Utility of 5'-o-2,7-dimethylpixyl for solid-phase synthesis of oligonucleotides containing acid-sensitive 8-aryl-guanine adducts

Article abstract of DOI:10.1021/jo4024842

To study the structural and biological impact of 8-aryl-2'-deoxyguanosine adducts, an efficient protocol is required to incorporate them site-specifically into oligonucleotide substrates. Traditional phosphoramidite chemistry using 5'- O-DMT protection can be limiting because 8-aryl-dG adducts suffer from greater rates of acid-catalyzed depurination than dG and are sensitive to the acidic deblock conditions required to remove the DMT group. Herein we show that the 5'-O-2,7- dimethylpixyl (DMPx) protecting group can be used to limit acid exposure and improve DNA synthesis efficiency for DNA substrates containing 8-aryl-dG adducts. Our studies focus on 8-aryl-dG adducts with 8-substituents consisting of furyl (FurdG), phenyl (PhdG), 4-cyanophenyl (CNPhdG), and quinolyl (QdG). These adducts differ in ring size and sensitivity to acid-promoted deglycosylation. A kinetic study for adduct hydrolysis in 0.1 M aqueous HCl determined that FurdG was the most acid-sensitive (55.2-fold > dG), while QdG was the most resistant (5.6-fold > dG). The most acid-sensitive FurdG was chosen for optimization of solid-phase DNA synthesis. Our studies show that the 5'-O-DMPx group can provide a 4-fold increase in yield compared to 5'- O-DMT for incorporation of FurdG into DNA substrates critical for determining adduct impact on DNA synthesis and repair.

Full text of DOI:10.1021/jo4024842

Article
pubs.acs.org/joc
Utility of 5′‑O‑2,7-Dimethylpixyl for Solid-Phase Synthesis of
Oligonucleotides Containing Acid-Sensitive 8‑Aryl-Guanine Adducts
Michael Sproviero, Katherine M. Rankin, Aaron A. Witham, and Richard A. Manderville*
Departments of Chemistry and Toxicology, University of Guelph, Guelph, Ontario N1G 2W1, Canada
S
* Supporting Information
ABSTRACT: To study the structural and biological impact of
8-aryl-2′-deoxyguanosine adducts, an efficient protocol is
required to incorporate them site-specifically into oligonucleo-
tide substrates. Traditional phosphoramidite chemistry using 5′-
O-DMT protection can be limiting because 8-aryl-dG adducts
suffer from greater rates of acid-catalyzed depurination than dG
and are sensitive to the acidic deblock conditions required to
remove the DMT group. Herein we show that the 5′-O-2,7-
dimethylpixyl (DMPx) protecting group can be used to limit
acid exposure and improve DNA synthesis efficiency for DNA
substrates containing 8-aryl-dG adducts. Our studies focus on 8-aryl-dG adducts with 8-substituents consisting of furyl (FurdG),
phenyl (PhdG), 4-cyanophenyl (CNPhdG), and quinolyl (QdG). These adducts differ in ring size and sensitivity to acid-promoted
deglycosylation. A kinetic study for adduct hydrolysis in 0.1 M aqueous HCl determined that FurdG was the most acid-sensitive
(55.2-fold > dG), while QdG was the most resistant (5.6-fold > dG). The most acid-sensitive FurdG was chosen for optimization
of solid-phase DNA synthesis. Our studies show that the 5′-O-DMPx group can provide a 4-fold increase in yield compared to 5′-
O-DMT for incorporation of FurdG into DNA substrates critical for determining adduct impact on DNA synthesis and repair.
which contains the recognition sequence of the NarI Type II
restriction endonuclease.6 This sequence represents a “hotspot”
for mutagenicity mediated by nitrogen-linked 8-aryl-dG adducts
produced by arylamine carcinogens.10 The 12-mer sequence
permits analysis of adduct impact on duplex DNA structure.
For N-linked arylamine adducts such duplex structures have
been used to predict mutagenic outcome10 and propensity for
DNA repair.11 A second goal is to incorporate 8-aryl-dG
adducts into longer DNA substrates that can be used for
primer-extension assays using DNA polymerases to assess the
biological impact of these lesions. In an effort to achieve these
goals and generate adducted oligonucleotide substrates
containing 8-aryl-dG lesions, we developed a postsynthetic
strategy using Suzuki cross-coupling,12 which avoids synthesis
of the modified phosphoramidite and exposure of the 8-aryl-dG
lesion to acidic deblock (3% dichloroacetic acid (DCA) for
removal of the 5′-O-DMT protecting group). This approach
can be used to incorporate a single adduct into relatively short
substrates (3−15mers).6,12,13 However, yields can be low,6 and
the strategy becomes problematic when either incorporating
multiple adducts into strands or generating longer adducted
DNAs required to assess the biological impact of the lesion
using DNA polymerases and DNA repair enzymes.
INTRODUCTION
■
Aryl radical species derived from the metabolism of
polyaromatic hydrocarbons (PAHs),1 arylhydrazines,2 estro-
gens,3 and phenolic toxins4 can react at the 8-position of 2′-
deoxyguanosine (dG) to afford carbon-linked 8-aryl-dG
adducts. If left unrepaired, these lesions may initiate carcino-
genesis. The 8-aryl-dG adducts are also highly emissive and are
useful fluorescent nucleobase probes for detecting G-quad-
ruplex folding5 and for monitoring adduct conformation within
duplex DNA.6
Attachment of the aryl substituent to the 8-position of dG
can also increase nucleobase sensitivity to acid-catalyzed
deglycosylation.7 The acid-catalyzed hydrolysis of dG proceeds
by a stepwise mechanism.8 The first step involves protonation
at N7; the pKa for N7H+-dG is 2.34.9 The second step is rate-
limiting and involves unimolecular cleavage of the glycosidic
bond to release protonated guanine as a good leaving group.
Direct attachment of the aryl ring to afford the C-linked 8-aryl-
dG adduct has little impact on protonation at N7.7 However,
removal of the sugar moiety in the second step releases steric
strain provided by the bulky aryl substituent to increase the rate
of depurination. Electron-withdrawing substituents attached to
the 8-aryl moiety also increase the rate of hydrolysis due to
stabilization of the developing negative charge at N9 during
glycosidic bond cleavage.7
The limitations of the Suzuki cross-coupling approach
prompted our laboratory to seek a solid-phase assisted strategy
for incorporating 8-aryl-dG adducts into oligonucleotide
To study the structural, photophysical and biological
properties of 8-aryl-dG adducts in oligonucleotide substrates,
our initial goal is to incorporate them into the G3-site of the 12-
mer sequence 5′-CTCG1G2CG3CCATC (G3 = 8-aryl-dG),
Received: November 8, 2013
Published: January 3, 2014
© 2014 American Chemical Society
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The Journal of Organic Chemistry
Article
Scheme 1. Synthesis of Phosphoramidites
substrates. Inspired by recent efforts of the Yan laboratory,14
the 2,7-dimethylpixyl (DMPx) protecting group was deemed an
attractive alternative to the widely used 5′-O-DMT. The DMPx
group is more acid-labile than DMT15 (because of the release
of an aromatic carbocation versus a benzylic carbocation) and
can be efficiently removed using 0.5% DCA in dry dichloro-
methane (DCM).14,15 This suggested that the DMPx group
may offer a general protocol for efficient solid-phase synthesis
of oligonucleotides containing acid-sensitive 8-aryl-dG adducts.
In this paper we present our efforts to optimize solid-phase
synthesis of NarI substrates containing 8-aryl-dG adducts and
demonstrate the utility of the 5′-O-DMPx protecting group for
efficient synthesis of oligonucleotide substrates containing acid-
sensitive 8-aryl-dG adducts.
Attachment of the electron-withdrawing cyano group to afford
CNPhdG generates a nucleoside with donor−acceptor character.
