Mechanism of Charge Transport in DNA
J. Am. Chem. Soc., Vol. 121, No. 40, 1999 9409
flash column chromatography (silica gel, CH2Cl2/MeOH ) 7:1, Rf )
reorganization). These internal motions might be some combi-
nation of changes in winding or inclination angle and shifts in
the location of protons involved in hydrogen bonding. We refer
to these motions collectively as phonons. Thus, this mechanism
for radical cation migration is termed phonon-assisted polaron
hopping.
1
0.3). Yield 3.30 g (95%). H NMR (DMSO-d6) δ 3.55-3.70 (m, 2H,
H2′ and H3′), 3.75 (q, 1H, H4′), 4.00-4.12 (m, 2H, H5′), 5.10 (d, 1H,
C2′-OH), 5.18 (t, 1H, C5′-OH), 5.50 (d, 1H, C3′-OH), 5.77 (d, 1H, H1),
5.95 (d, 1H, uracil H5), 7.63, 7.78 and 7.97 (m, 7H, AQ), 8.18 (d, 1H,
uracil H6).
2′,3′-O-(Dibutylstannylene)-N3-benzoyluridine (2). N3-Benzoyl-
uridine (1, 2.80 g, 8.1 mmol) and dibutyltin oxide (2.03 g, 8.2 mmol)
were heated at reflux in anhydrous methanol (400 mL) for 2 h. When
the solution turned clear, the solvent was removed under reduced
pressure and the residue was dried under vacuum to give the desired
crude product in ∼95% purity. 1H NMR (DMSO-d6) δ 0.80-1.60 (m,
18H, alkyl chain), 3.45-3.80 (m, 3H, H2′, H3′ and H4′), 4.10-4.20 (m,
2H, H5′), 5.10 (t, 1H, C5′-OH), 5.60 (d, 1H, H1′), 5.95 (d, 1H, uracil
H5), 7.50-8.00 (m, 7H, AQ), 8.12 (d, 1H, uracil H6).
In principle, the rate of polaron hopping must depend on the
local base sequence. Examination of Figures 7 and 9 show that
for UAQDNA(71)/DNA(5), there is no conspicuous sequence
dependence in the case examined. There is no regularity in the
sequence between the five GG steps of this compound, but the
distance dependence revealed in Figure 9 appears to be
essentially constant. We have suggested that this apparent
insensitivity of the hopping rate is a consequence of sequence
averaging. Structural distortion of the DNA around the radical
cation delocalizes the electron deficiency over several base pairs.
Evidence in support of this proposal is found in Saito’s
calculation of sequence-dependent ionization potentials for
guanines.37 Similarly, polaron-hopping steps of varying length
average differences that would be encountered if the radical
cation hopped exclusively from base to base. This model does
not require that averaging eliminate all differences between
DNA sequences.
The experiments described above incorporating 8-OxoG in
the DNA sequence reveal a limit to how much averaging of
DNA structure in the polaron model is possible. A polaron in
which an 8-OxoG replaces a G will has a significantly reduced
free energy. Evidently, this stabilization of the polaron reduces
the rate of its hopping and increases the probability that the
radical cation will be irreversibly trapped at this site.
BzU(2′-UAQ) (3). 2′,3′-O-(Dibutylstannylene)-N3-benzoyluridine (2,
2.8 g, 4.8 mmol), bromomethyl anthraquinone (2.71 g, 9.0 mmol), and
CsF (1.8 g, 11.8 mmol) were placed in a 250 mL round-bottom flask
containing anhydrous DMF (50 mL). The mixture was stirred for 48 h
at room temperature. Ethyl acetate (200 mL) was added to the reaction
and the mixture washed with water (2 × 50 mL) and dried over Na2-
SO4. The solution was concentrated to a minimum volume until a
precipitate formed. The precipitate was filtered, and the filtrate (majority
3′-isomer) was further concentrated and applied to a silica gel column
(4.5 × 30 cm) and eluted with CH2Cl2/MeOH ) 20:1. The desired
product (3), Rf 0.35, and 3′-isomer (4), Rf 0.40, were collected without
separation: 1.36 g (50%). 1H NMR (DMSO-d6) δ 3.50-3.80 (m, 2H,
H2′ and H3′), 4.00, 4.08, 4.15, 4.25 and 4.40 (m, 3H, H4′ and H5′), 4.70-
5.00 (m, 2H, CH2), 5.30 and 5.45 (m, 2H, C2′-OH, C3′-OH and C5′-
OH), 5.75-6.00 (m, 2H, uracil H5 and H1′), 7.50-8.30 (m, 8H, AQ
and uracil H6).
DMT-BzU(2′-UAQ) (5). The two isomers (3,4, 1.04 g, 1.8 mmol)
and DMT-Cl (0.74 g, 2.2 mmol) were dissolved in dry pyridine (20
mL) at room temperature. Concentrated ammonium hydroxide (5 mL)
was added to the solution and the solution stirred for 16 h. The mixture
was concentrated to dryness under reduced pressure and dissolved in
the minimal volume of CH2Cl2 and applied to a silica gel column (3 ×
45 cm). Elution with CH2Cl2/MeOH (20:1) gave two major fractions
which were separated as 5′-DMT-U(2′-AQ) (5, 905 mg, 1.01 mmol)
and 5′-DMT-U(3′-AQ) (6, 592 mg, 0.68 mg). Total yield 1.50 g, 95%.
