Metal-stabilized rare tautomers of nucleobases. 8. Promotion of rare cytosine tautomer upon complex formation with (dien)M2+ (M=Pt, Pd)

Inorganica Chimica Acta 339 (2002) 400 /410 www.elsevier.com/locate/ica

Metal-stabilized rare tautomers of nucleobases. 8. Promotion of rare cytosine tautomer upon complex formation with (dien)M2
(M  Pt, Pd) /

Wolfgang Bru¨ning a, Ivana Ascaso a,1, Eva Freisinger a,2, Michal Sabat b, Bernhard Lippert a,* b

a Fachbereich Chemie, Universita¨t Dortmund, 44221 Dortmund, Germany Department of Chemistry, University of Virginia, Charlottesville, VA 22901, USA

Received 4 December 2001; accepted 21 March 2002

Abstract Reactions of (dien)M2
(M /Pt, Pd) with cytosine (CH) lead to mixtures of linkage isomers with N3 and N1 bonded metal entities as well as N3,N1 bridged species, as demonstrated by 1H NMR spectroscopy. The N3 linkage isomer is preferred over the N1 linkage isomer throughout the pH range studied (2.5 /9), but the ratio does not parallel the equilibrium constant between the aminooxo tautomer (I) (proton at N1) and the aminooxo tautomer (II) (proton at N3), which has been estimated to be close to 1000 in solution. Rather, the minor tautomer (II) is complexed to a much higher extent. This finding is potentially significant for any metal /nucleobase interaction in that it suggests that metal complexes of rare tautomers may be formed more readily than generally assumed. X-ray data of four (CH
/N 3) complexes as well as of the first example of a (C
/N1 ,N3 ) bridged compound are reported. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Cytosine complexes; (dien)Pt(II); (dien)Pd(II); Nucleobase tautomerism

1. Introduction Nucleobases, regardless if present as biologically relevant building blocks of the nucleic acids (nucleotides) or in the form of their parent compounds (without sugar entity), exist in solution in an equilibrium of two or more tautomers. The preferred tautomer usually exceeds the second most abundant one by a factor of 103 /106 [1]. The predominance of the major tautomer in solution is absolutely essential in ensuring proper pairing with the correct complementary nucleobase during DNA replication or DNA transcription. The temporary existence of a rare nucleobase tautomer may

* Corresponding author. Tel.:
/49-231-755 3840; fax:
/49-231-755 3797. E-mail address: [email protected] (B. Lippert). 1 Exchange student from the University of Zaragoza, Spain. 2 Present address: Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, NY 11794, USA.

be responsible for base mispairing which, if undetected by repair enzymes, could lead to a point mutation. Many additional scenarios for mutagenic events are feasible [2], several of which include the assistance of heavy metal ions [3]. If metal binding to nucleobases is considered, one is tempted to assume that complex formation with a minor tautomer is irrelevant due to the extremely low abundance of the latter. This is, however, not necessarily the case. Others and ourselves have found evidence in a number of instances that reactions of metal ions with nucleobases, hence with mixtures containing an excess of a preferred major tautomer and a tiny amount of a rare tautomer, can spontaneously lead to a product containing exclusively the minor tautomer [4 /9]. Alternatively, such complexes have occasionally been prepared in a programmed, sequential way [10,11]. This feature has not always been viewed under the aspect of minor tautomer complexation, however, in particular not if the metalated nucleobase was present in a deprotonated form. Recently, when studying the inter-

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W. Bru¨ning et al. / Inorganica Chimica Acta 339 (2002) 400 /410

actions between unsubstituted cytosine and trans a2Pt2
(a /NH3 or methylamine), we noticed that the rare aminooxo tautomer of cytosine (II) is complexed (via N1) to an extent which is considerably higher than is expected on the basis of the tautomer distribution of (I) and (II) in the absence of trans -a2Pt2
[12] (Chart 1). Hence, the minor tautomer was ‘‘titrated out’’ by the metal entity. Following up on this observation, we have extended this work now to other metal entities and report here on cytosine complex formation with (dien)Pt2
and (dien)Pd2
(dien /diethylenetriamine). We are fully aware that our findings with cytosine are not immediately biologically relevant, simply because we are not dealing with a N1 blocked model or the corresponding nucleotide. Nevertheless, the mere fact that tautomer distribution does not necessarily parallel the situation in the metal-complexed form is of considerable interest with regard to metal-caused mutagenicity, for example.

401

Fig. 1. pH* dependence (d , ppm) of H5 (bottom) and H6 (top) resonances of cytosine in D2O. Only the major tautomer (I) of neutral cytosine is depicted. Changes at strongly alkaline pH indicate the beginning of dianion formation (deprotonation at exocyclic amino group).

2.2. Reactions of (dien)Pt2
with cytosine 2. Results and discussion

2.1. Solution equilibrium of cytosine Of the various possible tautomers of cytosine, the two aminooxo forms I and II are most relevant in condensed media (solution; solid state) with I estimated to exceed II by a factor of 102.9 [13]. For isolated cytosine molecules (gas phase calculations [14]; matrix isolation [15]), the 4amino,2-hydroxo form has been reported to exceed the 4-amino,2-oxo form (I) slightly. Neutral cytosine (CH) can be protonated (pKa of CH2
is 4.58 [16]) or deprotonated (pKa of CH is 12.15 [16]). A second deprotonation step, that of the exocyclic amino group, is expected to take place only in strongly basic medium (pKa estimated to be
/17 [17]). The first two pKa values have been reproduced in this work by applying pH*dependent 1H NMR spectroscopy (Fig. 1).

