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Crosslinking of Adjacent Guanine Residues in an Oligonucleotide by cis-[Pt(NH3)2(H2O)2]2+ Kinetic Analysis of the Two-Step Reaction.

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Crosslinking of Adjacent Guanine Residues
in an Oligonucleotide by C~S-[P~(NH,),(H,O),]~
Kinetic Analysis of the Two-step Reaction**
By Florence Gonnet, Jifi Kozelka,*
and Jean-Claude Chottard
The antitumor drug cis-diamminedichloroplatinum(Ir)
(“cisplatin”) binds to DNA to form preferentially the
d(GpG) intrastrand crosslink.[’] This macrochelate is formed
in a two-step process (Scheme I), and the reactive platinum
species is one of the hydrolyzed cisplatin forms, that is,
cis-[PtCI(NH,),(H,O)]+ (X = CI- ; Y = H,O) or cis[Pt(NH,),(H,0),]2f (X = Y = H,O). Between 60 and 65 %
of the DNA-bound platinum appears as the G G crosslink, as
shown both by in vitro and by in vivo
percentage significantly exceeds the probability of finding a
guanine residue adjacent to another, and since the reactions
in Scheme 1 are irreversible under the experimental condi-
k,, and k,, and apply it to the reaction between the singlestranded oligonucleotide d(TpGpG) and the diaqua form of
cisplatin, c~~-[P~(NH,),(H,O),]~+.
The method consists of
treating known amounts of oligonucleotide with approximately stoichiometric platinum complex in aqueous solution
and, at intervals, taking aliquots which are quenched by
addition of a saturated KC1 solution and cooled to liquid
nitrogen temperature. The conserved aliquots are subsequently analyzed by HPLC. Figure 1 displays a typical chromatogram showing peaks for all four species N, 11, 12, and
C defined in Scheme 1 for d(TGG). The high resolution of
our system allows a precise integration of peaks, and thus an
accurate determination of the concentrations. A computer
program written in our laboratory calculates for each time ti
the four concentrations “Ii, [Ill;, [I2Ii, and [C];, based on
assumed rate constants k,, , k,,, k , , ,and k,,, and optimizes
the fit by iteration on the constants as described in the appendix.
Scheme 1. Binding of a cis-[Pt(X)(Y)(NH,),] complex (X, Y
to a GpG sequence of an oligonucleotide.
= leaving groups)
tions, the guanine residues in GG sequences must be more
reactive than in other positions. The origin of this differential guanine reactivity is unknown. One possible factor is the
enhancement of the negative potential at the guanine N7
atom caused by a neighboring guanine, which is predicted by
ab initio calculations.[81However, as we have recently shown
using molecular modeling,[” nonbonding interactions in the
transition state,Iglthat is, those between the platinum ligands
and the DNA residues adjacent to the guanine being platinated, could also play a role. Such a mechanism would be
ligand-dependent, which opens the interesting possibility of
influencing the binding selectivity of the platinum complex
by an appropriate choice of ligands.
One question that arises is whether the 5’-guanine of a
d(GpC) sequence enhances the reactivity of the 3’-guanine,
or vice versa. No answer to this question could be given so
far, since it was not possible to measure the rate constants for
the platination of the two guanines ( k l l and k,, in Scheme 1)
In this paper, we describe a technique that enables the
measurement not only of the two primary platination rate
constants k , ,and k12but also of the two chelation constants
Dr. J. Kozelka. F. Gonnet, Dr. J.X. Chottard
URA 400, Laboratoire de Chimie et Biochimie Pharmacologiques
et Toxicologiques
45 rue des Saints Pkres, F-75270 Pans Cedex 06 (France)
We thank Dr. J. Igolen for supplying the d(TGG) tnnucleotide. The authors acknowledge EEC support (grant no. ST2J-0462-C) allowing regular
scientific exchange with the group of Prof. J. Reedijk, Leiden (The Netherlands).
Angew. Chem. I n [ . Ed. Engl. 1992, 31. N o . 11
Fig. 1. A typical HPL chromatogram for a stoichiometric reaction of d(TGG)
M) with cis-[Pt(NH,),(H,O),)(NO,), in 0.1 M NaCIO,, after quenching
by addition of an excess of KCI. T = 293 K ; pH = 4.4; I = 12 min (X =
impurity; for the other identifiers, see Scheme 1).
