Core level photoelectron spectroscopy on the lanthanide-inducedhydrolysis of DNA
Hidemi Shigekawa,a),b) Hiroyuki Ikawa, and Ryozo Yoshizakic)
Center for Tsukuba Advanced Research Alliance, and Institute of Materials Science, University of Tsukuba,Tsukuba 305, Japan

Yoshitoki Iijima
Application and Research Center, JEOL Limited, Akishima, Tokyo 196, Center for Tsukuba Advanced
Research Alliance, University of Tsukuba, Tsukuba 305, JapanJun Sumaoka and Makoto Komiyamaa)
Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Tokyo113, Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Tsukuba 305, Japan~Received 6 November 1995; accepted for publication 2 January 1996!
The electronic structures of the complexes of diphenyl phosphate ~DPP!, a model compound ofDNA, with lanthanide ions have been investigated to shed light on the mechanism of the cerium~IV!-induced nonenzymatic hydrolysis of DNA. Binding energies of the P 2 p core level of DPP
were 134.2 eV for the complexes with La~III!, Eu~III!, and Lu~III!, and was 134.4 eV for the Ce~IV!
complex, when the metal/DPP molar ratio was 1:1. When the molar ratio was increased, onlyCe~IV!, the most active metal ion for DNA hydrolysis, showed a chemical shift of ;0.5 eV toward
the higher binding energy region. The chemical shift of ;0.5 eV toward the higher binding energy
region. The chemical shift was due to the systematic increase in the intensity of the higher binding
energy component. The observed change in the electronic structure of the DPP-Ce~IV! complex may
be related to the superb ability of Ce~IV! for the hydrolysis of DNA. © 1996 American Institute ofPhysics. @S0003-6951~96!03110-0#

Nonenzymatic scission of DNA has been attracting interest, because of potential application to molecular biology,biotechnology and therapy.1 However the phosphodiester
linkages therein are enormously resistant to hydrolysis: thehalf-life of the linkages at pH 7 and 25 °C is estimated to be
200 million years. It was only in 1992 that the first nonenzymatic hydrolysis of linear DNAs was achieved by the authors by use of lanthanide ions.2–5 The cerium~IV! ion is themost active.6 – 8 It decreases the half-life to a few hours corresponding to an acceleration around 1011 fold. Quite interestingly, the remarkable catalysis is totally specific to lanthanide ions, especially to the Ce~IV! ion. In addition, anartificial nuclease, which selectively hydrolyzes the target
phosphodiester linkage in DNA and thus is a useful tool formolecular biology, has been synthesized by the attachment ofCe~IV! ion ~in the form of iminodiacetate complex! to aDNA oligomer.9 However, it has not yet been understood
why the Ce~IV! ion overwhelmingly exceeds the other lanthanide ions more than 100-fold in the activity.
In order to understand the scission mechanism, information about the change in the electronic structure of the phosphodiester linkage of nucleic acids during complex formation with lanthanide ions, is urgently required. In this letter,
we present the results of core level photoelectron
spectroscopy10 on the complexes of diphenyl phosphate~DPP!, a model sample of nucleic acids lanthanide. As
shown in Fig. 1, DNA and DPP have similar phosphodiester
linkages.

The specimens were prepared by mixing DPP with thecorresponding metal salt @Ce~NH4!2~NO3!6 , LaCl3 , EuCl3 ,or LuCl3# in water at a required molar ratio. After the mixtures were centrifugalized, the sediment was collected anddried in vacuum, and was then directly subjected to XPSanalysis. The electronic structure of the phosphodiester linkage in the DPP molecules in the presence of lanthanide ions
was studied by measuring the P 2p core level spectra. X-ray
used for excitation was Mg K a ~hn51253.6 eV!, and energy
resolution was 0.9 eV at Ag 3d 5/2 . 11 The obtained binding
energies were calibrated using the CO bonding state ~O 1s,533.2 eV! existing in all the samples. The measurements
were performed in a vacuum of 531027 Pa. Since absenceof the hydrolysis of DPP during the spectroscopy has been
confirmed by a reversed-phase high performance liquid chro-a!

Authors to whom correspondence should be addressed.
Electronic mail: hidemi@mat.ims.tsukuba.ac.jpc!
Permanent address: Institute of Applied Physics, and Cryogenics Center,
University of Tsukuba, Tsukuba, Japan.
b!

FIG. 1. Chemical structures of the phosphodiester linkage in DNA and DPP.

