C8-heteroaryl-2′-deoxyguanosine adducts as conformational fluorescent probes in the narl recognition sequence

Article abstract of DOI:10.1021/jo302164c

The optical, redox, and electronic properties of C⁸-heteroaryl- 2′-deoxyguanosine (dG) adducts with C⁸-substituents consisting of furyl (^FurdG), pyrrolyl (^PyrdG), thienyl ( ^ThdG), benzofuryl (^Bfu

Full text of DOI:10.1021/jo302164c

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pubs.acs.org/joc
C8‑Heteroaryl-2′-deoxyguanosine Adducts as Conformational
Fluorescent Probes in the NarI Recognition Sequence
Katherine M. Rankin,† Michael Sproviero,† Keegan Rankin,‡ Purshotam Sharma,§ Stacey D. Wetmore,*,§
and Richard A. Manderville*,†
†Departments of Chemistry and Toxicology, University of Guelph, Guelph, Ontario N1G 2W1, Canada
‡Department of Chemistry, University of Toronto, Toronto, Ontario M5S 3E4, Canada
§Department of Chemistry & Biochemistry, University of Lethbridge, Lethbridge, Alberta T1K 3M4, Canada
S
* Supporting Information
ABSTRACT: The optical, redox, and electronic properties of
C8-heteroaryl-2′-deoxyguanosine (dG) adducts with C8-sub-
stituents consisting of furyl (FurdG), pyrrolyl (PyrdG), thienyl
(ThdG), benzofuryl (BfurdG), indolyl (InddG), and benzothienyl
(
BthdG) are described. These adducts behave as fluorescent
nucleobase probes with emission maxima from 379 to 419 nm
and fluorescence quantum yields (Φfl) in the 0.1−0.8 range in
water at neutral pH. The probes exhibit quenched fluorescence
with increased solvent viscosity and decreased solvent polarity.
The FurdG, BfurdG, InddG, and BthdG derivatives were
incorporated into the G3 position of the 12-mer oligonucleo-
tide 5′-CTCG1G2CG3CCATC-3′ that contains the recognition
sequence of the NarI Type II restriction endonuclease. This
sequence is widely used to study the biological activity (mutagenicity) of C8-arylamine−dG adducts with adduct conformation
(anti vs syn) playing a critical role in the biological outcome. The modified NarI(X = FurG, IndG, BfurG, or BthG) oligonucleotides
were hybridized to the complementary strand containing either C (NarI′(C)) or G (NarI′(G)) opposite the probe. The duplex
structures were characterized by UV melting temperature analysis, fluorescence spectroscopy, collisional fluorescence quenching
studies, and circular dichroism (CD). The emission of the probes showed sensitivity to the opposing base in the duplex, and
suggested the utility of fluorescence spectroscopy to monitor probe conformation.
tricyclic ring systems have proven useful17,18 and have been
INTRODUCTION
■
converted into fluorescence resonance energy transfer (FRET)
pairs to distinguish between distance and orientation changes.19
Attachment of aryl groups to the commercially available
pyrrolo-dC20 or to C5 of deoxyuridine (dU)21,22 has also
been employed to generate solvent-sensitive emissive probes.
Within this division of analogues, pyrimidine bases bearing
heteroaryl ring systems (furan,21 or benzothiophene22) show
excellent photophysical properties for determining the micro-
environment of the fluorescent probe.
Generation of emissive purine nucleobase probes has also
attracted attention with the C8 position being a usual site of
modification.23−29 However, in this case, the attachment of π-
systems to generate emissive purine probes can shift the
conformational equilibrium of the glycosidic bond from anti to
syn.23 When the C8-modified purine adopts the syn-
conformation, Watson−Crick (W−C) base pairing with its
pyrimidine partner cannot take place and the probe can disrupt
the helical structure. Under this scenario, the emissive
Fluorescent nucleobase analogues have a wide range of
applications that include use as reporters of nucleic acid
structure and function;1−3 investigating nucleobase damage;4
detecting single nucleotide polymorphism (SNP);5 under-
standing protein−DNA interactions,6 generation of fluorescent
aptasensors that provide an optical signal upon molecular target
binding,7 and use as oligonucleotide-based therapies.8 The
prototypical fluorescent base analogue is 2-aminopurine (2AP),
which causes little perturbation to DNA structure.9 The
fluorescence of 2AP is strongly quenched when incorporated
into single-strand oligonucleotides and quenched further still
when hybridized to a complementary strand.10 The decrease in
quantum yield of 2AP also varies with oligonucleotide
sequence, temperature, and helical conformation, and these
characteristics have been exploited to understand structural
dynamics,11 protein−DNA interactions,12 and charge transfer
within duplex and hybrid (i.e., RNA:DNA) helical struc-
tures.13,14
Fluorescent nucleobase probes that retain high quantum
yields upon oligonucleotide incorporation have also been
developed.15,16 Here, modified cytosine analogues with fused
Received: October 2, 2012
Published: November 21, 2012
© 2012 American Chemical Society
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nucleobase is a poor model for the unmodified purine. Thus, in
order to generate purine analogues that minimally disturb the
structure and function of nucleic acids, a small vinyl group has
been attached to C8 of both dA24 and dG,25 and the resulting
probes have been found to be nonperturbing with improved
sensitivity over 2AP. Larger aromatic systems have also been
attached to C8 of dA26 and dG27−29 where dG derivatives are
useful for monitoring G-quadruplex folding, since the preferred
syn-conformation of the modified C8-dG nucleobase can
stabilize the G-tetrad.28,29
capability of BthdG was ascribed to changes in π-stacking and
charge-transfer character between the benzothienyl moiety with
the natural DNA bases in the duplex structure.
We have now extended our studies on C8-heteroaryl−dG
adducts and have used density functional theory (DFT)
calculations to help define the electronic and structural
properties of both the 5-membered ring derivatives (PyrdG,
FurdG, and ThdG, Figure 1) and the bulkier benzoheterocyclic
series of adducts (InddG, BfurdG, and BthdG, Figure 1). The
calculations provide a rationale for their photophysical
properties in solvents of differing polarity and rigidity. The
C8-heteroaryl−dG adducts 2FurdG, InddG, BfurdG, and BthdG
were incorporated into G3 of the 12-mer oligonucleotide 5′-
CTCG1G2CG3CCATC that contains the recognition sequence
of the NarI Type II restriction endonuclease. This sequence
was chosen for study because the G3 site represents a “hotspot”
for mutagenicity mediated by N-linked C8-dG adducts.33,35,39
Our goal is to derive structure−activity relationships for C8-dG
adducts, and emissive C8-heteroaryl−dG probes that mimic the
structural impact of C-linked adducts within duplex structures
could thereby help elucidate mechanisms of mutagenesis
through the use of luminescence-based assays.42 The experi-
ments described herein outline the role of the heteroatom (X)
and size of the C8-heteroaryl group in observing changes in
probe microenvironment using fluorescence spectroscopy. This
information has provided a deeper understanding of the
reporting capabilities of these emissive probes.
Our interest in C8-aryl−dG adducts stems from their
toxicological relevance. A number of chemical mutants generate
radical species that undergo direct addition reactions at C8 of
dG to afford carbon (C)- and oxygen (O)-linked adducts.30,31
These adducts are related, in terms of dG modification site, to
nitrogen (N)-linked derivatives formed by arylamine32 and
heterocyclic aromatic amines33 that are known human
carcinogens. For N-linked adducts, the syn-conformation is
regarded as promutagenic and plays a critical role in their
biological activity.32−35 These lesions are generally non-
fluorescent, and therefore, analytical tools with generally
lower limits of detection have been employed to distinguish
conformation, including circular dichroism (CD),36 UV
38
32,39
absorption,37 19F NMR, and H NMR
spectroscopies.
1
However, for C-linked adducts where conformation is also
expected to play an important biological role, fluorescence
spectroscopy is an attractive alternative for probing conforma-
tional preference.
