Sulfurizing Reagent Ii And Its Use In Synthesizing

SULFURIZING REAGENT II AND ITS USE IN SYNTHESIZING
OLIGONUCLEOTIDE PHOSPHOROTHIOATES
Marc M. Lemaître, Andrew S. Murphy and Robert L. Somers
Glen Research Corporation, 22825 Davis Drive, Sterling, VA 20164, USA
ABSTRACT
VOLUME 18
NUMBER 1
SUPPLEMENTARY
MATERIAL
DECEMBER 2006
Preparation of oligoribonucleotides with a
phosphorothioate backbone requires a sulfurizing
reagent that is efficient and stable in solution.
One of the most efficient sulfurizing reagents, 3H1,2-benzodithiol-3-one-1,1-dioxide (Beaucage
Reagent), is stable in solution but lacks longterm stability on the DNA synthesizer. This
report evaluates Beaucage Reagent and 3((N,N-dimethyl-aminomethylidene)amino)-3H1,2,4-dithiazole-5-thione (DDTT) (Sulfurizing
Reagent II) as sulfurizing reagents when
using TOM-protected and TBDMS-protected
RNA phosphoramidites, and standard DNA
phosphoramidites for oligonucleotide synthesis.
The product oligonucleotides were evaluated
by ion exchange HPLC, ESI-Mass, and 31P NMR.
It was confirmed that Ion Exchange HPLC can
resolve oligonucleotides containing a single
phosphodiester linkage from those containing all
phosphorothioate linkages.
INTRODUCTION
The replacement of one non-bridging oxygen
atom in the phosphodiester linkage of DNA or
RNA by sulfur creates a phosphorothioate (PS)
linkage. This is one of the oldest and most studied
backbone modifications of nucleic acids (Figure
1). Eckstein synthesized the first dinucleoside
containing a phosphorothioate linkage1. Interest
in phosphorothioate oligonucleotides (PSO) as
biological tools began with the discovery by De
Clercq et al. that PS RNA was more resistant to
RNases that natural RNA2.
Elemental sulfur was one of the first
sulfurization reagents used by Burgers and Eckstein
to sulfurize phosphite triesters3. Since then, a
number of sulfurizing reagents have been used for
chemically modifying the phosphate backbone of
FIGURE 1: PHOSPHOROTHIOATE LINKAGE
(1)
oligonucleotides. Some are shown in Figure 2, Page
2, Dibenzyl tetrasulfide4 (2a), Beaucage Reagent5
(2b), 3-Ethoxy-1,2,4-dithiazolidin-5-one (EDITH)6
(2c), 1,2,4-Dithiazolidine-3,5-dione (DtsNH)6 (2d),
3-Amino-1,2,4-dithiazole-5-thione7 (2e).
After this initial work, thousands of papers
describe activities and properties of such modified
oligonucleotides in cell culture as well as in vivo.
Indeed, PSO is considered to be the first-generation
antisense oligonucleotide (ASO)8-10.
Though not perfect, the phosphorothioate
modification offers several advantages:
∗ Increased nuclease resistance compared to
normal phosphodiesters
∗ Regular Watson-Crick pairing
∗ RNase H compatible
∗ Inexpensive and synthesis-friendly
And the use of chimeric oligos containing
several different modifications along with a normal
phosphodiester segment reduces side effects in total
PS modification11,12.
In addition to their use as DNA analogues in
ASO, PS modifications have some advantages for
RNA as well13. Recently, Overhoff and Sczakiel14
showed that full PSO could promote the delivery
of siRNAs in cell culture. This siRNA uptake is
(Continued on Page 2)
(Continued from Front Page)
2
sequence-independent and the optimal
length seems to vary between 30 and 70
nucleotides, depending on the cell line. Even
though this method is not yet as efficient
as the cationic lipids, it opens the way to
possible new methods.
Another recent paper15 describes a
method for the inactivation of miRNA that
may help to elucidate their functions. It uses
oligonucleotides called antagomirs (23mers, complementary to a target miRNA)
consisting of 2´-OMe bases, a cholesteryl
group at the 3´ terminus, phosphorothioates
at positions 1 and 2 at the 5´ end, and at
the last four at the 3´ end. These molecules
promote the cleavage of complementary
miRNAs and thus should allow analysis
of their function. Presumably the role of
the PS linkages is the stabilization against
degradation in the mouse experiments,
which is consistent with its function in the
antisense field in such in vivo situations.
