Synthesis of N 2-Alkyl-8-oxo-7,8-dihydro-2′-deoxyguanosine derivatives and effects of these modifications on RNA duplex stability

Article abstract of DOI:10.1021/jo102187y

N²-Alkyl analogues of 8-oxo-7,8-dihydro-2′-deoxyguanosine (OG) were synthesized (alkyl = propyl, benzyl) via reductive amination of the protected OG nucleoside and incorporated into various positions of an RNA strand. Thermal stability studies of duplexes containing A or C opposite a single modified base revealed only moderate destabilization. Both OG as well as its N²-alkyl analogues can pair opposite A or C with nearly equal stability, potentially offering a new means of modulating RNA-protein interactions in the minor vs major grooves.

Full text of DOI:10.1021/jo102187y

pubs.acs.org/joc
probing RNA structure and function,9-11 and exploring
Synthesis of N2-Alkyl-8-oxo-7,8-dihydro-20-
deoxyguanosine Derivatives and Effects of These
Modifications on RNA Duplex Stability
interactions between RNA and proteins or small molecules.1
Both sugar and backbone modifications have been explored
to improve nuclease stability and target identification in
antisense and siRNA approaches;12,13 however, examples
of base modification in these applications are few by com-
parison.14-16 The ability to alter the hydrophobicity and
steric properties of the major and minor groove appeared to
us as a possible means of modulating both interstrand inter-
actions as well as nucleic acid-protein interactions,7,17,18
thereby expanding the range of nucleic acid modifications in
the design of DNA and RNA therapeutics.
Arunkumar Kannan and Cynthia J. Burrows*
Department of Chemistry, University of Utah, 315 South 1400
East, Salt Lake City, Utah 84112-0850, United States
burrows@chem.utah.edu
Received November 2, 2010
Inspiration for the present work came from one of the
major products of oxidative damage to the DNA, namely
8-oxo-7,8-dihydro-20-deoxyguanosine (OG).19-21 Introduc-
tion of an oxo group at C8 of the purine increases the
propensity of the purine to flip from the normal anti con-
formation to syn, where it exposes the Hoogsteen face of the
purine to base pairing. Like its parent guanine, OG(anti)
accepts cytosine as a Watson-Crick partner, while the
complementary base for OG(syn) is adenosine (Figure 1).
During the anti-syn conformational change, the N2-amino
group and the C8-oxo groups exchange positions between
the minor and major grooves (Figure 1). As a result, addition
of an alkyl or aryl group to the exocyclic amine should enable
the placement of the substituent in either groove, in a way
that will be governed by the identity of the base opposite.
OG can be synthetically incorporated into DNA and
RNA oligomers via the corresponding phosphoramidite.
While we, among others, have been studying OG and its
incorporation into oligomers, including chemical character-
ization and enzymology with DNA processing enzymes,22,23
OG is rarely studied in RNA. Thermal denaturation
studies,22,24,25 NMR studies,26 and X-ray crystallographic
N2-Alkyl analogues of 8-oxo-7,8-dihydro-20-deoxygua-
nosine (OG) were synthesized (alkyl = propyl, benzyl)
via reductive amination of the protected OG nucleoside
and incorporated into various positions of an RNA
strand. Thermal stability studies of duplexes containing
A or C opposite a single modified base revealed only
moderate destabilization. Both OG as well as its N2-alkyl
analogues can pair opposite A or C with nearly equal
stability, potentially offering a new means of modulating
RNA-protein interactions in the minor vs major grooves.
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2619.
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sugars of DNA and RNA and have also been introduced into
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Published on Web 12/30/2010
DOI: 10.1021/jo102187y
r
2010 American Chemical Society

Kannan and Burrows
JOCNote
the modified OGs. The caspase-2 sequence selected has Us
situated near the 50 end, near the 30 end and in the middle of
the guide strand sequence. Accordingly, we chose positions 4
(50 seed region), 11 (middle; at the Argonaute2 cleavage site)
and 16 (near the 30 terminus) for incorporation of modified
bases (vide infra). These three positions would allow us to
understand the effects of the modified nucleosides on stabil-
ity of RNA duplexes in detail. Furthermore, by forming the
RNA duplex with either A or C opposite modified guano-
sines, we could evaluate the duplex stability with both
Watson-Crick (OG:C) and Hoogsteen (OG:A) base pairs
in these three regions. The 20-deoxynucleoside of OG was
selected for study because the absence of the 20-OH group
might readily facilitate both the syn and anti conformations
in comparison to the ribonucleoside analogue.30 Previous
studies have shown that the presence of a few 20-deoxy-
nucleosides in an RNA strand does not dramatically alter its
physical or biological properties.14 In the current work, the
N2 substituents are propyl or benzyl in comparison to H to
evaluate a small vs medium-sized hydrophobic group added
to either the major or minor groove.
