Synthesis and biochemical activity of new oligonucleotide analogs

Article abstract of DOI:10.1080/10426500701734745

Borane phosphonate deoxyoligonucleotides are synthesized from 5′-O-benzhydroxybis(trimethylsilyloxy)silyl-2′-deoxynucleoside- 3′-phosphoramidites. The exocyclic amines of adenine and cytosine are protected with dimethoxytrityl and trimethoxytrityl, respec

Full text of DOI:10.1080/10426500701734745

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Synthesis and Biochemical
Activity of New Oligonucleotide
Analogs
Heather Brummel McCuen a , Mary S. Noe b
,
Magdalena Olesiak c , Agnieszka B. Sierzchala a
Marvin H. Caruthers c & Adrian P. Higson d
a Agilent Laboratories, Boulder, Colorado, USA
,
b University of San Diego, San Diego, California, USA
c Department of Chemistry and Biochemistry,
University of Colorado Boulder, Boulder, Colorado,
USA
d Ultrafine, Manchester, United Kingdom
Published online: 21 Jun 2008.
To cite this article: Heather Brummel McCuen , Mary S. Noe , Magdalena Olesiak ,
Agnieszka B. Sierzchala , Marvin H. Caruthers & Adrian P. Higson (2008): Synthesis and
Biochemical Activity of New Oligonucleotide Analogs, Phosphorus, Sulfur, and Silicon
and the Related Elements, 183:2-3, 349-363
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Phosphorus, Sulfur, and Silicon, 183:349–363, 2008
Copyright © Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426500701734745
Synthesis and Biochemical Activity of New
Oligonucleotide Analogs
Heather Brummel McCuen,1 Mary S. Noe,2
Magdalena Olesiak,3 Agnieszka B. Sierzchala,1
Marvin H. Caruthers,3 and Adrian P. Higson4
1Agilent Laboratories, Boulder, Colorado, USA
2University of San Diego, San Diego, California, USA
3Department of Chemistry and Biochemistry, University of Colorado
Boulder, Boulder, Colorado, USA
4Ultrafine, Manchester, United Kingdom
Borane phosphonate deoxyoligonucleotides are synthesized from 5ꢀ-O-
benzhydroxybis(trimethylsilyloxy)silyl-2ꢀ-deoxynucleoside-3ꢀ-phosphoramidites.
The exocyclic amines of adenine and cytosine are protected with dimethoxytrityl
and trimethoxytrityl, respectively, whereas guanine protection is with N2-(9-
fluorenylmethoxycarbonyl) or N2-trimethoxytrityl. Thymine is protected with
N3-anisoyl. Using these synthons and under standard conditions via activation
with tetrazole, condensations in excess of 99% are observed. Oxidation with
•
either THF BH or a peroxyanion solution followed by cleavage of the silyl ether
3
with fluoride completes a cycle. Following synthesis of an appropriate oligomer,
protecting groups are removed using sequentially acetic acid, a dithiolate and
ammonium hydroxide. Oligodeoxynucleotide 10 mers and 12 mers having any
combination of borane phosphonate and phosphate internucleotide linkages as
well as all four 2ꢀ-deoxynucleotides are synthesized in isolated yields of 70–80%
and characterized by phosphorus NMR and mass spectrometry.
Keywords Borane phosphonate DNA
INTRODUCTION
Oligodeoxyribonucleotides (ODNs) bearing internucleotide borane
phosphonate linkages (Figure 1, compound 1) have been of consider-
able interest for applications in diagnostic and therapeutic areas as
This work was supported by the University of Colorado. The technical assistance of
Richard Shoemaker in the NMR Laboratory is gratefully acknowledged.
Address correspondence to Marvin H. Caruthers, Department of Chemistry and
Biochemistry, University of Colorado Boulder, Boulder, CO 80309-0215, USA. E-mail:
Marvin.Caruthers@colorado.edu
349

350
H. B. McCuen et al.
FIGURE 1 Borane phosphonate (a) and chimeric borane phospho-
nate/phosphate (b) ODNs. B = thymine, cytosine, adenine, and guanine. X,
Y = combinations of phosphate and borane phosphonate linkages.
they mimic natural DNA in various biological processes.1 The chal-
lenge over the years has been the development of a synthesis strategy
that generates ODNs having all four bases and in yields sufficient to
allow at least the preparation of 10mers. Until recently, the most suc-
cessful strategy was the use of unprotected bases where 10 mers can
be prepared in 20–30% yield.2 An alternative approach that may prove
useful involves the initial formation of mononucleotide borane phos-
phonates where these monomers are then joined via a phosphotriester
condensation route to generate dimers with yields from 72–92%.3 Both
methods must be improved considerably in order to generate a route
for preparing oligomers rapidly in high yields on supports.
RESULTS
The synthetic challenge with this analog is the development of an or-
thogonal synthesis strategy. Specifically there are several key consid-
erations. Of utmost importance, all base and phosphorus protecting
groups must be stable toward boronation under conditions where these
2ꢀ-deoxynucleoside bases are not reduced or form irreversible complexes
with borane. A second is the design of a transient 2ꢀ-deoxynucleoside hy-
droxyl protecting group, preferably 5ꢀ, which can be removed following