Finally, two recent papers16,17 show
that modifications including PS do not
systematically abolish siRNA activity,
opening the road for some potentially less
expensive stabilization of such molecules.
Incorporation of 2’-OMe or 2’-F (at various
positions in the sense as well as in the
antisense strand) in combination with PS
linkages should confer increased resistance
to degradation by nucleases, as well as
prolonged serum retention. And it is also
possible that such an easy modification of
siRNA may increase specificity by reducing
or eliminating sense strand recruitment in
the RISC complex and thus reducing a source
of off-target effect.
The most common method for making
PSO today is to use 3H-1,2-Benzodithiol3-one-1,1-dioxide5,18 (Beaucage Reagent)
(2b) as the sulfurizing reagent during
oligonucleotide synthesis in place of the
iodine-based oxidizer. Despite its ease
of use and long history, this product has
some disadvantages such as its limited
stability in solution while on a synthesizer.
Also, it may not display optimal kinetics
for the sulfurization of RNA in solid phase
phosphoramidite synthesis. This work
compares Beaucage Reagent with an
alternative sulfurizing reagent, 3-((N,N-di
methylaminomethylidene)amino)-3H-1,2,4dithiazole-5-thione (DDTT) (Sulfurizing
Reagent II) (Figure 3) to evaluate kinetics for
sulfurizing RNA, performance for sulfurizing
DNA, and stability in storage and while on
the synthesizer.
FIGURE 2: STRUCTURES OF VARIOUS SULFURIZING REAGENTS
(2a)
(2b)
(2c)
(2d)
MATERIALS AND METHODS
(2e)
FIGURE 3: STRUCTURE OF DDTT
Chemicals
Synthesis columns. phosphoramidites,
and synthesis reagents were from Glen
Research, Sterling, VA. TRIS was from JT
Baker. HPLC grade acetonitrile was from
B&J. Ammonium hydroxide, butanol, DMSO,
sodium perchlorate, and triethylammonium
trihydrofluoride were from Sigma-Aldrich.
Alcoholic methylamine and aqueous
methylamine were from Fluka. Reagent
alcohol and sterile water were from Fisher
Scientific. HPLC water and sodium acetate
were from EMD.
gently washed twice with reagent alcohol,
dried under vacuum, and dissolved in 1mL
of sterile water. The concentration of each
oligo was calculated using Beer's law and
the extinction coefficient at 260 nm.
RNA Oligonucleotide synthesis
DNA Oligonucleotide synthesis
RNA oligonucleotides and PSO RNA
were prepared using an ABI 394 DNA
synthesizer using standard protocols. The
sulfurizing reagent was loaded onto port 10
of the synthesizer. Sulfurization times and
coupling times were modified as indicated.
RNA synthesis was carried out on a T-CPG
or a T-Q-support. After synthesis, columns
were dried under a stream of argon and the
contents transferred to a 4mL glass vial.
The oligos were cleaved and deprotected
using 1mL of 40% aqueous methylamine:
33% ethanolic methylamine (1:1) at room
temperature overnight. The oligos were then
filtered into a 2mL centrifuge tube and dried
under vacuum in a Speedvac. The oligos
were dissolved in 100µL dry DMSO and
125µL triethylammonium trihydrofluoride
was added to the vial. The tube was
heated to 65°C for at least one hour and
then cooled briefly in a freezer. 25µL of
3M sodium acetate was added and mixed
thoroughly before 1mL of 1-butanol was
added and mixed. The tube was cooled on
dry ice for 30 minutes and centrifuged at
13,000rpm for 10 minutes. The pellet was
DDTT (Sulfurizing Reagent II)
DNA was prepared using an ABI 394 DNA
synthesizer using standard protocols and
Ac-dC phosphoramidite. Oligonucleotides
that required base deprotection were
deprotected and cleaved as described19.