Initially, attempts were made to follow a synthetic scheme
similar to that of alkyl-substituted 2-aminopurines by
substitution of the diacylated-2-bromo-20-deoxyinosine
nucleoside.17 Unfortunately, substitution of 2-bromoinosine
with ethyl, propyl and benzyl amine all proceeded in low
yields. Furthermore, diazotization of deoxyguanosine to
yield the 2-bromo-20-deoxyinosine base led to many different
products as in previous work.34,35
As a second approach, we attempted reductive amination
of aldehydes with guanosine; reaction of 20-deoxyguanosine
with the respective aldehyde in the presence of NaBH3CN led
to a ∼60% yield along with a small amount of dialkylated
side product (∼20%). However, subsequent conversion into
8-oxo-7,8-dihydro-20-deoxyguanosine yielded many side
products complicating the purification.
Thus, the most promising procedure was to install the
8-oxo moiety first and then carry out reductive amination to
prepare the N2-alkyl derivatives of OG; these were then
finally converted into the corresponding phosphoramidites.
The overall procedure, shown in Scheme 1, involved conver-
sion of 8-bromo-20-deoxyguanosine (2) to 8-O-benzyl-20-
deoxyguanosine (3) in the presence of sodium benzylate
and DMSO. Compound 3 is highly unstable in acidic con-
ditions and yields OG (4) upon stirring with 1 M HCl for 2 h.
Reductive amination of OG in the presence of sodium
cyanoborohydride in 20% aqueous methanol and the appro-
priate aldehyde yielded the respective N2-alkylated products
(5) in good yield. Modest changes in the concentration of
NaBH3CN or the aldehyde did not alter the yield of the
monoalkylated or dialkylated products (5 vs 6) significantly;
however, a large excess of the aldehyde and longer reaction
times yielded predominately the dialkylated compound. The
mono-N2-alkylated product was purified chromatograph-
ically, and the 50-OH was protected by DMTrCl in pyridine
to yield (7). This compound was then converted into its phos-
phoramidite (8) using triethylamine as base in CH2Cl2 under
anhydrous conditions.36 The modified phosphoramidites
FIGURE 1. 8-Oxo-7,8-dihydro-20-deoxyguanosine (OG) pairing
with A and C.
structures23,27,28 of the OG(syn):A(anti) base pair show that
the B-form DNA helix is only very slightly perturbed by the
presence of this noncanonical pair. Because the DNA-RNA
duplex synthesized during transcription is A-form, the
transcription studies suggest that both the Watson-Crick
OG(anti):C(anti) and the OG(syn):A(anti) base pairs can be
stably accommodated in A-form RNA.29,30
In the G:C or OG:C Watson-Crick base pair, only one
hydrogen on the N2 nitrogen of the purine is involved in
hydrogen-bonding to the opposite base; the other hydrogen
projects into the minor groove. In the OG-A base pair, the
entire exocyclic amino group of OG projects into the major
groove. Thus, replacement of one of the hydrogens of OG’s
N2 group by an alkyl should have a very minimal effect on the
hydrogen bonding ability in a duplex. However, the presence
of a bulky substituent could have other effects on local
solvation or steric interactions that are not easily predicted.
Therefore our current focus was to study the effects of
different substitutions that vary in steric bulk at the N2
position of OG on the stability of duplex formation in
RNA. Chemical modifications in siRNA are an important
way to address issues such as stability, delivery and reducing
off-target effects.14,31,32 The ability of OG to base pair in two
alternative conformations, syn and anti, could play a major
role in siRNA therapeutics. The switching of steric bulk
between minor (OG(anti)) vs major (OG(syn)) grooves may
prevent undesirable interactions depending on pairing with
C or A, respectively, in the complementary strand.