Synthesis and Biochemical Activity of New Oligonucleotide Analogs
351
each condensation cycle under conditions that do not modify the grow-
ing, support bound borane phosphonate oligonucleotide. Additionally
the ideal synthesis strategy would not only generate borane phospho-
nate ODNs but also chimeric oligomers having other internucleotide
linkages such as phosphate (compound 2).
To overcome these challenges, we have developed a new strategy
for synthesizing borane phosphonate DNA. Previous research has
shown that the 5ꢀ-dimethoxytrityl group, which transiently protects
each synthon during natural DNA chemical synthesis, is incompati-
ble with the preparation of borane phosphonate DNA.4 This observa-
tion forced us to explore alternative strategies. On the basis of earlier
research,5 we prepared 2ꢀ-deoxynucleosides having 5ꢀ-O-[benzhydroxy
bis(trimethylsilyloxy) silyl protection (Figure 2, compounds 3a–e) and
discovered that the silyl ether could be removed under conditions com-
patible with the synthesis of borane phosphonate DNA. As a result,
this group has been substituted for dimethoxytrityl as a transient 5ꢀ-
protecting group.
Another serious challenge is protection of the 2ꢀ-deoxynucleoside
bases. This is because borane reagents reduce the commonly used
amide protecting groups to N-alkyl or aryl exocyclic amines, which
cannot be removed from the bases, or form stable, irreversible base
adducts. This problem has been solved by using N-trityl protection
on the exocyclic amines—a strategy that is common in the pep-
tide field6 but used only sparingly in polynucleotide synthesis.7,8
Thus for adenine and cytosine, the exocyclic amines are protected
with dimethoxytrityl and trimethoxytrityl, respectively. For guanine,
two strategies have been studied. Initially we successfully used the
FIGURE 2 5ꢀ-O-Silyl-2ꢀ-deoxynucleoside-3ꢀ-phosphoramidites.
B
=
(3a)
N3-anisoylthymine; (3b) N4-trimethoxytritylcytosine; (3c) N6-dimethoxy-
trityladenine; (3d) N2-(9-fluorenylmethoxycarbonyl) guanine; and (3e) N2-
trimethoxytritylguanine. R = diphenylmethyl.

352
H. B. McCuen et al.
N2-(9-fluorenylmethoxycarbonyl)protecting group. However, overall
yields are low (15% to the completely protected 2ꢀ-deoxynucleoside)
and there is a risk, although minimal, of borane reduction. We, there-
fore, turned to the use of N2-trimethoxytrityl protection7 where the
overall yield is much higher (45–50% to the completely protected 2ꢀ-
deoxynucleoside) and there is no risk of base reduction or complex for-
mation with borane. We also introduced anisoyl on the N3 of thymine
in order to prevent N3 methylation via the methylphosphate protecting
group.
Using these appropriately protected 2ꢀ-deoxynucleoside phospho-
ramidites (Figure 2, compounds 3a–e), a new high yielding synthe-
sis cycle has been developed (Figure 3 and Table I). Starting with an
appropriately protected 2ꢀ-deoxynucleoside attached to polystyrene
(compound 4), the first step is condensation with 3a–e in anhydrous
acetonitrile and tetrazole to generate a family of dimers (compound
5) having a phosphite triester internucleotide linkage. These dimers
8
•
are then reacted with either THF BH3 or a peroxyanion solution. Re-
moval of 5ꢀ-silyl protection with triethylammonium hydrogen fluoride
(TEAHF) generates a family of dinucleotides and having any of the four
TABLE I Synthesis Cycle for Borane Phosphonate ODNs
Reaction
Coupling
Wash/reagents/solvents
Time (sec)
0.1 M Compound 3 in acetonitrile and 0.45 M
tetrazole in acetonitrile (1:1)
Acetonitrile
5 to column,
60 wait
30
30
Wash
Boranation
25 mM BH3·THF in THF
Peroxyanion Solutiona
or
Oxidation
Wash
120
30
30
45
Tetrahydrofuran
Dichloromethane,
Acetonitrile
Dimethylformamide
35
5ꢀ-Deprotection 1.1 M HF/1.1 M Triethylamine/0.02 M
25 to column,
45 wait
N-methyldiethanolamine in dimethylformamide
(pH 9)b
Wash
Dimethylformamide
Acetonitrile
40
60
3
0.4 M Tetrazole in Acetonitrile
aPeroxyanion Solution: Solution A = 3% (w/v) aqueous LiOH (10 mL), 1.5 M
2-amino-2-methyl-1-propanol in water (15 mL), and dioxane (17.5 mL). Solution B =
m-chloroperbenzoic acid (1.78 g), dioxane (32.5 mL), and aqueous 30% hydrogen
peroxide (12 mL). Equal volumes are mixed just prior to synthesis.