Biphenyl systems with D−A character produce charge-transfer
(CT) states in the emission spectra that are very sensitive to
solvent polarity,19 thereby permitting the use of fluorescence
spectroscopy to help define adduct conformation within duplex
Q
DNA. The bulky 8-quinolyl-dG derivative dG is a model for
C-linked naphthalene-dG adducts that when compared to PhdG
permits assessment of aryl ring size in promoting mutagenicity.
The electron-withdrawing ring nitrogen in QdG also provides
D−A character.
Hydrolysis Kinetics. Our previous study on acid-catalyzed
hydrolysis of 8-phenyl-dG adducts bearing different para and
ortho substituents showed that all adducts are more prone than
dG to acid-catalyzed deglycosylation.7 8-Phenyl-dG adducts
bearing o-substituents underwent hydrolysis at a significantly
slower rate than their p-substituent counterparts. These results
suggested that relief of steric strain upon sugar removal played a
key role in the observed rate constants. For p-adducts, a
decrease in twist angle of 40° was observed upon removal of
the sugar moiety to generate the planar nucleobase. However,
for o-adducts, such as 8-o-CH3Ph-dG, the neutral nucleobase is
also highly twisted so the relief of strain is diminished upon
sugar removal, and hence the rate of hydrolysis was also
reduced. The most acid-sensitive adducts were those bearing
electron-withdrawing p-substituents.7
RESULTS AND DISCUSSION
■
Nucleoside Adducts and Phosphoramidite Synthesis.
To test the utility of the 5′-O-DMPx group and draw
comparison to 5′-O-DMT protection, four representative 8-
aryl-dG adducts were synthesized using Suzuki cross-coupling
procedures16 and were converted into phosphoramidites using
standard protocols (Scheme 1). The nucleoside adducts chosen
for study serve different purposes. The 5-membered heteroaryl-
dG analogue (FurdG) has been employed to study probe
conformation in duplex DNA.6 Furan-decorated nucleobase
analogues serve as efficient fluorescent nucleobase mimics17
and are precursors for oxidative-promoted DNA cross-link
formation.18 The 8-phenyl-dG nucleoside (PhdG) is an
authentic DNA adduct produced by arylhydrazine carcinogens.2
In the present study the nucleoside adducts (FurdG, PhdG,
CNPhdG, and QdG) also possess very different sensitivity to acid-
catalyzed deglycosylation (Table 1). First order rate constants
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To test the efficiency of solid-phase DNA synthesis, the two
phosphoramidites 6a and 4a (Scheme 1) were used to initially
incorporate FurdG into a thymidine decamer (5′-T7XTT, X =
FurdG). This model decamer was employed because thymidine
is the most difficult nucleoside to detritylate.22 The reverse-
phase (RP) HPLC profiles of the synthesis (1 μmol-scale) are
shown in Figure 1. For synthesis using 4a (Figure 1a) and a
Table 1. Summary of First-Order Rate Constants (kobs) and
Half-Lives (t1/2) for Hydrolysis of 8-Aryl-dG Adducts
a
adduct
kobs (min−1), t1/2 (min)
kobs/kobs(dG)
FurdG
PhdG
CNPhdG
QdG
2.16 0.08, 0.321
0.79 0.008, 0.877
1.78 0.02, 0.389
0.22 0.002, 3.16
55.2
20.2
45.5
5.6
b
dG
0.0391, 17.7
1
a
Determined in 0.1 M aqueous HCl at 37 °C from the average of six
b
kinetic runs. Data for dG taken from ref 8.
(kobs) were determined using UV−vis by monitoring in 0.1 M
aqueous HCl at 37 °C the appearance of a peak corresponding
to the deglycosylated nucleobase as a function of time, as
previously described.7,20 Under these conditions dG undergoes
hydrolysis with a half-life (t1/2) of ∼18 min.8 Surprisingly, the
FurdG adduct was the most sensitive to acid and hydrolyzed 55
times faster than dG. Interestingly, the bulky quinolone adduct
(QdG) was the least sensitive to acid with a hydrolysis rate of
only 5.6-fold faster than dG. This suggested that preferential
protonation of the quinoline ring N atom (pKa 4.921), as
opposed to N7 of the nucleobase, helps protect the nucleoside
Q
from acid-catalyzed hydrolysis. Since dG was not particularly
sensitive to acid, its phosphoramidite containing 5′-O-DMPx
Figure 1. RP-HPLC traces of T7XTT (X = FurdG) synthesis using
phosphoramidites 4a (3% DCA deblock) (a) or 6a (0.5% DCA
deblock) (b), solid black trace, UV detection at 258 nm, dashed red
trace, FLD (λex = 310 nm, λem = 380 nm).
was not prepared (Scheme 1).
Optimization of DNA Synthesis. For optimization of
solid-phase DNA synthesis, the most acid-sensitive FurdG was
utilized. Initial experiments examined hydrolysis rates of FurdG
(1a), its N2-dimethylformamidyl derivative 2a, and the 5′-O-
DMT analogue (3a) (Scheme 1) in DCA as a function of acid
concentration (3, 1, and 0.5% DCA) and solvent (H2O vs
CH3CN; hydrolysis product FurG is not soluble in DCM, which
limited the use of DCM for determination of rate data). The
rate data (Table 2) demonstrated the impact of DCA
deblock solution of 3% DCA in DCM (50 s detritylation time,
continuous delivery to the column) the RP-HPLC trace
suggested that full length decamer was prepared and eluted at
∼22 min. This peak contained the characteristic fluorescent
properties of the modified nucleobase FurdG (dashed red trace
is with fluorescence detection (FLD)). However, a dominant
peak was also present at 13.6 min that lacked fluorescence. This
peak either resulted from incomplete coupling of 4a or acid-
promoted depurination of FurdG followed by hydrolysis of the
abasic site during ammonolysis.14 Synthesis of the truncated
strand 5′-DMT-TXTT (Supporting Information, Figure S1)
showed that coupling efficiency was not the problem. For the
synthesis of T7XTT using 6a, normal deblock (3% DCA) and
commercially available 5′-O-DMT thymidine phosphoramidites
were employed prior to coupling with 6a. At this point the
synthesis was halted, and the deblock solution was changed to
0.5% DCA in DCM. The synthesis was then allowed to proceed
to completion using 6a and the corresponding 5′-O-DMPx
phosphoramidite of T (9b, Scheme 1), which was also
prepared, as this amidite is not commercially available. HPLC
analysis (Figure 1b) showed the fluorescent peak for full length
product. Importantly, the use of 0.5% DCA deblock eliminated
formation of the decomposition peak at 13.6 min.
Table 2. Summary of First-Order Rate Constants (kobs) and
Half-Lives (t1/2) for Hydrolysis of FurdG and Its Derivatives
b
adduct
solvent
3% DCA
1% DCA
0.5% DCA
a
1a
H2O
0.40, 1.7
0.35, 2.0
0.84, 0.82
0.85, 0.81
0.91, 0.76
0.80, 0.9
0.21, 3.3
0.15, 4.6
0.40, 1.7
0.114, 6.1
0.40, 1.7
0.097, 7.1
0.12, 5.9
0.06, 11.4
0.22, 3.15
0.042, 16
0.22, 3.15
0.041, 17
a
a
CH3CN
a
2a
3a
H2O
CH3CN
a
H2O
CH3CN
a
b
Contains 0.25% DMSO. kobs (min−1), t1/2 (min) determined by
UV−vis at 21 °C from the average of six kinetic runs; errors <5%
concentration on hydrolysis rate and the need for dry solvents
as rates in H2O were ∼5-fold faster than rates in CH3CN. For
the 5′-O-DMT analogue (3a) a t1/2 value of 17 min was
determined in CH3CN with 0.5% DCA at 21 °C, while the
corresponding value in 3% DCA was 0.9 min (19.5-fold
reduction in rate of hydrolysis). The rate data also
demonstrated the protecting effect of the N2-dimethylforma-
midyl group in CH3CN. In both 1 and 0.5% DCA, hydrolysis
rates for 2a were slower compared to the unprotected FurdG
(1a). Since 3% DCA is required to efficiently detritylate 5′-O-
DMT, the rate data suggested that utility of the 5′-O-DMPx
protecting group coupled with a deblock solution of 0.5% DCA
in dry DCM could enable efficient incorporation of FurdG into
oligonucleotides.
