Fluorescent properties and conformational preferences of C-linked phenolic-DNA adducts

ARTICLE

pubs.acs.org/crt

Fluorescent Properties and Conformational Preferences of C-Linked

Phenolic-DNA Adducts

Alireza Omumi,^†,§Andrea L. Millen,^‡,§Stacey D. Wetmore,*^,‡and Richard A. Manderville*^,†

^†Departments of Chemistry and Toxicology, University of Guelph, Guelph, ON, Canada N1G 2W1

^‡Department of Chemistry & Biochemistry, University of Lethbridge, Lethbridge, AB, Canada T1K 3M4

S Supporting Information

b

ABSTRACT: Phenolic toxins and mutagenic diazoquinones generate C-linked adducts

at the C8 site of 2⁰-deoxyguanosine (dG) through the intermediacy of radical species.

We have previously reported the site-speciﬁc incorporation of these adducts into

oligonucleotides using a postsynthetic palladium-catalyzed cross-coupling strategy

[Omumi et al. (2011) J. Am. Chem. Soc. 133, 42ꢀ50]. We report here the structural

impact of these lesions within two decanucleotide sequences containing either 5⁰- and

3⁰-ﬂanking pyrimidines or purines. In the complementary strands, the base opposite

(N) the C-linked adduct was varied to determine the possibility of mismatch

stabilization by the modiﬁed nucleobases. The resulting adducted duplex structures

were characterized using UV thermal denaturation studies, circular dichroism, ﬂuorescence spectroscopy, and molecular dynamics

(MD) simulations. The experimental data showed the C-linked adducts to destabilize the duplex when base paired with its normal

partner C but to increase duplex stability within a G:G mismatch. The stabilization within the G:G mismatch was sequence

dependent, with ﬂanking purine bases playing a key role in the stabilizing inﬂuence of the adduct. MD simulations showed no large

structural changes to the B form double helix, regardless of the (anti/syn) adduct preference. Consideration of H-bonding and

stacking interactions derived from the MD simulations together with the thermal melting data and changes in ﬂuorescent emission

of the adducts upon hybridization to the complementary strands implied that the C-linked phenolic adducts preferentially adopt the

syn-conformation within both duplexes regardless of the opposite base N. Given that biological outcome in terms of mutagenicity

appears to be strongly correlated to the conformational preference of the corresponding N-linked C8-dG adducts, the potential

biological implications of phenolic C-linked adducts are discussed.

’ INTRODUCTION

hydrazines,^15ꢀ17polycyclic aromatic hydrocarbons (PAHs),¹⁸

estrogens,¹⁹and nitroaromatics.²⁰Given the importance of

N-linked adducts in arylamine carcinogenesis,^21ꢀ23O-linked

and C-linked adducts are expected to play a role in phenol

toxicity.

Phenols are ubiquitous substances that exhibit both anti-

oxidant and pro-oxidant properties. Vitamin E¹and polyphenols²

are beneﬁcial antioxidants, while chlorophenols³are deleterious

pro-oxidants. Interestingly, both activities can stem from oxida-

tion of the parent phenol to generate a phenoxyl radical.⁴Vitamin

E and polyphenols quench reactive oxygen species (ROS), such

as hydroxyl radical (HO^•), to generate relatively stable phenoxyl

radicals.^1,2Electron-withdrawing chlorine substituents decrease

phenoxyl radical stability,⁵leading to oxidative stress in biological

systems.⁶

Phenoxyl radicals can also react with the nucleobases to aﬀord

bulky DNA adducts.^7ꢀ10Structural evidence shows a tendency

for attachment at the C8 position of 2⁰-deoxyguanosine (dG), as

noted with HO^•in the formation of 8-oxo-dG.¹¹The phenoxyl

radicals also display ambident [oxygen (O) vs carbon (C)]

electrophilicity in reactions with dG, as outlined in Scheme 1.^12,13

Oxidation of pentachlorophenol (PCP) with peroxidase enzymes

generates an O-linked adduct,^7,8while metabolism of the chloro-

phenolic mycotoxin ochratoxin A favors C-linked adduct forma-

tion.^9,10The speciﬁc C-linked adducts shown in Scheme 1 are

also generated by mutagenic diazoquinones¹⁴and are structurally

related to a family of 8-aryl(Ar)-dG adducts produced by aryl

To study the properties of DNA adducts, synthetic methods

for their preparation and insertion into oligonucleotides are critical.

For N-linked adducts, Pd-catalyzed cross-coupling between

arylamine and protected 8-Br-dG can be used to synthesize the

nucleoside,^24,25which is then converted into a phosphoramidite

for incorporation into oligonucleotides using solid-phase DNA

synthesis.^26ꢀ28Structural studies have shown that C8 attach-

ment of the bulky N-linked arylamine can shift the conforma-

tional equilibrium of the glycosidic bond from anti to syn,

leading to distinct mutation and repair outcomes.²⁹For example,

the N-linked 8-acetylaminoﬂuorene-G (AAF-G) lesion can

adopt three diﬀerent conformers in duplex DNA.³⁰When

WatsonꢀCrick base-paired with C, anti-AAF-G adopts a non-

distorting B type structure.³⁰In the syn-conformation, a base-

displaced intercalated of “stacked” (S) structure is formed in

Received: June 13, 2011

Published: September 09, 2011

r

2011 American Chemical Society

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Scheme 1

both orientations may be present in duplex DNA.⁴¹The eﬀect of

H-bonding interactions on the anti/syn conformational stability⁴²

shows that o-PhOH-dG forms signiﬁcantly more stable com-

plexes with the WatsonꢀCrick face than with the Hoogsteen face

and that C forms the most stable base pair with o-PhOH-dG

regardless of the H-bonding face involved. However, for p-PhOH-

dG, Hoogsteen interactions are stronger than those with natural

dG, and the para-adduct binds with nearly equal stability to both

C and G in the Hoogsteen orientation.⁴²

The present work expands our understanding on the conformation

and binding preference of C-linked phenolic adducts and con-

siders the structural impact of p-PhOH-dG, o-PhOH-dG, and 8-phen-

yl-dG (Ph-dG) within the two decanucleotides oligonucleotide 1

(ODN1) and ODN2 shown in Figure 1. In ODN1, the C-linked

adducts (X) are ﬂanked by pyrimidine bases, while in ODN2, they

are ﬂanked by purines. In the complementary strands, ODN1⁰(N),

and ODN2⁰(N), the base opposite (N) the adduct has been varied

(N = C, G, A, T) to determine the possibility of mismatch

stabilization by the modiﬁed C-linked nucleobases. Our work

highlights the ability of these lesions to stabilize a G:G mismatch

in duplex DNA and represents a ﬁrst step in understanding the

potential biological signiﬁcance of C-linked phenolic-DNA adducts.

’ EXPERIMENTAL PROCEDURES

1

General. H and ¹³C NMR spectra were recorded at room tem-

perature on a Bruker Avance 300 DPX at 300.1 and 75.5 MHz,

respectively. NMR spectra were referenced to the solvent signal of the

deuterated solvent. Mass spectra were conducted at the Biological Mass

Spectrometry Facility (BMSF) at the University of Guelph and were

obtained from a Micromass/Waters Global Ultima quadrupole TOF

using electrospray ionization. UVꢀvis and fluorescence emission spec-

tra were recorded on a Cary 300-Bio UVꢀvisible Spectrophotometer

and Cary Eclipse Fluorescence Spectrophotometer with built-in Peltier

temperature controllers. Standard 10 mm light path quartz glass cells

from Hellma GmbH & Co. (Concord, ON) were used for both UVꢀvis

and fluorescence measurements. All UVꢀvis and fluorescence emission

spectra were recorded with baseline correction and stirring. Any water

used for buffers or spectroscopic solutions was obtained from a Milli-Q

filtration system (18.2 MΩ). pH measurements were taken at room

temperature with an Accumet 910 pH meter with an Accumet pH

Combination Electrode with stirring.

Unless otherwise noted, commercial compounds were used as received.

Arylboronic acids (phenyl, 4-hydroxyphenyl, and 2-hydroxyphenyl) were

purchased from Frontier Scientiﬁc (Logan, UT), Pd(OAc)₂was from

Sigma-Aldrich (Oakville, ON), dG was from ChemGenes (Wilmington,

MA), and 3,3⁰,3⁰⁰-phosphinidynetris(benzenesulfonic acid) trisodium salt

(TPPTS) was from Alfa Aesar (Ward Hill, MA). The synthesis of 8-bromo-

2⁰-deoxyguanosine (8-Br-dG) was performed according to literature pro-

cedures by treating dG with N-bromosuccinimide in water-acetonitrile.⁴³

SuzukiꢀMiyaura cross-coupling reactions of8-Br-dGwitharylboronicacids

were conducted according to the literature⁴⁴and NMR, and MS spectra for

p-PhOH-dG,^37,38o-PhOH-dG,^36,38and Ph-dG^34,44were identical to those

previously published.

