrium of thetwo tautomers was postulated.39 For quite some time the questionof additional minor guanine tautomers has rested in the hands ofcomputational chemists until, more recently, experimental spec-troscopic methods have become available.40 These experiments,together with theoretical calculations,41,42 now permit a critical

evaluation of this question and provide a more unified pictureof guanine tautomerism. According to these findings, the fourmost abundant tautomers of guanine in the gas phase are 1H,7H-keto > 1H,9H-keto > 6H,9H-enol (trans) > 6H,9H-enol (cis).Relative abundances are ca. 40 : 32 : 16 : 12 (Scheme 9), consistentwith rather small (<4 kJ mol-1) differences in energies.41 Energiesof other tautomers (e.g. of 6H,7H-enol, 3H,7H-keto, 3H,9H-keto,and 7H,9H-keto) become increasingly larger. Microhydration(one or two water molecules included) does not change thetautomer equilibrium substantially. Surprisingly, however, bulkwater “promotes” a truly minor tautomer in the gas phase—the3H,7H-keto form—strongly over 7H,9H, 3H,9H, 1H,7H and eventhe canonical 1H,9H tautomer! Consequently, the two dominanttautomers in water have been proposed to be the 3H,7H-andthe 3H,9H-keto forms. The authors41 provide a rationale as towhy a tautomer with a rather modest dipole moment (4.34 D)nevertheless is capable of outweighing other tautomers which, inthe gas phase, have higher dipole moments.

Scheme 9 Experimentally studied tautomers (i)–(iv) of neutral guanine inthe gas-phase (He nanodroplets)40 and examples of metal complexes (v)43

and (vi).46 While (v) is established by X-ray crystal structure, the structureof (vi) has been derived from DFT calculations of a complex detected byPI-MS.

There are numerous reports in the literature on metal complexesof unsubstituted guanine. However, in the absence of X-raystructural data it is frequently difficult to properly assign the metalbinding site and the location of the two imino protons. In fact,there are only a few examples of X-ray structurally characterizedexamples of guanine complexes with a metal at N9,43,44 and onlytwo in which the guanine is neutral, namely a Cu(II) complex and a(dien)PtII compound with the metals bonded to N9 of the 1H,7H-keto tautomer.43c,43d This tautomer, although the preferred one inthe gas phase (see above), is strongly disfavoured over three othertautomers in water, where formation of the PtII complex takes

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place! For neutral hypoxanthine, which may be similar to guanine,the crystal structure of a (NH3)5RuIII complex is available, whichshows the metal bonded to N7 and the purine in a 1H,9H-tautomerstructure.45 Another interesting case of “tautomer promotion”by a metal ion is that of a complex of Al3+ with guanine inwhich, according to photoionization mass spectrometry and DFTcalculations, the guanine nucleobase adopts a truly rare (in gasphase!) 7H,9H-tautomer with the metal chelated via N1 and O6.46

Sketches of the mentioned Pt and Al complexes are included inScheme 9.

Guanine accepts a proton in moderately acidic medium (pKa �3.0).47 According to gas phase calculations, the five most favouredguaninium tautomers are within a range of less than 21 kJ mol-1

(Scheme 10).42 The two keto–amino tautomers 1H,7H,9H and1H,3H,7H are most stable, followed by the enol–amino tau-tomers 1H,6H,9H; 6H,3H,7H; and 6H,7H,9H. In water, only thetwo keto–amino tautomers are favourably stabilized by solventmolecules. Few metal complexes have been X-ray structurally char-acterized, a CuII complex48 and a RuIII compound.44 Both show theguaninium ligand in the 1H,3H,7H-keto–amino tautomer form,hence the second most abundant tautomer in the gas phase, andthe metal coordinated to N9. In both compounds the proton at N3is involved in a hydrogen bonding interaction. Again, despite itsapparent similarity with guaninium, the protonated hypoxanthinedoes not display an analogous structure with Ru(III), but ratheradopts a 1H,7H,9H-tautomer form, with the metal bonded toN3!49

Scheme 10 The two dominant keto–amino tautomers of monoproto-nated guanine (i) and (ii) and examples of metal complexes with the metalat N9 (iii).

Finally, the guanine monoanion (pKa1 = 9.3 for deprotonationof neutral guanine)47 can act as a ligand in metal complexes. Atpresent, only a few examples appear to be characterized by X-rayanalysis,43a one of which shows two (dien)PtII moieties bondedto N7 and N9, with a proton residing at N1 of the amino–keto-tautomer (Scheme 11).43d

We are not aware of any other structurally characterizedmetal complexes containing a guanine dianion (pKa2 = 12.3 fordeprotonation of guanine monoanion) or even a trianion (aminogroup also deprotonated).

