Unprecedented mild acid-catalyzed desilylation of the 2'-O-tert-butyldimethylsilyl group from chemically synthesized oligoribonucleotide intermediates via neighboring group participation of the internucleotidic phosphate residue

Article abstract of DOI:10.1021/ja961959i

Hydrolytic removal of the 2'-tert-butyldimethylsilyl (TBDMS) group from a 2'-O-TBDMS protected UpU dimer [U(2'-Si)pU] (1) (Si = TBDMS) and related derivatives under various acidic conditions was studied in detail. First, desilylation of 1 by use of acetic acid was examined. Consequently, we made the unprecedented discovery that cleavage of the 2'-silyl ether linkage occurred fastest at a very low concentration of acetic acid within the range of 5-10%, depending on the temperature. Formic acid could cleave the silyl ether much faster than acetic acid, but the relationship between the reaction rate and the concentration of acid was different from that of acetic acid. The use of 20-40% formic acid resulted in very effective elimination of the 2'-TBDMS group. Moreover, diluted HCl solution (pH 2.0) could cleave the Si-O bond faster than acetic acid at 30°C. In contrast, the 2'-silyl gloup of the corresponding methylphosphonate derivative [U(2'-Si)p(Me)U] (3) was much more stable than that of 1. In the case of a diastereomeric mixture of the phosphorothioate dimer [U(2'-Si)psU] (2), a big difference in reaction rate between the Rp- and Sp-diastereomers was observed. These results strongly suggest that neighboring group participation of the 3'-5' phosphorodiester group is involved in the present acid-catalyzed 2'-desilylation. These conditions were successfully applied to the deprotection of the 2'-TBDMS group of an RNA intermediate which was chemically synthesized by the conventional phosphoramidite approach on a CPG gel.

Full text of DOI:10.1021/ja961959i

VOLUME 118, NUMBER 40
OCTOBER 9, 1996
© Copyright 1996 by the
American Chemical Society
Unprecedented Mild Acid-Catalyzed Desilylation of the
2′-O-tert-Butyldimethylsilyl Group from Chemically
Synthesized Oligoribonucleotide Intermediates Via Neighboring
Group Participation of the Internucleotidic Phosphate Residue
Shun-ichi Kawahara, Takeshi Wada, and Mitsuo Sekine*
Contribution from the Department of Life Science, Tokyo Institute of Technology, Nagatsuta,
Midoriku, Yokohama 226, Japan
ReceiVed June 10, 1996X
Abstract: Hydrolytic removal of the 2′-tert-butyldimethylsilyl (TBDMS) group from a 2′-O-TBDMS protected UpU
dimer [U(2′-Si)pU] (1) (Si ) TBDMS) and related derivatives under various acidic conditions was studied in detail.
First, desilylation of 1 by use of acetic acid was examined. Consequently, we made the unprecedented discovery
that cleavage of the 2′-silyl ether linkage occurred fastest at a very low concentration of acetic acid within the range
of 5-10%, depending on the temperature. Formic acid could cleave the silyl ether much faster than acetic acid, but
the relationship between the reaction rate and the concentration of acid was different from that of acetic acid. The
use of 20-40% formic acid resulted in very effective elimination of the 2′-TBDMS group. Moreover, diluted HCl
solution (pH 2.0) could cleave the Si-O bond faster than acetic acid at 30 °C. In contrast, the 2′-silyl group of the
corresponding methylphosphonate derivative [U(2′-Si)p(Me)U] (3) was much more stable than that of 1. In the case
of a diastereomeric mixture of the phosphorothioate dimer [U(2′-Si)psU] (2), a big difference in reaction rate between
the Rp- and Sp-diastereomers was observed. These results strongly suggest that neighboring group participation of
the 3′-5′ phosphorodiester group is involved in the present acid-catalyzed 2′-desilylation. These conditions were
successfully applied to the deprotection of the 2′-TBDMS group of an RNA intermediate which was chemically
synthesized by the conventional phosphoramidite approach on a CPG gel.
Introduction
biology,5 the chemical synthesis of RNA oligomers has been
rapidly developed during the past 10 years and is now
established by use of the phosphoramidite and H-phosphonate
approaches.6-8 In the current RNA synthesis, the tert-butyldi-
Chemically synthesized oligonucleotides1 have proved to be
useful not only for basic studies of biochemistry2 and molecular
recognition3 but also for medicinal applications.4 With the
increasing demand for synthetic RNAs in the field of molecular
(4) Murray, J. A. H., Ed. Antisense RNA and DNA; Wiley-Liss, Inc.:
New York, 1992; and references cited therein.
(5) Davis, R. H. Curr. Opin. Biotechnol. 1995, 6, 213-217.
X Abstract published in AdVance ACS Abstracts, September 1, 1996.
(1) (a) Engels, J. W.; Uhlmann, E. Angew. Chem. Int. Ed. Engl. 1989,
28, 716-734. (b) Englisch, U.; Gauss, D. H. Angew. Chem. Int. Ed. Engl.
1991, 30, 613-722.
(2) (a) Steitz, T. Q. ReV. Biophys. 1990, 23, 205. (b) Harrison, S. Nature
1991, 353, 715-719.
(3) (a) He´le`ne, C.; Lancelot, G. Prog. Biophys. Mol. Biol. 1983, 41, 43.
(b) Nguyen T.; He´le`ne, C. Angew. Chem. Int. Ed. Engl. 1993, 32, 666-
690.
(6) For reviews of RNA synthesis see, Gait, M. J.; Pritchard, C.; Slim,
G. In Oligonucleotides and Analogues, A Practical Approach; Eckstein,
F., Ed.; IRL Press: Oxford, 1991; pp 25-48. (b) Gait, M. J. Curr. Opin.