Synthesis of NarI Substrates. To incorporate FurdG into
the 12-mer NarI recognition sequence 5′-CTCGGCXCCATC
(NarI(12)) using the 5′-O-DMPx phosphoramidite 6a, 5′-O-
DMPx phosphoramidites of dG (7b) and dC (8b) were also
synthesized (Scheme 1). Figure 2 shows HPLC traces for the
synthesis of NarI(12) using 4a and commercially available 5′-
O-DMT phosphoramidites (Figure 2a) versus the synthesis
using 6a, 7b, and 8b (with 0.5% DCA in DCM after the
coupling of 6a). The synthesis of NarI(12) using 4a produced
full length product6 but contained hydrolysis peaks (Figure 2a)
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ammonium hydroxide. Any attempts to purify the modified
oligonucleotide with the final 5′-O-DMPx group attached (to
serve as a hydrophobic handle) resulted in degradation of the
oligonucleotide through exposure of the 8-aryl-dG adduct to
both acid and water using commercially available solid-phase
extraction cartridges for DNA purification. A number of
examples in the literature have reported use of solid-phase
DNA synthesis for incorporation of 8-aryl-dG adducts into
oligonucleotides using standard 5′-O-DMT phosphoramidites.
For probe development, these include insertion of 8-pyridyl-
dG,5 where preferential protonation of the pyridyl group (pKa
5.221) would be expected to protect the base from
depurination. In other examples the aryl ring is separated
from the dG nucleobase by a vinyl5b,23 or alkynyl group.24 This
reduces steric strain between the aryl ring and sugar moiety,
thereby diminishing the sensitivity of the nucleoside to acid-
promoted hydrolysis.7 For DNA adduction by chemical
mutagens, nucleophilic aryl radicals also attach to the 8-
position of 2′-deoxyadenosine (dA).2 In terms of sensitivity to
acid-catalyzed deglycosylation, dA is 2.2 times more reactive
than dG at 30 °C.25 No information is available on the
hydrolytic stability of 8-aryl-dA adducts, and very little has been
reported on incorporation of 8-aryl-dA adducts into oligonu-
cleotide substrates. A notable exception for probe development
is the click-chemistry-based 8-triazole-dA,26 which again
contains ring nitrogen atoms in the 8-aryl-substituent. Chemical
mutagens such as PAHs lack ring nitrogen atoms and attach
directly to the 8-position of purine nucleosides, making them
acid-sensitive. PAHs are ubiquitous environmental pollutants
and are present in cigarette smoke and in vehicle exhaust
condensate.27 Currently, very little is known about the
biological implications of C-linked 8-purine adducts produced
by PAHs. We plan to use the solid-phase assisted synthesis
strategies reported herein to prepare a variety of adducted
NarI(22) substrates to assess the differing propensities of 8-
aryl-dG adducts to induce mutagenesis when the adducted
DNA is replicated.
Figure 2. RP-HPLC traces of 5′-CTCGGCXCCATC (X = FurdG)
synthesis using phosphoramidites 4a (3% DCA deblock) (a) or 6a
(0.5% DCA deblock) (b), solid black trace, UV detection at 258 nm,
dashed red trace, FLD (λex = 310 nm, λem = 380 nm).
and afforded an isolated yield of 49 nmol for NarI(12). Using
6a, 7b, and 8b, the hydrolysis peaks were eliminated (Figure
2b), and an isolated yield of 190 nmol was obtained, for a 4-fold
increase in yield of NarI(12). The 5′-O-DMPx phosphor-
amidites 6b and 6c (Scheme 1) were then employed to
incorporate PhdG and CNPhdG into the G3-site of NarI(12). A
longer 22-mer substrate 5′-CTCGGCXCCATCCCTTA-
CGAGC (NarI(22)) suitable for primer-extension assays
using DNA polymerases was also prepared. For these NarI
sequences ESI-MS spectra are available in the Supporting
Information, and the results of the ESI-MS analysis and isolated
yields of oligonucleotide product are summarized in Table 3.
Table 3. Summary of Yields and ESI-MS Data for 8-Aryl-dG-
Modified NarI Oligonucleotides
a
yield
calc
mass
exptl m/z
b
exptl
mass
CONCLUSIONS
ODN
X
(nmol)
(ESI−)
■
c
NarI(12)
FurdG
FurdG
PhdG
49
190
216
170
147
42
3647.6
3647.6
3657.6
3682.6
3708.6
6695.2
6705.2
6756.2
M6− = 607.0
M7− = 520.2
M9− = 405.4
M7− = 525.1
M7− = 529.0
M11− = 607.7
M11− = 608.6
M9− = 749.7
3648.0
3648.4
3657.6
3682.7
3710.0
6695.7
6705.6
6756.3
The results of our studies demonstrate the utility of 5′-O-2,7-
dimethylpixyl (DMPx) as a protecting group of 8-aryl-dG
phosphoramidites for solid-phase assisted synthesis of
oligonucleotide substrates. 8-Aryl-dG adducts are produced
by a number of chemical mutagens that undergo bioactivation
to afford aryl radical species that attach to the 8-position of
purine nucleosides. These adducts also possess impressive
fluorescent properties that make them desirable for use as
fluorescent nucleobase probes. Unfortunately, 8-aryl-dG
adducts can be sensitive to acid-catalyzed deglycosylation,
which limits the utility of standard solid-phase synthesis using
5′-O-DMT protection and a deblock solution consisting of 3%
dichloroacetic acid (DCA) in dry dichloromethane (DCM).
The 5′-O-DMPx group can be efficiently removed using 0.5%
DCA in DCM, which limits exposure of the modified 8-aryl-dG
lesion to acid during solid-phase synthesis. Using 8-furyl-dG
CNPhdG
c
QdG
NarI(22)
FurdG
PhdG
53
c
QdG
26
a
Isolated yield from 1 μmol-scale synthesis after oligonucleotides were
purified via RP-HPLC and quantified using ε260 for the corresponding
unmodified oligonucleotide (NarI(12) = 102,100; NarI(22) = 185,700
b
c
M−1 cm−1). Measured m/z from mass spectrum. Oligonucleotide
synthesized with 5′-O-DMT protection, while all others were
synthesized using 5′-O-DMPx protection.
Applications for the 5′-O-DMPx Protecting Group.
The results of our studies demonstrate the utility of the 5′-O-
DMPx protecting group for solid-phase DNA synthesis of
oligonucleotide substrates containing acid-sensitive 8-aryl-dG
adducts. For the NarI substrates reported in Table 3 it was
critical to remove the final 5′-OH protecting group on-column
(using acidic deblock in dry DCM) prior to cleavage of the
oligonucleotide from the solid support using aqueous
(
FurdG) as a representative acid-sensitive 8-aryl-dG adduct (55-
fold more reactive than dG in 0.1 M HCl at 37 °C), our results
demonstrate that FurdG is much more stable in 0.5% DCA in
CH3CN versus 3% DCA (19.5-fold reduction in hydrolysis
rate). This increased stability provides a 4-fold increase in
oligonucleotide solid-phase synthesis using 5′-O-DMPx phos-
phoramidites. Thus, the 5′-O-DMPx protection strategy
provides a more general method for insertion of 8-aryl-dG
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using 2 mL of 30% ammonium hydroxide solution at 55 °C for 12 h
and purified by RP-HPLC.
adducts into oligonucleotide substrates and does not alter the
reagents used for the traditional 5′-O-DMT solid-phase
synthesis. These results demonstrate a real application for 5′-
O-DMPx protection.