353

354
H. B. McCuen et al.
bases and either a P-IV phosphonium borane adduct or a phosphate tri-
ester linkage to oligonucleotides (compound 6). These dimers can then
be extended using the same repetitive cycle to generate a chimeric ODN
having any defined sequence and length, as well as borane phosphonate
and phosphate internucleotide linkages. For the preparation of a simi-
lar ODN having only borane phosphonate internucleotide linkages, the
synthesis proceeds as outlined in Figure 3 except that oxidation is ex-
•
clusively with THF BH3.
The removal of protecting groups so as to generate compounds 1
or 2 proceeds via a three step procedure. Initially and with the ODN
attached to the support (compound 6), 80% acetic acid is used to elimi-
nate trityl groups from adenine, cytosine, and guanine (the P-IV borane
adduct is compatible with acetic acid). Next the oligomer is treated with
a dithiolate9 to remove internucleotide methyl protection. Finally am-
monium hydroxide eliminates carbamate, if compound 3d is used in the
synthesis cycle, the anisoyl group from thymine and generates 1 or 2 by
cleavage of the ODN from the support. Purification is by reverse phase
HPLC. Typical results for total reaction mixtures are shown in Figure 4
(10 mer) and 5 (12 mer) for ODNs having all four bases and borane
phosphonate internucleotide linkages. The major peaks in each case
(excluding the first, anisic acid peak) are the products (99% coupling
yields, isolated yields 75–88%). As expected from the many P-chiral
FIGURE 4 Reverse Phase HPLC analysis of the total crude reaction mixture
from the synthesis of compound 14. (A) Gel electrophoresis results from total,
crude reaction mixtures. Lanes 1–5, d(TpTp)4TpT, 11, 12, 13 and 14, respectively.
(B) Phosphorus NMR of compound 14.

Synthesis and Biochemical Activity of New Oligonucleotide Analogs
355
centers and the resulting large number of stereoisomers, these product
peaks are broad.
These ODNs have been fully characterized by NMR, mass spectral
analyses, and polyacrylamide gel electrophoresis. Phosphorus NMR
analyses display a broad signal at 96 ppm (borane phosphonate) and
a sharp peak at -2 ppm when some of the internucleotide linkages are
phosphate (Figure 1, compound 2). By phosphorus NMR, when all in-
ternucleotide linkages are borane phosphonate (Figure 1, compound
1), phosphate cannot be detected. For example, the insets to Figures 4
and 5 display the phosphorus NMR spectra of the 10 mer and 12 mer,
respectively, as prepared from the pooled product fractions of the re-
verse phase HPLC column eluates. In each case, phosphate peaks are
not observed. 11B NMR spectra for all oligomers synthesized consist
of a broad signal at −40 ppm which is characteristic of the borane
phosphonate linkage. Table II lists mass data for ODNs having vari-
ous combinations of 2ꢀ-deoxynucleoside bases and internucleotide phos-
phate and borane phosphonate linkages. The observed masses for all
ODNs correspond to those as calculated. These results show that ODNs
as prepared by this method have the predicted structures and are free
of any boronated bases or sugars. When further characterized by gel
electrophoresis (Figure 4A), only one major band for each ODN, which
corresponds to the product, is observed. This result is especially strik-
ing as total reaction mixtures are analyzed. Since only one major, in-
tense band is observed for each reaction mixture, very few oligomers of
lesser length are present. These results, as do the reverse phase HPLC
FIGURE 5 Reverse Phase HPLC analysis of the total crude reaction mixture
from the synthesis of compound 15. The inset is the phosphorus NMR of com-
pound 15.