Since, in general, only the concentration of the platinum
complex can be determined precisely (that of the oligonucleotide is measured by means of UV absorbance), the excess
(or shortfall) of oligonucleotide with respect to the platinum
complex is used as a fifth variable. The correct functioning of
the program has been tested in two control experiments in
which a defined large excess of oligonucleotide was employed. The excess determined by the program deviated by
up to 10 % from the value determined by UV spectroscopy,
which corresponds to the error of the concentration measurement. The four rate constants determined in these two
control experiments were similar to those found in the other
Figure2 shows a typical fit of the four concentration
curves, after optimization of the five variables, for the reac. The following
tion of d(TGG) with ~~s-[P~(NH,),(H,O),]~
rate constants were obtained for this system at 293 K (mean
values of five experiments):
= 0.90 k0.07 M - ~ S - ~
= (1.76 & 0.09) 10-3s-1
= 0.89 f0.08 M - ~ s - ~
= (1.27 f 0.09) 10-3s-1
Apparently, in this trinucleotide the two guanine residues are
equally reactive. The chelation of the 3’-intermediate I1 is
slightly faster than that of the 5’-intermediate 12.
Our ultimate goal in this research is to compare the rate
constants for reactions of oligonucleotides of different
lengths and sequences (including duplexes) with different
Verlagsgesellschafi mbH, W-6940 Weinheim, 1992
1 ooy
reaction; this decrease correlated roughly with the formation of the chelate C .
The concentration of C based o n peak integration was therefore corrected by
a factor of 1.04.
Identification of the monoadducts was achieved by using enzymatic digestion
of the corresponding fractions by the exonuclease Venum phosphodiesterase
(VPD. Sigma). VPD digests the 5'-monoadduct (12) to cis-[PtCl(NH,),d(TpGN7)] and dpG, whereas the 3'monoadduct (11) remains undigested [13].
The integration of the differential equations describing the kinetics of the reaction system shown in Scheme 1 yields the following expressions for the four
concentrations [N], [Ill, [12], and [C], at the time f ( W = excess of cis[Pt(NH,),(H,O),](NO,), over the oligonucleotide N):
Fig. 2. Calculated curves and observed values for the four relative concentrations IN] (x). [Ill (A), [I21 ( 0 ) . and [C] (+).
= e(x'r+A'W - 1
= l n ( y 0 +I); k , = k,,
[Ill = k,,e-*z~'f[N]([N] + W)ek"'di
[I21 = k,2e-k22'~[N]([N] W)ekz2'dt
platinum complexes. An understanding of the high affinity
of cisplatin towards GG sequences, and specifically, of the
role that the ligands play in this affinity, will possibly provide
a means to modify this affinity by appropriate changes in the
ligands. In this way, it should be possible to test whether
there is a relationship between sequence-specific DNA binding and antitumor activity of platinum complexes.
The kinetic analysis of a reaction between TGG and cis[PtCI(NH,),(H,O)]+ is currently being investigated in our
laboratory. The experimental procedure is exactly the same
as with the diaqua complex C~S-[P~(NH,),(H,O),]~+,
the quenched products are the same. However, the evaluation of the rate constants is more complicated because of the
equilibrium (a).
+ k,'; w = [Pt] - [hq
- "1
= "10
[I11 - [I21
The integrals in the expressions for [Ill and [I21 were calculated numerically
with time steps of 1 s. The Optimization procedure was based on perpendicular
distances of the experimental points from the calculated curves (p in Fig. 3).
rather than on vertical distances corresponding to the difference between calculated and experimental concentrations (v in Fig. 3). This approach, which takes
into account the experimental errors for both time and concentration, is. of
course, meaningful only if both variables are scaled in units corresponding to
the uncertainty in their measurement.
I n the minimized function (1) pr are the perpendicular deviations of the experimental points i from the calculated curve, determined as from Equation (2).
{[Xp- [X]r"C)cos a
- [X];ai'}(l
with tan
+ tan'
[X]P* and [X]?'"are the observed and calculated concentrations of the product
~ i s - [ P t C l ( N H ~ ) ~ ( H ~ 0 )H,O
1 + S c~~-[P~(NH,),(H,O),]~+
CI- (a)
The determination of the rate constants for the reaction
system with C~S-[P~(NH,),(H,O),]~~
is prerequisite for the
more complex system involving cis-[PtC1(NH3),(H,O)]+. As
far as other platinum complexes are concerned, their reactions with oligonucleotides can be analyzed by our method,
provided that the starting, intermediate, and final products
can be quenched in a simple way and separated by HPLC.
