Ion-tagged synthesis of an oligoribonucleotide pentamer - The continuing versatility of TBDMS chemistry

Article abstract of DOI:10.1139/V07-022

An oligoribonucleotide has been synthesized in solution, using an ionic-liquid-based soluble tag at a scale several hundred times that of a standard solid-phase synthesis approach. Ogilvie's 2′-TBDMS strategy was adopted, and because of the resultant increase in lipophilicity, it allowed an easier purification of the growing oligomer compared with the previously observed for DNA, which does not require 2′ protection. The procedure is illustrated by the synthesis of the pentaribonucleotide sequence AGAUC, corresponding to a segment of the tRNA^fMet from E. coli.

Full text of DOI:10.1139/V07-022

274
Ion-tagged synthesis of an oligoribonucleotide
pentamer — The continuing versatility of TBDMS
chemistry1
Robert A. Donga, Tak-Hang Chan, and Masad J. Damha
Abstract: An oligoribonucleotide has been synthesized in solution, using an ionic-liquid-based soluble tag at a scale
several hundred times that of a standard solid-phase synthesis approach. Ogilvie’s 2′-TBDMS strategy was adopted, and
because of the resultant increase in lipophilicity, it allowed an easier purification of the growing oligomer compared
with the previously observed for DNA, which does not require 2′ protection. The procedure is illustrated by the synthe-
sis of the pentaribonucleotide sequence AGAUC, corresponding to a segment of the tRNAfMet from E. coli.
Key words: solution-phase RNA synthesis, ionic-liquid tag.
Résumé : Nous avons réalisé la synthèse d’un oligoribonucléotide en solution en utilisant un support soluble à base
d’un liquide ionique dont la quantité correspond à plusieurs centaines de fois celle qui serait utilisée dans une approche
basée sur une synthèse en phase solide normale. On a adopté la stratégie 2′-TBDMS d’Ogilvie et on a trouvé que, en
raison de son caractère lipophile, la purification de l’oligomère croissant se fait beaucoup plus facilement que ce qui a
été observé avec l’ADN qui ne requiert pas de protection en 2′. La méthode est illustrée par la synthèse de la séquence
du pentaribonulcléotide AGAUC qui correspond à un segment de la t-ARNfMet du E. coli.
Mots-clés : synthèse d’ARN en solution, traceur ionique liquide.
[Traduit par la Rédaction] Donga et al. 282
Introduction
a solution-phase approach (5). A conventional solution-
phase approach, where the 3′-terminal nucleoside is not
linked to a support but simply protected at the 3′ position
(and 2′ for RNA), is intrinsically arduous because of the ne-
cessity of chromatographic purification after each coupling
cycle. An alternative approach, developed by Bonara et al.
(6), uses a soluble polyethylene glycol tag for DNA solu-
tion-phase synthesis. In an effort to improve this strategy,
which requires an energy-intensive cooling step during puri-
fication, and to adapt it to use in RNA, we have been explor-
ing the utility of ionic liquids as soluble supports in
oligonucleotide synthesis (7). Herein, we report the prepara-
tion of a pentaribonucleotide in solution, requiring only sim-
ple precipitations at ambient temperature for purification
during the chain-extension cycles. The process retains the
versatile TBDMS group as the 2′-protecting group. The
pentaribonucleotide was prepared on a 1 g scale and was as-
sembled in 32 h (an average of 8 h per coupling cycle).
Since it was first introduced by Ogilvie and co-workers in
1974 (1), the tert-butyl dimethylsilyl (TBDMS) group has
become the standard 2′-protecting group in both solid- and
solution-phase oligomerizations of ribonucleotides (2).
Along with the phosphoramidite approach (3) to ribonucleo-
side coupling, the TBDMS group remains one of the most
widely used 2′-protecting groups for commercially available
ribo-phosphoramidites. With the advent of RNAi and an in-
creasing number of RNA-based therapeutics progressing to-
ward clinical development (4), the TBDMS approach will
continue to be an important resource as more commercially
viable processes are developed for the large-scale synthesis
of oligoribonucleotides.
However, the limitation in RNA chemistry, thus far, has
been the cost-efficient preparation of oligomers at a suffi-
ciently large scale. A solid-phase approach to large-scale
DNA and RNA synthesis is expensive because of the cost of
the solid supports and the need for a large excess of
phosphoramidite compared with what is usually required in
Discussion
Received 10 January 2007. Accepted 28 February 2007.
Published on the NRC Research Press Web site at
canjchem.nrc.ca on 18 April 2007.
The sequence of the selected pentaribonucleotide repre-
sents base positions 45 to 49 of an unpaired coil region of N-
formyl methionine tRNA from E. coli. It was selected pri-
marily because it contains all four common ribonucleosides.
It is also a sequence that has been previously synthesized in
solution by Damha using the phosphite triester approach (8),
which required several weeks to achieve. The latter com-
pound serves as a good reference standard for the penta-
ribonucleotide made by the process described here.
R.A. Donga, T.-H. Chan, and M.J. Damha.2 Department of
Chemistry, McGill University, Montreal, QC H3A 2K6
Canada.
1This article is part of a Special Issue dedicated to Professor
Kelvin K. Ogilvie.
2Corresponding author (e-mail: masad.damha@mcgill.ca).