Biotechnol. 1991, 2, 61-8. (c) Damha, M. J.; Ogilvie, K. K. In Protocols
for Oligonucleotides and Analogs, Synthesis and Properties; Agrawal, S.,
Ed.; Humana Press: Totowa, 1993; Chapter 5, pp 81-114. (d) Uhlmann,
E.; Peyman, A. Chem. ReV. 1990, 90, 543-584. (e) Damha, M. J.; Ogilvie,
K. K. Methods Mol. Biol. 1993, 20, 81-114.
S0002-7863(96)01959-2 CCC: $12.00 © 1996 American Chemical Society

9462 J. Am. Chem. Soc., Vol. 118, No. 40, 1996
Kawahara et al.
Figure 1. The time dependent HPLC profiles of hydrolysis of U(2′-Si)pU (1) in 20% AcOH at 30 °C.
Scheme 1
methylsilyl (TBDMS) group has been most widely used as a
reliable protecting group,8a,9 because of the easy preparation of
2′-O-TBDMS-ribonucleoside derivatives10 and facile removal
by the action of fluoride ion sources11 such as tetrabutylam-
monium fluoride (TBAF)12 and Et3N‚3HF.13
It has long been realized in nucleic acid chemistry that the
TBDMS group attached to the 2′-hydroxyl group of RNA
oligomers would be rather resistant to acids, since the acid
hydrolysis of secondary TBDMS ethers usually requires strongly
acidic conditions8b such as aqueous HCl14,15 or prolonged
reaction times of as much as 20-25 h when aqueous acetic
acid was employed.12,16 This fixed idea might also be deduced
from the well-known fact that treatment of 2′,3′,5′-O-tris(tert-
butyldimethylsilyl)ribonucleosides with 80% acetic acid gives
2′,3′-O-bis(tert-butyldimethylsilyl)ribonucleosides selectively in
high yields.10b Therefore, to date, no papers have been published
on the hydrolytic property of the 2′-TBDMS group in protected
RNA oligomer intermediates.
Results and Discussion
This study was started after our finding that a 2′-O-TBDMS-
protected UpU dimer 1,9a which was synthesized as a reference
material for the study of the synthesis of antisense nucleic acids,
gradually decomposed upon storage in aqueous solution to
release the TBDMS group, thus giving rise to a considerable
amount of UpU. To elucidate the mechanism of this extremely
easy elimination reaction in more detail, we studied the
hydrolytic cleavage of 1 under various conditions.
Acidic Hydrolysis of the TBDMS Group of 2′-O-TBDMS-
Protected UpU Dimer [U(2′-Si)pU)] (1). First, desilylation
of 1 under acidic conditions was investigated (Scheme 1).
Compound 1 was treated with an aqueous acetic acid solution
at 30 and 50 °C, an aqueous formic acid solution at 30 °C, or
0.01 M HCl (pH 2.0) at 30 °C. The reactions were monitored
by reverse-phase HPLC. Figure 1 shows the typical HPLC
profiles of the reaction mixture obtained by treatment of 1 with
20% acetic acid at 30 °C.
These acid-catalyzed desilylations were found to obey pseudo-
first-order kinetics as demonstrated by linear relationships
between the log of the amount of the remaining 1 and the time
of incubation (Figure 2). Figure 3 shows the relationship
between the reaction rate and the concentration of acetic acid
or formic acid at 30 and 50 °C. Interestingly, the hydrolysis of
the 2′-O-TBDMS ether linkage was rather slow in 80% acetic
acid which has been used frequently to remove the 5′-O-TBDMS
group from 5′-O-TBDMS-nucleosides.11 In contrast, the fastest
cleavage of the 2′-silyl ether was observed at rather low
concentrations of ca. 4% and 8% acetic acid at 50 and 30 °C,
respectively (Figure 3).17 On the other hand, the 2′-silyl ether
was cleaved much faster by treatment with formic acid than
acetic acid and the fastest desilylation was achieved at a
In this paper, we wish to report for the first time an
unexpectedly facile acid-catalyzed elimination of the 2′-TBDMS
group from 2′-O-(tert-butyldimethylsilyl)uridylyl(3′-5′)uridine
[U(2′-Si)pU] (1) and related derivatives U(2′-Si)psU (2) and
U(2′-Si)p(Me)U (3) and its successful application to the
deprotection of chemically synthesized RNA intermediates. The
mechanism of the present desilylation involving neighboring
group participation of the internucleotidic phosphodiester group
is also proposed.
(7) Scaringe, S. A.; Francklyn, C.; Usman, N. Nucleic Acids Res. 1990,
18, 5433-5441.
(8) (a) Rozners, E.; Westman, E.; Stro¨mberg, R. Nucleic Acids Res. 1994,
22, 94-9. (b) Stawinski, J.; Stro¨mberg, R.; Westman, E. Nucleic Acids
Symp. Ser. 1991, 24, 228.
(9) (a) Stawinski, J.; Stro¨mberg, R.; Thelin, M.; Westman, E. Nucleic
Acids Res. 1988, 16, 9285-9298. (b) Wu, T.; Ogilvie, K. K. J. Org. Chem.
1990, 55, 4717-4724.
(10) (a) Ogilvie, K. K. Can. J. Chem. 1973, 51, 3799-3807. (b) Ogilvie,
K. K.; Beaucage, S. L.; Schifman, A., L.; Sadana, K., L. Can. J. Chem.
1978, 56, 2768-2780.