Mixtures of cytosine and [(dien)Pt(D2O)](NO3)2 were prepared and the samples studied by 1H NMR spectroscopy. Ratios of the two reactants were varied and reactions were carried out both without pH* adjustment and with addition of acid (DNO3) or base (NaOD) at the start of the reaction. Typically, samples were kept at 50 8C for 5 /7 days until no further changes took place in the 1H NMR spectra. At this point, spectra usually displayed four sets of aromatic H5 and H6 resonances (doublets each, 3J /6.7 /7.3 Hz, c.f. Table 1), occasionally also only three sets. The resonances were assigned by their pH* dependence, hence samples were treated with acid and/or base over a wide pH* range and 1H NMR spectra were recorded. In one case a 1H,1H COSY Table 1 1 H NMR chemical shifts (ppm) and 3J values (Hz) of cytosine H5 and H6 doublets (D2O, pH* 3) of [(dien)Pt(CH
N1 )]
(A), [(dien)Pt(CH
N3 )]
(B), [(dien)Pt(C
N1 ,N3 )Pt(dien)]3
(C), and CH/CH2
(D) and of the corresponding (dien)PdII complexes (A?) / (C?) H5 A B C D

5.93 6.02 5.84 6.12

A? B? C?

5.96 5.97 5.80

a

Chart 1.

b

a

b

H6

3

7.84 7.51 7.63 7.72

7.2 7.3 6.7 7.2

7.74 7.50 7.53

6.8 7.2 6.6

J (H5,H6)

b

Unresolved 195Pt satellites occasionally seen, 4J  10 Hz. Shifts and 3J value taken from spectrum at higher pH*.

402

W. Bru¨ning et al. / Inorganica Chimica Acta 339 (2002) 400 /410

spectrum was also used to confirm correlations between individual H5 and H6 doublets. In no instance was a rapid change in species distribution observed as a consequence of an altered pH*. Because of their complexity, resonances of the dien ligands (ca. 2.5 /3.3 ppm) were not analyzed. Out of the many experiments carried out, three will be subsequently discussed in more detail. 2.2.1. (i) 1:1 mixture of (dien)Pt2
and cytosine Fig. 2 shows a typical spectrum, obtained after allowing a 1:1 mixture of [(dien)Pt(D2O)]2
and CH (0.01 M each) in D2O to react for 7 days at 50 8C. The pH* had not been adjusted and was found to be 6.3 after that time. The four sets of doublets (A) /(D) were assigned on the basis of their pH* dependence. The chemical shifts of doublets (C) were pH* independent and for this reason this species was assigned to the dinuclear compound [(dien)Pt(C
/N1 ,N3 )Pt(dien)]3
. Signals (A) proved to be those of the N1 linkage isomer, [(dien)Pt(CH
/N1 )]2
/[(dien)Pt(C
/N1 )]
, based on their pKa value of approximately 6.5. It is close to the average of the pKa values of approximately 6.1 and 7.1 previously observed by us for trans -[Pt(CH3NH2)2(CH
/ N1 )2]2
[12]. As in the latter case, the cytosine H6 doublet of the N1 linkage isomer A appears furthest downfield when the cytosine base is neutral and is hence readily recognized. Resonances (B) were identified as those of the N3 linkage isomer, [(dien)Pt(CH
/N3 )]2
/ [(dien)Pt(C
/N3 )], both on the basis of their pKa value of approximately 9.29/0.2 (c.f. average pKa of trans [Pt(CH3NH2)2(CH
/N3 )2]2
, 9.1 [12]) and from comparison with the spectrum of the isolated compound 1 (vide infra), the structure of which has been established by X-ray crystallography. Finally, signals (D) were those of free cytosine (CH2
/CH/C ). In Table 1, cytosine chemical shifts of all four species (A) /(D) at

Fig. 2. 1H NMR spectrum (D2O, pH* 6.3, cytosine resonances only) of reaction mixture of [(dien)Pt(D2O)]2
and cytosine (0.01 M each) after 7 days at 50 8C. Assignment of resonances (A) /(D) was made on the basis of their pH* dependence (not shown).

pH 3.3 are listed. Relative (A):(B):(C):(D) /1:0.8:18:12.

intensities

are

2.2.2. (ii) Excess of cytosine With a slight excess of cytosine over (dien)Pt2
applied (2:1) and initial pH* adjustment to 9.4 (pH* dropped to 7.8 within 7 days at 50 8C), again resonances of the four species (A) /(D) are observed. As expected, resonances due to free cytosine are the most intensive ones (Fig. 3). Relative intensities are (A):(B):(C):(D) / 1:4:1:6. 2.2.3. (iii) Excess of (dien)Pt2
In an experiment carried out with a 3:1 ratio between (dien)Pt2
and cytosine (no base or acid added; pH* after 7 days at 50 8C ca. 2.6), no free cytosine is left and resonances due to species (C) have strongly increased in intensity (Fig. 4). Relative intensities are (A):(B):(C)/ 1:22:8. The experiments carried out with (dien)Pt2
unambiguously prove that throughout the pH range where cytosine is present in its neutral form, hence 6 B/pHB/ 10, but even in more acidic medium, when cytosine is becoming protonated (experiment (iii)), the N1 linkage isomer is formed. Its relative abundance clearly exceeds that of the 4-amino,2-oxo tautomer (II) in the absence of a metal entity by far. Thus, in experiment (ii) the N3 linkage isomer (B) is more abundant than the N1 linkage isomer (A) by a factor of approximately 4:1, even though the ratio of the two tautomers in solution is approximately 1000:1! One might argue that at higher pH (e.g. initial pH 9.4 in experiment (ii)) already traces of the cytosine anion are present in solution, which indeed could react preferentially at N1. However, even at lower pH (c.f. experiment (i)) the N1 linkage isomer (A) is more abundant by a factor of 100 than the corresponding free tautomer.