In previous work, we reported the stability of OG and its
oxidation products in DNA strands.22 The Beal laboratory
has shown that chemical modification of Gs in caspase-2
siRNAs can reduce their ability to bind to off-target proteins
such as Protein Kinase R (PKR) while maintaining caspase-2
gene knockdown capability.33 Hence, in our current experi-
ments, we have synthesized various N2-alkyl derivatives
of OG phosphoramidites and incorporated them into a
caspase-2 siRNA sequence by replacing selected uracils with
(27) Brieba, L. G.; Eichman, B. F.; Kokoska, R. J.; Doublie, S.; Kunkel,
T. A.; Ellenberger, T. EMBO J. 2004, 23, 3452–61.
(28) Hsu, G. W.; Ober, M.; Carell, T.; Beese, L. S. Nature 2004, 431, 217–
221.
(29) Kim, S. K.; Lee, S. H.; Kwon, O. S.; Moon, B. J. J. Biochem. Mol.
Biol. 2004, 37, 657–662.
(30) Kim, S. K.; Yokoyama, S.; Takaku, H.; Moon, B. J. Bioorg. Med.
Chem. Lett. 1998, 8, 939–944.
(31) Chiu, Y.-L.; Ali, A.; Chu, C.-y.; Cao, H.; Rana, T. M. Chem. Biol.
2004, 11, 1165–1175.
(32) Chiu, Y. L.; Rana, T. M. Mol. Cell 2002, 10, 549–561.
(33) Puthenveetil, S.; Whitby, L.; Ren, J.; Kelnar, K.; Krebs, J. F.; Beal,
P. A. Nucleic Acids Res. 2006, 34, 4900–4911.
(34) Suzuki, T.; Yamaoka, R.; Nishi, M.; Ide, H.; Makino, K. J. Am.
Chem. Soc. 1996, 118, 2515–2516.
(35) Glaser, R.; Rayat, S.; Lewis, M.; Son, M.-S.; Meyer, S. J. Am. Chem.
Soc. 1999, 121, 6108–6119.
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Kannan and Burrows
JOCNote
SCHEME 1. Preparation of N2-Alkyl-8-oxo-7,8-dihydro-20-
deoxyguanosine Phosphoramidites
FIGURE 2. TM values of RNA duplexes singly modified at posi-
tions 4, 11, or 16 of the caspase-2 guide sequence compared to native
U:A base pairs in these positions. The passenger strand has either
Y = C (green bars), A (blue bars), or G (red bars) opposite the
modified site X, in which the R group on N2 of OG is H (X = O),
propyl (X = P), or benzyl (X=B).
were introduced at either one, two or three positions via
standard solid-phase synthesis. Coupling efficiencies for the
modified phosphoramidites were similar to those of unmod-
ified bases. The deprotected oligonucleotides were purified
by ion-exchange chromatography, analyzed by negative ion
ESI-MS, and hybridized to form RNA duplexes.
The effect of the 20deoxy-OG nucleosides with and with-
out N2-alkyl substituents on duplex stability was investigated
via thermal denaturation (TM) studies at pH 7.4 in 100 mM
NaCl. In the initial set of experiments, the nucleoside analogs
were incorporated into the duplex at each of three different
positions (4, 11 or 16) of the guide strand sequence from
caspase 2 with C, A and G in the strand opposite (Figure 2).
The modified strands containing OG and its analogues were
also compared with the unmodified strand containing a U:A base
pair. In the second set of experiments, the nucleoside analogs
were incorporated into the duplex at more than one position,
that is, (4,11), (11,16), (4,16), or (4,11,16) of the antisense
strands opposite A or C (Figure 3).
FIGURE 3. TM values of RNA duplexes doubly and triply mod-
ified at positions 4, 11 and/or 16 of the caspase-2 guide sequence
compared to native U:A base pairs in these positions. The passenger
strand has either Y = C (green bars) or A (blue bars) opposite the
modified site X, in which the R group on N2 of OG is H (X = O),
propyl (X = P), or benzyl (X = B).
TM measurements showed that replacement of a single
U:A base pair with a single O:C (O = OG with R=H) at
positions 16 or 11 decreased the TM by ∼2 °C whereas at
position 4 it decreased by 5 °C. Indeed, position 4 proved to
be consistently more sensitive to the effects of modified
bases. Introduction of O:A at the same positions showed a
further slight decrease in TM of ∼1 °C. However, on intro-
duction of guanosine opposite the modified bases, which is
expected to be a destabilizing mispair, the TM decreased
considerably (ΔTm ≈ 8 °C). In DNA, the OG(syn):G(anti)
base pair can form, but is somewhat less stable;37 therefore, it
was of interest to know the likelihood of mispairing with G if
OG was present in the guide strand of an siRNA.