Oligonucleotide Purification. The 8-aryl-G-modified NarI(12)
and NarI(22) 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, a 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 carried out at 50 °C using a 5 μm
reversed-phase (RP) semipreparative C18 column (100 × 10 mm)
with a flow rate of 3.5 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).
Collected DNA samples were lyophilized to dryness and redissolved in
EXPERIMENTAL SECTION
■
Materials and Methods. Boronic acids, Pd(OAc)2, 3,3′,3″-
phosphinidynetris-(benzenesulfonic acid) trisodium salt (TPPTS),
DCA and other commercial compounds were used as received.
Pyridine, DCM, and acetonitrile were distilled over CaH2 and stored
under nitrogen. The synthesis of 8-bromo-2′-deoxyguanosine (8-Br-
dG) was performed as described previously.6 Suzuki cross-coupling
reactions of boronic acids with 8-Br-dG to afford FurdG,6 PhdG,7
CNPhdG,7 and QdG were performed as described previously by Western
and co-workers.16 NMR spectra were recorded on 300, 400, or 600
MHz spectrometers in either DMSO-d6, CDCl3, CD2Cl2, or CD3OD
referenced to TMS (0 ppm) or the respective solvent. All UV−vis
were recorded with baseline correction and stirring using 10 mm light
path quartz glass cells. 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 μL/min detecting positive
ions.
18.2 MΩ water for quantification by UV−vis measurement using ε260
.
Extinction coefficients were obtained from the following Web site:
http://www.idtdna.com/analyzer/applications/oligoanalyzer. The 8-
aryl-G modified oligonucleotides were assumed to have the same
extinction coefficient as the natural NarI(12) (102 100 M−1 cm−1) and
NarI(22) (185 700 M−1 cm−1) oligonucleotides.
MS Analysis of Oligonucleotides. MS experiments for DNA
identification were conducted on a quadrupole ion trap SL
spectrometer. Masses were acquired in the negative ionization mode
with an electrospray ionization source. Oligonucleotide samples were
prepared in 90% Milli-Q filtered water/10% methanol containing 0.1
mM ammonium acetate. Full scan MS spectra were obtained by direct
infusion at a rate of 5−10 μL/min.
Phosphoramidite Synthesis. Synthesis of the phosphoramidite
with 5′-O-DMT protection for FurdG (4a)6 has been previously
published using the synthetic strategy outlined in Scheme 1.
Unmodified dmf-dG, ac-dC, and T nucleosides were purchased and
protected with the 5′-O-DMPx protocol described below before
conversion to their corresponding phosphoramidite (Scheme 1).
i. N2 Protection. 8-Aryl-2′-deoxyguanosine (3.3 mmol) was placed
in a round-bottom flask (RBF) and reverse filled with argon. Dry DMF
(15 mL) was added, followed by dimethylformamide-diethyl-acetal
(2.7 mL, 13.5 mmol), and the mixture was allowed to stir until
completion overnight at room temperature. The reaction mixture was
then evaporated to dryness, and the solid washed with MeOH and
dried to yield product.
Kinetics. The kinetic study was carried out using a UV−vis
spectrophotometer equipped with a constant temperature water bath.
Hydrolysis reactions were followed by monitoring formation of the
deglycosylated nucleobase at its absorption maximum, as previously
outlined.7,20 For hydrolysis of the nucleoside adducts FurdG, PhdG,
CNPhdG, and QdG in 0.1 M HCl at 37 °C, the reactions were initiated
by injection of 20 μL of a 4 mM stock solution (DMSO) of the
nucleoside adduct into a Teflon-capped 10 mm UV cell containing
1980 μL of aqueous 0.1 M HCl that had been incubating at 37 °C for
15 min. For hydrolysis of FurdG (1a), N2-(dimethylformamidyl)-8-(2″-
furyl)-2′-deoxyguanosine (2a), and 5′-O-(4,4′-dimethoxytrityl)-N2-
(dimethylformamidyl)-8-(2″-furyl)-2′-deoxyguanosine (3a) in water
or dry CH3CN containing 3, 1, or 0.5% DCA, stock solutions (4 mM)
of 1a, 2a, and 3a were prepared in DMSO for kinetic measurements in
water. The same DMSO stock solutions of 1a and 2a were employed
for reactions carried out in dry CH3CN, while a separate stock solution
of 3a in CH3CN (4 mM) was used for kinetic measurements of 3a in
CH3CN containing various concentrations of DCA. Reactions were
initiated as outlined for the kinetic runs in 0.1 M HCl. All
measurements were conducted in parallel using the multicell changer,
with six first-order rate constant values for hydrolysis obtained for each
nucleoside in each set of conditions to allow for determination of
mean standard deviation.
Oligonucleotide Synthesis. Oligonucleotide synthesis of T7XTT,
5′-DMT-TXTT (X = FurdG) and 8-aryl-dG modified NarI(12) (5′-
CTCGGCXCCATC) and NarI(22) (5′-CTCGGCXCCATC-
CCTTACGAGC) with X = 2FurdG, PhdG, CNPhdG, or QdG was carried
out on a 1 μmol scale using an automatic DNA synthesizer. For all
modified phosphoramidites with 5′-O-DMT protection, synthesis was
carried out using standard β-cyanoethylphosphoramidite chemistry
(unmodified phosphoramidites (bz-dA-CE, ac-dC-CE, dmf-dG-CE,
and dT-CE), activator (0.25 M 5-(ethylthio)-1H-tetrazole in CH3CN),
oxidizing agent (0.02 M I2 in THF/pyridine/H2O, 70/20/10, v/v/v),
deblock (3% DCA in dry DCM), cap A (THF/2,6-lutidine/acetic
anhydride), cap B (methylimidazole in THF), and solid supports (5′-
DMT-dC(Ac), 1000 Å controlled pore glass (CPG), or 5′-DMT-dT
1000 Å CPG), as outlined previously.6 For phosphoramidites with 5′-
O-DMPx protection, standard DNA synthesis conditions and 5′-O-
DMT phosphoramidites were utilized prior to coupling with the
modified phosphoramidite. At this point the synthesis was halted, and
the normal deblock solution (3% DCA in dry DCM) was replaced
with 0.5% DCA in dry DCM. The synthesis was then allowed to
proceed to completion using 5′-O-DMPx phosphoramidites (modified
(6a−c), and unmodified (7b, 8b, 9b), Scheme 1). Following synthesis,
oligonucleotides were cleaved from the solid support and deprotected
ii. DMT Protection. N2-(Dimethylformamidyl)-8-aryl-2′-deoxygua-
nosine (2.7 mmol) was coevaporated from dry pyridine (3 × 5 mL) in
a RBF. The RBF was then fitted with a dropping funnel and reverse
filled with argon, and 7 mL of dry pyridine was added to the RBF.
DMT-Cl (1.28 g, 3.78 mmol) was placed in the dropping funnel and
dissolved in 3 mL of dry pyridine. The 3 mL of DMT-Cl/pyridine
solution was then allowed to add dropwise over 30 min. The reaction
was allowed to stir at room temperature under argon and was
monitored by TLC. Upon completion, the mixture was diluted with
DCM (10 mL) and washed with water (2 × 10 mL). TEA (1 mL) was
added, and the mixture was evaporated to yield an oil. The oil was then
loaded onto a silica column and eluted with MeOH:CH2Cl2:TEA
(5:90:5) to afford the product.
iii. DMPx Protection. N2-(Dimethylformamidyl)-8-aryl-2′-deoxy-
guanosine (1.67 mmol) was placed in a RBF, coevaporated from dry
pyridine (3 × 5 mL), and reverse filled with argon. The RBF was then
fitted with a dropping funnel containing DMPx-Cl (0.68 g, 1.91
mmol). Dry pyridine (10 mL) was then added to the RBF as well as 4
mL of dry pyridine to the dropping funnel. The RBF was then placed
in an ice bath and allowed to cool to 0 °C. The DMPx-Cl/pyridine
solution was then delivered to the RBF over 30 min, and the reaction
was monitored by TLC. Upon completion, the mixture was diluted
with ethyl acetate (10 mL) and washed with water (2 × 10 mL). TEA
(1 mL) was added, and the mixture was evaporated to dryness. The
solid was then recrystallized by first dissolving in DCM (3 mL),
followed by the addition of hexanes (10 mL). The resulting suspension
was filtered, and if the product was sufficiently pure it was carried
forward; otherwise it was loaded onto a silica column and eluted with
MeOH:DCM:TEA (5:90:5) to afford the product.