Can. J. Chem. 85: 274–282 (2007)
doi:10.1139/V07-022
© 2007 NRC Canada

Donga et al.
275
Scheme 1. Nucleoside succinylation.
Scheme 2. Coupling of ionic tag.
Synthesis began with the attachment of the 3′-cytidine to
the ionic ethyl-methyl-imidazolium tag through a succinyl
linker. Thus, N-benzoyl-2′-tert-butyldimethylsilyl-5′-dimeth-
oxytrityl-cytidine was reacted with succinic anhydride in the
presence of 4-dimethylaminopyridine (DMAP). The pres-
ence of the base, DMAP, caused the 2′-TBDMS protecting
group to isomerize, yielding a statistical mixture of 2′-
TBDMS- and 3′-TBDMS-protected cytidine with no alter-
ation of the other protecting groups (Scheme 1). However,
this isomerization is inconsequential, since the 2′/3′-silyl
group and the 3′/2′-succinyl linker of this terminal
nucleoside will be removed to release the required 2′- and 3′-
OH groups during the final deprotection step of the desired
full-length pentamer. In addition, it would be expected that
the solubility properties of the two regioisomers, an impor-
tant consideration for the purification steps, would be very
similar. Succinylation at the free 3′- or 2′-hydroxyl group
provided the 2′/3′-succinylated nucleoside along with some
unreacted nucleoside. This crude mixture was then esterified
with the free hydroxyl group of the ionic tag (Scheme 2). In
our previous work (7), dicyclohexylcarbodiimide (DCC) was
used as the condensing reagent, but long reaction times were
required (3–7 d), and the yield of the desired succinate ester
was only 65%–85%. An alternative coupling reagent,
O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetra-
fluoroborate (TBTU), proved to be more suitable for this re-
action. In addition, its counterion, tetrafluoroborate, matched
that of the ionic liquid employed as the tag. Thus, the
succinylated nucleoside 1 and TBTU were first mixed in the
solid state, followed by the addition of the ionic liquid [3-
(2′-hydroxyethyl)-1-methylimidazolium tetrafluoroborate],
triethylamine, and finally the solvent. The reaction was com-
plete in less than 90 min, yielding the desired product 2 and
a small amount of nucleoside dimer; the latter formed by the
reaction of 2 with a small amount of 5′-DMT rCNBz TBDMS
that was carried forward from the succinylation step. At this
point, a water–chloroform extraction was performed to re-
move any excess ionic liquid, the excess coupling reagent,
and the resulting tetramethylurea by-product of the reaction,
leaving a mixture of nucleoside dimer and the desired prod-
uct 2. The ion-tagged nucleoside 2 was easily separated
from the dimer and any other remaining impurities by two
simple precipitations. In our previous work on DNA oligo-
mers (7), the preferred precipitation medium consisted of a
mixture of ethyl acetate and diethyl ether. This yielded purer
precipitates than diethyl ether alone by effectively
solubilizing the undesired by-products while generating the
desired tagged oligomer as a finely divided precipitate.
However, when this media was used here, it was found that
the nucleoside 2 was slightly soluble, resulting in a loss of
the desired product and collapse of the precipitate into a
slightly impure waxy solid. Therefore, the system was
changed to neat diethyl ether as this was found to restore the
desired properties of the precipitation medium with no ap-
preciable loss of the tagged materials.
The synthesis cycle (Scheme 3) requires the removal of
the DMT group at the 5′-terminus of the growing oligo-
nucleotide chain. Generally, this is achieved by treatment
with trifluoroacetic acid in dichloromethane. In our earlier
work on DNA, this step was often problematic, especially
for removing the DMT group from the nucleoside unit di-
rectly linked to the ionic tag. We ascribe the slower rate of
DMT cleavage to the proximal imidazolium ion, which
makes protonation of the O5′ rate limiting. A similar phe-
nomenon is generally invoked to explain the slower rate of
+
esterification of glycine (NH3 –CH2–COOH), relative to
propanoic acid under acidic conditions (9). Consistent with
this notion, the rate of detritylation increases as the site of
© 2007 NRC Canada

276
Can. J. Chem. Vol. 85, 2007
Scheme 3. Oligoribonucleotide coupling cycle (illustrated as one regioisomer).
protonation is spatially removed from the positively charged
ion tag (i.e., rate of detritylation of trimer > monomer; see
later). Alternate detritylation methods employing Lewis ac-
ids, such as zinc bromide, were tested and found to be some-
what successful, but Lewis acids were more difficult to
remove via precipitation. Thymine and cytosine, and to a
greater extent adenine and guanine, are sensitive to acid-
mediated depyrimidation/depurination (10), rendering the
application of stronger acids troublesome in DNA synthesis.
Because of the inductive effects of 2′-OH groups, RNA is
not as prone to acid-mediated depurination; therefore, a
stronger acid may be employed in the detritylation step.
