Synthesis of novel C4-linked C2-imidazole ribonucleoside phosphoramidite and its application to probing the catalytic mechanism of a ribozyme

Article abstract of DOI:10.1021/jo802556s

The synthesis of a novel C4-linked C₂-imidazole ribonucleoside phosphoramidite (ICN-C₂-PA 1) with a two-carbon linker between imidazole and ribose moieties is described. In the phosphoramidite, POM and 2-cyanoethyl groups were select

Full text of DOI:10.1021/jo802556s

Synthesis of Novel C4-Linked C2-Imidazole Ribonucleoside
Phosphoramidite and Its Application to Probing the Catalytic
Mechanism of a Ribozyme
Lisa Araki,† Keiji Morita,† Maho Yamaguchi,† Zheng-yun Zhao,‡ Timothy J. Wilson,§
David M. J. Lilley,§ and Shinya Harusawa*,†
Osaka UniVersity of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan,
School of Chemistry, UniVersity of Southampton, Highfield, Southampton SO17 1BJ, U.K., and Cancer
Research UK Nucleic Acid Structure Research Group, MSI/WTB Complex, UniVersity of Dundee, Dundee
DDI 5EH, U.K.
harusawa@gly.oups.ac.jp
ReceiVed NoVember 18, 2008
The synthesis of a novel C4-linked C2-imidazole ribonucleoside phosphoramidite (ICN-C2-PA 1) with a
two-carbon linker between imidazole and ribose moieties is described. In the phosphoramidite, POM
and 2-cyanoethyl groups were selected to protect the endocyclic amine function of imidazole and the
2′-hydroxyl function of D-ribose, respectively. The C2-imidazole nucleoside, a flexible structural mimic
of a purine nucleobase, was successfully incorporated using ICN-C2-PA 1 into position 638 of the VS
ribozyme through 2′-TBDMS chemistry to study the role of G638 in general acid-base catalysis. The
modified VS ribozyme (G638C2Imz) exhibited significantly greater catalytic activity than observed with
the C0-imidazole that has no carbon atoms linking the ribose and the C4-imidazole. Imidazole nucleoside
analogues with variable spacer lengths could provide a valuable general methodology for exploring the
catalytic mechanisms of ribozymes.
Introduction
ribozymes.2 Guanine nucleobases are important in the ham-
merhead3 and GlmS4 ribozymes, and guanine and adenine bases
act jointly in the hairpin5 and VS6 ribozymes. In the HDV
ribozymes, a cytosine base appears to play a similar role.1c,7
One way to investigate the suspected role of a given nucleobase
is to seek restoration of activity when the nucleoside in question
Chemogenetic methods have proved to be powerful in the
study of RNA function and especially in the mechanistic
dissection of RNA catalysis.1 A significant body of evidence
now points to the direct role of nucleobases in ribozyme
mechanism, and general acid-base catalysis appears to be a
major contributor to rate enhancement in the nucleolytic
(2) Ribozymes and RNA Catalysis; Lilley, D. M. J., Eckstein, F., Eds.; Royal
Society of Chemistry: Cambridge, 2008.
(3) (a) Han, J.; Burke, J. M. Biochemistry 2005, 44, 7864–7870. (b) Martick,
M.; Scott, W. G. Cell 2006, 126, 309–320.
(4) Klein, D. J.; Ferre´-D’Amare´, A. R. Science 2006, 313, 1752–1756.
(5) Rupert, P. B.; Massey, A. P.; Sigurdsson, S. T.; Ferre´-D’Amare´, A. R.
Science 2002, 298, 1421–1424.
(6) Wilson, T. J.; McLeod, A. C.; Lilley, D. M. J. EMBO J. 2007, 26, 2489–
2500.
* To whom correspondence should be addressed. Phone: +81-72-690-1086.
Fax: +81-72-690-1086.
† Osaka University of Pharmaceutical Sciences.
‡ University of Southampton.
§ University of Dundee.
(1) (a) Ryder, S. P.; Strobel, S. A. J. Mol. Biol. 1999, 291, 295–311. (b)
Lebruska, L. L.; Kuzmine, I.; Fedor, M. J. Chem. Biol. 2002, 9, 465–473. (c)
Das, S. R.; Piccirilli, J. A. Nat. Chem. Biol. 2005, 1, 45–52.
(7) (a) Ferre´-d’Amare´, A. R.; Zhou, K.; Doudna, J. A. Nature 1998, 395,
567–574. (b) Nakano, S.; Chadalavada, D. M.; Bevilacqua, P. C. Science 2000,
287, 1493–1497.
