2′-Deoxy-1-methylpseudocytidine, a stable analog of 2′-deoxy-5-methylisocytidine

Article abstract of DOI:10.1016/j.bmc.2009.03.047

2′-Deoxy-5-methylisocytidine is widely used in assays to personalize the care of patients infected with HIV, hepatitis C, and other infectious agents. However, oligonucleotides that incorporate 2′-deoxy-5-methylisocytidine are expensive, because of its in

Full text of DOI:10.1016/j.bmc.2009.03.047

Bioorganic & Medicinal Chemistry 17 (2009) 3728–3732
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Bioorganic & Medicinal Chemistry
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20-Deoxy-1-methylpseudocytidine, a stable analog
of 20-deoxy-5-methylisocytidine
à
*
Hyo-Joong Kim , Nicole A. Leal , Steven A. Benner
Westheimer Institute for Science and Technology, Foundation for Applied Molecular Evolution, PO Box 13174, Gainesville, FL 32604, United States
a r t i c l e i n f o
a b s t r a c t
20-Deoxy-5-methylisocytidine is widely used in assays to personalize the care of patients infected with
HIV, hepatitis C, and other infectious agents. However, oligonucleotides that incorporate 20-deoxy-5-
methylisocytidine are expensive, because of its intrinsic chemical instability. We report here a C-glyco-
side analog that is more stable and, in oligonucleotides, pairs with 20-deoxyisoguanosine, contributing
to duplex stability about as much as a standard 20-deoxycytidine and 20-deoxyguanosine pair.
Ó 2009 Elsevier Ltd. All rights reserved.
Article history:
Received 29 December 2008
Revised 20 March 2009
Accepted 22 March 2009
Available online 27 March 2009
Keywords:
iso-Cytidine
Pseudocytidine
Synthetic biology
Expanded genetic alphabets
Clinical diagnostics
1. Introduction
ent hydrogen bonding scheme. This gives DNA molecules contain-
ing isoC and isoG the property known as ‘orthogonality’; they pair
One success story arising from efforts to create artificially ex-
panded genetic information systems (AEGIS) come from the use
of 20-deoxy-5-methylisocytidine (isoC, 1, Fig. 1) and 20-
deoxyisoguanosine (isoG, 2, Fig. 1) as fifth and sixth ‘letters’ in
the DNA genetic alphabet.1,2 The isoC presents a hydrogen bond
‘acceptor–acceptor–donor’ hydrogen bonding pattern on a small
pyrimidine; isoG presents a hydrogen bond ‘donor–donor–accep-
tor’ hydrogen bonding pattern on a large purine nucleobase. There-
fore, isoC and isoG can form a Watson–Crick pair having the same
geometry as the T:A and C:G base pairs, but are joined by a differ-
with each other without pairing with DNA built only from natural
dA, T, dC, and dG. At the same time, the close structural similarity
of the nonstandard and standard pairs means that polymerases
and other enzymes used as standard tools in molecular biology
can also be used with this synthetic genetic system.
Orthogonality enabled by a fifth and sixth letters of a genetic
alphabet can, in principle, support low-noise microarrays, multi-
plexed PCR, and low cost re-sequencing of the human genome,
among other applications. In commerce, the orthogonality pro-
vided by the isoC:isoG pair underlies the high sensitivity and broad
dynamic range of the branched DNA Versant diagnostics systems
that received FDA approval in 2003 and 2004.3 Developed by scien-
tists at Chiron and Bayer and now sold by Siemens Medical Solu-
tions, these permit the very sensitive detection of the nucleic
acids from infectious agents in complex biological media. These
tools personalize the care of some 400,000 patients infected with
the HIV, hepatitis B, and hepatitis C viruses.4
The isoC:isoG pair has also been used as a fifth and sixth letter in
a PCR-amplifiable artificial genetic system.5,6 This represents one
of the first artificial chemical systems capable of Darwinian evolu-
tion, meeting some of the criteria for artificial life.