356
H. B. McCuen et al.
TABLE II Mass Spectrum Analysis of Oligodeoxynucleotides
Molecular weight
No
ODNa
d[(TpTpTb)4TpT]
Calculated
Observed
7
8
9
10
11
12
13
14
15
4185.7
4179.8
4360.8
4354.9
2960.8
3005.9
2885.8
2973.9
3609.5
4183.4b
4177.5b
4360.7b
4355.2b
2954.5c
3000.7c
2881.5c
2967.5c
3617.0c
d[(TbTp)6TbT]
d[(GpTpGbTpGpTb)2GpT]
d[(GbTpGbTp)3GbT]
d(Tb)9T
d[(AbTb)4AbT
d[(CbTb)4CbT]
d(TbCbTbTbAbCbTbGbAbT)
d(AbAbCbGbAbTbAbTbCbGbTbT)
ap = phosphate; b = borane phosphonate. bPerseptive Biosystems Voyager
Biospectrometry Workstation using a previously published procedure.
cHPLC-ESI-Q-TOF-MS Instrument System.
analyses, further indicate that coupling yields are extremely high and
lead primarily only to the product ODNs.
Preliminary results suggest that borane phosphonate ODNs may
prove useful for both biochemical research and therapeutic applications.
For example, borane phosphonate ODNs are resistant to degradation
by exonucleases and DNase I. Results with DNase I are summarized in
Figure 6. Under conditions where natural DNA is completely degraded,
ODNs having borane phosphonate linkages (fully modified, every other
or every third linkage) are essentially resistant to degradation. Similar
results not shown were obtained with snake venom phosphodiesterase.
When borane phosphonate ODNs are allowed to form duplexes with
complementary RNA and then treated with E. coli RNase H, the RNA is
degraded (Figure 7 summarizes several experiments). Under conditions
where natural DNA stimulates complete RNA degradation by RNase
H, similar experiments with fully modified borane phosphonate ODNs
lead to 38% RNA hydrolysis. Oligomers having borane phosphonate
linkages at every other or every third position are almost as satisfactory
as natural DNA for stimulating RNase H degradation of RNA.
DISCUSSION
These results demonstrate that a new, high yielding method has been
developed for the synthesis of borane phosphonate DNA. The synthe-
sis strategy also demonstrates that chimeras containing both borane
phosphonate and phosphate internucleotide linkages can be prepared
by these procedures. Because the approach involves a simple oxidation

Synthesis and Biochemical Activity of New Oligonucleotide Analogs
357
FIGURE 6 A summary of experiments addressing the lability of borane phos-
phonate DNA to DNase I. dTBH3 FM, dTBH3 EO, dTBH3 E3: Borane phos-
phonate ODNs having fully modified (FM), every other (EO) and every third
(E3) borane phosphonate internucleotide linkages. dTSI FM, dTSI EO, dTSI
E3: phosphorothioate ODNs having fully modified (FM), every other (EO) and
every third (E3) phosphorothioate internucleotide linkages. All other linkages
are phosphate. All ODNs including the unmodified oligomer (dT) and comple-
mentary dA are twelve nucleotides in length.
step as the key variable for the synthesis of this chimera, other analogs
containing phosphorothioate or selenophosphate should also be possi-
ble. Of considerable importance is the observation that borane phospho-
nate ODNs are active with RNase H. An especially exciting observation
FIGURE 7 A summary of experiments addressing the ability of borane phos-
phonate ODNs to stimulate the degradation of complementary RNA by E. coli
RNase H. All oligomers are 12 nucleotides in length. See the legend to Figure
6 for abbreviations.

