nihms106735.pdf

Ping-Pong Electron Transfer through DNA

Benjamin Elias [Prof.]

Chimie Organique et Médicinale, Université catholique de Louvain (Belgium)

Joseph C. Genereux

, and

Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA

91125 (USA)

Jacqueline K. Barton

Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA

91125 (USA)

DNA charge transport (CT) chemistry has been characterized in detail[1] owing to its utility

in the construction of DNA-based sensors and nanoscale devices.[2–4] Interestingly, recent

evidence also points to the application of this chemistry within the cell in the context of DNA

damage and repair.[5] Both DNA hole transport (HT) and electron transport (ET) have been

explored using spectroscopy,[6] biochemical assays,[7] and DNA electrochemistry.[8] In

reporting on DNA-mediated HT,

2-cyclopropylguanine and

6-cyclopropyladenine (

have been useful hole traps owing to their low oxidation potentials and their fast rates of ring

opening (10

−

s) upon one-electron oxidation (Scheme 1).[

9] Similarly, owing to their redox

potentials,[10

] the pyrimidines

4-cyclopropylcytosine (

C)[11

] and 5-bromouridine (

U),

[12] have been sensitive probes for DNA-mediated ET.

Recently, direct comparisons of HT with ET were carried out using a charge injector strongly

coupled to the base stack and able to oxidize or reduce bases from a distance within the same

DNA assembly.[13,14] Taking advantage of the powerful photochemistry of Ir

III

biscyclometalated complexes, we have shown that the [Ir(ppy)

(dppz

′

)]

, functionalized

through a modified dipyrido[3,2-

′

]phenazine (dppz

′

; ppy=2-phenylpyridine; Scheme

1), can serve both as a photooxidant and reductant of distal DNA bases.[

13,15

] Strikingly, we

found that DNA HT and ET have similar characteristics; both show a remarkably shallow

distance dependence in their reactions as well as an equal sensitivity to perturbations in stacking

of the intervening DNA bridge.

Significantly, although the excited state of the Ir

III

complex is sufficiently potent to oxidize

guanine or adenine,[15] efficient electron injection into DNA required the use of the flash-

quench technique.[16] Indeed, the excited state of the Ir

III

complex is not sufficiently long-

lived (

<10 ns) in water to photoreduce

U from a distance in substantial yield.[13] When

sodium ascorbate is used as an external reducing agent, however, photolysis of the tethered

III

complex generates a long-lived ground-state reductant, which is in turn able to induce

significant

U decomposition from a distance.

Herein we describe a novel Ir system that is able to promote the reduction of pyrimidine bases

from a distance without the presence of an external quencher. Instead, DNA-mediated ET is

triggered by DNA-mediated HT. Thus, photoactivation of these Ir assemblies results in both

This work was supported by the NIH (GM49216).

*Fax: (+1)6265774976, E-mail: jkbarton@caltech.edu.

NIH Public Access

Author Manuscript

Angew Chem Int Ed Engl

. Author manuscript; available in PMC 2009 September 22.

Published in final edited form as:

Angew Chem Int Ed Engl

. 2008 ; 47(47): 9067–9070. doi:10.1002/anie.200803556.

NIH-PA Author Manuscript

a forward and a reverse pattern for charge migration, which we term “ping-pong” electron

transfer through DNA (Scheme 1).

We have designed Ir–DNA conjugates containing two modified bases embedded in an AT

tract,[17] a

A for hole injection, and either a

U or a

C as an electron trap (Scheme 2).

For synthetic reasons, we required three different strands, one short strand (5-mer) covalently

tethered to the Ir

III

complex, one strand (13-mer) containing a

A-modified base, and one

complementary strand (18-mer) containing either a

U or

C. It should be noted that 1) the

presence of a nick in the phosphate backbone of the DNA helix does not affect the CT process,

[18] 2) the intercalative dppz

′

unit stabilizes the short Ir strand in the duplex,[19] and 3) the

irreversible ring-opening of

A precludes back ET.

Figure 1 shows the decomposition percentage of

A and

U obtained after 30 min irradiation

of the DNA assemblies at 365 nm as a function of the

A position (see the Experimental

Section). Clearly, both modified bases undergo a decomposition reaction upon photolysis. No

decomposition of

U is observed without both

A and tethered Ir

III

complex in the assembly.

Moreover, the extent of

U decomposition varies with the position of

A. We therefore see

that the

U decomposition is correlated with

A ring-opening. We ascribe this correlation to

the ping-pong reaction, where the highly oxidizing Ir

III

excited state[13,15] is reductively

quenched in an intraduplex reaction by the distal

A (migration of a “ping” electron); the

ground-state Ir reductant, formed by the intraduplex flash-quench scheme, is identical to the

one generated using an external quencher[13] and is capable of reducing

U by ET through

the DNA stack (migration of a “pong” electron).

