Postsynthetic guanine arylation of DNA by Suzuki-Miyaura cross-coupling

Article abstract of DOI:10.1021/ja106158b

Direct radical addition reactions at the C⁸-site of 2′-deoxyguanosine (dG) can afford C⁸-Ar-dG adducts that are produced by carcinogenic arylhydrazines, polycyclic aromatic hydrocarbons, and certain phenolic toxins. Such modified nuc

Full text of DOI:10.1021/ja106158b

Published on Web 11/10/2010
Postsynthetic Guanine Arylation of DNA by Suzuki-Miyaura
Cross-Coupling
Alireza Omumi, Daniel G. Beach, Michael Baker, Wojciech Gabryelski, and
Richard A. Manderville*
Departments of Chemistry and Toxicology, UniVersity of Guelph, Guelph, Ontario, Canada N1G 2W1
Received July 12, 2010; E-mail: rmanderv@uoguelph.ca
Abstract: Direct radical addition reactions at the C8-site of 2′-deoxyguanosine (dG) can afford C8-Ar-dG
adducts that are produced by carcinogenic arylhydrazines, polycyclic aromatic hydrocarbons, and certain
phenolic toxins. Such modified nucleobases are also highly fluorescent for sensing applications and possess
useful electron transfer properties. The site-specific synthesis of oligonucleotides containing the C8-Ar-G
adduct can be problematic. These lesions are sensitive to acids and oxidants that are commonly used in
solid-phase DNA synthesis and are too bulky to be accepted as substrates for enzymatic synthesis by
DNA polymerases. Using the Suzuki-Miyaura cross-coupling reaction, we have synthesized a number of
C8-Ar-G-modified oligonucleotides (dimers, trimers, decamers, and a 15-mer) using a range of arylboronic
acids. Good to excellent yields were obtained, and the reaction is insensitive to the nature of the bases
flanking the convertible 8-Br-G nucleobase, as both pyrimidines and purines are tolerated. The impact of
the C8-Ar-G lesion was also characterized by electrospray ionization tandem mass spectrometry, UV melting
temperature analysis, circular dichroism, and fluorescence spectroscopy. The C8-Ar-G-modified oligo-
nucleotides are expected to be useful substrates for diagnostic applications and understanding the biological
impact of the C8-Ar-G lesion.
Introduction
as their formation is highly correlated with mutagenesis and
cancer.6 In this regard, our group is interested in the properties
Modified nucleic acids are of great interest because of their
applications in diagnostics, catalysis, or nanotechnology and
material science.1 Modified nucleobases with new fluorescent,2
electrical,3 magnetic,4 and metal ion binding5 properties have
been used to expand the scope of applications for modified
nucleic acids. They may also have detrimental consequences
of C8-aryl (Ar)-purine adducts that are formed by phenolic
toxins.7 Following oxidative activation to phenoxyl radicals, both
O- and C-linked adducts at the C8-site of 2′-deoxyguanosine
(dG) are produced as a result of the ambident (O- vs C-attack)
electrophilicity of phenoxyl radicals. Pentachlorophenol (PCP)
generates an O-linked C8-dG adduct,8 while the natural fungal
carcinogen ochratoxin A forms a C-linked C8-dG adduct.9
Related C-linked adducts, which are the focus of the present
study, are shown in Figure 1. The isomeric para (p) and ortho
(o) C-phenolic adducts (C-(p-OHPh)-8-dG and C-(o-OHPh)-8-
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42 J. AM. CHEM. SOC. 2011, 133, 42–50
10.1021/ja106158b  2011 American Chemical Society

Synthesis of C8-Ar-G-Modified Oligonucleotides
A R T I C L E S
ent excited-state intramolecular proton transfer (ESIPT) process
that may be used to sense the local solvent environment within
duplex DNA.18 (Pyren-1-yl)-8-dG (Py-8-dG) has been touted
as a duplex-sensitive optical probe and as a fluorescent donor
for the investigation of charge transfer processes between DNA
and peptides.19 The C8-dimethylanilide-G adduct p-NMe2Ph-
8-G has been used for the study of electron transfer in a donor-
bridge-acceptor (DBA) ensemble.20
While C8-Ar-purine adducts are of wide interest in the current
literature, their incorporation into oligonucleotides has been
problematic. Some studies have reported solid-phase DNA syn-
thesis of oligonucleotides bearing C8-Ar-purine adducts,19,21-23
while others have pointed out the limitations of this strategy.24
One of the problems may stem from their sensitivity to acid.
Our studies show that C8-Ar-dG adducts can have k1 values for
acid-catalyzed deglycosylation that are 200-fold larger than k1
for dG.25 Such adducts are also more prone than dG to
oxidation,26 suggesting that chemical incompatibility with syn-
thesis conditions may present a problem for certain C8-Ar-dG
adducts.
Modified nucleic acids can also be prepared enzymatically
by polymerase incorporations of functionalized nucleoside
triphosphates (dNTPs).27 This approach has been used for
incorporation of 8-NH2-dGTP28 and the bulkier 8-[(2-imidazol-
4-ylethyl)amino]-dGTP29 for construction of novel DNAzymes.
However, recent efforts from the Hocek laboratory30 show that
8-substituted purine derivatives bearing groups larger than
amino, bromo, or methyl are generally poor substrates for DNA
polymerases and that 8-Ph-dATP is too bulky for the polymerase
to accept as a substrate.30b,c
Figure 1. Structures of C8-Ar-dG adducts derived from chemical carcino-
gens and structural analogues used for fluorescence sensing, G-quadruplex
formation, and the study of electron transfer processes.
dG) are formed from reactions of DNA with mutagenic
diazoquinones.10 Carcinogenic arylhydrazines that produce aryl
radical intermediates generate Ph-8-dG and other C8-Ar-purine
adducts bearing various para-substituents.11 The polycyclic
aromatic hydrocarbons (PAHs) benzo[a]pyrene (BP)12 and
3-nitrobenzanthrone (NBA)13 generate 6-BP-8-dG and N-Ac-
ABA-8-dG, respectively. Collectively, these adducts may play
a role in the biological activity of these toxins.