HPLC analysis
The oligos were analyzed on Ion
Exchange (IEX) HPLC using a concentration
range of 5-20 A260/mL. IEX was performed
on a Beckman System Gold HPLC using
a Dionex DNAPac PA200 polymeric ion
exchange column (4.6x250mm). Buffer A
- 25mM TRIS, 10mM Sodium Perchlorate, pH
7.4, 20% acetonitrile, Buffer B - 25mM TRIS,
600mM Sodium Perchlorate, pH 7.4, 20%
acetonitrile. Simple RNA sequences were
analyzed at room temperature and longer
sequences were analyzed at 60°C using a
column heater. All analyses were run with
a gradient elution; 0-60% B in 30 minutes
at 1mL/min, monitored at 260nm.
ESI-Mass preparation
Samples for ESI-Mass were converted
from the sodium salt to the ammonium
salt as follows. Aliquots of each oligo
were evaporated to dryness in a Speedvac.
Samples were dissolved in 100µL of water.
25µL of 3M ammonium acetate was added
to each vial. Samples were vortexed before
adding 1 mL of butanol and the samples
were then vortexed again. The samples
were chilled on dry ice for at least 30
minutes. The samples were centrifuged for
10 minutes, washed with ethanol and dried
under vacuum.
FIGURE 4: COUPLING EFFICIENCIES DURING SULFURIZATION WITH TOM-PROTECTED RNA
RESULTS
Seven sequences were used to evaluate
the performance of the sulfurizing reagents.
TOM protected and TBDMS protected
RNA phosphoramidites were evaluated
in this study since we offer both types of
phosphoramidites.
Initial studies used the sequence,
5’-UUU UUU UUT T-3’, to compare the
performance of 0.05M Beaucage Reagent,
0.03M DDTT, and 0.05M DDTT. This sequence
offered the advantage in that there was no
need for the overnight base deprotection
step. These sulfurizing reagents were
evaluated using increasing times of 30, 60,
120, and 240 secs for sulfurization with
TOM RNA phosphoramidites. Results are
shown in Figure 4. The activator solution
was 0.25M 5-(ethylthio)-1H-tetrazole (ETT)
with a coupling time of 4 minutes. The
coupling efficiency was calculated based
on the purity by ion exchange and length
of oligonucleotide.
Setting the minimum coupling efficiency
at ≥98%, then a sulfurizing time between
one minute and four minutes was suitable for
similar sequences using 0.05M Sulfurizing
Reagent II. A four minute sulfurizing time
was also sufficient while using the 0.03M
Sulfurizing Reagent II for this oligo. When
using the 0.05M Beaucage Reagent, only a
four minute sulfurization time was suitable
under the same conditions. Figure 5 shows
the IEX chromatograms of oligos sulfurized
with 0.05M DDTT versus Beaucage Reagent
with sulfurization time of 60 seconds for
each.
The performance of the 0.05M
Sulfurizing Reagent II and Beaucage
Reagent was also evaluated on TBDMS-RNA
phosphoramidites. Results are in Figure 6
(Page 4). The 30 second sulfurizing time was
omitted for TBDMS-RNA phosphoramidites
based on the decreasing coupling efficiency
results for 60 and 120 seconds. Coupling time
FIGURE 5: IEX HPLC OF SULFURIZED RNA
(A)
(B)
IEX HPLC analysis of oligo U8T2 using TOM-protected RNA monomers with sulfurization as follows:
A) 0.05M DDTT, 60 sec and B) 0.05M Beaucage Reagent, 60 sec.
was also 4 minutes using ETT as the activator.
Using the TBDMS phosphoramidites, the
0.05M Sulfurizing Reagent II performed well
for all time periods from 1 to 4 minutes.
0.05M Beaucage Reagent performed well
only when using a sulfurizing time of 4
minutes.
Additional sequences were prepared
that represented the n-1 oligonucleotide
and a mixed oxidation sequence with a
single internal PO linkage. A sulfurization
time of 60 seconds was used for these
oligonucleotides. These sequences were
compared to the full-length oligo on ion
exchange HPLC. See Table 1, Page 4 for the
n-1 and mixed oxidation sequences.