2350 J. Org. Chem. 2009, 74, 2350–2356
10.1021/jo802556s CCC: $40.75 2009 American Chemical Society
Published on Web 02/16/2009

Synthesis of NoVel C4-Linked C2-Imidazole Ribonucleoside
FIGURE 1. C4-linked C2- and C0-imidazole ribonucleoside PAs 1 and
2.
is substituted, or the nucleobase ablated, by the addition of an
exogenous base in the medium.8 This approach provided
convincing evidence for general acid-base catalysis in the HDV
ribozyme, using exogenous imidazole bases.9 However, success
requires free access to the active center from the solvent, and
that is not always possible. We therefore introduced an
alternative approach, using a nucleoside analogue in which the
nucleobase is replaced by imidazole. This may be incorporated
as a phosphoramidite into RNA at a chosen site to investigate
the role of the nucleobase normally residing at that position. If
the substituted nucleobase participates in acid-base chemistry
in the unmodified ribozyme, it is possible that imidazole assumes
this role, leading to restoration of cleavage and ligation reactions.
Moreover, the pH dependence of the reaction should change to
reflect the pKa of the imidazole nucleoside.
Introduction of a C-imidazole nucleoside provides a useful
approach to study ribozyme function, as the pKa values of the
nucleobases are not optimal for general acid and base catalysis.
Imidazole, having a pKa close to 7, is an effective proton donor
or acceptor in general acid and base catalysis. We previously
reported the synthesis of a novel 2′-O-tert-butyldimethylsilyl
(tBDMS) C4-linked imidazole ribonucleoside phosphoramidite
(ICN-C0-PA 2)10 (Figure 1). In that study, pivaloyloxymethyl
(POM) was first introduced as an effective protecting group for
the imidazole endocyclic amine that can be readily removed
under mildly basic conditions [28% aq NH3-EtOH (1:3, v/v)],
compatible with 2′-O-tBDMS chemistry.10 Using this method,
we demonstrated the importance of a critical adenine base
(A756) in VS ribozyme, showing that A756Imidazole ribozyme
was active in both cleavage and ligation.11 In another study,
we showed that G8Imidazole hairpin ribozyme was active,
exhibiting a rate that is pH dependent, consistent with proton
transfer at the nucleobase at position 8.12
FIGURE 2. Superposition of molecular graphics structures of guanine
and C2-linked imidazole. The imidazole nucleoside was constructed
so that the ethylene linker and imidazole nucleus were planar, and the
ribose rings of both molecules were superimposed. This shows that
the ring N of the imidazole readily achieves a similar position to that
of guanine N1. By contrast, the imidazole of the C0-linked species
superimposes with the five-membered ring of the purine nucleus (not
shown) so that the ring N is at least 2 Å from guanine N1. The structures
are displayed using PyMol.
of VS ribozyme,11,14 we recently identified a guanine (G638)
in the substrate loop of the VS ribozyme as a second important
nucleobase for general acid-base catalysis.6 Functional group
substitution at position 638 showed that the N1 position of the
guanine base was critical, and the pH profile of the reaction
reflected the pKa of the base located at this position.6 Thus the
chemical mechanism appears to involve general acid-base
catalysis via the combination of G638 and A756 nucleobases.
These are juxtaposed with the scissile phosphate in our recent
model of the VS ribozyme derived from small-angle X-ray
scattering.15
In the course of these studies, we substituted our C0-linked
imidazole nucleoside at position 638 using ICN-C0-PA 2 to
probe the role of G638 in general acid-base catalysis by
the VS ribozyme. However, the modified VS ribozyme
(G638C0Imz) exhibited virtually no restoration of cleavage
activity, suggesting that neither of the two nitrogen atoms of
the imidazole ring could be superimposed with the critical N1
position of the guanine base at position 638. This result was in
contrast to the analogous substitution at position 756, suggesting
that the environment at position 638 was more constrained,
preventing the structural rearrangement that would be required
to bring the imidazole base to the position normally occupied
by the guanine N1. We therefore reasoned that the addition of
methylene carbon atoms between the ribose and the imidazole
might permit the base more readily to extend into the position
that would allow it to participate in proton transfer with the
RNA. Further, molecular graphics shows that the ring N atom
of imidazole C2-nucleoside and N1 of guanine can readily be
superimposed (Figure 2), whereas these atoms are separated by
g2 Å for the C0-nucleoside in the absence of significant
distortion.
The VS ribozyme is the largest of all known nucleolytic
ribozymes. It catalyzes the site-specific cleavage of a phos-
phodiester linkage to generate products containing 2′,3′-cyclic
phosphate and 5′-OH termini.13 Unfortunately, the VS ribozyme
is the only member of the class of nucleolytic ribozymes for
which there is no crystal structure at the present time, and
therefore mechanistic investigation cannot be guided by struc-
tural data. In our ongoing studies of the catalytic mechanism
Consequently, we designed and synthesized a two-carbon-
elongated homologue (ICN-C2-PA 1) that may be a better
structural mimic of purine nucleobase, as shown in Figure 1.