Some C8-Ar-purine adducts also have desirable properties.
Attachment of (m-acetylphenyl) to yield m-APh-8-dG expands
the Hoogsteen edge of dG and stabilizes G-quadruplex formation
that may have biomedical applications.14 In contrast, the Sessler
laboratory demonstrated that the pyrrole-8-G analogue forms a
three-point Hoogsteen-type interaction with G that can disrupt
G-quadruplex formation.15 C8-Ar-purines also act as fluoro-
phores,16 and phenolic adducts structurally related to C-(p-
OHPh)-8-dG act as pH-sensing fluorescent probes,17 while the
isomeric C-(o-OHPh)-8-dG adduct undergoes a solvent-depend-
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J. AM. CHEM. SOC. VOL. 133, NO. 1, 2011 43

A R T I C L E S
Omumi et al.
Scheme 1
Chart 1. Building Blocks for the Synthesis of C8-Ar-G
Oligonucleotides
Many C8-Ar-purine nucleoside adducts are prepared using
palladium-catalyzed (Suzuki-Miyaura) cross-coupling between
8-Br-purine and the arylboronic acid.31 This suggested that a
postsynthetic strategy32 with 8-Br-purine being the convertible
nucleoside may be a general approach for the synthesis of C8-
Ar-purine-modified oligonucleotides. The Pd-catalyzed cross-
coupling reaction is noted for its wide tolerance toward a variety
of functionalities and applicability to extremely labile systems
such as nucleoside monophosphates and triphosphates.30 Thus,
an extension of this application to oligonucleotide substrates
containing 8-Br-G, as shown in Scheme 1, seemed feasible and
would provide access to oligonucleotides bearing bulky acid-
labile C8-Ar-purine nucleobases. This postsynthetic strategy
differs from previous Pd-catalyzed cross-coupling reactions for
synthesis of uracil-alkynyl oligonucleotides,33,34 in which the
modified base is not sensitive to the solid-phase DNA synthesis
reagents. In these examples the modified oligonucleotide is
prepared using a semiautomated strategy that avoids lengthy
phosphoramidite synthesis, but exposes the uracil-alkynyl base
to acid and oxidant.33,34
Herein we demonstrate, for the first time, the feasibility of
Suzuki-Miyaura cross-coupling for the synthesis of C8-Ar-G-
modified oligonucleotides. The ease and versatility of this
protocol should allow synthesis of a wide range of C8-Ar-purine-
modified oligonucleotides that are difficult or impossible to
prepare using automated solid-phase synthesis with highly
functionalized C8-Ar-purine phosphoramidites. The impact of
the C8-Ar-dG lesion on duplex stability and structure is also
described and found to be a useful marker of C8-Ar-G incor-
poration. The C8-Ar-G-modified oligonucleotides are expected
to act as substrates for diagnostic applications and understanding
the biological impact of the C8-Ar-dG adduct.
ing group (hydrogenolysis or acid35) proved to be incompatible
with DNA, so phosphoramidite 2 containing the acetyl protect-
ing group was prepared, in which the acetyl is removed by
treatment with aqueous ammonium hydroxide after solid-phase
DNA synthesis.
Postsynthetic arylation reactions were initially investigated
on the dinucleotide ODN1 and trinucleotide ODN2. This work
was then extended to include the pyrimidine-rich decanucleotide
ODN3 and the purine-rich decanucleotide ODN4. In ODN4 the
convertible nucleoside 8-Br-G is flanked by purine bases, and
it was important to establish specificity for 8-Br-G in such a
sequence, as adenine bases are known to undergo direct metal-
mediated arylation (although Cu(I) plays a key role in this
reaction).36 The 15-mer substrate ODN5 served as a further test
for the utility of the Pd-catalyzed postsynthetic arylation
reaction. This 15-mer is an extension of the purine-rich
decanucleotide ODN4 and has greater relevance in biochemical
studies, as short oligonucleotides are poor substrates for DNA
polymerases and can be difficult to extend in ligation reactions
due to low thermal duplex stability coupled with the effects of
the adducts in reducing base pairing.37
Results and Discussion
Building Blocks for Oligonucleotide Synthesis. The building
blocks for synthesis of C8-Ar-G-modified oligonucleotides are
shown in Chart 1. The modified phosphoramidites 1 and 2 were
synthesized using literature procedures (Supporting Information).21-23
These phosphoramidites were prepared in an effort to incorpo-
rate 8-(p-OHPh)-dG (Figure 1) into DNA. Our synthetic efforts
demonstrated the need for protection of the phenolic OH that
competes with the sugar 3′-OH for reaction with the phosphi-
tylating reagent. Thus, the O-benzyl (Bn)-protected derivative
1 was synthesized first and served to optimize solid-phase DNA
synthesis. However, strategies for removal of the O-Bn protect-
Initial work also focused on the use of p-OBnPh-BA and
p-OHPh-BA for reaction with the 8-Br-G-modified DNA
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44 J. AM. CHEM. SOC. VOL. 133, NO. 1, 2011

Synthesis of C8-Ar-G-Modified Oligonucleotides
A R T I C L E S
substrates. This provided a direct comparison between the
postsynthetic arylation reaction with automated DNA synthesis
using the modified phosphoramidites 1 and 2. The arylation
reactions were then extended to include reactions of o-OHPh-
BA, Ph-BA, and benzothiophene (Bth)-2-BA. These boronic
acids (BAs) were chosen for study because the products c and
d (Chart 1) are formed by chemical carcinogens10,11 and their
insertion into oligonucleotides generates substrates that can be
tested for biological activity, while the nucleoside adduct of
product e (Chart 1) has recently been synthesized38 and shows
promise as a fluorescent probe.
