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Bioconlugate Chem. 1904, 5, 316-320
Platinum(I1)-Adenosine Phosphothiorate Complexes: Kinetics of Formation and Phosphorus-31 NMR Characterization Studies Lori L. Slavin,+ Elizabeth H. COX,*and Rathindra N. Bose' Chemistry Department, Kent State University, Kent, Ohio 44242. Received October 5, 1993"
Reactions of chloro(diethylenetriamine)platinum(II) chloride with adenosine 5'-O-thiomonophosphate, adenosine 5'-0-(2-thiodiphosphate), and adenosine 5'-0-(3-thiotriphosphate) yielded exclusively (phosphothiorato)platinum(II) complexes. Phosphorus-31 NMR data for the coordinated phosphothiorate phosphorus atom exhibited about 15-20 ppm upfield chemical shift compared to chemical shifts for, free nucleotides. Uncoordinated phosphate groups exhibited insignificant changes in the chemical shift upon complexation. Likewise, proton NMR data indicate no significant changes in chemical shift for the purine or ribose protons. Reactions between phosphothiorates and the platinum complex predominately take place through a second-order process, first order with respect to each of the reactants indicating that the aquated pathway contributes insignificantly toward complexation. The second-order rate constants, 1.9 f 0.1 M-' s-l for the AMP-S, 2.4 f 0.2 M-l s-l for the ADP-p-S, and 2.7 f 0.2 M-' s-1 for the ATP-7-Sreactions were evaluated at pH 6.5 and at 25 "C. These rate data were compared with those reactions of adenosine 5'-monophosphate (AMP) and guanosine 5'monophosphate (GMP) with the same platinum(I1) complex. These reactions proceed through the direct interaction between the starting platinum complex and nucleotides as well as through the reaction between the aquaplatinum complex and nucleotides. The rate constant for the aquation process was evaluated to be (2.0 f 0.1)X lo4 s-1 for both AMP and GMP reactions. Second-order rate constants M-ls-l for the for the direct reaction with the chloro complex were calculated to be (1.5 f 0.1) X GMP and (6.0 f 0.3) X lo3 M-ls-l for the AMP reaction at 40 OC.
INTRODUCTION A great deal of effort has been devoted to understanding the role of metal ions in phosphate hydrolyses catalyzed by many metalloenzymes-ranging from phosphokinases to polymerases (1-5). Inert metal ions such as Cr(III), Co(III), and Rh(II1) have been utilized to understand the roles played by metals in ATP hydrolysis (1-5, 6, 7).All these metal ions promote hydrolysis in highly basic solutions, but none of these metals catalyze hydrolysis significantly at or near physiological pH (6). Earlier, we showed that platinum(I1) catalyzes the hydrolysis of inorganic polyphosphates in acidic solutions (8). Recently, it has been demonstrated that platinum complexes promote hydrolytic cleavages of peptides (9). Since platinum(I1) aqua complexes are fairly acidic, presumably these hydrolyses are initiated through the hydroxyl transfer reactions from the metal center to the phosphate moiety. These coordinated acidic water molecules offer a unique advantage over the other tripositive inert metal centers in that mechanistic ambiguities between the coordinated hydroxyl transfer and involvement of the free hydroxide can be removed. Platinum(II), however, cannot be utilized in promoting the hydrolysis of NTP since nitrogen of the purine and pyrimidine bases favor coordination over the phosphate and coordination to the phosphate moiety is a prerequisite for hydrolysis. In order to circumvent the base coordination, a phosphothiorate group can be introduced to the adenosine nucleoside to facilitate platinum binding through the sulfur atom. Here, we report the kinetics of formation and the phosphorus3 1NMR characterization of platinum(I1)phosphothiorato
* To whom correspondence should be addressed.
