7574
J. Phys. Chem. B 2000, 104, 7574-7576
Photoinduced Electron Transfer in an Oligodeoxynucleotide Duplex: Observation of the Electron-Transfer Intermediate Mark T. Tierney, Milan Sykora, Shoeb I. Khan, and Mark W. Grinstaff* Department of Chemistry, Paul M. Gross Chemical Laboratory, Duke UniVersity, Durham, North Carolina 27708 ReceiVed: April 21, 2000; In Final Form: June 9, 2000
Kinetics of a photoinduced electron-transfer reaction between phenothiazine (PTZ) and Ru(bpy)2(4-m-4′-pabpy)2+ [bpy ) 2,2′-bipyridine and 4-m-4′-pa-bpy ) 4-methyl-4′-carbonylpropargylamine] covalently bound to a DNA duplex is investigated by transient absorption spectroscopy. The electron donor, PTZ, is attached to the 5′ terminal of one oligodeoxynucleotide strand, and the chromophore, Ru(bpy)2(4-m-4′-pa-bpy)2+, is covalently linked to a uridine of the complementary strand. Electron transfer between the donor, PTZ, and photoexcited acceptor, *Ru(bpy)2(4-m-4′-pa-bpy)2+, occurs, and the product of the forward electron-transfer reaction, PTZ+•, is observed. The rate of the back electron-transfer reaction (kb ) 3.94 × 106 s-1; ∆G ∼ 2.0 eV) is determined by monitoring the decay of the electron-transfer intermediate, PTZ+•, by transient absorption spectroscopy.
Electron-transfer reactions occur in oxidative phosphorylation and respiration, drug metabolism, nitrogen fixation, photosynthesis, and in deoxyribonucleic acid (DNA). Since understanding these reactions is of fundamental importance, the factors that control the dynamics of electron transfer are being investigated and recognized. Systematic studies of proteins and peptides, modified to contain electron donors and acceptors, have elucidated these factors and led to important observations and conclusions for protein-mediated electron transfer.1-3 In comparison, DNA-mediated electron transfer is less understood. The effects of oligodeoxynucleotide sequence, base-pairing, π-stacking, secondary structure, the donor-acceptor distance, labeling site, and driving force on electron-transfer rates are of interest, and experiments are being performed to address many of these issues.4-26 Additional electron-transfer measurements in welldefined oligodeoxynucleotides will aid in interpreting, modeling, and predicting the factors that control the dynamics of electrontransfer reactions in DNA.27-29 Herein, we report the electrontransfer dynamics between a donor and acceptor covalently attached at site-specific locations on complementary strands of an oligodeoxynucleotide duplex. The electron-transfer between the phenothiazine (PTZ) electron donor and the photoexcited acceptor chromophore, *Ru(bpy)2(4-m-4′-pa-bpy)2+ (bpy ) 2,2′bipyridine and 4-m-4′-pa-bpy ) 4-methyl-4′-carbonylpropargylamine), occurs on the subnanosecond time scale, and the product of the electron-transfer reaction, PTZ+•, is spectroscopically detected. Of the three general methods (i.e., postmodification, phosphoramidite, and on-column modification)30-33 to derivatize oligodeoxynucleotides with probes, the phosphoramidite approach is used to covalently attach the Ru(bpy)2(4-m-4′-pabpy)2+-linked uridine and PTZ to the DNA duplex. This allows site-specific labeling of the oligodeoxynucleotide using an automated DNA synthesizer.34 The ruthenium nucleoside and PTZ phosphoramidites of interest are shown in Scheme 1. These phosphoramidites are synthesized following our earlier published procedures,35,36 whereby 1 and 3 are treated with 2-cyanoethyl* Corresponding author, email
[email protected].
SCHEME 1. Synthesis of Ru(bpy)2(4-m-4′-pa-bpy)2+uridine, 2, and Phenothiazine, 4, Phosphoramidites for Automated Solid-Phase DNA Synthesis.