Solid-Phase versus Postsynthetic DNA Synthesis. To optimize
the Pd-catalyzed postsynthetic reaction, the short DNA substrates
ODN1 and ODN2 (0.1 µmol) were utilized first and reacted
with 1.2 equiv of p-OBnPh-BA in the presence of Pd(OAc)2,
P(C6H4-3-SO3Na)3 (TPPTS), and Na2CO3 in a mixture of water
and CH3CN (typical conditions for C8-Ar-purine nucleoside
synthesis31) and were heated at 70 °C for 24 h. The expected
products ODN1_a and ODN2_a from the arylation reactions
were also obtained from solid-phase synthesis using phosphora-
midite 1. These reactions were carried out on a 1 µmol scale
with an extended coupling time of 4 min (standard 1 min) using
standard deblock (3% CHCl2COOH in CH2Cl2 for 40 s) and
oxidant (I2/THF/pyridine/H2O for 1 min). Reversed-phase HPLC
results for reaction with ODN1 are shown in Figure S1
(Supporting Information). Solid-phase synthesis produced 28
OD of the dinucleotide ODN1_a that eluted at ∼9.5 min and
showed fluorescence (λex ) 280 nm, λem ) 390 nm), as expected
for attachment of the C8-Ar-G base.16 ODN1_a (29 OD) was
also produced from Pd-catalyzed coupling of ODN1 (48 OD)
with p-OBnPh-BA. This afforded an isolated yield of ∼60%.39-41
Both strategies were also successful in the synthesis of the
trinucleotide ODN2_a. The solid-phase synthesis afforded 16
OD of ODN2_a, while 22 OD (∼41%) was isolated from Pd
coupling of p-OBnPh-BA with 53 OD of ODN2.
Having established that the Pd-catalyzed arylation chemistry
is compatible with DNA, efforts were made to prepare longer
strands, and the initial focus was placed on the pyrimidine-rich
decanucleotide substrate ODN3. Figure 2 shows reversed-phase
HPLC results from attempted solid-phase synthesis of ODN3_a
and Suzuki-Miyaura coupling of p-OBnPh-BA with ODN3.
In this case, solid-phase synthesis using phosphoramidite 1
(Figure 2A) generated four peaks. The major products eluting
at ∼2 and 5.5 min lacked fluorescence for incorporation of the
C8-Ar-G base and gave singly charged ions at m/z 517 and 1427
that suggested formation of truncated strands. We speculate that
repetitive treatment of the modified base with acid and/or oxidant
(five deblock and five oxidant steps) led to the failed sequences
suggested by Figure 2A. In an effort to generate the decanucle-
otide using solid-phase synthesis, the deblock time was reduced
to 20 s, the normal oxidant was replaced with 10-camphorsul-
fonyloxaziridine, and in one instance 0.5 M 2-mercaptoethanol
Figure 2. Reversed-phase HPLC traces of the (A) product mixture from
attempted solid-phase DNA synthesis using 1, (B) product mixture following
Pd-catalyzed coupling of ODN3 with 1.2 equiv of p-OBnPh-BA, and (C)
product mixture following Pd-catalyzed coupling of ODN3 with 10 equiv
of p-OBnPh-BA using an alternative HPLC gradient protocol for slower
product elution.
was added to NH4OH during deprotection. However, no full-
length decanucleotide ODN3_a was produced. We were then
eager to determine the number of bases that can be placed after
8-(p-OBnPh)-G. These studies led to successful solid-phase
synthesis of the octanucleotide 5′-CCG-8-(p-OBnPh)-CTACC
(17 OD, Supporting Information) containing two bases after the
modified base.
In contrast to the solid-phase synthesis results, the Pd-
catalyzed coupling reaction between ODN3 and p-OBnPh-BA
(1.2 equiv) generated a single product peak at ∼6 min for
ODN3_a, as evidenced by the HPLC trace in Figure 2B that
exhibited the expected fluorescence for incorporation of the C8-
Ar-G base.16 Integration of the HPLC trace was used to establish
a yield of 15% ODN3_a.39 The HPLC trace shown in Figure
2C was obtained following the Pd-catalyzed reaction of ODN3
with 10 equiv of p-OBnPh-BA using a slightly altered HPLC
gradient protocol for slower product elution. The yield from
the 10 equiv reaction was 45%, and all further Pd-catalyzed
postsynthetic arylation reactions were carried out using 10 equiv
of Ar-BA.
The spectral properties of the isolated product ODN3_a are
shown in Figure 3. Its UV spectrum (Figure 3A) showed an
absorbance at ∼310 nm for the modified base. We have shown
that C8-Ar-dG nucleotides adopt a twisted structure with λmax
≈ 290 nm, while the nucleobase lacking the deoxyribose moiety
is planar and absorbs at ∼310 nm,18b,25 suggesting a planar 8-(p-
OBnPh)-G within the decanucleotide. Its ESI- (ESI ) electro-
spray ionization) spectrum (Figure 3B) showed the expected
cluster of multiply charged peaks for the full-length decanucle-
otide with molecular formula C108H133N34O59P9 and the mass
of the most abundant isotope peak at 3129.6. An expansion of
the isotopic peaks representing [M - 2H]2- ions is shown in
(38) Hobley, G.; Gubala, V.; Rivera-Sa´nchez, M. D. C.; Rivera, J. M. Synlett
2008, 1510, 1514.