+ Present
address: Department of Chemistry, Austin Peay State University, Clarksville, TN. t Undergraduate participant. Abstractpublishedin Advance ACSAbstracts, June 15,1994. @
complexes utilizing AMP-S, ADP-8-S, and ATP-$3 (Figure 1)and compare these rate data with those of AMP and GMP coordinations. Platinum(I1)-phosphothiorate chemistry has been exploited in several areas of cell and molecular biology. Lippard and co-workers (IO)have explored the application of platinum-bound phosphothiorate nucleotides for DNA sequencing by using electron microscopy. Chou and Orgel (11) have used trans-diamminedichloroplatinum(I1) to cross-link double-stranded oligonucleotides. These workers also used PtC1d2- to promote covalent cross-linking between double-stranded DNA and protein. The extent of platinum(I1)-phosphothioratecomplex formation and the roles played by this metal center to cross-link proteins and nucleotides are not understood. Phosphorus-31 NMR presented here would be useful to characterize platinumphosphothiorate complexes, and their possible roles in cross-linking oligonucleotides and nucleotide-protein can be explored. EXPERIMENTAL METHODS
Reagents. Chloro(diethylenetriamine)platinum(II)chloride was synthesized following the literature method (23). The corresponding aqua complex was prepared in situ by adding 2 equiv of AgN03 or AgC104. This aqua complex 2) to avoid was generated in acidic solution (pH dimerization. All nucleotides, AMP, GMP, AMP-S, ADP0-s,and ATP-7-S (Sigma) were of the highest quality available and used without further purification. Nuclear magnetic resonance experiments were carried out in DzO (99% atom) (Sigma). Sodium perchlorate was prepared by neutralization of HC104 with Na~C03. Physical Measurements. Rate Measurements. Reactions between nucleotides and platinum complexes were followed on a UV-vis spectrophotometer (Perkin-Elmer, Lambda 600) at 260 and 300 nm. The temperature was
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Platinum(1I)-Adenosine Phosphothlorate Complexes
Table 1. Rate Data. for the Reaction of Pt(dien)Cl+with Adenosine Phosphothiorateb Nucleotides thio-nucleotide [Ptl, mM [Nul, mM k p , M-l s-l AMP-S 1.0 4.0 1.7 2.0 2.0 4.0 2.0 1.9 6.0 2.5 4.0 ADP-8-S 2.0 2.2 2.0 4.0 2.6 5.0 15.0 2.7 ATP?-S 2.0 4.0 2.0 6.0 2.6 5.0
15.0
2.8
Determined by a nonlinear least-squares computer fit of eq 2. *At pH 6.8, T = 25.0 O C , p = 0.50 M NaClOd.
Table 2. Rate Dataa for the Reactions of Pt(dien)Cl+with AMP and GMP at 40 OC, pH = 6.8, p = 0.50 M (NaClO4) nucleotide [nucleotide], X 109 M k. X l(r,s-l AMP 10.0 2.7 20.0
GMP
I
0- P O - P - O I I 0 0
O
?d
A-oc;
3‘
2
OH OH
(C)
Figure 1. Structures of the phosphothiorato analogues of adenosine nucleotides: (A) adenosine 5’-O-thiomonophosphate (AMP-S), (B)adenosine 5’-0-(2-thiodiphosph) (ADP-I%), and (C) adenosine 5’-0-(34hiotriphosphate) (ATP-?-SI.