RESULTS AND DISCUSSION
Recently, we used thermal melting (Tm) studies, CD,
molecular dynamics (MD), and notably, fluorescence spectros-
copy to study the conformational preferences of phenolic C-
linked C8-dG adducts in duplex DNA.40 The use of
fluorescence spectroscopy in defining adduct conformation
did present limitations, as the excitation maximum of the
adducts (∼280 nm) overlapped with that of natural DNA
(∼260 nm). However, we have shown that the indole-linked
(InddG) C8-dG nucleoside (Figure 1) can act as a fluorescent
■
Structural, Photophysical, and Redox Properties of
C8-Heteroaryl−dG Adducts. The optical and redox proper-
ties of the C8-heteroaryl−dG nucleoside adducts and
unmodified dG43 are given in Table 1. Absorption and
Table 1. Photophysical and Redox Parameters for C8-
Heteroaryl−dG Adducts
d
λmax (nm),
Δν
b
b
c
e
f
adduct
log ε
λem (nm), ϕfl
(cm−1
)
brightness
Ep/2
PyrdG
FurdG
ThdG
InddG
BfurdG
BthdG
292, 4.36
292, 4.31
284, 4.24
321, 4.48
323, 4.41
315, 4.27
253, 4.14
379, 0.10
384, 0.49
414, 0.79
390, 0.78
405, 0.76
419, 0.46
7861
8205
11057
5512
6268
7880
2291
10005
13729
23600
19500
8570
0.78
0.91
1.05
0.89
0.92
1.06
1.14
a
dG
334, 9.7 × 10−5
9620
1.33
a
b
Photophysical data for dG taken from ref 43. Determined in 10 mM
MOPS buffer, pH 7, μ = 0.1 M NaCl. Determined using the
c
comparative method with quinine bisulfate in 0.5 M H2SO4 (ϕfl =
d
e
0.55). Stokes shift (Δν) is calculated as (1/λmax − 1/λem). Brightness
f
factor is calculated as ελmax × ϕfl. Half-peak potentials in volts vs SCE
Figure 1. Structures of C8-heteroaryl−dG nucleosides and NarI
oligonucleotide sequence considered in the present study. The
dihedral angle χ (∠(O4′−C1′−N9−C4)) defines the glycosidic
bond orientation to be anti (χ = 180 90°) or syn (χ = 0 90°),
and θ (∠(N9−C8−C10−X11)) defines the degree of twist between
the nucleobase and the C8-substituent.
using cyclic voltammetry with 0.1 M TBAF in anhydrous DMF using a
glassy carbon working electrode.
emission spectra were recorded in aqueous MOPS buffer,
while the redox properties were determined using cyclic
voltammetry (CV) in anhydrous DMF, as outlined previ-
ously.44−46 All C8-heteroaryl−dG nucleoside adducts have
lower 1-electron half-peak oxidation potentials (Ep/2) than
dG, indicating that the heteroaryl moiety enhances the electron
donor character of the purine nucleoside. The pyrrole-linked
derivative PyrdG possesses the lowest Ep/2 value of 0.78 V vs
reporter of syn- versus anti-structures on the basis of its
sensitivity to H-bonding in aprotic solvents and possesses an
excitation maximum (∼320 nm) distinct from DNA.41 The
benzothienyl derivative (BthdG, Figure 1) was also incorporated
into two decanucleotide sequences and was found to act as an
emissive conformational probe.42 In this case, the reporting
saturated calomel electrode (SCE), followed by InddG (Ep/2
=
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0.89 V), which is closely followed by the oxygen-containing
analogues (FurdG and BfurdG ∼0.92 V), and finally the sulfur-
containing derivatives (ThdG, BthdG ∼1.05 V). This indicates
that thienyl and benzothienyl substituents are the least
electron-donating groups. The sulfur analogues also show the
largest Stokes’ shift (Δν), displaying blue-shifted absorption
values and red-shifted emission wavelengths compared to the
other nucleosides. In fact, a clear trend is apparent between Ep/2
and emission wavelength where increased Ep/2 values cause the
emission to shift to longer wavelengths. The solvatochromic
properties of the C8-heteroaryl−dG nucleosides are given in
Tables S1−S6 (Supporting Information (SI)). As previously
presented for BthdG,42 the probes tend to show an increase in
Δν with increased solvent polarity, consistent with the excited
state possessing a dipole moment greater than the ground
state.47
Insight into the structural features and electronic properties
of the C8-heteroaryl−dG adducts was obtained from DFT
calculations, as presented previously for PyrdG, InddG,41 and
various C8-Ar−dG adducts.23,48 The C8-heteroaryl−dG adducts
adopt the syn-conformation, as predicted from their NMR
chemical shifts in DMSO (Table S7, SI)29 and from DFT
calculations where all syn minima contain an O5′−H···N3 H-
bond and are ∼25 kJ mol−1 more stable than anti minima.41
Fully optimized syn minima and transition states on the
potential energy surfaces for the C8-heteroaryl−dG adducts are
displayed in Figures S1−S6 (SI). The degree of twist angle (θ)
between the heteroaryl ring with respect to the nucleobase is
dependent on the nature of the heteroatom X. For PyrdG the
twist angle θ is 14.4°, while θ for ThdG is ∼32°, which is similar
to θ calculated for BthdG (∼31°, Figure 2). The HOMO and
LUMO of PyrdG (Figure 2) consist of delocalized π orbitals
with almost equal density on the nucleobase and pyrrole ring
system. The HOMO of BthdG also consists of delocalized π
orbitals, while the LUMO has greater density on the
benzothienyl ring system, which is consistent with push−pull
character from the donor nucleobase to the acceptor C8-
substituent.49
Orbital energies for the C8-heteroaryl−dG adducts obtained
from the DFT calculations are summarized in Table 2. The
a
Table 2. Electronic Properties of C8-Heteroaryl−dG
Adducts
b
HOMO E LUMO E ΔE calcd Vex
E
ΔE optical (eV)
c
adduct
(eV)
(eV)
(eV)
(eV)
[λabs (nm)]
PyrdG
FurdG
ThdG
InddG
BfurdG
BthdG
−5.12
−5.32
−5.50
−5.24
−5.35
−5.51
−0.52
−0.94
−1.09
−1.09
−1.32
−1.33
4.60
4.38
4.41
4.15
4.03
4.18
4.16
4.11
4.04
3.86
3.77
3.83
4.20 [295]
4.25 [292]
4.32 [287]
3.82 [325]
3.86 [321]
3.86 [321]
a
Optimized at the B3LYP/6-31G(d) level, with values corresponding
b
to a ground state (S0) with a relative energy of 0 kJ/mol. Vertical
excitation energy values determined from the corresponding TD-
c
B3LYP/6-31G(d) calculations. Determined experimentally for C8-
heteroaryl−dG adducts from UV absorption data in acetonitrile.
benzoheterocyclic series have lower HOMO−LUMO gaps than
the 5-membered ring analogues, and TD-B3LYP/6-31G(d)
vertical excitation energies (Vext) tend to correlate better than
the calculated HOMO−LUMO gaps with λmax values
determined in CH3CN. Changes in structural and electronic
(dipole moment) properties of the nucleoside probes from the
ground state (S0) to excited state (S1), as predicted from DFT
calculations, are given in Table 3. For all nucleosides, the
Table 3. Change in Structural Properties and Dipole
a
Moments from Ground (S0) to Excited State (S1)b of C8-
Heteroaryl−dG Adducts
adduct χ (S0)/χ (S1) (deg) θ (S0)/θ (S1) (deg) μ (S0)/μ (S1) (Debye)
PyrdG
FurdG
ThdG
InddG
BfurdG
BthdG
47.3/40.4
50.1/48.5
67.2/64.9
46.9/44.4
49.9/49.8
66.8/69.7
165.6/181.4
343.8/0.16
212.3/185.6
166.5/178.9
345.6/359.7
211.1/188.1
3.64/4.46
4.27/4.86
5.18/6.05
4.83/5.63
4.37/5.08
5.45/6.41
a
Optimized at the B3LYP/6-31G(d) level, with values corresponding
to a S0 with a relative energy of 0 kJ/mol. Optimized at the CIS/6-
31G(d) level, with S1 state optimization starting from the
corresponding S0-state geometry.
calculations predict twisted S0 structures that become almost
planar (θ ∼ 0, 180, 360°) in S1, which is consistent with the
behavior of biphenyl systems.50 All of the adducts possess
greater dipole moments in S1, which is consistent with their
solvatochromic properties (Tables S1−S6, SI).42 The sulfur
analogues (ThdG and BthdG) have the most twisted S0 structures
and the largest dipole moments in S0 and S1 states and show
the greatest increase in dipole moment on going from S0 to S1.