Figure 1. Structures of C-linked nucleoside adducts and oligonucleo-

tide sequences.

which the bulky AAF group intercalates into the helix and

displaces C.^30,31When mismatched with purine bases, a minor

groove binding “wedged” (W) structure is formed with syn-AAF-

G forming a Hoogsteen bonding arrangement with the purine

base.³⁰

For the C-linked adducts derived from phenolic toxins, we

have recently developed a postsynthetic Pd-catalyzed cross-

coupling strategy for their insertion into oligonucleotides.³²This

strategy was prompted by our ﬁndings that C-linked phenolic

adducts are sensitive to acids³³and oxidants,³⁴making solid-

phase DNA synthesis problematic,³²and that they are too bulky

to be used as substrates for DNA polymerases,³⁵precluding an

enzymatic strategy for their synthesis. Puriﬁcation of the ad-

ducted oligonucleotides using HPLC was facilitated by the

ﬂuorescent nature of the C-linked phenolic adducts.^36ꢀ38Fluor-

escent DNA adducts are also sensitive probes for gaining insight

into the conformational preference of the adduct within DNA

helices.^39,40Our previous studies^38,41on the conformations of

8-(4⁰⁰-hydroxyphenyl)-dG (p-PhOH-dG) and 8-(2⁰⁰-hydroxy-

phenyl)-dG (o-PhOH-dG) (Figure 1) imply that the energy

diﬀerences between anti and syn are likely quite small and that

Photophysical Properties. Stock solutions of p-PhOH-dG, o-

PhOH-dG, and Ph-dG were prepared in DMSO (due to sparing

solubility in other solvents) to a concentration of 4 mM. Spectroscopic

solutions were prepared with 5 μL of stock solution and 1995 μL of

either 10 mM MOPS (pH 7.0, μ = 0.1 M NaCl) or CHCl₃. UVꢀvis

spectral measurements were observed from 220 to 400 nm, with

fluorescence spectra recorded at an excitation wavelength of 290 nm,

with emission recorded from 320 to 500 nm. Excitation and emission slit

widths were kept constant at 2.5 nm.

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Quantum Yield Measurement. The quantum yield value for Ph-

dG was determined in 10 mM MOPS buffer (pH 7.0, μ = 0.1 M NaCl)

using the comparative method.⁴⁵Quinine bisulfate (ϕ_fl= 0.546) in 0.5 M

H₂SO₄was used as the fluorescence quantum yield standard.⁴⁶The

following equation was used to calculate fluorescence quantum yields:

found, 3039.6. ODN1_b (m/z) calcd, 3039.6; observed [M ꢀ 3H]^3ꢀ

=

1012.2; found, 3039.6. ODN1_c (m/z) calcd, 3023.6; observed

[M ꢀ 3H]^3ꢀ= 1006.9; found, 3023.7. ODN2_a (m/z) calcd, 3239.6;

observed [M ꢀ 4H]^4ꢀ= 808.9; found, 3239.6. ODN2_b (m/z) calcd,

3239.6; observed [M ꢀ 4H]^4ꢀ= 808.9; found, 3239.6. ODN2_c (m/z)

calcd, 3023.6; observed [M ꢀ 3H]^3ꢀ= 804.9; found, 3223.6.

ϕ_flðxÞ ¼ ðA_s=A_xÞðF_x=F_sÞðη_x=η_sÞ₂ϕ_flðsÞ

Thermal Melting. UV melting studies were carried out on a Cary

300-Bio UVꢀvisible spectrophotometer equipped with a 6 ꢁ 6 Multicell

Block Peltier, stirrer, and temperature controller with Probe Series II.

Equal amounts (0.5 A₂₆₀/mL each) of ODN1 or ODN2 and the

complementary strands ODN1⁰(N) or ODN2⁰(N) (N = C, G, T, A)

were dissolved in 0.4 mL of buffer (200 mM NaCl, 50 mM triethylamine

acetate, pH 7.2, and 1 mM EDTA). The UV absorption at 260 nm was

monitored as a function of temperature. Samples were heated to 85 °C

for 3 min, and then, the temperature was decreased at a rate of 0.5 °C/

min from 85 to 5 °C. A typical melting experiment consisted of forward/

reverse scans and was repeated three times. Reverse temperature scans

showed no hysteresis. The T_mvalues were calculated by determining the

first derivative of the melting curve and were reproducible within

(1.0 °C of the reported values.

Circular Dichroism (CD) and Emission Spectra of Oligo-

nucleotides. CD measurements were obtained on a Jasco J-815 CD

spectrometer at 5 °C in 10 mM triethylamine acetate, pH 7.1, 200 mM

NaCl, and 0.5 A₂₆₀/mL of each oligonucleotide. Samples were scanned

from 400 to 200 at 0.5 nm intervals averaged over 1 s in a 1 mm light path

quartz cuvette. Fluorescence emission spectra were recorded at 5 °C on a

Cary Eclipse Fluorescence Spectrophotometer in 10 mM triethylamine

acetate, pH 7.1, 200 mM NaCl, containing 1 μM duplex using excitation

at 280 nm. All fluorescence spectra were recorded with excitation and

emission slit widths kept constant at 2.5 nm.

Computational Details. In molecular dynamics (MD) simula-

tions of the oligonucleotides, parameters for the adducts were taken

from the parmbsc0⁴⁷force field with the exception of certain dihedral

parameters for the phenoxyl moiety, which were adopted from previous

literature (see the Supporting Information for additional details).⁴⁸

ANTECHAMBER 1.4⁴⁹was used to assign atom types. The R.E.D.v.

III.4⁵⁰program was used to calculate the partial charges of the modified

bases using a multiconformation and multiorientation approach, as well

as the rigid-body reorientation algorithm (see the Supporting Informa-

tion for partial charges).⁵¹Two input structures corresponding to the

lowest energy anti and lowest energy syn structures previously identified

in the literature⁴¹were used in the multiconformation approach.

Intermolecular charge constraints between the nucleoside sugar moiety

and a dimethlyphosphate group were used, as well as intermolecular

charge equivalencing procedures,⁵⁰to obtain partial charges for the

sugar moiety in the adducts analogous to the four natural nucleosides in

the parm99⁵²force field. Partial charges were obtained from a two stage

RESP fitting and HartreeꢀFock single-point calculations with the

6-31G(d) basis set using the RED code interfaced with Gaussian 03⁵³

and ANTECHAMBER 1.4.⁴⁹

Simulations were carried out using the SANDER module of the

AMBER 10⁵⁴or 11⁵⁵software packages. The parmbsc0 modiﬁcation⁴⁷

to the parm99⁵²force ﬁeld was used since this is the most recently

developed force ﬁeld for nucleic acids. Initial structures were prepared

using the NAB program⁵⁴and GaussView⁵⁶to modify the C8-PhOH-

dG residue (see the Supporting Information for initial duplex

structures). The LEaP module of AMBER 10 was used to prepare the

systems for MD. The (natural or modiﬁed) DNA strand was neutralized

with 18 sodium ions and solvated with an 8 Å TIP3P octahedron water

box.⁵⁷Coulombic interactions were approximated using the particle-

mesh Ewald method,⁵⁸bonds involving hydrogen atoms were con-

strained using the SHAKE option,⁵⁹and a 2 fs time step was used

throughout the simulation. A 10 Å cutoﬀ was applied to LennardꢀJones

interactions, and periodic boundary conditions were implemented. Five

where s is the standard, x is the unknown, A is the absorbance at the

excitation wavelength, F is the integrated area under the emission curve,

η is the refractive index of the solvent, and ϕ_flis the quantum yield. The

refractive index corrective term was not included due to the similar

refractive indices of H₂O and 0.5 M H₂SO₄. Excitation and emission slit

widths were kept constant for all fluorescence measurements at 2.5 nm.

Absorbance readings were kept below 0.06 to avoid inner-filter and self-

absorbance phenomena.

Cyclic Voltammetry (CV). Electrochemical oxidation measure-

ments for o-PhOH-dG and Ph-dG were conducted in a three-electrode

glass cell under nitrogen. Measurements were carried out in a solution of

0.1 M N,N-dimethylformamide (DMF)/tetrabutylammonium hexa-

fluorophosphate (TBAF). The working electrode used was glassy

carbon, 2 mm in diameter. The electrode was polished and ultrasonically

rinsed with ethanol. A silver wire placed in a 0.1 M DMF/TBAF solution

was used as the reference electrode and was separated from the main

solution by a fine porosity frit. The reference electrode potential was

calibrated in situ against 1 mol equiv of 9,10-anthraquinone (ꢀ0.800 V

vs SCE). The counter electrode used was platinum wrapped in foil. For

all CVs, the starting potential was 0 V, and the potential was first scanned

1.8 V toward positive potentials and then scanned 1.8 V toward negative

potentials. The scanning rate used was 0.2 V/s. Peak picking was

achieved by correlation of values obtained from automatic software

methods and manual assignment.

SuzukiꢀMiyaura Coupling Reactions of Arylboronic Acids

with 8-Br-G-Modified Oligonucleotides. The oligonucleotide

substrates containing 8-Br-dG for the synthesis of ODN1_a,b,c and

ODN2_a,b,c were custom-made by Sigma Genosys (Oakville, ON) in

1 μmol scale using standard phosphoramidites and 8-Br-dG-CE phos-

phoramidite purchased from Glen Research (Sterling, VA). Oligonu-

cleotides were cleaved from the solid support and deprotected in

ammonium hydroxide at room temperature (to lessen debromination)

for 24 h. For SuzukiꢀMiyaura coupling of arylboronic acids with the

brominated oligonucleotides (Br-DNA) (0.1 μmol), the conditions

outlined previously were employed.³²The resulting adducted oligonu-

cleotides were purified by HPLC using an Agilent 1200 series HPLC

equipped with an autosampler, autocollector, diode array detector

(monitored at 258 and 280 nm), and fluorescence detector (λ_ext

=

280 nm; λ_em= 390 nm). Separation was carried out using a Phenomenox

(Torrance, CA) Clarity 3 μ Oligo-RP C18 column (50 mm ꢁ 4.60 mm,

3 μ) and various gradients of buffer B in buffer A, in which buffer A was

aqueous 50 mM triethylammonium acetate (TEAA), pH 7.2:acetonitrile

(95:5) and buffer B was aqueous 50 mM TEAA, pH 7.2:acetonitrile

(25:75). Yields (ODN1_a = 79%, ODN1_b = 83%, ODN1_c = 55%,

ODN2_a = 77%, ODN2_b = 83%, and ODN2_c = 97%) were

estimated by quantification of the isolated product by UV using ε₂₆₀

(nearest neighbor method) for the unmodified oligonucleotides,

87700 cm^ꢀ1M^ꢀ1for unmodified ODN1 and 106000 cm^ꢀ1M^ꢀ1for

unmodified ODN2 (i.e., X = G).

