Design of a hole-trapping nucleobase: Termination of DNA-mediated hole transport at N2-cyclopropyldeoxyguanosine [5]

Full text of DOI:10.1021/ja010479a

J. Am. Chem. Soc. 2001, 123, 9681-9682
9681
Scheme 1
Design of a Hole-Trapping Nucleobase: Termination
of DNA-Mediated Hole Transport at
N2-Cyclopropyldeoxyguanosine
Kazuhiko Nakatani,* Chikara Dohno, and Isao Saito*
Department of Synthetic Chemistry and
Biological Chemistry, Faculty of Engineering
Kyoto UniVersity, CREST, Japan Science and
Technology Corporation (JST), Kyoto 606-8501, Japan
ReceiVed February 22, 2001
photoexcited riboflavin induces homolytic cyclopropane ring
opening as evidenced by the formation of N2-(3-hydroxypro-
panoyl)dG, 2. With the use of a dCPG-containing duplex, we have
demonstrated that dCPG efficiently terminates DNA-mediated hole
transport at its own site.
While the mechanism of DNA-mediated hole transport is a
subject of controversy,1-6 it is now evident from remote guanine
(G) oxidation of duplex DNAs containing a tethered oxidant that
G radical cation (hole) migrates a distance through the DNA
π-stack.5-9 Due to ionization potentials of GG and GGG lower
than that of single G,2d,10,11 these stacked G sites function as a
thermodynamic sink of holes eventually producing piperidine
labile sites. In principle, hole migration from hole donor to
acceptor competes with hole trapping by water and/or oxygen.
Therefore, overall efficiency of hole transfer is primarily deter-
mined by the rates of hole migration and hole trapping. When
the rate of hole trapping is much slower than that of hole
migration, equilibration of hole between donor and acceptor can
be achieved.8d While the rate of hole migration can be attenuated
by changing the potential energy gap between hole donor and
acceptor,7c the modulation of hole-transport efficiency by changing
the rate of hole trapping has never been demonstrated. We report
a novel hole-trapping nucleoside N2-cyclopropyl-2′-deoxy-
guanosine, 1 (dCPG), which possesses a cyclopropyl group on N2
as a radical-trapping device. One-electron oxidation of dCPG by
It is known that radical cations of cyclopropylamine12 and
N-alkyl- and N-arylcycloprpylamines13 rapidly undergo homolytic
cyclopropane ring opening to produce â-iminium carbon radicals.
The rate of homolytic ring opening of the cyclopropylamine
radical cation is believed to be larger than that of the correspond-
ing ring opening of the neutral N-alkylcycloprpylaminyl radical
(7.2 × 1011 s-1).14 While the magnitude of the rate is unknown,
the cyclopropane ring opening of dCPG radical cation is expected
to be rapid. We first examined one-electron oxidation of dCPG
with photoexcited riboflavin in aqueous solution.15 dCPG was
rapidly consumed by photoirradiation at 366 nm in the presence
of riboflavin, producing two major products after subsequent
incubation of the photoirradiated mixture (Figure S1). These
products were identified as dG and N2-(3-hydroxypropanoyl)dG
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2000, 41, 10041-10045.
1
1
2 by H NMR and high-resolution FABMS. Comparison of H
NMR spectra of 2 with those of the authentic sample unambigu-
ously confirmed the structure.16 Formation of dG by incubating
the photoirradiated mixture suggested that one-electron oxidation
of 1 activated transformation of the cyclopropyl group at N2 to a
group being highly susceptible to hydrolysis (Scheme 1).17
Having established that 1 radical cation undergoes a very rapid
cyclopropane ring opening, we examined the hole trapping by 1
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(15) dCPG was synthesized from 2-fluorodeoxyinosine by substitution of
fluorine with cyclopropylamine. (a) Acedo, M.; Fa`brega, C.; Avin˜o, A.;
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10.1021/ja010479a CCC: $20.00 © 2001 American Chemical Society
Published on Web 09/07/2001