These properties provide a rationale for the photophysical
parameters given in Table 1. Within each class of C8-
heteroaryl−dG nucleosides (5-membered rings and benzohe-
terocyclic series), the sulfur analogues have the most blue-
shifted absorption spectra because they have the highest degree
of twist in S0 due to the larger S-atom. This disfavors
conjugation between the nucleobase and the C8-substituent.
Upon conversion to a planar structure in S1, the sulfur
Figure 2. Ground-state B3LYP/6-31G(d) global minima and orbital
density plots of PyrdG and BthdG. The HOMO and LUMO energy
levels (eV) are given in Table 2.
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derivatives have the most red-shifted emission, as they can
accept electron density in their excited states and as a
consequence the nucleosides have the greatest S1 dipole
moments.
the 5-membered derivatives were expected to overlap with
DNA. However, FurdG has a red-shifted maximum compared to
ThdG and was significantly brighter than PyrdG. For this reason
FurdG52 was chosen from the 5-membered derivatives and its
incorporation into NarI provides a direct comparison to BfurdG
in terms of ring size. Our choice of NarI as the oligonucleotide
substrate stems from its extensive use as a substrate for related
N-linked C8-dG adducts derived from arylamine carcino-
gens.33,35,37,39
The calculated properties of C8-heteroaryl−dG adducts
(Table 3) also provide a rationale for their changes in emission
with increased solvent viscosity and temperature. Figure 3
The C8-heteroaryl−dG adduct FurdG was incorporated into
NarI using standard β-cyanoethyl phosphoramidite chemistry
according to published protocols.53,54 Specifically, the nucleo-
side FurdG was converted into a phosphoramidite for use on a
DNA synthesizer as per the synthetic strategy outlined in
Scheme 1. The benzoheterocyclic series were incorporated into
Scheme 1. Synthesis of FurdG Phosphoramidite
Figure 3. Changes in fluorescence emission spectra (dashed traces) of
InddG upon (a) increasing the glycerol content from 0% (solid trace,
100% 10 mM MOPS, pH 7, μ = 100 mM NaCl) up to 100% (added in
20% increments), recorded at 20 °C, and (b) increasing the
temperature in 10 °C increments from 20 °C (solid trace) in 100%
glycerol.
shows changes in emission for InddG in glycerol−water mixtures
at 20 °C (Figure 3a) and with increased temperature in 100%
glycerol (Figure 3b). The spectral changes shown in Figure 3
for InddG are representative of emission changes for C8-
heteroaryl−dG nucleoside adducts, which show a 3−4-fold
decrease in emission intensity from buffered water to 100%
glycerol, accompanied by a blue-shift in emission wavelength of
2−6 nm. Increasing the temperature from 20 to 80 °C in 100%
glycerol increases emission intensity 2−3-fold. With increased
viscosity, excited states have geometries similar to that of the
ground state,51 because increased solvent rigidity increases
barriers to rotation. Thus, a possible explanation for the
emissive behavior of C8-heteroaryl−dG nucleosides with
increased viscosity and temperature could stem from the
generation of twisted S1 structures, which causes a blue-shift in
emission wavelength and a decrease in intensity. Increased
temperature in 100% glycerol decreases the barrier to rotation
and more emissive planar structures in S1 can be generated.
Synthesis of Modified NarI. To test the emissive
properties of these probes in a helical environment, the
benzoheterocyclic series InddG, BfurdG, and BthdG along with the
5-membered derivative FurdG were incorporated into the 12-
mer NarI sequence at the G3 position (Figure 1). Our choice of
these adducts stemmed from their photophysical properties
given in Table 1. The bulky benzoheterocyclic series all exhibit
excitation maxima distinct from DNA and permit the role of
the heteroatom X to be determined. The excitation maxima of
NarI using our recently developed postsynthetic method55
involving palladium (Pd)-catalyzed Suzuki−Miyaura coupling
reactions with brominated NarI oligonucleotides and 10 equiv
of the requisite boronic acids. Negative electrospray ionization
mass spectrometry (ESI-MS) analysis confirmed the identity of
all C8-heteroaryl-G modified oligonucleotides, synthesized by
Suzuki−Miyaura coupling or standard phosphoramidite chem-
istry. The ESI-MS spectra showed the expected clusters of
multiply charged peaks for the modified oligonucleotides, with
the results of this analysis summarized in Table S8 (SI). Mass
and UV spectra for all C8-heteroaryl-G modified oligonucleo-
tides, including MS spectra of the starting brominated NarI
oligonucleotide, are also included in the SI. Integration of
HPLC traces55 allowed for determination of yields from
Suzuki−Miyaura couplings, which ranged from 10 to 21%
(Table S8, SI).
UV Thermal Melting Studies. The C8-heteroaryl-G
modified oligonucleotides NarI(X = FurG, IndG, BfurG or BthG)
were hybridized to the complementary strand NarI′(C) or
NarI′(G) (Figure 1). NarI′(G) refers to the complementary
strand in which a G has been inserted at the appropriate site to
allow for positioning opposite the site of C8-heteroaryl-G
modification upon duplex formation. Our previous studies have
shown C-linked C8-aryl−dG adducts to stabilize the G:G
mismatch,40,42,55 and molecular dynamics (MD) simulations
predict a syn-conformation for the modified C8-dG adduct.40,42
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In the present work, we determined the melting temperatures
(Tm’s) using UV−vis spectroscopy by monitoring the
absorbance at 260 nm versus temperature, and results are
summarized in Table 4.
Interestingly, when the modified NarI oligonucleotides were
hybridized to the NarI′(G) complement, thereby introducing a
mismatch, the C8-heteroaryl−dG adduct was observed to
increase duplex stability, in the range of +5.6 to +7.2 °C,
compared to the unmodified duplex. This stabilization effect
was previously observed to occur with both phenolic40 and
benzothienyl42 C8-dG modified 10-mers, hybridized to the
complement with G opposing the adduct, with observed
increases in Tm of ∼+10 °C. These C-linked phenolic40 and
benzothienyl42 C8-dG modified duplexes, in addition to N-
linked C8-arylamine−dG modified duplexes,34,36,38 are known
to favor a wedge (W-type) motif, with the adduct in the syn-
conformation, when the adduct is mismatched with a purine. In
this conformation, the lipophilic C8-heteroaryl moiety is located
in the minor groove in a nonpolar environment, and is thus
favorably protected from the aqueous environment surrounding
the duplex. Duplex stabilization also comes as a result of an
increase in π-stacking due to interactions between the modified
base and its flanking bases and increases in the H-bonding
stability as a result of additional interactions between the C8-
substituent and its base-pairing partner.40,42
Photophysical Studies. In order to definitively classify the
C8-heteroaryl−dG adducts as probes in the NarI recognition
sequence, their photophysical properties upon oligonucleotide
incorporation were examined. Emission and excitation spectra
of the adducted NarI oligonucleotides in the single-strand state,
and upon hybridization to the complementary strands NarI′(C)
and NarI′(G) were obtained and are shown in Figure 4, with
photophysical parameters given in Table 5.