358
H. B. McCuen et al.
is that ODNs having every other linkage or every third linkage as
borane phosphonate are very active in stimulating RNase H activity.
These ODNs are essentially resistant to degradation by nucleases as
well. When taken together, these results strongly suggest that borane
phosphonate chimeras having a minimum number of borane phospho-
nate linkages may prove extremely useful for cell biology research and
for various therapeutic drug applications.
EXPERIMENTAL
Synthesis of N6-Dimethoxytrityl-2ꢀ-deoxyadenosine
2ꢀ-Deoxyadenosine (2.69 g, 10 mmol) was coevaporated three times with
pyridine and dried in vacuo for 12 h. Anhydrous pyridine (50 mL) and
chlorotrimethylsilane (50 mmol) were added. After the mixture had
been stirred at room temperature for 2 h, dimethoxytrityl chloride (3.7
g,11 mmol) was added. The reaction was stirred overnight (∼16 h) at
room temperature. Water (60 mL) and aqueous ammonium hydroxide
(2 mL, 28–30%) were added and the reaction mixture was stirred for
30 min. The crude product was extracted into dichloromethane. The
organic layer was washed two times with a 5% aqueous solution of
sodium bicarbonate and dried with anhydrous sodium sulfate. The or-
ganic layer containing product was filtered from salts and purified by
column chromatography using chloroform/pyridine (99.9:0.1) and a gra-
dient of methanol (0–6%).
Yield 99%. 1H NMR (DMSO-d6) δ 8.42 (s, 1H), 8.32 (s, 1H), 7.28–7.26
(m, 5H), 7.19 (d, 4H), 6.84 (d, 4H), 6.32 (t, 1H), 5.31 (d, 1H), 5.12 (t,
1H), 3.86–3.84 (m, 1H), 3.71 (s, 6H), 3.61–3.47 (m, 2H), 2.79–2.74 (m,
1H), 2.27–2.22 (m, 1H); 13C NMR (DMSO-d6) δ 157.69, 153.68, 151.16,
148.07, 145.34, 140.37, 137.27, 129.77, 128.39, 127.71, 126.47, 121.05,
113.00, 88.04, 84.12, 70.92, 69.61, 61.83, 54.99; HRMS (FAB) calcd. for
C31H31N5O5 (M+) 553.2325. found 553.2309.
Synthesis of N4-Trimethoxytrityl-2ꢀ-deoxycytidine
2ꢀ-Deoxycytidine (2.64 g, 10 mmol) was coevaporated three times with
pyridine and dried in vacuo for 12 h. Anhydrous pyridine (50 mL) and
chlorotrimethylsilane (50 mmol) were added. After the mixture was
stirred at room temperature for 2 h, trimethoxytrityl chloride (3.85 g,
10.5 mmol) was added. The reaction was stirred overnight (∼16 h) at
room temperature. Water (60 mL) and aqueous ammonium hydrox-
ide (2 mL, 28–30%) were added, and the reaction mixture was stirred
for 30 min. The crude product was extracted into dichloromethane,

Synthesis and Biochemical Activity of New Oligonucleotide Analogs
359
the organic layer was washed two times with 5% aqueous solution
of sodium bicarbonate, and dried with anhydrous sodium sulfate. The
product was filtered and purified by column chromatography using chlo-
roform/pyridine (99.9:0.1) with a gradient of methanol (0–6%).
1
Yield 95%. H NMR (DMSO-d6) δ 8.29 (s, 1H), 7.69 (d, 1H), 7.10 (d,
4H), 6.82 (d, 4H), 6.21 (d, 1H), 6.04 (t, 1H), 5.17 (d, 1H), 4.94 (t, 1H),
4.16–4.14 (m, 1H), 3.72 (s, 9H), 3.51–3.48 (m, 2H), 2.05–1.86 (m, 2H);
13C NMR (DMSO-d6) δ 163.29, 157.37, 154.07, 139.53, 137.26, 129.78,
112.70, 96.38, 87.18, 84.67, 70.65, 68.92, 61.51, 54.98; HRMS (FAB)
calcd. for C31H33N3O7 (M+) 559.2319, found 559.2331.
Synthesis of N2-(9-Fluorenylmethoxycarbonyl)-
2ꢀ-deoxyguanosine
2ꢀ-Deoxyguanosine (3.11 g, 11.0 mmol) was twice co-evaporated with
50 mL pyridine and then suspended in 80 mL pyridine. The reac-
tion was started by the dropwise addition of chlorotrimethylsilane
(6.5 mL, 75 mmol) with a syringe. The reaction proceeded for 1 h dur-
ing which the deoxynucleoside was taken into solution. At this point
9-fluorenylmethyl chloroformate (3.5 g, 14.3 mmol) was added and the
solution stirred for another 1.5 h. When complete, the reaction mix-
ture was quenched with 20 mL water and stirring for 1 h. Follow-
ing an aqueous work-up, the crystalline product was dissolved into
dichloromethane, filtered and washed with chloroform. The product is
a white solid.
1
Yield 65%. H NMR (DMSO-d6): δ 2.6 (m, 2H), 3.5 (m, 2H), 3.8 (d,
J = 2.5 Hz), 4.3 (m, 1H), 4.4 (d, J = 6.2 Hz, 2H), 5.3 (m, 1H), 6.2 (t, 1H),
7.3 (t, 2H), 7.4 (t, 2H), 7.8 (d, J = 7.0 Hz, 2H), 7.9 (d, J = 7.6 Hz, 2H).
MS: calcd. = 489, Found (ESI+) = 512 (M+Na+).
Synthesis of 5ꢀ-O-[benzhydroxy-bis (trimethylsilyloxy]silyl-2ꢀ
-deoxynucleoside
N-protected 2ꢀ-deoxynucleoside (10 mmol) was dried in vacuo for 6 h and
then dissolved in anhydrous N,N-dimethylformamide (100 mL). Imida-
zole (20 mmol) was added to the mixture and the flask was placed on ice
and stirred. Benzhydroxy-bis(trimethylsilyloxy)silyl chloride (10 mmol)
was added slowly over 1 h via syringe. The flask was then removed from
ice and allowed to stir at room temperature for ∼4 h. The reaction was
monitored by TLC and additional aliquots of the silyl chloride (1 mmol)
were added until there was no presence of the starting material. Dis-
tilled water (60 mL) was added and the solvent was removed in vacuo
to a final volume of 50 mL. The remaining solution was dissolved in