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Article
iv. Phosphoramidite Reaction. The 5′-O-DMT(DMPx)-N2-
(dimethylformamidyl)-8-aryl-2′-deoxyguanosine (0.706 mmol) was
coevaporated from dry THF (3 × 5 mL), reverse filled with argon,
and dissolved in 10 mL of dry, degassed DCM. To this was added dry,
degassed TEA (0.4 mL, 2.83 mmol) and 2-cyanoethyl N,N-
diisopropylchlorophosphoramidite (0.24 mL, 1.06 mmol). The
reaction was monitored via TLC and, upon completion (20−40
min), was washed successively with saturated, degassed sodium
bicarbonate solution. The organic phase was separated, dried with
Na2SO4, and either purified by recrystallization by initially dissolving in
DCM and dripping into hexanes at −78 °C or loaded onto a flash
chromatography column eluting with 92:5:3 DCM:MeOH:TEA.
Phosphoramidites were isolated as their corresponding diastereomers,
which were typically off-white foams.
to a 95% yield: mp 218−220 °C; 1H NMR (300 MHz, DMSO-d6) δ =
11.46 (s, 1H), 8.49 (s, 1H), 7.65−7.53 (m, 5H), 6.09 (t, J = 6 Hz, 1H),
5.21 (d, J = 6.0 Hz, 1H), 4.88 (t, J = 6.0 Hz, 1H), 4.42 (bs, 1H), 3.81
(bs, 1H), 3.67 (m, 1H), 3.61 (m, 1H) 3.33 (m, 1H), 3.22 (s, 3H), 3.04
(s, 3H) 2.08 (s, 1H); 13C NMR (300 MHz, DMSO-d6) δ = 158.1,
157.5, 156.8, 150.7, 148.1, 130.1, 129.5, 129.1, 128.7, 120.1, 87.7, 84.8,
71.0, 62.0, 40.8, 37.0, 34.6.
5′-O-DMPx-N2-(dimethylformamidyl)-8-phenyl-2′-deoxy-
guanosine (5b). Isolated 0.68 g (1.0 mmol) as a white powder
corresponding to a 60% yield: mp 195−198 °C; 1H NMR (600 MHz,
CD2Cl2) δ = 9.11 (bs, 1H), 8.12 (s, 1H), 7.70 (d, J = 7 Hz, 2H), 7.43−
7.38 (m, 3H), 7.31 (d, J = 7.4 Hz, 2H), 7.21 (t, J = 7.4 Hz, 2H), 7.13−
7.08 (m, 2H), 7.02 (m, 2H), 6.93 (m, 3H), 6.24 (t, J = 6.4 Hz, 1H),
4.43 (m, 1H), 4.14 (m, 1H), 3.52 (t, J = 9.2 Hz, 1H), 3.34 (dd, J = 2.8,
9.4 Hz, 1H), 3.11 (m, 1H), 3.07 (s, 3H), 2.85 (s, 3H), 2.20 (s, 3H),
2.11 (s, 3H) 2.10 (m,1H); 13C NMR (600 MHz, CD2Cl2) δ = 158.2,
158.0, 156.1, 151.6, 149.9, 149.8, 149.7, 149.6, 133.4, 133.3, 130.9,
130.5, 130.4, 130.1, 129.7, 129.5, 129.0, 128.4, 127.0, 126.5, 123.0,
121.0, 116.7, 116.2, 86.7, 85.0, 76.3, 73.4, 65.5, 41.9, 38.5, 35.5, 21.1,
v. Synthesis of DMPx-Cl. Following a published protocol,15 tolyl
ether (1 g, 0.01 mol), benzoic acid (0.75 g, 0.01 mol), zinc chloride
(2.0 g, 0.02 mol), and phosphorus oxychloride (1.5 mL, 0.02 mol)
were heated at 95 °C for 2 h. The mixture was cooled to room
temperature and ethyl acetate (2.5 mL) was added to form a
suspension. The suspension was poured into stirring water at room
temperature and heated under reflux for 15 min and then allowed to
cool to room temperature overnight. Mixture was filtered and washed
with water. The damp cake was suspended with 30 mL of methanol
and stirred to boil for 2 or 3 min. The resultant suspension was
allowed to cool to room temp over a period of 3 h and was then
filtered, washed with methanol, and dried to give DMPx-OMe as a
+
20.7; HRMS calcd for C40H39N6O5 [M + H+] 683.2982; found
683.2985.
3′-O-[(2-Cyanoethoxy)(diisopropylamino)phosphino]-5′-O-
DMPx-N2-(dimethylformamidyl)-8-phenyl-2′-deoxyguanosine
(6b). Isolated as an off-white foam in 80% yield (0.5 g, 0.57 mmol):
1H NMR (300 MHz, CD2Cl2) δ = 9.16 (bs, 1H), 8.19−8.11 (m, 1H),
7.61−6.86 (m, 16H), 6.13 (m, 1H), 4.52 (m, 1H), 4.11 (m, 1H), 3.90
(m, 2H), 3.80−3.51 (m, 4H), 3.46 (s, 3H), 3.43 (s, 3H), 2.68−2.50
(m, 3H), 2.15 (m, 1H), 2.03 (s, 3H), 2.00 (s, 3H), 1.05−0.99 (m,
12H); 13C NMR (300 MHz, CD2Cl2) δ = 158.4, 158.2, 156.3, 151.8,
150.1, 150.0, 149.9, 149.8, 133.54, 133.49, 131.1, 130.7, 130.6, 130.3,
129.8, 129.7, 129.2, 128.6, 127.2, 126.7, 123.2, 123.19, 121.2, 116.9,
116.4, 86.9, 85.1, 76.5, 73.6, 65.7, 58.5, 43.9, 43.85, 42.1, 38.7, 35.6,
23.1, 23.0, 22.9, 21.3, 20.9, 20.2; 31P NMR δ = 148.74, 148.67; HRMS
calcd for C49H56N8O6P+ [M + H+] 883.4055; found 883.4060.