(11) (a) Colvin, E. W. Silicon Reagents in Organic Synthesis; Academic
Press: London, 1988. (b) Greene, T. W.; Wuts, P. G. M. ProtectiVe Groups
in Organic Synthesis; John Wiley and Sons Inc.: New York, 1991.
(12) (a) Corey, E. J.; Venkateswawarlu, A. J. Am. Chem. Soc. 1972, 94,
6190-6191. (b) Ogilvie, K. K.; Beaucage, S. L.; Entwistle, D. W.
Tetrahedron Lett. 1976, 17, 1255-1256. (c) Lalonde, M; Chan, T. H.
Synthesis 1985, 817-845.
(13) Wincott, F.; DiRenzo, A.; Shaffer, C.; Grimm, S.; Tracz, D.;
Workman, C.; Sweedler, C.; Gonzalez, C.; Scaringe, S.; Usman, N. Nucleic
Acids Res. 1995, 23, 2677-2684.
(14) Cunico, R. F.; Bedell, L. J. Org. Chem. 1980, 45, 4797-4798.
(15) Wetter, H.; Oertle, K. Tetrahedron Lett. 1985, 26, 5515-5518.
(16) Ogilvie, K. K.; Thompson, E. A.; Quillam, E. A.; Westmore, J. B.
Tetrahedron Lett. 1974, 2865-2868.
(17) When 1 was incubated in acetic acid at concentrations higher than
40% at 50 °C or in formic acid at concentrations higher than 60% at 30
°C, significant decomposition occurred, so the reaction rate could not be
estimated.

Mild Acid-Catalyzed Desilylation of Oligoribonucleotide
J. Am. Chem. Soc., Vol. 118, No. 40, 1996 9463
no papers have appeared concerning desilylation of the TBDMS
group in secondary TBDMS ethers by the use of weak acids at
low concentrations such as 4% acetic acid.11 Since the acid
hydrolysis of the 2′-TBDMS group of 1 proceeded unexpectedly
fast even in such a weak acid, neighboring group participation
of the vicinal internucleotidic phosphate residue20 seems to be
important (Scheme 2). Similar rate enhancements have been
reported in the deprotection of 2′-tetrahydropyranylated and 2′-
Ftmp-protected UpU derivatives.21,22 In such reactions involv-
ing the neighboring group participation, the charge distribution
of the reaction species changes from “A-B + C” to “A-
+
B-C+” through “[Aδ-‚‚‚B‚‚‚Cδ+]” where A-B and C refer to
RO-SiMe2tBu and H2O. Thus, it is likely that the reaction
should proceed faster in polar solvents owing to the solvation
effect than in nonpolar solvents if the attack of water on the
silyl ether is a rate-determining step. Since the specific
permittivities of acetic acid, formic acid, and water are 58.5,
6.15, and 78.5, respectively, the reaction in acetic acid is more
affected by [H2O] than in formic acid.
Figure 2. The kinetic data of hydrolytic cleavage of the TBDMS group
of U(2′-Si)pU (1) under acidic conditions using acetic acid, formic acid,
and HCl.
Acidic Hydrolysis of the TBDMS Group of 2′-O-TBDMS-
Protected UpU Dimer Derivatives [U(2′-Si)psU) (2) and U(2′-
Si)p(Me)U (3)]. To ascertain whether the participation of the
internucleotidic phosphodiester residue is involved in the present
desilyalation, the phosphorothioate derivative [U(2′-Si)psU, 2]23
was synthesized. If hydrolysis of the 2′-TBDMS group of 1
does not involve neighboring group participation, no difference
in the reaction rates between the diastereomers of 2 should be
observed. This is because the elimination abilities of the
TBDMS groups in the diastereomers are not so different from
each other. In contrast, if the reaction proceeds with neighboring
group participation, the rates of desilylation of the diastereomers
should be significantly different from each other because the
hydrogen bonding between the oxygen of the 2′-silyl ether and
the hydrogen of the protonated internucleotidic phosphodiester
[(RO)(R′O)P(O)(OH)] can be formed favorably for only one
of the diastereomers.
Figure 3. The relationship between the reaction rates and the acid
concentration in acid-catalyzed desilylation of U(2′-Si)pU (1).
concentration of ca. 30% formic acid. The 2′-TBDMS group
of 1 can be cleaved fast by treatment with 0.01 M HCl (pH
2.0, 30 °C), which usually is employed for removal of acid-
labile 2′-protecting groups in oligoribonucleotide synthesis,18
so that 4 h was required for completion. Moreover, the reaction
proceeded without any side reactions even after 24 h as
evidenced by HPLC.19
The difference in the reaction rate of hydrolytic desilylation
between acetic acid and formic acid might be due to not only
the difference in pKa between formic acid (pKa 3.75) and acetic
acid (pKa 4.74) but also the solvation effect of the acids. For
example, the proton concentration of 5% formic acid (1.33 M,
pH 1.8) is almost twice that of 10% acetic acid (1.75 M, pH
2.3), but the rate constants of the desilylations using 5% formic
acid and 10% acetic acid were determined to be 2.7 × 10-2
and 7.7 × 10-3 min-1, respectively, showing that the desilylation
of 1 occurred not 2 but 3.5 times faster in the former than in
the latter. The profile of the reaction rates in acetic acid seems
to obey fourth order kinetics regarding [H2O] (Figure 4A), but
the reaction rate profile in formic acid seems first order to [H2O]
(Figure 4B), from the mathematical consideration.19 These
results indicate that the solvation network in this reaction is
important. Therefore, the solvation effect is discussed as
follows.