kept constant at 25.0 f 0.1 OC by an Isotemp thermostat (Fisher). Reactions were followed under both pseudofirst- and -second-order conditions utilizing excess nucleotides over the platinum complex. For the first-order kinetic curves, the rate constants were evaluated from an iterative nonlinear least-squares fit of absorbance as a function of time according to the equation
+
A = ( A , - A,)e-kot A ,
k 2 [ B ] ( Am - A,)
e-(k2[B1 -kZ[A])t
k,[Bl ekZfBlt - k 2 [ A1
10.0 20.0 30.0
3.8 4.9 6.4
points were selected. Acquisition times lie in the range 200-500 ms, and 500 transients were usually necessary to observe a signal with S/N > 10 using 2.0 mM nucleotide solutions. A line-broadening factor of 2 or 3 Hz was introduced before Fourier transformation. A much longer delay time between pulses was employed to ensure >95% relaxation when P-31 resonances were subject to integration. For the proton spectra, smaller pulse widths of 12 ps with a narrower window of 2000 Hz were utilized; 32-64 accumulations were sufficient to generate spectra of S/N > 10 for solutions containing 2.0 mM nucleotides. The pH of the reaction mixture was adjusted with dilute NaOD and DN03 solutions in D2O. The pH correction was made by applying the relationship (14) pH = pD + 0.4, where pD is the pH meter reading in D2O solution consisting of 99% deuterium atom. The pK, values of thionucleotide complexes were estimated from the pH-6 profile according to (15) (3)
(1)
where A,, A , and A , are absorbances at time t = 0, at time t , and at infinite time, respectively, and k , is the firstorder rate constant. For the second-order reactions, eq 2 A=A,+
2.9 3.5
DPlatinum concentrations lie in the range (1.0-2.0) X 1W M. Nucleotide concentrations were a t least 10-fold excess over the platinum complex.
H+$JH
I
30.0
-1 (2)
was utilized to fit the absorbance-time data. Platinum and nucleotide concentrations are expressed by [AI and [ B ] ,and k2 is the second-order rate constant. N M R Measurements. Nuclear magnetic resonance spectra were obtained on a GE 300-MHz (GN 300) instrument. Proton signalswere internally referenced with respect to the H-O-D resonance at 4.67 ppm, and the P-31 signals were referenced with respect to 85% H3P04. For a typical P-31 experiment, usually a 90’ pulse for 25 ps was applied with a delay time interval of 1.0 s. Frequency windows of 4000-10 000 Hz with 8-16 K data
where 6 is the observed chemical shift, and 61 and 62 are the chemical shifts of the protonated and deprotonated forms. Equation 3 was derived assuming a monoprotic behavior (15). RESULTS
Reactions of Pt(dien)Cl+ with AMP-S, ADP-84, and ATP-7-S exhibited increases in absorbance at 300 and 260 nm. The absorbance-time traces with 2-3-fold excess of thionucleotides over the platinum complex can be adequately fitted to the second-order rate expression, eq 2. Second-order rate constants (Table 1) for the phosphothiorate reactions with the platinum complex are evaluated to be 1.9 f 0.1 M-l s-l, 2.4 f 0.2 M-ls-l, and 2.7 f 0.2 M-l s-1 for the AMP-S, ADP@, and ATP-+ reactions. Reactions of Pt(dien)Cl+ with AMP and GMP were carried out under pseudo-first-order conditions utilizing excess nucleotide. First-order rate constants, k,, as
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Bloconjugate Chem., Voi. 5, No. 4, 1994
A
I
Table 3. Phosphorus-31 Chemical Shifts for the Pt(dien) (AMP-S) Complex as a Function of pH chemical shift of coordination chemical PH the complex, ppm shift,"ppm 1.04
1.10 1.30 1.70 2.08 2.96 4.30 5.79 6.23 7.34 9.14
40.85 40.68 40.44 40.05 37.45 33.43 32.64 32.70 32.70 32.71 32.17
Coordination chemical shift =,1,,6, appeared as a broad signal.