In this article, we describe the chemical synthesis of the novel
ICN-C2-PA 1. We have introduced a 2-cyanoethyl (CE) group
(8) Peracchi, A.; Beigelman, L.; Usman, N.; Herschlag, D. Proc. Natl. Acad.
Sci. U.S.A. 1996, 93, 11522–11527.
(9) Perrotta, A. T.; Shih, I.; Been, M. D. Science 1999, 286, 123–126.
(10) (a) Araki, L.; Harusawa, S.; Yamaguchi, M.; Yonezawa, S.; Taniguchi,
N.; Lilley, D. M. J.; Zhao, Z.; Kurihara, T. Tetrahedron Lett. 2004, 45, 2657–
2661. (b) Araki, L.; Harusawa, S.; Yamaguchi, M.; Yonezawa, S.; Taniguchi,
N.; Lilley, D. M. J.; Zhao, Z.; Kurihara, T. Tetrahedron 2005, 61, 11976–11985.
(11) Zhao, Z.; McLeod, A.; Harusawa, S.; Araki, L.; Yamaguchi, M.;
Kurihara, T.; Lilley, D. M. J. J. Am. Chem. Soc. 2005, 127, 5026–5027.
(12) Wilson, T. J.; Ouellet, J.; Zhao, Z.; Harusawa, S.; Araki, L.; Kurihara,
T.; Lilley, D. M. J. RNA 2006, 12, 980–987.
(14) (a) Lafontaine, D. A.; Wilson, T. J.; Norman, D. G.; Lilley, D. M. J. J.
Mol. Biol. 2001, 312, 663–674. (b) Lafontaine, D. A.; Wilson, T. J.; Zhao, Z.;
Lilley, D. M. J. J. Mol. Biol. 2002, 323, 23–34.
(15) Lipfert, J.; Ouellet, J.; Norman, D. G.; Doniach, S.; Lilley, D. M. J.
Structure 2008, 16, 1357–1367.
(13) Saville, B. J.; Collins, R. A. Cell 1990, 61, 685–696.
J. Org. Chem. Vol. 74, No. 6, 2009 2351

Araki et al.
SCHEME 1. Synthesis of ICN-C2-PA 1
recently developed by Sekine and co-workers16 as a suitable
protecting group for the 2′-hydroxyl function in RNA, together
with the POM group that we developed earlier for the endocyclic
amine of imidazole. With this compound in hand, we demon-
strate the efficient syntheses of four RNA oligonucleotides using
PA 1 and present preliminary results showing cleavage activity
of G638C2Imz-substituted VS ribozyme.
quant). Introduction of a POM group at the imN position of 7
and subsequent debenzylation and reduction of double bond with
Pd(OH)2-C/cyclohexene produced N-POM-imidazole C2-ribo-
nucleoside 8 (77%), which was a partially protected derivative
of ICN-C2-PA 1. Further, 3′,5′-O-TIPDS-protection (TIPDS )
1,1,3,3-tetraisopropyldisiloxanediyl) gave 9 (quant) that allowed
selective introduction of the 2′-hydroxyl protecting group.22
We expended significant effort to find an appropriate 2′-O-
protecting group (Scheme 2). Whereas the 4,4′-dimethoxytrityl
(DMT) group is generally used to protect the 5′-OH of
nucleoside 3′-O-phosphoramidites,23 the selection of a 2′-O-
protecting group was crucial in the chemical synthesis. Various
2′-O-protecting groups have been reported in the last ∼30
years.24 Whereas the tBDMS group is the most widely used
protecting group for the 2′-hydroxyl function, our initial
approach was the selective protection of the 5′-OH of 8 to give
the 5′-O-DMT compound 13, followed by silylation with
tBDMS triflate (Scheme 2, eq 1). However, this resulted in an
inseparable 1:1 mixture 14ab of 2′-O-tBDMS and 3′-O-tBDMS
isomers arising from facile interconversion of the two isomers,
as in the previous synthesis of ICN-C0-PA 2.10 On the other
hand, bis(2-acetoxyethoxy)methyl (ACE)25 and triisopropylsi-
lyloxymethyl (TOM)26 groups have emerged recently as 2′-
hydroxyl-protecting groups in practical RNA chemical synthesis.