(39) Yields of C8-Ar-G-modified oligonucleotides were estimated by
quantification of the isolated product by UV using ε260 for the
unmodified oligonucleotides or from integration of the HPLC trace
assuming similar absorption of the product and 8-Br-G-modified
substrate. These estimations are reasonable given that log ε values of
dG (4.14)40 and 8-Br-dG (4.18)41 are similar to that of 8-Ar-dG
(∼4.26).
(40) Onidas, D.; Markovitsi, D.; Marguet, S.; Sharonov, A.; Gustavsson,
T. J. Phys. Chem. B 2002, 106, 11367–11374.
(41) Holmes, R. E.; Robins, R. K. J. Am. Chem. Soc. 1964, 86, 1242–
1245.
9
J. AM. CHEM. SOC. VOL. 133, NO. 1, 2011 45

A R T I C L E S
Omumi et al.
(see the Supporting Information for spectral properties of
modified DNAs). These results further highlighted the difficulty
of the automated solid-phase DNA synthetic procedure for
synthesis of C8-Ar-G-modified oligonucleotides24 and demon-
strated the ease and applicability of the Suzuki-Miyaura cross-
coupling procedure. This suggested that the postsynthetic
strategy may be employed for insertion of a range of C8-Ar-G
adducts into DNA.
Scope of Pd-Catalyzed Arylation of Oligonucleotides. To test
the general applicability of the Pd-catalyzed postsynthetic
arylation procedure, the boronic acids shown in Chart 1 were
reacted with DNA substrates ODN2, ODN3, and ODN4.
Representative HPLC chromatograms for reactions of the
purine-rich decanucleotide ODN4 with p-OBnPh-BA, o-OHPh-
BA, Ph-BA, and Bth-2-BA are shown in Figure 4. In each case
the product peaks labeled in the chromatograms showed the
expected fluorescence for insertion of C8-Ar-G16 and gave the
expected m/z values for full-length decanucleotide (Supporting
Information), confirming specificity for arylation at 8-Br-G. In
Figure 4A for reaction of ODN4 with p-OBnPh-BA, integration
of the HPLC peaks for unreacted ODN4 and the product
ODN4_a afforded a yield of 71%. A similar yield (77%) was
obtained for ODN4_b from reaction with p-OHPh-BA. The
results for the isomeric o-OHPh-BA shown in Figure 4B were
very pleasing given our interest in adducts derived from phenolic
toxins. Virtually no peak for starting material was observed,
and a yield of 87% for ODN4_c was determined. A remarkably
clean chromatogram was obtained from reaction of ODN4 with
Ph-BA (Figure 4C) with almost complete conversion into the
ODN4_d product (97%). In contrast, reaction of Bth-2-BA with
ODN4 (Figure 4D) showed a significant amount of unre-
acted ODN4 and gave the lowest yield of 39% for ODN4_e.
The variability in yield was not surprising given that arylation
of halonucleosides with TPPTS/Pd(OAc)2 range from 60% to
99%.31 The results with ODN4 demonstrated the specificity of
the Ar-BAs for the convertible nucleoside 8-Br-G when flanked
by purine bases.
Figure 3. Spectral properties of the decanucleotide ODN 3_a generated
by Pd coupling of ODN3 with p-OBnPh-BA: (A) UV spectrum (λmax
)
270 and 310 nm (shoulder), (B) ESI- spectrum, (C) expansion of the cluster
of peaks containing [M - 2H]2- ) 1563.8.
Figure 3C. The most abundant isotopic peak contains a single
13C and 107 12C atoms.
With phosphoramidite 2 (Chart 1), only the dinucleotide
ODN1_b could be prepared using solid-phase DNA synthesis,
as attempted syntheses of the trinucleotide ODN2_b and
decanucleotide ODN3_b generated failed sequences (data not
shown). The failed trinucleotide synthesis suggested that the
labile acetylphenolic protecting group in 2 may have presented
a problem and that further optimization would be required for
successful synthesis. However, both ODN2_b and ODN3_b
were prepared in yields of 60% and 79%, respectively, using
the postsynthetic arylation route with 10 equiv of p-OHPh-BA
As a further test of the Pd-catalyzed arylation reaction, the
longer 15-mer substrate ODN5 was reacted with the parent Ph-
BA. Figure S2 (Supporting Information) shows HPLC chro-
matograms of the product mixture following Suzuki-Miyaura
cross-coupling of ODN5 with 10 equiv of Ph-BA. In Figure
S2A, the bottom trace shows UV detection at 258 nm, while
Figure 4. Reversed-phase HPLC traces of product mixtures following Pd-catalyzed coupling of ODN4 with 10 equiv of Ar-BA: (A) ODN4 + p-OBnPh-
BA, (B) ODN4 + o-OHPh-BA, (C) ODN4 + Ph-BA, (D) ODN4 + Bth-2-BA.
