Coordination of 99Methyladenine to [cis-Pt(NH,),]*’ and [Pt(dien)]“’ as Studied by Proton NMR

Jeroen H. J. den Hartog, Hans van den Elst, and Jan Reedijk Department of Chemistry, Gorlaeus Laboratories, State University Leiden, L&den, The Netherlands

ABSTRACT

Reaction products of 9-methyladenine (mAde) with [Pt(dienJCl]CI and cis-RfNH3)$I~ have been

separated using CM-Sephadex C25 cation exchange chromatography. NMR and UV charaeter-

istics are presented; the platinum binding sites were established by studying the pH dependence of

the 1 H-NMR chemical shifts and of UV difference absorption. It is shown that the Nl atom of the

ligand can be protonated in Pt(mAde-iV7) adducts. while the N7 atom can be protonad in

Pt(mAde-N/J.

INTRODUCTION

The study of the binding of metal ions to nucleic-acids constituents is a field of incnasing

interest [ I]. Apart from the preference of certain sites in the qucleobases for certain metal ions, the kinetics of the binding are of interest, especially for slowly reacting metal ions, such as Pt(II), Co(W), and Cr(lII).

Nucleobases c& bind not only metal ions, but protons as well. The study of the metal binding as a function of pH is therefore of particular inte=st. After metal-ion binding, some sites may still be susceptible to protonation or deprotonation, and the study of the structure with spectroscopic techniques, such as NMR and UV, is more useful when performed at varying pH values.

Platinum compounds with purine bases are of special interest in view of the fact that this binding is generally accepted as playing a major role in the binding of the anti-tumor drug, cis-Pt(NH9)2C12 (cis-DDP) to DNA in vivo [2]. Not only the well-studied gua- nosine derivatives, but adenine fragments as well, may bind to platinum ions [2,3].

Address requests for reprints to Dr. Reedijk, Department of Chemistry. State University of L&en, P.O. Box 9502,230O RA Leiden. The Netherlands.

Jovnal oflnorganic Biochmhy 21,83-92 ( 1%) @ 1984 Elscvier science Publiig co., Inc.

52 Vaderbiit Ave., New Yodc, NY 10017

83

0162-0134@4/S3.00

04 J. H. J. den Hartog, H. van den Elst, and J. Reedijk

As early as 1973, Mansuy et al. [4], using UV difference spectroscopy, reported that either N7 or Nl can coordinate (they suggested a bidentate coordination with the exocyclic NH*, but that has not been observed in crystal structures [5]). UV difference spectroscopy was also used by Scovell and O’Connor for the determination of stability constants [6]. Kong and Theophanides showed N 1 or N7 coordination of adenosine to platinum using ’ H-NMR spectroscopy [7], and a study by Inagaki et al. [ 8) showed that possible adducts of cis-DDP or [Pt(NH,),CI]CI with adenosine (Ado) can be separated with the use of HPLC, and that the binding places can be established with UV difference spectroscopy at several pH-values.

Lim and Martin [9] used “C and ‘H-NMR chemical shifts of palladium diethylene- triamineadenosine complexes to assign the binding sites. Palladium compounds have been proven to be useful analogues in the study of platinum-nucleic acids interactions. Because of their high reactivity, equilibria are rapidly established and reliable stability constants can be gathered [lo]. Separation of possible adducts, however, may not be convenient, and at very high or very low pH the palladium complexes may decompose.

In this paper, we describe the study on the (separated) reaction products of 9- methyladenine (mAde) and cis-DDP or [Pt(diethylenetriamine)Cl]Cl, [Pt(dien)CI]Cl, (see Fig. I). The ligand mAde is used as a model compound for Ado. The structural difference between Ado and mAde is the replacement of the sugar moiety by a methyl group in the latter. We are especially interested in the behavior of the ’ H-NMR chemical shifts as a function of pH, because “pH-dependent NMR” is now the main method used to prove the binding sites of platinum compounds in oligonucleotides [I I]. In addition, we studied Pt(dien)(mAde) complexes with UV difference spectroscopy at pH values between - 1 and 15. because we wanted to gain more insight into the titration behavior of the compounds at extreme pH values. N 1 and N7 protonation in uncomplexed mAde and in N7 or N 1 platinated mAde are shown. The exocyclic NH2 deprotonation is strongly influenced by di-platinum binding, as reported earlier by Inagaki et al. [8] in Ado compounds.