Scheme 11 Guanine monoanion, complexed to two (dien)PtII entities viaN7 and N9.43d

5.3 Cytosine

Of the 14 possible tautomers (with imino–NH and hydroxo–OHrotamers included), six are within 40 kJ mol-1 in the gas phase, andthree of these, the 2H-enol (trans) tautomer, the 1H-keto–amino,and the 2H-enol (cis) forms (trans and cis refer to orientation of 2Hrelative to N3) are within 4 kJ mol-1 of each other (Scheme 12).42,50

Experimental IR data (matrix isolation, 15 K) are consistentwith the view that the 2H-enol and the 1H-keto–amino are thepredominant species in the gas phase.51

Scheme 12 Three most stable cytosine tautomers in gas phase (i–iii) andthree tautomers which follow up in energy (iv–vi).

In water, a marked destabilization of four of the six most stablegas phase tautomers of cytosine takes place. The canonical 1H-keto–amino form is by far the most stable and consequently thedominant one. Relative stabilities of imino and enol tautomers arereversed, and the 3H-keto–amino tautomer becomes the secondmost stable one. However, it is still some 24.4 kJ mol-1 less stablethan the 1H-keto–amino tautomer.42

In the solid state unsubstituted cytosine adopts the 1H-keto–amino tautomer structure.52,53 Cytosine can be protonated to givethe cytosinium cations (pKa = 4.58 ± 0.01) and deprotonated togive the cytosine anion (pKa = 12.15 ± 0.05).54 Both protonationand deprotonation occur at the endocyclic ring N atoms.55

There are numerous examples reported in the literature of metal-cytosine complexes, in which the 1H-keto–amino tautomer (whichis strongly preferred in the solid state and in water, yet onlysecond in gas-phase) is bonded to a metal ion through N3,56,57

or alternatively through O2,58 or even simultaneously through N3and O2.42b,59 These possibilities are depicted in Scheme 13.

Compared to them, the number of structurally characterizedexamples of metal complexes with the minor (vi) in row of gasphase tautomers, cf. Scheme 12 3H-keto–amino tautomer is rare.

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Scheme 13 X-Ray structurally characterized examples of metal com-plexes of 1H-keto–amino tautomer of cytosine (i–iii), of 3H-keto–aminotautomer (iv), and of cytosine anion (v, vi).

With the exception of a CH3Hg(II) complex, which has beenpreliminarily characterized,60 there are only a few cases of PtII

complexes which display N1 coordination.57 Solution 1H NMRspectroscopy verifies the existence of cytosine nucleobases bondedto Pd(II) or Pt(II) via N1 at a ratio that outruns the “natural”tautomer distribution by far. So N1 metal coordination is not justa solid state effect, but rather a reality also in solution.

5.4 Thymine and uracil

Despite considerable research on thymine and uracil tautomerism,at present there appears to be agreement on the predominant(gas phase, solution, solid state) species only. It is the 1H,3H-diketo tautomer for both nucleobases.61,62 The picture regardingthose tautomers which follow up, “still remains unclear”.61 Thereare eight additional enol and four dienol tautomers which, inthe gas-phase, are higher in energy than the canonical diketotautomer by ca. 39–118 kJ mol-1 for both parent nucleobases.The uncertainty stems in part from the fact that some of theminor enol tautomers have large dipole moments—up to 10D—which inevitably will promote these tautomeric forms inthe presence of polar solvent molecules such as water. Still,the unfavourable energy destabilization relative to the preferred1H,3H-diketo tautomer in the gas phase is sufficiently large toprevent any substantial population of the other forms. The onlyminor tautomer of uracil and thymine in bulk water with a freeenergy below 40 kJ mol-1 relative to the 1H,3H-diketo tautomer,is the 2H,4H-dienol form (with 2H cis to N1 and 4H cis to N3).61

These two structures are depicted in Scheme 14((i), (ii)).Interestingly, and particularly relevant to the question of

point mutations arising from nucleobase tautomerization, is afinding of Rak et al.,63 according to which the presence ofother (bio)molecules, rather than of water, may have a profoundinfluence on tautomer distribution. Thus, a molecule capable ofdifferentially interacting (via hydrogen bonds) with an individual

Scheme 14 Largely preferred uracil (R = H) and thymine (R = CH3)tautomer in gas phase, water, and solid state (i) and second moststable 2H,4H-dienol tautomer in solution (ii). In (iii) a situation isdepicted in which hydrogen bonding with zwitterionic glycine (left) causestautomerization to the 4H-enol tautomer (right).

rare tautomer may promote this one selectively over others. In thisparticular case the authors demonstrate, applying DFT methods,that the amino acid glycine in its zwitterionic structure “selects”the 1H-keto,4H-enol(cis) tautomer, thereby increasing the amountof this otherwise negligible tautomer by almost five orders ofmagnitude (Scheme 14, (iii)). This raises the general question asto whether there may indeed exist many more molecules capableof influencing tautomer equilibria of nucleobases.