Thus, p-toluenesulphonic acid was selected as the detrityl-
ation reagent, as it was found that a single treatment was
sufficient to achieve complete detritylation of the first
nucleoside. Weaker acids, such as trifluoroacetic acid, re-
quired multiple treatments and longer reaction times to
achieve the same result.
protected) 3′-cyanoethylphosphoramidite, using 4,5-
dicyanoimidazole (DCI) as the activator (0.06 mol/L in
anhyd. acetonitrile). After 2 h of stirring, excess tert-butanol
(approximately 6 equiv.) was added to quench the excess
phosphoramidite and to enhance the solubility of the result-
ing nucleoside 3′-(tert-butyl-2-cyanoethyl) phosphite triester
by-product in the precipitation medium. The intermediate
UpC dimer was isolated and purified by adding the reaction
mixture dropwise to stirred diethyl ether. The resultant fine
precipitate was filtered over celite and recovered from the
filter by dissolving in acetone.
Once purified, the intermediate UpC phosphite triester
was redissolved in anhydr. acetonitrile, and a capping step
was performed using acetic anhydride and DMAP. In our
DNA synthesis (7), oxidation of the intermediate phosphite
triester to the phosphate was achieved using iodine as the
oxidant. This procedure necessitated a difficult extraction to
remove the generated salts but precluded the need for a cap-
ping step, since any unreacted oligomer would be removed
with the aq. phase. The difficulty of the extraction (because
of the formation of strong emulsions) outweighed the advan-
With the 5′ position of the 3′-nucleoside deblocked, the
oligomerization cycle could begin (Scheme 3). The nucleo-
side 3 was mixed with 1.66 equiv. of ribouridine (2′-TBDMS-
© 2007 NRC Canada

Donga et al.
277
Table 1. Physical data.
Recovery
(%)
m/z
(Calculated)
m/z
Compound
Formulaa
31P NMR (ppm)
(Experimental)
73
93
972.4
670.3
972.3
5-DMT rC Succ-IL (2)
C53H62N5O11Si+
C32H44N5O9Si+
C71H90N8O19PSi2
C50H72N8O17PSi2
—
5-HO rC Succ-IL (3)
—
670.2
+
+
5-DMT rUC Succ-IL (4)
5-HO rUC Succ-IL (5)
93
—
1445.6
1143.4
1034.4
1743.6
1311.5
1160.4
1611.6
1460.5
1446.0
1143.5
1034.5
1744.4
1311.7
1161.0
1611.8
1460.8
>100
91
0.082 to –0.513
5-DMT rAUC Succ-IL (6)
5-HO rAUC Succ-IL (7)
5-DMT rGAUC Succ-IL (8)
5-HO rGAUC Succ-IL (9)
5-DMT rAGAUC Succ-IL (10)
5-HO rAGAUC Succ-IL (11)
C97H123N14O26P2Si3Na2+
—
+
>100
99
C76H105N14O24P2Si3
0.510 to –0.508
—
C118H154N20O34P3Si4Na2+
C97H136N20O32P3Si4Na2+
C144H187N26O41P4Si5Na2+
C123H169N26O39P4Si5Na2+
91
0.637 to –0.466
—
95
96
0.599 to –1.318
tage of the removal of the capping step, especially since the
capping step overcomes the undesirable O6 extension some-
times observed for guanosine (11). Thus, in the present pro-
tocol for RNA, the capping step was reinstated, and the
oxidation procedure was changed to employ a reagent that
would not require extractions. tert-Butylhydroperoxide
(6 mol/L) in decane was chosen and found to completely ox-
idize the phosphite triester in less than 15 min. A further ad-
vantage is that it could easily be removed, together with its
by-product tert-butanol, since it was completely soluble in
the precipitation medium. At this point, it was observed by
mass spectroscopy that the original tetrafluoroborate
counteranion of the tag had been exchanged for dicyano-
imidazolide, an effect that was not observed in our previous
work on DNA synthesis. Indeed, anion exchange occurred
several times during the synthesis cycles, alternating be-
tween dicyanoimidazolide and p-toluenesulphonate, but this
did not appear to adversely affect the solubility properties of
the intermediate di (UpC), tri (ApUpC), tetra (GpApUpC),
and pentanucleotides (ApGpApUpC), isolated during and af-
ter each coupling step (see later). It had been previously ob-
served in other experiments (data not shown) that the
solubility properties of these types of molecules were ad-
versely affected when exchanges occurred for halides; thus,
the identity of the counterion is of importance, since a
change in solubility can have a dramatic impact on the qual-
ity and yield of the synthesis cycle. Therefore, this must be
taken into account during the design of the oligomerization
cycle such that if anion exchanges occur, the resulting ion-
paired molecule maintains an acceptable level of solubility
in the solvents used for synthesis and recovery, while also
maintaining its insolubility in the precipitation medium.
detritylation was observed at the coupling stage, and precipi-
tations alone were sufficient to remove the acid. Thus, while
it has been found that weaker acids may be used for the
detritylation step of the oligomerization cycle shortly after
the initial nucleoside is deprotected, multiple treatments are
still required so it would be advantageous to use stronger ac-
ids if their complete removal could be assured.