9
46 J. AM. CHEM. SOC. VOL. 133, NO. 1, 2011

Synthesis of C8-Ar-G-Modified Oligonucleotides
A R T I C L E S
Table 1. Yields and ESI- MS Analysis of Pd-Catalyzed Postsynthetic DNA Synthesis of C8-Ar-G-Modified Oligonucleotidesa
DNA
Ar-BA (amt, equiv)
product formula
yield (%)
calcd massd
exptl m/z (ESI-)e
exptl massd
ODN1
ODN2
ODN3
ODN3
ODN4
ODN2
ODN3
ODN4
ODN2
ODN3
ODN4
ODN2
ODN3
ODN4
ODN5
ODN2
ODN3
ODN4
p-OBnPh- (1.2)
p-OBnPh- (1.2)
p-OBnPh- (1.2)
p-OBnPh- (10)
p-OBnPh- (10)
p-OHPh- (10)
p-OHPh- (10)
p-OHPh- (10)
o-OHPh- (10)
o-OHPh- (10)
o-OHPh- (10)
Ph- (10)
Ph- (10)
Ph- (10)
Ph- (10)
Bth-2- (10)
Bth-2- (10)
Bth-2- (10)
C32H35N8O11P
C41H47N11O17P2
60b
41b
15c
45c
71c
60b
79b
77c
39b
83b
87b
49b
55b
97c
83b
47b
78b
39c
738.2
1027.3
3129.6
3129.6
3329.6
937.2
3039.6
3239.6
937.2
3039.6
3239.6
921.2
3023.6
3223.6
4669.8
977.2
[M - H]- ) 737.2
[M - H]- ) 1026.1
[M - 2H]2- ) 1563.8
[M - 2H]2- ) 1563.3
[M - 5H]5- ) 664.9
[M - H]- ) 936.2
[M - 3H]3- ) 1012.2
[M - 4H]4- ) 808.9
[M - H]- ) 936.3
[M - 2H]2- ) 1012.2
[M - 4H]4- ) 808.9
[M - H]- ) 920.3
[M - 3H]3- ) 1006.9
[M - 4H]4- ) 804.9
[M - 5H]5- ) 932.95
[M - H]- ) 976.1
[M - 2H]2- ) 1538.7
[M - 4H]4- ) 818.9
738.2
1027.1
3129.6
3129.6
3329.5
937.2
3039.6
3239.6
937.3
3039.6
3239.6
921.3
3023.7
3223.6
4669.8
977.1
C
C
C
108H133N34O59P9
108H133N34O59P9
113H133N44O59P9
C34H41N11O17P2
C
C
101H127N34O59P9
106H127N44O59P9
C34H41N11O17P2
C
C
101H127N34O59P9
106H127N44O59P9
C34H41N11O16P2
C
C
C
101H127N34O58P9
106H127N44O58P9
151H187N59O88P14
C36H41N11O16P2S
C
C
103H127N34O58P9S
108H127N44O58P9S
3079.5
3279.6
3079.4
3279.6
a Conditions: molar ratios Br-DNA:Ar-B(OH)2 ) 0.8 or 0.1, Br-DNA:TPPTS ) 15, TPPTS:Pd(OAc)2 ) 25, and Na2CO3:Br-DNA ) 2 for a total
volume of 350 µL of 2:1 water/acetonitrile, heated under argon at 70 °C for 24 h. b Isolated yield derived using ε260 for the unmodified oligonucleotides.
c Yield derived from integration of the HPLC trace. d Monoisotopic mass of most abundant isotopologue. e Measured m/z (spectra presented in the
Supporting Information).
the top trace shows fluorescence detection (λex ) 290 nm, λem
) 390 nm). The product ODN5_d eluted as a sharp peak at
19.3 min, as readily apparent in the fluorescence trace. From
the UV trace, no discernible peak for unreacted ODN5 was
observed. Spiking the sample with ODN5 gave a new sharp
peak eluting at 18.7 min that did not overlap with the product
peak (Figure S2B). The excess Ph-BA was noted as a broad
peak at 17.2 min in both the UV and fluorescence traces. In the
UV trace the broad peak eluting just before Ph-BA at 16.7 min
was not identified. The fluorescence trace also showed peaks
at 28-30 min that were not identified; these peaks showed no
appreciable UV absorbance at 258 nm. To determine the yield
of ODN5_d, the product peak was isolated and quantified by
UV spectroscopy.39 This provided an excellent yield of 83%
for ODN5_d. ESI- MS analysis provided an ion at [M - 5H]5-
) 932.95 corresponding to the full-length 15-mer with a mono-
isotopic mass of 4669.8 for the most abundant isotopologue.
The results of these studies are summarized in Table 1. Using
10 equiv of Ar-BA, the yields of the Pd-catalyzed reactions
ranged from 39% to 97%. Surprisingly, some of the lowest
yields were obtained using the trinucleotide substrate ODN2
Figure 5. Product ion spectrum of ESI-produced [M - 2H]2- of ODN3_a
(5′-CCATG-8-(p-OBnPh)-CTACC), where X ) G-8-(p-OBnPh).
neutral molecule to give the [M - 2H - X]2- ) 1397.2 ion.
This base loss then promotes breakage of the 3′-C-O bond at
the sugar moiety to give the singly charged fragment ions at
m/z 1501.2 (a w5 fragment according to the nomenclature of
McLuckey et al.42) and m/z 1292.2 (an a5-B fragment42). The
ion at m/z 1292.2 still contains the sugar moiety from the
modified nucleobase, but it is now present as a furan ring.42
Other ions at m/z 1390.1, 1212.2, and 1083.2 are fragment ions
of m/z 1501.2. These results are very similar to those observed
previously by Zhang and Gross43 for oligonucleotides containing
abasic sites, in which complementary fragment ions (an and wn)
are produced at the abasic site to allow its location to be
determined. In this case, the labile G-8-Ar nucleobase is lost
first to initiate fragmentation of the oligonucleotide at the
modified base position. This type of positional mapping,
depurination followed by cleavage of the phosphodiester
(39-60%). Variation in the nature of the bases flanking the
convertible 8-Br-G nucleoside had little effect on the reaction
yield. ESI- MS spectra of the starting 8-Br-G substrates
ODN1-5 along with UV-vis and ESI- MS spectra of the
modified oligonucleotides are given in the Supporting Informa-
tion. The MS data confirmed the identity of the product oligo-
nucleotides and provided a check for purity, as did HPLC
analysis of the final isolated product.