Since the 31P NMR resonance signals of the diastereomeric
phosphorothioate dimers Rp-[U(2′-Si)psU] (2b) and Sp-[U(2′-
Si)psU] (2a) have been assigned by Stawinski et al.,23 the
desilylations of 2 were monitored by 31P-NMR. Figure 5B
shows the HPLC profile of the mixture obtained by the reaction
of 2 with 0.01 M HCl-D2O (9:1, v/v) at 23 °C for 8 h. As
described in the hydrolysis of 1, these diasteroisomers have been
shown to undergo pseudo-first-order hydrolysis. On the other
hand, a marked difference in hydrolytic rate between these
diastereomers was observed as shown in Figure 6: The Rp
isomer 2b, -2.7 × 10-3 min-1; the Sp isomer 2a, -5.3 × 10-3
min-1
.
Next, desilylation of the methylphosphonate derivative [U(2′-
Si)p(Me)U] (3)24 was carried out to compare with that of 1.
Since the methylphosphonate diester bond is neutral, the
possibility of neighboring group participation is ruled out and
simple hydrolysis of the 2′-TBDMS ether linkage should be
expected. When the methylphosphonate dimer 3 was treated
with 0.01 M HCl (pH 2.0) at 30 °C under the same conditions
as prescribed for the hydrolysis of the TBDMS ether of 1, the
desilylation proceeded much more slowly (t1/2 ) 7 h) than for
(20) Tsuruoka, H.; Shohda, K.; Wada, T.; Sekine, M. submitted for
publication.
(21) Rao, T. S.; Reese, C. B.; Serafinowska, H. T.; Takaku, H.; Zappia,
G. Tetrahedron Lett. 1984, 28, 4897-4900.
It is known that hydrolysis of TBDMS ethers derived from
secondary alcohols requires stronger conditions than that of
TBDMS ethers from primary alcohols.10 In addition, to date
(22) Reese, C. B. Nucleosides Nucleotides 1991, 10, 81-97.
(23) Stawinski, H. A. J.; Stro¨mberg, R.; Thelin, M. J. Org. Chem. 1992,
57, 6163-6169.
(18) Griffin, B. E.; Jarman, M.; Reese, C. B. Tetrahedron 1968, 24, 639-
(24) (a) Jager, A.; Engels, J. Tetrahedron Lett. 1984, 25, 1437-1440.
(b) Helinski, J.; Dabkowski, W.; Michalski, J. Tetrahedron Lett. 1991, 32,
498l-4984.
622.
(19) See supporting information.

9464 J. Am. Chem. Soc., Vol. 118, No. 40, 1996
Kawahara et al.
Scheme 2
Figure 5. The 31P NMR analysis of the desilyation of a mixture of
Rp- and Sp-isomers of U(2′-Si)psU (2) in 0.01 M HCl-D2O (9:1, v/v)
at 23 °C.
Figure 4. Plots of the calculated (line) and observed (dot) reaction
rates.
1 (t1/2 ) 32 min), as clearly shown in Figure 7. In this case, a
diastereomeric mixture of 3 was used for the estimation of the
reaction rate. As a result, a linear relationship between the time
and logarithm of the progress of the reaction was observed, as
shown in Figure 7. It turned out that the rate of hydrolysis of
the 2′-TBDMS ether in the Rp isomer was similar to that of the
Sp isomer. On the basis of the above results, a plausible
mechanism is depicted in Scheme 2.
Monte Carlo Simulation25 of a Diastereomeric Pair of the
Protonated Form of U(2′-Si)pU. Provided that the hydrolysis
of the 2′-TBDMS ether in 1 is assisted by the participation of
the internucleotidic phosphodiester group in the protonation
process, a favorable conformer of 1 for hydrogen bonding
between the acidic hydrogen of the 3′-5′-phosphodiester group
and the oxygen of the 2′-silyl ether group must exist. Therefore,
conformational analysis using Monte Carlo simulations of a
diastereomeric pair of protonated U(2′-Si)pU (1a and 1b, Figure
8) was investigated.
If the neighboring group participation exists, the hydrogen-
bonding ability of 1a is different from that of 1b, and the
reaction pathway is limited to be Via conformers which are
suitable for the hydrogen bonding. Figure 8 shows the
relationship between the energy (vertical axis) and the atomic
Figure 6. The kinetic data of hydrolytic cleavage of the TBDMS group
of the Sp and Rp isomers of U(2′-Si)psU (2) in 0.01 M HCl-D2O
(9:1, v/v) at 23 °C.
distance between the oxygen atom of the 2′-TBDMS ether and
the acidic hydrogen atom of the protonated internucleotidic
phosphorodiester (horizontal axis) of the 100 conformers which
have lower energy structures of 1a and 1b. Obviously, 1a was
favorable to form the hydrogen bond, because the distances
between the oxygen atom and the hydrogen atom of most of
(25) Allen, M. P., Tildesley, D. J. Computer Simulation of Liquids,
Clarendon Press: Oxford, 1987.