Appl. Phys. Lett. 68 (10), 4 March 1996
0003-6951/96/68(10)/1433/3/$10.00© 1996 American Institute of Physics
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FIG. 2. Core level spectra of P 2p obtained for the DPP complex with
La~III!, Ce~IV!, Eu~III!, and Lu~III! for the molar ratio of ~a! 1:1 and ~b!
10:1.

matography, the observed spectra are related to the electronic
structures of the DPP-Ce~IV! complex at the stage immediately before the phosphodiester linkage is broken.
Figure 2 shows the core level spectra of P 2 p for themixtures of lanthanide ions and DPP with a molar ratio of
1:1 and 10:1.12 When the metal/DDP molar ratio was 1:1, thebinding energy of the P 2p core level was observed at 134.2
eV for the complexes with La~III!, Eu~III!, and Lu~III! ions.
For the Ce~IV! complex, however, the P 2 p core level had aslightly higher binding energy of 134.4 eV. Quite significantly, only Ce~IV! induced a chemical shift of ;0.5 eV
toward the higher binding energy region as indicated by thearrows in Fig. 2~b!, when the molar ratio was increased from
1:1 to 10:1. The activity of Ce~IV! for the modulation of theelectronic structure of the phosphodiester linkage is remarkably greater than those of other lanthanide ions.12 This is, toour best knowledge, the first spectroscopic evidence showing
a clear-cut difference between Ce~IV! and other lanthanide
ions in the interaction with phosphodierster linkages.
Figure 3 shows a series of P 2p core level spectra for thesamples of Ce~IV!/DPP molar ratios of ~a! 0:1, ~b! 1:1, ~c!
2:1, ~d! 5:1, and ~e! 10:1. The spectra of Figs. 3~b!–3~e!
could be decomposed into two spin doublet components
P H and P L , which have higher and lower binding energies,respectively. When the molar ratio was 1:1, the main component was the peak P L as shown in Fig. 3~b!. However,with the increase of the molar ratio, the intensity of peak
P H increased @Figs. 3~c!–3~e!#. The change in the relative

FIG. 3. Core level spectra of P 2p observed for the Ce~IV!/DPP mixtures,with the molar ratios of ~a! 0:1, ~b! 1:1, ~c! 2:1, ~d! 5:1, and ~e! 10:1. P H andP L correspond to the doublet peaks of higher and lower binding energies.

intensity of the two components in Fig. 3 is quantitatively
presented in Fig. 4. A possible explanation for the observed
P 2p core level spectra is that, component P L corresponds tothe bonding of the phosphodiester linkage with Ce~IV! ion,and the component P H corresponds to the simultaneous
bonding with two Ce~IV! ions. The full width at halfmaximum ~FWHM! increased with increasing molar ratio;from 0.31 to 0.90 eV for peak P H , and 0.93 to 1.08 eV forpeak P L @Figs. 3~b!–3~e!#. From the observed chemical shift
and the change in the FWHM of the two components, thechemical reactivity of the phosphodiester linkage in a DPP

FIG. 4. Change in the relative intensity of the two P 2p components P H andP L in Fig. 3.

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FIG. 5. Schematic model for the structure of the 2:1 complex betweenCe~IV! ion and DPP.

molecule seems to be increased, on the complex formation
with two Ce~IV! ions.
Proposed structure of the 2:1 complex between Ce~IV!
ion and DPP is schematically depicted in Fig. 5. In addition
to the usual coordination of a Ce~IV! ion to the phosphate,
another Ce~IV! ion in the hydroxide cluster interacts with
one of the other three oxygen atoms around the phosphorus
atom ~the interaction with the phenoxide oxygen atom ispresented in the figure!. The mutual orientation of the twoCe~IV! ions in the hydroxide cluster should be favorable forthe multiple interactions. Because of the significant electron
withdrawal by the cooperation of the two Ce~IV! ions, theelectron density on the phosphorus atom is greatly decreased,
exactly as observed in the present photoelectron spectroscopy ~the peaks P H in Fig. 3!. This effect renders the highly
stable phosphodiester linkage susceptible to hydrolysis under
mild conditions. The activation of the linkage by the complex formation with only one metal ion ~corresponding to thepeak P L in Fig. 3! is insufficient. The DNA hydrolysis proceeds via nucleophilic attack by the Ce~IV!-bound hydroxide
toward the phosphorus atom.8 The superiority of Ce~IV! overother lanthanide ions and nonlanthanide ions is presumably
ascribed to a better orientation of two metal ions in the hydroxide cluster and/or larger acidity, which makes the synergetic catalysis much more effective. According to a recent ab
initio calculation, bonding of a single Ce~IV! atom to DPPdoes not result in breaking of the phosphodiester linkage.13
Further information on the chemical structure of the Ce~IV!
complexes is necessary to clarify the details.
The spectroscopic evidence is totally consistent with thefollowing kinetic results showing that the rate of the Ce~IV!induced DNA hydrolysis monotonously decreases with decrease in the molar ratio of the Ce~IV! ion to the substrate
DNA. The rate constants of the hydrolysis ofthymidylyl~38 –58!thymidine at pH 7 and 50 °C are 0.33,0.03, and 0.00 h21 , respectively, when the Ce~IV!/substrate
ratios are 100, 10, and 1. Apparently, two or more Ce~IV!