Results and Discussion
Synthesis of ICN-C2-PA 1. Although the useful intermediate
ꢀ-D-ribofuranosyl carbaldehyde 5 is a known compound,17 we
employed an alternative feasible approach (51.5% overall yield)
starting from commercially available 2,3,5-tri-O-benzyl-D-ribose
(3)18 involving four steps. Acetylation of 3 and subsequent
treatment with TMSCN/BF3OEt2 gave ꢀ-D-ribofuranosyl cya-
nide 4ꢀ (56%)19b and its R-epimer 4r (28%), which were easily
separated by column chromatography using a modification of
Yokoyama’s procedure19 (Scheme 1). Two-step conversion of
the desired cyanide 4ꢀ into aldehyde 5 was carried out via the
methyl ester according to Koert’s procedure.20 In this case, direct
DIBAL reduction of the cyanide 4ꢀ into the aldehyde caused
partial epimerization at the C1 position of 5. Wittig olefination
of 5 using N-1-trityl-4-imidazolylmethylphosphonium chloride
621 followed by acid treatment to remove the trityl group
provided two-carbon-elongated vinylimidazole 7 (E/Z ) 2/1;
(21) (a) Harusawa, S.; Kawamura, M.; Koyabu, S.; Hosokawa, T.; Araki,
L.; Sakamoto, Y.; Hashimoto, T.; Yamamoto, Y.; Yamatodani, A.; Kurihara, T.
Synthesis 2003, 2844–2850. (b) Recrystallized phosphonium salt 6 (mp 264-
265 °C, MeOH/EtOAc ) 1:1) should be used to sustain the reproducibility of
this reaction.
(16) (a) Saneyoshi, H.; Ando, K.; Seio, K.; Sekine, M. Tetrahedron 2007,
63, 11195–11203. (b) Saneyoshi, H.; Seio, K.; Sekine, M. J. Org. Chem. 2005,
70, 10453–10460.
(22) Markiewicz, W. T. J. Chem. Res. (S) 1979, 24–25.
(17) (a) Dondoni, A.; Formaglio, P.; Marra, A.; Massi, A. Tetrahedron 2001,
57, 7719–7727. (b) Dondoni, A.; Scherrmann, M.-C. J. Org. Chem. 1994, 59,
6404–6412. (c) Dondoni and co-workers reported a synthetic method17a,b to
generate aldehyde 5 from 2,3,5-tri-O-benzyl-D-ribono-1,4-lactone by three-step
route in 44% overall yield.
(18) 2,3,5-Tri-O-benzyl-D-ribofuranose 3 was purchased from Carbosynth
Ltd.
(19) (a) Togo, H.; Ishigami, S.; Fujii, M.; Ikuma, T.; Yokoyama, M. J. Chem.
Soc., Perkin Trans. 1 1994, 2931–2942. (b) Yokoyama and co-workers reported
a cyanation giving an anomeric mixture (88%) of 4r and 4ꢀ in 75:25 ratio at 0
°C.19a When we carried out the same reaction at-48 °C, 4ꢀ was obtained in
56% yield.
(23) Seliger, H. In Current Protocols in Nucleic Acid Chemistry; Beaucage,
S. L., Bergstrom, D. E., Glick, G. D., Jones, R. A., Eds.; John Wiley & Sons:
New York, 2000; pp 2.3.1-2.3.34.
(24) Reese, C. B. In Current Protocols in Nucleic Acid Chemistry; Beaucage,
S. L., Bergstrom, D. E., Glick, G. D., Jones, R. A., Eds.; John Wiley & Sons:
New York, 2000; pp 2.2.1-2.2.24.
(25) (a) Scaringe, S. A.; Wincott, F. E.; Caruthers, M. H. J. Am. Chem. Soc.
1998, 120, 11820–11821. (b) Scaringe, S. A. Methods Enzymol. 2000, 317, 3–
19.
(26) (a) Pitsch, S.; Weiss, P. A.; Jenny, L.; Stutz, A.; Wu, X. HelV. Chim.
Acta 2001, 84, 3773–3795. (b) Pitsch, S.; Weiss, P. A. In Current Protocols in
Nucleic Acid Chemistry; Beaucage, S. L., Bergstrom, D. E., Glick, G. D., Jones,
R. A., Eds.;John Wiley & Sons: New York, 2001; pp 3.8.1-3.8.15.
(20) Koert, U.; Stein, M.; Wagner, H. Liebigs Ann 1995, 1415–1426.
2352 J. Org. Chem. Vol. 74, No. 6, 2009

Synthesis of NoVel C4-Linked C2-Imidazole Ribonucleoside
31P NMR data of 1 indicated a pair of P diastereoisomers at
δ 149.2 and 150.0 ppm (CDCl3). We recently reported a reliable
MS measurement of nucleoside or non-nucleoside phosphora-
midites using a matrix system [triethanolamine (TEOA)-NaCl]
on LSIMS or FABMS equipped with a double-focusing mass
spectrometer.28 The present method successfully revealed the
molecular-related ion of 1 at m/z 920.4336, leading to the
chemical formula C49H64N5O9P + Na. To check the stability
of PA 1 in CH3CN used in automated RNA synthesis, a CD3CN
solution of 1 was allowed to stand at rt in an NMR tube. After
SCHEME 2. Studies of 2′-O-Protecting Group for
ICN-C2-PA 1
1
1 week, H, 13C, and 31P NMR measurements of the solution
did not show any significant decomposition, indicating that the
solution of 1 in CH3CN is stable. Furthermore, phosphoramidite
1 could be stored as a white foam in a vial at rt for nearly 1
month without significant spectral changes.
