Synthesis and biological activity of borane phosphonate DNA

Article abstract of DOI:10.1080/10426507.2010.538456

Borane phosphonate oligodeoxyribonucleotides are synthesized from 5'-O-[benzhy droxybis(trimethylsilyloxy)silyl]-2'-deoxyribonucleoside-3'-O- methylphosphoramidites. The exocyclic amine functions of adenine, guanine, and cytosine are protected with trimet

Full text of DOI:10.1080/10426507.2010.538456

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Phosphorus, Sulfur, and Silicon and the
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Synthesis And Biological Activity of
Borane Phosphonate DNA
Magdalena Olesiak a , Angelika Krivenko a , Heera Krishna a & Marvin
H. Caruthers a
a Department of Chemistry and Biochemistry , University of
Colorado–Boulder , Boulder , Colorado , USA
Published online: 25 Apr 2011.
To cite this article: Magdalena Olesiak , Angelika Krivenko , Heera Krishna & Marvin H. Caruthers
(2011) Synthesis And Biological Activity of Borane Phosphonate DNA, Phosphorus, Sulfur, and Silicon
and the Related Elements, 186:4, 921-932, DOI: 10.1080/10426507.2010.538456
To link to this article: http://dx.doi.org/10.1080/10426507.2010.538456
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Phosphorus, Sulfur, and Silicon, 186:921–932, 2011
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426507.2010.538456
SYNTHESIS AND BIOLOGICAL ACTIVITY OF BORANE
PHOSPHONATE DNA
Magdalena Olesiak, Angelika Krivenko, Heera Krishna,
and Marvin H. Caruthers
Department of Chemistry and Biochemistry, University of Colorado–Boulder,
Boulder, Colorado, USA
Abstract Borane phosphonate oligodeoxyribonucleotides are synthesized from 5ꢁ-O-[benzhy
droxybis(trimethylsilyloxy)silyl]-2ꢁ-deoxyribonucleoside-3ꢁ-O-methylphosphoramidites. The
exocyclic amine functions of adenine, guanine, and cytosine are protected with trimethoxytrityl,
and thymine is unprotected. Using these synthons and under standard conditions via activation
with S-ethylthiotetrazole, condensations on a highly crosslinked polystyrene support are in
excess of 99%. Following the complete synthesis of the oligodeoxyribonucleotide phosphite
triester, oxidation with THF·BH3 yields the oligodeoxyribonucleotide borane phosphonate.
Further treatment with 80% aqueous acetic acid followed by disodium 2-carbamoyl-2-
cyanoethylene-1,1-dithiolate removes trimethoxytrityl from the 2ꢁ-deoxyribonucleoside bases
and the methyl protecting group from the internucleotide phosphate triester, respectively.
Cleavage from the support with ammonium hydroxide and purification by reverse phase HPLC
affords the pure oligodeoxyribonucleotide borane phosphonate. These oligomers are taken up
by cells in the absence of cationic lipids and transport biologically active interfering RNA into
cells.
Keywords Borane; DNA; phosphonate
INTRODUCTION
The search for DNA and RNA analogs that may potentially fulfill the necessary
requirements for classification as a therapeutic drug has led to a wide range of backbone
oligonucleotide modifications. These criteria include stability toward nucleases, low toxic-
ity, ease of transport across cell membranes, and biological activity. A promising member
of this class, in which a nonbridging oxygen has been replaced by a borane group in DNA,
is shown in Figure 1. The synthesis of borane phosphonate DNA has been reported by
Shaw et al.1 and the RNA analog by Chen et al.2 This group is closely related to natu-
ral phosphodiesters and methylphosphonates, as it is isoelectronic and isostructural with
methylphosphonates and carries a negative charge. Moreover, this linkage is nuclease re-
sistant,1,3 and as DNA containing borane phosphonate internucleotide linkages, it forms
Received 7 September 2010; accepted 4 November 2010.
We thank Rich Shoemaker for assistance with the NMR facility and University of Colorado Central Analytical
Laboratories for the mass spectral facility. This research was supported by the University of Colorado at Boulder.
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
921