We have further tested the ping-pong reaction using

C as a reductive probe, and here

substantial yields of decomposition of the modified base are observed.[

11,15] While

C can

be either photoreduced or photooxidized by the Ir

III

complex, it has been shown that this

modified base is reductively ring-opened by excited Ir when embedded in a thymine stack.

[15] Figure 2 shows the decomposition percentages of

A and

C as a function of

position in Ir–DNA conjugates, which contain

A within an A tract and

C on the

complementary strand. Here, a higher relative yield is obtained for the decomposition of the

reductive probe compared to that of

U. In fact, the

C decomposition is stoichiometric

with

A decomposition. Importantly, in the absence of

A, little

C decomposition is

obtained; the presence of

A is required for significant

C yields of decomposition. Also,

no decomposition is observed without the presence of tethered Ir

III

complex. Furthermore, no

change in

C decomposition is found in assemblies where

C is base-paired to an inosine

(Figure 2); if decomposition of

C were to occur through photooxidation, an enhancement in

yield would be evident without the competitive oxidative guanine sink.[20] Thus

C ring-

opening here results from reduction by DNA-mediated ET and not from a photooxidation

process. The ping-pong reaction is, then, highly efficient. Figure 2 shows also the

decomposition yields for

C in the absence of

A, both without (gray bars) and with (white

bars) an external reductive quencher (sodium ascorbate) known to reductively quench the

III

excited state.[13] The ground-state reduced metallic species, generated through

oxidation, can effectively reduce the distal

C, indeed comparably to an external reductive

quencher.

The sensitivity of DNA CT to DNA structure and dynamics is illustrated also for the ping-pong

reaction through variations in the DNA sequence. Despite equal overall energetics, the

decomposition efficiency in these assemblies is seen to depend upon the position of the redox

trap in the double helix. For instance, exchanging the

A and

C and the AT tract from one

strand to the other significantly affects the overall yield, especially for reduction (Figure 3).

This result is consistent with sequence variations seen earlier for DNA-mediated ET, and more

generally for CT in DNA with variations in the intervening base stack.[13,21]

Elias et al.

Page 2

Angew Chem Int Ed Engl

. Author manuscript; available in PMC 2009 September 22.

NIH-PA Author Manuscript

Thus, for the first time, DNA-mediated HT and ET have been triggered consecutively within

the same DNA duplex by irradiation of a unique charge injector. The ping-pong reaction likely

involves hole migration primarily through the purine strand with electron migration facilitated

by stacked pyrimidines. Critically, the analogous parameters govern both HT and ET through

the DNA base-pair stack.

Experimental Section

All DNA oligonucleotides were synthesized using standard phosphoramidite chemistry.

Strands containing a

A or

C were synthesized by placing an

6-phenylinosine or 4-

thiouridine at the target

A or

C site, respectively. After 16 h incubation at 60°C in 6

aqueous cyclopropylamine leading to simultaneous substitution reaction, cleavage from the

solid support, and deprotection, the strands were purified using reversed-phase HPLC and

characterized by MALDI mass spectrometry. [Ir(ppy)

(dppz)

′

]

was synthesized and

covalently tethered to DNA oligonucleotides according to previously described methods.[13,

15] Ir–DNA conjugates were prepared by combining equimolar amounts (1:1:1) of the desired

DNA single strands. After annealing (solution was heated to 90°C for 5 min, then slowly cooled

down to 15°C over a period of 3 h), all of the resulting duplexes showed melting temperatures

above ambient temperature.

Aliquots (30 μL) of the Ir–DNA conjugates (10 μ

in 50 m

TrisHCl, pH 7.0) were irradiated

for 30 min at 365 nm (Hg/Xe lamp, 1000W, with monochromator). Subsequent cleavage into

deoxynucleosides upon digestion with phosphodiesterase I and alkaline phosphatase was

carried out and the results analyzed by HPLC. The percentage of base decomposition, that is,

the amount of decomposed

C, or

U, was determined by subtracting the ratio of the

area under the peak of the undecomposed base in an irradiated sample over that in a non-

irradiated sample from unity, with adenine or inosine as an internal HPLC standard. Irradiation

experiments were repeated three times and the results averaged.

References

1. a) Carell T, Behrens C, Gierlich J. Org. Biomol. Chem 2003;1:2221. [PubMed: 12945689]O’Neill,

MA.; Barton, JK. Charge Transfer in DNA: From Mechanism to Application. Wagenknecht, HA.,

editor. New York: Wiley; 2005. p. 27 c) Conwell EM. Proc. Natl. Acad. Sci. USA 2005;102:8795.