MS Spectra of Oligonucleotides. Modified oligonucleotides

ODN1_a,b,c and ODN2_a,b,c were dissolved in 50% water in methanol

with 0.1 mM ammonium acetate for mass spectral analysis. Full scan

mass spectra were acquired by infusion through an electrospray ioniza-

tion source in negative mode (ESI^ꢀ) using a Waters Q-TOF Micro

quadrupole time-of-flight instrument. Low voltages were used for the

cone (14 V) and extraction cone (1 V) for oligonucleotide detection.

ESI^ꢀMS ODN1_a (m/z) calcd, 3039.6; observed [M ꢀ 3H]^3ꢀ= 1012.2;

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Table 1. Photophysical and Redox Parameters for C-Linked Nucleoside Adducts

c

e

adduct

λ_max(nm), log ε^b

λ_em(nm),^bΦ_ﬂ

λ_em(nm),^dI_rel

E_p/2(V/SCE)^f

p-PhOH-dG

o-PhOH-dG

Ph-dG

278, 4.26

276, 4.25

253, 4.14

390, 0.47

371, 0.03

0.85

0.98

1.06

1.14

395, 0.44

375, 467, 0.25

375, 0.07

393, 0.44

334, 9.7 ꢁ 10^ꢀ5

dG^a

^aOptical data for dG taken from ref 64. ^bDetermined in aqueous 10 mM MOPS buﬀer, pH 7.0, μ = 0.1 M NaCl. ^cDetermined using the comparative

method with quinine bisulfate in 0.5 M H₂SO₄(ϕ_ﬂ= 0.55). ^dEmission data recorded in CHCl₃. ^eRelative ﬂuorescence emission intensity in CHCl₃as

compared to MOPS buﬀered water. ^fHalf-peak potentials in volts vs SCE using CV in 0.1 M TBAF in anhydrous DMF with a glassy carbon working

electrode.

Scheme 2

hundred steps of steepest descent minimization followed by 500 steps of

conjugate gradient minimization were performed with the solute held

ﬁxed using a force constant of 500 kcal mol^ꢀ1Å^ꢀ2. Subsequently, 1000

steps of steepest descent minimization followed by 1500 steps of

conjugate gradient minimization were performed on the entire system.

The system was heated from 0 to 300 K using the Langevin thermostat

with the solute restrained using a force constant of 10 kcal mol^ꢀ1Å^ꢀ2

over 20 ps under constant volume conditions. Following heating, a total

of 20 ns of unrestrained MD simulations was carried out on each system

under constant temperature (300 K) and pressure (1 atm) conditions.

Snapshots were taken every 500 fs, and average structures were

calculated from the ﬁnal 2 ns of the simulation. Plots of rmsd vs time

are available in the Supporting Information to show the stability of the

simulations.

with the emitting excited state possessing a dipole moment

greater than the ground state.⁶⁵The o-PhOH-dG adduct also

showed a peak in the emission spectrum recorded in CHCl₃at

467 nm that was absent in aqueous buffer. As discussed in detail

previously,^36,38this red-shifted emission is due to the presence of

the keto tautomer resulting from an excited state intramolecular

proton transfer (ESIPT) process, as outlined in Scheme 2. A

requirement for observation of the keto tautomer is the intra-

molecular H-bond between the phenolic OH and the N7 of o-

PhOH-dG in the ground state enol structure shown in Scheme 2.

That o-PhOH-dG shows only enol emission at 395 nm in water

indicates that the intramolecular H-bond required for ESIPT is

disrupted in the H-bonding solvent.

The electron donor properties of the nucleoside adducts and

dG were determined using CV in anhydrous DMF, as outlined

previously.^34,37The nucleoside analogues showed irreversible

1-electron oxidation peaks withhalf-peak potentials (E_p/2) ranging

from 0.85 V/SCE for p-PhOH-dG to 1.06 V for Ph-dG. Under

these experimental conditions, dG gave E_p/2= 1.14 V/SCE,^34,37

indicatingthatattachmentof the aryl moiety enhances the electron

donor characteristics of the purine nucleoside, especially when a

hydroxyl group is included in the para-position.

DNA Melting Studies. The adducted decanucleotides ODN1_a,b,c

and ODN2_a,b,c were hybridized to the complementary strands

ODN1⁰(N) and ODN2⁰(N), where N is the base opposite the

adduct X (Figure 1). The impact of the C-linked adducts on

thermal stability of the duplex was measured using UV melting

temperature analysis (T_m) with the T_mdata summarized in

Table 2. When N = C, the T_mdata showed the C8ꢀAr-dG

adduct to decrease helix stability (i.e., ΔT_m= ꢀ6 to ꢀ17 °C) as

compared to the control unmodified duplex (T_m≈ 44 °C).

Introduction of a mismatch (N = G, A, and T) within the control

duplexes also decreased helix stability with T_mvalues ranging

from 25 to 31 °C (ΔT_m= ꢀ13 to ꢀ19 °C). Within the

mismatched sequences, the C-linked adducts could not form

stable duplexes when mismatched with A or T but could cause a

stabilizing influence on the G:G mismatch. The only exception to

this trend was o-PhOH-dG within ODN1 where a destabilization

for ODN1_b:ODN1⁰(G) was observed (ΔT_m= ꢀ6 °C). How-

ever, for p-PhOH-dG and Ph-dG in both decanucleotides and

H-bonding interactions were calculated for MD structures obtained

by averaging the coordinates of the frames from the ﬁnal 2 ns of the

trajectory, and replacing the backbone and surrounding bases with a

hydrogen atom. ΔE_Hbondis calculated using counterpoise-corrected

B3LYP/6-311+G(2df,p) energies. Stacking interactions were similarly

calculated for MD structures obtained by averaging the coordinates of

the frames that occupy the range of θ values in the most populated

minimum region (shown in histograms of θ distributions, see the

Discussion) of the adduct and replacing the backbone and surrounding

bases with an H atom. Stacking binding strengths were calculated with

M06-2X/6-31+G(d,p) since this combination has been shown to

produce reliable interaction energies for noncovalent interactions with

large dispersion components.⁶⁰ΔE_Int5is deﬁned as the interaction

0

energy between X and the 5⁰-ﬂanking base (G for ODN2 or T for

0

ODN1), while ΔE_Int3is the interaction energy between X and the

3⁰-ﬂanking base (A for ODN2 or C for ODN1). All (gas phase)

interaction energies are calculated as the energy diﬀerence between

the dimer and the individual monomers in the dimer geometry. All DFT

calculations were performed with Gaussian 03⁵³or Gaussian 09.⁶¹

’ RESULTS

Photophysical and Redox Properties of Nucleoside

Adducts. The optical and redox properties of p-PhOH-dG, o-PhOH-

dG, and Ph-dG are given in Table 1. Absorption and emission

spectra were recorded in aqueous MOPS buffer, pH 7.0, and in

CHCl₃to model the solvatochromic properties of the adducts in

the interior of the DNA helix, given that the dielectric constant

(ε = 4.9) ofCHCl₃issimilar to that induplex DNA (ε = 3ꢀ5).^62,63

Optical data for unmodified dG in aqueous media⁶⁴are also

included for comparison. The results show blue-shifted

(∼20 nm) and quenched emission for the C-linked adducts

in CHCl₃as compared to aqueous buffer. However, the

change in solvent had little influence on the absorption

maxima of the nucleoside adducts, indicating a decrease in Stokes'

shift in CHCl₃as compared to water. This observation is consistent

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Table 2. Emission Properties and T_mValues Derived from

UV Melting Experiments

a

b

c

d

e

ODN

T_m

ΔT_m

λ_em

407

Δλ_em

I_rel

ODN1_a

+17

ꢀ21

ꢀ23

ꢀ7

ꢀ44

ꢀ2

ꢀ3

ꢀ8

ꢀ4

ꢀ9

ꢀ2

+14

ꢀ1

ꢀ15

ꢀ13

+6

0.60

0.31

0.22

0.74

0.45

0.69

0.19

0.16

0.32

0.40

0.32

1.66

0.25

2.23

1.78

0.71

0.42

0.182

0.33

0.29

0.88

0.05

0.52

0.75

0.15

1.15

0.50

1.37

1.46

ODN1_a:1⁰(C)

ODN1_a:1⁰(G)

ODN1_a:1⁰(A)

ODN1_a:1⁰(T)

ODN1_b

ODN1_b:1⁰(C)

ODN1_b:1⁰(G)

ODN1_b:1⁰(A)

ODN1_b:1⁰(T)

ODN1_c

ODN1_c:1⁰(C)

ODN1_c:1⁰(G)

ODN1_c:1⁰(A)

ODN1_c:1⁰(T)