Figure 1. The chemical structures of 20-deoxyisocytidine (1), 20-deoxyisoguanosine
(2) and 20-deoxy-5-methylpseudocytidine (3).
As one of its disadvantages, however, 20-deoxy-5-methylisocyti-
dine undergoes facile cleavage of the glycosidic bond under acidic
conditions. This propensity for acid-catalyzed depyrimidinylation
can be used as a tool to detect isoC in oligonucleotides5 and is man-
aged during phosphoramidite synthesis by careful handling of
formamidine protected phosphoramidite precursors. Nevertheless,
* Corresponding author. Tel.: +1 352 271 7005; fax: +1 352 271 7076.
E-mail addresses: hkim@ffame.org (H.-J. Kim), nleal@ffame.org (N.A. Leal),
sbenner@ffame.org (S.A. Benner).
Tel.: +1 386 418 8085; fax: +1 352 271 7076.
Tel.: +1 352 375 8680; fax: +1 352 271 7076.
à
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.03.047

H.-J. Kim et al. / Bioorg. Med. Chem. 17 (2009) 3728–3732
3729
these requirements increase the cost of oligonucleotides that con-
tain 20-deoxy-5-methylisocytidine, especially in human diagnostics
where regulatory standards are rigorous. An analog of isoC that still
pairs with 20-deoxyisoguanosine but does not suffer depyrimidiny-
lation is desirable.
then with ammonium hydroxide (75% yield). The structure of 9
was established by 1H and 13C NMR and high resolution mass
spectrometry.
With the 20-deoxy-1-methylpseudocytidine 9 in hand, the phos-
phoramidite derivative 13 suitable for oligonucleotide synthesis
was prepared as shown in Figure 3. Compound 9 was treated with
benzoyl chloride in pyridine/CH2Cl2 to give dibenzoylated com-
pound 10. Treatment of 10 with excess triethylamine trihydrofluo-
ride in THF at 45 °C for 2 days gave 11 in 85% yield. The
phosphoramidite derivative 13 for DNA synthesis was synthesized
via dimethoxytritylation of 11 and subsequent phosphitylation of
12 using a standard procedure.
One analog that might have these properties is 20-deoxy-1-
methylpseudocytidine (w
C, 3, Fig. 1), the C-glycoside analog of 20-
deoxy-5-methylisocytidine. Further, a methyl group on the pyrim-
idine nitrogen should prevent tautomerization and epimerization,
undesired reactions seen with other AEGIS components.7 However,
given the known propensity of C-glycosides to change the confor-
mation of the 20-deoxyribose ring, we were concerned that incorpo-
rating a significant number of C-glycosides would significantly alter
the conformation of a DNA duplex that is made by this strand.
Very recently, we reported experiments that showed that the
conformation of a duplex is not altered even if twelve C-glycosides
are incorporated sequentially.8 Further, we showed that certain
polymerases could incorporate five consecutive C-glycosides in
template-directed synthesis from triphosphates. This provided the
motivation to examine the analogous C-glycoside of iso-cytidine.
We report here the synthesis of this species, a new compound,
in protected form, as well as the synthesis of a test set of oligonu-
cleotides. We report that when incorporated into an oligonucleo-
tide at the appropriate position, 20-deoxy-1-methylpseudocyti-
dine forms nucleobase pairs with isoG that contribute to duplex
stability about as well as the natural C:G nucleobase pair. Further,
discrimination against mismatches is at the same level as seen
with standard nucleobases. Therefore, this compound is a cost-
effective replacement for a 20-deoxyisocytidine that will support
the synthesis and use of AEGIS-containing oligonucleotides at low-
er cost.
Oligonucleotides were constructed to permit melting studies
with both perfectly matched duplexes and duplexes containing
mismatches. The standard sequence was identical to that used by
Geyer et al.11 and by their predecessor, Horn et al.12 This allowed
direct comparison of the results obtained here with their earlier re-
sults. Melting temperatures are illustrated in Table 1.