922
M. OLESIAK ET AL.
Figure 1 Chemical structure of borane phosphonate DNA. B = adenine, cytosine, guanine, and thymine (A,C,G,T,
respectively); n = 0–20.
duplexes with complementary natural RNA. The resulting heteroduplex is recognized by
RNase H, which catalyzes degradation of the unmodified RNA strand.3 In related research
Shaw and co-workers4 have demonstrated that α-borane 2ꢁ-deoxynucleoside triphosphates
are recognized by the polymerases that catalyze the formation of borane phosphonate inter-
nucleotide linkages. These results have encouraged us to continue to develop the chemistry
associated with synthesizing borane phosphonate oligodeoxyribonucleotides (ODNs) and
to study their biochemistry. In this article we report a new method for synthesis of these
ODNs and demonstrate that the resulting oligomers are taken up by cells in culture. We also
show that these oligomers, in complex with natural, interfering RNA, stimulate degradation
of mRNA presumably through interacting with the RISC complex.
RESULTS
The challenge with this analog is to develop an orthogonal synthesis strategy. There
are several key considerations. Of utmost importance, all base and phosphorus protecting
groups must be stable toward boranation under conditions where the 2ꢁ-deoxynucleoside
bases are not reduced or form irreversible complexes with borane. A second is the design
of a transient 2ꢁ-deoxyribonucleoside hydroxyl protecting group, preferably 5ꢁ, which can
be removed following each condensation cycle under conditions that do not modify the
growing, support-bound borane phosphonate oligodeoxyribonucleotide.
Although several approaches have been proposed,4 a viable methodology, which
can be used to prepare heterologous constructs, has only recently been developed.3,5 This
approach yields oligomers 10–12 monomers in length with a combination of phosphate
and borane phosphonate internucleotide linkages. In order to extend this approach to the
synthesis of heterologous ODNs having at least 20 monomers and exclusively borane
phosphonate internucleotide linkages, the synthesis strategy outlined in Figures 2 and 3
was developed.

923

924
M. OLESIAK ET AL.
Figure 3 Schematic for the synthesis of borane phosphonate DNA.
The serious challenge that must be overcome in order to synthesize these ODNs
is to develop protecting groups that are stable toward boranation. In the previous ap-
proach,3,5 we used a combination of fluorenylmethoxycarbonyl, dimethoxytrityl, and
trimethoxytrityl on the amino groups of guanine, adenine, and cytosine, respectively.
However, the guanine and adenine protection strategies proved unsatisfactory, as both
the N2-(9-fluorenylmethoxycarbonyl) and N6-dimethoxytrityl groups proved to be diffi-
cult to remove completely. We therefore turned to the strategy shown in Figure 2, where the
amino groups on all three bases are protected with trimethoxytrityl. Because we planned
to use phosphoramidites as synthons, thymine does not require protection.1 We retained
the benzhydroxybis(trimethylsilyoxy)silyl for transient 5ꢁ-hydroxyl protection, as it could
be completely removed in 70 s. Additionally, we chose to continue the use of the 3ꢁ-O-
methylphosphoramidites, because condensation rates are rapid and lead to very high yield-
ing product (98–99% per nucleotide addition). Removal of the methyl protecting group from
the final product with disodium 2-carbamoyl-2-cyanoethylene-1,1-dithiolate is also rapid.8
Synthesis of these ODNs is carried out using the approach outlined in Figure 3
and Table 1. The first step is to condense an appropriately base-protected 5ꢁ-[benzhy
droxybis(trimethylsilyloxy)silyl]-3ꢁ-O-methylphosphoramidite-2ꢁ-deoxynucleoside and a
polystyrene-linked and base-protected 2ꢁ-deoxynucleoside using S-ethylthiotetrazole to
1Previous research has shown that the use of 2ꢁ-deoxynucleoside 3ꢁ-H-phosphonates as synthons requires
that N3 of thymine be protected preferably with N3-anisoyl. Otherwise, the combination of silylation to activate
an internucleotide H-phosphonate linkage toward boranation, followed by boranation, causes reduction of the
thymine base.6,7