ODN2_a

ODN2_a:2⁰(C)

ODN2_a:2⁰(G)

ODN2_a:2⁰(A)

ODN2_a:2⁰(T)

ODN2_b

ODN2_b:2⁰(C)

ODN2_b:2⁰(G)

ODN2_b:2⁰(A)

ODN2_b:2⁰(T)

ODN2_c

ODN2_c:2⁰(C)

ODN2_c:2⁰(G)

ODN2_c:2⁰(A)

ODN2_c:2⁰(T)

27

31

20

ꢀ17

+1

386

384

400

363

393

390

385

389

ꢀ5

ꢀ11

31

24

23

22

ꢀ13

ꢀ6

ꢀ2

ꢀ9

384, 485

391

405

390

376

378

396

382

395

396

388

383

380

386

395

382

388

386

391

386

385

31

32

23

26

ꢀ13

+2

ꢀ2

ꢀ5

28

39

23

21

ꢀ16

+9

ꢀ14

ꢀ1

0

ꢀ8

ꢀ7

ꢀ8

ꢀ12

ꢀ3

+3

38

39

21

23

ꢀ6

+9

ꢀ4

ꢀ8

+12

ꢀ1

ꢀ5

ꢀ2

+3

Figure 2. Fluorescence emission spectra of nucleoside adducts and

oligonucleotides recorded at 5 °C in 10 mM triethylamine acetate, pH

7.1, 200 mM NaCl with excitation at 280 nm. (a) p-PhOH-dG (dashed

black trace), ODN1_a (solid black trace), and ODN2_a (dotted black

trace); (b) o-PhOH-dG (dashed red trace), ODN2_b (solid red trace),

and ODN2-b (dotted red trace); and (c) Ph-dG (dashed blue trace),

ODN1_c (solid blue trace), and ODN2_c (dotted blue trace).

34

35

25

21

ꢀ10

+5

0

ꢀ2

ꢀ3

ꢀ10

^aIn °C, determined in 50 mM triethylamine acetate, pH 7.1, 200 mM

NaCl, 0.5 A₂₆₀/mL of each oligonucleotide. ^bChange in T_mrelative to

unmodiﬁed duplexes. ^cIn nm, determined at 5 °C in 10 mM triethyla-

mine acetate, pH 7.1, 200 mM NaCl. ^dChange in emission maximum for

ODN1 and ODN2 vs nucleoside adducts and duplexes vs ODN1

Figure 3 shows emission spectra for the adducted decanucleo-

tides hydridized to the complementary strands. The changes in

emission maxima (Δλ_em) and relative ﬂuorescence emission

intensity (I_rel) for the decanucleotide strands and duplexes are

included with the T_mdata in Table 2. Upon hybridization, blue

shifts in emission maxima were typically observed for ODN1_a-c

and ODN2_a-c duplexes. This was especially the case for p-

PhOH-dG in ODN1 (Figure 3a), where signiﬁcant blue shifts

were observed for duplexes involving N = C (Δλ_em= ꢀ21 nm),

G (Δλ_em= ꢀ23 nm), and T (Δλ_em= ꢀ44 nm), while only a blue

shift of 7 nm was observed for N = A (open circles, Figure 3a). Much

smaller blue shifts were observed for ODN2_a (Figure 3b),

although a blue shift of 14 nm was observed for ODN2_a:

ODN2⁰(C) (dashed trace). This general trend of greater blue

shifts for duplexes involving ODN1 was also observed for the

modiﬁed strands containing o-PhOH-dG (Figure 3c vs d) and

Ph-dG (Figure 3e vs f). Given that a decrease in solvent polarity

causes a blue shift in emission maxima for these adducts

(Table 1), the Δλ_emdata suggested a general decrease in solvent

exposure of the modiﬁed base upon duplex formation.

e

and ODN2. Relative ﬂuorescence emission intensity for ODN1 and

ODN2 vs nucleoside adducts and duplexes vs ODN1 and ODN2.

o-PhOH-dG within ODN2, T_mvalues ranging from 31 to 39 °C

were recorded for a maximum increase in T_mof 9 °C as compared to

the unmodified G:G mismatch (T_m= 30 °C). Stabilization of the G:

G mismatch was greater in ODN2 (ΔT_m5ꢀ9 °C) than ODN1

(ΔT_m1ꢀ2 °C), suggesting that the flanking purine bases in ODN2

play a key role in the observed stabilization.

Fluorescence Emission Studies. Figure 2 shows emission

spectra of ODN1_a,b,c and ODN2_a,b,c versus free nucleoside

adduct in aqueous buffer. In all instances, the oligonucleotides

showed quenched emission intensity as compared to the free

nucleoside adducts, especially for ODN1_c and ODN2_c vs Ph-

dG shown in Figure 3c (I_rel= 0.32 for ODN1_c, 0.15 for

ODN2_c). The observed emission quenching was ascribed to

π-stacking interactions of the emissive adduct with the flanking

bases. The greater quenching observed for ODN2_c (flanking

purine bases versus pyrimidine bases for ODN1_c) relative to

the free nucleoside Ph-dG was consistent with this hypothesis.

For the phenolic C-linked adducts (a and b), another notice-

able change in ﬂuorescence upon duplex formation was quench-

ing. This phenomenon was dependent on the base opposite (N)

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Figure 3. Fluorescence emission spectra of duplex oligonucleotides (ODN1:ODN1⁰and ODN2:ODN2⁰) recorded at 5 °C in 10 mM triethylamine

acetate, pH 7.1, 200 mM NaCl with excitation at 280 nm. (a) ODN1_a:ODN1⁰, (b) ODN2_a:ODN2⁰, (c) ODN1_b:ODN1⁰, (d) ODN2_b:ODN2⁰,

(e) ODN1_c:ODN1⁰, and (f) ODN2_c:ODN2⁰, where the speciﬁc complementary strands, ODN1⁰and ODN2⁰, are represented by N = C (dashed

trace), G (solid trace), A (long dash and dot), and T (dotted trace).

the adduct, as the quenched ﬂuorescence was typically more

prominent when N = C or G, which form the more stable duplex

structures. This was especially the case for ODN2_b opposite

N = C or G where I_rel= 0.05, while opposite N = A or T, I_rel= 0.52,

0.72. For both C-linked phenolic adducts, greater quenching was

observed within ODN2, suggesting more favorable stacking

interactions with the ﬂanking purine bases. In contrast, the

ﬂuorescence of duplexes containing Ph-dG (c) actually increased

when N = C, A, and T, as exempliﬁed for ODN1_c (N = C, I_rel=

1.66; N = A, I_rel= 2.23; and N = T, I_rel= 1.78).

Changes in the emission maxima also provided insight into the

(anti/syn) orientation of the adduct. Particularly diagnostic in

this regard was the emission spectra for o-PhOH-dG within

ODN1 shown in Figure 3c. When mismatched with T (dotted

trace), the nucleoside adduct undergoes an ESIPT process to

generate the keto tautomer (Scheme 2), as indicated by the red-

shifted emission peak at 485 nm. This ESIPT process for o-

PhOH-dG was not observed in the single-strand decanucleotides

(Figure 2b), nor was it observed for the other duplexes

(Figure 3c,d). The observed ESIPT conﬁrms the presence of

the intramolecular H-bond between the phenolic OH and the N7

of o-PhOH-dG within the duplex and suggests that the Hoogs-

teen face of the adduct is not solvent exposed.^36,38This observa-

tion argues strongly that o-PhOH-dG is in the syn-conformation

within ODN1_b:ODN1⁰(T). It is also informative that the keto

tautomer of o-PhOH-dG only occurs with the T mismatch; the

only base to lack an endocyclic H-bond donor N-atom. For the

other bases, Hoogsteen base pairing⁴²or ESPT from the

phenolic moiety to more basic nearby N-atoms could inhibit

the ESIPT process.

CD. CD spectra of ODN1:ODN1⁰(C) and ODN2:ODN2⁰(C)

are shown in Figure 4. The unmodified duplexes (dashed traces)

show characteristics of normal B form DNA with roughly equal

positive (275 nm) and negative (244 nm) bands and a crossover

at ∼260 nm.⁶⁶The CD spectra of the modified duplexes for

ODN2 shown in Figure 4b also show characteristics that closely

resemble the normal B form helix. Greater changes in CD

features were noted for the duplexes for ODN1 (Figure 4a).