Figure 7, Page 4 shows the ion exchange
HPLC of a) a mixture of the full-length
3
oligo and the mixed oxidation oligo, b) a
mixture of the full-length oligo and the
n-1 oligo, c) a mixture of the n-1 oligo and
the mixed oxidation oligo. Under these
conditions, ion-exchange HPLC can separate
phosphorothioates that contain one or more
PO linkages. Additionally, ion-exchange
HPLC can also separate failure sequences,
such as n-1, from the full-length PSO.
Samples were selected for analysis by
ESI-Mass and 31P NMR. See Tables 2 and
3, respectively, on Page 5. The mass was
confirmed for each sequence indicating
that no modifications of the bases occurred
during synthesis or the subsequent work up
of the sample. The 31P NMR can distinguish
between the PS linkage and PO linkage
as seen in Figure 8, Page 5. None of the
sulfurizing reagents showed any extraneous
oxidation of the phosphite linkages. The 31P
NMR of the mixed oxidation sequence shows
the presence of a phosphodiester linkage
and the phosphorothioate.
A mixed base sequence,
5’-UUA UUC UUG UUA UUC UUG TT-3’,
containing the standard RNA bases was
also evaluated using TOM-protected RNA
phosphoramidites and 0.05M DDTT as the
sulfurizing reagent. Under these conditions,
a sulfurizing time of 1-2 minutes is suitable.
Results are in Table 4, Page 5.
The final portion of the work used a
more complex sequence, the mammalian
LMNA sequence that contains more
purines than the previous sequences,
representing a real-life sequence,
5’-CUG GAC UUC CAG AAG AAC ATT-3’.
Results are listed in Table 5, Page 6. A
longer coupling time of 10 minutes was
FIGURE 6: COUPLING EFFICIENCIES WHEN USING TBDMS –PROTECTED RNA
TABLE 1: SEQUENCES PREPARED WITH TOM-PROTECTED RNA MONOMERS AND SULFURIZING REAGENT II
n-1 PSO
Mixed (1 PO)
Full length PSO
5’-UUU UUU UTT-3’
5’-UUU UOUU UUT T-3’
5'-UUU UUU UUT T-3'
Oligo
Sequence
CE %
Rt (min)
n-1 PSO
UUU UUU UTT
98.1
12.4
Mixed
(1 PO)
Full length
PSO
UUU UOUU UUT T
98.0
12.4
UUU UUU UUT T
97.9
13.3
FIGURE 7: IEX HPLC OF OLIGOS
(A)
A)
4
(B)
(C)
also used for this sequence to minimize
any effect from the coupling reaction. Our
results indicate that for RNA synthesis
using TBDMS-protected RNA monomers
and a standard coupling time of 10 minutes,
sulfurization with 0.05M DDTT for 360
seconds resulted in coupling efficiencies
approaching or even exceeding 99%.
TABLE 2: ESI-MASS DATA
Oligo
Expected
Observed
Description
U7T2
2815.12 Da 2815.13 Da
n-1 PSO
U7T2
3121.15 Da 3121.28 Da
Full-length, one PO
U8T2
3137.13 Da 3137.23 Da
Full-length PSO
DISCUSSION
Coupling Efficiency (CE) is a common
metric for assessing the quality of the
synthesis and is based on the overall purity
and length of the crude oligonucleotide.
Many factors affect the quality of synthesis
with the most important being the coupling
step of the incoming amidite to the growing
DNA chain. A failure to couple will result
in capping by the capping reagents and a
failure sequence will result at that stage
of synthesis. Repeated failures will result
in a ladder on the ion exchange or gel.
Incomplete oxidation or sulfurization of
the phosphite linkage results in cleavage of
the acid-sensitive phosphite bond during
the subsequent detritylation step and
produces a deletion in the sequence. These
oxidation failures are cumulative, resulting
in sequences that show up as n-1, n-2, etc.,
peaks in ion-exchange chromatography and
reduce the overall purity of the full-length
oligonucleotide. An important distinction
is that the coupling failure and subsequent
capping results in a truncated sequence
whereas an oxidation failure results in a
different sequence, a deletion sequence.