N,N′-diisopropylchlorophosphoramidite, in the presence of diisopropylethylamine, to yield 2 and 4. The site-specific incorporation of the ruthenium nucleoside and PTZ in a 16-mer oligodeoxynucleotide is accomplished using an automated ABI 392 DNA synthesizer (Scheme 2).37 This doubly labeled duplex possesses a thermal denaturation temperature similar to the corresponding nonlabeled duplex as well as the characteristic features for B-DNA in the CD spectra. The Ru(bpy)2(4-m-4′-pa-bpy)2+ covalently linked to uridine is a suitable chromophore for reductive quenching studies since it is photochemically stable, inert to ligand substitution reactions, and possesses an energetic excited state (0.84 eV) with a long lifetime.38 The electron-transfer quencher, phenothiazine, is
10.1021/jp001539m CCC: $19.00 © 2000 American Chemical Society Published on Web 07/19/2000
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J. Phys. Chem. B, Vol. 104, No. 32, 2000 7575
SCHEME 2. Automated Solid-Phase Synthesis of Ru(bpy)2(4-m-4′-pa-bpy)2+-uridine and PTZ Labeled Oligodeoxynucleotides.
known to reductively quench *Ru(bpy)32+.39,40 The bimolecular electron-transfer reaction between 1 and 3 is studied in solution by varying the quencher concentration. Stern-Volmer analysis yields a quenching rate constant (kq) of 1.3 × 109 M-1 s-1. Based on the reduction potential of PTZ+/0 (0.76 V), the driving force for this bimolecular electron-transfer reaction is ∼ 0.1 eV. In this electron-transfer system, the electron donor and acceptor are covalently attached to different oligodeoxynucleotide strands and separated by four base pairs.41 First, the complementary duplex containing only the ruthenium acceptor (5′-TCA ACA GTT TGU† AGC A-3′; 5′-TGC TAC AAA CTG TTG A-3′; Ru-DNA) is investigated (U† ) Ru(bpy)2(4-m-4′pa-bpy)2+ linked uridine). The visible absorption spectrum of this duplex contains the characteristic metal-to-ligand chargetransfer band (1MLCT-1A1), centered at 450 nm. Emission occurs with λmax ) 660 nm, a quantum yield of 0.041 ( 0.001, and an emission lifetime of 548 ns in phosphate buffer at 20 °C.42 Next, a duplex is formed with the ruthenium labeled oligodeoxynucleotide and the complementary oligodeoxynucleotide strand containing PTZ at the 5′ terminus (5′-TCA ACA GTT TGU† AGC A-3′; 5′-PTZ-TGC TAC AAA CTG TTG A-3′; Ru-DNA-PTZ; Figure 1). Introduction of the quencher, PTZ, decreases the steady-state emission by 75% (Φem ) 0.010 ( 0.001). Transient absorption (TA) spectroscopy of Ru-DNA and Ru-DNA-PTZ confirms the origin of the quenching to be electron transfer between the excited state *Ru(diimine)32+ complex and PTZ. Kinetic TA traces of Ru-DNA and RuDNA-PTZ at 499 nm (Ru-DNA isosbestic point) are shown in Figure 2. The absorption feature at 499 nm is observed only in the Ru-DNA-PTZ sample and corresponds to PTZ+•.39,40 The rate of PTZ+• formation cannot be directly determined (10 ns instrument resolution), but the back electron-transfer reaction occurs with τ ) 250 ns.43 The rate of PTZ+• formation is estimated to occur in less than 10 ns (kf > 108 s-1; ∆G ∼ 0.1 eV), whereas the back electron-transfer rate is relatively slow with kb ) 3.94 × 106 s-1 (∆G ∼ 2.0 eV). The electron-transfer pathway is either DNA-mediated or solution medium mediated. If the structure shown in Figure 1 is a realistic model in which the donor and acceptor are separated and fairly ridged, the electron transfer is likely DNA mediated. Alternatively, if the donor is aligned along the DNA groove, leading to a reduced donor-acceptor distance, then contributions from a through solution medium mechanism could become
Figure 1. Top: Photoinduced electron-transfer scheme. Bottom: Model of a 16-base pair covalently modified with Ru(bpy)2(4-m-4′pa-bpy)2+ (orange) and phenothiazine (blue).
