Synthesis of helix 69 of Escherichia coli 23S rRNA containing its natural modified nucleosides, m(3)Psi and Psi.

Article abstract of DOI:10.1021/jo026364m

The synthesis of 3-methylpseudouridine (m(3)Psi) phosphoramidite, 5'-O-[benzhydryloxybis(trimethylsilyloxy)silyl]-2'-O-[bis(2-acetoxyethoxy)methyl]-3-methylpseudouridine-3'-(methyl-N,N-diisopropyl)phosphoramidite, is reported. Selective pivaloyloxymethyl protection of the Psi N1 followed by methylation at N3 was used to generate the naturally occurring pseudouridine analogue. The m(3)Psi phosphoramidite was used in combination with pseudouridine (Psi) and standard base phosphoramidites to synthesize a 19-nucleotide RNA representing helix 69 of Escherichia coli 23S ribosomal RNA (rRNA) (residues 1906-1924), containing a single m(3)Psi at position 1915 and two Psi's at positions 1911 and 1917. Our synthesis of the fully modified helix 69 RNA demonstrates the ability to make milligram quantities of RNA that can be used for further high-resolution structure studies. Site-selective introduction of the methyl group at the N3 position of pseudouridine at position 1915 causes a slight increase in the thermodynamic stability of the RNA hairpin relative to pseudouridine; RNAs containing either uridine or 3-methyluridine at position 1915 have similar stability. One-dimensional imino proton NMR and circular dichroism spectra of the modified RNAs reveal that the methyl group does not cause any substantial changes in the RNA hairpin structure.

Full text of DOI:10.1021/jo026364m

Syn th esis of Helix 69 of Esch er ich ia coli 23S r RNA Con ta in in g Its
Na tu r a l Mod ified Nu cleosid es, m 3Ψ a n d Ψ
Helen M.-P. Chui, J ean-Paul Desaulniers, Stephen A. Scaringe,† and Christine S. Chow*
Department of Chemistry, Wayne State University, Detroit, Michigan 48202
csc@chem.wayne.edu
Received August 28, 2002
The synthesis of 3-methylpseudouridine (m3Ψ) phosphoramidite, 5′-O-[benzhydryloxybis(tri-
methylsilyloxy)silyl]-2′-O-[bis(2-acetoxyethoxy)methyl]-3-methylpseudouridine-3′-(methyl-N,N-diiso-
propyl)phosphoramidite, is reported. Selective pivaloyloxymethyl protection of the Ψ N1 followed
by methylation at N3 was used to generate the naturally occurring pseudouridine analogue. The
m3Ψ phosphoramidite was used in combination with pseudouridine (Ψ) and standard base
phosphoramidites to synthesize a 19-nucleotide RNA representing helix 69 of Escherichia coli 23S
ribosomal RNA (rRNA) (residues 1906-1924), containing a single m3Ψ at position 1915 and two
Ψ’s at positions 1911 and 1917. Our synthesis of the fully modified helix 69 RNA demonstrates the
ability to make milligram quantities of RNA that can be used for further high-resolution structure
studies. Site-selective introduction of the methyl group at the N3 position of pseudouridine at
position 1915 causes a slight increase in the thermodynamic stability of the RNA hairpin relative
to pseudouridine; RNAs containing either uridine or 3-methyluridine at position 1915 have similar
stability. One-dimensional imino proton NMR and circular dichroism spectra of the modified RNAs
reveal that the methyl group does not cause any substantial changes in the RNA hairpin structure.
In tr od u ction
IV appears to be involved with almost all of the RNA-
RNA contacts on the 50S subunit side of the 70S
intersubunit interface; thus, it is likely to play a critical
role in protein synthesis.4 The exact biological role of helix
69 is still unclear, and its structural role is also not
completely defined because of the dynamic nature of this
region. Helix 69 likely undergoes conformational changes
during protein synthesis because of its contacts with the
mobile tRNAs.
The site of protein synthesis in the ribosome is com-
prised of a set of specific structural motifs of the large
and small subunit rRNAs, known as 23S and 16S rRNA
in Escherichia coli. Interestingly, a large number of
nucleotides in this region (from 23S rRNA, 16S rRNA,
and tRNAs) are modified, including methylation and
pseudouridylation.1-3 Helix 69 of domain IV, or residues
1906-1924 in 23S rRNA, is centered at the heart of the
ribosome machinery, and recent crystallographic studies
revealed that this conserved, highly modified hairpin is
in close proximity to the decoding region of 16S rRNA
and tRNAs.4 More specifically, the minor groove of helix
69 interacts with the minor groove of the D stem of the
P-site tRNA, and the 1915 loop region interacts with the
D stem of the A-site tRNA.4 The loop portion of helix 69
was disordered in the Haloarcula marismortui 50S
subunit structure,5 which can be explained by the absence
of direct stacking or packing interactions with the 30S
subunit. In the Thermus thermophilus 70S ribosome
structure, helix 69 is clearly observed contacting the
decoding region near position 1408. Helix 69 of domain
The secondary structure of helix 69 is highly conserved
throughout phylogeny, and three of its modified residues
are pseudouridines or pseudouridine analogues. There-
fore, structural information obtained on this hairpin may
be relevant across a broad range of organisms. The
disorder of helix 69 in the 50S subunit X-ray structure
may be significant and suggests that interactions with
the 30S subunit and tRNAs in the peptidyltransferase
center may be coupled with RNA loop flexibility. Modified
bases such as pseudouridine may play a role in control-
ling RNA flexibility or in making direct contacts with
RNA motifs in its vicinity. Understanding the dynamic
nature of RNA can be approached by carrying out
solution NMR studies, which require milligram quanti-
ties of the RNAs with all of their natural modifications.
Our approach is to synthesize the 19-nucleotide helix 69
RNA (Figure 1) by using RNA phosphoramidite chemis-
try6 and then to carry out biophysical studies on the
isolated hairpin RNA. The resulting synthetic RNA
* To whom correspondence should be addressed. Phone: (313) 577-
2594. Fax: (313) 577-8822.
† Dharmacon Research Inc., Lafayette, CO 80026.
(1) Ofengand, J .; Bakin, A.; Wrzesinski, J .; Nurse, K.; Lane, B. G.
Biochem. Cell. Biol. 1995, 73, 915-924.
(2) Ofengand, J .; Bakin, A. J . Mol. Biol. 1997, 266, 246-268.
(3) Kowalak, J . A.; Bruenger, E.; Hashizume, T.; Peltier, J . M.;
Ofengand, J .; McCloskey, J . A. Nucleic Acids Res. 1996, 24, 688-693.
(4) Yusupov, M. M.; Yusupova, G. Z.; Baucom, A.; Lieberman, K.;
Earnest, T. N.; Cate, J . H. D.; Noller, H. F. Science 2001, 292, 883-
896.
contains three natural modified nucleotides, Ψ1911, m3Ψ1915
,
and Ψ1917, in which Ψ is pseudouridine and m3Ψ is
(5) Ban, N.; Nissen, P.; Hansen, J .; Moore, P. B.; Steitz, T. A. Science
2000, 289, 905-920.
(6) Scaringe, S. A.; Wincott, F. E.; Caruthers, M. H. J . Am. Chem.
Soc. 1998, 120, 11820-11821.