[PubMed: 15956188] d) Giese B. Top. Curr. Chem 2004;236:27.

2. a) Bandyopadhyay A, Ray AK, Sharma AK, Khondaker SI. Nanotechnology 2006;17:227. b) Porath

D, Cuniberti G, Di Felice R. Top. Curr. Chem 2004;237:183. c) Clever GH, Kaul C, Carell T. Angew.

Chem 2007;119:6340.Angew. Chem. Int. Ed 2007;46:6226. d) Guo X, Gorodetsky AA, Hone J, Barton

JK, Nuckolls C. Nat. Nanotechnol 2008;3:163. [PubMed: 18654489]

3. a) Drummond TG, Hill MG, Barton JK. Nat. Biotechnol 2003;21:1192. [PubMed: 14520405] b) Yang

Y, Wang Z, Yang M, Li J, Zheng F, Shen G, Yu R. Anal. Chim. Acta 2007;584:268. [PubMed:

17386614] c) Wang X, Ozkan CS. Nano Lett 2008;8:308.

4. a) Boon EM, Ceres DM, Hill MG, Drummond TD, Barton JK. Nat. Biotechnol 2000;18:1096.

[PubMed: 11017050] b) Boon EM, Salas JW, Barton JK. Nat. Biotechnol 2002;20:282. [PubMed:

11875430] c) Boal AK, Barton JK. Bioconjugate Chem 2005;16:312.

5. Merino EJ, Boal AK, Barton JK. Curr. Opin. Chem. Biol 2008;12:229. [PubMed: 18314014]

6. a) Wan C, Fiebig T, Kelley SO, Treadway CR, Barton JK, Zewail AH. Proc. Natl. Acad. Sci. USA

1999;96:6014. [PubMed: 10339533] b) Takada T, Kawai K, Cai X, Sigimoto A, Fujitsuka M, Majima

T. J. Am. Chem. Soc 2004;126:1125. [PubMed: 14746481] c) Lewis FD, Zhu H, Daublain P, Cohen

B, Wasielewski MR. Angew. Chem 2006;118:8150.Angew. Chem. Int. Ed 2006;45:7982.

7. a) Hall DB, Holmlin RE, Barton JK. Nature 1996;382:731. [PubMed: 8751447] b) Ghosh A, Joy A,

Schuster GB, Douki T, Cadet J. Org. Biomol. Chem 2008;6:916. [PubMed: 18292885] c) Nakatani

K, Saito I. Top. Curr. Chem 2004;236:163. d) Nunez ME, Hall DB, Barton JK. Chem. Biol 1999;6:85.

[PubMed: 10021416]

Elias et al.

Page 3

Angew Chem Int Ed Engl

. Author manuscript; available in PMC 2009 September 22.

NIH-PA Author Manuscript

8. a) Gorodetsky AA, Barton JK. Langmuir 2006;22:7917. [PubMed: 16922584] b) Kelley SO, Hill MG,

Barton JK. Angew. Chem 1999;111:991.Angew. Chem. Int. Ed 1999;38:941. c) Drummond TG, Hill

MG, Barton JK. J. Am. Chem. Soc 2004;126:15010. [PubMed: 15547981]

9. a) Nakatani K, Dohno C, Saito I. J. Am. Chem. Soc 2001;123:9681. [PubMed: 11572693] b) Musa

OM, Horner JH, Shahin H, Newcomb M. J. Am. Chem. Soc 1996;118:3862. c) O’Neill MA, Dohno

C, Barton JK. J. Am. Chem. Soc 2004;126:1316. [PubMed: 14759170] d) Dohno C, Ogawa A,

Nakatani K, Saito I. J. Am. Chem. Soc 2003;125:10154. [PubMed: 12926921]

10. a) Steenken S, Telo JP, Novais LP, Candeias LP. J. Am. Chem. Soc 1992;114:4701. b) Seidel CAM,

Schultz A, Sauer MHM. J. Phys. Chem 1996;100:5541.

11. a) Lu W, Vicic DA, Barton JK. Inorg. Chem 2005;44:7970. [PubMed: 16241147] b) Shao F, O’Neill

MA, Barton JK. Proc. Natl. Acad. Sci. USA 2004;101:17914. [PubMed: 15604138]

12. a) Manetto A, Breeger S, Chatgilialoglu C, Carell T. Angew. Chem 2006;118:325.Angew. Chem.