Mild Acid-Catalyzed Desilylation of Oligoribonucleotide
J. Am. Chem. Soc., Vol. 118, No. 40, 1996 9465
Table 1. Atomic Distances, Bond Angles, and Diheadral Angles
of Each Moleculea
atom distance
(Å)
bond angle
(deg)
diheadral angle
(deg)
(MeO)2POS- (7)
S1-P2 1.9980 S1-P2-O3 118.4173
P2-O3 1.4677 S1-P2-O4 109.8489
P2-O4 1.6100 P2-O4-C5 119.7189
O4-C5 1.3993 O4-C5-H6 110.9188 S1-P2-O4-C5 -74.9066
C5-H6 1.0857 O4-C5-H7 107.2660 P2-O4-C5-H6
67.1413
186.2137
C5-H7 1.0839 O4-P2-O9
94.9998 P2-O4-C5-H7
C5-H8 1.0851 O4-C5-H8 110.9382 P2-O4-C5-H8 -54.4184
(MeO)2POSH (8)
S1-P2 2.0931 S1-P2-O3 109.7857
P2-O3 1.4455 S1-P2-O4 106.3407
P2-O4 1.5635 P2-O4-C5 122.2591 S1-P2-O4-C5 -86.4018
O4-C5 1.4246 O4-C5-H6 110.2105 P2-O4-C5-H6
C5-H6 1.0831 O4-C5-H7 106.1714 P2-O4-C5-H7
67.3490
186.2303
C5-H7 1.0783 O4-C5-H8 110.1343 P2-O4-C5-H8 -54.1737
C5-H8 1.0806 O4-P2-O9
S1-H14 1.3321 P2-S1-H14
99.6096 H14-S1-P2-O4 -52.7855
95.8045 O3-P2-S1-H14 179.9622
(MeO)2PSOH (9)
Figure 7. Comparison of the hydrolytic rate of the TBDMS group of
U(2′-Si)pU (1) with that of U(2′-Si)p(Me)U (3).
S1-P2 1.9190 S1-P2-O3 111.9424
P2-O3 1.5860 S1-P2-O4 117.6054
P2-O4 1.5678 P2-O4-C‘ 124.3526 S1-P2-O4-C5 -35.8750
O4-C5 1.4234 O4-C5-H6 110.0554 P2-O4-C5-H6
C5-H6 1.0803 O4-C5-H7 106.0366 P2-O4-C5-H7
66.3792
185.7560
C5-H7 1.0785 O4-C5-H8 110.5645 P2-O4-C5-H8 -55.0603
C5-H8 1.0827 O4-P2-O9
98.8210 H14-O3-P2-O4
51.6633
O3-H14 0.9449 P2-O3-H14 114.6540 S1-P2-O3-H14 179.9975
a Numerical labels used in this table are shown in Figure 9.
Figure 9. The numerical labels of 7, 8, and 9 used in MO calculation.
Scheme 3
Figure 8. Relationship between the O(2′)-H(phosphate) atom distance
and the energy of the conformers of the protonated diastereomers 1a
and 1b generated by Monte Carlo calculations.
the conformers are short enough to form the hydrogen bond,
and the energies of the conformers are relatively low. On the
other hand, the distance between the oxygen atom and the
hydrogen atom of the conformer of 1b is too long to form the
hydrogen bond, and the energy of each conformer is too high.
Therefore, the present hydrolysis seems to occur in favor of
1a.
Molecular Orbital (MO) Calculation of Phosphorothioate
Diesters. In the case of the phosphorothioate derivative 2, it
is important to see which atom is protonated, sulfur or oxygen.
In neutral water, most of the phosphorothioate is dissociated
and its sulfur atom is negatively charged (Scheme 3, 7a and
7b).26 However, in acidic solution, it is unknown which is
predominant, the sulfur protonated form or the oxygen proto-
nated form. The MO calculation of a phosphorothioate triester
was reported by Katagi,27 but there are no papers regarding the
phosphorothioate diester. Therefore, MO calculations (structure
optimization, HF/6-311G**; single point energy calculation,
MP2/6-311G**) of dimethyl phosphorothioate anion (7) and
two equilibrium forms of dimethyl phosphorothioate (8 and 9)
were carried out. Table 1 shows the calculated values of the
atomic distances, bond angles, and dihedral angles of each
structure.28 Table 2 shows the total energy of each structure,
atomic charge of each atom, energy levels of HOMO and
LUMO, and the atomic orbitals having large coefficients at
HOMO and LUMO. In the case of 7, the sulfur atom (S1) has
a larger atomic charge than that of the oxygen atom (O3), and
(26) (a) Frey, P. A.; Sammons, R. D. Science 1985, 228, 541-545. (b)
Frey, P. A.; Reimschussel, W.; Paneth, P. J. Am. Chem. Soc. 1986, 108,
1720-1722.

9466 J. Am. Chem. Soc., Vol. 118, No. 40, 1996
Kawahara et al.
the p-orbital of S1 has a large coefficient of HOMO. This result
suggests that the oxygen-protonated form 7a is predominant
(Scheme 3), being in good agreement with the experimental
result.26 On the other hand, from the total energy values of 8
and 9, the oxygen-protonated form 9 is 1.23 kcal/mol more
stable (1 au ) 627.5 kcal/mol) than the sulfur-protonated form
8. According to the energy difference between 8 and 9, the
ratio of 8 and 9 is calculated to be 11:89 at 25 °C on the basis
of the Boltzmann distribution.
Mechanism and Kinetics of Desilylation of Phospho-
rothioate Diastereomers. From consideration of the result of
the Monte Carlo simulations and MO calculations, the difference
in the reaction rates between the Sp and Rp isomers of 2 is
explained by the participation of the phosphorothioate diester
group in the protonation process of the 2′-silyl ether group. The
2′-TBDMS group in the Sp isomer of 2, which is hydrolyzed
faster, is protonated in the same direction as 1a, which is suitable
for neighboring group participation based on the Monte Carlo
simulation. On the other hand, the 2′-TBDMS group of the Rp
isomer, which is hydrolyzed more slowly, is protonated in the
same direction for 1b, which is unfavorable for neighboring
group participation.
Application to Deprotection of the 2′-TBDMS Group of
a Chemically Synthesized Oligoribonucleotide Intermediate.