BORANE PHOSPHONATE DNA
925
Table 1 Synthesis cycle for borane phosphonate DNA
Reaction
Coupling
Wash
Wash/reagents/solvents
Time (s)
Wait: 180 s
0.1M phosphoramidites (13–16) and 0.25 M
S-ethylthiotetrazole in acetonitrile (1:1)
Acetonitrile
40 s
30 s
Dichloromethane
Capping
Cap Mix A (Tetrahydofuran/pyridine/acetic anhydride) and
Cap Mix B (16% 1-methylimidazole in tetrahydrofuran,
Equal volumes were mixed at port 14 just prior to synthesis
Acetonitrile
14 to column: 10 s
Wait: 30 s
Wash
40 s
Dichloromethane
45 s
Dimethylformamide
45 s
5ꢁ- Deprotection
Wash
1.1M HF/ 1.1 Triethylamine/0.2 M NMDEAa in
Dimethylformamide (pH 9.2)
Dimethylformamide,
10 to column: 25 s
Wait: 45 s
45 s
Acetonitrile
55 s
aNMDEA = N-methyl diethanolamine.
activate the condensation. This reaction is followed by capping with a solution of acetic
anhydride and removal of the 5ꢁ-silyl group with fluoride at pH 9.2. This cycle (condensa-
tion with an appropriately protected 2ꢁ-deoxynucleoside-3ꢁ-phosphoramidite, capping with
acetic anhydride, and removal of the 5ꢁ-silyl group with fluoride) can be repeated until the
synthesis of the desired oligodeoxyribonucleotide phosphite triester is complete.
Further steps involving boranation and removal of protecting groups generate the final
product. Boranation with BH3·THF converts the internucleotide linkage to a PIV analog.
Relative to the total synthetic scheme, this P(IV) compound is extremely important, as it is
stable to aqueous acetic acid, which is used as the next step to remove the N-trimethoxytrityl
groups from cytosine, adenine, and guanine. Moreover, unlike the borane phosphonate in-
ternucleotide linkage,6 the P(IV) adduct does not react with the trimethoxytrityl cation
(present during the aqueous acetic acid step). Thus, in order to complete deprotection
of the oligodeoxyribonucleotide analog, the P(IV) borane adduct is treated with aque-
ous acetic acid to remove the trimethoxytrityl group from the 2ꢁ-deoxynucleoside bases.
This is followed first with disodium 2-carbamoyl-2-cyanoethylene-1,1-dithiolate to remove
the methyl group8 and convert the internucleotide linkage to borane phosphonate and fi-
nally conc. ammonium hydroxide to remove the completely deprotected ODN from the
polystyrene support. Purification is by reverse phase HPLC.3,5
The products of this chemistry have been fully characterized by NMR and mass
spectral analyses. Phosphorus NMR analyses display a broad signal at 94.4–94.8 ppm
(borane phosphonate) and a sharp peak at –2 ppm, indicating that a small fraction (3–4%)
of the internucleotide linkages are phosphate (Figure 4A and 4B). B11 NMR spectra display
a broad signal at –40 ppm, which is characteristic of the borane phosphonate linkage. As
found for all ODNs synthesized by this procedure, the observed mass corresponds to those
as calculated. The mass spectral analyses for two oligomers are included in the legend to
Figure 4. These results demonstrate that oligomers as prepared by this method have the
predicted structures and are free of any bases or sugars complexed with borane.
These ODNs form duplexes with complementary RNA but, when compared to the
unmodified oligomers, have a reduced melting temperature (Tm). For example the duplex