360
H. B. McCuen et al.
dichloromethane and rinsed with an aqueous solution of 5% sodium bi-
carbonate saturated with sodium chloride. The organic layer was dried
over anhydrous sodium sulfate. The product was filtered and purified by
column chromatography. Elution initially was with chloroform/benzene
(9:1) followed by a gradient of methanol in chloroform (for N-trityl ana-
logues, 0.1% pyridine was added to the eluting system). The product
eluted in 5–10% methanol.
5ꢀ-O-[benzhydroxy-bis(trimethylsilyloxy)silyl]-N3anisoyl-2ꢀ-
deoxythymidine
Yield 65.8%; 1H NMR (CDCl3) δ 7.89 (d, 2H), 7.60 (s, 1H), 7.37–
7.22 (m, 10H), 6.94 (d, 2H), 6.31 (t, 1H), 5.94 (s, 1H), 4.32–4.29 (m,
1H), 3.90–3.87 (m, 1H), 3.86 (s, 3H), 3.82–3.76 (m, 2H), 2.26–2.20 (m,
1H), 1.98–1.92 (m, 1H), 1.88 (s, 3H), 0.10 (s, 18H); 13C NMR (CDCl3) δ
168.06, 165.30, 163.08, 149.53, 143.97, 143.90, 135.64, 133.26, 128.53,
127.64, 127.60, 126.51, 126.41, 124.44, 114.66, 111.03, 86.78, 85.11,
77.44, 72.18, 63.26, 55.83, 36.71, 13.05, 1.73; HRMS (ESI) calcd. for
C37H47N2O10Si3 (M+-H) 763.2544, found 763.2533.
5ꢀ-O-[benzhydroxy-bis(trimethylsilyloxy)silyl]-
N4trimethoxytrityl-2ꢀ-deoxycytidine
Yield 43.5%; 1H NMR (DMSO-d6) δ 8.40 (bs, 1H), 7.48 (d, 1H), 7.39–
7.22 (m, 10H), 7.15 (d, 6H), 6.83 (d, 6H), 6.27 (d, 1H), 6.06 (t, 1H),
5.96 (s, 1H), 5.26 (d, 1H), 4.11–4.05 (m, 1H), 3.80–3.78 (m, 1H), 3.71
(s, 9H), 3.68–3.61 (m, 2H), 2.05–1.98 (m, 1H), 1.74–1.68 (m, 1H), 0.05
(s, 18H); 13C NMR (DMSO-d6) δ 163.28, 157.39, 153.95, 144.06, 138.67,
137.25, 129.81, 129.56, 128.27, 127.16, 125.80, 112.69, 96.48, 86.15,
84.73, 76.07, 70.84, 68.97, 63.28, 54.95, 40.05, 1.43; HRMS (ESI) calcd.
for C50H62N3O10Si3 (M++H) 948.3737, found 948.3725.
5ꢀ-O-[benzhydroxy-bis(trimethylsilyloxy)silyl]-
N6dimethoxytrityl-2ꢀ-deoxyadenosine
Yield 67.9%; 1H NMR (DMSO-d6) δ 8.30 (s, 1H), 7.89 (s, 1H), 7.34–7.15
(m, 19H), 6.83 (d, 4H), 6.31 (t, 1H), 5.89 (s, 1H), 5.37 (d, 1H), 4.39–4.34
(m, 1H), 3.86–3.83 (m, 1H), 3.80–3.77 (m, 1H), 3.70 (s, 6H), 3.62–3.58
(m, 1H), 2.79–2.74 (m, 1H), 2.28–2.23 (m, 1H), −0.03 and −0.04 (2xs,
18H); 13C NMR (DMSO-d6) δ 157.67, 153.54, 151.14, 148.13, 145.29,
144.00, 140.02, 137.21, 129.66, 128.30, 128.09, 127.61, 126.99, 126.41,
125.75, 120.97, 112.94, 86.58, 83.72, 75.94, 70.61, 69.55, 63.21, 54.93,
38.39, 1.27; HRMS (ESI) calcd. for C50H60N5O8Si3 (M++H) 942.3744,
found 942.3773.