10.1021/jo026364m CCC: $22.00 © 2002 American Chemical Society
Published on Web 11/15/2002
J . Org. Chem. 2002, 67, 8847-8854
8847

Chui et al.
hydryloxybis(trimethylsilyloxy)silyl; ACE, bis(2-acetoxy-
ethoxy)methyl). Previous studies have been carried out
in our laboratories to synthesize pseudouridine12 and its
corresponding 5′-O-BzH-2′-O-ACE-protected phosphora-
midite.8 In nature, pseudouridines (Ψ) are synthesized
through isomerization of uridines within the RNA motifs
by one of many specific pseudouridine synthases, such
as RluD.13 Methylations of pseudouridines are presumed
to occur through the action of specific RNA methyltrans-
ferases. Although the natural enzymes appear quite
capable of specific methylation of N1 or N3 of Ψ, such
specific modification is considerably more challenging to
the organic chemist. Luyten et al. reported that pseudo-
uridine N1 is only slightly more acidic (pKa ) 9.3) than
N3 (pKa ) 9.6).14 Thus, one can infer that the chemical
reactivities of N1 and N3 in solution will be similar. To
generate 1-methylpseudouridine or 3-methylpseudouri-
dine, an efficient method to protect selectively either the
N1 or N3 position is necessary. Several approaches to
synthesize methylated pseudouridines, such as 1-meth-
ylpseudouridine and 1,3-dimethylpseudouridine, in high
yields have been reported previously.15-17 One group has
produced 3-methylpseudouridine and the corresponding
phosphoramidite; however, details of the syntheses and
compound characterizations were not provided in their
reports.10,18 Other reports on 3-methylpseudouridine syn-
theses have lacked in complete characterization of the
product(s), or the reactions proceeded with poor regiose-
lectivity.11,19 For example, Cohn showed that N-alkylation
of pseudouridine in the presence of diazomethane formed
multiple methylated products.19 Matsuda et al. reported
an 80% yield of 3-methylpseudouridine by using tri-
methylsilylated pseudouridine and generating the 1-acetyl
derivative.11 The 1-acetylpseudouridine product was me-
thylated to give 3-methylpseudouridine; however, the
assignment of the regiochemistry of the methyl group at
N3 was ambiguous. The reported chemical shift of the
methyl group in DMSO was shifted by ∼0.8 ppm (2.52
ppm) compared to those in other spectra of methylated
pseudouridine taken in the same solvent (3.29 ppm).11,20
The synthesis reported by Kowalak et al. was motivated
by the desire to identify 3-methylpseudouridine in E. coli
23S rRNA by mass spectrometry.3 For that reason, their
synthesis was only carried out on the milligram scale,
and the product was not characterized by NMR.
F IGURE 1. Secondary structure of the 19-nucleotide sequence
of helix 69 (1920 loop) of E. coli 23S rRNA domain IV and the
structure of modified nucleoside m3Ψ, which occurs at position
1915.
3-methylpseudouridine. The advantage of the synthetic
approach is that the effects of pseudouridine and the
methyl substitution can be assessed individually, but
within the context of the natural 23S rRNA helix 69
sequence.
Although Ψ and U are structural isomers, the presence
of the additional NH group in pseudouridine can dra-
matically influence the overall structure of an RNA
oligonucleotide.7 The effects are more subtle in the helix
69 RNA,8 and methylation of a uridine at position 1915
does not have much influence on the stability or second-
ary structure of the RNA hairpin.9 This study focused
on the synthesis of the natural nucleoside at position
1915 of E. coli 23S rRNA, 3-methylpseudouridine, and
its conversion into the corresponding phosphoramidite
and incorporation into a 19-nucleotide helix 69 RNA. This
modified nucleoside has been synthesized previously, but
not in quantities sufficient for phosphoramidite synthesis
or high-resolution NMR structure studies.3,10,11 Our
synthetic approach involved the selective protection of
N1 of the uracil base with pivaloyloxymethyl (POM),
followed by N3 methylation. Helix 69 RNAs (also referred
to as 1920 loop RNAs) containing one or all of the natural
modifications were synthesized, and circular dichroism,
UV melting, and NMR studies were carried out to
determine the effects of m3Ψ on the RNA structure and
stability.
Here we report on a facile and selective method for
production of 3-methylpseudouridine and preparation of
the corresponding phosphoramidite. Our approach in-
volved an N1 protection-deprotection strategy in which
the N3 position can then be selectively methylated. The
Resu lts a n d Discu ssion
(12) Grohar, P. J .; Chow, C. S. Tetrahedron Lett. 1999, 40, 2049-
2052.
(13) Raychaudhuri, S.; Conrad, J .; Hall, B. G.; Ofengand, J . RNA
1998, 4, 1407-1417.
m 3Ψ P h osp h or a m id ite Syn th esis. The first goal of
this work was to generate the natural modified nucleo-
side 3-methylpseudouridine (m3Ψ) and the corresponding
5′-O-BzH-2′-O-ACE-protected phosphoramidite (BzH, benz-
(14) Luyten, I.; Pankiewicz, K. W.; Watanabe, K. A.; Chatto-
padhyaya, J . J . Org. Chem. 1998, 63, 1033-1040.
(15) Bhattacharya, B. K.; Devivar, R. V.; Revankar, G. R. Nucleo-
sides Nucleotides 1995, 14, 1269-1287.
(7) Newby, M. I.; Greenbaum, N. L. RNA 2001, 7, 833-845.
(8) Meroueh, M.; Grohar, P. J .; Qiu, J .; SantaLucia, J ., J r.; Scaringe,
S. A.; Chow, C. S. Nucleic Acids Res. 2000, 28, 2075-2083.
(9) Chui, H. M.-P.; Meroueh, M.; Scaringe, S. A.; Chow, C. S. Bioorg.
Med. Chem. 2002, 10, 325-332.
(10) Agris, P. F.; Malkiewicz, A.; Kraszewski, A.; Everett, K.;
Nawrot, B.; Sochacka, E.; J ankowska, J .; Guenther, R. Biochimie 1995,
77, 125-134.
(11) Matsuda, A.; Pankiewicz, K.; Marcus, B. K.; Watanabe, K. A.;
Fox, J . J . Carbohydr. Res. 1982, 100, 297-302.
(16) Hirota, K.; Watanabe, K. A.; Fox, J . J . J . Heterocycl. Chem.
1977, 14, 537-538.
(17) Reichman, U.; Hirota, K.; Chu, C. K.; Watanabe, K. A.; Fox, J .
J . J . Antibiot. 1977, 30, 129-131.
(18) Yarian, C. S.; Basti, M. M.; Cain, R. J .; Ansari, G.; Guenther,
R. H.; Sochacka, E.; Czerwinska, G.; Malkiewicz, A.; Agris, P. F. Nucleic
Acids Res. 1999, 27, 3543-3549.
(19) Cohn, W. E. J . Biol. Chem. 1960, 235, 1488-1498.
(20) Pankiewicz, K.; Watanabe, K. A.; Takayanagi, H.; Itoh, T.;
Ogura, H. J . Heterocycl. Chem. 1985, 22, 1703-1710.