X at the time ti, and s(r) and s([X]) are the errors in the determination of time
and concentration, estimated to be 4 s and 2 % of the initial oligonucleotide
concentration, respectively. The individual sums in Equation (1) were taken
as the second power to prevent the program from fitting one curve considerably
worse than the others. Our optimization procedure used agrid search algorithm
Received: May 14, 1992 [ Z 5348 IE]
German version: Angew,. Chem. 1992, 104, 1494
Experimental Procedure
The reactions were typically initiated by mixing a solution of about 2 pmol of
d(TGG) (measured by UV spectrophotometry, czi* = 22 170 ~ - l c m ')- [lo] in
0.1 M NaCIO, with a solution containing 2 pmolbf cis-[Pt(NO,),(NH,),j (prepared according to Ref. [ l l ] )in 2 mL H,O. The pH of the mixture was adjusted
to pH = 4.4 by addition ofHC10,. After an initial increase to pH = 4.5 resulting from liberation of coordinated water, the pH decreased slowly to a value of
4.3, presumably due to dissolution of CO, from the atmosphere. The reaction
temperature was kept at 293 k 0.2 K. In initial intervals of 30 s (beginning) to
15 min (end), ahquots of 30 pL were withdrawn and quenched with 30 gL of a
saturated KCI solution in an Eppendorf tube, and after 1.75 min incubation at
293 K (conversion of monoaqua to monochloro adducts) [12], dipped into
liquid nitrogen and stored at 77 K until they were analyzed by HPLC. The
cooling to 77 K is important, since at 193 K (dry ice temperature), the monocoordinated species I1 and I2 (Scheme 1) still slowly convert to the chelate C,
despite KCI excess. As the subsequent HPLC analyses are quite time-consuming, and thus some of the samples have to be conserved for several days, this
slow conversion significantly modifies the relative concentrations.
The HPLC analyses were performed on a Spectra Physics SP8800 Chro.
matograph with a Nucleosil C38 column ( 2 5 0 x 4 . 6 m m ID, 5 ~ m lO0A;
Macherey Nagel). Standard operating conditions were: mobile phase, ammonium acetate buffer (Merck, 0.01 M, pH = 4.70) and acetonitrile (Merck, 95:5
v k ) ; flow rate 0.8mLmin-'; T = 29XK. At the detection wavelength of
255 nm. the absorbance of the reaction mixture decreased by 4 % during the
VCH Verlugsgeseils~~huJi
inhH, W-6940 Wrinhehn. 1992
point i
x 1gbs
calculated curve
f [minl
Fig. 3. Two distances relating an experimental point with the calculated curve:
v, the vertical distance; p, the normal to the curve through the experimental
point. In the linear approximation, p = v cos a. The variables are scaled in units
of their inherent experimental errors. Whereas v reflects only the uncertainty in
the concentration measurement, p takes into account the imprecision of both
0570-OS33i92/1111-14843 3.50i.2510
Angew. Chrm. 1111.Ed. Engl. 1992, 31, N o . 11
CAS Registry numbers:
N, 34727-11-2; C, 344017-98-1; 11, 144041-54-3; 112. 144017-99-2: cis[Pt(NH,),(H,O),lZ+ .2NO;, 52241-26-6; cis-[PtCI(NH,), ,(H,O)]+, 5386142-0.
A. Laoui, J. Kozelka, J. C. Chottard, h o r g . Chem. 1988, 27, 2751 -2753.
A. Eastman, Biochemistry 1983, 22, 3927-3933.
A. Eastman, Biochemistry 1985, 24, 5027-5032.
A. Eastman, M. A. Barry, Biochemistr~.1987, 26, 3303-3307.
[S] A. Eastman, N. Schulte, Biochemistry 1988. 27, 4730-4734.
161 A. M. J. Fichtinger-Schepman, P. H. M. Lohmdn. J. Reedijk, Nucleic
Acids Res. 1982, 10, 5345-5356.
[7] A. M. J. Fichtinger-Schepman. J. L. Van der Veer, J. H. J. Den Hartog,
P. H. M. Lohman, J. Reedijk, Biochemis~ry1985, 24, 707-713.
(81 A. Pullman, C. Zakrzewska, D. Perahia. Int. J. Quanrum Chem. 1979, 16.
191 The modeled species was, in fact, the pentacoordinated intermediate, since
the assumption was made that the structure of the transition state would
not be very different.