Synthesis and Biochemical Activity of New Oligonucleotide Analogs
361
5ꢀ-O-[benzhydroxy-bis(trimethylsilyloxy)silyl]-N2-(9-
fluorenylmethoxycarbonyl)-2ꢀ-deoxyguanosine
Yield 24.3%; 1H NMR (DMSO-d6) δ 11.68 (s, 1H), 11.33 (s, 1H), 7.95
(s, 1H), 7.92 (d, 2H), 7.82 (d, 2H), 7.44 (t, 2H), 7.37–7.27 (m, 10H),
7.22–7.19 (m, 2H), 6.22 (t, 1H), 5.92 (s, 1H), 5.37 (d, 1H), 4.52–4.46 (m,
2H), 4.36–4.31 (m, 2H), 3.86–3.83 (m, 1H), 3.77–3.64 (m, 2H), 2.48–
2.25 (m, 2H), 0.00 and −0.01 (2xs, 18H); 13C NMR (DMSO-d6) δ 155.02,
154.44, 148.60, 147.30, 144.02, 143.22, 140.73, 136.92, 128.14, 127.79,
127.06, 125.75, 125.40, 120.13, 119.91, 86.64, 82.88, 75.98, 70.48, 67.39,
63.28, 46.08, 39.62, 1.30; HRMS (ESI) calcd. for C44H52N5O9Si3 (M++H)
878.3067, found 878.3051.
Synthesis of 5ꢀ-O-silyl-N
protected-2ꢀ-deoxynucleoside-3ꢀ-O-phosphoramidites
Protected 2ꢀ-deoxynucleoside (2 mmol) was dried in vacuo for 6 h and
then dissolved in anhydrous dichloromethane (20 mL). Methyl tetraiso-
propylphosphorodiamidite (2.1 mmol) was added and the mixture was
stirred. Tetrazole (2 mmol) was added slowly over 1 h and the solution
was allowed to stir for an additional 3 h. A small amount of triethy-
lamine (approximately 0.4 mL) was added to neutralize the solution
and the solvent was removed in vacuo. The crude product was isolated
by chromatography with benzene followed by a gradient of ethyl ac-
etate (0–40 or 100%) in benzene containing 0.1% triethylamine. Tri-
ethylamine was excluded during purification of compound 1d in order
to prevent the elimination of the Fmoc protecting group.
Compound 3a (Bꢀ = Thy An). Yield 67.7%; 31P NMR (CDCl3) δ
150.51, 149.50; 13C NMR (CDCl3) δ168.10, 165.26, 163.07, 149.60,
144.03, 143.93, 135.62, 133.24, 128.52, 128.50, 127.65, 127.57, 126.56,
126.41, 126.39, 124.63, 114.65, 111.06, 86.90, 86.54, 86.48, 85.32, 77.28,
74.07, 63.39, 55.82, 50.65, 50.49, 43.28, 43.22, 43.16, 43.10, 40.21,
24.83, 24.76, 13.04, 1.76, 1.72; HRMS (ESI) calcd. for C44H65N3O11Si3P
(M++H) 926.3659, found 926.3632.
Compound 3b (Bꢀ = C TMT). Yield 61.3%; 31P NMR (CDCl3) δ 150.3,
150.06; 13C NMR (CDCl3) δ 165.40, 158.70, 155.46, 144.00, 140.88,
136.83, 129.89, 128.36, 128.33, 127.40, 127.33, 126.43, 126.38, 126.36,
113.64, 94.79, 86.16, 86.11, 76.94, 73.73, 73.56, 73.47, 73.30, 69.69,
62.96, 62.89, 55.28, 50.67, 50.51, 43.15, 43.08, 43.02, 42.96, 40.85,
23.08, 23.06, 1.61; HRMS (ESI) calcd. for C57H78N4O11Si3P (M++H)
1109.4707, found 1109.4667.
Compound 3c (Bꢀ = A DMT). Yield 86.4%; 31P NMR (CDCl3) δ 149.77,
δ 149.64; 13C NMR (CDCl3) δ 158.42, 154.30, 152.41, 148.80, 145.72,