Synthesis of Oligoribonucleotides Containing C2-Imidazole
Substitution. The efficiency of ICN-C2-PA 1 in automated RNA
synthesis was examined. Four RNA species of lengths between
25 and 36 nt were synthesized, each containing a single C2-
linked imidazole nucleotide at a given position. The RNA was
synthesized using t-BDMS-protected phosphoramidites,29 using
a DNA/RNA synthesizer, with a coupling time of 12 min for
the imidazole nucleoside phosphoramidite. Measurement of trityl
cation conductivity indicated a step yield of >95% for the
coupling of the C2-linked imidazole phosphoramidite. Gel
electrophoretic separation under denaturing conditions of the
crude synthesis following deprotection showed substantial full-
length products with no evidence of strong bands that would
correspond to failure at the position of the modified nucleotide
(Figure 3).
The RNA sequences synthesized were (1) hairpin ribozyme
a strand G8C2Z (CCGACAGAZAAGUCAACCAGAGAAA-
CACACUUGCGg), (2) hairpin ribozyme b strand A38C2Z
( C C G C A A G U G G U Z U A U U A C C U G G U A C G C G -
UUCACGg),(3)VSsubstrateG638C2Z(GCGCGAAGGGCGUC-
GUCGCCCCZAt), (4) VS lower strand A756C2Z (AACG-
CAGUAUUGCZGCACAGCACAAGCCCGCUUGc), where Z
designates the C2-imidazole nucleoside, and letters shown in
lower case are 3′-deoxyribonucleosides arising from the support.
Ribozyme Activity of G638C2Imz. One of the RNA oligo-
nucleotides synthesized was based on the substrate of the VS
ribozyme, with a single imidazole substitution at position 638.
G638 is one of the nucleobases proposed to act in general
acid-base catalysis in the catalytic mechanism of the ribozyme.6
After purification, we tested the ribozyme activity of the
modified substrate in a cleavage reaction with unmodified VS
ribozyme in trans. The C2-linked imidazole-containing substrate
was incubated with 1 µM trans VS ribozyme under single
turnover conditions, in the presence of 50 mM MES-HCl (pH
6.5), 200 mM MgCl2, 25 mM KCl at 37 °C, and the products
were analyzed by gel electrophoresis (Figure 4A). Extended
incubation under these conditions resulted in 70% ribozyme
cleavage at the normal site. Reaction progress is plotted in Figure
Unfortunately, the reaction of 3′,5′-O-protected compound 9
with tris(2-acetoxyethoxy)orthoformate under standard condi-
tions did not proceed (Scheme 2, eq 2). Furthermore, the reaction
of 3′,5′-O-di-tert-butylsilanediyl (DTBS) protected precursor
1527 with TOMCl in the presence of N,N-diisopropylethylamine
provided 2′-O-TOM nucleoside 16 in only low yield (eq 3).
From these results, it appeared that the introduction of bulky
protecting groups to the ICN-PAs is responsible for the reaction
failures, unlike in the cases of conventional N-nucleosides.
Consequently, there is a need for small temporary substituents
with little steric hindrance for 2′-O-protection.
During the course of our investigation, Sekine and co-workers
reported a new method for the synthesis of oligoribonucleotides
using the CE group, that is, the smallest of the available
protecting groups for the 2′-hydroxyl group.16 Migration of the
protecting group between 2′- and 3′-positions is inhibited, and
it can be removed using 1 M TBAF in THF. Therefore, our
attention was directed to the synthesis of novel 2′-O-CE-
protected PA 1 (Scheme 1). Cyanoethylation of 9 with acry-
lonitrile in t-BuOH in the presence of Cs2CO3 at room
temperature (rt) successfully afforded fully protected intermedi-
ate 10 in excellent yield (97%). The TIPDS group of 10 was
selectively removed by treatment with Et3N·3HF to give 3′,5′-
O-unprotected ribonucleoside derivative 11 (91%).16 Although
the imN-POM group was readily removed under the mildly basic
conditions as described previously, it remained intact during
cyanoethylation and removal of the TIPDS group, showing the
added advantage of using this group. After dimethoxytritylation
of 11, 3′-hydroxyl derivative 12 was subjected to phosphityla-
tion. Treatment of 12 with 2-O-cyanoethyl-N,N,N′,N′-tetraiso-
propylphosphodiamidite in the presence of 4,5-dicyanoimidazole
(DCI) in dichloroethane proceeded smoothly at 40 °C for 20
min to give the final product ICN-C2-PA 1. Purification of the
crude product was easily carried out on basic column chroma-
tography to yield phosphoramidite 1 (72%) as a white foam.