One of the advantages of solution-phase over solid-phase
synthesis of oligomers is the capacity to directly monitor
chain elongation by various means, including mass spec-
trometry (MS). Indeed, ion-tagged oligomers are especially
amenable to this technique, since the molecules contain per-
manent positive charges. This means that ion-tagged mole-
cules only need to have their solutions nebulized in an
electrospray ionization (ESI) source to give very strong MS
signals. Indeed, when using an octopole analyzer, short oli-
gomers provided excellent results. Long oligomers quickly
exceed the upper limits of the mass detector; however, in
these cases multiple charging occurred more readily, pulling
the analyte back into an observable window (Fig. 1). This is
noticed by comparing the mass spectrum for longer oligo-
mers to that of UpC (Fig. 2). For the dimer, the ion tag cre-
ates a high-energy barrier to protonation and likewise to
formation of these adducts. As the chain length increases,
the site of protonation is spatially removed from the posi-
tively charged ion tag, and adducts are readily observed.
Using this technique, the identities of all post-detritylation
compounds were confirmed by HR-ESIMS (Table 2).
Finally, the pentamer 11 was deprotected to the free nu-
cleotide in parallel with a sample of the original sequence 8,
made by the original solution-phase approach (8). This con-
sisted of treatment of 1–2 mg of each detritylated (but other-
wise fully protected oligomer) with 1 mL of 40% (v/v) aq.
methylamine and incubating at 60 °C for 20 min. The resul-
tant solution was cooled to –78 °C and dried under vacuum.
The resultant solid was treated with 300 µL of 1.5:0.75:1
Chain extension proceeded as previously outlined, but it
was found that the yields exceeded 100% after the
detritylation steps, as determined by the weight of the prod-
uct (Table 1). This was due to the incomplete removal of p-
toluenesulphonic acid, which subsequently interfered with
the next coupling steps (e.g., premature detritylation follow-
ing the next coupling step). Once this effect was realized,
the detritylating reagent was substituted with the volatile
trifluoroacetic acid, which could be more readily removed
during the precipitation step or afterwards, by evaporation if
necessary. Again, this approach necessitated two acid treat-
ments per detritylation step, but no further premature
(v/v/v) triethylamine trihydrofluoride
–
1-methyl-2-
pyrrolidinone – triethylamine solution and incubated for
90 min at 60 °C (12). The reaction mixture was then cooled
to ambient temperature and 1 mL of anhyd. ethanol was
added. The samples were mixed and cooled at –78 °C for
30 min, resulting in precipitates. The samples were then cen-
trifuged at 14000 r/min at 4 °C for 30 min, and the
supernatant was removed. The precipitates were washed
© 2007 NRC Canada

278
Can. J. Chem. Vol. 85, 2007
Fig. 1. Low resolution ESIMS of 9.
Fig. 2 Low resolution ESIMS of 3.
with 200 µL of –78 °C ethanol, and then the residual solvent
was removed under vacuum. The precipitates were dissolved
in 1 mL of autoclaved, deionized water and loaded onto Wa-
ters® Sep-Pak™ C18 cartridges for desalting. The cartridges
were rinsed with water, and then the desired products were
eluted with 1:1:1 (v/v/v) water–methanol–acetonitrile. The
solvent was removed under vacuum to yield the crude,
deprotected AGAUC pentamers.
The identity and purity of the pentamers were assessed us-
ing a triethylammonium acetate ion-pairing reverse-phase
© 2007 NRC Canada

Donga et al.
279
Table 2. HR-ESIMS data.
m/z
(Calculated)
m/z
Compound
Formulaa
(Experimental)
670.29069
572.21727
872.31268
1149.39748
966.66590
670.29028
572.21795
872.31383
1149.39934
966.66546
5-HO rC Succ-IL (3)
C32H44N5O9Si+1
5-HO rUC Succ-IL (5)
5-HO rAUC Succ-IL (7)
5-HO rGAUC Succ-IL (9)
5-HO rAGAUC Succ-IL (11)
C50H73N8O17Si2P+2
C76H106N14O24Si3P2
C97H137N20O32Si4P3
+2
+2
+3
C123H171N26O39Si5P4
aExcluding counterion.
Fig. 3. HPLC chromatogram of the co-injection of 11 and solution-phase synthesized standard.
HPLC analysis on a Phenomenex® Clarity™ semipreparatory-
scale column. The samples were injected individually, and
the major peaks in the chromatograms were collected and
analyzed by low resolution ESIMS in the negative mode.
The major ion found in both cases corresponded exactly to
the expected mass of the fully deprotected AGAUC
pentamers missing a single proton (1551 daltons). The two
samples were also co-injected at a 1:1 ratio in the HPLC col-
umn and found to overlap perfectly (Fig. 3). As a final test
to show that the deprotected compounds were identical, they
were analyzed side by side, co-loaded on a 24% polyacryl-
amide gel, and electrophoresed (data not shown). In all
cases, single bands with the same electrophoretic mobility
were found, showing that indeed the synthesis using the
ionic tag yielded, after deprotection, an identical compound
to that of the standard solution-phase-derived molecule.
purifications. Thus, the purification of RNA using this
approach is superior to that of DNA, as observed in our pre-
vious work (7). We believe that the soluble ionic tag we
have developed, in conjunction with Ogilvie’s RNA method,
enable the synthesis of short RNA oligomers as rapidly as
DNA. We will further explore the limits of this method with
target sequences corresponding to those of short interfering
RNA (19–23 nucleotides).