While still fluorescent, all modified NarI oligonucleotides
exhibited significantly quenched emission intensity compared
to the free C8-heteroaryl−dG nucleosides, with relative
emission intensity (Irel) values of ∼0.05−0.06 (Table 5).
Many fluorescent base analogues, including 2AP, exhibit
quenched fluorescence when incorporated into DNA. For
2AP, several mechanisms have been proposed for fluorescence
quenching that include base stacking effects,56 photoinduced
electron transfer,57 and the presence of dark states.58 Recent
theoretical calculations reveal two different pathways that can
lead to fluorescence quenching.10 One involves conversion of
the bright (ππ*) state into a dark minimum (nπ*) involving
lone pair orbitals on 2AP. A second pathway involves charge
transfer (CT) between the bases, which can lead to
radiationless decay.
The wavelengths of emission for the modified oligonucleo-
tides do not distinctly differ from those of the free nucleosides,
with only very subtle shifts, or no change, in wavelength
observed and no discernible pattern in the shift present (Table
5). The excitation spectra of the modified NarI oligonucleotides
did display other features distinct from the spectra of the free
modified nucleosides. In all spectra, a new excitation band was
present between ∼270 and 280 nm. In particular, this new band
was clearly visible in the spectra of NarI(X = IndG) (Figure 4b,
bold trace) and NarI(X = BfurG) (Figure 4c, bold trace). This
peak occurs at the red-edge of DNA absorbance and can be
ascribed to CT from the natural DNA nucleobases to the
adduct.42 Similar bands have been observed in the emission
spectra of 2AP modified oligonucleotides, in the 260−270 nm
region.13,14 The wavelength of excitation of NarI(X = IndG) was
considerably blue-shifted (15 nm) compared to the free
nucleoside, suggesting a more twisted IndG structure within
NarI.
Table 4. Tm Studies of C8-Heteroaryl-G Modified NarI
a
b
Oligonucleotides Hybridized to NarI′(C) or NarI′(G)
c
duplex
Tm (°C)
ΔTm
NarI(X = G):NarI′(C)
NarI(X = G):NarI′(G)
NarI(X = FurG):NarI′(C)
NarI(X = FurG):NarI′(G)
NarI(X = IndG):NarI′(C)
NarI(X = IndG):NarI′(G)
NarI(X = BfurG):NarI′(C)
NarI(X = BfurG):NarI′(G)
NarI(X = BthG):NarI′(C)
60.5 1.3
49.6 1.0
51.0 0.9
56.8 1.6
50.4 2.6
55.3 0.4
48.7 0.5
57.5 1.0
54.5 1.0
55.2 0.9
−9.5
+7.2
−10.1
+5.7
−11.8
+7.9
−6.0
+5.6
NarI(X = BthG):NarI′(G)
a
b
c
5′-GATGGCGCCGAG. 5′-GATGGGGCCGAG. Tm’s of duplexes
(1.25 μM of each oligonucleotide) were measured in 50 mM sodium
phosphate buffer, pH 7, with 0.1 M NaCl. Absorbance was monitored
at 260 nm, with a heating rate of 1 °C min−1.
In each instance, when the C8-heteroaryl-G probe was paired
with its normal pyrimidine partner C, the duplex was
considerably destabilized, in the range from −6.0 to −11.8
°C, compared to the unmodified duplex. Clearly, these probes
are perturbing and are not good emissive models for
unmodified dG. In this regard, the results with X = FurG were
somewhat surprising because the relatively small furan ring was
not expected to have such a destabilizing influence on duplex
stability (ΔTm = −9.5 °C). However, overall these findings are
in agreement with previous studies showing that C-linked C8-
aryl-G (ΔTm = −6 to −17 °C),40,55 C8-pyrenyl-G (ΔTm ∼ −10
°C),27 and N-linked C8-arylamine-G (ΔTm = −8 to −13 °C)37
lesions significantly decrease duplex stability when paired with
C, suggesting that the C8-heteroaryl−dG adducts are reasonable
emissive models of bulky adducts generated by chemical
mutants.
For N-linked C8-dG adducts base-paired with C, anti- and
syn-conformations are in equilibrium and both are destabiliz-
ing.29−32 In the anti-conformation, a major groove (B-type)
structure is generated, placing the N-linked aryl ring system in
the major groove. In the present case, this would result in
exposure of the lipophilic C8-heteroaryl moiety to the aqueous
extrahelical environment and, subsequently, duplex destabiliza-
tion and a decrease in Tm, compared to the unmodified
duplex.42 Additionally, steric clash between the major groove
located C8-heteroaryl substituent and the phosphate backbone
and sugar moieties may be a factor in duplex destabilization.26
By contrast, duplex destabilization could be the result of syn-
conformer adaptation of the C8-heteroaryl−dG adduct, which
for N-linked C8-dG adducts favors a stacked, intercalated (S-
type) structure.32−39 In this structure, the N-linked C8-aryl
moiety is intercalated between its flanking nucleobases, in turn
flipping the opposing pyrimidine (C) out of the helix.39 The
resulting helical distortion and loss of W−C H-bonding
accounts for the decreased duplex stability and decrease in
Tm, compared to the unmodified duplex.37 NMR evidence is in
favor of the S-type structure for an N-linked heteroaryl adduct
in NarI at G3.39
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Table 5. Photophysical Parameters of C8-Heteroaryl-G
a
Modified NarI Oligonucleotides Hybridized to NarI′(C) or
b
NarI′(G)
a
λ
Δ λex
λem (nm)
Δ λem
b
ex a
b
c
oligonucleotide
NarI(X = FurG)
(nm)
(nm)
(Irel)
(nm)
306
305
+2
386 (0.045)
385 (0.63)
+2
NarI(X =
−1
−1
FurG):NarI′(C)
NarI(X =
306
387 (0.18)
+1
FurG):NarI′(G)
NarI(X = IndG)
308
308
−15
−1
389 (0.057)
391 (1.05)
−1
+2
NarI(X =
IndG):NarI′(C)
NarI(X =
307
387 (0.17)
−2
IndG):NarI′(G)
NarI(X = BfurG)
320
335
−3
+15
403 (0.060)
411 (0.67)
NarI(X =
+8
BfurG):NarI′(C)
NarI(X =
325
+5
395 (0.62)
−8
BfurG):NarI′(G)
NarI(X = BthG)
317
328
+2
413 (0.062)
419 (1.41)
−4
+6
NarI(X =
+11
BthG):NarI′(C)
NarI(X =
319
+2
408 (0.65)
−5
BthG):NarI′(G)
a
All spectra of single-strand oligonucleotides (1.25 μM) and duplexes
(equivalent amounts (1.25 μM) of Nar1 and its complementary
strand) were recorded in 50 mM sodium phosphate buffer, pH 7, with
b
0.1 M NaCl at 10 °C. Change in excitation or emission maximum for
single-strand Nar1 is versus the free C8-heteroaryl−dG adduct, while
c
change for the duplex is versus single-strand Nar1. Irel for single-
strand Nar1 is determined as Isingle‑strand/Iadduct. Iadduct was determined at
a concentration of 1.25 μM. Irel for the duplex is determined as Iduplex
/
Isingle‑strand. All intensity values for determination of Irel were measured
at the same wavelength.
that the modified bases are in a polar environment. Red-shifts
in wavelength were also observed in the excitation spectra of
NarI(X = BfurG):NarI′(C) and NarI(X = BthG):NarI′(C), of 15
and 11 nm, respectively. Negligible shifts in emission and
excitation wavelengths were observed for the NarI(X =
FurG):NarI′(C) duplex, which may be indicative of a bulky
C8-moiety as a requirement of notable changes in fluorescence
wavelength parameters, such as those observed for the NarI
oligonucleotides with InddG, BfurdG, or BthdG modifications.