Introduction of alkyl substituents onto the N2 position of
OG, either propyl (P) and benzyl (B), showed similar TM
patterns, of which the benzyl modification was somewhat
more destabilizing than the propyl analogue or OG itself.
In brief, the order of stability of these duplex oligonucleo-
tides can be listed as: Unmodified>O>P>B. From the
pattern observed from the data, it was clear that the larger
the N2 group, the greater the destabilizing effect, although
all duplexes with single modifications were still quite stable
(TM ≈ 60 °C). The small difference in TM between the pairing
against A and C further supports the concept that OG can
switch conformations according to the complementary base,
projecting the N2 substituent into either the major or minor
groove. Interference with RNA-protein contacts at these
sites is expected to disrupt complexes with double-stranded
RNA binding motifs in key proteins.
(36) Bodepudi, V.; Shibutani, S.; Johnson, F. Chem. Res. Toxicol. 1992, 5,
608–617.
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Kannan and Burrows
JOCNote
In the next set of experiments (Figure 3), the alkylated
OGs were incorporated into the duplex at 2 or 3 positions of
the caspase-2 guide strand sequence with either As or Cs in
the strand opposite. As expected, this decreased the stability
even further with most of the TM values of strands having
modifications at more than one position being around 55 °C.
These strands still form stable duplexes, however, and the
differences in TM values between pairing against A or C
remained very similar.
In conclusion, we have developed an optimized route to
N2-alkyl derivatives of 8-oxo-7,8-dihydro-20deoxyguanosine
phosphoramidites and incorporated them into the caspase-2
siRNA guide sequence at one, two or three positions in which
a U site was replaced by an OG nucleotide. The thermal
stabilities of duplexes containing OG(anti):C(anti) vs
OG(syn):A(anti) were consistent with the conclusion that
OG can exist in either the syn or anti orientation depending
on the identity of the base opposite. Introduction of alkyl
groups onto the exocyclic amino group led to only modest
decreases in stability. No major sequence effects were evident
although modification at position 4, a 50GXU-30 sequence
context, led to slightly greater destabilizing effects than at
positions 11 (50-UXG-30) or 16 (50-UXC-30). The overall
stabilities of the duplexes and the small differences in stabil-
ities of N2-alkyl-8-oxo-20-deoxyguanosine on pairing with
A and C suggests that these modifications can be successfully
incorporated into siRNA duplexes with a view to switching
between modes that might prevent unwanted protein inter-
actions while permitting silencing activity.
(500 mg, 1.47 mmol). The cooling bath was removed and stirring
was continued at RT for 15 min, followed by cooling in ice and
quenching with water (50 mL). The solution was extracted with
CH2C12 (5 ꢀ 20 mL), and the combined organic layers were
washed with H2O (2 ꢀ 20 mL) and then dried over MgSO4. The
solvent was evaporated under reduced pressure, and the crude
residue was purified by column chromatography over silica gel
using MeOH-CH2Cl2 containing 2% triethylamine to inhibit
detritylation yielding 7a (385 mg, 50%). 1H NMR (CD2Cl2): δ
10.22 (2 H), 7.50-6.89 (13 H, m), 6.41(1H, bs), 6.21 (l H, t, J =
6.05 Hz), 4.55-4.51 (l H, m), 4.01-3.98 (l H, m), 3.88- 3.80 (1 H, m],
3.86 and 3.85 (6 H, 2 s), 3.57-3.30 (2 H, 2 m), 3.18-3.06
(2 H, m), 2.23-2.19 (2 H, m), 1.50-1.43 (2 H, m), 0.89 (3H, t,
J = 7.5 Hz); 13C NMR (CD2Cl2): δ 176.4, 154.5, 152.4, 147.6,
140.6, 128.9, 128.8, 127.7, 127.5, 127.3, 99.5, 87.6, 81.4, 71.9,
63.0, 44.4, 43.4, 36.0, 25.3. ESI-MS (m/z): calcd for C34H37N5O7
Na (M þ Naþ) 650.2591; found 650.2604.