DNA Sequence. In addition to confirmation of oligonucleotide
structure by ESI- MS, it was found that ESI MS/MS was
particularly useful for confirming the site of the C8-Ar-G lesion
within the oligonucleotide. Due to the lability of the C8-Ar-G
nucleobase, product ion spectra show loss of the modified base
with subsequent fragmentation of the resulting abasic site to
allow the site of the C8-Ar-G adduct to be determined. This is
highlighted in Figure 5, which shows the product ion spec-
trum of [M - 2H]2- from ODN3_a (5′-CCATG-8-(p-OBnPh)-
CTACC). The spectrum is considerably simpler than that of
the corresponding unmodified decanucleotide (not shown) and
is dominated by loss of the G-8-(p-OBnPh) nucleobase as a
(42) (a) McLuckey, S. A.; Habibi-Goudarzi, S. J. Am. Soc. Chem. Soc.
1993, 115, 12085–12095. (b) Wu, J.; McLuckey, S. A. Int. J. Mass
Spectrom. 2004, 237, 197–241.
(43) Zhang, L.-K.; Gross, M. L. J. Am. Soc. Mass Spectrom. 2002, 13,
1418–1426.
9
J. AM. CHEM. SOC. VOL. 133, NO. 1, 2011 47

A R T I C L E S
Omumi et al.
Table 2. Melting Temperatures (Tm) of Decanucleotidesa
decanucleotide
Tm (°C)
∆Tm (°C)
5′-CCATGCTACC
44
39
27
27
31
31
33
CCATG-8-Br-CTACC (ODN3)
-5
CCATG-8-(p-OBnPhG)-CTACC (ODN3_a)
CCATG-8-(p-OHPh)-CTACC (ODN3_b)
CCATG-8-(o-OHPh)-CTACC (ODN3_c)
CCATG-8-Ph-CTACC (ODN3_d)
-17
-17
-13
-13
-11
CCATG-8-(2-Bth)-CTACC (ODN3_e)
a Conditions: 200 mM NaCl, 50 mM triethylamine acetate, pH 7.2, 1
mM EDTA, 0.5 260/mL of each oligonucleotide (modified and
A
complementary strand 5′-GGTAGCATGG).
backbone, is established for carcinogens that undergo metabolic
activation to form DNA adducts at the N7 position of G and
can be used to determine the chemical stability of DNA
adducts.44 That C8-Ar-G-modified oligonucleotides behave in
a similar fashion highlights the lability of the C8-Ar-G lesion.
Duplex Stability and Structure. The modified pyrimidine-
rich ODN3 decanucleotides were hybridized to the comple-
mentary strand 5′-GGTAGCATGG, and the effect of the C8-
Ar-G adduct on the thermal stability of the DNA duplex was
measured using UV melting temperature analysis (Tm) by
monitoring the absorbance at 260 nm versus temperature. These
data are summarized in Table 2 and demonstrate that the C8-
Ar-G adduct destabilizes the duplex considerably (11-17 °C),
while the 8-Br-G adduct shows a more modest 5 °C destabiliza-
tion for the decanucleotide. These findings are in accord with
previous results showing bulky C8-Ar-G lesions to significantly
decrease duplex stability.19,45
Figure 6. CD spectra of the unmodified duplex (closed circles) versus
adducted duplexes (open circles) [(A) ODN3_b and (B) ODN3_e] obtained
at 5 °C in 10 mM triethylamine acetate, pH 7.1, 40 mM NaCl, 0.5 A260/mL
of each oligonucleotide (complementary strand 5′-GGTAGCATGG).
The melting temperature analysis showed 8-(p-OHPh)-G to
have a dramatic impact on helix stability (∆Tm ) -17 °C), while
ODN3_e bearing 8-(2-Bth)-G generated the most stable helix
of the C8-Ar-G-modified duplexes (∆Tm ) -11 °C). To
determine the impact of these C8-Ar-G lesions on the duplex
structure, circular dichroism (CD) measurements of decanucle-
otides ODN3_b and ODN3_e were carried out. Figure 6 shows
CD spectra for the modified and unmodified decanucleotide
duplexes. The unmodified duplex shows roughly equal positive
(275 nm) and negative (244 nm) bands, with a crossover at 260
nm that is characteristic of normal B-form DNA.46 The duplex
of ODN3_b (Figure 6A) shows a broad positive band at 271
nm and a negative band at 224 nm that is decreased in intensity
compared to the negative band at 244 nm for the unmodified
duplex. These changes in CD features suggest more A-like
character for the right-handed duplex of ODN3_b.47 In contrast,
the duplex of ODN3_e shows characteristics that closely
resemble the normal B-form DNA of the unmodified duplex
(Figure 6B). The positive induced CD signal at ∼325 nm in
Figure 6B (open circles) is assigned to the benzothiophene group
and results from the chromophore residing in the chiral
environment of the DNA duplex. These CD measurements show
that the 8-(p-OHPh)-G lesion has a greater impact on the duplex
structure than the 8-(2-Bth)-G adduct, which correlates with the
impact of these lesions on the duplex stability.
Figure 7. Normalized emission spectra of (A) ODN3_b (blue) +
complementary strand (red) and (B) ODN3_e (blue) + complementary
strand (red) obtained at 5 °C in 10 mM triethylamine acetate, pH 7.1, 40
mM NaCl, 0.5 A260/mL of each oligonucleotide (complementary strand 5′-
GGTAGCATGG).
Fluorescence emission spectroscopy was utilized to probe the
environment of 8-p-OHPh-G and 8-(2-Bth)-G in more detail.