Fig. 3. Section of 1H NMR spectrum (D2O) of sample prepared by reacting cytosine (0.024 M) with [(dien)Pt(D2O)]2
(0.012 M) (initial pH* 9.4) for 7 days at 50 8C, then brought to pH* 3 (DNO3) to achieve optimum signal separation.

W. Bru¨ning et al. / Inorganica Chimica Acta 339 (2002) 400 /410

403

their relative intensities, very much as previously reported for nucleotide mixtures [19]. Interestingly, the N1 linkage isomer (A?) exists even at a pH* /2.5. In contrast to (dien)Pt2
, the N1,N3 bridged cytosinato species (C?) is not observed below pH* 4, not even when (dien)Pd2
is present in excess (e.g. 3:1, pH* 3.3). This suggests that the dinuclear Pd species is not thermodynamically stable at acidic pH. 2.4. X-ray crystallographic studies of cytosine complexes with (dien)Pt2
, (dien)Pd2
, and (en)Pt2
and NMR spectra of isolated species Fig. 4. Cytosine resonances of sample obtained by reacting [(dien)Pt(D2O)]2
and cytosine in a 3:1 ratio in D2O. The pH* of the sample displayed had been adjusted to pH* 3.4. No resonances due to free cytosine/cytosinium are detected.

2.3. Comparison with (dien)Pd2
Similar 1H NMR experiments were also carried out with [(dien)Pd(D2O)]2
. A major advantage of the use of the Pd species over the corresponding Pt species is its faster reaction kinetics which permits the equilibrium to be reached within minutes after sample preparation [18]. A typical 1H NMR experiment (1:1, no pH* adjustment; pH* 7.2, subsequently lowered) is given in Fig. 5. Again, the assignment of the various species (A?)/(C?) was based on the pH* sensitivity of the H5 and H6 resonances of cytosine. As evident from Table 1, cytosine chemical shifts of corresponding species of (dien)Pt2
and (dien)Pd2
differ slightly. A major difference with the (dien)Pt2
system discussed above is that the species distribution with (dien)Pd2
is pH* dependent, viz. that during changes of pH* not only chemical shifts of individual species are affected but also

Fig. 5. Cytosine resonances of 1:1 mixtures of [(dien)Pd(D2O)]2
and cytosine at pH* 7.2 (top), 5.3 (middle), and 2.5 (bottom). (A?) refers to N1 linkage isomer, (B?) to N3 linkage isomer, (C?) to N1,N3 bridged species, and (D) to free nucleobase.

Preparative work has yielded a series of complexes of cytosine, all of which contain the cytosine base bonded to the metal via N3, hence cytosine in its preferred tautomer structure (I). The following compounds have been isolated: [(dien)Pt(CH
/N3 )](ClO4)2 ×/CH3OH (1), [(dien)Pd(CH
/N3 )](ClO4)2 ×/H2O (2), [(en)Pt(CH
/ N3 )Cl]X ×/n H2O (X /Cl0.75, (NO3)0.25, n /1 (3a); X / NO3, n /1 (3b); X /ClO4, n/0 (3c)) and [(en)Pt(CH
/ N3 )(ONO2)]Cl0.25(NO3)0.75 (4). X-ray crystal structures were obtained for 1, 2, 3a and 4. Crystallographic data and experimental details are compiled in Table 2. It also contains data on a fifth compound (5) to be described below. Selected bond distances and angles of these compounds are listed in Tables 3 /5. The cations of 1 and 2 are rather similar (Fig. 6). Metal coordination takes place via N3 , while the N1 position carries a proton, as evident from the internal ring angle at N1 (C2
/N1
/C6, 122.9(1)8 (1), 122.2(2)8 (2)) and confirmed by the hydrogen bonding pattern between pairs of cations of 1 and 2 (Fig. 7). The dien ligands in both complexes adopt a ‘‘stingray’’-like folding [20] with no unusual structural features [21]. The geometry of the cytosine ring is likewise in agreement with data for other Pt(II) [12,22] and Pd(II) [23] complexes with this coordination pattern. Although isolated from different solvents, MeOH (1) and H2O (2), both compounds crystallize in the triclinic space group P 1¯ with very similar cell constants (c.f. Table 2). Both the hydrogen bonding pattern, hence formation of dimers, and the crystal packing are very similar. Dimer formation takes place through a pair of H bonds around a center of inversion involving N1 and ˚ in the case of 1, 2.798(3) A ˚ in the O2 atoms (2.88(1) A case of 2). The OH group of MeOH in 1 forms a hydrogen bond both with O2 of the cytosine and N11 of the dien ligand (Table 3). In compound 2 the water molecule occupies the same position as does the OH group of MeOH, again forming these two H bonds. An interesting detail of the 1H NMR spectrum of 1 in D2O (pH* 6.7) is the fact that below 15 8C cytosine H5 and H6 resonances undergo a reversible signal splitting, leading to two sharp sets of resonances of relative intensities of approximately 4:6. Similarly, the 195Pt

W. Bru¨ning et al. / Inorganica Chimica Acta 339 (2002) 400 /410

404

Table 2 Crystallographic data and details of refinement for compounds 1, 2, 3a, 4 and 5