An overall yield of 23% was achieved in 13 steps from readily
available starting material 2′,3′,5′-O-tribenzyl D-ribose 3, making
it a feasible approach for scaling up synthesis of 1.
(28) (a) Fujitake, M.; Harusawa, S.; Araki, L.; Yamaguchi, M.; Lilley,
D. M. J.; Zhao, Z.; Kurihara, T. Tetrahedron 2005, 61, 4689–4699. (b) Fujitake,
M.; Harusawa, S.; Zhao, Z.; Kurihara, T. Bull. Osaka UniV. Pharm. Sci. 2007,
1, 107–112. (c) Harusawa, S.; Fujitake, M.; Kurihara, T.; Zhao, Z.; Lilley,
D. M. J. In Current Protocols in Nucleic Acid Chemistry; Beaucage, S. L.,
Bergstrom, D. E., Herdewijn, P., Matsuda, A., Eds.; John Wiley & Sons: New
York, 2006; pp 10.11.1-10.11.15.
(29) (a) Beaucage, S. L.; Caruthers, M. H. Tetrahedron Lett. 1981, 22, 1859–
1862. (b) Usman, N.; Pon, R. T.; Ogilvie, K. K. Tetrahedron Lett. 1985, 26,
4567–4570. (c) Damha, M. J.; Ogilvie, K. K. In Methods in Molecular Biology.
Protocols for Oligonucleotides and Analogs; AgrawalS., Ed.; Humana Press:
Totowa, NJ, 1993; Vol. 20, Chapter 5.
(27) Furusawa, K.; Ueno, K.; Katsura, T. Chem. Lett. 1990, 97–100.
J. Org. Chem. Vol. 74, No. 6, 2009 2353

Araki et al.
FIGURE 4. Cleavage activity of a VS ribozyme substrate with C2-
linked imidazole replacing guanine at position 638. (A) Electrophoretic
separation of substrate and product during the course of the incubation
with VS ribozyme over a time course of 27 h. Times of incubation
(min) are indicated over each track. Radioactive 5′-32P-labeled substrate
RNA was incubated with 1 µM VS ribozyme in 50 mM MES-HCl
(pH 6.5), 200 mM MgCl2, and 25 mM KCl at 37 °C; aliquots were
removed at the times indicated, and the reaction was terminated with
EDTA. These were separated by electrophoresis in a 20% polyacry-
lamide gel. The substrate and product were visualized and quantified
by phosphorimage analysis. (B) Plot of the extent of cleavage as a
function of time, using the data from (A). The data (points) have been
fitted (line) to a rate equation with two exponential functions, from
which rates of 0.0076 and 0.0007 min-1 were calculated.
FIGURE 3. Analysis of the crude products of RNA synthesis by gel
electrophoresis. After deprotection, ∼120 µg aliquots of the synthetic
RNA were applied to a 16% polyacrylamide gel containing 7 M urea
in 90 mM Tris-borate (pH 8.3), 10 mM EDTA. The RNA was
visualized by UV shadowing. The arrowheads mark the position
corresponding to the incorporation of C2-linked imidazole. The RNA
species were (1) hairpin ribozyme strand a, 36 nt; (2) hairpin ribozyme
strand b, 35 nt; (3) VS ribozyme substrate, 25 nt (C2-linked imidazole
is located three nucleotides from the 3 terminus, which is not resolved
by the gel and therefore not observable in the image); and (4) VS
ribozyme 6-2 strand, 35 nt.
4B. Fitting the data required two exponential functions, corre-
sponding to rates of 0.0076 and 0.0007 min-1 (Table 1). The
faster rate is 15-fold greater than that achieved with ribozyme
containing the C0-linked imidazole at the same position.
Incubations were also performed in 50 mM Tris-HCl (pH 8.0),
10 mM MgCl2, 25 mM KCl, and 2 mM spermidine. Rates of
cleavage were 10-fold slower, but the ratio of rates between
the two imidazole substituents was closely similar at 14. Thus
the additional linker length between the ribose and imidazole
has resulted in significantly greater catalytic activity of the
ribozyme.