926
M. OLESIAK ET AL.
Figure 4 Phosphorus NMR of two ODNs having borane phosphonate internucleotide linkages.
(A) d (GbAbTbTbAbTbGbTbCbCbGbGbTbTbAbTbGbTbAbTbT); (B) d(AbAbCbCbAbTbAbCbAbAbCbCbTbAb
CbTbAbCbCbTbCbT). The major peak corresponding to borane phosphonate is 94.4–94.8; the minor peak is
phosphate. Compound A, calc mass 6414.69, observed mass 6415.7; Compound B, calc mass 6522.78, observed
mass 6526.8.
formed between the oligomer whose sequence is shown in the legend to Figure 4A and
a complementary unmodified RNA with two uridine nucleotides at the 3ꢁ-end (i.e., both
strands in the duplex have two unpaired nucleotides at the 3ꢁ-ends) has a Tm of 55◦C,
whereas the Tm of the natural RNA/RNA duplex (both oligomers have two unpaired uridine
nucleotides at the 3ꢁ-ends) is 79◦C. (Tm studies were completed in siRNA buffer × 1, as this
buffer was used for studying cell uptake of ODNs.) This difference corresponds to about
1.18◦C drop in the Tm per borane phosphonate internucleotide linkage. The corresponding
Tm reduction for oligomers having phosphorothioate is 0.6◦C per linkage.
Our previous research has demonstrated that esterified phosphonoacetate analogs of
DNA are taken up by cells in culture.9 Most importantly, and unlike previous research with
other analogs such as phosphorothioate DNA, transfection with these oligomers takes place
in the absence of cationic lipids. This is a major advantage, as cationic lipids are toxic to
cells and their uptake must be carefully controlled. The toxicity also varies considerably
for various cell lines. This earlier work has encouraged us to investigate the possibility that
borane phosphonate DNA, either as single strands or as a duplex, could be taken up by cells
in the absence of cationic lipids. Our first experiment involved synthesizing the ODN whose
sequence is shown in Figure 4A and attaching a 5ꢁ-fluoroscein label in order to monitor
results. This ODN is then added to cells in culture at a concentration of 1µM. After 16 h,
the cells are rinsed and uptake examined with fluorescence microscopy and fluorescence
assisted cell sorting (FACS) as a quantitative method. Figure 5 shows representative data
with HeLa cells as acquired from FACS analysis. The left panel shows results with untreated
cells where a small amount of background fluorescence is observed primarily from flavins
and other small molecules found in these cells. The right panel shows that all HeLa cells, in
the absence of cationic lipid, are transfected by this 5ꢁ-fluorescein labeled ODN. Controls
such as 5ꢁ-fluorescein labeled phosphorothioate DNA are not taken up by HeLa cells in the
absence of cationic lipid. When examined by fluorescence microscopy, the 5ꢁ-fluorescein
labeled ODN is primarily localized throughout the cytoplasm (data not shown).
We then wanted to assess if a complementary, unmodified RNA strand hybridized
to the ODN could also be transfected into cells without cationic lipids. This experiment

BORANE PHOSPHONATE DNA
927
Figure 5 FACS analysis of 5ꢁ-fluorescein labeled d(GbAbTbTbAbTbGbTbCbCbGbGbTbTbAbTbGbTbAbTbT).
(A) HeLa cells; (B) HeLa cells tranfected for 16 h with ODN at 1 µM.
was carried out using 5ꢁ-fluorescein labeled RNA (no internucleotide modifications) that
is complementary to the ODN whose sequence is shown in Figure 4A, as illustrated in
the legend to Figure 6. This duplex has two unpaired 2ꢁ-deoxythymidine nucleotides at the
3ꢁ-ends of both the ODN and the unmodified RNA strands. The duplex that is formed is
then added to HeLa cells at 5 µM (as presented previously, this duplex would be stable at
37◦C in cell culture as the Tm is 55◦C). Results by FACS analysis are shown in Figure 6.
The left panel displays only the low level of background fluorescence as observed in HeLa
cells. When the duplex is added in the absence of cationic lipid (results shown in the
right panel), approximately 85% of the cells show an uptake of the complementary RNA
strand. When a fluorescein-labeled, noncomplementary RNA is added with the borane
phosphonate oligomers, there is no RNA uptake. Thus a duplex must form in order for
transfection to occur. When uptake is examined by fluorescence microscopy (data not
shown), the fluorescein-labeled RNA is localized in the cytoplasm.
In order to complete this research, we wanted to test whether this RNA/borane phos-
phonate DNA duplex is biologically active (Figure 7). The RNA of this duplex corresponds
in sequence to a small interfering RNA (siRNA) that, when taken up by the RISC complex,
degrades luciferase RNA.10 Therefore, we added this duplex at 5 µM to HeLa cells and
allowed transfection to proceed for 24 h. The media containing the duplex was removed,
Figure 6 FACS analysis of 5ꢁ-fluorescein labeled natural RNA as a duplex with d(GbAbTbTbAbTb
GbTbCbCbGbGbTbTbAbTbGbTbAbTbT). The RNA sequence was 5ꢁ-(Fl-U-A-C-A-U-A-A-C-C-G-G-A-C-A-U-
A-A-U-C-U-U). (A) HeLa cells; (B) HeLa cells transfected for 24 h with the duplex at 5 µM.