Although metal complexes containing neutral uracil have beenprepared with numerous transition elements,64 X-ray crystalstructure analyses are scarce. O4 binding to the canonical tautomerhas been proven for HgCl2 in HgCl2(UH2)2 (UH2 = neutraluracil).65 According to ab initio calculations, [Pt(NH3)3]2+ bindsto neutral thymine likewise through one of the exocyclic oxygenatoms.66

Uracil deprotonates with a macroscopic pKa = 9.33 ± 0.05,67

and thymine with a pKa = 9.75.68 Monoanion formation can occurin both cases via two pathways, namely deprotonation of the N1or the N3 site, leading to two major tautomers (Scheme 3).8 Asdiscussed in detail for uracil,67a the proton at N3 is somewhat moreacidic than the one at N1, leading to microscopic pKa values of9.43 ± 0.05 for N(3)H and 10.02 ± 0.31 for N(1)H, which is ingood agreement with DFT calculations.69 The N3 deprotonatedanion is considerably more polar than the N1 deprotonated andconsequently favoured in polar environment. In water (alkalinepH), the two tautomers co-exist in comparable amounts.

Again, metal complexes of uracil and thymine monoanionsconvincingly characterized crystallographically, or otherwise (1HNMR, IR, Raman, UV-vis spectroscopy),70,71 are not all thatnumerous. Rather than listing here all X-ray structurally char-acterized examples [for a summary of early examples, see ref. 64]only examples of two metal ions, ZnII and PtII, shall be discussed.Zn complexes of uracil (and in part of thymine) are known in whicheither the 3H-tautomer (via N1)72 or the 1H-tautomer (via N3)73 iscoordinated, or even both isomers are bonded to a single Zn ion.74

In the case of N1 coordination the low polarity of the solvent

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mixture (MeOH–CH2Cl2) and in addition the hydrophobicity ofthe pocket containing the Zn(II) appear to be accountable for thisbinding pattern while in the case of N3 coordination both thehigher polarity of the solvent (water) and the efficient stabilizationof the N3 bonded ligands by a pair of short hydrogen bondsbetween the exocyclic O atoms and the macrocyclic amine co-ligand of the metal favour this other binding pattern. E. Kimuraand coworkers have intensively taken advantage of this principlewhen designing Zn complexes which selectively recognize uracil–N3 and thymine–N3 sites in nucleic acids.75 In the unique caseof simultaneous N1 and N3 coordination in Zn(HU-N1)(HU-N3)(NH3)2 “the interactions in intermolecular space”74 hence anefficient hydrogen bonding scheme and favourable electrostaticinteractions in the crystal, have been proposed to be responsiblefor this particular binding scheme.

As pointed out elsewhere,70,71 a differentiation of uracil andthymine anion tautomers when bonded to PtII is possible bynumerous spectroscopic techniques, with 1H NMR spectroscopybeing particularly useful. 195Pt–1H coupling with H6 and H5protons of uracil or H6 of thymine is highly diagnostic with regardto a differentiation. Because of their good accessibility,76 PtII

complexes containing the N1 bonded tautomers have been studiedintensively.70,71,77–79 In contrast, N3 bonded tautomers are moredifficult to obtain.79 Interestingly, the latter form at acidic pH,hence under conditions where the nucleobase is not deprotonatedat all. This raises the question of how its formation proceeds andthe authors tentatively suggest that initial binding to a carbonyloxygen atom takes place, followed by base deprotonation andmetal migration to N3. Research with [Pt(HU-N1)(en)H2O)]+ hasalso yielded a cyclic, tetranuclear complex of composition [Pt(HU-N1,N3)]4(NO3)4, which contains a truly minor uracilate tautomer,which has its movable proton at O2 and O4 (Scheme 15).79 Theexistence of this tautomer has hitherto not been considered.

Scheme 15 Pt binding to two major tautomers of uracil monoanion ((i),(ii)) and to a minor tautomer ((iii)).

5.5 Isocytosine

Isocytosine (2-aminopyridin-4-(3H)-one) is a structural isomer ofcytosine, occasionally used in artificial oligonucleotide duplexeswith the aim of expanding the genetic code.80 It exists as twomajor tautomers,81a the keto form with the N1 position and theN3 position carrying the acidic proton, respectively (Scheme 16).The two tautomeric forms crystallize in a 1 : 1 ratio and forma hydrogen bonded adduct81b in the solid state. A third, minortautomer (not shown) adopts an enol structure. A number oftemperature dependent solution studies82 have been carried out,which suggest that isocytosine exists in solution as a mixture ofthe two forms. However, these studies were not able to distinguishwhich tautomer predominates. It has also been observed that

Scheme 16 Two tautomeric forms of isocytosine in the solid state.

the electronic absorption spectra55,83 of isocytosine change ongoing from aqueous to nonaqueous solution. Therefore, it waspostulated that this behaviour of isocytosine might be dependenton the change of tautomeric ratio caused by the nature of solvent,the temperature and that isocytosine exists predominately in 3H-keto form in ethanol and diethyl ether, while in aqueous solutionthe two tautomers co-exist in comparable amounts. It has also beenreported84 that certain bands in the Raman spectra of isocytosinealso shift to higher wavenumbers, i.e. from 789 cm-1 to 791 cm-1

and from 1214 cm-1 to 1232 cm-1, as DMF is successively dilutedwith water, though there was no separation into individual peaksas is the case for isocytosine in the solid state and in pure DMF.