In a variety of contexts, the isoC:isoG base pair contributes more
to duplex stability than any of the other standard nucleobase pairs.
The reason for this is unclear, but the isoC:isoG pair appears to be
the strongest of any joined by three hydrogen bonds.
At least in the context in the two test oligonucleotides, the pair
between 20-deoxy-1-methylpseudocytidine and 20-deoxyisoguano-
sine contributes to duplex stability like the standard dC:dG. This
result is consistent with other results obtained by Geyer et al.,11
who found that an oligonucleotide duplex having a C-glycoside
had a melting temperature 1–2 degrees lower than an analogous
duplex with an N-glycoside.
To demonstrate the improved stability of oligonucleotides con-
taining 20-deoxy-1-methylpseudocytidine above that displayed by
analogous oligonucleotides containing 20-deoxyisocytidine, four
oligonucleotides were prepared (Table 2) and purified by PAGE.
Treatment of the oligonucleotides with gamma-labeled ATP
generated 50-kinased oligonucleotides. These oligonucleotides
were treated in parallel experiments with (a) heat at 95 °C for var-
ious times (Fig. 4) and (b) 0.05 M acetic acid followed by 0.1 M
ammonium hydroxide (Fig. 5).
2. Results and discussion
Pseudothymidine (7), prepared from pseudouridine,9 is poten-
tially a precursor for protected 20-deoxy-1-methylpseudocytidine
(9). As pseudouridine is expensive and the N(1)-methylation of
pseudouridine requires 5 days at reflux, an alternative route was
sought. Coupling of glycal (4) and 5-iodo-1-methyluracil (5) via a
palladium-catalyzed Heck reaction, followed by desilylation and
diastereoselective reduction, gave pseudothymidine (7) in 51%
yield (Fig. 2), a result consistent with results obtained similar
palladium-mediated glycal-aglycone couplings.10
Pseudothymidine having both sugar oxygens protected (8) was
converted to the correspondingly protected 20-deoxy-1-methyl-
pseudocytidine (9) by treatment first with 2,4,6-triisopropyl-
benzenesulfonyl chloride, DMAP and triethylamine in CH3CN, and
Treatment of oligonucleotides containing 20-deoxyisocytidine
(oligo 1 and 2 in Fig. 4) with heat at 95 °C gave fragmentation of
the oligonucleotides and the intensity of fragments were increased
with prolonged treatment time. However, treatment of oligonucle-
otides containing 20-deoxy-1-methylpseudocytidine instead of iso-
cytidine (oligo 4 and 5 in Fig. 4) with heat at 95 °C gave no cleavage
up to 90 min treatment. This result showed 20-deoxy-1-meth-
ylpseudocytidine is more stable than 20-deoxyisocytidine under
thermal condition at neutral pH.
Figure 2. Synthesis of protected 20-deoxypseudocytidine (9). Conditions: (a) Pd(OAc)2, Ph3As, Bu3N, DMF, 60 °C, 18 h; (b) TBAF, HOAc; (c) NaBH(OAc)3, HOAc, CH3CN, 0 °C, 2 h
(51% for three steps); (d) TBDMSCl, imidazole, DMF (93%); (e) 2,4,6-triisopropylbenzenesulfonyl chloride, DMAP, TEA, CH3CN, rt, 24 h; (f) NH4OH, rt, 4 h (75% for two steps).

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H.-J. Kim et al. / Bioorg. Med. Chem. 17 (2009) 3728–3732
Figure 3. Synthesis of 20-deoxypseudocytidine phosphoramidite (13). Conditions: (a) BzCl, pyridine (74%); (b) Et3Nꢀ3HF, THF, 45 °C, 2 days (86%); (c) DMTrCl, pyridine (66%);
(d) 2-cyanoethyl diisopropylchlorophosphoramidite, DIPEA, CH2Cl2 (78%).