ions, with respect to the phosphodiester linkage, are required
for the efficient DNA hydrolysis.
Finally, it is noteworthy that the Zr~IV! ion, which is not
active for DNA hydrolysis, caused virtually no change to theP 2p binding energy of DPP at the metal/DPP ratio 10 ~data
not presented!. Thus, the significant change by the Ce~IV!
ion is never simply ascribed to its large positive charge. It isstrongly indicated that some specific interactions between theorbital~s! of the Ce~IV! ion and those of DPP is responsible
for the change ~and also presumably for the remarkable catalytic activity of the Ce~IV! ion for DNA hydrolysis!.
In conclusion, the binding energies of the P 2 p corelevel were rather similar for the DPP mixtures with La,Ce~IV!, Eu, and Lu, when the metal/DPP molar ratio was
1:1. However, when the molar ratio was increased, only
Ce~IV! induced an apparent change in the spectra. The
P 2 p core level spectra of the Ce~IV! complex were deconvoluted into two components, the intensity ratio andFWHM of which were found to change systematically withthe increase in the Ce~IV!/DPP molar ratio. Since the DPP
complex has a chemical structure similar to that of the DNAcomplex, the characteristic P 2 p core level spectra, observed
only with Ce~IV!, is expected to be related to the scission
mechanism of DNA.
This work was supported in part by Grants-in-Aid for
Scientific Research on Priority Areas ~New Development ofRare-Earth Complexes!, from the Ministry of Education, Science and Culture of Japan. Support from Shigekawa projectof TARA, University of Tsukuba is also acknowledged.
P. B. Dervan, Science 232, 464 ~1986!.
Y. Matsumoto and M. Komiyama, Nucl. Acids Symp. Ser. 27, 33 ~1992!.
3M. Komiyama, K. Matsumura, K. Yonezawa, and Y. Matsumoto, Chem.
Express 8, 85 ~1993!.
4
T. Shiiba, K. Yonezawa, N. Takeda, Y. Matsumoto, M. Yashiro, and M.
Komiyama, J. Mol. Catal. 84, L21 ~1993!.
5
M. Komiyama, Y. Matsumoto, T. Shiiba, N. Hayashi, and K. Watanabe,
Polymer J. 25, 1211 ~1993!.
6
M. Komiyama, T. Kodama, N. Takeda, J. Sumaoka, T. Shiiba, Y. Matsumoto, and M. Yashiro, J. Biochem. 115, 809 ~1994!.
7
B. Takasaki and J. Chin, J. Am. Chem. Soc. 116, 1121 ~1994!.
8M. Komiyama, N. Takeda, Y. Takahashi, H. Uchida, T. Shiiba, T. Kodama,and M. Yashiro, J. Chem. Soc. Perkin Trans. 2, 269 ~1995!; and references
therein.
9
M. Komiyama, T. Shiiba, H. Uchida, Y. Takahashi, N. Takeda, K. Matsumura, and T. Kodama, Supramol. Chem. 4, 31 ~1994!.
10
Y. Ma, C. T. Chen, G. Meigs, G. Illing, and H. Shigekawa, Phys. Rev. B
45, 5961 ~1992!.
11Y. Iijima, K. Muramoto, M. Uemura, and K. Hirooka, Jpn. J. Appl. Phys.
32, 5080 ~1993!.
12The chemical shift change due to the 1:1 complex formation could not be
estimated here, since the spectrum for the free anion of DPP is not available at present. The specimen for the free DPP used here was preparedfrom acidic solutions, and DPP therein is protonated. Thus, we focus thepresent analysis to the comparison of the spectral change of DPP on thecomplex formation with the Ce~IV! ion with the corresponding values forother lanthanide ions.
13K. Hirao ~private communication!.
12Appl. Phys. Lett., Vol. 68, No. 10, 4 March 1996
Shigekawa et al.
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