8848 J . Org. Chem., Vol. 67, No. 25, 2002

Synthesis of Ψ- and m3Ψ-Containing RNAs
SCHEME 1. Syn th esis of th e m 3Ψ P h osp h or a m id itea
a
Reagents and conditions: (a) TIPDSCl2, pyridine, 0 °C f rt, 94% yield of 1; (b) TMSCl, Et3N, CH2Cl2, 0 °C f rt, 99% yield of 2; (c)
POMCl (9 equiv), Et3N, pyridine, rt, 91% yield of 3; (d) DMF-DMA, benzene, reflux, 98% yield of 4; (e) (i) pTSA‚H2O, Et3N, THF, 95%
yield of 5; (ii) NH3/CH3OH, rt, 94% yield of 6; (f) excess NH3/CH3OH, rt, 92% yield of 6; (g) tris(2-acetoxyethoxy)orthoformate, pyridinium
p-toluenesulfonate, 4-(tert-butyldimethylsilyloxy)-3-penten-2-one, dioxane, rt f 40 °C, 69-80% yield of 7; (h) TMEDA/HF, CH3CN, 0 °C,
68% yield of 8 from 6; (i) BzHCl, diisopropylamine, CH2Cl2, 0 °C, 68% yield of 9; (j) methyl tetraisopropyl phosphorodiamidite, 1H-
tetrazole, CH2Cl2, rt, 77% yield of 10; (k) TMEDA/HF, CH3CN, 0 °C, 85% yield of 11.
synthetic route is illustrated in Scheme 1. The first step
involved simultaneous protection of the 3′- and 5′-
hydroxyl groups of pseudouridine using dichlorotetra-
isopropyldisiloxane in the presence of pyridine at 0 °C
to give compound 1 (94% yield). Compound 1 was treated
with trimethylsilyl chloride in the presence of triethyl-
amine to give compound 2 in 99% yield. The final
phosphoramidite will contain 2′-O-ACE and 5′-O-BzH
protective groups; however, the ACE group was found to
be unstable under the conditions of the N1 protection
reaction. For this reason, the TMS group was employed
for 2′-hydroxyl protection. The TMS group can also be
removed simultaneously or selectively in the presence of
the N1 protective group POM under mild conditions.
Following protection of the ribose hydroxyl groups, the
N1 position was selectively protected by reaction with
triethylamine/pyridine (6:1) and excess (>9 equiv) piv-
aloylmethyl chloride (POMCl) for 5 days to give com-
pound 3 in 91% yield. The addition of a single portion of
POMCl (9 equiv) gave a lower yield of 3 (71%), and we
found that it was necessary to add the reagent in smaller
aliquots (∼3 equiv) in 24 h intervals.
tics.22 In polar solvents (DMSO, CH3CN, and DMF), the
formation of N1-POM-protected pseudouridine (3) oc-
curred in a relatively short time in low yield with
competing formation of the N1,N3-bis-POM product.
Reactions with stoichiometric amounts of POMCl (1.1
equiv) and K2CO3 in DMF at room temperature gave near
equimolar mixtures of N1-POM- and N1,N3-bis-POM-
protected pseudouridines along with ∼40% starting
material (2). In nonpolar solvents (Et3N, CHCl3, and
CCl4), the formation of N1-POM required longer reaction
times with little production of N1,N3-bis-POM-protected
pseudouridine. The use of THF as a solvent led to
production of N1-POM-protected pseudouridine in low
yield, even with long reaction times. As more POMCl was
added (in THF), the formation of N1,N3-bis-POM product
increased and the amount of N1-POM-protected pseudo-
uridine remained the same. When THF/K2CO3 or DMF/
NaH was used, only the formation of N1-POM-protected
pseudouridine (3) in ∼30% and 70% yields, respectively,
was observed after 24-72 h at room temperature. Thus,
the N1-POM-protected pseudouridine appears to be the
thermodynamically favored product, and the selectivity
of POM for N1 or N3 is dependent on the solvent polarity.
The complete details of the solvent conditions, temper-
ature, and bases employed are currently under further
investigation.
We observed that the selectivity of the POM reaction
with N1 or N3 was solvent dependent and for the reaction
to be driven to completion a large excess of POMCl (∼9-
15 equiv) was required. These reaction conditions were
based on an earlier report by Pieles et al. in which the
POM group was employed to protect both N1 and N3 of
pseudouridine.21 Some of the results for solvent effects
are summarized in Table 1. Solvent polarity was based
on dielectric constant and dipole moment characteris-
Following successful protection of N1, compound 3 was
methylated under neutral conditions by refluxing in N,N-
dimethylformamide dimethyl acetal (DMF-DMA) in ben-
zene for 6 h to obtain a single product, 4, in 98% yield.
Stepwise removal of 2′-O-TMS and N1-POM protective
groups can be achieved by treatment with p-toluene-
(21) Pieles, U.; Beijer, B.; Bohmann, K.; Weston, S.; O’Loughlin, S.;
Adam, V.; Sproat, B. S. J . Chem. Soc., Perkin Trans. 1 1994, 3423-
3429.
(22) Carlson, R.; Lundstedt, T.; Albano, C. Acta Chem. Scand. B
1985, 39, 79-91.
J . Org. Chem, Vol. 67, No. 25, 2002 8849

Chui et al.
TABLE 1. Rea ction of 2 w ith P OMCl
product distribution (%)
amt of
POMCl
(equiv)
time
(h)
R ) POM,
R′ ) H
R ) H,
R′ ) POM
R ) R′ )
POM
solvent
base
2
DMSO
CH3CN
DMF
THF
THF
K2CO3
K2CO3
K2CO3
K2CO3
K2CO3
K2CO3
none
none
K2CO3
NaH
1.1
1.1
1.1
1.1
1.1
5.0
1.1
9.0
1.1
1.1
12
12
12
24
60
60
48
96
48
48
∼30a
∼10a
31b
∼30a
∼30a
∼80a
25b
0
∼10a
∼10a
44b
0a
0
0
0
100b
58b
29b
0
0
THF
∼33a
45b
0a
0
∼33a
∼33a
Et3N/py
Et3N/py
CHCl3
CCl4
0
35b
91b
0
0
0
<5a
∼15a
∼15a
4b
∼70a
∼15a
∼70a
∼15a
Reaction yields are estimated by TLC analysis (2:1 hexane/EtOAc). b Isolated yields. All reactions were carried out at room temperature.
a
sulfonic acid (pTSA) followed by methanolic ammonia to
give 5 and then 6 (89% for two steps), or compound 6
can be obtained in one step by reacting 4 with excess
methanolic ammonia (92%). The 3′,5′-O-TIPDS-protected
(TIPDS ) 1,1,3,3-tetraisopropyldisiloxanediyl) 3-meth-
ylpseudouridine can be obtained in an overall 76% yield
from pseudouridine on a gram scale. The products were
fully characterized by NMR spectroscopy and mass
spectrometry. The 1H NMR spectrum of 4 in CDCl3
exhibited a methyl signal at δ 3.34 ppm. The unprotected
product 3-methylpseudouridine (11) was obtained in 85%
yield by treatment of 6 with TMEDA/HF and examined
by NOE spectroscopy to confirm the identity and location
of the methyl group. No NOEs between base protons were
observed upon irradiation of either the N3-CH3 or H6
peaks. The regioisomer 1-methyl-3-pivaloyloxymethyl-
3′,5′-O-(1,1,3,3-tetraisopropyl-1,3-disiloxanediyl)-2′-O-(tri-
methylsilyl)pseudouridine was also synthesized and char-
to drive the reaction to completion; we presume that this
reagent works by reacting with the primary alcohol that
is formed upon ionization of the tris(2-acetoxyethoxy)-
orthoformate. In our previous studies, the ACE protection
reaction was carried out at 55 °C or room temperature.6,8,9
For the 3-methylpseudouridine analogue, ACE protection
was carried out at 40 °C for 48 h to obtain 7 in 69-80%
yield (reactions at 55 °C were not successful). After 2′-
O-ACE protection, the 3′,5′-O-TIPDS group was removed
under mild and neutral conditions (TMEDA/HF at 0 °C;
68% yield). The free 5′-hydroxyl was then protected with
BzH by using BzHCl and diisopropylamine at 0 °C to
afford a 68% yield of 9. Finally, compound 9 was reacted
with freshly prepared methyl tetraisopropyl phospho-
rodiamidite and 1H-tetrazole to generate the target
3-methylpseudouridine phosphoramidite 10 in 77% yield
(27% yield in nine or ten steps from pseudouridine).