Experimental
N-benzoyl-2′/3′-tert-butyldimethylsilyl-3′/2′-succinyl-5′-
dimethoxytrityl-cytidine (1)
N-benzoyl-2′-tert-butyldimethylsilyl-5′-
dimethoxytrityl-cytidine (1 g, 1.31 mmol) was mixed with
succinic anhydride (0.39 g, 3.93 mmol) and 4-
dimethylaminopyridine (0.16 g, 1.34 mmol), and the mixture
was dissolved in 30 mL of dry acetonitrile. The reaction was
stirred for 4 d at room temperature, and the progress was de-
termined by TLC (9:1 dichloromethane–methanol), which
showed the disappearance of the starting material and the
appearance of two products (2′-3′ isomerization of the
TBDMS-protecting group). The solvent was removed under
vacuum, and the resulting solid was taken up in chloroform
and extracted with water. The organic layer was dried with
anhyd. sodium sulphate, filtered, and the solvent was re-
moved under vacuum. The resulting crude product (1.1 g,
Conclusion
In summary, a functional method has been developed to
rapidly synthesize oligoribonucleotides in solution and in
excellent yields. The method utilizes simple precipitations as
the sole purification method during chain elongation and is
amenable to scales several hundred times that of a standard
solid-phase synthesis. Although 5′-detritylation continues to
pose a challenge, the presence of the lypophilic TBDMS
group greatly enhances the solubility of the phosphoramidite
monomers in the medium used for the precipitation-based
© 2007 NRC Canada

280
Can. J. Chem. Vol. 85, 2007
97%) was used without further purification. C47H54N3O11Si+
(low resolution ESIMS) calculated: 864.3; found: 863.9.
ether, and collected from the filter by dissolution in acetone
(0.77 g, 93%) and was used without further purification.
C71H90N8O19PSi2 (low resolution ESIMS) calculated: 1445.6;
+
found: 1446.0, dicyanoimidazolide counterion by ESIMS.
N-benzoyl-2′/3′-tert-butyldimethylsilyl-3′/2′-(1-succinyl-4-
[3-(2′′-oxyethyl)-1′′-methylimidazolium tetrafluoro-
borate])-5′-dimethoxytrityl-cytidine (2)
5′-HO-rU-(2′-TBDMS)-3′-5′-rC-(N-Bz)-2′/3′-TBDMS-
Succ-IL (as p-toluenesulphonate salt) (5)
Compound 1 (1.1 g, 1.31 mmol) was mixed with TBTU
(0.5 g, 1.58 mmol), 3-(2′-hydroxyethyl)-1-methylimidazo-
lium tetrafluoroborate (0.34 g, 1.58 mmol) and triethylamine
(0.4 mL, 2.62 mmol). The mixture was dissolved in 20 mL
dry acetonitrile and stirred for 90 min. The solvent was then
removed under vacuum, and the resulting oil was taken up in
chloroform and extracted with water. The organic layer was
dried over sodium sulphate. The solvent was removed under
vacuum, and the resulting solid was dissolved in 5 mL of ac-
etone. The solution was added dropwise to stirred diethyl
ether, resulting in a finely divided precipitate. The precipi-
tate was filtered off, rinsed with diethyl ether, taken up in
acetone, and precipitated again. The solid was filtered off,
rinsed, and collected from the filter by dissolution in acetone
(1.01 g, 73%) and was used without further purification.
C53H62N5O11Si+ (low resolution ESIMS) calculated: 972.4;
found: 972.3, tetrafluoroborate counterion by ESIMS.
Compound 4 (0.77 g, 0.49 mmol) was dissolved in 10 mL
of 0.1 mol/L p-toluenesulphonic acid solution in acetonitrile
and stirred for 10 min. Methanol (5 mL) was added to
quench the trityl cation, and the reaction mixture was added
dropwise to stirred diethyl ether, resulting in a finely divided
precipitate. The precipitate was filtered off, rinsed with di-
ethyl ether, taken up in acetone, and precipitated again. The
solid was filtered off, rinsed and collected from the filter by
dissolution in acetone (0.68 g, 0.65 g theoretical yield, ex-
cess thought to be residual salts), dried under vacuum, and
was used without further purification. 31P NMR (ppm):
0.082 to –0.513. C50H73N8O17Si2P+2 (HR-ESIMS) required:
572.21727; found: 572.21795, p-toluenesulphonate counterion
by ESIMS.
5′-DMT-rA-(N-Bz, 2′-TBDMS)-3′-5′-rU-(2′-TBDMS)-3′-5′-
rC-(N-Bz)-2′/3′-TBDMS-Succ-IL (as dicyanoimidazolide
salt) (6)
N-benzoyl-2′/3′-tert-butyldimethylsilyl-3′/2′-(1-succinyl-4-
[3-(2′′-oxyethyl)-1′′-methylimidazolium p-toluene-
sulphonate])-cytidine (3)
Compound 5 (0.60 g, 0.46 mmol) was mixed with N-
benzoyl-2′-tert-butyldimethylsilyl-5′-dimethoxytrityl-adenosine
(1.65 g, 1.67 mmol) and dicyanoimidazole (0.12 g, 1.0 mmol),
and the mixture was dissolved in 10 mL of dry acetonitrile.