EXPERIMENTAL

Adenine was purchased from Sigma Biochemical Co. and was methylated at N9 according to Kruger [ 121. cis-DDP and [Pt(dien)CI]Cl were prepared as described in the literature [ 13, 14). The latter was precipitated from water by adding a highly concen- trated solution of [Pt(dien)Cl]+, Cl-, N03-, Na+ to a large volume of acetone, prevent- ing the inclusion of NO,- as the anion.

The reaction of mAde and [Pt(dien)Cl]Cl was performed with a slight excess of mAde (mAde:Pt = I .2: 1.1); the reaction with cis-DDP and mAde was performed with a large excess of mAde (mAde:cis-DDP = 5: 1). The reaction mixtures were stirred and heated to 50°C. After 24 hr at 50°C (pH between 6 and 7.5. IO-) M/ 1 mAde), the mixtures were concentrated to IO ml and the adducts were separated on a CM Sephadex C25 cation exchange resin column (15 x 2.5 cm) eluted with 0.0-1.0 M/I triethylammonium hydrogencarbonate (TEAB) as a linear gradient. TEAB is a volatile salt and can be removed from Pt(mAde) products by evaporation. Unfortunately, products with a charge of 4+ or higher, such as {Pt(dien)}2(p-mAde), cannot be eluted from the column, not even with 2M TBAB, so these can only be eluted by at least 0.5 M/I NaCl.

NMR spectra were recorded on a Bruker WM-300 spectrometer. Samples were prepared by lyophilizing the products 2 times from 99.8% DZO and dissolving them in

9-mAde Coordination to c&F? and Pt(dien)

H HzN

J

85

Cl

FIGURE 1. Schematic sfructures of mAde. cis-DDP and (Pt(dien)Cl]Cl

99.95% D,O. The residual HDO signal was reduced by selective irradiation. The pH of the samples was adjusted in the sample tubes by adding small quantities of concentrated NaOD or DC1 solutions and was measured before and after each NMR experiment. The pH, reported as pH*, is not corrected for the deuterium isotope effect because pK,s measured in H,O and D,O are virtually the same [ 15). At the end of each titration experiment, the pH* was brought to 10.5, and after heating for one hour at 70°C the H8 protons were largely exchanged with D20, allowing their assignment. Tetramethylam-

monium chloride was added as an internal reference (chemical shift 3. I8 ppm relative to DSS (4,Cdimethyl+silapentanesulphonic acid)), but chemical shifts are reported with respect to DSS.

UV difference spectra were recorded on a Beckman DB spectrophotometer with a reference cell at pH 6, and the sample cell at various pH, within the range I 5 pH I 13. After each run, the pH in the sample cell was brought again to pH 6 in order to check that the compound had not decomposed, and that the UV difference was not due to dilution in one of the cells. Exteme low or high pH (pH zz 1, pH 2 13) was achieved by adding a

concentrated (small volume) solution of Pt(dien)(mAde) compound to a IOM, IM. or 0. IM HCI or NaOH solution. The results were checked extensively by measuring water against concentrated HCI or NaOH solutions with and without added Pt(dien)(mAde) product.

Nomenclature

The common nomenclature for inorganic compounds is combined with the one accepted last year on nucleic acid constituents and abbreviations [ 161, i.e., the metal is listed first in formulae, followed by the ligands. The coordinating atom of the ligand, in case of ambiguity, is indicated-after the ligand-separated by a hyphen and underlined or in italics, according to ref. 16. Bridging ligands are indicated with the prefix cc-. cis-Pt is used as an abbreviation for a coordinating c~~-P~(NH,),~+.

RESULTS AND DISCUSSION

Reaction of [Pt(dien)CI]Cl with mAde

The UV profile achieved during the separation of the reaction products of mAde and Pt(dien) is shown in Figure 2. As can be seen, species II and III in the middle peak are not

J. H. J. den Hat-tog, H. van den Elst, and J. Reedijk

FIGURE 2. Elution pattern of separation of reaction products of

mAde and [Pt(dienKIlCI on a Sephadex C25 column. (eluens

0.0-0.7 M NaCl in doubly distilled water)