N2-(Dimethylformamidyl)-8-(4″-cyanophenyl)-2′-deoxygua-
nosine (2c). Isolated 1.33 g (3.14 mmol) of a yellow powder
corresponding to a 95% yield: mp 179−182 °C; 1H NMR (600 MHz,
DMSO-d6) δ = 8.48 (s, 1H), 8.00 (d, J = 8.3 Hz, 2H), 7.85 (d, J = 8.3
Hz, 2H), 6.11 (t, J = 7.7 Hz, 1H), 5.75 (bs, 1H), 5.30 (bs, 1H), 4.44
(m, 1H), 3.86 (m, 1H), 3.67 (dd, J = 3.5, 12 Hz, 1H), 3.55 (dd, J = 3.8,
11.9 Hz, 1H), 3.24 (m, 1H), 3.09 (s, 3H), 3.00 (s, 3H), 2.07 (m, 1H);
13C NMR (600 MHz, DMSO-d6) δ = 157.7, 151.6, 145.0, 142.5, 141.9,
1
white solid in 64% yield: mp 135−137 °C; H NMR (300 MHz,
CDCl3) δ = 7.57−7.53 (m, 2H), 7.45−7.40 (m, 2H), 7.35−7.33
(m,1H), 7.24 (m, 4H), 7.14 (s, 2H), 3.09 (s, 3H), 2.39 (s, 6H); 13C
NMR (300 MHz, CDCl3) δ =149.9, 149.3, 132.7, 130.1, 129.9, 129.4,
129.3, 128.0, 126.6, 126.57, 122.7, 116.1, 76.3, 51.2, 21.0. DMPx-OMe
(2.0 g, 6.9 mmol) was azeotroped with dry toluene (2 × 10 mL), and
the residue was dissolved in dry toluene (10 mL). Acetyl chloride (5.8
mL, 81.8 mmol) was added, and the reaction was left to stir for 16 h at
room temperature under argon. Upon completion the reaction was
evaporated to dryness and coevaporated with dry toluene (3 × 10
mL). The product was further dried under a vacuum at 40 °C for 3 h
to afford DMPx-Cl in quantitative yield as an orange/brown solid: mp
108−109 °C.1H NMR (300 MHz, CD2Cl2) δ = 7.63−7.60 (m, 2H),
7.45−7.42 (m, 2H), 7.29−7.25 (m, 4H), 7.05 (s, 2H), 2.26 (s, 6H);
13C NMR (300 MHz, CDCl3) δ = 149.3, 143.5, 134.4, 133.1, 130.4,
129.2, 128.5, 125.3, 117.1, 85.7, 21.0.
5′-O-DMPx-N2-(dimethylformamidyl)-8-(furan-2-yl)-2′-deox-
yguanosine (5a). Isolated 0.76 g (1.13 mmol) as an off-white powder
corresponding to a 68% yield: mp 225−227 °C; 1H NMR (600 MHz,
CD2Cl2) δ = 8.99 (bs, 1H), 8.12 (s, 1H), 7.44 (d, J = 1.0 Hz, 1H),
7.28−6.87 (m, 12H), 6.63 (dd, J = 6.0 Hz, 7.8 Hz, 1H), 6.50 (m, 1H),
4.55, (m, 1H), 4.14 (m, 1H) 3.45 (m,1H), 3.31 (m,1H), 3.15 (m,1H),
3.05 (s, 3H), 2.86 (s, 3H), 2.26 (m, 1H), 2.19 (s, 3H), 1.84 (s, 3H);
13C NMR (600 MHz, CD2Cl2) δ = 157.84, 157.81, 156.2, 151.1, 149.7,
135.0, 133.0, 132.6, 129.7, 128.1, 121.3, 118.6, 111.4, 88.3, 85.2, 71.3,
+
62.3, 40.5, 37.3, 34.4; HRMS calcd for C20H22N7O4 [M + H+]
424.1733, found 424.1724.
5′-O-DMPx-N2-(dimethylformamidyl)-8-(4″-cyanophenyl)-
2′-deoxyguanosine (5c). Isolated 0.87 g (1.23 mmol) of a yellow
powder corresponding to a 74% yield: mp 170−173 °C (d); 1H NMR
(600 MHz, DMSO-d6) δ = 11.37 (bs, 1H), 7.99 (s, 1H), 7.95 (d, J =
8.2 Hz, 2H), 7.83 (d, J = 8.2 Hz, 2H), 7.19−7.04 (m, 7H), 6.87−6.83
(m, 2H), 6.75−6.72 (m, 2H), 6.05 (m, 1H), 5.24 (m, 1H), 4.40 (m,
1H), 4.03 (m, 1H), 3.4 (t, J = 9.5 Hz, 1H), 3.26 (m, 1H), 3.08 (m,
1H), 3.01 (s, 3H), 2.78 (s, 3H), 2.11 (s, 3H), 2.04 (m, 1H), 1.62 (s,
3H); 13C NMR (600 MHz, DMSO-d6) δ =157.6, 157.4, 156.4, 150.5,
149.3, 148.5, 148.4, 146.2, 134.6, 132.6, 132.2, 131.7, 130.1, 129.7,
129.5, 128.5, 128.4, 127.9, 126.4, 125.5, 122.3, 122.26, 120.7, 118.6,
116.2, 115.5, 111.7, 86.7, 84.6, 74.9, 71.3, 64.7, 40.9, 40.4, 37.1, 34.7,
149.6, 149.4, 145.3, 144.0, 140.1, 133.22, 133.17, 130.4, 130.3, 129.5,
129.3, 128.2, 126.9, 126.4, 122.9, 122.8, 121.2, 116.5, 116.1, 112.9,
112.1, 86.3, 84.7, 76.2, 73.3, 65.3, 46.5, 41.7, 39.0, 35.3, 20.9, 20.5;
+
HRMS calcd for C38H37N6O6 [M + H+] 673.2776, found 673.2775.
3′-O-[(2-Cyanoethoxy)(diisopropylamino)phosphino]-5′-O-
DMPx-N2-(dimethyl-formamidyl)-8-(furan-2-yl)-2′-deoxygua-
nosine (6a). Isolated as an off-white foam in 72% yield (0.44 g, 0.51
1
+
20.4, 19.9; HRMS calcd for C41H38N7O5 [M + H+] 708.2929; found
mmol): H NMR (300 MHz, CD2Cl2) δ = 9.20 (bs,1H), 8.21 (m,
1H), 7.54 (m, 1H), 7.42−6.87 (m, 12H), 6.67 (m, 1H), 6.56 (m, 1H),
4.81 (m, 1H), 4.27 (m, 1H), 3.80−3.34 (m, 6H), 3.19−3.10 (m, 4H),
2.92 (s, 3H), 2.56 (m, 2H), 2.42 (m,1H), 2.21 (m, 3H), 1.89 (m, 3H),
1.19−1.09 (m, 12H); 13C NMR (300 MHz, CD2Cl2) δ = 158.0, 157.9,
156.3, 151.2, 149.8, 149.7, 148.6, 145.5, 144.1, 140.3, 133.4, 133.3,
130.5, 130.4, 129.6, 129.5, 127.0, 123.0 122.9, 121.3, 117.7, 116.2,
113.0, 112.2, 86.5, 84.8, 76.3, 73.4, 65.4, 57.9, 46.6, 43.3, 41.9, 39.1,
35.4, 23.4, 23.2, 21.0, 20.6, 20.2; 31P NMR (300 MHz, CD2Cl2) δ =
149.3, 149.0; HRMS calcd for C47H54N8O7P+ [M + H+] 873.3848;
found 873.3852.
708.2932.
3′-O-[(2-Cyanoethoxy)(diisopropylamino)phosphino]-5′-O-
DMPx-N2-(dimethylformamidyl)-8-(4″-cyanophenyl)-2′-deoxy-
guanosine (6c). Isolated as a white foam in 65% yield (0.42 g, 0.46
1
mmol): H NMR (300 MHz, CD3OD) δ = 8.21 (s, 1H), 7.95−7.84
(m, 4H), 7.29−6.78 (m, 11H), 6.15 (m, 1H), 4.95 (m, 1H), 4.37−4.20
(m, 1H), 3.84−3.47 (m, 6H), 3.30−3.17 (m,1H), 3.16 (m, 3H), 2.97
(m, 3H), 2.62 (m,2H), 2.16 (m, 3H), 2.15 (m,1H), 1.75 (m, 3H), 1.30
(d, J = 5.2 Hz, 6H), 1.27 (d, J = 5.2 Hz, 6H); 13C NMR (300 MHz,
CD3OD) δ = 160.3, 159.0, 158.1, 152.5, 151.5, 150.8, 150.5, 149.0,
135.4, 133.9, 133.7, 133.4, 131.4, 131.2, 130.9, 130.6, 128.8, 127.6,
127.4, 127.3, 124.0, 123.8, 121.7, 119.5, 119.3, 117.3, 116.8, 114.5,
N2-(Dimethylformamidyl)-8-phenyl-2′-deoxyguanosine
(2b).28 Isolated 1.25 g (3.14 mmol) of a white powder corresponding
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87.5, 86.4, 77.2, 70.0, 65.9, 60.3, 59.7, 59.5, 46.7, 46.65, 44.7, 44.6,
44.5, 42.0, 35.6, 25.1, 25.0, 23.2, 20.8, 20.3; 31P NMR δ = 149.40,
149.12; HRMS calcd for C50H55N9O6P+ [M + H+] 908.4007; found
908.4013.