Int. Ed 2006;45:318. b) Cook GP, Greenberg MM. J. Am. Chem. Soc 1996;118:10025. c) Ito T,

Rokita SE. J. Am. Chem. Soc 2003;125:11480. [PubMed: 13129334]

13. Elias B, Shao F, Barton JK. J. Am. Chem. Soc 2008;130:1152. [PubMed: 18183988]

14. Valis L, Wang Q, Raytchev M, Buchvarov I, Wagenknecht HA, Fiebig T. Proc. Natl. Acad. Sci. USA

2006;103:10192. [PubMed: 16801552]

15. a) Shao F, Elias B, Lu W, Barton JK. Inorg. Chem 2007;46:10187. [PubMed: 17973372] b) Shao F,

Barton JK. J. Am. Chem. Soc 2007;129:14733. [PubMed: 17985895]

16. a) Chang IJ, Gray HB, Winkler JR. J. Am. Chem. Soc 1991;113:7056. b) Stemp EDA, Arkin MR,

Barton JK. J. Am. Chem. Soc 1997;119:2921.

17. The A tract was chosen because of its resistance to inherent charge trapping. See a)Kawai K, Osakada

Y, Fujitsuka M, Majima T. J. Phys. Chem. B 2007;111:2322. [PubMed: 17291027] b)Augustyn KE,

Genereux JC, Barton JK. Angew. Chem 2007;119:5833. Angew. Chem. Int. Ed 2007;46:5731.

18. Liu T, Barton JK. J. Am. Chem. Soc 2005;127:10160. [PubMed: 16028914]

19. Duplexes show higher melting temperature in the Ir band (365 nm) than in the DNA band (260 nm)

by 9–10°C. This indicates that the complex prevents the two end strands from separating. This

observation is characteristic of a DNA complex containing an intercalated planar ligand and has

already been observed in other systems. See a)Kitamura Y, Ihara T, Okada K, Tsujimura Y, Shirasaka

Y, Tazaki M, Jyo A. Chem. Commun 2005:4523. b)Ossipov D, Pradeepkumar PI, Holmer M,

Chattopadhyaya J. J. Am. Chem. Soc 2001;123:3551. [PubMed: 11472126]

20. Inosine is structurally similar to guanosine but has a higher oxidation potential. Therefore, if

C is

oxidized, a higher extent of decomposition is expected when

C is base-paired to inosine rather

than to guanosine, as less competition for hole formation is expected. No decomposition enhancement

with inosine versus guanine indicates that a different process other than hole formation must be

involved. See Refs. [11] and [13] for further discussion.

21. Shao F, Augustyn KE, Barton JK. J. Am. Chem. Soc 2005;127:17445. [PubMed: 16332096]

Elias et al.

Page 4

Angew Chem Int Ed Engl

. Author manuscript; available in PMC 2009 September 22.

NIH-PA Author Manuscript

Figure 1.

Decomposition percentage for

A (red) in position 7, 9, 11, or 13 from Ir attachment

and

U (blue) in position 5 from Ir after 30 min irradiation (365 nm).

Elias et al.

Page 5

Angew Chem Int Ed Engl

. Author manuscript; available in PMC 2009 September 22.

NIH-PA Author Manuscript

Figure 2.

Decomposition percentage for

A (red) in position 9, 11, or 13 from Ir attachment and

(blue) in position 7 from Ir after 30 min irradiation (365 nm). The

C is base-paired either to

a guanine (left) or to an inosine (right). In both cases, the gray and white bars show the

decomposition percentage for

C without the presence of

A, with (white) or without (gray)

an external reductive quencher (sodium ascorbate, 200 m

Elias et al.

Page 6

Angew Chem Int Ed Engl

. Author manuscript; available in PMC 2009 September 22.

NIH-PA Author Manuscript

Figure 3.

Decomposition percentage for

A (red) in position 9, 11, or 13 from Ir attachment and

(blue) in position 7 from Ir after 30 min irradiation (365 nm). Both the AT tract and the

and

C have been flipped compared to sequences presented in Figure 2.

Elias et al.

Page 7

Angew Chem Int Ed Engl

. Author manuscript; available in PMC 2009 September 22.

NIH-PA Author Manuscript

Scheme 1.

Representation of the ping-pong reaction generated through photoactivation of an Ir–DNA

assembly along with modified base traps for hole (

A) and electron (

U and

C) transport.

Upon excitation, the excited Ir

III

complex irreversibly oxidizes a

A base from a distance.

The subsequent reduced metallic species is, in turn, able to reduce distal

U or

C bases.

Elias et al.

Page 8

Angew Chem Int Ed Engl

. Author manuscript; available in PMC 2009 September 22.

NIH-PA Author Manuscript

Scheme 2.

The assembly of three modified DNA single strands annealed to generate the Ir–DNA duplex

for the ping-pong reaction.

Elias et al.

Page 9

Angew Chem Int Ed Engl

. Author manuscript; available in PMC 2009 September 22.

NIH-PA Author Manuscript