Table 1
Melting temperatures (°C) for nucleobase pairs
Matched nucleobase pairs
Mismatched nucleobase pairs
N1–N2
Measured
Geyer (2003)11
N1–N2
Measured
Geyer (2003)11
iG–iC
62.5
60.2
59.6
61.3
59
63.3
—
59.5
61.5
—
iG–C
iG–A
iG–T
iG–G
52.5
42.6
51.9
53.2
52.6
43.4
52.3
52.7
iG–
G–C
iC–iG
C–iG
C–G
w
C
w
60.1
58.5
The sequence of the DNA duplex used in the thermal denaturation experiments are
5’—C A C N1 A C T T T C T C C T-3’
3’-TG T G N2 T G A A AG A G G—5’
Table 2
Oligonucleotides used in thermal and acidic stability study
Oligomer no.
Sequence
Comment
1
2
4
5
50-GGAGAAAGT-iC-GTGT-30
50-CAC-iC-ACTTTCTCCT-30
iC at position 10
iC at position 4
50-GGAGAAAGT-
W
C-GTGT-30
WC at position 10
C at position 4
50-CAC- C-ACTTTCTCCT-30
W
W
The acid-stability of oligonucleotides containing 20-deoxyisocy-
ti- dine and 20-deoxy-1-methylpseudocytidine was investigated by
the incubation of the oligonucleotides in 0.05 M acetic acid at 90 °C
for 0–90 min. Treatment of oligonucleotides containing 20-
deoxyisocytidine (oligo 1 and 2 in Fig. 5) in 0.05 M acetic acid gave
fragmentation of the oligonucleotides and complete disappearance
of the starting 14-mer after 60 min. The acid treatment of oligonu-
cleotides containing 20-deoxy-1-methylpseudocytidine instead of
iso-cytidine (oligo 4 and 5 in Fig. 5) in 0.05 M acetic acid gave frag-
mentation of the oligonucleotides but even after 90 min, small
fraction of the starting 14-mer of oligo 4 and 5 was not cleaved.
This result showed 20-deoxy-1-methylpseudocytidine is more
resistant to acidic depyrimidinylation.
Figure 4. Thermostability of oligonucleotides containing 20-deoxyisocytidine and
20-deoxy-5-methylpseudocytidine. Samples (0.3 pmol) were resolved on
PAGE.
a 20%
it joins with the 6-aza-20-deoxyisocytidine, which is also reported
to display acid stability.13 Further, it supports duplex pairing well
with complementary nucleotides as part of an expanded genetic
information system. Oligonucleotides that incorporate it are consid-
erably less expensive to prepare in high quality than oligonucleo-
tides that incorporate 20-deoxy-5-methylisocytidine. Therefore, we
expect 20-deoxy-1-methylpseudocytidine to become an important
new addition to synthetic genetic systems, especially in practical
application.