By contrast, hybridization to the NarI′(G) complement, and
thereby induction of mismatch formation, resulted in different
changes to the emission and excitation properties. The energy
transfer assigned excitation band previously viewed for the
single-strand oligonucleotides and NarI′(C) hybridized du-
plexes was not observed, or was relatively indiscernible, in
NarI′(G)-hybridized duplexes. Furthermore, mismatch to the
opposing G resulted in a decrease in emission intensity, as
compared to the corresponding single-strand oligonucleotide
with the largest decrease in intensity observed for the
NarI(FurG):NarI′(G) and NarI(IndG):NarI′(G) duplexes, with
Irel = 0.18 and 0.17, respectively. It is of interest to note the
decrease in degree of emission intensity correlates with
decrease in intensity observed for the C8-heteroaryl−dG
nucleosides in going from water to the more viscous glycerol
solvent (i.e., Figure 3) or to the more nonpolar CHCl3 (Tables
S1−S6, SI). A negligible shift in the wavelength of the
fluorescence was again observed for the FurG modified duplex,
while the wavelength of emission for NarI(IndG), NarI(BfurG)
Figure 4. Excitation and emission spectra of C8-heteroaryl-G modified
NarI oligonucleotides, in the single-strand state (solid line), or
hybridized to its complementary strand, NarI′(C) (dashed line) or
NarI′(G) (dotted line), with (a) NarI(X = FurG), (b) NarI(X = IndG),
(c) NarI(X = BfurG) or (d) NarI(X = BthG). All spectra of single-strand
oligonucleotides (1.25 μM) and duplexes (equivalent amounts (1.25
μM) of NarI and its complementary strand) were recorded in 50 mM
sodium phosphate buffer, pH 7, with 0.1 M NaCl at 10 °C.
The spectral changes upon hybridization were markedly
different depending on the nature of the base opposing the
probe in the duplex. For all modified NarI oligonucleotides
upon hybridization to NarI′(C), an additional excitation band
was observed at ∼275 nm, as was observed in the excitation
spectra of the single-strand oligonucleotides. Little change was
observed in emission intensity, with Irel values ranging from
∼0.6−1.4, as compared to the single-strand oligonucleotide.
The wavelength of emission for NarI(X = IndG), NarI(X =
BfurG), and NarI(X = BthG) was notably red-shifted upon base-
pairing to C, by increments of 2, 8, and 6 nm, respectively,
compared to the single-strand oligonucleotides. This suggests
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and NarI(BthG) was blue-shifted upon mismatch formation, by
increments of 2, 8, and 5 nm, respectively, compared to the
single-strand oligonucleotides.
provide the Stern−Volmer quenching constant for different
adducts, Ksv, which are listed in Table 6. Quenching constants
Table 6. Stern−Volmer Quenching Constants, Ksv, for
Collisional Fluorescence Quenching of C8-Heteroaryl−dG
Adducts, or C8-Heteroaryl-G Modified NarI
Collisional Fluorescence Quenching. Collisional fluo-
rescence quenching studies provided further confirmation of
adduct conformation in the duplex. Collisional quenching has
proved useful for study of aqueous accessibility of fluorescent
groups, with changes in sensitivity, exposure and accessibility
following ligand binding indicative of a change in conformation
of the fluorophore.59 Collisional quenching, using quenchers
such as acrylamide and deoxyuridine (neutral), iodide ion
(negatively charged), and cesium ion (positively charged) has
been used to provide insight into the conformation of
proteins,59,60 benzopyrene diol epoxide (BPDE)−DNA ad-
ducts61 and 2AP.62
Collisional quenching experiments were first conducted with
the free C8-heteroaryl−dG adducts, using KI as quencher. Plots
of Fo/F versus [KI] of the fluorescence quenching process of
FurdG (a) and NarI(X = FurG) hybridized to its complementary
strand (b) NarI′(C) or (c) NarI′(G) are shown in Figure 5.
Slopes of the plots derived from KI quenching experiments
Oligonucleotides Hybridized to NarI′(C) or NarI′(G)
a
compd
Ksv (M−1
)
FurdG
BthdG
10.2 1.2
11.8 1.1
2.51 0.08
0.50 0.03
2.5 0.4
NarI(X = FurG):NarI′(C)
NarI(X = FurG):NarI′(G)
NarI(X = BthG):NarI′(C)
NarI(X = BthG):NarI′(G)
0.64 0.03
a
Ksv values were determined from the slope of the plot of Fo/F vs [KI]
(quencher concentration), expressed as values fitting error.
reflect the extent of adduct accessibility toward the quencher.62
Examination of the Ksv values determined for the quenching of
modified duplexes by KI reveals that the values are much
smaller than those determined for the corresponding adducts,
reflecting a lower accessibility of the fluorophores to the
quencher when located in the helical environment. The Ksv
values also show that when base-paired to C, the fluorophore is
more accessible to the aqueous environment than when
mismatched to G, as larger Ksv values were derived for the
modified oligonucleotides hybridized to NarI′(C) than when
hybridized to NarI′(G).
The Tm data for C8-heteroaryl-G modified NarI oligonucleo-
tides hybridized to NarI′(C) pointed toward two possible
destabilizing adduct conformations; an anti-preferred con-
formation, with a B-type duplex motif, or a syn-preferred
conformation with an S-type duplex motif. The Ksv values
obtained following collisional quenching of NarI′(C) annealed
duplexes allows for a defining classification of the adduct
conformation. The larger quenching constants for fluorescence
quenching of NarI(X = FurG):NarI′(C) and NarI(X =
BthG):NarI′(C) duplexes, compared to NarI(X = FurG):Nar-
I′(G), and NarI(X = BthG):NarI′(G), can be equated to a more
readily accessible fluorophore to the aqueous environment
surrounding the double helix, and thus the quencher. In order
for the fluorophores to be solvent exposed, the adduct must be
in an anti-conformation, since in this conformation, the C8-
heteroaryl moiety is located outside of the helix in the major
groove. From the corroboration of thermal melting, emission
and excitation spectra, and finally collisional fluorescence
quenching studies, the C8-heteroaryl−G adduct likely exists in
the anti-conformation when base-paired to C.
Conversely, the smaller slopes from plots of Fo/F versus [KI]
determined from the quenching of NarI(X = FurG):NarI′(G)
and NarI (X = BthG):NarI′(G) suggest that the fluorophore has
become more shielded and is likely stacked within the interior
of the helix. This implies a syn-preferred conformation for the
C8-heteroaryl−dG adduct upon mismatch formation, placing
the C8-heteroaryl moiety in a more rigid environment, which is
also more nonpolar in nature. The solvatochromic, including
viscosity-related, properties of the C8-heteroaryl−dG adducts
can account for its dramatic blue-shift and decrease in emission
intensity upon base-pairing with G, and subsequently
conversion of glycosyl bond conformation. This assumption
is also consistent with the known syn-preference of N-linked
adducts within a purine mismatch.34,36,38
Figure 5. Plots of Fo/F vs [KI] for the collisional fluorescence
quenching of (a) FurdG and the C8-heteroaryl-G modified oligonucleo-
tide NarI (X = FurG) hybridized to its complementary strand (b)
NarI′(C) or (c) NarI′(G). Spectra were recorded in 50 mM sodium
phosphate, pH 7, with 100 mM NaCl, using 1.25 μM of the adduct or
equivalent amounts (1.25 μM) of NarI and its complementary strand.
KI was added to the adduct sample in aliquots of 0.05 M and to the
duplex samples in 0.025−1.0 M aliquots.
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CD Measurements. CD spectra of NarI(X = FurG) and
NarI(X = BFurG) hybridized to their complementary strands (a)
NarI′(C) or (b) NarI′(G) are shown in Figures S7 and S8 (SI).