Upon addition of solvent, the solution turned pale orange,
indicating that the excess mass appreciated in the previous
step was likely p-toluenesulphonic acid, causing some
detritylation of the incoming phosphoramidite. The reaction
was stirred for 1 h, and then 0.5 mL of tert-butanol was
added. The mixture was stirred a further 15 min and was
then precipitated from diethyl ether, as with previous com-
pounds. The precipitate was isolated from the filter by dis-
solving in acetone and then removing the solvent under
vacuum. The resulting solid was dissolved in 5 mL of dry
acetonitrile and 250 µL each of a 17.5% (v/v) solution of
acetic anhydride in anhyd. acetonitrile and saturated
dimethylaminopyridine in 25% (v/v) anhyd. pyridine –
acetonitrile. This reaction mixture was stirred for 10 min,
and then 0.5 mL of 6 mol/L tert-butylhydroperoxide in dec-
ane was added. After a further 10 min of stirring, the reac-
tion mixture was added dropwise to stirred diethyl ether,
resulting in a finely divided precipitate. The precipitate was
filtered off, rinsed with diethyl ether, collected from the fil-
ter by dissolution in acetone (0.89 g, 91%), and was used
without further purification. C97H123N14O26P2Si3Na2+ (low
resolution ESIMS) calculated: 1034.4; found: 1034.5,
dicyanoimidazolide counterion by ESIMS.
Compound 2 (0.69 g, 0.65 mmol) was dissolved in 10 mL
of 0.1 mol/L p-toluenesulphonic acid solution in acetonitrile
and stirred for 10 min. Methanol (5 mL) was added to
quench the trityl cation, and the reaction mixture was added
dropwise to stirred diethyl ether, resulting in a finely divided
precipitate. The precipitate was filtered off, rinsed with di-
ethyl ether, taken up in acetone, and precipitated again. The
solid was filtered off, rinsed, and collected from the filter by
dissolution in acetone (0.51 g, 93%) and was used without
further purification. C32H44N5O9Si+1 (HR-ESIMS) required:
670.29069; found: 670.29028, p-toluenesulphonate counter-
ion by ESIMS.
5′-DMT-rU-(2′-TBDMS)-3′-5′-rC-(N-Bz)-2′/3′-TBDMS-
Succ-IL (as dicyanoimidazolide salt) (4)
Compound 3 (0.44 g, 0.53 mmol) was mixed with 2-tert-
butyldimethylsilyl-5-dimethoxytrityl-uridine-3-cyanoethyl-
phosphoramidite (0.75 g, 0.88 mmol) and dicyanoimidazole
(0.14 g, 1.2 mmol), and the mixture was dissolved in 20 mL
of dry acetonitrile. The reaction mixture was stirred for 2 h,
and then 0.5 mL of tert-butanol was added. The mixture was
stirred a further 15 min and was then precipitated from di-
ethyl ether as with previous compounds. The precipitate was
isolated from the filter by dissolving in acetone and then re-
moving the solvent under vacuum. The resulting solid was
dissolved in 5 mL of dry acetonitrile and 250 µL each of a
17.5% (v/v) solution of acetic anhydride in anhyd.
acetonitrile and saturated dimethylaminopyridine in 25%
(v/v) anhyd. pyridine – acetonitrile. This reaction mixture
was stirred for 10 min, and then 0.5 mL of 6 mol/L tert-
butylhydroperoxide in decane was added. After a further
10 min of stirring, the reaction mixture was added dropwise
to stirred diethyl ether, resulting in a finely divided precipi-
tate. The precipitate was filtered off, rinsed with diethyl
5′-HO-rA-(N-Bz, 2′-TBDMS)-3′-5′-rU-(2-TBDMS)-3′-5′-
rC-(N-Bz)-2′/3′-TBDMS-Succ-IL (as p-toluenesulphonate
salt) (7)
Compound 6 (0.89 g, 0.41 mmol) was dissolved in 5 mL
of 0.1 mol/L p-toluenesulphonic acid solution in acetonitrile
and stirred for 10 min. Methanol (1 mL) was added to
quench the trityl cation, and the reaction mixture was added
dropwise to stirred diethyl ether, resulting in a finely divided
© 2007 NRC Canada

Donga et al.
281
precipitate. The precipitate was filtered off, rinsed with di-
ethyl ether, dissolved off the filter with acetone, and dried
under vacuum. TLC analysis indicated that detritylation was
incomplete, so the solid was dissolved in a further 5 mL of
0.1 mol/L p-toluenesulphonic acid solution in acetonitrile
and stirred for 10 min. Methanol (1 mL) was again added to
quench the trityl cation, and the reaction mixture was precip-
itated as before. The precipitate was filtered off, rinsed with
diethyl ether, and collected off the filter by dissolution in ac-
etone. The solution was concentrated under reduced pressure
to an approximate volume of 5 mL and was again precipi-
tated from diethyl ether. The solid was filtered off, rinsed
with diethyl ether and collected from the filter by dissolution
in acetone (0.81 g, 0.79 g theoretical yield, excess thought to
be residual salts), dried under vacuum, and was used without
further purification. 31P NMR (ppm): 0.510 to –0.508.
tate precipitation, and the reaction mixture was added
dropwise to stirred diethyl ether, resulting in a finely divided
precipitate. The precipitate was filtered off, rinsed with di-
ethyl ether, dissolved off the filter with acetone, and dried
under vacuum. The solid was redissolved in trifluoroacetic
acid solution and stirred for 10 min. Acetone (1 mL) was
again added, and the reaction mixture was precipitated as
before. The precipitate was filtered off, rinsed with diethyl
ether, and collected off the filter by dissolution in acetone.