RNA Syn th esis. The modified helix 69 RNA (1920
loop) shown in Figure 1 was synthesized using building
block 10 and the G, C, A, U, and Ψ phosphoramidites.
The modified base m3Ψ was incorporated at position
1915, along with two Ψ’s at positions 1911 and 1917 (this
RNA is referred to as Ψm3ΨΨ). A second RNA containing
only m3Ψ modification at position 1915 and uridines at
position 1911 and 1917 was also synthesized (referred
to as Um3ΨU). The two RNAs were synthesized in high
yields (∼4 mg/µmol synthesis or 1 mg/0.2 µmol synthesis),
deprotected, and purified by 20% polyacrylamide gel
electrophoresis. The isolated RNAs were desalted and
used for further biophysical studies. Electrospray mass
spectrometry was used to confirm the identity of the
modified RNAs (Supporting Information). The presence
of the modified nucleosides was also confirmed by P1
nuclease digestion and alkaline phosphatase treatment
of each RNA, followed by HPLC analysis (data not
shown). In addition, the methyl group of m3Ψ is clearly
1
acterized by NMR. In this case, the H NMR spectrum
taken in CDCl3 exhibited a methyl signal at δ 3.37 ppm,
and a strong NOE between H6 and N1-CH3 was observed
(Supporting Information). We have also observed that
upon methylation of the Ψ NH position, the two meth-
ylene protons of the POM group on compound 4 or its
regioisomer exhibit major differences in their J coupling
patterns in the proton NMR spectra. The methylene
group of POM showed different spin-coupling patterns
depending on its location on Ψ. For 4, the spin coupling
between the two geminal protons (δ 5.46 (d) and 5.76 (d)
ppm) exhibited a typical first-order AX pattern (where
∆ν/J ) 12.5). In contrast, the spin coupling between the
two methylene protons (δ 5.92 (d) and 5.95 (d) ppm) on
the regioisomer exhibited an AB splitting pattern (where
∆ν/J ) 1.2) (Supporting Information).
To complete the synthesis of the 3-methylpseudouri-
dine phosphoramidite, compound 6 was reacted with tris-
(2-acetoxyethoxy)orthoformate in the presence of pyri-
dinium p-toluenesulfonate and 4-(tert-butyldimethylsilyl-
oxy)-3-penten-2-one (TBDMS-acac) to achieve 2′-O-ACE
protection. The addition of TBDMS-acac was necessary
1
visible in the 1D H NMR spectrum of the Ψm3ΨΨ RNA
in D2O at δ 3.04 ppm (Supporting Information).
Effects of m 3Ψ on Helix 69 Sta bility a n d Str u c-
tu r e. Previous studies have shown that Ψ at position
8850 J . Org. Chem., Vol. 67, No. 25, 2002

Synthesis of Ψ- and m3Ψ-Containing RNAs
F IGURE 2. CD spectra of the 3-methylpseudouridine-modi-
fied RNAs taken in 15 mM NaCl, 20 mM sodium cacodylate,
0.2 mM Na2EDTA, pH 7.0. The molar ellipticities are normal-
ized to RNA concentrations (6.4 × 10-6 and 4.9 × 10-6 M in
molecules of RNA for Ψm3ΨΨ (closed circles) and Um3ΨU
(open circles), respectively). Each spectrum is the average of
four scans.
F IGURE 3. 1D imino proton (guanine N1H/uridine N3H/
pseudouridine N1H and N3H) NMR spectrum of Ψm3ΨΨ RNA
taken in 90% H2O/10% D2O, 30 mM NaCl, 10 mM sodium
phosphate, 0.2 mM Na2EDTA, pH 6.5, at 3 °C. The nucleotide
assignments are based on the 1D NOE difference spectra and
2D NOESY spectrum.
1915 slightly destabilizes helix 69 by 0.5 kcal/mol com-
pared to uridine, whereas m3U has no effect.9 For the
newly synthesized RNA Ψm3ΨΨ, methylation of pseudo-
uridine at position 1915 leads to a 0.5 kcal/mol stabiliza-
tion relative to pseudouridine; RNAs with uridine and
3-methylpseudouridine at position 1915 have similar
stability. The ∆G°37 values are -5.3,9 -5.3,9 -4.8,8 and
-5.2 kcal/mol for ΨUΨ, Ψm3UΨ, ΨΨΨ, and Ψm3ΨΨ,
respectively, in which the three letters for the RNAs
designate the nucleosides at positions 1911, 1915, and
1917. Similar results were observed with the Um3ΨU
RNA (∆G°37 values are -4.9,8 -5.0,9 -4.2,8 and -4.7 for
UUU, Um3UU, UΨU, and Um3ΨU, respectively) in
which m3Ψ at position 1915 has little effect on helix 69
RNA stability relative to uridine or 3-methyluridine, but
stabilizes the RNA by ∼0.5 kcal/mol relative to pseudo-
uridine. The CD spectrum of Um3ΨU in Figure 2 is
essentially identical to the previously reported spectra
for UUU and Um3UU RNAs.8,9 Similarly, the CD spec-
trum of Ψm3ΨΨ overlaps completely with the spectra
for ΨUΨ and Ψm3UΨ RNAs. Thus, the observed differ-
ences between the Um3ΨU and Ψm3ΨΨ spectra appear
to arise from the presence of Ψ modifications at positions
1911 and 1917. The imino proton NMR spectrum of
Ψm3ΨΨ shown in Figure 3 indicates that the effect of
m3Ψ on the RNA structure is also very subtle. Seven
major imino proton peaks are observed (G1922, Ψ1911 N3H,
changes in the loop region due to the presence of m3Ψ.
Our biophysical studies are still preliminary; however,
the thermodynamic and 1D NMR data are both consis-
tent with the methyl group playing a minor structural
role when present on pseudouridine. This result is in
contrast to previous studies in which methylation of
uridine had negligible effects on structure or stability.
Even slight changes in RNA structure resulting from the
presence of a methyl group may have important biological
implications such as correct positioning of the 1920 loop
in the ribosome. Further high-resolution structure stud-
ies on the fully modified helix 69 RNA are necessary to
understand these effects more clearly.
In conclusion, we have reported on the synthesis of
3-methylpseudouridine and its corresponding phosphora-
midite. Our approach involved an N1 protection-depro-
tection strategy in which the N3 position of pseudouridine
can be efficiently modified by methylation. A similar
strategy should also be possible for selective N3 alkyla-
tion, aminoylation, 15N isotopic enrichment, or fluorescent
labeling of pseudouridine. Furthermore, the protection-
deprotection strategy presented will also allow for specific
combinations of modifications at the N1, N3, and 2′-O
positions of pseudouridine. Thus, large quantities of
pseudouridine derivatives can be synthesized for applica-
tions in RNA structure-function studies or as novel
nucleoside inhibitors.