Here, the positive bands are slightly blue-shifted (271 nm) and

broader, while the negative band is decreased in intensity and

blue-shifted (224 nm). This observation suggests more distor-

tion for duplexes involving ODN1 than ODN2. Unfortunately,

the C-linked adducts absorption maxima overlap with DNA

absorption, and it was not possible to resolve ellipticities due

to the modified bases

Computational Studies. MD simulations on the unmodified

ODN1:1⁰(C) and ODN2:2⁰(C) sequences (X = G) yield

DNA duplexes with average B-DNA backbone dihedral angles

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Table 3. DFT Interaction Energies (kJ mol^ꢀ1) in Average

Conformations Obtained from MD Simulations

a

₀b

₀c

ΔE _Int3

conformation

X

N

C

strand

ΔE_Hbond

ΔE_Int5

anti

G

ODN1

ODN2

ODN1

ODN2

ODN1

ODN2

ODN1

ODN2

ODN1

ODN2

ODN1

ODN2

ODN1

ODN2

ODN1

ODN2

ODN1

ODN2

ODN1

ODN2

ODN1

ODN2

ODN1

ODN2

ꢀ106.7

ꢀ108.7

ꢀ49.9

ꢀ50.5

ꢀ105.6

ꢀ106.4

ꢀ22.7

ꢀ49.0

ꢀ105.3

ꢀ107.1

ꢀ26.3

ꢀ55.7

N/A

ꢀ12.2

ꢀ6.0

ꢀ50.1

ꢀ24.8

ꢀ42.9

ꢀ34.4

ꢀ51.6

ꢀ37.7

ꢀ46.3

ꢀ33.6

ꢀ59.7

ꢀ39.1

ꢀ54.2

ꢀ33.7

N/A

G

C

G

C

G

C

G

C

G

C

G

ꢀ22.5

ꢀ13.5

ꢀ17.3

ꢀ18.6

ꢀ15.5

ꢀ12.6

ꢀ10.1

ꢀ14.9

ꢀ19.7

ꢀ13.0

N/A

a

b

G

a

syn

N/A

ꢀ50.5

ꢀ49.9

ꢀ35.5

ꢀ3.6

ꢀ46.9

ꢀ48.2

6.0

ꢀ13.8

ꢀ27.9

ꢀ16.6

ꢀ55.8

ꢀ16.5

ꢀ35.5

ꢀ13.5

ꢀ36.5

ꢀ16.8

ꢀ44.0

ꢀ6.3

ꢀ13.4

ꢀ7.0

ꢀ18.2

ꢀ14.2

ꢀ19.9

ꢀ6.6

ꢀ27.3

ꢀ7.9

ꢀ20.4

Figure 4. CD spectra of duplex oligonucleotides (N = C) recorded at

5 °C in 10 mM triethylamine acetate, pH 7.1, 200 mM NaCl. (a)

Unmodiﬁed ODN1:ODN1⁰(dashed), ODN1_a:ODN1⁰(black),

ODN1_b:ODN1⁰(red), and ODN1_c:ODN1⁰(blue); (b) unmodiﬁed

ODN2:ODN2⁰(dashed), ODN2_a:ODN2⁰(black), ODN2_b:ODN2⁰

(red), and ODN2_c:ODN2⁰(blue).

b

ꢀ24.7

ꢀ45.5

ꢀ41.3

^aΔE_Hbondis calculated as the counterpoise-corrected B3LYP/6-311

(Table S1 in the Supporting Information) and three highly

occupied (∼99%) WatsonꢀCrick H-bonds between X and C.

The average structure of the X:C base pair has a stability of ꢀ106.7

kJ/mol for ODN1 and ꢀ108.7 kJ/mol for ODN2 (Table 3).

When X = anti (syn) G is mispaired with syn (anti) G, two strong

G:G (∼50 kJ/mol) Hoogsteen bonds form with 68 (93) and 96

(93)% occupancy in ODN1:1⁰and 93 (68) and 93 (96)%

occupancy in ODN2:2⁰(Table S2 in the Supporting In-

formation). The strands containing the mispair are likely less

stable than native DNA helices due to weaker G:N H-bond

strengths (by approximately 50 kJ/mol, Table 3). The strength of

stacking interactions between X = G and the 5⁰-flanking base is

ꢀ12 kJ/mol in ODN1:1⁰(C) and ꢀ6 kJ/mol in ODN2:2⁰(C),

while the interactions with the 3⁰-flanking base are ꢀ50 kJ/mol in

ODN1:1⁰(C) and ꢀ25 kJ/mol in ODN2:2⁰(C). For G:G mis-

matches, the stacking interactions range between ꢀ13 and ꢀ27

kJ/mol for the 5⁰-flanking base and ꢀ6 and ꢀ42 kJ/mol for the

3⁰-flanking base. The calculated interaction strengths for these

unmodified bases compare very well to values reported in the

literature^42,67ꢀ71and, therefore, provide a benchmark for com-

parison to the binding strengths of the adducts.

When X is modiﬁed to the o-PhOH- or p-PhOH-dG adduct,

no large structural changes occur to the double helix. The strands

retain average values of B-DNA backbone dihedral angles with

the exception of some local distortion at the site of the adduct

(Table S1 in the Supporting Information). All natural and

modiﬁed bases remain within the helix regardless of the (anti

or syn) conformation adopted (Figure 5). Thus, although inter-

calation of related bulky adducts that adopt the syn-conformation

into the helix can displace the opposing base,^30,31the relatively

small size of the C8-phenyl group is well accommodated opposite

+G(2df,p) H-bond strength between the dimer consisting of X and N.

^bΔE_Int5is deﬁned as the M06-2X/6-31+G(d,p) interaction energy

0

between X and the 5⁰-ﬂanking base (G for ODN2 or T for ODN1).

^cΔE_Int3is deﬁned as the M06-2X/6-31+G(d,p) interaction energy

0

between X and the 3⁰-ﬂanking base (A for ODN2 or C for ODN1).

another base within the helix. However, the structure of the

adduct nucleotide can signiﬁcantly deviate from that predicted by

smaller (nucleoside and nucleotide) computational models.⁴¹In

particular, the dihedral angle θ describing the relative orientation

of the phenoxyl group and the dG moiety can vary widely with

the adduct, the sequence, and the (anti/syn) orientation. There-

fore, further details of the structure of each adduct in each

sequence when paired with C or mismatched with G will be

discussed below. H-bond stabilities and occupancies (percentage

of the total simulation time) will also be presented, where greater

occupancies represent more stable H-bonds. In addition, the

strength of (intrastrand) stacking interactions will be discussed

to aid the determination of sequence eﬀects on adduct structure

and stability.

anti-Conformation of Adducts (X = a or b) for N = C. When

the phenolic adducts (a and b) adopt the anti-conformation in

ODN1:1⁰(C) and ODN2:2⁰(C), the bulky phenoxyl group is

located in the major groove (Figure 5, far left). Three strong

(∼ꢀ100 kJ/mol, Table 3) WatsonꢀCrick H-bonds are intact

between X:C (98ꢀ100% occupancy, Tables 4 and 5 and

Figure 6a). Stacking between X and the flanking bases is generally

stronger as compared to the unmodified sequences (by up to

14 kJ/mol, Table 3). However, the adduct structure is sequence

dependent. For ODN2, the average θ of both adducts through-

out the simulation (∼220ꢀ225°, Figures 7 and 8) is similar to

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Figure 5. Examples of MD snapshots showing the view into the major groove of the B-DNA structure of the modiﬁed ODN1 and ODN2 duplexes with

X = p-PhOH-dG (a) or o-PhOH-dG (b) (blue) paired opposite N = C or G (red) (purine or pyrimidine ﬂanking bases highlighted in green).

Table 4. Hydrogen-Bond Occupancies for X = a over the

Duration of MD Simulations

Table 5. Hydrogen-Bond Occupancies for X = b over the

Duration of MD Simulations

N

C

strand

adduct conformation

H-bond^a

%

N

C

strand

adduct conformation

H-bond^a

%

ODN1

anti

N2ꢀH1 O2

100.0

98.9

72.4

43.1

99.7

99.9

99.0

39.3

23.2

92.3

56.5

45.0

96.5

94.9

82.7

86.1

94.0

92.9

ODN1

anti

N1ꢀH N3

99.9

99.6

98.5

65.4

95.1

61.1

36.6

99.8

99.9

89.8

98.4

91.9

72.7

22.0

75.6

49.2

47.7

97.2

95.9

76.7

57.2

89.2

73.7

45.4

30.7

76.9

68.2

20.9

39.3

35.2

3 3 3

N1ꢀH N3

N2ꢀH O2

3 3 3

N4ꢀH1 O6

3 3 3

syn

N4ꢀH2 O6

3⁰-N4ꢀH O11

3 3 3

N4ꢀH1 N7

syn

OꢀH N7

3 3 3

ODN2

ODN1

ODN2

anti

N2ꢀH1 O2

N4ꢀH1 O6

3 3 3

N1ꢀH N3

N4ꢀH2 O6

3 3 3

N4ꢀH1 O6

ODN2

ODN1

ODN2

anti

N2ꢀH O2

3 3 3

syn

N4ꢀH1 O6

N1ꢀH N3

3 3 3

N4ꢀH1 N7

OꢀH N7

3 3 3

G

anti

N2ꢀH1_aN7

N4ꢀH O6

3 3 3

N1ꢀH_aO6

syn

anti

syn

OꢀH N7

3 3 3

N1ꢀH_aN7

N4ꢀH1 O6

3 3 3

syn

anti

syn

N1ꢀH O6_a

N4ꢀH2 O6

3 3 3

N2ꢀH N7_a

G

N2ꢀH1_aN7

3 3 3

N1ꢀH_aO6

3 3 3

N2ꢀH1_aN7

N1ꢀH_aN7

3 3 3

N1ꢀH O6_a

3 3 3

N2ꢀH1 N7_a

OꢀH N7

3 3 3

^aData are provided for H-bonds within 3.40 Å heavy atom separation

and 120° XꢀHꢀX angle for greater than 20% of the total simulation

time. To distinguish between X:G mismatches, atoms belonging to the

adduct are denoted with a subscript “a”.