The present reaction conditions were applied to the deprotection
of a chemically synthesized RNA intermediate. A 2′-O-
TBDMS-protected 12mer having the sequence of UACGUA-
CGUACG was synthesized on a highly cross-linked polystyrene
resin29 by the standard phosphoramidite method developed by
Andrus et al.30 The base protecting groups of A, C, and G used
were phenoxyacetyl, isobutyryl, and dimethylformamidine,
respectively.31 A commercially available polystyrene resin
carrying an appropriately protected G unit Via the usual succinate
linker was chosen, and the chain elongation was carried out as
usual. Release of the protected oligomer from the resin and
deprotection of the N-protecting groups were done by treatment
with concentrated ammonia-ethanol (3:1, v/v). The 2′-O-
TBDMS-protected 12mer having the DMTr group at the 5′-
end was treated with 0.01 M HCl (pH 2.0)-dioxane (1:1, v/v)
at 35 °C for 12 h. The ion-exchange HPLC profile of the
deprotected product thus obtained is shown in Figure 10A.
Further purification using ion-exchange HPLC followed by
desalting using gel filtration gave the pure oligomer in an overall
yield of 54% (Figure 10B,C).
Table 2. Total Energy, Atomic Charge, and HOMO and LUMO
Energies Representative Orbital Coefficients
total energy
(Hartree)
atomic charge
orbital energy
(Hartree)
(e)
(MeO)2POS- (7)
-1043.4293
1
2
3
4
5
6
7
8
9
10
11
12
13
S
P
-0.814820
1.351960
-0.804731
-0.640237
0.032888
0.096339
0.057313
0.087505
-0.640209
0.032849
0.087569
0.057299
0.096274
HOMO
orbital coefficient
0.5580
0.3359
-0.15128
O
O
C
H
H
H
O
C
H
H
H
(S1 10PZ)
(S1 11PZ)
1.4482
1.4458
-1.0774
-1.0755
(C5 4S)
(C10 4S)
(H7 3S)
(H12 3S)
(MeO)2POSH (8)
-0.188419
1.370418
-1043.9608
1
2
3
4
5
6
7
8
9
10
11
12
13
14
S
P
HOMO
orbital coefficient
0.6010
0.2714
-0.39848
O
O
C
H
H
H
O
C
H
H
H
H
-0.725247
-0.630023
0.017687
0.110636
0.113376
0.125895
-0.630009
0.017645
0.125913
0.113375
0.110629
0.068125
(S1 10PX)
(S1 11PX)
LUMO
orbital coefficient
-0.8934
0.9939
-0.6813
-0.6811
0.13435
(S1 6S)
(S1 11PZ)
(C5 4S)
(C10 4S)
(MeO)2PSOH (9)
-0.551246
1.371840
-1043.9628
1
2
3
4
5
6
7
8
9
10
11
12
13
14
S
P
HOMO
orbital coefficient
0.5753
0.2716
LUMO
orbital coefficient
1.1747
-0.35127
O
O
C
H
H
H
O
C
H
H
H
H
-0.602344
-0.635231
0.015077
0.134919
0.111782
0.107469
-0.635266
0.015108
0.107486
0.111770
0.134901
0.313736
(S1 10PZ)
(S1 11PZ)
0.13590
(H14 3S)
(O3 4S)
(H11 3S)
-0.5188
0.5621
using a high range scale in HPLC for detection of four kinds
of 2′-5′ linked dimers (UA, AC, CG, and GU) which would be
theoretically formed as nuclease P1 resistant fragments by
random phosphoryl migration. In this case, however, we could
not observe these byproducts clearly. Digestion of the isolated
12mer with nuclease P1 followed by alkaline phosphatase gave
an equimolar mixture of A, G, U, and C (see supporting
information). The pH of the mixture of 0.01 M HCl-dioxane
(1:1, v/v) used in our study was ca. 2.3, which is a little milder
than the pH 2.0 usually employed in RNA synthesis. Accord-
ingly, the cleavage and phosphoryl migration of internucleotidic
linkages would be better avoided than when pH 2.0 was used.
Quite recently, Reese32 and Hecht33 have independently
reported detailed studies of cleavage and migration of inter-
nucleotidic linkages of oligoribonucleotides to acids to check
the optimum conditions for removal of the acid-labile Ftmp
group. They recommended the use of pH higher than 3 to
circumvent such side reactions but also suggested that the
phosphoryl migration is highly sequence dependent. Therefore,
we carefully checked if such byproducts could be observed by
(27) Katagi, T. Theochem 1990, 68, 61-67.
(28) The structural parameters of O9 to H13 are not shown in Table 1
because of the symmetry of these molecules (O9 to H13 are equal to O4 to
H8, respectively).
(29) McCollum, C.; Andrus, A. Tetrahedron Lett. 1991, 32, 4069-4072.
(30) Vu, H.; McCollum, C.; Jacobson, K.; Theisen, P.; Vinayak, R.;
Spiess, E.; Andrus, A. Tetrahedron Lett. 1990, 31, 7269-7272.
(31) (a) Schulhof, J. C.; Molko, D.; Teoule, R. Tetrahedron Lett. 1987,
28, 51-54. (b) Schulhof, J. C.; Molko, D.; Teoule, R. Nucleic Acids Res.
1987, 17, 397-416. (c) Sinha, N. D.; Davis, P. Usman, N.; Perez, J.; Hodge,
R.; Kremsky, J. Casale, R. Biochemie 1993, 75, 13-23. (d) Vhaix, C.;
Molko, D.; Teoule, R. Tetrahedron Lett. 1989, 30, 71-74. (e) Wu, T.
Ogilvie, K. K.; Pon, R. T. Nucleic Acids Res. 1989, 17, 3501-3517.
(32) Capaldi, D. C.; Reese, C. B. Nucleic Acids Res. 1994, 22, 2209-
2216.