30-O-[(Diisopropylamino)-(2-cyanoethoxy)phosphino]-50-O-
(4,40-dimethoxytrityl)-N2-propyl-8-oxo-7,8-dihydro-20-deoxy-
guanosine (8a). To a mixture of 7a (0.112 g, 0.18 mmol) dried
over P2O5 in a vacuum desiccator for 24-48 h and then
coevaporated with 1:1 dry CH2Cl2 and benzene prior to reac-
tion, plus dry Et3N (0.044 g, 0.43 mmol) in dry CH2C12 (1 mL)
under N2 was added 2-cyanoethyl N,N-diisopropyl-phosphor-
amidochloridite (0.050 g, 0.25 mmol). The reaction was mon-
itored by TLC analysis for 20 min; the solvent was evaporated,
and a mixture of dry THF-benzene (1:4; 25 mL) was introduced,
stirred for 10 min and filtered under N2 to remove the
Et3N HCl. The process was then repeated twice using dry
3
benzene as the solvent. The material was purified by column
chromatography using 5% MeOH-CH2Cl2 containing 2%
triethylamine. The resultant viscous foamy material, when dried
over P2O5 in a vacuum desiccator at RT overnight, led to a yield
of 105 mg of 8a (70%). 1H NMR (CD2Cl2): 7.42-6.74 (13H, m),
6.25 (1H, t, J = 6.05 Hz), 4.72 (1H, m), 4.21-4.08 (2H, m), 3.79
(6H, s), 3.59-3.52 (4H, m,), 3.42-3.14 (2H, m), 3.12-3.01 (4H,
m), 2.75-2.70 (2H, m), 2.40-2.23 (2H, m), 1.48-1.05 (14H, m),
0.85, (3H, t, J = 7.5). 13C NMR (CD2Cl2): δ 158.8, 152.9, 149.2,
147.9, 136.3, 130.3, 128.5, 128.4, 127.9, 126.9, 113.2, 98.6, 86.2,
81.3, 64.2, 58.6, 55.6, 55.4, 45.5, 43.3, 24.6, 22.6, 20.7, 11.6.
31P NMR (CD2Cl2): 147.69, 147.27. ESI-MS (m/z): calcd for
C43H54N7O8PNa (M þ Naþ) 850.3669; found 850.3662.
Experimental Section
N2-Propyl-8-oxo-7,8-dihydro-20-deoxyguanosine (5a). To a
suspension of 43,31 (950 mg, 3.3 mmol) and NaBH3CN (628 mg,
9.9 mmol) in methanol (80 mL) was added propanal (8.16 mL,
114 mmol) in one portion, and the mixture was heated at 50 °C
overnight under nitrogen. After removal of the solvent under
reduced pressure, the residue was purified by column chroma-
tography using 20% MeOH-CH2Cl2 to yield 805 mg (75%) of
1
5a. H NMR ((CD3)2SO): δ10.4-9.9 (2 H, bs), 6.95 (1 H, bs),
6.03 (l H, t, J = 6.04 Hz), 5.17 (1 H, bs), 5.7 (1 H, bs), 4.36-4.30
(1H, m), 4.31-4.27 (1H, m), 3.58-3.47 (1H, m), 3.65-3.52
(2H), 3.42-3.31 (1 H, m), 3.04-2.76 (1H, m), 1.88-1.88 (1 H,
m), 0.89 (3H, t, J = 7.5 Hz) 13C NMR ((CD3)2SO): δ 151.7,
146.9, 124.2, 98.5, 94.2, 87.1, 80.7, 71.2, 64.1, 62.4, 42.2, 35.6,
22.1, 11.3. ESI-MS (m/z): calcd for C13H19N5O5Na (M þ Naþ)
348.1284; found 348.1286.
Acknowledgment. We thank Prof. P. A. Beal (UC, Davis)
for valuable comments and the NIH for financial support
(GM080784).
Supporting Information Available: Additional experimental
1
procedures, copies of H, 13C and 31P NMR spectra for new
50-O-4,40-Dimethoxytrityl-N2-propyl-8-oxo-7,8-dihydro-20-
deoxyguanosine (7a). To a solution of 5a (400 mg, 1.23 mmol) in
dry pyridine (6 mL) cooled in ice water was added 4,40-DMTrCl
nucleoside derivatives, sequence information, Tm curves and
mass spectral data for oligomers. This material is available free
of charge via the Internet at http://pubs.acs.org.
J. Org. Chem. Vol. 76, No. 2, 2011 723