N2ꢀH1 O6_a

3 3 3

N2ꢀH1 N7_a

3 3 3

anti

syn

N1ꢀH1_aO6

3 3 3

N2ꢀH1_aN7

3 3 3

OꢀH N7

3 3 3

the minimum identified using smaller computational models,

which predict a small twist (by 20°)⁴¹for the ortho adduct due to

N2ꢀH1_aO6

3 3 3

N1ꢀH O6_a

a strongly stabilizing (OꢀH N7) intramolecular H-bond, and

3 3 3

only a slightly greater twist (by 31°)⁴¹for p-PhOH-dG. As a

result in ODN2, the hydroxyl group of o-PhOH-dG remains

H-bonded to N7 for 90% of the simulation (Table 5). In ODN1,

p-PhOH-dG has three minimum conformations of θ (θ = ∼40,

∼220, and ∼310°, respectively, Figure 7), which differ from o-

PhOH-dG since the location of the hydroxyl group does not

afford the same H-bonds. For ODN1, the θ angle in o-PhOH-dG

is predominantly highly twisted (θ ≈ 100° for the dominant

conformation) throughout the majority of the simulation

(Figure 8), which corresponds to a structure where the OH

group is directed away from the dG nucleobase. Correspondingly,

N2ꢀH1 N7_a

3 3 3

OꢀH N7

3 3 3

N2ꢀH1 O6_a

3 3 3

5⁰-N2ꢀH2 O11

3 3 3

^aData are provided for H-bonds within 3.40 Å heavy atom separation

and 120° XꢀHꢀX angle for greater than 20% of the total simulation

time. To distinguish between X:G mismatches, atoms belonging to the

adduct are denoted with a subscript “a”.

the OꢀH N7 H-bond is disrupted (<20% occupancy).

3 3 3

Instead, there is a very strong (ꢀ50 kJ/mol, Table 3) interaction

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between the phenoxyl oxygen in the adduct and the flanking 3⁰-

dC amino group for 65% of the simulation (Table 5). The

difference in θ, and the corresponding difference in H-bonding

patterns for o-PhOH-dG, within ODN1 versus ODN2 suggests

that the adduct structure is dependent on the sequence. Regard-

less, the similarity in H-bonding and stacking between the anti-

orientation of the adducts and the unmodified sequences may

result in similar stability between the natural and the modified

strands.

syn-Conformation of the Adducts (X = a or b) for N = C.

When o-PhOH-dG adopts the syn-conformation within ODN1:1⁰(C)

and ODN2:2⁰(C), the phenolic ring is located in the minor

groove (Figure 5), and there is much greater distortion in the

base pairs as compared with the corresponding anti pairs. This

is due to the decreased stability of Hoogsteen as compared to

WatsonꢀCrick H-bonds for X:C. In fact, the (syn) adduct base

pair is repulsive in ODN1:1⁰(C) (by 6 kJ/mol, Table 3) and only

weakly stable in ODN2:2⁰(C) (by ꢀ24 kJ/mol, Table 3), due to

the presence of only one (or a maximum of two) H-bonds (see,

for example, Figure 6b). Specifically, N4ꢀH of C H-bonds with

O6 of the adduct for 61ꢀ73% of the simulation (Table 5). The

second amino hydrogen also interacts with O6 in some config-

urations (22ꢀ37% occupancy, Table 5). However, a Hoogsteen

H-bond involving N7 cannot form since a stable intramolecular

Figure 6. Average structures of H-bonded base pairs from MD simula-

tions: (a) ODN1_a:1⁰(C) with the anti-conformation of the adduct, (b)

ODN1_b:1⁰(C) with the syn-conformation of the adduct, and (c)

ODN1_a:1⁰(C) with the syn-conformation of the adduct.

Figure 7. Percent distribution of θ (degrees) throughout the 20 ns trajectories for the p-PhOH-dG (a) adduct.

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Figure 8. Percent distribution of θ (degrees) throughout the 20 ns trajectories for the o-PhOH-dG (b) adduct.

OꢀH N7 H-bond is intact for 92ꢀ95% of the simulation.

H-bond contacts occur between the WatsonꢀCrick face of the

adduct and the Hoogsteen face of G. Specifically, the N2 amino

group of the adduct bonds to N7 of G (74ꢀ92%, Tables 4 and 5)

and N1ꢀH of the adduct bonds to O6 of G (49ꢀ89%).

However, as compared to the unmodified G:G mismatch, the

Hoogsteen base pair strength is considerably weaker for the

modified ODN1:1⁰(G) (ꢀ23 to ꢀ26 kJ/mol as compared to

ꢀ50 kJ/mol, Table 3) but comparable for ODN2:2⁰(G) (ꢀ49 to

ꢀ56 kJ/mol, Table 3). As found when the anti-adducts are paired

with C, the θ twist is dependent on both the sequence and the

adduct. For ODN1_a:1⁰(G), three minimum conformations of

θ occur at θ = ∼280, 220, and 50°, and there is a single confor-

mation (θ = 256.5°, Figure 7) for ODN2_a:2⁰(G). These values

correspond to a highly twisted structure regardless of sequence

for p-PhOH-dG. For o-PhOH-dG, an average θ value of ∼80°

occurs for ODN1:1⁰(G) (Figure 8), which corresponds to

a highly twisted structure (similar to p-PhOH-dG) with no

3 3 3

Because of the poor H-bonding between the bases, the strands

are expected to be destabilized when the syn-conformation of the

adduct is paired with C as compared with strands containing

natural dG or the anti-adduct paired with C.

When p-PhOH-dG adopts the syn-conformation opposite C,

there are some diﬀerences in Hoogsteen bonding as compared to

o-PhOH-dG. Speciﬁcally, the Hoogsteen bond between the

N4ꢀH of C and O6 in the adduct is present for less time for

p-PhOH-dG (8% for ODN1 and 39% for ODN2, Table 4) as

compared to o-PhOH-dG (61ꢀ73%, Table 5). Instead, the domi-

nant binding conformation of p-PhOH-dG in ODN1 involves one

or two H-bonds between the N4 amino group of C and O6 (72%)

and, interestingly, sometimes an interaction between N4ꢀH of C

and N7 (43%) (Figure 6c). This increased Hoogsteen bonding to

N7 in ODN1 is likely due to a lack of competing OꢀH N7

3 3 3

H-bond. This results in a base pair stability of ꢀ35 kJ/mol in ODN1

(Table 3). There is less Hoogsteen bonding to N7 in ODN2 (23%,

Table 4), which leads to weaker H-bonding (ꢀ3.6 kJ/mol, Table 3),

although the reason for this is currently unclear.

OꢀH N7 H-bond (within the cutoff criteria, Table 5). For

3 3 3

ODN2:2⁰(G), two minima θ conformations occur (Figure 8). In

the first (θ ∼ 230°), the OꢀH N7 H-bond is intact (45%,

3 3 3

anti-Conformation of Adducts (X = a or b) for N = G. When

the adducts adopt the anti-conformation opposite syn-G, two

Table 5), while the phenoxyl group rotates (θ ∼ 270°) in the

second conformation, which breaks or significantly weakens the

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Figure 10. H-bonding in ODN2:2⁰(G) from MD simulation. (a) The

syn-conformation of p-PhOH-dG paired with G. (b) The syn-conforma-

tion of o-PhOH-dG paired with G. (c) The H-bonding interaction

between o-PhOH-dG and the 5⁰-ﬂanking G base.

Figure 9. H-bonding in ODN1:1⁰(G) from MD simulation. (a) The

syn-conformation of p-PhOH-dG paired with G. (b) A representative

structure of the H-bonding pattern for the syn-conformation of o-PhOH-

dG paired with G. (c) The average H-bonding pattern of the syn-

conformation of o-PhOH-dG paired with G that overestimates the

strand stability.

disrupted and the phenoxyl group forming a N2ꢀH O inter-

3 3 3

action (35%, Table 5) with the 5⁰-flanking G base (Figure 10c).

This reduces the occupation of the OꢀH N7 contact (to 21%)

3 3 3

and frees N7 to form Hoogsteen bonds.

’ DISCUSSION

OꢀH N7 H-bond. These changes in θ lead to adduct

Structural Impact of C-Linked Phenolic Adducts on

Duplex DNA. The data summarized in Table 2 show the C⁸-

Ar-dG adduct to destabilize the duplex when base paired with its

normal partner C. This observation is in agreement with previous

studies showing bulky C-linked and N-linked C⁸-Ar-G lesions to

decrease duplex stability. The Wagenknecht laboratory carried

out T_mstudies on the C8-(pyren-1-yl)-dG modification and

recorded a ΔT_mvalue of ∼ꢀ10 °C for the adduct when placed in

the middle of the 16-mer duplex 5⁰-GCATCTGXATCACTGA,

where X = Py-8-dG.⁷²The N-linked C8-dG adducts of 2-amino-

fluorene (AF) and AAF also destabilize the duplex considerably

when base paired with C. For the N-linked AF adduct, Elmquist

and co-workers incorporated the lesion into three 12-mer

oligonucleotides and showed the adduct to lower the T_mby

8ꢀ13 °C relative to the unmodified duplex.⁷³For the N-linked

AAF adduct, Koehl and co-workers⁷⁴incorporated the adduct

into the three G sites of the NarI oligonucleotide 5⁰-AC-

CGGCGCCACA (underlined Gs were replaced with the

N-linked AAF adduct). The AAF-modified oligonucleotides

showed a large destabilization with ΔT_mvalues of ꢀ10ꢀ13 °C.⁷⁴

Factors thought to cause the destabilizing inﬂuence of the

C-linked and N-linked C⁸-Ar-dG lesions when base paired with C

depend on the conformation of the adduct. If the adduct is

present in the anti-conformation, it can form a WatsonꢀCrick

base pair with C. This base pairing places the bulky C8-Aryl

group outside the helix in the major groove, and it is not involved

in base-stacking interactions. Solvent exposure of the lipophilic

aryl group is thought to account for the lesion-induced duplex

destabilization in the anti-conformation.^73,74If the C⁸-Ar-dG

adduct is present in the syn-conformation, then the loss of

WatsonꢀCrick base pairing with C coupled with helix distor-

tions decreases the stability of the modiﬁed duplex.^29ꢀ31

3 3 3

stacking interactions ranging between ꢀ13 and ꢀ20 kJ/mol

with the 5⁰-flanking base and ꢀ34 and ꢀ54 kJ/mol with the 3⁰-

flanking base (Table 3). In general, the magnitudes of the

stacking interactions for the adducts and the unmodified G:G

mismatch are similar, although o-PhOH-dG shows a greater

interaction with the 3⁰-flanking base within ODN1 as compared

to the G:G mismatch (ꢀ54 vs ꢀ43 kJ/mol, Table 3). These

calculated structures for anti-adduct:syn-G within ODN1 predict

a more stable structure for o-PhOH-dG versus p-PhOH-dG and

that both should be less stable than the unmodified G:G

mismatch due primarily to the significant drop in H-bonding

stability.

syn-Conformation of the Adducts (X = a or b) for N = G.