Figure 2. Kinetic TA trace at 499 nm for Ru-DNA and Ru-DNAPTZ. The spectra are corrected for emission.
significant. Likewise, if there is flexibility in the relative orientations of the redox pair, a through solution medium mechanism could also dominate. In this last case, however, such
7576 J. Phys. Chem. B, Vol. 104, No. 32, 2000 flexibility should be reflected by nonexponential dynamics, which are not observed. Sequence dependence electron-transfer and fluorescence anisotropy experiments are underway to address this issue. In this novel DNA assembly, the redox probes Ru(bpy)2(4m-4′-pa-bpy)2+ linked uridine and PTZ, are covalently attached at known site-specific locations. This doubly labeled duplex forms a stable B-form DNA structure at room temperature and is similar to the analogous unlabeled duplex. A photoinduced subnanosecond electron-transfer reaction occurs from PTZ to *Ru(bpy)2(4-m-4′-pa-bpy)2+, and the product of this forward electron-transfer reaction, PTZ+•, is observed. A slower electrontransfer rate is measured for the highly favorable back electrontransfer reaction, which is likely in the Marcus inverted region.44 This is the first observation of an electron-transfer product in a donor-DNA-acceptor system, where the two redox/spectroscopic probes are covalently attached to DNA. In summary, the Ru(bpy)2(4-m-4′-pa-bpy)2+ linked uridine and phenothiazine phosphoramidites expand the current repertoire of DNA electrontransfer probes available for study, and provide a new means to systematically investigate electron-transfer dynamics. Acknowledgment. This work was supported in part by NSF (CAREER AWARD), and the Army Research Office. M.W.G. thanks the Pew Scholar Program in the Biomedical Sciences, the Camille Teacher-Scholar Program, and the Alfred P. Sloan Foundation. We thank Professor T. J. Meyer for instrumentation use. References and Notes (1) Gray, H. B.; Winkler, J. R. Annu. ReV. Biochem. 1996, 65, 537561. (2) Winkler, J. R.; Gray, H. B. Chem. ReV. 1992, 92, 369-379. (3) McLendon, G. Acc. Chem. Res. 1988, 21, 160-167. (4) For recent reviews see: (a) Schuster, G. B. Acc. Chem. Res. 2000, 33, 253-260. (b) Kelly, S. O.; Barton, J. K. In Metal Ions in Biological Systems; Sigel, A., Sigel, H., Eds.; Marcel Dekker: New York, 1999; Vol. 36, pp 211-249. (c) Holmlin, R. E.; Dandliker, P. J.; Barton, J. K. Angew. Chem., Int. Ed. Engl. 1997, 36, 2714-2730. (d) Netzel, T. L. In Organic and Inorganic Photochemistry; Ramamurthy, V., Schanze, K. S., Eds.; Marcel Dekker: New York, 1998; pp 1-54. (5) For recent highlights see: (a) Ratner, M. Nature 1999, 397, 480481. (b) Taubes, G. Science 1997, 1420-1421. (c) Netzel, T. L. J. Chem. Educ. 1997, 74, 646-651. (d) Diederichsen, U. Angew. Chem., Int. Ed. Engl. 1997, 36, 2317-2319. (e) Grinstaff, M. W. Angew. Chem., Int. Ed. Engl. 1999, 38, 3629-3635. (6) Murphy, C. J.; Arkin, M. R.; Jenkins, Y.; Ghatlia, N. D.; Bossmann, S. H.; Turro, N. J.; Barton, J. K. Science 1993, 262, 