928
M. OLESIAK ET AL.
Figure 7 Luciferase activity of HeLa cells following transfection. Lane 1, siRNA/borane phosphonate DNA
duplex at 5 µM. Lane 2, siRNA/RNA duplex at 5 µM plus lipofectin. Lane 3, pGL 3 firefly luciferase plasmid
plus lipofectin (no duplex). For all experiments, firefly luciferase activity was measured 24 h after transfection
with plasmid. siRNA/borane phosphonate duplex: 5ꢁ-(U-A-C-A-U-A-A-C-C-G-G-A-C-A-U-A-A-U-C-U-U)/5ꢁ-
d(GbAbTbTbAbTbGbTbCbCbGbGbTbTbAbTbGbTbAbTbT); siRNA/RNA duplex: 5ꢁ-(G-A-U-U-A-U-G-U-C-C-
G-G-U-U-A-U-G-U-A-U-U)/ 5ꢁ-(U-A-C-A-U-A-A-C-C-G-G-A-C-A-U-A-A-U-C-U-U).
and the cells were washed three times with fresh media to ensure removal of the duplex from
the cell culture plate. These cells were then transfected in fresh media with pGL 3 plasmid
and lipofectin (Dharmafect 1, Thermofisher was essential for this step in order for cells to
take up plasmid DNA). After 24 h, analysis for luciferase activity clearly shows that the
siRNA/borane phosphonate DNA duplex reduces luciferase activity by approximately 60%
relative to controls having no duplex (100% active luciferase) or the natural siRNA/RNA
duplex (no luciferase activity).
DISCUSSION
These results demonstrate that a new high-yielding procedure has been devel-
oped for the synthesis of borane phosphonate DNA. The method is based on the
3ꢁ-O-methylphosphoramidites in combination with a series of base protecting groups
(trimethoxytrityl) that are completely stable to borane but are rapidly removed by mild
acid. The oligomers are obtained in high yield with a minimum (3–4%) contamination with
phosphate internucleotide linkages. Previous results have shown that borane phosphonate
ODNs are stable toward nuclease and stimulate RNase H activity.3,5 Here we demonstrate
that these analogs are readily taken up by cells and, in complex with complementary siRNA,
show biological activity. Because borane phosphonate DNA stimulates RNase H activity,
the possibility exists that these analogs degrade the siRNA in situ in the presence of endoge-
nous RNase H. This RNase H activity would, as a consequence perhaps, partially counter
the expected reduction in luciferase activity. Such a possibility might be responsible for
incomplete inhibition of luciferase activity by this duplex. Of interest would be to prepare
2ꢁ-O-methyl RNA as the borane phosphonate oligomer and test its ability, in complex with
siRNA, to reduce luciferase activity. Because 2ꢁ-O-methyl RNA does not stimulate RNase
H activity, a 2ꢁ-O-methyl RNA borane phosphonate oligomer should deliver siRNA without
endogenous degradation by RNase H to cells and thus exhibit enhanced activity.

BORANE PHOSPHONATE DNA
929
EXPERIMENTAL
Chemical Synthesis
Unless otherwise noted, all materials were obtained from commercial suppliers and
used without further purification. Dichloromethane was distilled over P2O5. Commercially
available DNA synthesis reagents and 5ꢁ-fluorescein phosphoramidite were purchased from
Glen Research (Sterling, VA, USA). Medium-pressure, preparative column chromatogra-
phy was performed using 60 Å standard grade silica gel from Sorbent Technologies. The
silica gel was dried overnight in the oven before being used for phosphoramidite purifica-
tion. Thin-layer chromatography was performed on aluminum-backed silica 60 F254 plates
from EMD Chemicals, USA. 1H and 31P NMR spectra were recorded on Bruker Avance-III
300 NMR and Varian INOVA 400 NMR spectrometers using a CDCl3 lock, and TMS was
used as an internal reference.
HPLC Methods
Analytical HPLC injections were carried out using an Agilent Hypersil ODS 5 µm
column, 4.0 mm i.d. × 250 mm, eluting at 1.5 mL/min with a gradient of acetonitrile/50 mM
triethylammonium bicarbonate buffer, pH 8.5. Preparative HPLC (Agilent Zorbax SB-C18,
5 µm column 9.4 mm i.d. × 250 mm) was performed on an Agilent Technologies Model
HPLC 1100, eluting at 1.5 mL/min with a gradient of acetonitrile/50 mM triethylammonium
bicarbonate buffer, pH 8.5. The eluent was monitored for absorption at 280 nm. Mass
spectral and analytical data were obtained via the PE SCIEX/ABI API QSTAR Pulsar i
Hybrid LC/MS/MS at the University of Colorado Central Analytical Laboratories.
General Procedure for the Synthesis of N-Trimethoxytrityl Protected
2ꢀ-Deoxyribonucleosides [2ꢀ-Deoxyriboadenosine (5), 2ꢀ-Deoxyriboguanosine
(6), 2ꢀ-Deoxyribocytidine (7)]
The 2ꢁ-deoxyribonucleoside (0.02 mol) was co-evaporated three times with pyridine
and dried under vacuum for 12 h. The 2ꢁ-deoxyribonnucleoside was transferred to a round-
bottomed flask equipped with a magnetic stir-bar, and anhydrous pyridine (350 mL) was
added with stirring. To this suspension, chlorotrimethylsilane (0.1 mol, 5 equiv.) was
added. After the mixture had been stirred at room temperature for 2 h, trimethoxytrityl
chloride (0.03 mol, 1.5 equiv.) was added. The reaction mixture was stirred for 16 h at
room temperature. The precipitated salts were removed by filtration using Whatman filter
paper. To the filtrate, 60 mL of water and aqueous ammonium hydroxide (10 mL, 28–30%)
were added, and the reaction mixture was stirred until complete deprotection of silyl groups
from the 5ꢁ and 3ꢁ positions of the N-trimethoxytrityl protected 2ꢁ-deoxyribonucleoside was
observed by TLC [CHCl3/MeOH (9:1)]. The above mixture was concentrated on a rotary
evaporator to approximately 30–40 mL. The crude mixture was dissolved in ethyl acetate,
and this solution was washed first with a 5% aqueous solution of sodium bicarbonate. The
organic phase was separated and extracted from ethyl acetate/water. The crude product in
ethyl acetate was dried over anhydrous magnesium sulfate. The product was further purified
by column chromatography using hexane/chloroform/pyridine (48:48:4) and a gradient of
chloroform/methanol (100:0–6%). Average yields: 85–90%.