Formula Asymmetric unit Formula weight Crystal color and habit Crystal size (mm) Temperature (K) Crystal system Space group ˚) a (A ˚) b (A ˚) c (A a (8) b (8) g (8) ˚ 3) V (A Z Dcalc (g cm 3) m (Mo Ka) (mm 1) F (000) 2u Range (8) Number of reflections collected Number of reflections observed Number of parameters refined Rint R1 a (observed data) wR2 a (observed data) ˚ 3) Residual rmax, rmin (e A a

1

2

3a

4

5

C9H22N6O10Cl2Pt C9H22N6O10Cl2Pt 640.32 colorless block 0.38  0.38  0.25 293 triclinic P 1¯ 7.654(2) 11.250(2) 11.749(2) 102.91(3) 93.48(3) 102.72(3) 955.5(3) 2 2.226 7.686 620 9.5 /51.4 2328 1455 (I ] 2s (I )) 218 0.051 0.0476 0.0920 0.752, 1.374

C8H20N6O10Cl2Pd C8H20N6O10Cl2Pd 537.60 colorless stick 0.58 0.25 0.24 293 triclinic P 1¯ 7.504(2) 10.937(2) 11.749(2) 101.24(3) 95.78(3) 104.10(3) 906.1(3) 2 1.971 1.382 536 4.7 /55.1 4187 3488 (I ] 2s (I )) 285 0.021 0.0245 0.0554 0.462, 0.408

C6H15N5.25O2.75Cl1.75Pt C6H15N5.25O2.75Cl1.75Pt 461.85 colorless needle 0.44  0.25  0.24 153 monoclinic P 21/c 8.105(4) 12.426(3) 12.601(1)

C6H13N6.75O6.25Cl0.25Pt C6H13N6.75O6.25Cl0.25Pt 483.68 pale yellow plate 0.44 0.19 0.13 293 monoclinic P 21/n 8.040(2) 6.849(1) 24.107(5)

C18H44N14O18Cl4Pt3 C9H22N7O9Cl2Pt1.5 1471.74 yellow plate 0.33 0.31  0.25 293 monoclinic C 2/c 34.326(7) 9.099(2) 13.446(3)

92.47(2)

94.86(3)

99.94(3)

1267(1) 4 2.419 11.556 870 4 /50.0 2344 1737 (I ] 3s (I )) 158 0.005 0.0278 0.0362 0.52, 0.48

1322.7(5) 4 2.429 10.700 914 9.1 /51.3 2492 1939 (I ] 2s (I )) 200 0.055 0.0306 0.0731 0.940, 1.100

4136.6(15) 8 2.363 10.463 2784 9.3 /51.4 3585 2108 (I ] 2s (I )) 259 0.060 0.0407 0.0856 0.928, 0.666

R1  S½½Fo½½Fc½½/S½Fo½; for 3a: Rw  [Sw (½Fo½½Fc½)2/Sw (Fo2)1/2; for 1, 2, 4, 5: wR2  [Sw (Fo2Fc2)2/Sw (Fo2)2]1/2.

Table 3 ˚ ) and angles (8) for 1 and 2 Selected interatomic distances (A 1

2

Pt(1)/Pd(1)
N(11) Pt(1)/Pd(1)
N(12) Pt(1)/Pd(1)
N(13) Pt(1)/Pd(1)
N(3)

2.04(1) 2.02(1) 2.04(1) 2.05(1)

2.051(2) 2.042(2) 2.004(2) 2.043(2)

N(11)
Pt(1)/Pd(1)
N(12) N(11)
Pt(1)/Pd(1)
N(13) N(11)
Pt(1)/Pd(1)
N(3) N(12)
Pt(1)/Pd(1)
N(13) N(12)
Pt(1)/Pd(1)
N(3) N(13)
Pt(1)/Pd(1)
N(3)

168.3(5) 84.4(4) 97.3(4) 85.2(4) 93.1(4) 178.2(4)

N(1)


O(2) a,b O(30)/O(1w) c


O(2) N(11)


O(30)/O(1w) d

2.88(1) 2.79(2) 2.93(2)

a b c d

Table 4 ˚ ) and angles (8) for 3a and 4 Selected interatomic distances (A 3a

4

Pt(1)
N(11) Pt(1)
N(14)/N(12) Pt(1)
N(3) Pt(1)
Cl(1)/O(11)

2.031(7) 2.035(7) 2.036(6) 2.313(2)

1.989(6) 2.038(5) 2.034(5) 2.082(5)

166.77(8) 84.51(8) 97.03(8) 84.42(8) 94.01(8) 178.42(8)

N(11)
Pt(1)
N(14)/N(12) N(11)
Pt(1)
N(3) N(11)
Pt(1)
Cl(1)/O(11) N(14)/N(12)
Pt(1)
N(3) N(14)/N(12)
Pt(1)
Cl(1)/O(11) N(3)
Pt(1)
Cl(1)/O(11)

83.8(3) 175.5(3) 93.7(2) 91.7(3) 177.1(2) 90.8(2)

84.2(2) 93.7(2) 176.1(3) 177.5(2) 96.1(2) 85.9(3)

2.798(3) 2.903(3) 3.055(4)

N(1)


O(2) a N(4)


O(2) b N(1)


O(11) c N(12)


O(2) d N(12)


O(2) e

2.767(8) 2.931(8)

x , y
1, z
1. x
1, y
2, z
1. x , y1, z . x , y
1, z .

a b c d

NMR spectrum displays two resonances at /2906 and /2920 ppm. This observation is reminiscent of findings of Arpalahti et al. who reported a doubling of 195Pt NMR resonances (4:6 and 3:7, Dd 11 /14 ppm) for N1 and N7 linkage isomers of (dien)Pt2
with adenosine at

e

2.943(9) 2.953(9) 3.055(9)

x
1, y
1, z . x , y
0.5, z
0.5. x , y1, z . x
1.5, y
0.5, z
1.5. x , y
1, z .