3. Conclusion
20-Deoxy-1-methylpseudocytidine (1-deaza-5-aza-5-methyliso-
cytidine) is more robust chemically than the 20-deoxy-5-
methylisocytidine that is widely used in assays to personalize the
care of patients infected with HIV, hepatitis, and other infectious
agents, assay for genetic disease, and support advanced architec-
tures for human genome sequencing and analysis. In one respect,
4. Experimental
4.1. Pseudothymidine (7)
A mixture of Pd(OAc)2 (36 mg, 0.16 mmol) and AsPh3 (98 mg,
0.32 mmol) in anhydrous DMF (10 mL) was stirred at rt for

H.-J. Kim et al. / Bioorg. Med. Chem. 17 (2009) 3728–3732
3731
rt and stirred for 24 h. The mixture was treated with saturated
NH4OH (7 mL) and stirred at rt for 4 h. Volatiles were removed
by rotary evaporation under reduced pressure and the residue
was dissolved in CH2Cl2 (40 mL) and washed with brine. The or-
ganic layer was collected, dried and evaporated. The product was
purified from the residue by flash chromatography (silica, EtOAc/
hexane = 1:1 to CH2Cl2/MeOH = 20:1) to give 9 as a light yellow so-
lid (150 mg, 75%). TLC (CH2Cl2/MeOH, 20:1): Rf 0.15. 1H NMR
(300 MHz, CDCl3) d 7.20 (s, 1H), 4.82 (dd, 1H, J = 11.1, 5.4 Hz),
4.39 (d, 1H, J = 6.6 Hz), 3.7–4.0 (m, 3H), 3.41 (s, 3H), 2.2–2.3 (m,
1H), 1.75–1.85 (m, 1H), 0.90 (s, 18H), 0.08 (m, 12H). 13C NMR
(75 MHz, CDCl3) d 163.99, 144.87, 103.93, 88.71, 77.84, 74.04,
63.51, 41.27, 37.65, 26.09, 26.00, 25.23, 18.74, 18.23, ꢁ4.37,
ꢁ4.49, ꢁ5.28, ꢁ5.31. HRMS calcd for C22H43N3O4Si2 + H+:
470.2865, found: 470.2907.
4.4. 20-Deoxy-30,50-di-O-tert-butyldimethylsilyl-1-methyl-N,N0-
dibenzoyl-pseudocytidine (10)
A
mixture of 20-deoxy-30,50-di-O-tert-butyldimethylsilyl-1-
methylpseudocytidine (9) (75 mg, 0.16 mmol), benzoyl chloride
(74 L, 0.64 mmol) and anhydrous pyridine (0.5 mL) in CH2Cl2
l
(5 mL) was stirred at rt overnight. Volatiles were removed by ro-
tary evaporation under reduced pressure. The product was purified
by flash chromatography (silica, ethyl acetate/hexane = 1:5 to 1:3)
to give 10 as a white solid (80 mg, 74%). TLC (EtOAc/hexane, 1:3):
Rf 0.3. 1H NMR (300 MHz, CDCl3) d 8.28 (m, 2H), 8.12 (m, 2H), 7.36–
7.64 (m, 7H), 5.35 (dd, 1H, J = 10.2, 5.4 Hz), 4.38 (m, 1H), 3.98 (m,
1H), 3.60–3.80 (m, 2H), 3.43 (s, 3H), 2.59 (m, 1H), 1.70 (m, 1H),
0.97 (s, 9H), 0.91 (s, 9H), 0.14 (s, 3H), 0.12 (s, 3H), 0.10 (s, 6H).
13C NMR (75 MHz, CDCl3) d 179.44, 158.85, 148.94, 141.75,
137.34, 133.90, 132.63, 130.41, 130.10, 128.69, 128.33, 116.91,
87.82, 74.51, 74.16, 63.87, 43.22, 37.07, 26.14, 26.02, 18.56,
18.24, ꢁ4.40, ꢁ4.47, ꢁ5.11, ꢁ5.13.
Figure 5. Acid-catalyzed hydrolysis of oligonucleotides containing 20-deoxyisocyti-
dine and 20-deoxy-5-methylpseudocytidine. Starting 14-mers moved in electro-
phoresis at slightly different rates.
30 min. The solution was then added to a mixture of glycal 4
(280 mg, 0.80 mmol), 5-iodo-1-methyluracil (5) (200 mg,
0.80 mmol), and tributylamine (0.28 mL, 0.12 mmol) in DMF
(10 mL). The resulting mixture was stirred at 60 °C for 18 h. After
being cooled, the mixture was treated with HOAc (0.2 mL) and
TBAF (1 M in THF, 2 mL) and stirred at rt for 1 h. Volatiles were re-
moved by rotary evaporation under reduced pressure. The product
was purified by flash chromatography (silica, gradient CH2Cl2/
MeOH = 15:1 to 10:1). The appropriate fraction (by TLC) was col-
lected, evaporated and dissolved in acetic acid/acetonitrile (7 mL/
7 mL). To this solution were added NaBH(OAc)3 (370 mg,
1.75 mmol) at 0 °C. The mixture was stirred for 2 h, volatiles were
removed by rotary evaporation under reduced pressure, and the
residue was resolved by flash chromatography (silica, gradient
CH2Cl2/MeOH = 7:1 to 4:1) to give 7 as a white solid (100 mg,
51%). TLC (CH2Cl2/MeOH, 5:1): Rf 0.25.