930
M. OLESIAK ET AL.
General Procedure for the Synthesis of
5ꢀ-O-[Benzhydroxybis(trimethylsilyloxy)silyl]-2ꢀ-deoxyribonucleosides (8–11)
The 5ꢁ-O-silyl-N-trimethoxytrityl-2ꢁ-deoxyribonucleosides (8–10) and 5ꢁ-O-silyl-2ꢁ-
deoxythymidine (11) were prepared according to published procedures.5 The N-protected
2ꢁ-deoxyribonucleoside (0.02 mol) was dried in vacuo for 16 h and then dissolved in anhy-
drous N,N-dimethylformamide (600 mL). Imidazole (0.06 mol, 3 equiv.) was added to the
mixture, and the flask was placed on ice and stirred. Benzhydroxybis(trimethylsilyloxy)silyl
chloride (0.022 mol, 1.1 equiv.) was added slowly over 1 h via syringe. The flask was al-
lowed to stir at room temperature for ∼16 h. Distilled water (60 mL) was added, and the
solvent was removed in vacuo to a final volume of 50 mL. This solution was dissolved in
dichloromethane and rinsed with an aqueous solution of 5% sodium bicarbonate 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
CHCl3/hexane (1:1) followed by a gradient of 2–10% methanol in chloroform. (For N-
trityl analogs, 2% pyridine was added to the eluting system.) The product eluted in 5–10%
methanol. Average yields: 55–65%.
General Procedure for the Synthesis of Protected
2ꢀ-Deoxyribonucleoside 3ꢀ-O-Methyl Phosphoramidites
Protected 2ꢁ-deoxyribonucleosides (8–11; 3.6 mmol each) were dried in vacuo for 12
h and dissolved in anhydrous dichloromethane (30 mL). Methyl tetraisopropylphosphor-
diamidite (4 mmol, 1.1 equiv.) was added with stirring. Then 0.4 M solution of 1H-tetrazole
in anhydrous acetonitrile (3.6 mmol, 1.0 equiv.) was added slowly over 2 h, and the reaction
mixture was stirred for an additional 2 h. A small amount of triethylamine (approximately
0.4 mL) was added to neutralize the solution, and the solvent was removed in vacuo. The
products were isolated by column chromatography using a 0–100% gradient of ethyl acetate
in benzene, containing 1% triethylamine.
Solid-Phase Synthesis
Low volume (LV) polystyrene columns (0.2-µmol synthesis scale) were purchased
from Glen Research (Sterling, VA). Prior to synthesis, the 5ꢁ-DMT group on the
support-bound 2ꢁ-deoxyribonucleoside was removed with 3% dichloroacetic acid in
dichloromethane. DNA synthesis was carried out on an Applied Biosystems Model
392 automated DNA synthesizer (Applied Biosystems, Foster City, CA, USA) opti-
mized for fluoride-ion chemistry. Using appropriately protected 2ꢁ-deoxyribonucleoside
3ꢁ-O-methylphosphoramidites and the synthesis cycle outlined in Table 1, an oligodeoxyri-
bonucleotide having phosphite triester internucleotide linkages was generated. This
oligomer on the solid support was next treated twice (90 s each) with a 25 µM complex of
THF·BH3 in THF followed by a wash with THF (30 s).
For oligomers having 5ꢁ-fluorescein, the 5ꢁ-fluorescein phosphoramidite was con-
densed with the oligomer using standard synthesis methods (Table 1) to generate the
phosphite triester. These oligomers, including the fluorescein phosphite linkage, were then
converted to borane phosphonate using 25 µM complex of THF·BH3 in THF (2 × 90 s)
followed by THF wash (30 s).