1025-1029. (7) Arkin, M. R.; Stemp, E. D. A.; Holmlin, R. E.; Barton, J. K.; Hormann, A.; Olson, E. J. C.; Barbara, P. F. Science 1996, 273, 475-480. (8) Hall, D. B.; Holmlin, R. E.; Barton, J. K. Nature 1996, 382, 731735. (9) Hall, D. B.; Barton, J. K. J. Am. Chem. Soc. 1997, 119, 50455046. (10) Dandliker, P. J.; Holmlin, R. E.; Barton, J. K. Science 1997, 275, 1465-1468. (11) Kelley, S. O.; Holmlin, R. E.; Stemp, E. D. A.; Barton, J. K. J. Am. Chem. Soc. 1997, 119, 9861-9870. (12) Kelley, S. O.; Barton, J. K. Science 1999, 283, 375-381. (13) Kelley, S. O.; Barton, J. K. In Metal Ions in Biological Systems; Sigel, A., Sigel, H., Eds.; Marcel Dekker: New York, 1999; Vol. 36, pp 211-249. (14) Nunez, M. E.; Hall, D. B.; Barton, J. K. Chem. Biol. 1999, 6, 8597. (15) Wan, C.; Fiebig, T.; Kelly, S. O.; Treadway, C. R.; Barton, J. K.; Zewail, A. H. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 6014-6019. (16) Lewis, F. D.; Wu, R.; Zhang, Y.; Letsinger, R. L.; Greenfield, S. R.; Wasielewski, M. R. Science 1997, 277, 673-676. (17) Meade, T. J.; Kayyem, J. F. Angew. Chem., Int. Ed. Engl. 1995, 34, 352-354. (18) Lewis, F. D.; Zhang, Y.; Liu, X.; Xu, N.; Letsinger, R. L. J. Phys. Chem. B. 1999, 103, 2570-2578. (19) Ly, D.; Kan, Y.; Armitage, B.; Schuster, G. B. J. Am. Chem. Soc. 1996, 118, 8747-8748.
Letters (20) Gasper, S. M.; Schuster, G. B. J. Am. Chem. Soc. 1997, 119, 12762-12771. (21) Fukui, K.; Tanaka, K. Angew. Chem., Int. Ed. Engl. 1998, 37, 158161. (22) Fink, H. W.; Schonenberger, C. Nature 1999, 398, 407-410. (23) Meggers, E.; Kusch, D.; Spichty, M.; Wille, U.; Giese, B. Angew. Chem., Int. Ed. Engl. 1998, 37, 460-462. (24) Meggers, E.; Michel-Beyerle, M. E.; Giese, B. J. Am. Chem. Soc. 1998, 120, 12950-12955. (25) Giese, B.; Wessely, S.; Spormann, M.; Lindermann, U.; Meggers, E.; Michel-Beyerle, M. E. Angew. Chem., Int. Ed. Engl. 1999, 38, 996998. (26) Harriman, A. Angew. Chem., Int. Ed. Engl. 1999, 38, 945-949. (27) Jortner, J.; Bixon, M.; Langenbacher, T.; Michel-Beyerrle, M. E. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 12759-12765. (28) Priyadarshy, S.; Risser, S. M.; Beratan, D. N. J. Biol. Inorg. Chem. 1998, 3, 196-200. (29) Sugiyama, H.; Saito, I. J. Am. Chem. Soc. 1996, 118, 7063-7068. (30) Beilstein, A. E.; Tierney, M. T.; Grinstaff, M. W. Comments Inorg. Chem., in press. (31) Holmlin, E. R.; Dandliker, P. J.; Barton, J. K. Bioconjugate Chem. 1999, 10, 1122-1130. (32) Khan, S. I.; Grinstaff, M. W. J. Am. Chem. Soc. 1999, 121, 47044705. (33) Khan, S. I.; Beilstein, A. E.; Tierney, M. T.; Sykora, M.; Grinstaff, M. W. Inorg. Chem. 1999, 38, 5999-6002. (34) Caruthers, M. H. Acc. Chem. Res. 1991, 24, 278-284. (35) Khan, S. I.; Beilstein, A. E.; Grinstaff, M. W. Inorg. Chem. 1999, 38, 418-419. (36) Tierney, M. T.; Grinstaff, M. W. J. Org. Chem., in press. (37) The oligodeoxynucleotides were synthesized from the 3′ to 5′ end on the 1.0 µmol scale using standard automated DNA synthesis. Coupling of the ruthenium nucleoside phosphoramidite was 60% and the PTZ phosphoramidite was 95%. The