4.5. 20-Deoxy-1-methyl-N-benzoyl-pseudocytidine (11)
A
mixture of 20-deoxy-30,50-di-O-tert-butyldimethylsilyl-1-
methyl-N,N0-dibenzoyl-pseudocytidine (10) (310 mg, 0.46 mmol)
and triethylamine trihydrofluoride (0.6 mL) in THF (15 mL) was
stirred at 45 °C for two days. Volatiles were removed by rotary
evaporation under reduced pressure, and the product was purified
by flash chromatography (silica, CH2Cl2/MeOH = 15:1 to 12:1) to
give 11 as a white solid (137 mg, 86%). TLC (CH2Cl2/MeOH, 12:1):
Rf 0.3. 1H NMR (300 MHz, CDCl3/CD3OD) d 8.16 (d, 2H, J = 7.8 Hz),
7.70 (s, 1H), 7.3–7.5 (m, 3H), 5.24 (m, 1H), 4.21 (m, 1H), 3.84 (m,
1H), 3.5–3.7 (m, 2H), 3.37 (s, 3H), 2.4–2.6 (m, 1H), 1.8–1.9 (m, 1H).
4.2. 30,50-Di-O-tert-butyldimethylsilyl-pseudothymidine (8)
To a mixture pseudothymidine (7) (100 mg, 0.41 mmol) and
imidazole (113 mg, 1.65 mmol) in anhydrous DMF (4 mL) was
added TBDMSCl (154 mg, 1.03 mmol). The mixture was stirred at
rt overnight, volatiles were removed by rotary evaporation under
reduced pressure, and the product was isolated by flash chroma-
tography (silica, gradient ethyl acetate/hexane = 1:2 to 1:1) to give
8 as a colorless viscous liquid (180 mg, 93%). TLC (EtOAc/hexane,
1:1): Rf 0.6. 1H NMR (300 MHz, CDCl3) d 8.43 (br s, 1H), 7.32 (d,
1H, J = 1.2 Hz), 5.02 (m, 1H), 4.32 (m, 1H), 3.87 (m, 1H), 3.60–
3.72 (m, 2H), 3.34 (s, 3H), 2.31 (ddd, 1H, J = 12.8, 6.0, 2.4 Hz),
1.70–1.82 (m, 1H), 0.90 (s, 9H), 0.89 (s, 9H), 0.07 (s, 6H), 0.06 (s,
6H).
4.6. 20-Deoxy-50-dimethoxytrityl-1-methyl-N-benzoyl-
pseudocytidine (12)
A
mixture of 20-deoxy-1-methyl-N-benzoyl-pseudocytidine
(11) (129 mg, 0.37 mmol) and 4,40-dimethoxytritylchloride
(128 mg, 0.38 mmol) in anhydrous pyridine (6 mL) was stirred at
rt overnight. Volatiles were removed by rotary evaporation under
reduced pressure. The product was purified by flash chromatogra-
phy (silica, EtOAc/hexane = 1:2 to 1:1 then CH2Cl2/MeOH = 20:1) to
give 12 as a white solid (160 mg, 66%). TLC (CH2Cl2/MeOH, 20:1): Rf
0.2. 1H NMR (300 MHz, CDCl3) d 8.2–8.3 (m, 2H), 7.61 (d, 1H,
J = 1.5 Hz), 7.2–7.6 (m, 12H), 6.8–6.9 (m, 4H), 5.39 (t, 1H,
J = 7.5 Hz), 4.45 (m, 1H), 4.06 (m, 1H), 3.80 (s, 6H), 3.35 (m, 1H),
3.17 (s, 3H), 2.6–2.7 (m, 1H), 2.0–2.2 (m, 1H). 13C NMR (75 MHz,
CDCl3) d 158.82, 158.73, 148.78, 144.88, 142.36, 137.22, 135.95,
132.59, 130.23, 129.97, 128.34, 128.29, 128.12, 127.23, 116.74,
113.39, 86.55, 85.47, 73.80, 73.73, 63.99, 55.43, 42.47, 36.81.