When the adducts adopt the syn-conformation opposite anti-G,

there is not a dramatic drop in Hoogsteen H-bonding stability as

compared with the natural helices as discussed above for ODN1

strands with the anti-adduct opposite syn G. For p-PhOH-dG,

two strong (ꢀ46 to ꢀ48 kJ/mol, Table 3) Hoogsteen bonds

(92ꢀ96%, Table 4) form between the adduct and G regardless of

the sequence (Figures 9a and 10a), which are only slightly less

stable than the unmodified G:G mismatch (ꢀ50 kJ/mol,

Table 3). The duplexes containing o-PhOH-dG show a slightly

greater decrease in H-bonding stability (ꢀ41, ꢀ45 as compared

to ꢀ50 kJ/mol, Table 3). For ODN1, N1ꢀH of the Watsonꢀ

Crick face of G H-bonds to O6 of the ortho adduct (97%,

Table 5), and a second N2ꢀH O6 Hoogsteen bond is present

3 3 3

(77%), while a third Hoogsteen bond (N2ꢀH N7) is present

3 3 3

for less (57%) time (Figure 9b,c). The lower percentage of

Hoogsteen bonding to N7 may be related to the competing

(OꢀH N7) hydrogen bond (96%, Table 5). For ODN2, the

3 3 3

two Hoogsteen bonds to O6 of o-PhOH-dG are less occupied

(39ꢀ77%, Table 5), while the N2ꢀH N7 Hoogsteen bond is

For the phenolic C-linked adducts, our experimental and MD

simulations suggest that they adopt the syn-conformation with

the phenolic ring located in the minor groove, similar to the W

structure observed for N-linked adducts.³⁰Such a minor groove

conformation would account for the signiﬁcant drop in duplex

3 3 3

more occupied (68%) than ODN1 (Figure 10b). This can be

correlated to a much higher degree of θ twist in ODN2 than

ODN1 (predominantly 80° as compared to 225°, Figure 8). The

larger twist leads to a structure with the OꢀH N7 interaction

3 3 3

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stability (6ꢀ17 °C, Table 2) when these C8-Ar-dG lesions

base pair with C since the syn base is not WatsonꢀCrick paired.

For o-PhOH-dG, enhanced stacking interactions (Table 3) in

ODN2_b:2⁰(C) (syn-adduct) as compared to the unmodiﬁed

sequence (anti-G) may explain why ODN2_b:2⁰(C) is less

destabilized than ODN1_b:1⁰(C), which has weaker stacking

relative to the unmodiﬁed sequence. Furthermore, the X:C

H-bonding is more favorable in ODN2 than in ODN1 (Table 3).

Comparison of the MD simulations for ODN1_a and ODN2_a

to the T_mresults leads to similar conclusions noted for strands

containing o-PhOH-dG. Speciﬁcally, the decrease in stability

(by 17 and 16 °C, Table 2) observed for strands containing the

p-PhOH-dG:C pair implies that the syn-orientation of the adduct

is present in both duplexes. Unlike o-PhOH-dG, there is no

improved stability for ODN2_a as compared to ODN1_a, which

is likely due to a cancellation in improved stacking by weakening

of the H-bonds (Table 3). The fact that the adduct prefers to be

in the syn-conformation, despite loss in stability due to weaker

H-bonding, implies that other (sterics, major groove contacts,

etc.) factors push the adduct to adopt a syn-orientation regardless

of the base in the opposite strand.

(96%), N7 is not as available to form Hoogsteen bonds. In fact,

only one Hoogsteen bond (N1ꢀH O6) is occupied for the

3 3 3

majority of the simulation (97%), while G-N2ꢀH bonds to N7

of the adduct 57% of the time. For some of the simulation (77%),

G-N2ꢀH also H-bonds to O6 (Figure 9b). In addition, both of

the latter interactions involve >3 Å average distance for o-PhOH-

dG but less than 3 Å for p-PhOH-dG or natural G. Therefore,

p-PhOH-dG is expected to form a much more stable base pair

with G than o-PhOH-dG in ODN1:1⁰(G). This is not reﬂected

in the calculated values for H-bond strengths (Table 3) since

the average structure of ODN1_b:1⁰(G) contains three weak

H-bonds even though the dominant conformation contains only

one strong interaction (Figure 9c). The decrease in Hoogsteen

binding to N7 also explains why ODN1_b:1⁰(G) is destabilized

with respect to the natural G:G mismatch (by ꢀ6 °C, Table 2).

The stability of p-PhOH-dG:G and o-PhOH-dG:G base pairs

is also sequence dependent. ODN2_a:2⁰(G) and ODN2_b:2⁰-

(G) are stabilized to the same degree (+9 °C) with respect to the

unmodiﬁed G:G mismatched sequences. Close examination of

the base pairs throughout the simulation reveals that two Hoogsteen

N1ꢀH O6 (94%) and N2ꢀH N7 (93%) p-PhOH-dG

3 3 3

The impact of duplex formation on the ﬂuorescent properties

of the C-linked phenolic adducts (Figure 3 and Table 2) also

supports a syn-preference. Upon hybridization, blue shifts in

emission maxima were typically observed. This correlates with

changes in emission maxima of the nucleoside adducts in water

versus CHCl₃(Table 1), suggesting a decrease in solvent exposure

of the modiﬁed base upon duplex formation. The emission

spectrum of ODN1_b:1⁰(T) also gave rise to keto emission,

H-bonds are not aﬀected by the sequence (Figure 10a). How-

ever, o-PhOH-dG forms two strong Hoogsteen bonds in

ODN2:2⁰(Figure 10b) but only one in ODN1:1⁰(Figure 9b).

Speciﬁcally, N1ꢀH O6 (77%) and N2ꢀH N7 (68%) are

3 3 3

both present in ODN2, which is more similar to the bonding in

the p-PhOH-dG and unmodiﬁed duplexes (68ꢀ96%). In

ODN1_b:1⁰(G), N7 is blocked from forming strong bonds

due to the stability of OꢀH N7 (96%). However, in

3 3 3

conﬁrming the presence of the intramolecular OꢀH N7

ODN2_b:2⁰(G), the OꢀH N7 bond is present only 21% of

3 3 3

H-bond and suggesting that the Hoogsteen face of the adduct

the simulation, which allows for Hoogsteen bonding to occur

during the remainder of the simulation. This eﬀect is sequence

speciﬁc since the H-bond is broken due to formation of a weak

is not solvent exposed. ^36,38

The syn-adduct preference is also supported by the ability of

the adduct to stabilize the G:G mismatch. Experimental insight

into the stabilizing inﬂuence of the phenolic C-linked adducts on

the G:G mismatch comes from work on G-quadruplex forma-

tion. A G-quadruplex can form with alternating syn- and anti-

conformation of G bases.⁷⁵Because the addition of bulky groups

to C8-dG can shift the conformational equilibrium of the

glycosidic bond from anti to syn, replacement of syn Gs within

the G-quadruplex with C8-Ar-dG adducts that favor the syn-

conformation can be used to stabilize the quadruplex.^76,77

Incorporation of 8-Br-dG into G-quadruplexes has been shown

to stabilize the quadruplex by 4ꢀ13 °C.⁷⁶More recently, Dumas

and Luedtke⁷⁷incorporated a C8-pyridyl-dG into G-quadruplex-

folding oligonucleotides and demonstrated the utility of the

nucleobase to stabilize the G-quadruplex due to the syn-con-

formational preference of the C8-pyridyl adduct. The impact of

C8-pyridyl-dG on G-quadruplex formation (ΔT_m= +10 °C) was

found to correlate with the ΔT_mvalues recorded for the C-linked

phenolic adducts ODN2_b:2⁰(G) and ODN2_c:2⁰(G) (ΔT_m=

+9 °C) given in Table 2.

It is interesting that the T_mdata show that the G mismatch

with p-PhOH-dG is more stable than that with o-PhOH-dG in

ODN1:1⁰(G). The syn p-PhOH-dG:G base pair in ODN1:1⁰(G)

involves two H-bonds between N1ꢀH of G and O6 of the adduct

(96%) and N2ꢀH of G and N7 of the adduct (95%) (Figure 9a).