TABLE 1. Comparison of Cleavage Rates between G638C2Imz
and G638C0Imz
rates/min-1
natural
G638C2Imz G638C0Imz C2Imz/C0Imz
10 mM Mg2+
pH 8
- Rz
6 × 10-5
5 × 10-5
7 × 10-5
+ 1 µM Rz
0.61
0.001
14
15
200 mM
Mg2+ pH 6.5
- Rz
1 × 10-5
2 × 10-5
5 × 10-4
+ 1 µM Rz
6.2
0.008
Conclusions
synthesis using TBDMS methodology, it provided C2-imidazole
modified ribozymes in >95% single stepwise coupling yield
with other RNA bases coupling over 98% on average. This
promises to open a more practical pathway to this chemical
strategy for investigating RNA functionality. Of particular
interest is that the modified VS ribozyme (G638C2Imz) incor-
porated from ICN-C2-PA 1 showed significantly greater catalytic
activity than G638C0Imz. Further work on the application of
imidazole oligonucleotides is underway, and the characteristics
of the modified VS ribozyme will be published in due course.
ICN-C2-PA 1 having a two-carbon linker between imidazole
and ribose moieties was designed as a novel purine nucleobase
mimic and was successfully synthesized from 2,3,5-tri-O-
benzylribofuranose 3. We have demonstrated the feasibility of
preparing stable 2′-O-cyanoethylated PA 1, and we see no
impediment to increasing the scale of the synthesis to meet the
needs of RNA structural studies. Furthermore, we have clarified
that the CE group is an efficient protecting group for the 2′-
hydroxyl group and reinforced that the POM group could be
used for the protection of imidazole endocyclic amino function
of ICN-PAs. The phosphoramidites with these modifications are
compatible with 2′-TBDMS chemistry for RNA synthesis. The
synthetic method using the CE and POM protecting groups may
be generally applicable to the PAs of ribose-(CH2)n-imidazole
species. When ICN-C2-PA 1 was subjected to automated RNA
Experimental Section
Chemical Synthesis of Oligoribonucleotides. Oligoribonucle-
otides were synthesized using t-BDMS-protected phosphoramid-
ites29 implemented on an DNA/RNA synthesizer. LV200 columns
2354 J. Org. Chem. Vol. 74, No. 6, 2009

Synthesis of NoVel C4-Linked C2-Imidazole Ribonucleoside
(Applied Biosystems) introduced a deoxyribonucleotide at the 3′
end of each oligonucleotide. Imidazole phosphoramidites were
dissolved in anhydrous acetonitrile as a 0.1 M solution less than
30 min before coupling. An equal volume of 0.25 M 4,5-
dicyanoimidazole was employed as activator, and coupling times
of 12 min were used. A 0.02 M iodine/water solution was used for
phosphorus oxidation at each step of synthesis. RNA was depro-
tected using ammonia/ethanol (3:1, v/v) solution at 55 °C for 2 h,
followed by treatment with 1 M TBAF in THF under standard
conditions. RNA oligonucleotides were separated by electrophoresis
in a 15% polyacrylamide gel containing 7 M urea with 90 mM
Tris-borate (pH 8.3), 10 mM EDTA buffer, for 2.5 h at 20 W.
RNA was visualized by UV shadowing.
Preparation of VS Ribozyme by Transcription. Templates for
transcription of ribozymes were made by recursive PCR from
synthetic DNA oligonucleotides. RNA was synthesized using T7
RNA polymerase and purified by electrophoresis in 5% polyacry-
lamide gels containing 7 M urea. RNA was recovered by electro-
elution into 8 M ammonium acetate. The sequence of the trans-
acting ribozyme30 was GCGGUAGUAAGCAGGGAACUCACCU-
CCAAUUUCAGUACUGAAAUUGUCGUAGCAGUUGACUA-
CUGUUAUGUGAUUGGUAGAGGCUAAGUGACGGUAUUG-
GCGUAAGUCAGUAUUGCAGCACAGCACAAGCCCGCUUGC-
GAGAAU.
Analysis of VS Ribozyme Kinetics. Cleavage kinetics were
studied under single-turnover conditions.6 Ribozyme and 5′-32P-
labeled substrate were incubated separately at 37 °C for 20 min in
reaction buffer, and the reaction was initiated by adding an equal
volume of ribozyme to the substrate tube. The final reaction
contained 1 µM ribozyme and 10 nM substrate. Two buffer
conditions were used, either 50 mM Tris (pH 8), 10 mM MgCl2,
25 mM KCl, 2 mM spermidine, or 50 mM MES (pH 6.5), 200
mM MgCl2, 25 mM KCl. Mineral oil was layered on top to prevent
evaporation. Two microliter aliquots were removed at intervals,
and the reaction was terminated by addition to 8 µL of a mixture
containing 95% (v/v) formamide, 50 mM EDTA, and electrophore-
sis dyes. Substrate and product were separated by electrophoresis
in 20% polyacrylamide gels containing 7 M urea and quantified
by phosphorimaging. Progress curves were fitted by nonlinear
regression analysis to exponential functions using Kalaidagraph.