[lo] This value was approximated as czs4{d(TGG)}% &254(dT)
2&,,,{d(pG)); EZ5,(dT) and c,,,{d(pG)) were measured on Sigma products, and their analytically determined water content taken into account
as 5850 ~ - ‘ c m - ’and 8160 ~ - ‘ c m - ’ , respectively.
[Ill Y. N. Kukushkin. S. C. Dhara, hdian J. Chem. 1970, 8 , 184-185.
1121 I t is essential to allow the monoaqua products (I, X = H,O) to convert to
monochloro species (I, X = Cl-). otherwise the chelation reactions proceed during HPLC analysis, leading to smearing of the peaks. The minimum time necessary at 293 K is 1.75 min. Longer incubations should be
avoided, since the chelation reactions are not sufficiently inhibited at this
[I31 K. Inagaki, Y Kidani, Inorg. Chim. Arta 1985, 106, 187-191.
1141 J. A. McCammon, S. C. Harvey, Dynamics ofproteins and nucleic acrds,
Cambridge University Press, Cambridge, 1987, p. 48.
Scheme 1. a) [(CH,),Si],NLi; -[(CH,),Si],NH,
L = 1,4-dioxane.
-LiH; THF, 2h, 60°C.
Stable Cyclic Germanediyls (“Cyclogermylenes”):
Synthesis, Structure, Metal Complexes,
and Thermolyses**
By Wolfgang A . Herrmann,* Michael Denk, Joachim Behm,
Wolfgang Scherer, Franz-Robert Klingan, Hans Bock,
Bahman Solouki, and Matthias Wagner
Substituted ethylenediamine 2 a and its derivatives form
volatile, spirocyclic titanium amides, which precipitate from
the gas phase (produced thermally or plasma-induced) as
titanium carbonitrides.“] We discovered that this type of
ligand is generally suited for the stabilization of main group
and subgroup elements with unusual valencies and report
here on new applications in the chemistry of germanium.
The cyclic germanium(1v) diamide 3aIZ1was formed in
almost quantitative yields from GeCl, ( l a ) and diamine 2 a
in the presence of triethylamine. Reductive dehalogenation
of 3a then provided the new germanediyl (“germylene”) 3 b
(Scheme l).13]In another approach to 3 b the dilithium salt
2 b was allowed to react with germanium dichloride. 1,4dioxane (1 b). Surprisingly, the CC-unsaturated germanediyl
3 c obtained by a formal dehydrogenation was isolated as a
side product (Scheme 1). Compound 3 c can be prepared
[*] Prof. Dr. W A. Herrmann, Dr. M. Denk. Dr. J. Behm, W. Scherer,
F.-R. Klingan
Anorganisch-chemisches Institut der Technischen Universitat Miinchen
Lichtenbergstrasse 4, D-W-8046 Garching (FRG)
Prof. Dr. H. Bock, Dr. B. Solouki
Anorganisch-chemisches Institut der Universitat Frankfurt
Dr. M. Wagner
Anorganisch-chemisches Institut der Universitdt Munchen
[**I Cyclic Metal Amides, Part 2. This research was supported by the Bundesministerium fur Forschung und Technologie. Part 1: ref. [lh].
Angew. Chem. Int. Ed. Engl. 1992, 31, N o . 11
C Ib
Fig. 1. Top: Crystal structure of 3b at -70°C (ORTEP, without hydrogen
atoms, thermal ellipsoids at the SO% probability level). Middle: Model of the
disorder in 3b (SCHAKAL, see text). Selected distances [pm] and angles I”]:
Ge-NI 183.3(2),N1-CI 145.9(4),CI-Cla 157.1(7), N1-C2 146.7(4);Nl,Ge,Nl’
88.0(1), NI,CI,Cla 105.7(2), Ge,Nl,Cl 113.1(2), C2,Nl,Ge 128.2(2); shortest
Ge-Ge distance: 643 pm. Bottom: Crystal structure of 3c at -50°C. Selected
distances[pm]and angles[“]: Ge-NI 185.6(1), NI-CI 138.4(1), Ct-Cl’ 136.4(1),
Nl-C10 149.3(1); Nl,Ge,Nl‘ 84.8(1), Nl,Cl,CI‘ 144.3(1), Ge,Nl,Cl 113.3(1);
shortest Ge-Ge distance: 641 pm.
Verlagsgesellschaji mbH, W-6940 Weinheim, 1992
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two, step, reaction, residue, guanine, oligonucleotide, kinetics, h2o, cis, nh3, adjacent, analysis, crosslinking
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