The modified duplexes show characteristics of B-form DNA
with roughly equal positive (275 nm) and negative (244 nm)
bands and a crossover at ∼260 nm.63 It was not possible to
resolve ellipticities due to the C8-heteroaryl-G modified bases,
and so CD could not distinguish conformational preference by
the adducts.
using 10 mm light path quartz glass cells. Quantum yields for the
nucleoside adducts were determined, as outlined previously in detail,41
using the comparative method,68 with quinine bisulfate (Φfl = 0.546 in
0.5 M H2SO4) serving as the fluorescence quantum yield standard.69
pH measurements were taken at room temperature with stirring. Any
water used for buffers or spectroscopic solutions was obtained from a
filtration system (18.2 MΩ). High-resolution mass spectra were
recorded on a Q-Tof instrument, operating in nanospray ionization at
0.5 uL/min detecting positive ions. Electrochemical oxidation
measurements were conducted in a three-electrode glass cell under
nitrogen in a solution of 0.1 M DMF/TBAF, as previously outlined in
detail.44−46
Selection of C8-Heteroaryl−dG Probe for Future
Studies. All C8-heteroaryl−dG adducts described herein act
as fluorescent nucleobase probes for selective excitation (306−
320 nm) within the NarI oligonucleotide (Table 5). DFT
calculations indicate that the benzothienyl derivative BthdG
possesses the largest ground-state and excited-state dipole
moment (Table 3) and its emission proved sensitive to
conformation within the NarI duplex structure. When base-
paired with C (NarI(X = BthdG):NarI′(C), Table 5, Figure 4d),
the BthdG probe exhibited a red-shift of 6 nm and an increase in
emission intensity 1.4-fold compared to the emission of the
probe in the single-strand. In contrast, when base-paired with G
(NarI(X = BthdG):NarI′(G)), the probe exhibited a blue-shift of
5 nm and exhibited quenched fluorescence compared to the
probe emission in the single-strand. Overall, base-pairing with
C provided a 2-fold increase in emission intensity and an 11 nm
red-shift compared to the probe emission when base-paired
with G. This change in emission characteristics was attributed
to a change in conformation from anti to syn. The emission of
the benzofuryl derivative BfurdG was also sensitive to
conformation on the basis of wavelength (16 nm difference,
Table 5, Figure 4c), but not emission intensity. In contrast, the
emission of the indole-linked derivative InddG was strongly
quenched in the syn-conformation (Irel = 0.17 compared to the
single-strand), but it was not possible to distinguish base-
pairing of IndG with C from the single-strand NarI, and the
probe-incorporated duplexes failed to show changes in emission
wavelength (Figure 4b). The smaller furyl derivative FurdG
exhibited quenched emission with both duplex structures with
the emission wavelength lacking sensitivity to base-pairing.
Thus, BthdG was the only probe to show differences in both
emission intensity and wavelength upon change in conforma-
tion within the NarI duplex, making it the most sensitive probe
for monitoring conformation. For mutagenic C8-dG adducts,
conformation within the DNA duplex is directly correlated with
biological activity.32−39 The ability to employ fluorescence
spectroscopy to characterize adduct conformation should be
useful for determining structure−activity relationships for
adduct processing by the DNA polymerases.
Photophysical Properties of C8-Heteroaryl−dG Adducts.
Stock solutions were made in DMSO, due to sparing solubility in
other solvents, to a concentration of 4 mM. Spectroscopic solutions of
the modified nucleosides were prepared in 10 mM MOPS buffer, pH
7, with 100 mM NaCl, and made to a concentration of 50 μM for
UV−vis measurements or 20 μM for fluorescence measurements.
UV−vis spectra were recorded from 400 to 220 nm wavelength, using
quartz cells (100-QS) with a light path of 10 mm. Fluorescence spectra
were recorded at the excitation wavelength (absorbance maxima) for
the C8-heteroaryl−dG adducts, from 10 nm above the excitation
wavelength to 600 nm. Fluorescence spectra were recorded using
quartz cells (101-QS) with a light path of 10 × 10 mm, and excitation
and emission slit widths were kept constant at 2.5 nm.
Oligonucleotide Synthesis by Standard Phosphoramidite
Chemistry. Oligonucleotide synthesis for the C8-heteroaryl-G
modified NarI oligonucleotide (5′-CTCGGCXCCATC) with X =
2FurG was carried out on a 1 μmol scale on a DNA synthesizer using
standard β-cyanoethylphosphoramidite chemistry according to pub-
lished protocols.53,54 Following synthesis, oligonucleotides were
cleaved from the solid support, deprotected using 2 mL of 30%
ammonium hydroxide solution at 55 °C for 12 h and purified by
HPLC.
N2-(Dimethylformamidyl)-8-(2″-furyl)-2′-deoxyguansine (1). 8-
(2″-Furyl)-2′-deoxyguansine (1.1 g, 3.3 mmol) was placed in 15 mL
of dry DMF under argon. N,N-Dimethylformamide diethyl acetal (2.7
mL, 13.5 mmol) was then added and the mixture allowed to stir for 5
h. The reaction mixture was evaporated to dryness, washed with
MeOH, and dried to give 1 as a gray solid (1.28 g, 99.0% yield): mp
195−197 °C; 1H NMR (600.1 MHz, DMSO-d6) δ 11.59 (s, 1H), 8.57
(s, 1H), 8.01 (d, J = 1.2 Hz, 1H), 7.07 (d, J = 3.3 Hz, 1H), 6.78 (dd, J
= 1.8 Hz, 3.4 Hz, 1H), 6.51 (t, J = 7.2 Hz, 1H), 6.36 (d, J = 4.3 Hz,
1H), 4.95 (t, J = 5.1 Hz, 1H), 4.54 (m, 1H), 3.98 (m, 1H), 3.88 (m,
1H), 3.71 (m, 1H), 3.27 (m, 1H), 3.22 (s, 3H), 3.11 (s, 3H), 2.23 (m,
1H); 13C NMR (151 MHz, DMSO-d6) δ 159.2, 158.3, 158.0, 151.3,
145.6, 145.0, 139.8, 121.4, 113.3, 112.8, 88.6, 85.5, 71.9, 62.9, 41.8,
38.4, 35.6; HRMS calcd for C17H20N6O5 [M + H+] 389.1573, found
389.1568.