The solution was concentrated under reduced pressure to an
approximate volume of 10 mL and was again precipitated
from diethyl ether. The solid was filtered off, rinsed with di-
ethyl ether and collected from the filter by dissolution in
acetone (0.82 g, 91%), dried under vacuum, and was used
without further purification. 31P NMR (ppm): 0.637 to
–0.466. C97H137N20O32Si4P3+2 (HR-ESIMS) required: 1149.39748;
found: 1149.39934, p-toluenesulphonate counterion by ESIMS.
+2
C76H106N14O24Si3P2 (HR-ESIMS) required: 872.31268;
found: 872.31383, p-toluenesulphonate counterion by ESIMS.
5′-DMT-rA-(N-Bz, 2′-TBDMS)-3′-5′-rG-(N-Ac, 2′-
TBDMS)-3′-5′-rA-(N-Bz, 2′-TBDMS)-3′-5′-rU-(2-
5′-DMT-rG-(N-Ac, 2′-TBDMS)-3′-5′-rA-(N-Bz, 2′-
TBDMS)-3′-5′-rU-(2′-TBDMS)-3′-5′-rC-(N-Bz)-2′/3′-
TBDMS)-3′-5′-rC-(N-Bz)-2′/3′-TBDMS-Succ-IL (as p-
TBDMS-Succ-IL (as p-toluenesulphonate salt) (8)
toluenesulphonate salt) (10)
Compound 7 (0.71 g, 0.37 mmol) was mixed with N-
acetyl-2′-tert-butyldimethylsilyl-5′-dimethoxytrityl-guanosine
(0.72 g, 0.77 mmol) and dicyanoimidazole (0.10 g,
0.84 mmol), and the mixture was dissolved in 7 mL of dry
acetonitrile. Upon addition of the solvent, the solution
turned slightly orange, indicating that the excess mass appre-
ciated in the previous step was likely p-toluenesulphonic
acid, causing some detritylation of the incoming phosphor-
amidite. The reaction was stirred for 1 h, and then 0.5 mL of
tert-butanol was added. The mixture was stirred a further
15 min and was then precipitated from diethyl ether, as with
previous compounds. The precipitate was isolated from the
filter by dissolving in acetone and then removing the solvent
under vacuum. The resulting solid was dissolved in 5 mL of
dry acetonitrile and 250 µL each of a 17.5% (v/v) solution of
acetic anhydride in anhyd. acetonitrile and saturated
dimethylaminopyridine in 25% (v/v) anhyd. pyridine –
acetonitrile. This reaction mixture was stirred for 10 min,
and then 0.5 mL of 6 mol/L tert-butylhydroperoxide in dec-
ane was added. After a further 10 min of stirring, the reac-
tion mixture was added dropwise to stirred diethyl ether,
resulting in a finely divided precipitate. The precipitate was
filtered off, rinsed with diethyl ether and collected from the
filter by dissolution in acetone (1.02 g, 99%), and was used
without further purification. Low resolution MS indicated
that no anion exchange occurred in this cycle (i.e., counterion
Compound 9 (0.71 g, 0.29 mmol) was mixed with N-
benzoyl-2-tert-butyldimethylsilyl-5′-dimethoxytrityl-adenosine
(0.72 g, 0.77 mmol) and dicyanoimidazole (0.08 g,
0.65 mmol), and the mixture was dissolved in 7 mL of dry
acetonitrile. No colour change was observed upon solvent
addition this time. The reaction mixture was stirred for 2 h,
and then 0.5 mL of tert-butanol was added. The mixture was
stirred a further 15 min and was then precipitated from di-
ethyl ether, as with previous compounds. The precipitate was
isolated from the filter by dissolving in acetone and then re-
moving the solvent under vacuum. The resulting solid was
dissolved in 5 mL of dry acetonitrile and 250 µL each of a
17.5% (v/v) solution of acetic anhydride in anhyd.
acetonitrile and saturated dimethylaminopyridine in 25%
(v/v) anhyd. pyridine – acetonitrile. This reaction mixture
was stirred for 10 min, and then 0.5 mL of 6 mol/L tert-
butylhydroperoxide in decane was added. After a further
10 min of stirring, 1 mL of acetone was added, and the reac-
tion mixture was added dropwise to stirred diethyl ether, re-
sulting in a finely divided precipitate. The precipitate was
filtered off, rinsed with diethyl ether and collected from the
filter by dissolution in acetone (0.92 g, 95%), and was used
without further purification. C144H187N26O41P4Si5Na2+ (low
resolution ESIMS) calculated: 1611.6; found: 1611.8, p-
toluenesulphonate counterion by ESIMS.
remained p-toluenesulphonate).
(low resolution ESIMS) calculated: 1311.5; found: 1311.7,
p-toluenesulphonate counterion by ESIMS.