G
1910, G1921, U1923, G1907, and Ψ1911 N1H at 13.5, 13.0, 12.8,
12.4, 12.1, 11.7, and 10.3, respectively), which are similar
to those seen previously in 1D imino proton NMR spectra
of ΨΨΨ, ΨUΨ, and Ψm3UΨ RNAs.8,9 An additional peak
is observed at 11.2 ppm corresponding to G1906 (closing
base pair). The Ψ1911 N3H and N1H imino resonances
observed at 13.0 and 10.3 ppm, respectively, were con-
firmed by 1D NOE difference and 2D NOESY spectros-
copy and indicate base pairing with A1919. Three unas-
signed peaks occur at 12.6, 10.7, and 9.7 ppm, and the
G1910 and Ψ1911 N1H peaks both have shoulders, suggest-
ing possible conformational exchange in the region
containing the modified nucleotides. These peaks do not
occur in the corresponding ΨΨΨ spectrum; therefore, the
conformational exchange in this region likely results from
Exp er im en ta l Section
Gen er a l P r oced u r es. All reactions were carried out at
room temperature under an inert atmosphere and anhydrous
conditions unless otherwise noted. Tris(acetoxyethoxy)ortho-
formate, benzhydryloxybis(trimethylsilyloxy)silyl chloride, meth-
yl tetraisopropyl phosphorodiamidite, and 4-(tert-butyldime-
thylsilyloxy)-3-penten-2-one were prepared as described else-
where.6 All other reagents were obtained from commercial
sources. All solvents for RNA purification and HPLC analysis
were spectra grade. 1H, 13C, and 31P NMR spectra were
J . Org. Chem, Vol. 67, No. 25, 2002 8851

Chui et al.
obtained in deuterated solvents on either 400 or 500 MHz
spectrometers. The chemical shifts of the imino protons of the
1920 hairpin RNAs were reported relative to that of 3-(tri-
methylsilyl)propionate at 0.0 ppm. Nuclease digestions and
HPLC analyses for the modified RNAs were performed as
described previously.8 The RNA sample preparation and
experimental conditions for the thermal melting studies, CD
spectroscopy, and NMR studies were done as reported previ-
ously.8
to give a white crystalline solid: 1H NMR (CDCl3, 400 MHz)
δ (ppm) 0.19 (s, 9H), 0.92 (m, 28H), 1.20 (s, 9H), 3.94 (dd, 1H,
J ) 12.8, 1.6 Hz), 4.00-4.07 (m, 2H), 4.14 (d, 1H, J ) 3.2 Hz),
4.16 (d, 1H, J ) 13.6 Hz), 4.70 (s, 1H), 5.45 (d, 1H, J ) 10.4
Hz), 5.75 (d, 1H, J ) 9.6 Hz), 7.60 (d, 1H, J ) 1.6 Hz), 9.14 (s,
1H); 13C NMR (CDCl3, 400 MHz) δ (ppm) 0.29, 12.75, 12.85,
12.95, 13.37, 16.93, 17.04, 17.08, 17.19, 17.32, 17.34, 17.44,
17.47, 26.86, 38.82, 60.18, 69.64, 70.96, 75.80, 79.66, 80.82,
114.55, 140.89, 150.29, 162.16, 177.62; ESI-MS (ES+) m/z calcd
for C30H56N2O9Si3 672.3, found 673.2 (MH+), 695.1 (M + Na),
1367.2 (2M + Na).
3′,5′-O-(1,1,3,3-Te t r a isop r op yl-1,3-d isiloxa n e d iyl)-
p seu d ou r id in e (1). Pseudouridine (0.95 g, 3.89 mmol) was
dried by azeotropic removal of water with benzene in vacuo
overnight and then dissolved in 50 mL of freshly distilled
pyridine. The white suspension was stirred for 1 h to give a
clear solution and then cooled to 0 °C. 1,3-Dichloro-1,1,3,3-
tetraisopropyldisiloxane (TIPDSCl2) (1.37 mL, 4.28 mmol) was
added dropwise to the cooled solution and stirred for 1 h at 0
°C and then overnight at rt. The solvent was evaporated, and
the white residue was taken up in CH2Cl2, washed with 5%
NaHCO3, and then washed with water. The organic layers
were combined and washed with aqueous brine, dried over Na2-
SO4, filtered, and concentrated. The crude product was purified
by flash column chromatography on silica gel using a 0-5%
EtOH in CH2Cl2 gradient to give compound 1 as a white solid
(1.78 g, 94%): 1H NMR (CDCl3, 500 MHz) δ (ppm) 1.02-1.09
(m, 28H), 3.07 (s, 1H), 3.94-4.00 (m, 2H), 4.08 (dd, 1H, J )
12.5, 3, 3.5 Hz), 4.12 (dd, 1H, J ) 5.5, 2.0 Hz), 4.31 (dd, 1H, J
) 8.0, 5.5 Hz), 4.75 (s, 1H), 7.54 (s, 1H), 9.86 (s, 1H), 10.08 (s,
1H); 13C NMR (CDCl3, 500 MHz) δ (ppm) 12.57, 12.69, 13.05,
13.38, 16.93, 17.01, 17.03, 17.14, 17.28, 17.33, 17.41, 61.28,
70.85, 74.57, 79.99, 80.89, 112.83, 138.83, 152.51, 163.04; ESI-
MS (ES+) m/z calcd for C21H38N2O7Si2 486.2, found 487.0
(MH+), 509.0 (M + Na), 995.1 (2M + Na).
3-Meth yl-1-p iva loyloxym eth yl-3′,5′-O-(1,1,3,3-tetr a iso-
p r op yl-1,3-d isiloxa n ed iyl)-2′-O-(t r im et h ylsilyl)p seu d o-
u r id in e (4). To a solution of 3 (1.69 g, 2.51 mmol) in 30 mL of
benzene was added dropwise DMF-DMA (1.00 mL, 7.53 mmol).
The light yellow solution was refluxed for 6 h, and the solvent
was evaporated. The deep yellow crude product was purified
by flash chromatography on silica gel using a 10-25% EtOAc
in hexanes gradient to afford compound 4 as a thick colorless
oil in 98% yield (1.69 g): 1H NMR (CDCl3, 400 MHz) δ (ppm)
0.18 (s, 9H), 0.90-1.07 (m, 28H), 1.18 (s, 9H), 3.34 (s, 3H),
3.93 (dd, 1H, J ) 12.8, 2.4 Hz), 3.99-4.07 (m, 2H), 4.09 (d,
1H, J ) 3.2 Hz), 4.15 (d, 1H, J ) 12.8 Hz), 4.70 (s, 1H), 5.46
(d, 1H, J ) 9.6 Hz), 5.76 (d, 1H, J ) 9.6 Hz), 7.57 (s, 1H); 13
C
NMR (CDCl3, 400 MHz) δ (ppm) 0.36, 12.76, 12.85, 12.95,
13.40, 16.93, 17.05, 17.09, 17.19, 17.32, 17.35, 17.44, 17.47,
26.87, 27.75, 38.81, 60.18, 69.61, 71.93, 75.95, 79.60, 81.24,
113.63, 138.74, 150.85, 161.66, 177.56; ESI-MS (ES+) m/z calcd
for C31H58N2O9Si3 686.3, found 709.2 (M + Na).