Metal complex formation with isocytosine has been studiedby various groups,84,85 and there are examples of X-ray struc-turally characterized metal compounds with O4,85c N384,85d andN1,N385e coordination. Various substituted isocytosine derivatives(substituents at N1, N2, C5 positions) have likewise been studied.86

Reactions with a series of PdII and PtII species84 such as (dien)PdII,(dien)PtII and trans-(NH3)2PtII reveal, however, a distinct prefer-ence of these metals for the N3 site, as determined by 1H NMRspectroscopy. DFT calculations84,85e suggest that intramolecularhydrogen bonding between the isocytosine tautomers and theco-ligands at metal, while adding to the preference for N3coordination, is not the major determining factor. Rather it is theinherently stronger metal–N3 bond which favours complexationof 1H-keto. The excess of (dien)MII (M = PdII and PtII) hasalso afforded the dinuclear species [(dienM)2(IC-N1,N3)]3+.85e Instrongly acidic medium [(dienPt)2(IC-N1,N3)]3+ is converted to[Pt(ICH-N1)(dien)]2+, hence to the PtII complex of the 3H-ketotautomer. However, the experimental findings are in qualitativeagreement with the results of the computational studies whichsuggest an inherently stronger binding of isocytosine throughN3.84,85e

5.6 Nucleobase radicals

Nucleobase can form radicals, which occur as different tautomers.Two scenarios can be differentiated: first, a nucleobase traps alow-energy electron produced by high energy radiation, giving anucleobase anion radical.87 Second, a nucleobase [usually guanine,as it is the nucleobase most easily oxidized] is converted intoa radical cation initially,88 followed by proton dissociation as aconsequence of the low pKa (ca. 3.9 for guanosine radical cation89).If taking place in a base-paired arrangement, proton transfer maylead to unconventional base pairs.90 The proposal has been putforward that even CuII coordination to N7 of guanine mightlead to oxidation of this nucleobase, followed by H+ transferto cytosine and formation of Cu+.91 Based on known aqueoussolution chemistry of CuII with nucleobases, such a possibilitywould seem less likely when compared to alternative scenarios inwhich no redox chemistry is involved. Of course, generation of

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highly reactive oxygen species such as OH∑ radicals in the presenceof Cu+ and dioxygen is not questioned.

6. Tautomerism in metal complexes of nucleotidesand their models

With respect to the relevance of tautomerism for base mispairingand mutagenicity in biology, the behaviour of N9-substitutedpurines and N1-substituted pyrimidines rather than that ofthe parent nucleobases needs to be considered. Consequently,in the following, only systems with nucleobases blocked atthese positions (nucleotides, nucleosides, N9-alkylpurines, N1-alkylpyrimidines) will be considered and, as an additional simplifi-cation, differences in charge between anionic nucleotides (negativecharge at phosphate group) and neutral nucleosides as well asalkylated nucleobases are ignored.

Concerning the preferred tautomeric structures of these substi-tuted nucleobases, similar differences in gas phase and condensedphase are seen as for the parent nucleobases (see, e.g. ref. 92 for9-methylguanine).

There have been scattered reports on the existence of raretautomers of nucleotides in the solid state, e.g. in uridine 5¢-diphosphate,93 d(TMP) in DNA,94 or AMP in Z-DNA (in presenceof [Co(NH3)6]3+).95 Evidence for the existence of rare tautomers ofN9-blocked in aqueous solution is indirect only, relying on theso called “basicity method”.96,97 In brief, this method uses the factthat two different neutral tautomers form an identical cation uponprotonation or a common anion upon deprotonation. By applyingexperimentally observable pKa values of the two tautomers, inwhich a proton is substituted by a methyl group, thereby generatinga “fixed” tautomer structure, and with the assumption (to bechecked by spectroscopic means) that the substitution of H+ byCH3

+ does not substantially affect the electronic structure ofthe nucleobase, K t values can be estimated. In return, from amacroscopic acidity constant, obtained experimentally, and withK t known, the micro acidity constant of a rare tautomer canbe derived.98 This situation is summarized in Scheme 17. Asmentioned below (section 6.2.1), this method can, in principle,be applied to metal nucleobase complexes as well.

6.1 Mutations

It has been estimated that within 24 h, DNA in every cell isdamaged on average at least 10 000 times.99 If unrepaired, thisdamage can be the origin of a mutation.99 There are multipleways by which the genetic information stored in DNA canbe altered. Major processes causing mutations are oxidativenucleobase damage, non-enzymatic alkylation reactions of nu-cleobases, glycosidic bond breakage, base deamination reactions,etc. Alterations in nucleobase tautomer structure or simple non-complementary pairing are more subtle changes, even thoughthey can, in principle, be as serious as the other mentionedones.100 A change in tautomer structure can be spontaneous asa consequence of the inherent tautomer equilibrium constant K t

between preferred and rare tautomer forms, through an electronicexcitation of the nucleobase, by a chemical modification, or a“wrong” pairing partner,101 among others. A concerted protontransfer within a Watson–Crick pair generates two rare tautomerssimultaneously.102 Base mispairing may also be brought about by a