9682 J. Am. Chem. Soc., Vol. 123, No. 39, 2001
Communications to the Editor
Figure 2. HPLC profiles of a nucleoside mixture obtained by enzymatic
digestion of CPG-P21/C-21 after photoirradiation for (a) 0 min, (b) 15
min, and (c) 30 min. The ODN solution (50 µM strand concentration) in
sodium cacodylate buffer (pH 7.0) was irradiated at 312 nm at 0 °C for
the indicated period. Photoirradiated oligomers were treated with alkaline
phosphatase (33 u/mL), snake venom phosphodiesterase (0.1 u/mL), and
P1 nuclease (33 u/mL) at 0 °C for 12 h. Adenine (A) was added as an
internal standard.
Figure 1. Autoradiogram of a denaturing sequencing gel for photore-
actions of duplexes of 32P-5′ end-labeled G-P21, MeG-P21, and CPG-
P21 hybridized to C21. Duplex ODNs were irradiated at 312 nm (0 °C,
15 min). After piperidine treatment (90 °C, 20 min), the samples were
electrophoresed through a denaturing 15% polyacrylamide/7 M urea gel.
Lane 1, Maxam-Gilbert G+A sequencing reactions; lane 2, G-P21;
lane 3, MeG-P21; lanes 4-6, CPG-P21.
lower than that for the corresponding G-containing 10-mer duplex
(50 µM base concentration, 100 mM NaCl). CD spectrum of the
CPG-containing duplex shows a typical B-form structure (Figure
S2). These observations imply that the disruption of the π-stack
by incorporating dCPG is not the reason for the suppression of
hole transport. HPLC analysis of a nucleoside mixture obtained
by enzymatic digestion of photoirradiated CPG-P21/C21 clearly
showed that dCPG was completely consumed under the photo-
irradiation conditions used for the PAGE experiments, although
other nucleosides, including dCNBPU, remain largely unchanged
(Figure 2). These results suggest that suppression of G18 oxidation
in CPG-P21/C21 is most likely due to the selective destruction
of dCPG. Calculated ionization potential at B3LYP/6-31G(d) level
for N9-methyl-CPG/N9-methyl-C base pair is only 0.13 eV lower
than that for the normal G/C base pair, and 0.40 eV higher than
that for the GG/CC doublet. The oxidation potential of dCPG
measured in water containing 0.1 M LiClO4 was 0.93 V (vs SCE,
cf. G 1.07 V). Therefore, it is unlikely that dCPG functions as a
thermodynamic sink in hole migration. On the basis of these
observations, we propose that dCPG effectively terminates hole
transport through the DNA π-stack by a rapid and irreversible
cyclopropane ring opening of its radical cation.
in DNA-mediated hole transport. We have used 21-mer probe
ODNs d(ATT TAT AG8T XTG TAG15 GTA TTT) containing G
(G-P21), MeG (MeG-P21), and CPG (CPG-P21) as a base X.
The complementary strand d(AAA TAC CTA CAC ACCNBP
U
ATA AAT) (C21) contains cyanobenzophenone-substituted uri-
dine (dCNBPU) as a photoinducible one-electron oxidant.9 Guanine
radical cation was site selectively produced at G8 in duplexes
G-P21/C21, MeG-P21/C21, and CPG-P21/C21 by single-
electron transfer to photoexcited dCNBPU.9a The hole is able to
migrate up to G15G through
a bridge of d(TXTGTA)/
d(TACACA). Probe ODNs MeG-P21 and CPG-P21 were
obtained from 2-fluoroinosine containing 21-mer according to the
reported method.15 Photoirradiation of duplex G-P21/C21 at 366
nm for 15 min followed by subsequent piperidine heating
produced a distinct cleavage band at G15 selectively (Figure 1,
lane 2). This indicates an efficient hole migration from G8 to G15.
In marked contrast, G15 oxidation was significantly suppressed
in CPG-P21/C21 duplex, where dCPG was incorporated into the
bridge of d(TCPGTGTA)/d(TACACA) (lane 4). Normalized band
intensities at G15 relative to intact bands as determined by
densitometry were 1.00 for G-P21 (standard), 0.53 for MeG-
P21, and 0.09 for CPG-P21. While incorporation of the methyl
group at N2 of dG resulted in a modest reduction of G15 oxidation
via hole transport from G8 to G15, the incorporation of the
cyclopropyl group was dramatically effective for the suppression
of hole transport.
The pseudo-first-order rate of trapping of G•+ by water was
estimated to be 6 × 104 s-1, which is significantly smaller than
the rate for the hole migration between the two single Gs separated
by two AT base pairs (2.5 × 106 s-1).8d Discovery of a powerful
hole-trapping nucleobase possessing a trapping rate faster than
2.5 × 106 s-1 would change the current view of hole transport.
The studies described here clearly demonstrated that dCPG can
serve as a useful probe for gaining deeper insight into the kinetic
aspects of hole transport through the DNA π-stack.
The melting temperature for CPG-containing 10-mer duplex
d(GTC CAC TAT C)/d(GAT ACPGT GGA C) is only 1.3 °C
Supporting Information Available: Experimental protocol for the
synthesis of 2, HPLC analysis of the photoreaction of dCPG, and CD
spectra of CPG-containing duplex (PDF). This material is available free
of charge via the Internet at http://pubs.acs.org.
(16) Authentic sample of 2 was synthesized by acylation at N2 of dG with
3-benzyloxypropionyl chloride, followed by hydrogenolysis of the benzyl ether
(see Supporting Information).
(17) For a review of reactions of phenylcyclopropane radical cation, see:
Mizuno, K.; Ichinose, N.; Yoshimi, Y. J. Photochem. Photobiol., C. 2000, 1,
167-193.
JA010479A