4.3. 20-Deoxy-30,50-di-O-tert-butyldimethylsilyl-1-
methylpseudocytidine (9)
To a mixture of 30,50-di-O-tert-butyldimethylsilyl-pseudothymi-
dine (8) (200 mg, 0.42 mmol), 2,4,6-triisopropylbenzenesulfonyl
chloride (257 mg, 0.85 mmol) and DMAP (51 mg, 0.42 mmol) in
CH3CN (15 mL) was added triethylamine (0.24 mL, 1.70 mmol) at

3732
H.-J. Kim et al. / Bioorg. Med. Chem. 17 (2009) 3728–3732
4.7. 20-Deoxy-50-dimethoxytrityl-1-methyl-N-benzoyl-
pseudocytidine 30-(2-cyanoethyl diisopropylphosphoramidite)
(13)
4.10. Thermal DNA duplex denaturation
Thermal DNA duplex denaturation experiments were done in
0.45 M NaCl, 0.045 M sodium citrate buffer (pH 7.9). Each oligo-
nucleotide was present at 1.6 lM. Absorbance was monitored at
260 nm over a range of 25 °C to 85 °C with a change in tempera-
ture of 1 °C/minꢁ1 for five heating and cooling cycles on a Varian
Cary 300 (Palo Alto, CA). Initial heating and cooling cycles were
discarded and the Tm was determined by averaging the tempera-
ture of the maximum derivatives of each cooling and heating
cycle.
To a mixture of 20-deoxy-50-dimethoxytrityl-1-methyl-N-ben-
zoyl-pseudocytidine (12) (120 mg, 0.185 mmol) and DIPEA
(97
lL, 0.55 mmol) in anhydrous CH2Cl2 (5 mL) was added 2-cya-
noethyl diisopropylchlorophosphoramidite (62
l
L, 0.278 mmol)
at 0 °C. The mixture was allowed to warm to rt and stirred for
2 h. The mixture was poured into a saturated sodium bicarbonate
solution (10 mL) and extracted with CH2Cl2. The organic layer
was washed with brine, dried over anhydrous sodium sulfate and
evaporated. The product was purified from the residue by flash
chromatography (neutral silica, EtOAc/hexane = 1:3 to 1:1 with
0.5% Et3N) to give diastereomeric mixture of 13 as a white foam
(123 mg, 78%). TLC (EtOAc/hexane, 1:1): Rf 0.5, 0.53. 1H NMR
(300 MHz, CDCl3) d 8.2–8.3 (m, 2H), 7.2–7.8 (m, 14H), 6.8–6.9
(m, 4H), 5.39 (m, 1H), 4.57 (m, 1H), 4.19 (m, 1H), 3.79 (m, 6H),
3.5–3.7 (m, 3H), 3.2–3.5 (m, 2H), 3.15 (m, 3H), 2.7–2.9 (m, 1H),
2.4–2.6 (m, 2H), 2.0 (m, 1H), 1.6 (m, 1H), 1.0–1.3 (m, 12H). 31P
NMR (121 MHz, CDCl3) d 150.26 (s), 149.36 (s).