5′-O-(4,4′-Dimethoxytrityl)-N2-(dimethylformamidyl)-8-(2″-
furyl)-2′-deoxyguanosine (2). 4,4′-Dimethoxytrityl chloride (DMT-
Cl, 1.28 g, 3.78 mmol) was dissolved in 3 mL of dry pyridine. N2-
Dimethylformamide-8-(2″-furyl)-2′-deoxyguanosine 1 (923 mg, 2.70
mmol) was coevaporated from dry pyridine (3 × 5 mL) in a separate
flask and reverse filled with argon. To this flask was added 7 mL of dry
pyridine, followed by 3 mL of DMT-Cl/pyridine solution. The
reaction was allowed to stir at room temperature for 3 h under argon
and was monitored by TLC. Upon completion, the reaction mixture
was diluted with ethyl acetate (10 mL) and washed with water (2 × 10
mL). To the washed reaction mixture was added 1 mL of triethylamine
(TEA), and the mixture was then evaporated to dryness, with the
resulting solid dissolved in CH2Cl2 (3 mL). Hexanes (10 mL) were
added, and the reaction mixture was stirred overnight. The resulting
suspension was filtered and purified on silica gel and eluted with
MeOH/CH2Cl2/TEA (5:90:5) to afford 2 as a white solid (1.45 g,
EXPERIMENTAL SECTION
■
Materials and Methods. Boronic acids (1-N-Boc-pyrrole-2-,
furan-2-, thiophene-2-, 1-N-Boc-indole-2-, benzofuran-2-, and thio-
phene-2-boronic acid), Pd(OAc)2, 3,3′,3″-phosphinidynetris-
(benzenesulfonic acid) trisodium salt (TPPTS), and other commercial
compounds were used as received. The synthesis of 8-bromo-2′-
deoxyguanosine (8-Br-dG) was performed according to literature
procedures by treating dG with N-bromosuccinimide in water−
acetonitrile.64 Suzuki cross-coupling reactions of boronic acids with 8-
Br-dG to afford PyrdG,41 FurdG,65 ThdG,65 InddG,41 BfurdG,66 and
BthdG42,66 were performed as described previously by Western and co-
workers.67 NMR spectra were recorded on 300 and 600 MHz
spectrometers in either DMSO-d6, CDCl3 or CD3CN referenced to
TMS (0 ppm) or the respective solvent. All UV−vis and fluorescence
emission spectra were recorded with baseline correction and stirring
1
78.0% yield): mp 162−167 °C; H NMR (600.1 MHz, CD3CN) δ
9.44 (bs, 1H), 8.33 (s, 1H), 7.66 (s, 1H) 7.29 (m, 2H), 7.16 (m, 7H),
7.06 (d, J = 3.4 Hz, 1H) 6.7 (m, 4H), 6.62 (dd, J = 1.62 Hz, 3.24 Hz,
1H), 6.50 (dd, J = 4.5 Hz, 8 Hz, 1H), 4.77 (q, J = 5.9 Hz, 13.0 Hz,
1H), 3.97 (m, 1H), 3.71 (s, 3H), 3.70 (s, 3H), 3.46 (m, 1H) 3.25 (m,
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1H), 3.14 (m, 1H) 3.14 (s, 3H), 3.02 (s, 3H), 2.45 (q, J = 7.1 Hz, 14.3
Hz, 5H, TEA), 2.24 (m, 2H), 0.945 (t, J = 7.1 Hz, 8H, TEA); 13C
NMR (151 MHz, CD3CN) δ 160.0, 159.9, 159.4, 158.9, 157.9, 152.1,
146.7, 146.3, 145.6, 141.5, 137.6, 137.4, 131.4, 131.1, 129.4, 129.1,
128.1, 122.2, 118.8 (CD3CN), 114.3, 113.7, 113.2, 87.5, 87.1, 86.1,
72.9, 65.6, 56.32, 56.3, 47.5 (TEA), 42.2, 38.9, 35.7, 12.7 (TEA), 1.8
(CD3CN); HRMS calcd for C38H38N6O7 [M + H+] 691.2880, found
691.2866.
carried out at 50 °C using a 3 μm reversed-phase (RP) C18 column
(50 × 4.60 mm) with a flow rate of 0.5 mL/min, and various gradients
of buffer B in buffer A (buffer A = 95:5 aqueous 50 mM TEAA, pH
7.2/acetonitrile; buffer B = 30:70 aqueous 50 mM TEAA, pH 7.2/
acetonitrile. Yields were determined from integration of the HPLC
trace.55 Collected DNA samples were lyophilized to dryness and
unmodified and C8-heteroaryl-G modified NarI oligonucleotides were
dissolved in 18.2 MΩ water for quantification by UV−vis measure-
ment using ε260. Extinction coefficients were obtained from the
following website: http://www.idtdna.com/analyzer/applications/
oligoanalyzer. The C8-heteroaryl-G modified NarI oligonucleotides
were assumed to have the same extinction coefficient as the natural
NarI oligonucleotide.55 In all cases of hybridization, oligonucleotides
were annealed by heating at 80 °C for 10 min, cooling to room
temperature, and refrigerating until analysis.
MS Analysis of Oligonucleotides. Oligonucleotide samples were
prepared in 50:50 methanol/water containing 0.1 mM ammonium
acetate. Full scan MS spectra were obtained by direct infusion at a rate
of 5−10 μL/min into an ESI source operated in negative mode and
analyzed using triple quadrupole mass spectrometers. The capillary
and cone voltages were optimized for each analyte and varied from 2.5
to 3.5 kV and 25−35 V, respectively. A source offset of 60 V was used
for all samples. The desolvation temperature was between 250 and 350
°C. All data was acquired with 36−60 MCA and processed using mass
spectrometry software.
3′-O-[(2-Cyanoethoxy)(diisopropylamino)phosphino]-5′-O-(4,4′-
dimethoxytrityl)-N2-(dimethylformamidyl)-8-(2″-furyl)-2′-deoxy-
guanosine (3). 5′-DMT-N2-dimethylformamide-8-(2″-furyl)-2′-deox-
yguanosine 2 (0.25 g, 0.362 mmol) was coevaporated from dry toluene
(3 × 5 mL). Dry CH2Cl2 (10 mL) was added to a reaction flask
backfilled with argon. 2-Cyanoethyl N,N-diisopropylchlorophosphor-
amidite (0.14 mL, 0.398 mmol) was added to the flask along with 0.5
mL of dry TEA. The reaction was allowed to proceed to completion as
monitored by TLC (90:5:5 CH2Cl2/MeOH/TEA). The reaction
mixture was then reduced to dryness, immediately purified on silica
gel, and eluted with 90:5:5 CH2Cl2/MeOH/TEA. The phosphor-
amidite 3 eluted as the diastereomers, which were a white foam (241
1
mg, 76.0%): H NMR (600.1 MHz, CDCl3) δ 9.28−9.24 (m, 1H),
8.32−8.27 (m, 1H), 7.44−7.06 (m, 13H), 6.66−6.56 (m, 5H), 6.43
(bs, 1H), 5.03−4.90 (m, 1H), 4.12 (m, 1H), 3.73−3.62 (m, 7H),
3.53−3.42 (m, 3H), 3.34−3.20 (m, 3H), 2.99 (s, 3H), 2.90 (s, 3H),
2.69−2.50 (m, 2H), 2.40−2.27 (m, 2H), 1.46 (m, 2H), 1.11−1.05 (m,
9H), 1.00−0.98 (m, 3H); 13C NMR (151 MHz, CDCl3) δ 158.32,
158.3, 157.7, 157.6, 157.5, 155.8, 155.8, 150.52, 150.50, 144.82,
144.80, 144.7, 143.60, 143.55, 140.39, 140.35, 135.82, 135.76, 135.7,
130.0, 129.9, 129.87, 128.1, 128.0, 127.7, 127.6, 126.7, 126.6, 121.1,
121.0, 117.6, 117.5, 112.93, 112.88, 112.85, 112.8, 111.73, 111.71,
86.04, 85.99, 84.9, 84.85, 84.78, 84.2, 83.9, 74.7, 74.5, 73.5, 73.4, 63.8,
63.3, 58.4, 58.3, 58.1, 58.0, 57,9, 55.2, 55.1,46.7, 45.2, 43.3, 41.1, 37.5,
35.1, 30.9, 29.2, 24.6, 24.5, 24.4, 24.3, 22.9, 22.8, 20.4, 20.2, 20.1; 31P
NMR (121.4 MHz, CDCl3) δ 149.32, 148.93; HRMS calcd for
C47H56N8O8P [M + H]+ 891.3959, found 891.3964.
Thermal Melting. All melting temperatures (Tm’s) of oligonucleo-
tides were measured by UV−vis spectroscopy in 50 mM phosphate
buffer, pH 7, with 100 mM NaCl, using equivalent amounts (1.25 μM)
of the unmodified or C8-heteroaryl-G modified NarI oligonucleotide
and its complementary strand. The UV absorption at 260 nm was
monitored as a function of temperature using quartz cells (108.002-
QS) with a light path of 10 mm. The temperature was increased from
10 to 80 °C, or decreased from 80 to 10 °C, at a heating rate of 1 °C/
min. The Tm’s of the duplexes were calculated by determining the first
derivative of the melting curve.
Suzuki−Miyaura Coupling Reactions with 8-Br-G-modified
Oligonucleotides. Synthesis of C8-heteroaryl-G-modified NarI
oligonucleotides (5′-CTCGGCXCCATC) with X = IndG, BfurG, or
BthG was conducted using our recently developed postsynthetic
Circular Dichroism Measurements. Spectra were obtained on a
CD spectrophotometer equipped with a 1 × 6 multicell block thermal
controller and a water circulator unit. Measurements were carried out
in 50 mM phosphate buffer, pH 7, with 100 mM NaCl, using
equivalent amounts (1.25 μM) of the unmodified or C8-heteroaryl-G-
modified NarI oligonucleotide and its complementary strand. Quartz
glass cells (110-QS) with a light path of 1 mm were used for
measurements. Spectra were collected at 10 °C between 200 and 400
nm, with a bandwidth of 1 nm and scanning speed at 100 nm/min.