Conclusion
The TBDMS group used as the 2′-protecting group of
chemically synthesized RNA intermediates is easily hydrolyzed
in diluted acidic solutions. It was found that there is a
characteristic relation between the reaction rate and the acid
concentration. From the result of the extended studies of acid
hydrolysis of the 2′-TBDMS group of the phosphorothioate and
methylphosphonate derivatives 2 and 3, it was concluded that
the neighboring group participation of the internucleotidic
(33) Morgan, M. A.; Kazakov, S. A.; Hecht, S. M. Nucleic Acids Res.
1995, 23, 3949-3953.

Mild Acid-Catalyzed Desilylation of Oligoribonucleotide
J. Am. Chem. Soc., Vol. 118, No. 40, 1996 9467
Figure 10. HPLC profiles of the crude and purified RNA 12mer obtained by treatment with HCl (pH 2.0)-dioxane (1:1, v/v).
Scheme 4
Calculation. Monte Carlo simulations were carried out using Macro
phosphorodiester group is highly plausible in this reaction.
Finally, the reaction was successfully applied to deprotection
of the TBDMS groups of a chemically synthesized RNA
oligomer. This method can be used an alternative to the well-
known procedure using the fluoride ion.
Model34 (version 4.5) software on a Silicon Graphics Inc. Indigo2
workstation. Each structure, obtained on the simulation, was minimized
using the Amber* force field.35 The effect of solvation was included
in these simulations by the implicit treatment of solvent water with
the GB/SA model.36 The simulation was continued until the same
global minimum structure was found four or five times. The low-
energy structures within 20 kcal/mol from a global minimum were
sampled. Theoretical calculations were carried out using Gaussian 9437
on a Cray C-916/12256 super computer. The acidic proton (H1) was
placed outside of the S1 or O3 as shown in Figure 9. Although it seems
that the structure for which H14 was placed between S1 and O3 is more
stable than the structure used in the calculation, it is not unfavorable
to consider neighboring group participation of the internucleotidic
phosphodiester group from the result of Monte Carlo simulations. Each
structure was optimized using a 6-311G** basis set38 at the Hartree-
Fock level, and single point calculations were carried out at the MP2
level involving electronic correlation to obtain accurate energies and
atomic charges.
Experimental Section
General. Reverse-phase HPLC analyses were carried out on a
Waters LC module 1 with a µ-Bondasphere C-18 column (3.9 × 150
mm, Waters). Conditions: a linear gradient of 0-30% MeCN in 0.1
M aqueous AcONH4 in 30 min at a flow rate of 1 mL/min at 50 °C.
Ion-exchange HPLC analyses were carried out on a Waters LC module
1 with a Gen-Pak FAX column (4.6 × 100 mm, Waters). Conditions:
a linear gradient of 10-63% aqueous 1.0 M NaCl containing 25 mM
NaH2PO4 in 25 mM aqueous NaH2PO4 in 40 min at the flow rate of 1
mL/min at 50 °C. 1H-NMR spectra were recorded at 23 °C on a JEOL
EX-270 (270.00 MHz) with TMS or CHCl3 (δ ) 7.26) as an internal
reference. 31P-NMR spectra were measured at 23 °C on a JEOL EX-
270 (109.25 MHz) with 85% H3PO4 as an external reference. UV
spectra were measured at 23 °C on a Hitachi U-2000. Silica gel TLC
and column chromatography were performed on Merck Kieselgel 60F-
254 and Wakogel C-200, respectively. Reagents were purchased from
Tokyo Kasei Co. or Aldrich Chemical Co. unless noted. The organic
solvents were purified and dried by the appropriate procedure. Snake
venom phosphodiesterase and calf intestine alkaline phosphatase were
purchased from Boeringer Mannheim GmbH and Nuclease P1 was
purchased from Yamasa Co.
Synthesis of 2′-O-TBDMS-Protected UpU Derivatives. U(2′-Si)-
pU,9a U(2′-Si)psU,20 and U(2′-Si)p(Me)U24 were prepared by the
(34) Mohamadi, F.; Richards, N. G. J.; Guita, W. C.; Likskamp, R.;
Caufield, C.; Chang, G.; Hendrickson, T.; Still, W. C. J. Comput. Chem.
l990, 11, 440-467.
(35) Amber* is the force field involved in Macro Model version 4.5. (a)
Kollman, P. A. Chem. ReV. 1993, 93, 2395-2417. (b) Kollman, P. A.
Tetrahedron Lett. 1992, 33, 7793-7796.
(36) Still, W. C.; Tempczk, A.; Hawley, R. C.; Hendrickson, T. J. Am.
Chem. Soc. 1990, 112, 6127-6129.

9468 J. Am. Chem. Soc., Vol. 118, No. 40, 1996
Kawahara et al.
literature procedures and stored as 1 mM solution in MeCN at -30
°C. The 2′-silylated RNA 12mer intermediate was prepared by using
a Perkin-Elmer model 392 DNA/RNA synthesizer with commercially
available reagents (Perkin-Elmer Applied Biosystems) and released from
the solid support automatically by treatment four times with 25% NH3-
EtOH (3:1, v/v) at room temperature for 15 min. The 2′-O-TBDMS
protected dodecaribonucleotide UACGUACGUACG having the DMTr
group at the 5′-terminal end was obtained in an overall yield of 95%
with the average coupling yield of 99.5%.
Desilylation of 2′-O-TBDMS Dimer Derivatives. After 1 mL of
1 mM 1 or 3 solution was evaporated in Vacuo, 1 mL of acetic acid or
formic acid solution [acid concentration: 0, 1, 2, 5, 10, 20, 40, 60, 80
and 100% (v/v)] or 0.01 M HCI (pH 2.0) was added. These solutions
were incubated at 30 or 50 °C using a thermal controlled water bath.