l
i
'
i
'
50
/
'
'
i
'
l
'
i
'
30
40
'
l
'' ppm
I " " I ~ ' " I ' ' " I ' " ' I ' '
45
40
35
30
ppm Figure 2. (a) Proton-decoupledphosphorus-31NMR (121.5 Mz) spectrum of a reaction mixture containing 4.0 mM [Pt(dien)ClIC1 and 6.0 mM AMP-S at pH 6.5 after 10 min of mixing. The signal B is for the free AMP-S and A is for the Platinum(I1)S-AMP complex. (b) Spectrum of the same reaction mixture at equimolar concentration (10 mM each). Note that no unreacted AMP-S remained as evidenced by the complete depletion of the signal B. Platinum-195 satellites are exhibited around A. 25
functions of [AMP] and [GMP] exhibited a familiar twoterm rate law 16
k, = k, + k2[Nul
(4)
where [Nu] represents the nucleotide concentration. The value of k, obtained for the AMP reaction, (2.0 f 0.1) X 10-4 s-1, agrees closely with the same value evaluated for the GMP reaction. The constant, k,, can be taken as the rate constant for the equation of the platinum complex. The values of the second-order rate constants (k2)for GMP and AMP reactions, (1.5 f 0.1) X s-1 and (6.0 f 0.3) X 10-3 M-1 s-1 were evaluated. Note that the value of k2 for the GMP reaction is about 2.5 times higher than that of the AMP reaction. The magnitude of the second-order rate constant for the thionucleotide is about 50 times greater than that for the GMP and AMP reactions. Since kpl [thionucleotidel
-19.04 -18.40 -17.71 -17.09 -17.63 -20.72 -19.60 -15.10 -13.70 -13.30' -13.30'
- 8fTw ligand. Free ligand
at its lowest concentration is greater than k, by a factor of 30, inclusion of a parallel first-order path contributes insignificantly to the data-fitting procedure outlined earlier. Products were characterized by proton and phosphorus31 NMR spectroscopy. The proton NMR spectrum of GMP exhibits a signal at 8.02 ppm for the H(8) of the purine ring. When the reaction of Pt(dien)Cl+ with GMP was followed at various time intervals, a new signal at 8.56 ppm grew initially at the expense of the peak at 8.02 ppm and then leveled off after 6 h. This new signal at 8.56 ppm is taken as evidence of GMP coordination through N7 of the purine ring (17). Similarly, AMP complexation is accompanied by the appearance of a new resonance at 8.76 ppm for the H(8) of the coordinated AMP molecule at the expense of the H(8) resonance of free AMP at 8.15 PPm. The reactions of phosphothiorate were essentially over in the time required to mix the reactants, place samples in the NMR tube, and record spectra. Free AMP-S exhibits a P-31 resonance at 46.40 ppm at pH 6.63 (Figure 2a). When 10.0 mM Pt(dien)Cl+ was mixed with equal concentration of the phosphothiorate, a new signal at 32.70 ppm appeared, and no unreacted nucleotide was observed by P-31 spectroscopy (Figure 2b). When a 2-3-fold excess of nucleotide over the platinum complex was employed, no additional resonances for the product other than the one at 32.70 ppm were observed (Figure 2a). Furthermore, integrated peak areas indicate that the reaction essentially follows 1:l stoichiometry. The proton NMR spectra recorded at regular time intervals did not exhibit any alteration of the H(8) signal. No changes in chemical shifts of ribose protons were apparent during the reactions. Furthermore, a direct coordination through the phosphate oxygen in a monodentate fashion can be ruled out since such a coordination is accompanied by a 4-10 ppm downfield shift of the P atom of the coordinated phosphate group (18). Proton and phosphorus NMR data indicate that nitrogen atoms of the purines are not involved in coordination. The signal at 32.70 ppm can be attributed to the Pt(dien)-S-AMP complex in which the nucleotide is coordinated through the sulfur atom. The chemical shift of the P-31 resonance of Pt(dien)-S-AMP complex decreases with pH up to pH 5 but levels off above pH 5. These data were utilized to estimate the K, value of the complex by using eq 3. The pH chemical shift data are shown in Table 111. Figure 3 shows the P-31 spectrum recorded for ADPp-S (6 mM) and Pt(dien)Cl+(5 mM) 10 min after mixing. Two doublets, A and B, at 37.8 and -8.6 ppm are for the p- and a-phosphorus atoms of the unreacted nucleotide, and C and D at 20.23 and -9.09 ppm are the corresponding