site specifically labeled oligonucleotides were purified using reverse-phase HPLC methods (C18; 0.1 M TEAA/CH3CN). The isolated oligonucleotides were shown to be pure by analytical HPLC and characterized by mass spectroscopy. ESI: Ru-ssDN calc: 5526, found: 5527; and ESI PTZ-ssDNA calc: 5184, found: 5184. (38) Khan, S. I.; Beilstein, A. E.; Smith, G. D.; Sykora, M.; Grinstaff, M. W. Inorg. Chem. 1999, 38, 2411-2415. (39) Mecklenburg, S. L.; Peek, B. M.; Schoonover, J. R.; McCafferty, D. G.; Wall, C. G.; Erickson, B. W.; Meyer, T. J. J. Am. Chem. Soc. 1993, 115, 5479-5495. (40) Alkaitis, S. A.; Beck, G.; Gratzel, M. J. Am. Chem. Soc. 1975, 97, 5723-5729. (41) The model is based on the Ru(bpy)32+ and PTZ coordinates which were obtained from the Cambridge X-ray database and an NMR structure of DNA. A donor-acceptor distance (edge to edge) of ∼16 Å is determined from the model shown in Figure 1, whereas the distance is less than half that if the PTZ is aligned along the same face as the Ru(bpy)32+. The PTZ is likely end capped on the duplex and this is consistent with the increase in Tm and with similar work on anthraquinone labeled oligonucleotides (see refs 4a and 36). (42) The quantum yield of Ru(bpy)32+ in water is 0.042 ( 0.001. The nanosecond lifetime was measured using a Laser Photonics LN1000 Nitrogen Laser-LN102 dye laser (coumarin 460 dye). The emission was monitored at right angle with a Macpherson 272 monochromator and Hammamatsu R666-10 PMT at 22 °C. The signal was processed by a LeCroy 7200A transient digitizer interfaced with an IBM-PC. The excitation wavelength was 455 nm and the monitoring wavelength was 640 nm. The pulse width is ∼10 ns fwhm. Power at the sample was 60 W/pulse‚mm3 as measured by a Molectron J3-09 power meter. The acquired emission decay curves were analyzed by a locally written software based on the Marquardt algorithm. The data were fit to a monoexponential curve, and the residuals between the experimental and fitted curves were less than 2%. The RuDNA concentration was 1 × 10-6 M. (43) The TA spectra of the labeled oligonucleotides were measured on a system capable of measuring TA spectra on the time scale of microsecond to nanoseconds. A Surelite II-10 Nd:YAG-OPO system is used as the excitation source. The excitation beam from the laser irradiates the sample perpendicularly to an optical axis of an Applied Photophysics laser kinetic spectrometer with a 250W pulsed Xe lamp, f3.4 monochromator, and Hammamatsu PMT. The output from the PMT is coupled to LeCroy 7200 A oscilloscope and analyzed as described for the lifetime measurements. The excitation wavelength is 460 nm and the power at the sample is typically 3 mJ/pulse as measured by a Molectron J3-09 power meter. The Ru-DNA and Ru-DNA-PTZ concentrations were 5 × 10-5 M. The back electrontransfer data were fit to a monoexponential curve. The residuals between the experimental and fitted curves were less than 2%. (44) Marcus, R. A.; Sutin, N. Biochim. Biophys. Acta 1985, 811, 265322.