ambient temperature in D2O, and a similar doubling of aromatic adenine resonances in DMF-d7 at /50 8C [24]. Like these authors we suggest that either a conforma-

W. Bru¨ning et al. / Inorganica Chimica Acta 339 (2002) 400 /410

405

Table 5 ˚ ) and angles (8) for 5 Selected interatomic distances (A Pt(1)
N(3) Pt(1)
N(7) Pt(2)
N(11) Pt(2)
N(12) Pt(2)
N(13) Pt(2)
N(1)

2.025(7) 2.05(1) 2.035(9) 2.014(9) 2.000(8) 2.035(7)

N(3)
Pt(1)
N(7) a N(7)
Pt(1)
N(3) a N(11)
Pt(2)
N(12) N(11)
Pt(2)
N(13) N(11)
Pt(2)
N(1) N(12)
Pt(2)
N(13) N(12)
Pt(2)
N(1) N(13)
Pt(2)
N(1)

90.0(4) 90.0(4) 168.4(3) 84.0(4) 97.0(3) 85.1(4) 93.9(3) 179.0(4)

N(12)


O(2) N(4)


O(2) b

2.96(1) 2.71(1)

a b

x
1, y
1, z
1. x , y
1, z
0.5.

Fig. 6. View of cation of [(dien)Pd(CH
/N3 )](ClO4)2 ×/H2O (2) with atom numbering scheme. The cation of the corresponding Pt complex [(dien)Pt(CH
/N3 )](ClO4)2 ×/CH3OH (1) is very similar and is not shown.

tional change of the dien ligand or slow rotation of the nucleobase about the Pt
/N3 bond (with dien ligand fixed) are responsible for the observed doubling of resonances. Addition of free cytosine (2 equiv.) to an aqueous solution of 1 has no effect on the spectrum as long as the sample is kept at ambient temperature. However, prolonged heating (5 days, 50 8C) leads to formation of cytosine resonances of the N1 linkage isomer (ca. 5/ 6% of sum of 1 and free cytosine). Its abundance does not change further with time (24 days, 50 8C). Unlike 1, which displays 1H NMR signals of a single species at ambient temperature (resonances (B)) in D2O, the Pd analogue 2 equilibrates instantaneously with the N1 linkage isomer, the N1,N3 bridged species, and free cytosine. Although the N3 linkage isomer is by far the preferred one (
/90%), resonances of (A?), (C?) and free cytosine are clearly discernible in the 1H NMR spectrum (D2O, pD 5.4).

Fig. 7. Comparison of H bonding pattern between cations of 1 (top) and 2 (bottom) and solvent molecules CH3OH (top) and H2O (bottom). For details see text.

Figs. 8 and 9 give views of the cations of [(en)Pt(CH
/ N3 )Cl]Cl0.75(NO3)0.25 ×/H2O (3a) and [(en)Pt(CH
/ N3 )(ONO2)]Cl0.25(NO3)0.75 (4). Pt binding is again via the N3 site of the neutral cytosine ligand. Pt
/N, Pt
/Cl, and Pt
/O distances are normal; angles about the Pt atom deviate somewhat from an ideal geometry (Table 4). An interesting detail refers to the difference in Pt
/ ˚ ) and NH2(en) distances trans to Cl in 3a (2.035(7) A ˚ ), respectively, which trans to O NO2 in 4 (1.988(6) A reflects the differences in the trans -influence of Cl and O donors. Geometries of the cytosine ligands are as expected [12]. Cations of 3a form centrosymmetric pairs ˚ ). of H bonds between N1H and O2 (2.767(8) A Additional cation/cation hydrogen bonds involve O2

406

W. Bru¨ning et al. / Inorganica Chimica Acta 339 (2002) 400 /410

Fig. 8. View of cation of [(en)Pt(CH
/N3 )Cl]Cl0.75(NO3)0.25 ×/H2O (3a) with atom numbering scheme.

shifts. These resonances are tentatively assigned to species involving either N3,N4 bridging or exclusive binding to N4 [10f,10g]. There are many possibilities to explain the drop in pH*, e.g. formation of m-OH species, deprotonation at N1 and platination, deprotonation at N4 and platination, etc. An aged solution of [(en)Pt(HC
/N3 )(D2O)]2
reveals at least five new 195 Pt NMR resonances in addition to the resonance of the aqua species: Four of these new resonances are upfield from that of the starting species (/2376 ppm), namely at /2744, /2613, /2462, and /2589 ppm, while the fifth one is downfield (/2337 ppm). The latter is still in the range expected for a N3O coordination sphere, while the others are closer to N4 spheres (Fig. 10). No conclusive evidence concerning the nature of the condensation products formed is available at present. 2.5. N1,N3 bridged cytosinato complex 5

Fig. 9. View of cation of [(en)Pt(CH
/N3 )(ONO2)]Cl0.25(NO3)0.75 (4).