Scheme 17 K t for 1-methylcytosine can be estimated (upper branch)from macroscopic K1 (experimental value) and K I and/or K II (methylderivatives of I or II, e.g. 1,3-dimethylcytosine instead of tautomer II or1,4-dimethylcytosine instead of tautomer I). K ¢II, hence acidity of raretautomer II, can be estimated (lower branch) from macroscopic K2 andK t: pK ¢II = pK2 - pK t.

charged nucleobase (protonated or deprotonated). Such processescould in fact circumvent the necessity of tautomerization.103

Any point mutation (transition mutation or transversion muta-tion) proceeds via a central base mispairing step (AC or GT pairingin case of transition mutations; purine–purine or pyrimidine–pyrimidine mismatches in case of transversion mutations). Quite anumber of such DNA base pair mismatches have been X-ray struc-turally characterized,104 or determined by NMR spectroscopy.105

It may or may not involve a rare tautomer. For example, thenon-Watson–Crick pair between G and T (“wobble pair”) hasboth bases in their dominant tautomeric structures. In RNA,the corresponding GU pair is very common! Purine–purinemismatches such as those between G and A can come in differentforms, involving the dominant or a rare adenine tautomer of A ora protonated adenine (Scheme 18).104,106

6.2 The possible roles of metal ions in base mispairing stepsfollowing nucleobase tautomerization

It is beyond doubt that metal ions and metal-containing com-pounds are frequently mutagenic.107 The ways by which metalspecies exert their mutagenic potential, are poorly understood.The effects may be indirect (disturbance of polymerases, repairmachinery, etc.) or direct on the nucleobase level (e.g. hydrolyticdamage, blockage of hydrogen bonding sites, steric distortion ofDNA template, metal-mediated oxidative damage, H+ transfer asa consequence of nucleobase acidification, etc.). Among the latterdirect effects, which shall be dealt with here, the possibility of a shiftof the tautomer equilibrium of a nucleobase as a consequence ofmetal coordination is particularly intriguing. In further discussingsuch scenarios, it is helpful to differentiate two cases, (I) and (II).

6.2.1 Case I: Metal remote from base pairing site. The N7sites of the purine bases A and G are well accessible, located inthe major groove of B-DNA and consequently preferred metalbinding sites in DNA. C8 of A and G as well as C5 of C are

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Scheme 18 A–G mispairs involving the bases in their preferred tautomeric forms ((a), (b)), A in its protonated form (c), and A in its rare imino tautomericform (d).

likewise in the major groove, yet only rarely the target of metalions.108 N3 of A and G, located in the minor groove of DNA, arestill other potential binding sites of metal species (Scheme 19).

Scheme 19 Potential metal binding sites remote of the Watson–Crickbase pairing edges: Purine sites N7, C8, N3 (top) and cytosine-C5. Nodifferentiation between A and G is made.

An obvious question is whether binding of a strongly polarizingmetal ion to N7 of either A or G or replacement of an aromaticproton (e.g. H5 of cytosine) leads to a shift in K t (Scheme 20).For G, ab initio calculations have suggested that binding ofcis-[Pt(NH3)2Cl]+ to N7 has virtually no effect on the keto–enol tautomer equilibrium.109 This result does seem logical inview of the—admittedly simplistic—consideration that in an enoltautomeric structure, the metal at N7 and a proton at O6 repeleach other more strongly than if the proton resides at N1. If thecharge of the metal ions is neutralized by anionic co-ligands andif these co-ligands stabilize a proton at O6 in a hydrogen bond, thesituation is less unfavourable,110 however. Whether or not a shiftof the 1H form to a (zwitterionic) 3H form is possible, remains anopen question.

In contrast, and using similar simplistic arguments, a shift ofan amino to an imino tautomer would seem more likely in thecase of adenine metal coordination at N7. Indeed, theoreticalcalculations suggest that, at least in the gas phase and with thedipositive [Pt(NH3)3]2+ at N7, a shift towards the imino tautomerstructure of adenine takes place.110 A favourable hydrogen bond

Scheme 20 Potential shifts in tautomer equilibria of A, G, and C uponmetal binding to sites remote of Watson–Crick edge.

from an ammonia ligand at the Pt to the imino group N6 furtherstabilizes such a situation. In view of the established mutagenicityof cisplatin–adenine adducts, we had earlier attempted to demon-strate an amino → imino tautomer shift in aqueous solutionby application of the “basicity method” to PtII complexes of 9-methyladenine and related 1,9- as well as 1,6-dimethyladenineanalogues.111 This study proved ambiguous, however, possiblybecause the differential effects of protons on one side and methylgroups on the other cannot be neglected.

With C8-bound metal entities, proton shifts from C8 toN7 have been observed both for guanine,112 adenine,113 andhypoxanthine.114 Whether or not this proton shift also affects theWatson–Crick edge, hence causing a shift in tautomer structure(and consequently has an effect on base pairing), has not beeninvestigated.