3-Meth yl-1-p iva loyloxym eth yl-3′,5′-O-(1,1,3,3-tetr a iso-
p r op yl-1,3-d isiloxa n ed iyl)p seu d ou r id in e (5). To a clear
solution of 4 (190 mg, 0.28 mmol) in THF (8 mL) was added
p-toluenesulfonic acid monohydrate (pTSA‚H2O) (79 mg, 0.42
mmol). After the solution was stirred for 3 h, triethylamine
(84 µL, 0.6 mmol) was added. The reaction was quenched with
5% aqueous NaHCO3 and extracted with EtOAc. The organic
layers were combined, washed with brine, dried over Na2SO4,
concentrated, and purified by flash chromatography on silica
gel with a 10-33% EtOAc in hexanes gradient to give a
colorless thick oil. Drying in vacuo gave compound 5 as a white
foam (161 mg, 95%): 1H NMR (CDCl3, 500 MHz) δ (ppm) 1.01-
1.08 (m, 28H), 1.19 (s, 9H), 2.90 (s, 1H), 3.33 (s, 3H), 3.9-3.94
(m, 1H), 4.00 (dd, 1H, J ) 13.0, 3.0 Hz), 4.06 (dd, 1H, J )
13.0, 4.0, 4.5 Hz), 4.17 (dd, 1H, J ) 5.5, 2.5 Hz), 4.35 (dd, 1H,
J ) 8.0, 5.8 Hz), 4.70 (dd, 1H, J ) 2.3, 1.5 Hz), 5.58 (d, 1H, J
) 10.0 Hz), 5.60 (d, 1H, J ) 10.0 Hz), 7.53 (d, 1H, J ) 1.0 Hz);
13C NMR (CDCl3, 500 MHz) δ (ppm) 12.53, 12.63, 13.02, 13.31,
16.92, 16.99, 17.01, 17.14, 17.26, 17.30, 17.35, 17.40, 26.86,
27.77, 38.82, 61.48, 71.27, 71.34, 74.42, 80.82, 80.99, 112.45,
139.50, 150.88, 161.60, 177.99; ESI-MS (ES+) m/z calcd for
3′,5′-O-(1,1,3,3-Tetr a isop r op yl-1,3-d isiloxa n ed iyl)-2′-O-
(tr im eth ylsilyl)p seu d ou r id in e (2). A solution of 1 (1.78 g,
3.65 mmol) in 60 mL of CH2Cl2 was cooled to 0 °C. To the
stirring solution was added triethylamine dropwise (2.47 mL,
17.75 mmol). The clear solution was stirred for 30 min at 0 °C
followed by the addition of TMSCl (1.35 mL, 10.65 mmol). The
solution was stirred for 1 h at 0 °C and then overnight at rt.
The resulting red solution was poured over 100 mL of cold 5%
aqueous NaHCO3 and extracted twice with CH2Cl2. The
organic layers were combined, washed with brine, dried over
Na2SO4, and concentrated in vacuo to afford a light yellow
foam that was further purified by flash chromatography on
silica gel using a 0-5% EtOH in CH2Cl2 gradient to afford
compound 2 as a white solid (2.02 g, 99%). A 25-50% gradient
of EtOAc in hexanes was also successful and gave a compa-
rable yield of product: 1H NMR (CDCl3, 400 MHz) δ (ppm)
0.18 (s, 9H), 0.90-1.06 (m, 28H), 3.91 (dd, 1H, J ) 13.2, 1.6
Hz), 4.00-4.07 (m, 2H), 4.16 (d, 1H, J ) 4 Hz), 4.14 (d, 1H, J
) 5.6 Hz), 4.70 (s, 1H), 7.61 (dd, 1H, J ) 5.6, 1.6 Hz), 10.04 (s,
1H), 10.21 (d, 1H, J ) 5.2 Hz); 13C NMR (CDCl3, 400 MHz) δ
(ppm) 0.32, 12.68, 12.91, 12.97, 13.43, 16.93, 17.06, 17.14,
17.27, 17.30, 17.36, 17.41, 60.11, 69.29, 75.74, 79.65, 80.90,
113.91, 138.25, 152.84, 162.89; ESI-MS (ES+) m/z calcd for
C
28H50N2O9Si2 614.3, found 615.2 (MH+), 637.2 (M + Na),
1251.5 (2M + Na).
3-Me t h yl-3′,5′-O-(1,1,3,3-t e t r a isop r op yl-1,3-d isilox-
a n ed iyl)p seu d ou r id in e (6). Meth od A. The POM protective
group can be removed following 2′-O-TMS removal (from 5).
Compound 5 (1.19 g, 1.93 mmol) was dissolved in 35 mL of
methanolic ammonia (2.0 M in methanol) and stirred over-
night. The solvent was evaporated and the product isolated
by flash chromatography on silica gel using a 15-20% EtOAc
in hexanes gradient to afford 6 (0.91 g, 94%) as a white foam:
1H NMR (CDCl3, 500 MHz) δ (ppm) 1.00-1.09 (m, 28H), 2.94
(d, 1H, J ) 1.5 Hz), 3.31 (s, 3H), 3.95-4.00 (m, 2H), 4.07-
4.18 (m, 2H), 4.28 (dd, 1H, J ) 8.5, 5.0 Hz), 4.79 (dd, 1H, J )
1.5 Hz), 7.47 (dd, 1H, J ) 6.0, 1.0 Hz), 10.20 (d, 1H, J ) 5.5
Hz); 13C NMR (CDCl3, 500 MHz) δ (ppm) 12.53, 12.67, 13.00,
13.38, 16.89, 16.98, 17.02, 17.10, 17.26, 17.28, 17.34, 17.40,
27.08, 60.93, 70.40, 74.71, 80.25, 80.56, 112.45, 135.88, 152.91,
162.24; ESI-MS (ES+) m/z calcd for C22H40N2O7Si2 500.2, found
501.0 (MH+), 523.0 (M + Na), 1023.2 (2M + Na).
C
24H46N2O7Si3 558.3, found 559.0 (MH+), 581.0 (M + Na),
1139.2 (2M + Na).
1-P iva loyloxym eth yl-3′,5′-O-(1,1,3,3-tetr a isop r op yl-1,3-
d isiloxa n ed iyl)-2′-O -(tr im eth ylsilyl)p seu d ou r id in e (3).