˚ ), thereby resulting in layers and N4 sites (2.931(8) A which extend in y and z directions. Unlike in 1, 2, and 3a, there is no cytosine, cytosine pairing in 4. Rather, N1 of cytosine forms an intermolecular H bond with O11 of the coordinated nitrate ˚ ), while O2 of cytosine acts as an acceptor for (2.943(9) A two H bonds of dien-NH2 groups of two different ˚ and 3.055(9) A ˚ ). All three cations (N12


O2, 2.953(9) A oxygen atoms of the ionic nitrate are likewise involved in H bonds, to NH2 of cytosine and NH (bifunctional bridge) of the amino group N12 of the dien ring. 195 Pt NMR shifts of freshly prepared samples of 3a (3b, 3c) in D2O (d //2605 ppm) are as expected for a PtN3Cl coordination sphere [22b,25]. The 195Pt NMR resonance of 4 at d //2376 ppm is consistent with a N3O set of donor atoms about the Pt [25], with O being an aqua ligand. Samples of 4 or of aqua species derived from 3a/3c and AgNO3 in D2O are acidic (pH* 3.5 / 3.8) and 1H NMR spectra (H6, 7.53 ppm, d, 3J /7.1 Hz; H5, 6.04 ppm, d; 2.68 ppm, s, b) undergo changes upon aging (decrease in pH* below 3; new cytosine resonances). If base is added (samples brought to pH* 6 /7), the changes are more rapid. Among the new 1H NMR resonances formed, doublets at 6.81 and 5.80 ppm are particularly noticeable because of their marked upfield

Reaction of trans -[(CH3NH2)2Pt(CH
/N3 )2](ClO4)2 [12] with 2 equiv. of [(dien)Pt(H2O)]2
in the presence of 2 equiv. of NaOH gave trans -[(CH3NH2)2Pt{(N3
/ C
/N1 )Pt(dien)}2](ClO4)4 ×/H2O (5) in high yield. Fig. 11 provides a view of the trinuclear cation and selected structural features are given in Table 5. In the solid state structure, the two cytosinato ligands adopt a head /tail arrangement. As expected from the way of preparation, the trans -(CH3NH2)2Pt2
entity is bonded to the N3 positions of the two nucleobases, and the (dien)Pt2
entities are bonded at N1. The cytosine planes are almost perpendicular to the central Pt coordination plane (85.5(4)8), while the dihedral angle between the cytosinate and the PtN3 plane of the dien ligand is 55.7(2)8. The Pt
/N1 and Pt
/N3 vectors form an angle of 114.3(3)8. Intramolecular H bonding involves the amino group N12 of dien and O2 of the cytosinato ˚ ). ligand (2.96(1) A

Fig. 10. 195Pt NMR spectra of [(en)Pt(CH
/N3 )(D2O)]2
, prepared from 3a and AgNO3: Sample after 14 days at room temperature pH* 2.7 (bottom) and 3 days later (top) after raising the pH* to 6.7 with NaOD.

W. Bru¨ning et al. / Inorganica Chimica Acta 339 (2002) 400 /410

407

Since spectra were recorded with a 400-MHz NMR spectrometer, no 195Pt coupling with H5 and H6 protons was observed. We note that we have previously unambiguously identified a case of cytosinato bridging, in [(NH3)3Pt(N3
/C
/N1 )Pt(dien)]3
, on the basis of 195Pt coupling to both H5 and H6 in spectra recorded on a 80MHz spectrometer [22a]. In the 195Pt NMR spectrum of 5 (D2O, pH* 5.6), three resonances are observed which on their relative intensities are assigned to Pt1 (/2539 (ht ), /2634 (hh) ppm) and Pt2 (/2798 ppm), respectively.

3. Experimental 3.1. Starting materials Fig. 11. Trinuclear cation trans -[(CH3NH2)2Pt{(N3
/C
/ N1 )Pt(dien)}2]4
(5) with atom numbering scheme.

As evident from Fig. 12, cations form infinite chains along the z axis by means of pairs of H bonds between ˚ ). The N4 and O2 sites of cytosinato ligands (2.71(1) A ˚ Pt1


Pt1 separations amount to 6.723(2) A. Perchlorate anions are linked to the cations via additional H bonds which involve N7, N11, N12, and N13 sites. Comparison of bond lengths and angles of the cytosinato ligand in 5 with those of cytosinato complexes carrying a PtII at either N1 or N3 [12] does not reveal any significant (D
/3s ) differences. This may, however, also be due to the relatively high standard deviations in 5. Both the 1H and 195Pt NMR spectra reveal that 5 exists in aqueous solution in two rotamer forms. Thus, both H6 and H5 resonances are observed as two doublets (3J /6.9 Hz) in a ratio of approximately 10:3, which in analogy to the starting compound trans -[(CH3NH2)2Pt(CH
/N3 )2]2
are assigned to head /tail (preferred species) and head/head rotamers (minor species). Chemical shifts (d [ppm], D2O, pH* 5.6) are as follows: H6, 7.694 (ht ), 7.688 (hh ); H5, 5.93 (ht ), 5.89 (hh ); dien, 3.23 /2.83, m; CH3, 2.15 and 2.14.

Fig. 12. H bonding interactions between cations of 5.