The effect of a metal ion binding to C5 of a cytosine nucleobaseon its tautomer equilibrium and its base pairing properties has,to the best of our knowledge, not been studied either. Neither are

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acid–base properties of such an organometallic species available,although such data might provide a hint concerning tautomerism.A comparison with the situation of the RNA model nucleobase 1-methyluracil, carrying a PtII entity at C5, is instructive, however.115

There, C5 platination causes a reduction in acidity of N3H (pKa

is 12.2 as compared to 9.75 for the free 1-methyluracil), which islikely to suppress a movement of the proton at N3 to an oxygenatom, hence tautomerization. It is to be expected that replacementof the aromatic proton at C5 of a N1 blocked cytosine likewiseincreases the basicity of the heterocyclic ring. As a consequence,two trends might be expected: An increase in N3 basicity and adecrease in N4H2 acidity. The outcome with regard to the possibleformation of an imino tautomer appears to be open. Nevertheless,the electronic complementarity with the hydrogen binding partnerguanine is likely to be disturbed.

Coming back to guanine, it needs to be pointed out thatthe question of tautomeric change becomes irrelevant, once theproton at N1 is lost as a consequence of metal coordination toN7. Models for mispairs resulting from this process, have beendiscussed elsewhere.108a,116

6.2.2 Case II: Metal at a site normally involved in Watson–Crick pairing. Formally, any prototropic tautomerization pro-cess can be considered a sequence of a deprotonation and areprotonation step. If reprotonation of the anionic form takesplace at a site different from that of the starting molecule, atautomer of the initial molecule is generated. In the presenceof a metal species, reprotonation of the anionic species at theinitial site may be intercepted by metal coordination. In this case,reprotonation has to take place at a different site, and consequentlyleads to formation of a tautomer, which is “stabilized” by the metalentity (Scheme 21). Such cases are today established for all fourcommon DNA nucleobases86,117–120 and likewise for the RNA baseuracil.121

Scheme 21 Relationship between nucleobase tautomerization via depro-tonation/reprotonation and formation of metal-stabilized rare tautomerthrough metal coordination to anion.

Let us first consider thymine (or uracil) with the metal sitting atN3, and guanine, with the metal bonded to N1. Protonation of thenucleobase anions has to take place at either O4 or O2 (thymine,uracil), or N7, O6 or N3 (guanine), respectively. Ignoring the metalfor a moment, the nucleobase is then present as an oxo, hydroxotautomer (T, U) or in zwitterionic forms and as an enol tautomer(G), respectively (Scheme 22).

Scheme 22 Rare metalated iminol tautomer forms of N1-substitutedthymine (R¢ = CH3) and uracil (R¢ = H), respectively (i), and rareN9-substituted, N1-metalated guanine tautomers (ii).

Formation of the nucleobase anion and metal coordination ismore easily achieved than generally assumed. In particular, no highpH (to accomplish nucleobase deprotonation) is required! This isso because metal ions, which behave as Lewis acids in aqueoussolution, carry their own “base” –OH for ligand deprotonation,and/or acidify the position of the nucleobase to be deprotonatedby transient binding to other sites (e.g. exocyclic oxygen atoms ofT, U or, with G, also endocyclic N sites such as N7 or N3). Forexample, Zn2+ coordination to O4 of a uracil ligand causes a dropin pKa of the N3H by more than 3 log units,122 and simultaneousbinding of PtII to N7 of a G base and of PdII to N3 leads toa drop in pKa of N1H by 4 log units.123 Similarly, binding ofa dirhodium(II) entity to O6 and N7 of G lowers the pKa ofN1H to 5.7.124 A straightforward procedure to attach cisplatinto N3 of 1-methyluracil (pKa = 9.75) is to allow reaction of cis-Pt(NH3)2Cl2 with 1-MeUH at pH 4!125 Once the metal is bondedto the deprotonated ring N site, only a moderate drop in pH, if atall, is required to generate the “metal stabilized rare tautomer”!This is so, because the exocyclic oxygen atoms display increasedbasicity following the replacement of the proton at N3 by PtII.The pKa of the metalated rare tautomer, hence of the OH group,depends to some extent on the overall charge of the complexand the microenvironment, hence the co-ligands. Typically, for1-methyluracil complexes of PtII, pKa values are in the range of1–4.126 For 1-methylthymine complexes slightly higher values canbe expected. For guanine complexes of PtII, with the metal at N1and the acidic proton at N7, the pKa is in the range 3–5.118,127 Asthese (upper) values are close to or even identical with the pKa

values of numerous metal–aqua species, an attractive hypothesisof formation of rare tautomer complexes of T(U) and G mightbe that the acidic proton at the nucleobase is derived from anaqua co-ligand (Scheme 23).128 Theoretical calculations, carriedout for PtII complexes, confirm that complexes containing the 2-oxo-4-hydroxo tautomers of U and T can be more stable than thecomplexes containing these bases in their preferred dioxo tautomerstructure and the metal bonded to one of the two exocyclic oxygenatoms.128

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Scheme 23 Feasible ways of generating rare T* and G* tautomers via H+

transfer from an aqua ligand to the nucleobase anion.