2.37 (m,1H), 2.07 (m, 6H), 1.11−1.00 (m, 12H); 13C NMR (300
MHz, CD2Cl2) δ = 161.1, 161.0, 159.6, 153.0, 152.31, 152.28, 151.5,
135.8, 135.7, 133.2, 133.1, 132.3, 132.1, 130.8, 129.6, 129.3, 125.2,
125.0, 123.2, 119.0, 117.7 88.9, 86.3, 79.1, 75.4, 67.0, 57.2, 44.3, 43.8,
42.8, 38.0, 33.9, 23.74, 23.68, 22.7, 19.6; 31P NMR δ = 148.93, 148.72;
HRMS calcd for C43H52N8O6P+ [M + H+] 807.3748; found 807.3752.
5′-O-DMPx-N4-(acetyl)-2′-deoxycytidine (8a). Isolated 0.86 g
(1.55 mmol) of a white powder corresponding to a 93% yield: mp
8-(8″-Quinolyl)-2′-deoxyguanosine (QdG, 1d). Isolated 2.2 g
(5.6 mmol) of a yellow solid corresponding to a 73% yield: mp 215−
218 °C (d); 1H NMR (600 MHz, DMSO- d6) δ = 10.74 (s, 1H), 8.89
(d, J = 2.6 Hz, 1H), 8.50 (dd, J = 1.4, 8.3 Hz, 1H), 8.19 (dd, J = 1.0,
8.3 Hz, 1H), 7.92 (bs, 1H), 7.74 (t, J = 7.7 Hz, 1H), 7.61 (dd, J = 4.1,
8.3 Hz, 1H), 6.31 (bs, 2H), 5.51 (bs, 1H), 5.04 (bs, 1H), 4.90 (d, J =
4.1 Hz, 1H), 4.27 (bs, 1H), 3.55 (m, 2H), 3.41 (bs, 1H), 3.18 (bs,
1H), 2.09 (bs, 1H); 13C NMR (600 MHz, DMSO-d6) δ = 157.7,
157.2, 151.3, 150.5, 146.9, 146.3, 136.9, 132.8, 130.9, 129.7, 128.0,
126.6, 122.5, 120.8, 87.8, 85.5, 71.2, 62.4, 38.0; HRMS calcd for
1
115−118 °C (d); H NMR (300 MHz, CD2Cl2) δ = 9.03 (bs, 1H),
8.21 (d, J = 7.5 Hz, 1H), 7.36−7.11 (m, 10H), 6.92 (m, 2H), 6.22 (t, J
= 6.1 Hz, 1H), 4.35 (m, 1H), 4.06 (m, 1H), 3.24 (dd, J = 3.2, 10.6 Hz,
1H), 3.10 (dd, J = 4.1, 10.6 Hz, 1H), 2.78−2.70 (m, 1H), 2.22 (m,
10H); 13C NMR (300 MHz, CD2Cl2) δ = 170.6, 162.8, 155.6, 150.0,
149.9, 148.7, 144.6, 133.42, 133.37, 130.8, 129.5, 129.3, 128.3, 127.2,
126.8, 122.5, 122.2, 116.6, 96.3, 87.9, 87.0, 76.9, 71.9, 63.6, 42.4, 25.2,
+
C19H19N6O4 [M + H+] 395.1462; found 395.1468.
+
21.0; HRMS calcd for C32H32N3O6 [M + H+] 554.2294; found
N2-(Dimethylformamidyl)-8-(8″-quinolyl)-2′-deoxyguano-
sine (2d). Isolated 1.39 g (3.1 mmol) as a yellow powder
corresponding to a 94% yield: mp 188−192 °C; 1H NMR (300
MHz, DMSO-d6) δ = 11.45 (bs, 1H), 8.90 (m, 1H), 8.50 (m, 2H),
8.21 (d, J = 8.2 Hz, 1H), 7.95 (d, J = 6.7 Hz, 1H), 7.75 (t, J = 7.7 Hz,
1H), 7.62 (dd, J = 4.2, 8.2 Hz), 5.56 (bs, 1H), 4.97 (m, 1H), 4.89 (m,
1H), 4.30 (bs, 1H), 4.09 (m, 1H), 3.57−3.45 (m, 2H), 3.16−3.12 (m,
4H), 3.03 (s, 3H), 2.10 (bs, 1H); 13C NMR (300 MHz, DMSO-d6) δ
= 158.1, 157.4, 157.0, 151.1, 150.2, 146.7, 146.1, 136.6, 132.6, 130.5,
129.4, 127.7, 126.3, 122.0, 120.6, 87.5, 85.4, 70.9, 62.1, 48.6, 37.4, 34.5;
HRMS calcd for C22H23N7O4K+ [M + K+] 488.1449; found 488.1438.
5′-O-DMT-N2-(dimethylformamidyl)-8-(8″-quinolyl)-2′-deox-
yguanosine (3b). Isolated 1.12 g (1.49 mmol) of a yellow powder
corresponding to a 55% yield: mp 178−181 °C; 1H NMR (600 MHz,
DMSO-d6) δ = 11.39 (bs, 1H), 8.88 (bs, 1H), 8.50 (d, J = 7.8 Hz, 1H),
8.19 (m, 2H), 8.02 (bs, 1H), 7.78 (bs, 1H), 7.62 (m, 1H), 7.26 (m,
2H), 7.17−7.11 (m, 7H), 6.76 (m, 4H), 5.65 (bs, 1H), 5.08 (bs, 1H),
4.42 (bs, 1H), 3.70 (s, 6H) 3.61 (m, 1H), 3.21 (bs, 1H), 3.07 (m, 1H),
3.00 (m, 4H), 3.91 (s, 3H), 1.78 (bs, 1H); 13C (600 MHz, DMSO-d6)
158.0, 157.9, 157.6, 151.2, 150.2, 145.0, 136.7, 135.7, 135.6, 132.8,
130.5, 129.7, 129.4, 127.8, 127.6, 126.5, 122.1, 113.0, 112.95, 87.0,
86.5, 85.2, 71.0, 64.5, 55.0, 54.97, 45.7, 40.7, 40.1, 37.9, 34.6, 30.7;
554.2296.
3′-O-[(2-Cyanoethoxy)(diisopropylamino)phosphino]-5′-O-
DMPx-N4-(acetyl)-2′-deoxycytidine (8b). Product isolated in 91%
yield as a foam (0.48 g, 0.64 mmol): 1H NMR (300 MHz, CDCl3) δ =
10.25 (bs, 1H), 8.22−8.05 (m, 1H), 7.33−6.78 (m, 12H), 6.17 (m,
1H), 4.32 (m, 1H), 4.09 (m, 1H), 3.73−3.41 (m, 4H), 3.17 (m, 1H),
2.98 (m, 1H), 2.70 (m, 1H), 2.52 (t, J = 6.3 Hz, 2H), 2.32 (m, 1H),
2.22 (s, 3H), 2.16 (m, 6H), 0.95 (m, 12H);13C NMR (300 MHz,
CDCl3) δ = 169.1, 168.9, 161.4, 154.1, 148.4, 147.1, 143.11, 143.09,
131.9, 129.3, 128.0, 127.8, 126.8, 125.6, 125.3, 121.0, 120.7, 117.1,
115.0, 94.81, 94.79, 86.4, 85.4, 75.4, 70.3, 62.1, 58.1, 43.4, 43.4, 40.8,
23.7, 23.6, 23.2, 23.1, 20.6, 20.5, 19.5, 19.4; 31P NMR δ = 149.55,
149.23; HRMS calcd for C41H49N5O7P+ [M + H+] 754.3369 found
754.3373.