[(Dien)PtI]I [26], [(dien)PdI]I [27], (en)PtCl2 [28], and trans -[(CH3NH2)2Pt(CH
/N3 )2](ClO4)2 [12] were prepared as described in the literature; cytosine was purchased from Fluka. 3.2. Syntheses [(Dien)Pt(CH
/N3 )](ClO4)2 ×/CH3OH (1) was prepared by treating a suspension of [(dien)PtI]I (1.574 g, 2.85 mmol) in water (100 ml) with AgClO4 ×/H2O (1.272 g, 5.64 mmol) for 2 days at 40 8C with daylight excluded. Then the solution was cooled to room temperature, AgI filtered off, and cytosine (0.314 g, 2.82 mmol) was added. The mixture was stirred for 5 h at 80 8C and evaporated to near dryness. The precipitate was filtered off and recrystallized from CH3OH/H2O (9:1). Colorless crystals that formed were filtered off, briefly washed with cold CH3OH and dried in air (yield 24%). Anal. Calc. for C9H22Cl2N6O10Pt: C, 16.9; H, 3.5; N, 13.1. Found: C, 16.9; H, 3.4; N, 13.2%. 1H NMR (d ppm, D2O, pH* 6.1): 7.51 (d, 3J/7.3 Hz, H6), 6.02 (d, H5), 3.34 (CH3OH), 3.3 /2.8 (m, en). [(Dien)Pd(CH
/N3 )](ClO4)2 ×/H2O (2) was prepared analogously, with a shorter reaction time (1 h, 60 8C) for formation of the aqua species, however. The product was isolated upon concentration of the reaction mixture to 1/6 of its original volume and keeping the solution at 4 8C. Within 3 days the compound was isolated in 71% yield. Anal. Calc. for C8H20Cl2N6O10Pd: C, 17.9; H, 3.8; N, 15.7. Found: C, 18.1; H, 3.7; N, 16.1%. [(en)Pt(CH
/N3 )Cl]Cl0.75(NO3)0.25 ×/H2O (3a) was obtained by treating enPtCl2 (0.5 g, 1.53 mmol) with AgNO3 (260 mg, 1.53 mmol) in DMF (20 ml) for 2 days at 40 8C. After filtration of AgCl, cytosine (0.17 g, 1.53 mmol) was added and the mixture stirred for 5 days at room temperature. To the colorless, clear solution diethylether was added until the solution became turbid and then the solution was kept at /4 8C overnight. The precipitate that had formed then was filtered off, washed

408

W. Bru¨ning et al. / Inorganica Chimica Acta 339 (2002) 400 /410

with ether and dried in air. This crude material was recrystallized twice from 0.01 M HCl at 60 8C. Upon cooling (4 8C),colorless crystals of 3a were harvested (yield 14%). The composition of 3a (ratio of anions) was deduced from X-ray crystallography. Elemental analysis data were, however, in better agreement with the presence of Cl  and NO3 in a 1:1 ratio, hence [(en)Pt(CH
/N3 )Cl]Cl0.5(NO3)0.5 ×/H2O: Found: C, 15.5; H, 3.1; N, 16.4; Cl, 11.4% [(en)Pt(CH
/N3 )Cl]NO3 ×/H2O (3b) was prepared in an analogous way, with the crude product recrystallized from water (yield 8%). Anal. Calc. for C6H15ClN6O5Pt: C, 15.0; H, 3.1; N, 17.4. Found: C, 14.9; H, 3.0; N, 17.4% [(en)Pt(CH
/N3 )Cl]ClO4 (3c) was prepared in a similar fashion in DMF. Subsequently the DMF solution was concentrated on a rotavapor to small volume, ethanol was added in excess and after cooling (1 h, 4 8C) the precipitate was filtered off, washed with ethanol and diethylether and dried. Recrystallization from 0.01 M HClO4 (4 8C) gave colorless needles of 3c in 48% yield. Anal. Calc. for C6H13Cl2N5O5Pt: C, 14.4; H, 2.6; N, 14.0. Found: C, 14.3; H, 2.6; N, 14.0%. [(en)Pt(CH
/N3 )(NO3)](NO3)0.75Cl0.25 (4) was obtained in very low yield after treatment of 3c with 0.9 equiv. of AgNO3, filtration of AgCl and, upon concentration, subsequent filtration of some 3c from the acidified (HNO3, pH 0) filtrate. Crystals of 4 were characterized by X-ray crystallography. trans -[(CH3NH2)2Pt{(N3
/C
/N1 )Pt(dien)}2](ClO4)4 ×/ H2O (5) was prepared as follows: To a solution of trans [(CH3NH2)2Pt(CH
/N3 )2](ClO4)2 [12] (0.328 g, 0.48 mmol) was added [(dien)PtI]I (0.534 g, 0.96 mmol) in water (50 ml), followed by addition of AgNO3 (0.329 g, 1.92 mmol) and NaOH (9.7 ml of 0.1 M NaOH). The solution (pH 9.7) was stirred for 4 days at 50 8C in the dark, then AgI was filtered off, and the almost colorless solution was reduced to a volume of a few milliliters. Excess NaClO4 ×/H2O was added, which caused immediate formation of a precipitate. The precipitate was washed with a small amount of ice water and acetone and was then dried in air. Recrystallization of the crude material from a methanol/water mixture (4:1) gave 5 in 82% yield as pale yellow plates. Anal. Calc. for C8H46Cl4N14O19Pt3: C, 14.5; H, 3.1; N, 13.2. Found: C, 14.3; H, 3.1; N, 13.3%. 3.3. NMR spectroscopy 1

H NMR spectra were recorded on Bruker AC200 (200.13 MHz) and DRX400 (400.26 MHz) instruments in D2O with sodium-3-(trimethylsilyl)propanesulfonate (TSP) as internal reference. 195Pt NMR spectra were recorded on the AC200 (42.95 MHz) instrument with Na2PtCl6 (d /0 ppm) as external reference. pH* values refer to uncorrected pH meter readings (Metrohm 632).