The formation of “metal-stabilized” imino tautomers of C andA (in principle also for G), with the metal bonded to the exocyclicdeprotonated amino group, via an analogous deprotonation andreprotonation pathway is more difficult to achieve. This is so,because of the considerably lower acidity of the exocyclic aminogroup (pKa ca. 17) as compared to NH acidities of T, U, andG (pKa ca. 9–10). Nevertheless, there is ample evidence for suchbinding modes, both with mononuclear metal complexes of A119

and C,117 as well as with dinuclear ones of A.129 The types ofcompounds feasible or known to date are outlined in Scheme 24.

Scheme 24 Established or feasible metal-stabilized rare tautomers of Cand A, with different orientations of metal entities relative to N3 (cytosine)and N1 (adenine) positions. Mixed syn/anti arrangements of dinuclearadenine species are not considered.

Before discussing their formation, it needs to be emphasized thatmetal binding to an exocyclic amine group per se is not equivalentwith formation of a rare tautomer complex (Scheme 25). Only ifthe nucleobase is neutral, hence the proton that used to be at theamino group is bonded to another site, is the notion of a “raretautomer” valid. If the nucleobase is anionic, it just representsthe anion of the common, preferred tautomer!130 Computationalstudies on the situation in gas phase clearly confirm the ideathat metal coordination to the exocyclic nitrogen atom of iminotautomers stabilize these rare tautomers.131

Scheme 25 Mono- and di-metalated anions of major tautomers ofN1-blocked cytosine (i, ii) and metalated forms of neutral minor tautomer(iii, iv). Species (iv) is unlikely to occur in aqueous solution becauseof the expected strong repulsion between the two metal ions and theproton at O2.

The ways by which metalated imino tautomers of A and C(and G132) are generated, are probably numerous. It appears thata common way is initial anchoring of a metal at an endocyclicN atom, followed by metal migration to the exocyclic N atom.As twofold metal binding, e.g. simultaneously to N1 and N7 ofA, facilitates migration and at the same time causes a strongacidification of the amino group,133 it is reasonable to assumethat amino group deprotonation precedes nucleophilic attack ofthe NH- group onto the metal. Alternatively, the metal migrationcan proceed via a redox reaction (e.g. from PtII to PtIV), witha chelate involving both the endocyclic (N3 of C) and theexocyclic (N4 of C) positions representing the key intermediate.134

A third possibility might include a Dimroth rearrangement inthe case of initial metal binding to N1 of A and transientopening of the heterocyclic purine ring between positions N1and C2 analogous to the interconversion of 1-methyladenine to6-methyladenine. In fact, there may be even additional ways. Forexample, formation of trans-[PtCl2(1-MeC-N3)(1-MeC-N4)] froma precursor containing exclusively the 1-MeC-N3 linkage isomer,takes place at a surprisingly low pH.117

The dirhodium complexes of adenine nucleobase reported byChifotides and Dunbar,124 which display N7,N6 metal binding,are presumably formed via initial N7 metal anchoring, followedby a condensation reaction between N6H2 and a hydroxo ligandof the second Rh atom.

6.3 Consequences

The most obvious change in physico-chemical properties of metalbinding to a rare nucleobase tautomer is its strongly shifted pKa

value. If pKa values of closely related metal (e.g. PtII) nucleobasecomplexes of known stoichiometry with N1 blocked cytosineligands are determined, the species containing a rare cytosinetautomer (metal at N4) is immediately recognized by its pKa

of ca. 7–8. This value is more than 5 log units lower than thatof its counterpart containing the major tautomer bonded to Pt

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through N3. Or: a single PtII at either N1 or N7 of a N9-blockedadenine nucleobase shifts the pKa of the amino group at mostto 11, whereas PtII at N6 leads to a pKa of 4–5. Even if two PtII

atoms, at N1 and N7, bind to the major tautomer, the acidification(pKa as low as 7133a), the pKa of a single PtII at N6 is not reached.Similarly, two PtII atoms at guanine may decrease the pKa fromca. 9–10 to ca. 5–6, yet a guanine complex with a single PtII atN1 displays a pKa of the proton at N7 of ca. 3–5. Elsewhere, wehave discussed this feature in terms of the potential of “metal-stabilized rare nucleobase tautomers” to act as acid–base catalystsin catalytic RNA molecules.135

As has been shown by us,98,121 structural changes of nucleobasesupon engagement in metal coordination as rare tautomers arerather modest. By “subtracting” the effect of the metal, it ispossible to come up with an approximate geometry of the raretautomer, which otherwise can only be derived by theoreticalcalculations.