To a clear solution of 2 (31.6 mg, 0.057 mmol) in 1.2 mL of
freshly distilled triethylamine and 0.2 mL of dry pyridine was
added POMCl (25 µL, 0.173 mmol). The reaction was stirred
for 70 h at rt. An additional 25 µL of POMCl was added and
the reaction stirred for 24 h. This process was repeated with
a third aliquot of 25 µL of POMCl. The reaction was quenched
with 5% NaHCO3 and extracted with EtOAc. The organic layer
was washed with aqueous brine, dried over Na2SO4, filtered,
and concentrated under reduced pressure. The crude product
was purified by flash column chromatography on silica gel
using a 20-34% gradient of EtOAc in hexanes to yield a
colorless oil (34.8 mg, 91%) which coevaporated with hexanes
Meth od B. The POM and TMS groups can be removed
simultaneously from 4. Compound 4 (211 mg, 0.31 mmol) was
dissolved in 15 mL of methanolic ammonia (2.0 M in methanol)
8852 J . Org. Chem., Vol. 67, No. 25, 2002

Synthesis of Ψ- and m3Ψ-Containing RNAs
at 0 °C to give a clear solution. The solution was gradually
warmed to rt and stirred overnight. The resulting light blue
solution was concentrated, and the product was purified by
flash chromatography using a 0-5% EtOH gradient in hex-
anes/EtOAc (2:1 mixture) to afford 6 (142 mg, 92%) as a white
foam.
completion, the reaction was quenched with 5% NaHCO3 and
extracted with CH2Cl2. The combined organic layers were
washed with brine, dried over Na2SO4, concentrated, and
purified by flash column chromatography on silica gel with
33-50% EtOAc in hexanes and then 0-5% CH3OH in EtOAc
to afford compound 9 as a thick clear oil in 68% yield: 1H NMR
(CDCl3, 500 MHz) δ (ppm) 0.03 (s, 9H), 0.04 (s, 9H), 2.02 (s,
6H), 2.03 (s, 6H), 2.87 (d, 1H, J ) 8.5 Hz), 3.21 (s, 3H), 3.78-
3.85 (m, 4H), 3.87 (d, 1H, J ) 4.5 Hz), 3.88 (d, 1H, J ) 5.0
Hz), 3.96-4.01 (m, 2H), 4.12 (dd, 1H, J ) 5.0, 1.5 Hz), 4.18-
4.27 (m, 4H), 4.87 (s, 1H), 5.63 (s, 1H), 5.94 (s, 1H), 7.15-7.20
(m, 2H), 7.23-7.27 (m, 4H), 7.32-7.34 (m, 4H), 7.38 (d, 1H, J
) 5.0 Hz), 9.16 (d, 1H, J ) 5.0 Hz); 13C NMR (CDCl3, 500 MHz)
δ 1.26, 20.61, 20.64, 26.78, 61.32, 62.20, 62.39, 62.93, 63.02,
68.69, 76.56, 78.12, 78.55, 81.74, 111.53, 112.53, 126.14,
126.27, 127.12, 128.08 (2C), 136.03, 143.76, 143.85, 152.03,
162.16, 170.72, 170.76; ESI-MS (ES+) m/z calcd for C38H56N2O15-
Si3 864.3, found 887.3 (M + Na).
5′-O-[Ben zh yd r yloxyb is(t r im et h ylsilyloxy)silyl]-2′-O-
[bis(2-a cetoxyeth oxy)m eth yl]-3-m eth ylp seu d ou r id in e-3′-
(m eth yl-N,N-d iisop r op yl)p h osp h or a m id ite (10). To a so-
lution of 9 (452 mg, 0.52 mmol) in CH2Cl2 (3 mL) were added
methyl tetraisopropyl phosphorodiamidite (0.42 mL, 1.46
mmol) and 1H-tetrazole (90 mg, 0.52 mmol). The resulting
white suspension turned clear after being stirred for 1 h and
was then stirred for an additional 20 h. The reaction was then
quenched with 5% aqueous NaHCO3 and extracted with CH2-
Cl2. The organic layers were combined, dried over Na2SO4, and
concentrated. The crude product was purified by flash chro-
matography with 5-10% TEA in the eluents (15% CH2Cl2 in
hexanes followed by 20-33% acetone in hexanes) to afford
compound 10 as a colorless oil (413 mg, 77%): 1H NMR (CDCl3,
400 MHz) δ (ppm) (mixture of diastereomers) 0.02-0.06 (3s,
18H), 1.16, 1.17, 1.18, 1.19 (4s, 12H), 2.05, 2.060, 2.064, 2.07
(4s, 6H), 3.23 (s, 3H), 3.33, 3.37 (2s, 3H), 3.54-3.62 (m, 2H),
3.82-4.00 (m, 5H), 4.05-4.34 (m, 8H), 4.96 (s, 1H), 5.72, 5.79
(2s, 1H), 5.98 (1s, 1H), 7.24-7.38 (m, 10H), 7.42, 7.44 (2s, 1H);
31P NMR (CDCl3, 400 MHz) δ 150.45, 152.03; ESI-MS (ES+)
m/z calcd for C45H72N3O16PSi3 1025.4, found 1026.2 (MH+),
1048.1 (M + Na).
2′-O-[Bis(2-a cet oxyet h oxy)m et h yl]-3-m et h yl-3′,5′-O-
(1,1,3,3-t e t r a isop r op yl-1,3-d isiloxa n e d iyl)p se u d ou r i-
d in e (7). In a 100 mL round-bottom flask, compound 6 (0.91
g, 1.81 mmol) was dissolved in 11 mL of 1,4-dioxane to give a
clear solution. Tris(2-acetoxyethoxy)orthoformate (1.63 g, 5.07
mmol) and pyridinium p-toluenesulfonate (182 mg, 0.72 mmol)
were then added to give a clear solution. The reaction was
stirred for 1 h, and then 4-(tert-butyldimethylsilyloxy)-3-
penten-2-one (0.78 mL, 3.26 mmol) was added. The solution
was stirred for 12 h at rt and then at 40 °C for 24 h. The
reaction progress was monitored by TLC using a hexanes/
EtOAc (1:2) solution. Upon completion, the reaction was cooled
to rt, and the cloudy mixture was quenched by the addition of
TMEDA (0.15 mL, 1.0 mmol) and stirred for 15 min. The
resulting clear brown reaction solution was first purified by
flash column chromatography on silica gel using a 16-67%
EtOAc in hexanes gradient to give a partially purified com-
pound 7 (80%) that was further purified using a hexanes/
EtOAc (1:7) mixture to afford a white solid (898 mg, 69%): 1H
NMR (CDCl3, 500 MHz) δ (ppm) 0.83-0.88 (m, 4H), 0.91-
1.00 (m, 24H), 1.97 (s, 3H), 1.98 (s, 3H), 3.20 (s, 3H), 3.74-
3.86 (m, 5H), 3.89-3.95 (m, 2H), 4.04 (d, 1H, J ) 4.0 Hz),
4.07-4.12 (m, 3H), 4.21 (t, 2H, J ) 5 Hz), 4.80 (s, 1H), 5.70 (s,
1H), 7.45 (d, 1H, J ) 5.5 Hz), 10.23 (d, 1H, J ) 5.5 Hz); 13C
NMR (CDCl3, 500 MHz) δ (ppm) 12.43, 12.70, 13.18, 16.65,
16.78, 16.84, 16.96, 17.04, 17.07, 17.14, 17.19, 20.59, 20.67,
26.74, 59.43, 60.17, 63.11, 63.45, 65.56, 68.83, 76.97, 78.40,
79.61, 111.39, 112.21, 135.73, 152.47, 161.88, 170.70, 170.80.