The modified nucleosides are violet-blue fluorophores with
emission maxima at ∼380-420 nm.16,17 Figure 7A shows
emission spectra for ODN3_b in the absence and presence of
the complementary strand 5′-GGTAGCATGG, while the cor-
responding spectra for ODN3_e are shown in Figure 7B. The
emission of the 8-p-HOPh-G lesion in ODN3_b at 408 nm (λex
) 290 nm) is significantly quenched upon hybridization and
shows a blue shift to 390 nm. When ODN3_e (λex ) 315 nm,
λem ) 420 nm) is hybridized to the complementary strand, the
emission of the duplex also exhibits a blue shift (λem ) 400
nm), but its intensity is enhanced 3-fold compared to the
(44) Marzilli, L. A.; Wang, D.; Kobertz, W. R.; Essigmann, J. M.; Vouros,
P. J. Am. Soc. Mass Spectrom. 1998, 9, 676–682.
(45) (a) Elmquist, C. E.; Stover, J. S.; Wang, Z.; Rizzo, C. J. J. Am. Chem. Soc.
2004, 126, 11189–11201. (b) Takamura-Enya, T.; Ishikawa, S.; Mochizuki,
M.; Wakabayashi, K. Chem. Res. Toxicol. 2006, 19, 770–778.
(46) Gray, D. M.; Ratliff, R. L.; Vaughan, M. R. Methods Enzymol. 1992,
211, 389–406.
(47) (a) Gray, D. M.; Liu, J.-J.; Ratliff, R. L.; Allen, F. S. Biopolymers
1981, 20, 1337–1382. (b) Clark, C. L.; Cecil, P. K.; Singh, D.; Gray,
D. M. Nucleic Acids Res. 1997, 25, 4098–4105.
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48 J. AM. CHEM. SOC. VOL. 133, NO. 1, 2011

Synthesis of C8-Ar-G-Modified Oligonucleotides
A R T I C L E S
from the solid support and deprotected using 2 mL of 30%
ammonium hydroxide solution at 55 °C for 12 h. The solutions
were then concentrated under diminished pressure to ∼100 µL using
a ThermoSavant DNA 120 SpeedVac at medium drying rate. The
solutions were then purified using an Agilent 1200 series HPLC
instrument equipped with an autosampler, an autocollector, a diode
array detector (monitored at 258 nm), and a fluorescence detector
(λex ) 290 nm; λem ) 390 nm). Separation was carried out using
a Phenomenox Clarity 3 µm Oligo-RP C18 column (50 × 4.60
mm, 3 µm) and various gradients of buffer B in buffer A (buffer
A, aqueous 50 mM triethylammonium acetate (TEAA), pH 7.2/
acetonitrile (95:5); buffer B, aqueous 50 mM TEAA, pH 7.2/
acetonitrile (25:75).
emission of the single-strand ODN3_e. These studies show that
C8-Ar-G lesions are sensitive to the DNA microenvironment.
The ability of ODN3_e to report hybridization with significantly
increased emission, coupled with its relative ease of preparation
through postsynthetic arylation, suggests wide use of the
Suzuki-Miyaura methodology for generation of fluorescent
DNAs with diagnostic applications.
Conclusions
We have utilized Suzuki-Miyaura cross-coupling for the site-
specific synthesis of modified oligonucleotides containing the
C-linked C8-Ar-G lesion. The significance of this work stems
from the fact that certain C8-Ar-dG adducts are sensitive to acids
and oxidants, making solid-phase DNA synthesis using highly
functionalized C8-Ar-dG phosphoramidites problematic. These
adducts are also too bulky to be used as substrates for DNA
polymerases, precluding an enzymatic strategy for their syn-
thesis. Suzuki-Miyaura cross-coupling avoids the use of acids
and oxidants, and the chemistry is compatible with DNA. Our
studies demonstrate that good to excellent yields of C8-Ar-G-
modified decanucleotide product are obtained and that the
reaction in insensitive to the nature of the bases flanking the
convertible 8-Br-G nucleobase. The reaction can also be carried
out with 15-mer substrates that are more relevant for molecular
biology studies, including primer-extension assays using DNA
polymerases. Our studies also highlight the use of ESI MS/MS
to confirm the site of the C8-Ar-G lesion within the oligonucle-
otides and demonstrate the potential utility of the C8-Ar-G
nucleobase for fluorescent sensing applications within duplex
DNA. Our laboratory now plans to use Suzuki-Miyaura cross-
coupling to generate a range of modified DNA substrates,
including those resulting from postsynthetic C8-arylation of
adenine, to understand the biological and structural impact of
the C8-Ar-purine lesion.
Suzuki-Miyaura Coupling Reactions with 8-Br-G-Modi-
fied Oligonucleotides. Modified oligonucleotides containing 8-Br-
dG of the desired sequence and length on a 1 µmol scale were
custom-made by Sigma Genosys (Canada) using standard phos-
phoramidites and 8-Br-dG-CE phosphoramidite purchased from
Glen Research. Oligonucleotides were cleaved from the solid
support and deprotected in ammonium hydroxide at room temper-
ature for 24 h (to lessen debromination). The synthesized DMT-
ON oligonucleotides were desalted, purified, and deprotected on
reversed-phase cartridges and rechecked for correct mass and
bromine isotopic distribution by ESI MS at Guelph (see the
Supporting Information for ESI MS spectra of starting DNA
substrates ODN1-5). For Suzuki-Miyaura coupling of brominated
oligonucleotides (Br-DNA) (0.1 µmol), the other reaction compo-
nents were initially prepared as 1000× stock solutions in deoxy-
genated 2:1 water/acetonitrile. Through serial dilution the reagents
were added via syringe to the brominated oligonucleotides in
septum-capped vials at molar ratios Br-DNA:Ar-B(OH)2 ) 0.8 or
0.1, Br-DNA:TPPTS ) 15, TPPTS:Pd(OAc)2 ) 25, and Na2CO3:
Br-DNA ) 2 for a total volume of 350 µL of 2:1 water/acetonitrile.