5: mp 139 °C; TLC (CH2Cl2/MeOH ) 9:1) Rf 0.67; 1H NMR (DMSO-
d6) δ 3.30 (m, 2 H, H5′), 3.72 (s, 6 H, CH3O of DMT), 4.10(m, 2 H,
H2′ and H3′) 4.28 (ddd, 1 H, becoming dd with D2O, H3′), 4.92 (dd, 2
H, ArCH2), 5.24 (d, 1 H, uracil H5), 5.47 (d, 1 H, diminished with
D2O 3-OH), 5.95 (d, 1 H, H1), 6.87 and 7.27 (m, 13 H, DMT), 7.68 (d,
1 H, uracil H6), 7.90 and 8.20 (m, 7H, AQ), 12.40 (s, 1 H, amide). 6:
mp 141 °C; TLC (CH2Cl2/MeOH ) 20:1) Rf 0.55; 1H NMR (DMSO-
d6) δ 3.27 (m, 2 H, H5′), 3.68 (s, 6H, CH3O of DMT), 4.13 (m, 2 H,
H3′ and H4′), 4.40 (ddd, 1 H, becoming dd with D2O, H2′), 4.82 (dd,
ArCH2), 5.30 (d, 1 H, uracil H5), 5.72 (d, 1 H, diminished with D2O,
H2′) 5.78 (d, 1 H, H1′), 6.85 and 7.25 (m, 13 H, DMT), 7.75 (d, 1 H,
uracil H6′), 7.85 and 8.20 (m, 7 H, AQ).
UAQ Phosphoramidite (7). DMT-Bz(2′-UAQ) (5, 234 mg, 0.27
mmol) was dissolved in dry CH2Cl2 (3 mL), followed by addition of
DIEA (0.24 mL, 1.37 mmol). The mixture was stirred under N2 until
everything dissolved. To this solution, 2-cyanoethyldiisopropylchlo-
rophosphoramidite (0.12 mL, 0.60 mmol) was added dropwise, and
the reaction mixture was stirred at room temperature for an additional
30 min and then applied directly to a silica gel column. Elution with
CH2Cl2/EtOAc/Et3N (45:45:10) gave one major fraction with Rf 0.65.
The solvent was removed under reduced pressure to give pale yellow
oil, which was used directly in the DNA synthesis.
Anthraquinone-Oligonucleotide Conjugate Synthesis. The first
part of UAQ-DNA (before AQ incorporation) was performed on solid
phase synthesis following standard conditions, using cyanophophora-
midite monomers. When the sample was at the position to couple the
AQ monomer, the resin was thoroughly washed and the cartridge
(containing the resin) was removed from the synthesizer. The AQ-
phosphoramidite monomer was dissolved in 500 µL of a dry CH3CN
solution containing 0.1 M tetrazole. The monomer was taken into the
cartridge via a syringe, and the coupling reaction was allowed to proceed
Experimental Section
1
General. H and 13C NMR spectra were recorded on a Varian 300
MHz spectrometer. 31P NMR were recorded on a Bruker 400 MHz
spectrometer. Radioactive isotopes [γ-32ATP] and [R-32ddATP] were
purchased from Amersham Bioscience. T4 polynucleotide kinase and
terminal deoxynucleotidyl transferase (TdT) were purchased from
Pharmacia Biotech and stored at -20 °C. 8-OxoG-modified and
unmodified oligonucleotides (gel filtration grade) were obtained from
the Midland Certified Reagent Company. Internally linked anthraquino-
ne oligonucleotides were synthesized on an Applied Biosystems DNA
synthesizer and were purified by reverse-phase HPLC. The extinction
coefficients of the unmodified oligonucleotide were calculated using
the nearest-neighbor values, and the absorbance was measured at 260
nm. Anthraquinone-modified oligonucleotide solution concentrations
were determined the same way as that of the unmodified oligomers
except that an anthraquinone was replaced with adenine in the extinction
coefficient determination. Ion-exchange and reverse-phase HPLC were
performed on a Hitachi system using a Vydac column. Matrix assisted
laser desorption ionization time-of-flight (MALDI-TOF) mass spec-
trometry of the conjugate strands was performed at the Midland
Certified Reagent Company. The buffer solution used in all DNA
experiments was 10 mM sodium phosphate at pH ) 7.0. UV melting
and cooling curves were recorded on a Cary 1E spectrophotometer
equipped with a multicell block, temperature controller, and sample
transport accessory. Phosphorescence emission spectra were measured
on a Spec Fluorolog spectrofluorimeter.
N3-Benzoyluridine (1). Uridine (2.44 g, 10.0 mmol) was added to
a 100 mL round-bottom flask containing pyridine (40 mL, 495 mmol)
and stirred at room temperature until the uridine was completely
dissolved. To this solution, chlorotrimethylsilane (6.4 mL, 50.4 mmol)
was added while stirring (a white precipitate formed upon addition).
The mixture was stirred for 20 min after the addition was completed.
Benzoyl chloride (5.8 g, 41.3 mmol) was then added slowly, and the
mixture was stirred for 2 h, cooled in an ice bath, and then extracted
twice with ethyl acetate (2 × 200 mL). The organic fractions were
combined, washed with brine, and dried over Na2SO4. Purification by