I 1 I I 1

0 20 LO 60 60

fraction number )

completely separated, but the front and the back-sides of the total peak appear to have a different UV spectrum (see Table l), indicating at least two different species. Because the separation on the column is mainly based on charge differences, the compounds are to be expected in the following sequence: Peak I: “free,” unreacted, mAde (zero charge), Peak II/III: Pt(dien)(mAde-N7) and Pt(dien)(mAde-NI), both with charge 2+, Peak IV: {Pt(dien)}z(p-mAde-N7, Nf) with charge 4+. The UV characteristics are shown in Table I. A,,,, A,,, and A-/A,,, are typical for each compound, therefore the values in Table 1 can be compared with those reported by Inagaki et al. [8]. This makes clear that peaks I and IV indeed contain the species mAde and {Pt(dien)),(p-mAde- Nl ,N7), respectively, and that peak II is likely to be Pt(dien)(mAde-NT), while peak III contains Pt(dien)(mAde-NI).

The proposed structures have been proven by studying the pH dependence of the chemical shifts. The results are shown in Figure 3; the numbers I-IV in this figure correspond with those in the elution pattern. In between pH 5 and 10, no change in chemical shift is seen in any compound. Going to acidic pH, “free” mAde (Fig. 3-I) exhibits its well-known [ 171 N 1 protonation (pK, 2 4). In contrast to all other species,

{ Pt(dien)},(p-mAde-N I, N7) does not show any change in chemical shift down to pH*

TABLE 1. UV Characteristics (A in nm, A in OD) of the Species Isolated from the Reaction of mAde with [R(dien)CI]Cl

I 262 0.66 229 0.12 5.4 II 266 0.64 238 0.23 2.8 III 263 0.36 239 0.16 2.3 IV 270 0.63 247 0.43 1.5

“The values of A _tUldA,i,i3PEarbi~.

9-mAde Coordination to cis-F’t and Pt(dien) 87

6ippm)

9.0

6bpml

9.0

8.5 8.5

-*

-&-_.

s o-0-0 8.0 8.0

L.0

i

L.C

-\4--6 _j .-=

1 3 5 7 9 11 13pH*

III

L $1 ‘. ---_

8.5

t\

1 \ --a

8.0

t :=

i LO

1”-,y;, -_ I;

1 3 5 7 9 11 13pl-c

1

I 1 ‘-

1

/ :- L

1-e

,--

I--

:

1

/ = L

6 (ppml

9.0

8.5

8.0

4.C

: -r- --r--7

1 3 5 7 9 11 13pH*

1 3 5 7 9 11 13pH*

!V

AU&b-A-&A

“%,

FIGURE 3. I H-NMR chemical shift (6). relative to DSS, versus pH* of Pt(dieN products. Open symbols: HS, closed symbols: H2, symbol with stick: H3 -C9

peak symbol species

I P InAde

II 0 R(dien)(mAde-N7)

111 0 Pt(dien)(mAde-N/J

IV d {Pt(dien))*tr-mAde-NI,N7)

J. H. J. den Hartog, H. van den Elst, and J. Reedijk

0.6 (Fig. 3-W), as can be expected because the two basic sites are platinated. Species I1 and III (Figs. 3-11 and 3-111, respectively) both show a chemical shift change upon acidification, although species II protoriates clearly at a less acidic pH. This indicates that species II must be Pt(dien)(mAde-N7), because N I is known to be more basic than N7. Species III is now deduced to be Pt(dien)(mAde-NI). The protonation, occurring below pH* 2, is concluded to be the N7 protonation, because the H8 proton exhibits the largest downfield shift (Fig. 3-111).

At basic pH, free mAde is known to lose a proton from the exocyclic NH2 (pK, 2 14). which deprotonation is also influenced by platination [8]. Especially the di-platinated adduct {Pt(dien)}z(p-mAde-NI,N7) shows a dramatic lowering of this pK, to a pH* of about 1 I .3 (Fig. 3-IV). The Nl platinated adduct (Fig. 3-111) also exhibits at pH* > 12 a small upfield shift, most likely originating from the onset of deprotonation of the exocylic NH?, confirming its assignment. Both observations are in agreement with those of Inagaki et al. (81, on adenosine compounds.