2′-O-[Bis(2-a cet oxyet h oxy)m et h yl]-3-m et h ylp seu d o-
u r id in e (8). CH3CN (1.0 mL), TMEDA (0.22 mL, 6.12 mmol),
and HF (48% aq stock solution, 0.15 mL, 4.08 mmol) were
added dropwise via syringe to a 50 mL round-bottom flask at
0 °C. The HF/TMEDA mixture was stirred at 0 °C for 10 min
and then transferred to a clear solution of 7 (729 mg, 1.02
mmol, in 7 mL of CH3CN) at 0 °C dropwise over 5 min by
cannula. The resulting light yellow solution was stirred at 0
°C for 30 min and then at rt for 5 h. The reaction progress
was monitored by TLC analysis in a hexanes/EtOAc (1:2)
mixture. Upon completion of the reaction, the solvent was
evaporated, and the residue was taken up in a mixture of
hexanes/EtOAc (1:2) (5 mL). The crude product was purified
by flash chromatography on silica gel with 0.5% TMEDA in
the eluents (33-88% EtOAc in hexanes and then 0-5% CH3-
OH in EtOAc) to give a pale yellow oil (585 mg, 68% yield from
compound 6): 1H NMR (CDCl3, 500 MHz) δ (ppm) 2.05 (s, 3H),
2.07 (s, 3H), 3.09 (s, 1H), 3.29 (s, 3H), 3.66-3.82 (m, 6H), 3.85
(dd, 1H, J ) 12.0, 2.0 Hz), 4.07 (m, 1H), 4.16-4.28 (m, 5H),
4.55 (dd, 1H, J ) 6.5, 4.5 Hz), 4.60 (d, 1H, J ) 6.5 Hz), 5.39
(s, 1H), 7.41 (s, 1H), 10.17 (br s, 1H); 13C NMR (CDCl3, 500
MHz) δ (ppm) 20.74, 20.79, 27.25, 62.12, 62.72, 62.76, 63.03,
63.16, 71.59, 76.85, 79.39, 85.04, 109.48, 112.89, 138.97,
152.16, 162.91, 171.02 (2 carbonyls); ESI-MS (ES+) m/z calcd
for C29H28N2O12 476.2, found 499.2 (M + Na), 515.2 (M + K).
3-Meth ylp seu d ou r id in e (11). Removal of the TIPDS
protective group from compound 6 (70 mg, 0.14 mmol) in the
presence of a HF/TMEDA (3:1) mixture in CH3CN (as de-
scribed for compound 8) gave 11 as a white crystalline solid
(31 mg, 85%): 1H NMR (D2O, 500 MHz) δ (ppm) 3.23 (s, 3H),
3.68 (dd, 1H, J ) 12.0, 4.8, 4.8 Hz), 3.82 (dd, 1H, J ) 12.4,
3.2, 3.2 Hz), 3.97 (m, 1H), 4.10 (t, 1H, J ) 5.6, 5.6 Hz), 4.23 (t,
1H, J ) 5.4, 6.0 Hz), 4.67 (d, 1H, J ) 5.6, 5.6 Hz), 7.61 (s, 1H);
13C NMR (D2O, 500 MHz) δ (ppm) 27.20 (N3-CH3), 61.51,
70.80, 73.46, 79.81, 83.16, 110.06, 139.34, 153.16, 164.75; ESI-
MS (ES+) m/z calcd for C10H14N2O6 258.1, found 259 (MH+),
281 (M + Na)+, 539 (2M + Na).
Syn th esis, Dep r otection , a n d P u r ifica tion of Mod ified
R NA Oligon u cleot id es. Two RNAs containing 3-methyl-
pseudouridine (m3Ψ) at position 1915 and pseudouridines (Ψ)
or uridines at positions 1911 and 1917 were synthesized
chemically on 0.2 and 1.0 µmol scales using polystyrene
supports as described previously.6 The sequences of the two
RNAs are as follows, with the numbering based on the full-
5′-O-[Ben zh yd r yloxybis(tr im eth ylsilyloxy]-2′-O-[bis(2-
a cetoxyeth oxy)m eth yl]-3-m eth ylp seu d ou r id in e (9). Solu-
tion A was prepared by adding diisopropylamine (172 µL, 1.23
mmol) dropwise to a solution of 8 (0.59 g, 1.23 mmol, in 7 mL
of CH2Cl2) and cooling the resulting solution to 0 °C. Solution
B was prepared by adding diisopropylamine (172 µL, 1.23
mmol) dropwise to benzhydryloxybis(trimethylsiloxy)silyl chlo-
ride (BzHCl; 1.3 g, 3.08 mmol) in 3 mL of CH2Cl2 at 0 °C.
Aliquots of solution B (0.5 equiv and 0.33 equiv × 3) were
added to solution A at 0 °C dropwise over 2 min, and the
resulting solutions were stirred for 20 min after each addition.
The reaction progress was monitored by TLC (hexanes/EtOAc,
1:2). The light yellow solution was stirred for 2 h at 0 °C. Upon
length E. coli 23S rRNA: 5′-G1906GCCGU1911AACm3Ψ1915
-
AU1917AACGGUC1924-3′ (referred to as Um3ΨU; the names
of the RNAs correspond to the nucleotides at positions
1911, 1915, and 1917, respectively) and 5′-G1906GCCGΨ1911
-
AACm3Ψ1915AΨ1917AACGGUC1924-3′ (Ψm3ΨΨ). The crude RNAs
were divided into 0.20-0.25 µmol per microcentrifuge tube and
deprotected by adding 400 µL of 100 mM NaOAc buffer (pH
3.8) and heating the solution at 60 °C for 30 min. The
deprotected RNAs were then purified by gel electrophoresis
on 20% denaturing polyacrylamide gels followed by electro-
elution. The gel-purified RNA oligonucleotides were desalted
by dialysis against RNase-free deionized water (1 × 4 L), salt
solution (1 × 4 L of 50 mM NaCl, 0.1 mM EDTA), and RNase-
J . Org. Chem, Vol. 67, No. 25, 2002 8853

Chui et al.
free deionized water (1 × 4 L) for 12 h each. The dialyzed RNAs
were lyophilized to dryness, resuspended in RNase-free water,
and further purified over C-18 columns. Each column was
prepared by addition of 10 mL of CH3CN followed by 10 mL
of RNase-free deionized water. Approximately 10 OD units
(∼300 µg) of RNA was loaded onto each column and washed
with 5 mL of deionized water. The RNA eluted from the
column in three fractions with 60% CH3OH in water. The final
purified and desalted RNA oligonucleotides were dried under
reduced pressure in a Speed-Vac concentrator to give a white
solid. The single-stranded extinction coefficient (ꢀ) was calcu-
lated to be 188860 cm-1 M-1 for each RNA. The extinction
coefficient for uridine (1.0 × 104 cm-1 M-1 at pH 7.0)23 was
used for pseudouridine and 3-methylpseudouridine because the
nearest-neighbor extinction coefficients for those modified
bases are unknown.
Ack n ow led gm en t. This work is supported by the
National Institutes of Health (Grant GM54632). We
thank J . SantaLucia for the use of the Aviv spectrometer
and M. Ksebati, L. Hryhorczuk, J . Kieltyka, D. Kitchen,
and G. Haas for technical assistance and helpful dis-
cussions.
Su p p or tin g In for m a tion Ava ila ble: NMR data for com-
pounds 1-11 and RNA and mass spectral data for compounds
10 and 11 and RNA. This material is available free of charge
via the Internet at http://pubs.acs.org.
(23) Richards, E. G. Use of Tables in Calculation of Absorption,
Optical Rotary Dispersion, and Circular Dichroism of Polyribonucle-
otides. In Handbook of Biochemistry and Molecular Biology: Nucleic
Acids; Fasman, G. D., Ed.; CRC Press: Cleveland, OH, 1975; pp 596-
599.
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