5′-O-DMPx-2′-deoxythymidine (9a). Isolated 0.81 g (1.47
mmol) of a white powder corresponding to a 88% yield: mp 139−
141 °C (d); 1H NMR (600 MHz, DMSO-d6) δ = 11.33 (s, 1H), 7.53
(s, 1H), 7.27−7.24 (m, 4H), 7.16 (m, 3H), 7.11 (m, 2H), 7.04 (bs,
1H), 6.97 (m, 1H), 6.20 (t, J = 7 Hz, 1H), 5.32 (d, J = 4.4 Hz, 1H),
4.31 (m, 1H), 3.82 (m, 1H), 3.13 (dd, J = 2.8, 10.4 Hz, 1H), 3.06 (dd,
J = 4.0, 10.4 Hz, 1H), 2.27 (m, 1H), 2.18 (m, 1H), 2.14 (s, 3H), 3.10
(s, 3H), 1.42 (s, 3H); 13C NMR (600 MHz, DMSO-d6) δ = 163.6,
150.4, 149.0, 148.7, 148.4, 135.7, 132.5, 132.4, 130.4, 130.2, 128.3,
128.22, 128.18, 126.8, 125.6, 122.1, 121.9, 116.1, 109.5, 85.3, 83.6,
75.5, 70.8, 63.7, 20.3, 20.2, 11.7; HRMS calcd for C31H30N2O6Na+ [M
+ Na+] 549.2002; Found 549.1995.
3′-O-[(2-Cyanoethoxy)(diisopropylamino)phosphino]-5′-O-
DMPx-2′-deoxythymidine (9b). Product isolated in 93% yield as a
foam (0.48 g, 0.66 mmol): 1H NMR (400 MHz, CD2Cl2) δ = 9.22 (bs,
1H), 7.67 (s, 1H), 7.35−7.06 (m, 10H), 6.93 (m, 1H), 6.35 (m, 1H),
4.55 (m, 1H), 4.13−4.07 (m, 1H), 3.63−3.55 (m, 4H), 3.30−3.27 (m,
1H), 3.17−3.14 (m, 1H), 2.53 (m, 1H), 2.41−2.33 (m, 3H), 2.24 (s,
3H), 2.18 (s, 3H), 1.63 (s, 3H), 1.18 (d, J = 6.8 Hz, 12H); 13C NMR
(400 MHz, CD2Cl2) δ = 164.9, 164.8, 151.1, 150.1, 149.6, 149.0,
136.1, 133.33, 133.30, 130.8, 130.7, 129.3, 129.0, 128.4, 127.2, 126.6,
122.4, 122.1, 118.2, 111.4, 85.6, 85.0, 76.9, 74.8, 74.6, 63.7, 60.7, 58.8,
58.6, 43.6, 43.5, 40.4, 24.8, 24.7, 24.6, 24.5, 21.0, 20.8, 20.7, 14.3, 12.3,
1.12; 31P NMR δ = 149.23, 148.33; HRMS calcd for C40H48N4O7P+
[M + H+] 727.3260; found 727.3264.
+
HRMS calcd for C43H42N7O6 [M + H+] 752.3197; found 752.3191.
3′-O-[(2-Cyanoethoxy)(diisopropylamino)phosphino]-5′-O-
DMT-N2-(dimethylformamidyl)-8-(8″-quinolyl)-2′-deoxygua-
nosine (4b). Isolated as a white foam in 74% yield (0.5 g, 0.52
mmol): 1H NMR (300 MHz, DMSO-d6) δ = 11.40 (bs, 1H), 8.92 (m,
1H), 8.53 (m, 1H), 8.26−8.19 (m, 2H), 8.00 (bs, 1H), 7.77 (m, 1H),
7.66 (dd, J = 4.2, 8.3 Hz, 1H), 7.29−6.73 (m, 13H), 5.71 (bs, 1H),
5.10 (d, J = 4.5 Hz,1H), 4.41 (bs, 1H), 3.73−3.68 (m, 7H), 3.64 (m,
1H), 3.23−3.18 (m, 2H), 3.18−3.07 (m,3H), 3.06−2.94 (s, 6H), 2.12
(bs, 1H), 0.97−0.81 (m, 12H); 13C NMR (300 MHz, CDCl3) δ =
154.5, 154.4, 154.1, 147.6, 146.7, 141.5, 133.2, 132.2, 132.0, 129.2,
127.0, 126.1, 125.9, 124.3, 124.1, 123.0, 118.6, 117.5, 109.5, 109.4,
82.0, 81.1, 80.1 67.1, 61.0, 58.2, 51.5, 51.45, 43.3, 43.1, 42.1, 37.2, 36.5,
36.4, 35.6, 34.4, 31.1, 27.2, 23.8, 23.6, 20.9; 31P NMR δ = 149.32,
148.88; HRMS calcd for C52H59N9O7P+ [M + H+] 952.4275; found
952.4278.
5′-O-DMPx-N2-(dimethylformamidyl)-2′-deoxyguanosine
(7a). Isolated 0.79 g (1.30 mmol) of a white powder corresponding to
1
78% yield: mp 113−118 °C (d); H NMR (300 MHz, CDCl3) δ =
ASSOCIATED CONTENT
* Supporting Information
9.50 (bs, 1H), 8.43 (s, 1H), 7.65 (s, 1H), 7.31 (m, 2H), 7.22−6.95 (m,
7H), 6.90 (m, 2H), 6.33 (t, J = 6.9 Hz, 1H), 4.55 (m, 1H), 4.13 (m,
1H), 3.16 (m, 2H), 3.02 (s, 3H), 2.99 (s, 3H), 2.54 (m, 2H), 2.13 (s,
3H), 2.08 (s, 3H); 13C NMR (300 MHz, CDCl3) δ = 158.1, 158.0,
156.6, 150.1, 149.3, 149.28, 148.5, 132.8, 132.7, 130.2, 130.1, 129.3,
129.1, 127.9, 126.6, 126.3, 122.2, 122.0, 120.3, 116.0, 85.9, 83.3, 76.1,
72.5, 64.0, 45.7, 41.3, 40.8, 35.1, 30.9, 20.7, 20.68; HRMS calcd for
■
S
Figure S1 described in the text, NMR spectra of synthetic
samples, and ESI-MS spectra of modified NarI oligonucleo-
tides. This material is available free of charge via the Internet at
http://pubs.acs.org.
+
C34H35N6O5 [M + H+] 607.2669; found 607.2672.
AUTHOR INFORMATION
Corresponding Author
*E-mail: rmanderv@uoguelph.ca.
3′-O-[(2-Cyanoethoxy)(diisopropylamino)phosphino]-5′-O-
■
DMPx-N2-(dimethylformamidyl)-2′-deoxyguanosine (7b). Iso-
1
lated as a white foam in 91% yield (0.52g, 0.642 mmol): H NMR
(300 MHz, CD2Cl2) δ = 9.70 (bs, 1H), 8.45 (m, 1H), 7.60 (m, 1H),
7.26−6.86 (m, 11H), 6.21 (m, 1H), 4.58 (m, 1H), 4.16 (m, 1H),
3.74−3.37 (m, 5H), 3.12 (m, 2H), 3.01 (s, 6H), 2.43−2.39 (m, 2H),
Notes
The authors declare no competing financial interest.
698
dx.doi.org/10.1021/jo4024842 | J. Org. Chem. 2014, 79, 692−699

The Journal of Organic Chemistry
Article
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ACKNOWLEDGMENTS
■
Financial 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. M.S. and R.A.M. thank Dr.
Hongbin (Tony) Han, Brock University, for helpful discussions
and a sample of DMPx-Cl.
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Sturla, S. J.; Wetmore, S. D.; Manderville, R. A. J. Org. Chem. 2013, 78,
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