362
H. B. McCuen et al.
144.26, 144.21, 138.53, 137.76, 130.32, 129.02, 128.41, 128.39, 128.04,
127.34, 126.95, 126.57, 126.51, 126.49, 121.48, 113.30, 86.89, 86.67,
84.63, 84.58, 76.98, 74.37, 74.20, 74.06, 73.89, 70.76, 63.20, 55.41, 50.77,
50.71, 50.60, 50.54, 43.17, 43.04, 39.63, 24.93, 24.86, 24.80, 24.77, 1.72,
1.69; HRMS (ESI) calcd. for C57H76N6O9Si3P (M++H) 1103.4713, found
1103.4715.
Compound 3d (Bꢀ = GFmoc). Yield 37.1%; 31P NMR (CDCl3) δ 149.84,
149.69; 13C NMR (CDCl3) δ 155.86, 153.55, 153.49, 148.47, 148.42,
146.44, 146.38, 144.13, 144.06, 143.02, 142.99, 142.96, 141.53, 137.16,
128.44, 128.42, 128.27, 127.47, 127.39, 126.52, 126.39, 124.98, 121.25,
120.36, 87.07, 86.77, 84.10, 77.07, 74.51, 74.33, 74.06, 68.46, 63.31,
63.24, 50.70, 50.53, 46.81, 43.15, 43.02, 40.19, 24.89, 24.83, 24.76,
1.71, 1.67; HRMS (ESI) calcd. for C51H68N6O10Si3P (M++H) 1039.4036,
found 1039.4038.
Synthesis of N2-Trimethoxytrityl-2ꢀ-deoxyguanosine
2ꢀ-Deoxyguanosine (3.2 g; 11 mmol) was twice co-evaporated with 50 mL
pyridine and dried in vacuo for 12 h. Anhydrous pyridine (60 mL) and
chlorotrimethylsilane (7.1 mL; 56 mmol) were added. After the mix-
ture was stirred at room temperature for 2 h, trimethoxytrityl chlo-
ride (4.3 g; 12 mmol) was added. The reaction was stirred overnight
(∼16 h) at room temperature. Pyridine hydrochloride was filtered. Wa-
ter (60 mL) and aqueous ammonium hydroxide (2 mL) were added and
the reaction mixture was stirred for 30 min. The crude product was ex-
tracted into dichloromethane, the organic layer was washed two times
with 5% aqueous solution of sodium bicarbonate and dried with anhy-
drous sodium sulfate. The product was filtered and purified by column
chromatography using chloroform/pyridine (99.9:0.1) with a gradient of
methanol (0–25%). Yield 68.5%.
Synthesis of 5ꢀ-O-[benzhydroxy-bis(trimethylsilyloxy)silyl]
-N2-trimethoxytrityl-2ꢀ-deoxyguanosine
N-Trimethoxytrityl-2ꢀ-deoxyguanosine (4.6 g; 7.7 mmol) was dried in
vacuo for 12 h and then dissolved in anhydrous N,N-dimethylformamide
(80 mL). Imidazole (1.05 g; 15.4 mmol) was added to the mix-
ture and the flask was placed on ice and stirred. Benzhydroxy-
bis(trimethylsilyloxy)silyl chloride (4 mL; 9.9 mmol) was added slowly
over 2 h via syringe. The flask was then removed from ice and al-
lowed to stir at room temperature for 6 h. The reaction was moni-
tored by TLC. Distilled water (60 mL) was added and solvents were

Synthesis and Biochemical Activity of New Oligonucleotide Analogs
363
removed in vacuo to a final volume of 50 mL. The remaining solution
was dissolved in dichloromethane and rinsed with aqueous solution
of 5% sodium bicarbonate saturated with sodium chloride and dried
with anhydrous sodium sulfate. The product was filtered and purified
by column chromatography using chloroform/pyridine (99.9:0.1) with a
gradient of methanol (0-20%). Yield 47.4%.
Synthesis of 5ꢀ-O-silyl-N-trimethoxytrityl-2ꢀ-deoxyguanosine
3ꢀ-O-phosphoamidite (3e)
5ꢀ-O-[benzhydroxy-bis(trimethylsilyloxy)silyl]-2ꢀ-deoxyguanosine (3.6
g; 3.6 mmol) was dried in vacuo for 12 h and then dissolved in an-
hydrous dichloromethane (30 mL). Methyl tetraisopropylphosphorodi-
amidite (1.2 mL; 4 mmol) was added with stirring. 0.4 M solution of
tetrazole in acetonitrile (3.2 mL; 3.6 mmol of tetrazole) was added
slowly over 2 h and the reaction mixture was stirred for an addi-
tional 2 h. A small amount of triethylamine (approximately 0.4 mL)
was added to neutralize the solution and the solvents were removed
in vacuo. The crude product was isolated by column chromatography
with benzene/triethylamine (99:1) followed by gradient of ethyl acetate
(0–100%). Yield 75%.
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