pH* values were adjusted by addition of DNO3 or NaOD solutions; pKa values of cytosine and complexed cytosine were taken from plots of Dd versus pH* at the point of inflection. 3.4. X-ray crystallography Data collections of 1, 4, and 5 were performed on an Enraf /Nonius KappaCCD diffractometer [29] (Mo Ka, ˚ , graphite-monochromator). They covered l /0.71069 A the whole sphere of reciprocal space by measurement of 360 frames rotating about v in steps of 18. The detector was positioned in a distance of 28.6 (1), 26.8 (4), and 32.5 mm (5), respectively, to the crystal. Exposure times were 15 (1), 20 (4), and 40s (5) per frame. Preliminary orientation matrices and unit cell parameters were obtained from the peaks of the first ten frames, respectively, and refined using the whole data set. Frames were integrated and corrected for Lorentz and polarization effects using DENZO [30]. The scaling as well as the global refinement of crystal parameters were performed by SCALEPACK [30]. Reflections, which were partly measured on previous and following frames, are used to scale these frames on each other. Merging of redundant reflections in part eliminates absorption effects and also considers a crystal decay if present. Intensity data for 2 were collected on a Nicolet/ Siemens R3m/V diffractometer with graphite-mono˚ ) using the chromated Mo Ka radiation (l/0.71069 A v /2U-scan technique with variable scan speed (3 /158 min 1). Unit cell parameters were obtained from a least-squares fit of 20 reflections (5.2 B/U B/15.18). Three standard reflections measured every 100 data points showed no systematic variation in intensity. The data have been corrected for absorption, Lorentz and polarization effects using the data reduction program XDISK [31]. An empirical absorption correction was carried out via azimuth (c scans). X-ray measurements for 3a were carried out on a Rigaku AFC6S diffractometer using the same wavelength. Calculations were performed on a VAXstation 3520 and Silicon Graphics Indigo 2 Extreme computers using the teXsan 1.7 crystallographic software package [32]. Unit cell dimensions were determined applying the setting angles of 25 high-angle reflections. Three standard reflections were monitored during the data collection showing no significant variance. The intensities were corrected for absorption with c scans of several reflections with the transmission factors in the range 0.54 /1.00. All structures were solved by standard Patterson methods [32,33] and refined by full-matrix least-squares based on F 2 (1, 2, 4, and 5) using the SHELXTL-PLUS [31] and SHELXL-93 programs [34] or based on F (3a) using teXsan [32]. The scattering factors for the atoms in 1, 2, 4, and 5 were those given in the SHELXTL-PLUS program.

W. Bru¨ning et al. / Inorganica Chimica Acta 339 (2002) 400 /410

Transmission factors were calculated with SHELXL-97 [35]. Hydrogen atoms were included in calculated positions and refined with isotropic displacement parameters according to the riding model, except for the protons of the amine group in 5, which were assigned according to U (H)/1.5 U (Cbonded/Nbonded), and of all protons in 3a, which were not refined at all. Nonhydrogen atoms were refined anisotropically with the following exceptions: (a) The carbon atoms of the cytosine base and the three nitrogen atoms of the dien ligand as well as the two disordered oxygen atoms of one perchlorate anion (refined occupancies 35 and 65% each) in 1 in order to save parameters, because of the poor observed reflections to parameters ratio (7: 1), (b) the oxygen atoms of the nitrate anion in 3a, and (c) the oxygen atoms of all perchlorate anions in 5 (all with occupancies of 50%). Difference Fourier maps of structures 3a and 4 revealed three peaks, respectively, equidistant from the chloride anion, which resembled a nitrate moiety. It was then assumed that both the chloride and the nitrogen atom of the nitrate anion share the same position. Subsequent refinement resulted in population parameters of 75 and 25% for chloride and nitrate anions in 3a, and 25 and 75% for the anions in 4.

4. Summary This study on reactions of (dien)M2
(M /Pt, Pd) with unsubstituted cytosine (CH) confirms recent findings with trans -(CH3NH2)2Pt2
according to which N1 metal coordination occurs more frequently relative to N3 coordination than could have been expected on the basis of the tautomer equilibrium constant of the free bases [12]. With the free base the tautomer having a nonprotonated N3 site exceeds the one with a non-protonated N1 site by almost a factor of 1000 [13]. The fact that in the neutral pH range for the respective metal complexed forms this ratio is down to less than 10 implies that the N1 site is faster complexed that the N3 site. This is true both for (dien)Pt2
and (dien)Pd2
. It is likely that the larger steric hindrance of the N3 site (two exocyclic groups) over the N1 site (one exocyclic group) is important in this respect, very similar as demonstrated for a series of metal complexes of nucleobases carrying different substituents [36]. The situation is further complicated in that [(dien)M(H2O)]2
and [(dien)M(OH)]
species can be expected to react differently as well, with the hydroxo species capable of condensing with a NH site of the heterocycle, for example. Moreover, the distribution of these two species not only is pH-dependent (pKa of [(dien)M(H2O)]2
ca. 7.7 for M /Pd [37], and ca. 5.9 / 6.2 for M/Pt [38]) but also concentration */as well as (for M /Pt) time-dependent because of the possibility

409

of formation of unreactive m-hydroxo dimers [(dien)M(OH)M(dien)]3
[18b]. Although more quantitative studies in these systems are desirable, an important conclusion is evident from our findings already, namely that equilibrium constants of nucleobase tautomers are not necessarily a measure for the distribution of the respective metal complexes. Rather, metal binding to a rare tautomer may be kinetically promoted.

5. Supplementary material Crystal data have been deposited at the Cambridge Crystallographic Data Centre, CCDC Nos. 175000 / 175004. Copies of this information may be obtained free of charge from The Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax:
/44-1223-336033; e-mail: [email protected] or www: http:// www.ccdc.cam.ac.uk).

Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie. We thank Pablo Sanz for his help during the preparation of the manuscript and Michael Roitzsch for recording NMR spectra.

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