Each oligonucleotide sample was scanned nine times and background
corrected.
Suzuki-Miyaura cross-coupling strategy.55 The protocol is briefly
described here. 8-Br-G-modified NarI oligonucleotides (5′-
CTCGGCXCCATC) with X = 8-Br-G were prepared on a 1 μmol
scale using standard phosphoramidites and 8-Br-dG-CE phosphor-
amidite. The synthesized oligonucleotides were fully deprotected with
ammonium hydroxide for 24 h, desalted, and purified by reversed-
phase chromatography. The mass of NarI(X = 8-Br-G) was rechecked
prior to use in Suzuki−Miyaura coupling. For Suzuki−Miyaura
coupling, NarI(X = 8-Br-G) (500 nmol) was initially dissolved in
degassed 2:1 H2O/CH3CN. The appropriate boronic acid and sodium
carbonate were added to the solution at the molar ratios Ar-B(OH)2/
NarI(X = 8-Br-G) = 10, and Na2CO3/NarI(X = 8-Br-G) = 2. The
other reaction components were initially prepared as 100× stock
solutions in degassed water. Through serial dilution, the reagents were
added to the reaction mixture at molar ratios NarI(X = 8-Br-G)/
TPPTS = 15, and NarI(X = 8-Br-G)/Pd(OAC)2 = 37.5, for a total
volume of 700 μL 2:1 H2O/CH3CN, and the resulting solution was
heated under argon at 80 °C for 24 h. To the reaction mixture was
added 1 mL of 5 mM EDTA in 50 mM TEAA, pH 7.2. The resulting
solution was added to a Sep-Pak Vac C18 1 cc cartridge and washed
with 5% acetonitrile in 50 mM TEAA, pH 7.2 in order to remove
excess reagents. The product was eluted with 30% acetonitrile in 50
mM TEAA, pH 7.2, and further purified by HPLC.
Fluorescence Studies of C8-Heteroaryl-G Modified NarI
Oligonucleotides. All fluorescence spectra were recorded in 50
mM phosphate buffer, pH 7, with 100 mM NaCl. In each case, both
excitation and emission spectra were recorded for the C8-heteroaryl-G
modified NarI oligonucleotide hybridized to its complementary strand.
In addition, to allow for comparison, spectra were recorded for the C8-
heteroaryl−dG nucleoside adducts under the same conditions. All
adduct and single-strand oligonucleotide samples were prepared to a
final concentration at 1.25 μM, and duplex samples were prepared
using equivalent amounts (1.25 μM) of the C8-heteroaryl-G modified
NarI oligonucleotide and its complementary strand. All measurements
were made using quartz cells (108.002F-QS) with a light path of 10 ×
2 mm, and excitation and emission slit-widths were kept constant at 5
nm. All fluorescence excitation spectra were recorded at the emission
wavelength (maximum) of the C8-heteroaryl−dG adduct, from 200 to
10 nm below the emission wavelength, while fluorescence emission
spectra were recorded at the excitation wavelength (maximum) of the
adduct, from 10 nm above the excitation wavelength to 600 nm.
Spectra were initially recorded at 10 °C and then at increasing 10 °C
intervals to a maximum of 80 °C. Samples were held at each
temperature for 5 min prior to beginning measurement.
Oligonucleotide Purification and Sample Preparation. The
C8-heteroaryl-G-modified NarI oligonucleotide solutions were first
filtered using syringe filters (PVDF 0.20 μm), and concentrated under
diminished pressure. Purification was performed using an HPLC
instrument equipped with an autosampler, diode array detector
(monitored at 258 nm and λAbs of the incorporated modified
nucleoside), fluorescence detector (monitored at λex and λem of the
incorporated modified nucleoside), and autocollector. Separation was
Collisional Fluorescence Quenching Studies. All quenching
studies were carried out using KI as the quencher, following a
previously described method.59 A 5 M stock solution of KI was
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dx.doi.org/10.1021/jo302164c | J. Org. Chem. 2012, 77, 10498−10508

The Journal of Organic Chemistry
Featured Article
prepared in 50 mM sodium phosphate, pH 7, with 100 mM NaCl, and
0.1 mM Na2S2O3 was added to the stock solution to prevent I3
Notes
−
The authors declare no competing financial interest.
formation. Fluorescence measurements were carried out in 50 mM
sodium phosphate, pH 7, with 100 mM NaCl using 1.25 μM of the C8-
heteroaryl−dG adduct, or equivalent amounts (1.25 μM) of the C8-
heteroaryl-G modified NarI oligonucleotide and its complementary
strand. KI was added to the duplex sample as 0.025 or 0.05 M aliquots.
All measurements were made at 10 °C using quartz cells (108.002F-
QS) with a light path of 10 × 2 mm, and excitation and emission slit-
widths were kept constant at 5 nm. Fluorescence emission intensity
was measured at the emission wavelength maximum for the
corresponding C8-heteroaryl−dG adduct. Quenching data for the
homogeneous single fluorophores system were analyzed using the
Stern−Volmer equation
ACKNOWLEDGMENTS
■
Financial support for this research was provided by the Natural
Sciences and Engineering Research Council (NSERC) of
Canada, the Canada Research Chairs program, the Canada
Foundation for Innovation, and the Ontario Innovation Trust
Fund. This research has been facilitated by use of computing
resources provided by WestGrid and Compute/Calcul Canada.
We wish to thank Prof. Scott Mabury for providing use of the
MS facilities at University of Toronto. A helpful discussion with
Prof. Robert Hudson (University of Western Ontario) is also
acknowledged.
F /F = 1 + Ksv[Q]
o
where Fo and F are the fluorescence intensities in the absence and
presence of the quencher, respectively, [Q] is the concentration of the
quencher, KI, and Ksv is the Stern−Volmer quenching constant. Values
for Ksv were determined from the slope of Fo/F versus [Q].
DFT Calculations. The preferred conformations of the PyrdG
adduct were previously analyzed by scanning the potential energy
surface (PES) with respect to rotation about θ, the dihedral angle
controlling the relative orientation of the heterocyclic ring at the C8-
position and the nucleobase ring, and χ, the dihedral angle controlling
the orientation of the nucleobase about the glycosidic bond (Figure
1).41 Full optimizations were subsequently carried out to identify
minima and transition states on PES.
In the present work, all minima and transition states previously
identified for the PyrdG adduct41 were used to generate the initial
conformations of the adducts analyzed in the present work.
Specifically, the pyrrole ring of PyrdG was replaced with a furan,
thiophene, indole, benzofuran or benzothiophene ring to generate
FurdG, ThdG, InddG, BfurdG, and BthdG adducts, respectively. Since the
anti minima of PyrdG are at least 25 kJ/mol higher in energy than the
global syn minima, it is anticipated that all anti minima will be higher in
energy compared to syn minima for all adducts analyzed in the present
work. Therefore, only the syn minima and transition states were
considered for the adducts analyzed in the present work. These
conformations of the adducts were optimized at the B3LYP/6-31G(d)
level of theory.
Higher level (B3LYP/6-311+G(2df,p)) single-point as well as
B3LYP/6-31G(d) frequency calculations were performed on all fully
optimized structures for the nucleoside models, and scaled zero-point
vibrational energies (ZPVE) are included in the reported relative
energies. The global minimum identified for each nucleoside adduct
was used to calculate the B3LYP/6-31G(d) orbital energies and TD-
B3LYP/6-31G(d) vertical excitation energies. Geometry optimizations
of the first excited singlet state (S1 state) of the adducts were carried
out with CIS/6-31G(d) using the global minimum of the ground state
as starting geometries. All electronic structure calculations were
performed using Gaussian 09.70
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