In order to gain insight into the behavior of these compounds at an even more acidic or basic pH, UV difference spectroscopy was performed. The results are shown in Figure 4. Between pH 1 and 13, the titration curves obtained here, closely resemble those of the

FlGURE4. UV difference-pH profiles for Pt(dien) compounds (species I to IV). Symbols and numbers are the

same as used in Fig. 3: I = mAde: II = Pt(dien)(mAde-N7): Ill = Pt(dien)(mAde-NI); IV = {Pt(dien&

(Cc-mAde-N/N). On the left-hand side (pH < 7) in the figure. the signal intensities are reduced (multiplica-

tion factor n), on the right hand side (pH 17). the signal intensities are enhanced by a factorof 2 for species II

and IV.

d

A-A

“‘V

AA P A

OB- 0

0.6- \ .A-A-A-A-A-a-A-A'

OL-

0.2.

o.o-

-0.8

- 0.6

-0.C

-0.2

-0.0

\, I’/ ”

n=0.3

B, /

0 0

0

\

4 0

Q&O- O-O-O_O-O---”

II n:l q

n=0.3 1

v 0

\

/

i

)+. --- -0 o Do--

06

0.8-

06-

OL-

0 2-

002

I -1 1 3 5 7 9 11 i3 15 pH

9-mAde Coordination to cis-Pt and F’t(dien) 89

NMR experiments and are largely in agreement with those of Inagaki et al. [8] for Ado, although he did not observe a protonation at pH 5 2 in Pt(Ado-Nl) species. Below pH I, “free” mAde gives a UV difference which is tentatively assigned to the N7 protonation. The protonation of the N7 in species III: Pt(dien)(mAde-N1), can now be compared with

the protonation at N7 in mAdeH+ (already containing a proton at Nl). It appears that the replacement of the proton at NI in mAdeH+ by a less polarizing Pt(dien)I+ in Pt(dien)(mAde-N1) results in a more basic N7 position! It is noted that the protonation at the NI occurs at lower pH. when platinum is attached at N7, compared with “free” mAde [I. 81.

Another interesting feature is seen in the UV difference profile of species IV. The endpoint of the titration curve for the exocyclic NH2 deprotonation is even reached and the estimation for the concomitant pK, yields 1 I .O.

Reaction of ck-DDP with mAde

The UV profile obtained during the separation of the reaction products of mAde and cis-DDP, with the use of a 0.0-I .O M TEAB gradient is depicted in Figure 5a. The one peak II-IV was further separated using a OS-O.9 M TEAB gradient, which resulted in Figure 5b. Note that the numbers II, III, and IV refer to different species with respect to the former section on Pt(dien) compounds.

The UV characteristics of the four species are given in Table 2. Peak I contains mAde, which has zero charge. Peaks II. III, and IV are the three expected species: cis-Pt(mAde- N7),, cis-Pt(mAde-Nf), and cis-Pt(mAde-N/)(mAde-N7), all with charge 2+. Other

FIGURE 5. Elution pattern of separation of reaction products of mAde and cis-DDP on a Sephadex C25 column

(a) eluens O.&l .O M TEAB. (b) eluens 0.5-0.9 M TEAB in doubly distilled water.

I I I 1 I I I I I I

0 ‘LO 80 0 LO 80 FRACTION FRACTION NUMBER NUMBER

90 J. H. J. den Hartog, H. van den Elst, and J. Reedijk

TABLE 2. UV Characteristics (X in nm, A in OD) of the Species Isolated from the reaction of mAde with cis-DDP.

I 260 0.54 228 0.12 4.5

II 262 0.54 240 0.29 1.6

III 265 0.95 236 0.3 I 3.1

IV 261 0.62 240 0.3 I 2.0

aThe values of A _andA,i,marbitrary.

products are not expected because of the 5-fold excess of mAde over cis-DDP used in the reaction.

Definite assignments again are based on the behavior of the chemical shifts as influenced by the pH. The results are shown in Figure 6; the numbers in the figure correspond with the ones in Figure 5. In Figure 6-1, the titration curve of “free” mAde is shown. The curves of Pt(dien)(mAde-N7) in Figure 3-11 closely resemble cis-Pt(mAde- N7)2 in Figure 6-111 (right upper comer). The same holds for Pt(dien)(mAde-Nf) (Fig. 3-111) and cis-Pt(mAde-iVI)Z (Fig. 6-W), confirming these structures. In Figure 6-B (right lower comer) the spectral results of the mixed species cis-Pt(mAde-N/)(mAde- N7) are shown. Each mAde ligand clearly exhibits its own titration pattern; only chemical shifts are influenced by the other complexed base. Thus, in each base where platinum is bound via N 1, and the N7 is free for protonation, the N7 indeed protonates below pH* 2. In Ado, it might well be possible that the N7 protonation occurs at a different pK,, which explains why Inagaki et al. [8] did not observe this protonation. However, in a study by Scheller et al. [IS], the H8 of the Pd(dien)(Ado-Nf) adduct clearly shows a downfield shift change below pH* 2, suggesting that in Ado this protonation does indeed occur.