After 5 and 20 min and 1, 2, 3, 4, 5, 6, 7, 8, and 24 h, an aliquot of 10
µL was sampled from the mixture and poured into 200 µL of 0.1 M
ammonium acetate (pH 7.0). When the pH was less than 6, 10 µL of
25% NH3 was added so as to make the pH 6-7. The mixture was
analyzed by reverse-phase HPLC. The reaction rates were calculated
from the ratio of 1 to the sum (10 nmol) of 1 and 4. Compound 6 is
not stable and decomposed immediately after the TBDMS group of 3
was removed. The peak corresponding to 6 was not detected so that
the reaction rate was calculated from the sum of the decomposed
products.
the phosphate protecting groups were removed at the same time. This
solution was incubated at 60 °C for 1 h to remove the base-protecting
groups and evaporated in Vacuo. This 12mer intermediate was
dissolved in dioxane (5.0 mL) and 0.01 M HCI (5.0 mL) was added.
The mixture was incubated at 35 °C for 12 h. The reaction was
quenched by addition of 200 µL of 1.0 M ammonium acetate (pH 7.0).
This crude product was analyzed using ion-exchange HPLC. The
solution was lyophilized and dissolved in sterilized water (2.0 mL).
Part (1/10) of this crude product was purified using anion-exchange
HPLC and desalted by using gel filtration to give UACGUACGUACG
(5.4 A254 unit, 54% based on the assumption of 20% hypochromicity).
This product was analyzed by ion-exchange and reverse-phase HPLC.
Enzymatic Studies. The isolated RNA 12mer (1.0 A260 unit) was
dissolved in 50 mM Tris-HCl buffer (pH 8.0, 80 µL). Snake venom
phosphodiesterase (8 unit) and 1 M MgCl2 (4 µl) were added. The
reaction mixture was incubated at 37 °C for 3.5 h. After the enzyme
was inactivated by heat treatment (100 °C, 1 min), calf intestine alkaline
phosphatase (10 unit) was added. After the reaction mixture was
incubated at 37 °C for 3.5 h, the enzyme was inactivated by heat
treatment (100 °C, 1 min). The nucleoside ratio of the mixture was in
good agreement with the stoichiometric one (calcd for A:G:U:C ) 1:1:
1:1, found A:G:U:C ) 1:0.89:0.97:1.13) from the reverse-phase HPLC
analysis.
The isolated RNA 12mer (1.0 A260 unit) was dissolved in 50 mM
sodium acetate buffer (pH 5.4, 50 µL) containing ZnCl2 (0.1 mM).
Nuclease P1 (10 unit) was added and the mixture was incubated at 37
°C for 3.5 h. After the enzyme was inactivated by heat treatment (100
°C, 1 min), the mixture was analyzed by the reverse-phase HPLC. There
are essentially no phosphoryl-migrated products.
Compound 2 (Rp:Sp ) 5:1 mixture of the diastereomers, 32 µmol)
was dissolved in 0.01 M HCI-D2O (9:1, 500 µL). 31P-NMR spectra
were measured after 8.63, 28.88, 44.50, 59.83, 89.75, 116.5, 509.0,
and 1310 min, the midpoints between the starting and end times during
the NMR measurement).
Desilylation of 2′-Silylated Dodecaribonucleotide by Use of 0.01
M HCl-Dioxane. A fully protected RNA oligomer (1.0 µmol), which
was prepared according to the literature method, was released from
the solid support using 25% NH3-EtOH (3:1, v/v, 15 min × 4) and
(37) Gaussian 94, ReVision B. 3; Frisch, M. J.; Trucks, G. W.; Schlegel.
H. B.; Gill, P. M. W.; Johnson, B. G.; Robb, M. A.; Cheeseman, J. R.;
Keith, T.; Petersson, G. A.; Montfomery, J. A.; Raghavachari, K.; Al-Laham,
M. A.; Zakrzewski, V. G.; Ortiz, J. V.; Foresman, J. B.; Peng, C. Y.; Ayala,
P. Y.; Chen, W.; Wong, M. W.; Andres, J. L.; Replogle, E. S.; Gomperts,
R.; Martin, R. L.; Fox, D. J.; Binkley, J. S.; Defrees, D. J.; Baker, J.; Stewart,
J. P.; Head-Gordon, M.; Gonzales, C.; Pople, J. A. Gaussian, Inc.: Pittsburgh
PA, 1995.
Acknowledgment. This work was supported by a Grant-
in-Aid for Scientific Research on Priority Areas from the
Ministry of Education, Science, Sports and Culture, Japan. The
authors also thank the Toray Scientific Fundation for generous
financial support.
Supporting Information Available: Reverse-phase and ion-
exchange HPLC profiles of the dimers and oligomer, 31P-NMR
profiles of phosphorothioate dimer and the procedure for
calculation of the relationship between the reaction rates and
acid concentration (8 pages). See any current masthead page
for ordering and Internet access instructions.
(38) (a) Krishnan, R.; Binkley, J. S.; Seeger, R.; Pople, J. A. J. Chem.
Phys. 1980, 72, 650-654. (b) Collins, J. B.; Schleyer, P. von R.; Binkley,
J. S.; Pople, J. A. J. Chem. Phys. 1976, 64, 5142-5151. (c) Pietro, W. J.;
Francl, M. M.; Hehre, W. J.; DeFrees, D. J.; Pople, J. A. ; Binkley, J. S. J.
Am. Chem. Soc. 1982, 104, 5039-5048.
JA961959I