In this work, the coupling conditions were
held constant for each portion of the study,
so that this parameter does not affect the
coupling efficiency. This allows us to isolate
the effects of the sulfurizing/oxidation
reaction on coupling efficiency.
The work using the U-TOM
phosphoramidites shows that sulfurization
times of 1-2 minutes are suitable for these
poly-U sequences when using 0.05M DDTT,
and longer sulfurization times did not
improve the result. Beaucage Reagent
achieved results similar to DDTT on these
same poly-U sequences provided the
sulfurization time was longer.
Similar results were achieved using UTBDMS phosphoramidites with 0.05M DDTT
and a range of sulfurization times between
60 and 240 seconds. Beaucage Reagent
required a 240 second sulfurization time
to achieve the similar results when using
TABLE 3: 31P NMR DATA
Chemical Shift
PS
PO
Oligo
PSO
PSO
PSO
DNA PSO
PSO
PSO + 1 PO
PSO + 1 PO
57.03ppm
57.05ppm
57.04ppm
56.15ppm
57.00ppm
57.06ppm
57.01ppm
n/a
n/a
n/a
n/a
n/a
0.18ppm
0.21ppm
Sulfurizing
Reagent
Monomer
0.05M DDTT TBDMS-RNA
Beaucage TBDMS-RNA
0.03M DDTT TOM-RNA
0.05M DDTT
DNA
0.05M DDTT TOM-RNA
mixed oxd. TOM-RNA
mixed oxd. TOM-RNA
FIGURE 8: 31P NMR SPECTRA
(A)
(B)
A) 31P NMR of Poly-U RNA phosphorothioate, B) 31P NMR of Poly-U RNA phosphorothioate with a
single phosphodiester linkage
TABLE 4: COUPLING RESULTS MIXED BASE OLIGO
Sequence: 5’-UUA UUC UUG UUA UUC UUG TT-3
Reagent
CE %
Time
Monomer
0.05M DDTT
0.05M DDTT
0.05M DDTT
0.05M DDTT
0.05M DDTT
0.05M DDTT
Beaucage Reagent
Beaucage Reagent
94.9
94.8
98.4
97.9
97.2
98.1
96.8
97.0
60
60
120
120
240
240
240
240
TOM-RNA
TOM-RNA
TOM-RNA
TOM-RNA
TOM-RNA
TOM-RNA
TOM-RNA
TOM-RNA
5
U-TBDMS phosphoramidites.
We also synthesized some mixed base
PSO sequences using TOM phosphoramidites
at increasing sulfurization times and
compared them to a mixed base synthesis
using Beaucage Reagent at a 240 second
sulfurization time. The DDTT reagent was
a more efficient sulfurizing reagent with
higher coupling efficiency results than
Beacuage Reagent under these conditions.
Finally, the synthesis of the mammalian
LMNA sequence provided the highest
coupling efficiencies. The coupling
efficiencies even exceeded 99% when using
0.05M DDTT with a sulfurizing time of 360
seconds and TBDMS phosphoramitides. It is
important to note that coupling times were
increased from four minutes to ten minutes
to eliminate potential kinetic effects from
the coupling reaction. Further work is
warranted to fully optimize the conditions
to obtain the highest coupling efficiencies
for each type of phosphoramidite and the
shortest cycle times.
CONCLUSIONS
DDTT and Beaucage Reagent are both
suitable sulfurizing reagents for RNA and
DNA. DDTT offers the added benefits of
improved performance, extended stability
for use on a DNA synthesizer, and does not
require the use of silanized glassware.
6
TABLE 5: COUPLING EFFICIENCIES FOR THE LMNA SEQUENCE.
Sequence: 5’-CUG GAC UUC CAG AAG ATT-3’.
Reagent
CE %
Time
Monomer
Iodine Oxidation
Iodine Oxidation
0.05M DDTT
0.05M DDTT
0.05M DDTT
0.05M DDTT
Beaucage Reagent
96.3
96.2
97.9
97.7
98.6
99.1
98.2
30
30
240
360
360
360
360
TBDMS-RNA
TBDMS-RNA
TOM-RNA
TOM-RNA
TBDMS-RNA
TBDMS-RNA
TBDMS-RNA
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