NMR Chemical Shifts

’ H-NMR chemical shifts at pH* 7 of all described species are tabulated in Table 3. Scrutinizing the chemical shifts, one can say that binding of a platinum compound to mAde at N7 results in a downfield shift of the H2 proton of somewhat more than 0.1 ppm and of the H8 of almost 0.6 ppm. Binding to N 1 expectedly results in a strong downfield chemical shift change of H2 (approximately 0.5 ppm), except for cis-Pt(mAde-NI),. In this case, the downfield shift amounts to only 0.12 ppm. which difference is most probably due to ring currents of the neighboring aromatic six-membered ring. The downfield shifts of the H8 protons in the three Nl coordinated species amount to 0. I ppm. The chemical shift of the dinuclear species appears to be almost the additive of the mononuclear species, and is 0.69 ppm for H8 and 0.64 ppm for H2.

DISCUSSION

In the study presented here, the predominant reaction products of mAde and cis-DDP or

[Pt(dien)Cl]Cl are systematically described. Based on the pH dependence of the NMR chemical shifts one can discriminate between the complexes in which platinum is bound via N7 and/or N 1. The unbound N I nitrogen in Pt(mAde-N7) can be protonated below

9-mAde Coordination to cis-Pt and Pt(dien) 91

9.0 1 I

8.5

Z’\ \ .-.- ‘b-m

8.0

, , , 1 3 5 7 9 11 pH*

a.*, “1 O-0-0-^ 8.5 ‘\

\ .-.-.-.-.

6 (ppm 1

9.0--

8.5--

8.0--

. IV

L_____. L --

6hwm) 1

4

9.0 -- 11

--m o-o-d-

-.--

---0a 8.0--

,‘. 1

1 3 5 7 9 11 pH* 1 3 5 7 9--l<H’

FIGURE 6. 1 H-NMR chemical shift (6). relative to DSS, versus pH* of cis-pt products. open symbol, closed symbols. H2; symbol with stick, H&9.

Peak I III IV 11

species

mAde cis-Pt(mAde-N7)2

cis-Pt(mA&-NI),

cis-Pt(mAdexV/)(mAde.-N7)

92 J. H. J. den Hat-tog, H. van den Elst, and J. Reedijk

TABLE 3. ‘H-NMR Chemical Shifts (6) in ppm, relative to DSS, of the Base Protons of cis-Pt(mAde) and Pt(dien)(mAde) Compounds Measured at pH* 7”

Species Proton H2 H8

mAde Pt(dien)(mAde-NI)

Pt(dien)(mAde-N7)

{Pt(dien)}z(p-mAde-

Nl,N7) cis-F’t(mAde-NI),

cis-Pt(mAde-N7J2 cis-Pt(mAde-NI)-

(mAde-NT)

8.22 8.06 8.70 8.15 8.34 8.63 8.86 8.75

8.34 8.12

8.35 8.60 8.7Oi8.35 8.16/8.60

aRing numbering is given in Fig. I.

pH* 3, while the N7 in Ft( mAde-Nf ) can protonate below pH* 2. These observations are significant for the characterization of the Pt adducts with oligonucleotides containing

adenine as a base.

This research was supported by rhe Netherlands Foundarion for Chemical Research (S.O.N.) with$nancial

aid from rhe Netherlands Organisarion for rhe Advancement of Pure Research (Z.W.O.) and by grant

(MBL-83-l) from the Koningin Wilhelmina Fends (K. W.F., Dutch Organisarion for Fight Against Cancer).

The authors thank Mr. C. Erkelens for his assistance aI the 300 MHz NMR faciliry at Leiden. Experimenral

assistance by P. van Klaveren and L. J. Rinkel is gratefully acknowledged.

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2.

3.

4.

5.

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K. H. Scheller, V. Scheller-Kmttinger. and R. B. Martin, J. Am. Chem. Sot. 103.6833 ( 1981). ILJPAC-IUB Joint Commission on Biochemical Nomenclature, Recommendations (1982) Eur. J. Bio-

them. 131.9 (1983).

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