This is similar to the natural unmodiﬁed sequence, where N7 is

free to form Hoogsteen bonds. Thus, ODN1_a:1⁰(G) and the

unmodiﬁed G:G mismatch have similar stabilities (ΔT_m= +1 °C,

Table 2). However, the stability of ODN1_b:1⁰(G) is quite

N2ꢀH O contact (35%) between the 5⁰-ﬂanking G and the

3 3 3

phenoxyl oxygen (Figure 10c), which distorts θ (76.7°). This

provides a possible explanation for the observed relative stabi-

lities of ODN2_a:2⁰(G) and ODN2_b:2⁰(G) (Table 2).

The interaction of the phenolic OH in o-PhOH-dG with the

5⁰-ﬂanking G in ODN2 also provides a rationale for the inability

to detect keto emission for duplexes involving ODN2_b

(Figure 3d), even though o-PhOH-dG is predicted to be in the

syn-conformation and is not solvent exposed. A similar argument

has been proposed by the Romesberg laboratory for the inability

to detect keto emission in a 2-(2⁰-hydroxyphenyl)benzoxazole

(HBO) artiﬁcial nucleoside.⁷⁸When incorporated into the major

groove opposite an abasic site, the HBO nucleobase undergoes

ESIPT to generate keto emission. However, upon incorporation

of the HBO base with the enol moiety positioned in the minor

groove, formation of a stable H-bond between the phenolic OH

and the 3⁰-ﬂanking sugar O4⁰atom inhibits the ESIPT process to

generate only enol emission.⁷⁸

The increase in stability of the modiﬁed G:G mismatch within

ODN2 may be due to the enhanced stacking of the syn-adduct

with purine bases as compared with the pyrimidine bases

(Table 3). Stronger stacking arises since the phenoxyl group is

involved in stacking and T-shaped interactions with the ﬂanking

bases in ODN2 (Figure 11a,b) but to a lesser extent in ODN1.

Indeed, the pyrimidine bases are too small to overlap with the

entire adduct and, therefore, only interact with dG in a manner

similar to the natural strand (Figure 11c,d). The stacking strengths

calculated for the syn-orientations of the adducts (Table 3) support

enhanced stability of ODN2:2⁰upon incorporation of both

diﬀerent. Speciﬁcally, because OꢀH N7 is highly populated

3 3 3

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Figure 11. Face (left, center) and edge (right) views of the intrastrand stacking interactions between o-PhOH-dG and the 3⁰- (a, c) or 5⁰- (b, d) ﬂanking

base in the ODN2:2⁰(a, b) and ODN1:1⁰(c, d) sequences.

adducts. Greater ﬂuorescence quenching for the C-linked phe-

nolic adducts was also observed within ODN2, suggesting more

favorable stacking interactions with the ﬂanking purine bases.

Biological Implications of C-Linked Phenolic Adducts. For

the N-linked C8-dG adducts, biological outcome in terms of

mutagenicity appears to be strongly correlated to the conforma-

tional preference of the adduct.²⁹The duplex DNA structures

containing N-linked adducts base paired with C can be minimally

perturbed with the adduct present in the anti-conformation with

the N-linked aryl ring located in the major groove. Alternatively,

the syn-adduct structures have the N-linked aryl ring intercalated

into the helix with local unwinding and with displacement of the

modified G. The equilibrium between the two conformers is

highly sequence-dependent,^30,31,73,74which generates mutagenic

hotspots.²⁹Because DNA repair enzymes seek out distorting

lesions as substrates, the minimally perturbed anti-conformer

structures are less repair-prone.²⁹However, the syn-conformer

adduct structures are relevant to mutagenic replication and

provide a rationale for the propensity of N-linked C8-dG adducts

to induce frameshift mutations by stabilizing bulges through

formation of the base-displaced intercalated structures.²⁹The

syn-adduct structures also provide a rationale for misincorpora-

tion of A opposite the modified G base.²⁹

For the C-linked phenolic adducts in the syn-conformation,

our MD simulations suggest that the C8-phenyl group is located

in the minor groove within the helix and the C base is not ﬂipped

out, as noted for the corresponding N-linked adducts.²⁹This

observation is important in terms of biological activity, as it

suggests that the C-linked phenolic adducts will be less able to

stabilize bulges that are important for inducing frameshift muta-

tions. Our MD simulations also suggest that the C-linked

phenolic adducts minimally perturb the B-form duplex DNA

structure. These ﬁnding imply that the C-linked phenolic adducts

may be less repair-prone than the corresponding N-linked

adducts, which cause major distortion to the duplex in the

base-displaced intercalated structure.²⁹

the lesion ﬂanked by pyrimidine bases (C).⁷⁹UV thermal

melting experiments were also performed for 8-Ph-dG within

the 12-mer sequence 5⁰-d(GCGCCXGCGGTG), where X =

8-Ph-dG. Unfortunately, the thermal melting data contained the

lesion ﬂanked by C and G, which does not correlate with the

sequence used for the primer-extension assays.⁷⁹Within the 12-

mer, the T_mof the duplex was 5.6 °C lower than the normal G:C

base pair, while the T_mof 8-Ph-dG:G was 2.1 °C higher than that

of the mismatched G:G base pair, again highlighting the ability of

8-Ph-dG to stabilize a G:G mismatch. Klenow fragment from

E. coli incorporated predominately the correct base dCMP

opposite 8-Ph-dG, while primer extension by mammalian pol

α was strongly blocked opposite the lesion. Small amounts of

dGMP and dAMP were incorporated opposite the lesion; two-

base deletions were also observed, suggesting that 8-Ph-dG

is weakly mutagenic capable of generating GfC and GfT

transversions and deletions in cells.

The results of our studies suggest that C-linked phenolic

adducts could show hotspots for mutagenicity. The lesions are

better able to stabilize G:G mismatches when ﬂanked by purine

bases. Thus, purine-rich sites would be expected to show a

greater propensity for misincorporation of G, as the mismatched

G can be stably accommodated opposite the syn-adduct within

the duplex.

In summary, we have demonstrated the structural impact of

C-linked phenolic adduct within two decanucleotide substrates.

The C8-dG adducts decrease duplex stability (6ꢀ17 °C) when

base paired with C but show a sequence-dependent increase in

duplex stability when mismatched with G (5ꢀ9 °C). Stabiliza-

tion of the G:G mismatch by the C-linked phenolic adducts was

greater in the decanucleotide containing ﬂanking purine bases,

suggesting more favorable stacking interactions with the ﬂanking

purine bases, as opposed to the smaller pyrimidines. MD

simulations and changes in ﬂuorescent emission of the adducts

upon hybridization to the complementary strands implied a syn-

conformation preference for the adducts within both duplexes

regardless of the opposite base N. The syn-preference of the

adducts coupled with their ability to stabilize G:G mismatches in

a sequence speciﬁc fashion suggests the possibility for C-linked

While the biological activity of C-linked phenolic adducts has

yet to be addressed, primer-extension assays have been per-

formed for 8-Ph-dG within a single 24-mer oligonucleotide with

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phenolic adducts to display mutagenic hotspots, as noted for the

corresponding N-linked C8-dG adducts derived from arylamine

carcinogens.

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’ ASSOCIATED CONTENT

S

Supporting Information. Table S1 containing average

b

backbone values according ﬁnal snapshots from MD simulations,

Table S2 containing H-bond occupancies for the natural helices,

Tables S3 and S4 containing average distances and angles for

H-bond occupancies of the adducts, mol2 ﬁles for modiﬁed bases

with partial charges and atom types, additional parameters for

modiﬁed bases, HPLC traces and MS spectra for ODN1_a,b,c

and ODN2_a,b,c, ﬁgures of MD snapshots, and ﬁgures of rmsd

plots for MD simulations; ﬁles of duplex starting structures

available in separate ﬁle in pdb format; and Cartesian coordinates

for average MD structures used in DFT calculations available

upon request. This material is available free of charge via the

Internet at http://pubs.acs.org.

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’ AUTHOR INFORMATION

Corresponding Author

*E-mail: stacey.wetmore@uleth.ca (S.D.W.). E-mail: rmanderv@

uoguelph.ca (R.A.M.).

Author Contributions

^§These authors contributed equally to this work.

Funding Sources

Support for this research was provided by the Natural Sciences

and Engineering Research Council (NSERC) of Canada, the

Canada Research Chair program, the Canada Foundation for

Innovation, and the Ontario Innovation Trust Fund. A.L.M.

also thanks Alberta Ingenuity, now part of Alberta Innovates—

Technology Futures.

’ ACKNOWLEDGMENT

R.A.M. thanks Katherine M. Schlitt and Christopher K.

McLaughlin for carrying our preliminary experiments. This

research has been facilitated by use of computing resources

provided by WestGrid and Compute/Calcul Canada.

’ ABBREVIATIONS

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(1988) Synthesis and identiﬁcation of benzo[a]pyrene-guanine nucleo-

side adducts formed by electrochemical oxidation and by horseradish

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dG, 2⁰-deoxyguanosine; Ar, aryl; CD, circular dichroism; MD,

molecular dynamics; p-PhOH-dG, 8-(4⁰⁰-hydroxyphenyl)-dG;

o-PhOH-dG, 8-(2⁰⁰-hydroxyphenyl)-dG; Ph-dG, 8-phenyl-dG;

AAF-G, 8-acetylaminoﬂuorene-G; ODN, oligonucleotide; TPPTS,

3,3⁰,3⁰⁰-phosphinidynetris(benzenesulfonic acid) trisodium salt; DMF,

N,N-dimethylformamide; CV, cyclic voltammetry; TBAF, tetra-

butylammonium hexaﬂuorophosphate.

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Fluorescent properties and conformational preferences of C-linked phenolic-DNA adducts

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