The resulting solutions were heated under argon at 70 °C for 24 h
and then concentrated under diminished pressure and purified by
HPLC using the conditions outlined for the modified oligonucle-
otides. Yields were determined either by quantifying the isolated
oligonucleotide by UV using ε260 (nearest-neighbor model) for the
unmodified oligonucleotides or from integration of the HPLC
trace.39
Experimental Section
General Methods. Unless otherwise noted, commercial com-
pounds were used as received and, in general, were purchased from
Aldrich (Milwaukee, WI) with the exception of all boronic acids,
which were from Frontier Scientific (Logan, UT), dG from
ChemGenes (Wilmington, MA), and tris(2-sulfophenyl)phosphine
trisodium salt (TPPTS) from Strem Chemical (Newburyport, MA).
Pyridine was distilled from KOH and stored over 3 Å molecular
sieves. Water used for buffers and spectroscopic solutions was
obtained from a Milli-Q filtration system (18.2 MΩ). 8-Bromo-2′-
deoxyguanosine (8-Br-dG) was prepared by treating dG with
N-bromosuccinimide in water/acetonitrile.31 Moisture-sensitive
reactions were performed under an argon atmosphere using oven-
dried (120 °C) glassware, syringes, and needles. Compounds were
dried under vacuum and over P2O5 (12-24 h) prior to use in water-
sensitive reactions. NMR spectra were recorded on a Bruker Avance
300 DPX spectrometer (1H, 300.1 MHz; 13C, 75.5 MHz; 31P, 121.4
MHz) in DMSO-d6 or CDCl3. High-resolution mass spectra were
obtained with a Micromass/Waters Global Ultima quadrupole time-
of-flight (Q-TOF) instrument using electrospray ionization.
Oligonucleotide Synthesis. A MerMade 12 automatic synthe-
sizer (BioAutomation Corp.) was used for modified oligonucleotide
synthesis according to the manufacturer protocol for a 1 µmol scale
DNA synthesis optimized for MerMade CPG columns (standard
CPG loaded with 3′-C, 1000 Å pore size, loading 30 µmol/g). An
extended coupling time of 4 min (standard 1 min) was employed
using standard deblock (3% CHCl2COOH in CH2Cl2 for 40 s, which
was reduced to 20 s in some instances) and oxidant (I2/THF/
pyridine/H2O for 1 min). All normal phosphoramidites were
obtained from Glen Research, while DNA synthesis reagents were
from EMD. After the synthesis, the oligonucleotides were cleaved
MS of Oligonucleotides. Synthesized oligonucleotides 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 ESI source in negative mode using
either a Finnigan LCQ Deca ion trap or a Waters Q-TOF Micro
quadrupole time-of-flight instrument. Oligonucleotides are particu-
larly fragile molecules in the gas phase, and the use of gentle
conditions in the ion source and ion optics regions of the instruments
was essential for their detection by ESI. For the ion trap, a low
flow of sheath gas (20 arbitrary units), the absence of capillary
and tube lens voltages (0 V), and low multipole offset voltages
(5.5 and 8.0 V for offsets 1 and 2, respectively) were found to
give good detection of most of the oligonucleotides analyzed. In
the Q-TOF instrument, similarly low voltages were used for the
cone (14 V) and extraction cone (1 V) as well as a lower than
normal collision energy in the quadrupole collision cell (4 V).
Tandem mass spectra were collected using the Q-TOF instrument
with the above conditions and a cone voltage of 30 V that gave
slightly better transmission of the higher m/z lower charge states
and a collision energy of 20 V.
Thermal Melting. UV melting studies were carried out on a
Cary 300-Bio UV-vis spectrophotometer equipped with a 6 × 6
multicell block Peltier, stirrer, and temperature controller with Probe
Series II. Equal amounts of the adducted decanucleotide and the
complementary strand 5′-GGTAGCATGG (0.5 unit each) were
dissolved in 0.5 mL of buffer (200 mM NaCl, 50 mM triethylamine
acetate, pH 7.2, 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
9
J. AM. CHEM. SOC. VOL. 133, NO. 1, 2011 49

A R T I C L E S
Omumi et al.
of 0.5 °C/min from 85 to 5 °C.48 The melting temperatures of the
duplexes were calculated by determining the first derivative of the
melting curve.
Circular Dichroism and Emission Measurements. CD mea-
surements were performed on a Jasco J-815 CD spectrometer at 5
correction, and excitation and emission slit widths were kept con-
stant at 5.0 nm.
Acknowledgment. Support for this research was provided by
the Natural Sciences and Engineering Research Council (NSERC)
of Canada, the Canada Foundation for Innovation, and the Ontario
Innovation Trust Fund. R.A.M. thanks Mr. Christopher McLaughlin
for carrying out preliminary experiments and Dr. Hongbin Yan
(Brock University) for assistance with phosphoramidite synthesis.
°C in 10 mM triethylamine acetate, pH 7.1, 40 mM NaCl, 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 on a
Cary Eclipse fluorescence spectrophotometer equipped with a 1 ×
4 multicell block Peltier, stirrer, and temperature controller with
Probe Series II. Standard 10 mm light path quartz glass cells from
Hellma GmbH & Co. were used for fluorescence measurements.
All fluorescence spectra were recorded at 5 °C with baseline
Supporting Information Available: Experimental section for
phosphoramidite synthesis, NMR spectra of synthetic products,
MS spectra of starting substrates ODN1-5, UV and MS spectra
of C8-Ar-G-modified oligonucleotides, and Figures S1 and S2
described in the text. This material is available free of charge
via the Internet at http://pubs.acs.org.
(48) Wang, Y.; Rusling, D. A.; Powers, V. E. C.; Lack, O.; Osborne, S. D.;
Fox, K. R.; Brown, T. Biochemistry 2005, 44, 5884–5892.
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