C
118H154N20O34P3Si4Na2+
5′-HO-rA-(N-Bz, 2′-TBDMS)-3′-5′-rG-(N-Ac, 2′-
TBDMS)-3′-5′-rA-(N-Bz, 2′-TBDMS)-3′-5′-rU-(2′-
TBDMS)-3′-5′-rC-(N-Bz)-2′/3′-TBDMS-Succ-IL (as p-
toluenesulphonate salt) (11)
5′-HO-rG-(N-Ac, 2′-TBDMS)-3′-5′-rA-(N-Bz, 2′-
Compound 10 (0.92 g, 0.27 mmol) was dissolved in 5 mL
of 3% (v/v) trifluoroacetic acid in acetonitrile (~0.4 mol/L)
and stirred for 10 min. Acetone (1 mL) was added to facili-
tate precipitation, and the reaction mixture was added
dropwise to stirred diethyl ether, resulting in a finely divided
precipitate. The precipitate was filtered off, rinsed with di-
TBDMS)-3′-5′-rU-(2′-TBDMS)-3′-5′-rC-(N-Bz)-2′/3′-
TBDMS-Succ-IL (as p-toluenesulphonate salt) (9)3
Compound 8 (1.02 g, 0.37 mmol) was dissolved in 5 mL
of 3% (v/v) trifluoroacetic acid in acetonitrile (~0.4 mol/L)
and stirred for 10 min. Acetone (1 mL) was added to facili-
3 Because of the residual p-toluenesulphonic acid observed carrying over in the previous two coupling cycles, it was decided that a volatile
acid would be used for subsequent detritylations.
© 2007 NRC Canada

282
Can. J. Chem. Vol. 85, 2007
ethyl ether, dissolved off the filter with acetone, and dried
under vacuum. The solid was redissolved in trifluoroacetic
acid solution and stirred for 10 min. Acetone (1 mL) was
again added, and the reaction mixture was precipitated as
before. The precipitate was filtered off, rinsed with diethyl
ether, and collected off the filter by dissolution in acetone.
The solvent was removed under reduced pressure. TLC anal-
ysis indicated that detritylation was incomplete, so a third
acid treatment was performed following exactly the same
procedure as before. After precipitation and redissolution off
the filter with acetone, the solution was concentrated under
reduced pressure to an approximate volume of 10 mL and
was again precipitated from diethyl ether. The solid was fil-
tered off, rinsed with diethyl ether and collected from the fil-
ter by dissolution in acetone (0.81 g, 96%), and dried under
vacuum. No further purification was performed. 31P NMR
(ppm): 0.599 to –1.318. C123H171O39N26P4Si5+3 (HR-ESIMS)
required: 966.66590; found: 966.66546, p-toluenesulphonate
counterion by low resolution ESIMS.4
to acknowledge Alain Lesimple and Orval Mamer for the
HR-ESIMS measurements.
References
1. K.K. Ogilvie, K.L. Sadana, E.A. Thompson, M.A. Quilliam,
and J.B. Westmore. Tetrahedron Lett. 2861 (1974).
2. K.K. Ogilvie, N. Usman, K. Nicoghosian, and R.J. Cedergren.
Proc. Natl. Acad. Sci. USA. 85, 5764 (1988).
3. S.L. Beaucage and M.H. Caruthers. Tetrahedron Lett. 22, 1859
(1981).
4. K.K. Jain. Mol. BioSyst. 2, 523 (2006).
5. R.T. Pon, S. Yu, Z. Guo, R. Deshmukh, and Y.S. Sanghvi. J.
Chem. Soc., Perkin Trans. 1, 2638 (2001).
6. G.M. Bonora, C.L. Scremin, F.P. Colonna, and A. Garbesi.
Nucleic Acids Res. 18, 3155 (1990).
7. R.A. Donga, M. Khaliq-Uz-Zaman Syed, T.-H. Chan, and M.J.
Damha. J. Org. Chem. 71, 7907 (2006).
8. M.J. Damha. Developments in the chemical synthesis of nu-
cleic acids and their analogues. Ph.D. thesis, McGill Univer-
sity, Montreal. 1988.
9. A. Streiwieser, C.H. Heathcock, and E.M. Kosower. Introduc-
tion to organic chemistry. 4th ed. Macmillan Publishing Com-
pany, NY. 1992.
10. M. Roger and R.D. Hotchkiss. Proc. Natl. Acad. Sci. USA. 47,
653 (1961).
Acknowledgements
We acknowledge the financial support from the Natural
Sciences and Engineering Research Council of Canada
(NSERC) in the form of grants to M.J.D. and T.-H.C. This
work was also supported by a scholarship award from the
Chemical Biology Strategic Training Initiative of the Cana-
dian Institutes of Health Research to R.A.D. We would like
11. R.T. Pon, M.J. Damha, and K.K. Ogilvie. Nucleic Acids Res.
13, 6447 (1985).
12. L. Bellon and C. Workman. Deprotection of RNA. US Patent
6-054-576, 2000.
4 Supplementary data for this article are available on the journal Web site (http://canjchem.nrc.ca) or may be purchased from the Depository
of Unpublished Data, Document Delivery, CISTI, National Research Council Canada, Ottawa, ON K1A 0S2, Canada. DUD 5148. For more
information on obtaining material, refer to http://cisti-icist.nrc-cnrc.gc.ca/irm/unpub_e.shtml.
© 2007 NRC Canada