4.11. Acid stability
The 50-radiolabelled oligonucleotides (10
lL, 0.2 pmol/lL)
were mixed with an equal volume of 0.1 M acetic acid. Mixtures
were incubated for 0, 15, 30, 60, or 90 min at 95 °C in a thermo-
cycler with a heated lid. The tubes containing the mixtures were
then heated with the tubes open to let volatile materials evapo-
rate (2 min). Two volumes (20 lL) of ammonium hydroxide
(0.1 M) were then added, and heating continued at 95 °C for
5 min. The tubes were then opened, and the NH3 was evaporated
by heating at 95 °C for 2 min. Cleavage products were resus-
pended in formamide (3ꢂ) and resolved by PAGE (20%, 0.3 pmol
samples).
4.8. Oligonucleotide synthesis
Oligonucleotide synthesis was performed by the DNA synthe-
sizer ABI 394, at the 1.0
the manufacturer’s recommended procedure except extended
(600 s) coupling time for isoG, isoC and C. All natural (A, G, C,
lmol scale. The synthesis was done using
4.12. Thermal stability
w
and T) and unnatural (isoG and isoC) nucleobase phosphoramidites
were purchased from Glen Research. At the completion of the syn-
thesis, all protective groups in oligonucleotides were removed by
heating in 30% ammonium hydroxide at 55 °C overnight. The mix-
To determine stability to heating at near neutral pH, oligonucle-
otides (10 lL, 0.2 pmol/lL) in Tris buffer (10 mM, pH 8.5) were
incubated for 0, 15, 30, 60, or 90 min at 95 °C in a thermocycler
with a heated lid. The tubes containing the mixtures were then
heated with the tubes open to let volatiles evaporate (2 min).
Cleavage products were resuspended in formamide (3ꢂ) and
resolved by PAGE.
ture was then filtered through 0.2 lm filter and the remaining
ammonia was removed by rotary evaporation under reduced pres-
sure at rt. The sample was then diluted with water and lyophilized.
The residue after lyophilization was dissolved in water and filtered
through 0.2 lm filter. The oligonucleotide was purified from the
Acknowledgement
filtrate by ion exchange HPLC (Dionex DNAPac PA-100
9 ꢂ 250 mm Semi-Prep column, eluent A = 25 mM NaOH, eluent
B = 25 mM NaOH, 1.0 M NaCl, gradient from 10% to 80% B in
40 min, flow rate 3 mL/min). The appropriate fractions were col-
lected, neutralized by aqueous acetic acid, desalted by Sep-PaK car-
tridges (Waters) and lyophilized. The purified oligonucleotides
This work was supported by the National Institutes of Health
1R01GM081527-01 and a grant from SAB.
References and notes
were dissolved in water at a concentration of 50
lM.
1. Sismour, A. M.; Benner, S. A. Nat. Rev. Genet. 2005, 6, 533.
2. Rich, A. In Horizons in Biochemistry; Kasha, M., Pullman, B., Eds.; Academic
Press: NY, 1962; p 103.
3. Elbeik, T.; Markowitz, N.; Nassos, P.; Kumar, U.; Beringer, S.; Haller, B.; Ng, V. J.
Clin. Microbiol. 2004, 42, 3120.
4.9. HPLC profile of synthetic oligonucleotides
4. Benner, S. A. Acc. Chem. Res. 2004, 37, 784.
HPLC condition; Dionex DNAPac PA-100 4 ꢂ 250 mm analytical
column, eluent A = 25 mM NaOH, eluent B = 25 mM NaOH, 1.0 M
NaCl, gradient from 10% to 80% B in 50 min, flow rate 0.5 mL/min
5. Sismour, A. M.; Benner, S. A. Nucleic Acids Res. 2005, 33, 5640.
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5’—C A C N1 A C T T T C T C C T-3’
3’-TG T G N2 T G A A AG A G G—5’
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N1
Retention time
N2
Retention time
iG
iC
36.6
34.4
34.3
iG
iC
wC
43.5
41.8
41.8
w
C