As far as base pairing properties are concerned, and irrespectiveof the site of metal coordination, it is clear that a change in pKa

will influence the electronic complementarity between potentialbase pairing partners. Of course, base pairing will be hampered ifthe metal blocks sites normally needed for Watson–Crick pairing(e.g. blockage of N3 of T or N1 of G). This may also be thecase, when the metal sits at the exocyclic amino group and themetal is syn with respect to the N3 position of C or N1 of A(see Scheme 24). Even if the Watson–Crick face is not blocked(anti orientation of metal(s)), pairing with the proper comple-mentary nucleobase is prevented because the Watson–Crick facehas altered its tautomeric structure. In Scheme 26, a relevantmispairing scenario between a HgII-stabilized imino tautomer ofadenine and a guanine in its predominant tautomeric structure isdepicted. If not repaired, this mispair gives rise to transversionmutation.119c

Scheme 26 Potential mispair between metalated rare adenine tautomerA* and guanine base G in syn orientation (top) and sequence of pairingsteps leading to a transversion mutation (bottom).119a

The generation of a short-lived metal-free rare thymine tau-tomer from a metal-stabilized rare tautomer is likewise feasible(Scheme 27). Thus, excess H+ will eventually break the metal–N3bond in a metal–thymine complex with release of the neutral raretautomer. While in water, tautomerization to the preferred 2,4-dioxo-tautomer is expected to be instantaneous, in a non-aqueousenvironment the rare tautomer may be sufficiently long-lived topermit formation of a mispair.120a

Scheme 27 Generation of rare thymine tautomer (T*) from metal-stabilized rare tautomer, rapid conversion into major dioxo tautomer(top right), and potential mispair with guanine (bottom).

7. Summary

In this article, we have examined the roles coordinated metal ionscan play in stabilizing and/or generating tautomers of nucleobaseswhich, in the absence of the metal, are extremely rare. Both exam-ples with the parent nucleobases and with their biologically morerelevant relatives (N9-substituted purines and N1-substitutedpyrimidines) have been discussed. As a result, it is obvious thata metal ion can “titrate out” a rare tautomer by binding to it, orgenerate the rare tautomer in a reaction sequence which starts outfrom coordination to the major tautomer. Although not discussedhere, we do not wish to exclude the possibilities that “outer-sphere” metal binding or simple electrostatic interactions betweennucleobases and (several?) metal cations may have qualitativelysimilar effects. Given the fact that metal ions are “natural” counterions of the negatively charged nucleotides, there is a high likelihoodthat metal ions, and in particular non-physiological ones, arecapable of interfering with tautomer equilibria of nucleobases.In combination with a favourable microenvironment (co-ligands,solvent molecules, helper molecules called “tauterogens”136) itwould thus appear that nucleobase tautomerization in the presenceof coordinating metal ions is a process much more likely than gen-erally anticipated. Consequently, the proposal of an involvementof a rare nucleobase in ribozyme chemistry (rare enol tautomerin hammerhead cleavage reaction137) appears to be anything butunrealistic. Similarly, the suggestion that the xanthine oxidasereaction involves tautomerization steps of pteridines and purineswhen coordinated to Mo,138 makes sense. In protein chemistry(e.g. histidine tautomerism; action of “tautomerases” on smallmolecules139) the concept of minor tautomers playing a majorrole in reaction sequences is more widespread than in nucleic acidchemistry. Likewise, in general metal coordination chemistry140

and in organometallic chemistry,141 and especially in view ofcatalytic processes, the feature of ligand tautomerization in thecoordination sphere of a metal is commonly accepted. Withnucleobases, the very same principles work. It is possibly theHoly Grail of the Watson–Crick base pairing schemes, whichrequires the preferred tautomeric structures of the nucleobases,

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which makes us reluctant in anticipating ways by which minortautomers of nucleobases are formed.

Acknowledgements

The authors wish to thank the Fonds der Chemischen Industrie(FCI) for financial support, and Dr Pablo J. Sanz Miguel forhelpful discussions.

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125 A. Khutia, and B. Lippert, unpublished results.126 L. Holland, W.-Z. Shen, W. Micklitz and B. Lippert, Inorg. Chem.,

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130 For example of 9-methyladeninato complexes, see e.g.: (a) G. Trovo,G. Bandoli, M. Nicolini and B. Longato, Inorg. Chim. Acta, 1993,211, 95–99; (b) B. Longato, L. Pasquato, A. Mucci, L. Schenetti andE. Zangrando, Inorg. Chem., 2003, 42, 7861–7871.

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135 B. Lippert, Chem. Biodiversity, 2008, 5, 1455–1474.136 P. Strazewski, Nucleic Acids Res., 1988, 16, 9377–9398.137 M. Martick and W. G. Scott, Cell, 2006, 126, 309–320.138 P. Ilich and R. Hille, Inorg. Chim. Acta, 1997, 263, 87–93.139 See, e.g.: N. Metanis, A. Brik, P. E. Dawson and E. Keinan, J. Am.

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141 For recent examples, see: (a) E. P. Kundig, A. Enrıquez Garcia, T.Loberget and G. Bernardinelli, Angew. Chem., Int. Ed., 2006, 45, 98–101; (b) M. A. Esteruelas, F. J. Fernandez-Alvarez and E. Onate, J. Am.Chem. Soc., 2006, 128, 13044–13045; (c) M. Buil, M. A. Esteruelas,K. Garces, M. Ollivan and E. Onate, J. Am. Chem. Soc., 2007, 129,10998–10999.

4634 | Dalton Trans., 2009, 4619–4634 This journal is © The Royal Society of Chemistry 2009

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