BORANE PHOSPHONATE DNA
931
Postsynthesis, the polystyrene (PS) was washed with anhydrous acetonitrile for 60
s and then flushed with a stream of argon until dry. The bases were detritylated using
an 80% acetic acid solution for 24 h. The methyl groups were then removed from the
phosphate backbone using 2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate (30 min,
Acacia Lifesciences, IL, USA) in DMF. The polystyrene beads were thoroughly washed
with water followed by anhydrous acetonitrile and dichloromethane, and flushed with argon
until dry. The PS support was removed from the column and placed in a 1.5 mL screw-cap,
conical glass reaction vial. A conc. solution of ammonium hydroxide was added, and the
vial was sealed. The vial was vortexed to stir the contents, and the cleavage was allowed
to proceed for 15 h at 55◦C. The vial’s contents were transferred to a 1.5 mL Eppendorf
tube and evaporated to dryness in a Speedvac. The ODN product was redissolved in 5%
acetonitrile-water and purified by RP-HPLC.
Preparation of Cells
HeLa cells were obtained from American Type Culture Collection (Rockville, MD,
USA) and serially maintained at monolayer cultures in a humidified atmosphere of 5% CO2
at 37◦C in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine
serum, penicillin (100 U/mL), and streptomycin (100 U/mL) in 12 well culture plates, and
incubated for 24 h prior to oligonucleotide transfection. The cells were used at passages
12–18.
Incubation of Cells with ODNs
For uptake experiments, ∼1 × 105 cells/well (12 well plates) were incubated in
DMEM containing 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100
U/mL) for 24 h. The concentration of 5ꢁ-fluorescein-labeled boranephosphonate ODN
dissolved in siRNA buffer 1X (Thermoscientific) was measured by UV spectroscopy. The
media was removed, and the cells were transfected with the ODN in DMEM to give a final
concentration of 1 uM, 5 uM, or 10 uM. The cells were then incubated at 37◦C for 24 h.
After the incubation, the medium was removed from the wells, and cells were washed four
times with 0.5 mL PBS. Cells were then treated for 3 min at 37◦C with a pre-warmed
solution of trypsin-EDTA (1X) until all cells became round and detached from the plates.
The cells from each plate were taken up in 1 mL PBS and centrifuged at 1000 rpm for
5 min to form cell pellets. The pellets were washed and resuspended in PBS and kept at
0◦C in the dark for flow cytometric analysis.
Cell Viability
Viability of the cells was determined before and after incubation with ODNs by
trypan blue exclusion (conventional microscopy) or by propidium iodide exclusion (flow
cytometric analysis). By both experiments, greater than 86–90% cells were found to be
viable after 24 h of ODN transfection.
Flow Cytometry
Flow cytometric data on at least 10,000 cells per sample were acquired on a Moflow
flow cytometer (Beckman-Coulter) equipped with a single 488 nm argon laser, 530/40

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M. OLESIAK ET AL.
nm emission filter (fluorescein), and 630/30 nm emission filter (propidium iodide). In
two-color flow cytometric analyses (cell viability test), spectral overlap was corrected by
compensation. Raw flow cytometry data were manipulated and visualized using Summit 4.3
software (Beckman-Coulter). Fluorescence intensity of the 5ꢁ-fluorescein tag was analyzed
for cells presenting higher fluorescence than the background. The background was defined
as the auto fluorescence of cells incubated only with the vehicle (DMEM + siRNA buffer).
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