Bioconjugate Chem., Vol. 5, No. 4, lQ94 310
Platinum(II)-Adenosine Phosphothiorate Complexes
E
[
C
I A
G
J
w
I ' " ' I " ' ' l ' " ' l " " ~ " " I " '
40
30
20
10
0
-10
PPM
Figure 3. Protondecoupled121.5MHzphosphorus-31spectrum of [Pt(dien)Cl]Cl (5.0 mM) and ADP-fi-S (6.0 mM) reaction mixture at pH 6.5. Doublets A and B are for the free ligands, and C and D are for the Pt(I1)-S-ADP complex. doublets for the complex. Like the AMP-S system, coordination through the sulfur atom has shifted the resonance of the /?-phosphorusatom about 17 ppm upfield. Very little change in the chemical shift of a-phosphorus atom is observed since this phosphate group did not coordinate the platinum and was virtually unaffected by the /?-phosphothiorate coordination. We were unable to determine the pH-chemical shift profile for this complex owing to the precipitation at pH < 4 due to its limited solubility. The reaction of ATP-y-S also afforded a complex in which the sulfur atom of the phosphothiorate group is coordinated to platinum(I1) as evidenced by P-31 NMR data (Figure 4). Free ATP-y-S exhibits two doublets a t 37.57 and -8.08 ppm for the y- and a-phosphorus atoms a t pH 7.0. The /?-phosphorus atom shows doublet of doublets centered at -19.43 ppm due to the coupling with both a- and y-phosphorus atoms. As for the AMP-S and ADP-/?-S reactions with Pt(dien)Cl+, the signals for the coordinated y-phosphorus atom shifted 18 ppm upfield to 20 ppm. The a- and /?-phosphorus atoms show about 1 ppm upfield shift as well. DISCUSSION
The complexations of AMP and GMP with Pt(dien)Cl+ follow the familiar two-term rate law (eq 4), consistent with a sequence of reactions shown in eqs 5-7: Pt(dien)Cl+
+ H20
k,
Pt(dien)(H20I2+
-
Pt(dien)(H20)2++ Nu Pt(dien)Cl+ + Nu
(5)
fMt
Pt(dien)(Nu)
(6)
ka
Pt(dien)(Nu)
(7)
The value of k, was determined to be (2.0 f 0.1) X 1V s-1 for both the AMP and GMP reactions at 40 "C. This value can be compared with 5 X 106 s-l a t 25 OC reported by Belluco et al. (16). The magnitude of k2 for the GMP reaction is about 2.5 times larger than that for the AMP reaction which is consistent with the established kinetic preference toward guanine bases in DNA binding to platinum(I1) (17).
*I l
40
111 '
i
'
l
20
'
~
'
l
'
'
'
-20
0
l
'
l
i
"
Figure 4. Proton-decoupled 121.5-MHz phosphorus-31 NMR spectrum of [Pt(dien)Cl]Cl (5.0 mM) and ATP-y-S (6.0 mM) reaction mixture at pH 6.5. Peaks A, B, and C are for the y-, a-, and &phosphorus atoms of free ligand. Doublets D and E and doublet of doublets F are for the y-, CY-, and j3-phosphorus atoms of ATP-y-S bound to platinum(I1).Note that Pt-195 satellites are barely visible near the base of peak D. Peak G is due to an unidentified hydrolyzed product. The inset exhibitsthe expansion of signals abbreviated by B and E. In principle, phosphothiorate complexation should follow the same reactions as shown in eqs 5-7. However, these complexes are formed mainly through the direct reactions with the chloro complex. The contribution from the aquated pathway is insignificant, as can be judged by comparing the relative magnitudes of k, and k2. The values of k2 for all phosphothiorates lie in the range 1.9-2.7 M-l s-l. These values are close to those found for the cysteine and glutathione reactions with the same platinum(I1) complex (19). It is interesting to note that the value of k2 is largest for ATP-y-S complexation. This relatively higher reactivity may be associated with a higher nucleophilicity of the phosphothiorate group in the ATP-y-S due to significant deprotonation of the triphosphate moiety. Reactions a t lower acidities would have helped us to understand how deprotonated oxygens modulate the reactivity of the phosphothiorate group. Unfortunately, precipitation of the (phosphothiorato)platinumcomplexes at lower pH prevented us from establishing a pH-rate profile. All phosphothiorato complexes exhibited 15-20 ppm upfield chemical shifts compared to free nucleotides. This upfield shift of the coordinated phosphothiorate group may be due to the d?r-d?rPt S backbonding which in turn enriches the electron density around the phosphorus atom. There were no significant changes in the chemical shift of purine or ribose protons upon complexation with Pt(I1). This is primarily due to the fact that remote phosphothionate coordination has little or no influence to the electronic environment of these protons. In conclusion, the present study establishes that platinum(I1) exclusively coordinates to the phosphothiorate group of thionucleotides even when purine nitrogens are available for coordination. This selectivity toward phosphothiorate groups can be exploited in elucidating mechanisms of intramolecular hydroxyl transfer reactions by
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Bioconjugate Chem., Vol. 5, No. 4, 1994
utilizing platinum(I1) complexes that offer coordinated water molecules adjacent to the Pt-S bond. ACKNOWLEDGMENT
Funding of this research in part through the Biomedical is gratefully acknowledged. Research Support Grant (NIH) We also thank Prof. Roger Gregory for reading the manuscript and making valuable suggestion and Johnson Matthey, Inc., for the generous loan of KZPtC14. LITERATURE CITED
(1) Mertes, M. P.,and Mertes, K. B. (1990)Acc. Chem. Res. 23, 413. (2) Cornelius, R. D., and Cleland, W. W. (1978)Biochemistry 17,3279.Norman, P. R., and Cornelius, R. D. (1982)J. Am. Chem. SOC.104,2356. (3) Masoud, S.S.,and Milburn, R. M. (1990)J. Inorg. Biochem. 39,337-49. (4) Hendry, P., and Sargson,A. M. (1990)Inorg. Chem. 29,97104;(1990)InProgress in Inorganic Chemistry (S. J. Lippard, Ed.) Wiley, New York. (5) Kramer, P.,and Nowak, T. J. (1988)Inorg. Biochem. 32,135. and Chin, J. (1992)J . Am. Chem. SOC.29,97-104. (6) Kim, J. H., (1988) (7)Lu, Z., Shorter, A. L., Lin, I.,andDuhaway-Mariano,D. Inorg. Chem. 27,4135.
Slavin et al.
(8) Bose, R. N., Viola, R. E., and Cornelius, R. D. (1984)Znorg. Chem. 23, 1181;(1985)24,4403. (9) Burgeson, E. E., and Kostic, N. M. (1991)Inorg. Chem. 30, 4299. (10) Strothkamp, K.G., and Lippard, S. J. (1976)Proc. Natl. Acad. Sci. U.S.A. 2536-2540. (11) Szalda, S.J., Eckstein, F., Sternbach, H., and Lippard, S. J. (1979)J. Inorg. Biochem. 1 1 , 279-282. (12) Chu, B.C. F., and Orgel, L. E. (1992)Nucleic Acid. Res. 20, 2497-2502; (1990)DNA Cell Biol. 9,70-76. (13)Mahal, G., and Van Eldik, R. (1987)Inorg. Chim Acta 127, 203-208. (14) Glasoe, P. K., and Long, F.A. (1960)J . Phys. Chem. 64, 118. (15) Slavin, L. L., and Bose, R. N. (1990)J. Inorg. Biochem. 40, 339-347. (16) Belluco, U.,Cattalini, L., Basolo, F., Pearson, R. G., and Turco, A. (1965)J. Am. Chem. SOC.87,241. (17) See, for example: Sherman, S. E., and Lippard, S. J. (1987) Chem. Rev. 87,1153. (18) Bose, R. N.,Slavin, L. L., Cameron, J. W., Luellen, D. L., and Viola, R. E. (1993)Inorg. Chem. 32,1795.Slavin, L. L., Bose,R. N. (1990)J. Chem. Soc., Chem. Commun. 1256.Bose, R. N., Viola, R. E., and Cornelius, R. D. (1984)J . Am. Chem. Soc. 106, 3336. (19) Moghaddas, S.,Cox, E. H., and Bose, R. N. (1994)Inorg. Chem. (submitted).