4-[(E,Z)-2-(2,3,5-Tri-O-benzyl-ꢀ-D-ribofuranos-1-yl)vinyl]-1H-imi-
dazole [(E,Z)-7]. A 1.6 M BuLi solution in hexane (4.4 mL, 7.0
mmol) was added dropwise over a period of 15 min to a white
suspension of phosphonium salt 621b (4.34 g, 7.0 mmol) in dry
THF (60 mL) at -70 °C. The resulting yellow suspension was
stirred for 30 min at the same temperature, and a solution of
aldehyde 5 (1.51 g, 3.5 mmol) in THF (20 mL) was added slowly
to keep the temperature of the suspension at approximately -70
°C. The reaction mixture was elevated to rt, where it was
transformed into a yellow solution, and continued to stir for 1 h at
rt. The reaction was quenched by the addition of water (1 mL) and
evaporated. The residue was subsequently dissolved in CHCl3 (50
mL), and the organic layer was washed with water, dried over
anhydrous MgSO4, and evaporated. The residue was subjected to
column chromatography with EtOAc/hexane (40:60) as eluent using
the coated silica gel technique to give a crude pale yellow oil {ca.
2.60 g; 4-[(E,Z)-2-(2,3,5-tri-O-benzyl-ꢀ-D-ribofuranos-1-yl)vinyl]-
1-tritylimidazole containing a small amount of Ph3P ) O}. Aqueous
2 N HCl (20 mL) was added to the solution of partially purified
oil in EtOH (24 mL), and the solution was refluxed for 2 h. After
cooling to rt, the precipitated material (Ph3PdO) was removed by
filtration. The filtrate was evaporated to give a residue that was
subsequently diluted with water (20 mL) and neutralized by the
addition of saturated aq NaHCO3 solution. The mixture was
extracted with EtOAc (2 × 50 mL), and the combined organic layers
were washed with H2O, dried, and evaporated to yield a residue.
Chromatography purification on silica with EtOAc as eluent gave
a 2:1 mixture (1.74 g, quant) of (E)-7 and (Z)-7. Although the
separation of (E)-7 and (Z)-7 was not required for the following
experiment, they could be partially isolated by chromatography.
1
(E)-7: oil; Rf ) 0.52 (12% MeOH/EtOAc); H NMR (CDCl3) δ
3.55 (dd, 1H, J ) 10.3, 3.4 Hz), 3.59 (dd, 1H, J ) 10.3, 3.4 Hz),
3.76 (t, 1H, J ) 5.1 Hz), 3.93 (t, 1H, J ) 3.4 Hz), 4.24 (q, 1H, J
) 3.4 Hz), 4.44-4.60 (m, 7H), 6.08 (dd, 1H, J ) 15.5, 6.8 Hz,
CHdCH-Im), 6.54 (d, 1H, J ) 15.5 Hz, CHdCH-Im), 6.80 (s,
1H), 7.20-7.35 (m, 15H), 7.40 (s, 1H); HRMS calcd for
C31H32N2O4 (M+) 496.2360, found 496.2364. (Z)-7: oil; Rf ) 0.58
(12% MeOH/EtOAc); 1H NMR (CDCl3) δ 3.52 (dd, 1H, J ) 10.0,
2.5 Hz), 3.58 (dd, 1H, J ) 10.0, 2.5 Hz), 3.86 (dd, 1H, J ) 7.5,
5.0 Hz), 4.02 (dd, 1H, J ) 5.0, 2.5 Hz), 4.26 (q, 1H, J ) 2.5 Hz),
4.46-4.69 (m, 6H), 4.83 (t, 1H, J ) 7.5 Hz), 5.49 (dd, 1H, J )
11.6, 7.5 Hz, CHdCH-Im), 6.46 (d, 1H, J ) 11.6 Hz,
CHdCH-Im), 7.00 (s, 1H), 7.20 (s, 1H), 7.25-7.40 (m, 15H);
HRMS calcd for C31H32N2O4 (M+) 496.2360, found 496.2363. 13
C
NMR of a 2:1 mixture of (E)-7 and (Z)-7 was measured: 13C NMR
(CDCl3) of (E,Z)-7 δ 70.4, 72.0, 72.2, 72.8, 73.4, 73.6, 77.2, 77.6,
78.1, 80.9, 81.4, 81.9, 82.2, 82.3, 118.6, 120.1, 121.7, 125.2, 126.0,
127.2, 127.3 (127.28), 127.3 (127.31), 127.4 (127.37), 127.4
(127.40), 127.5, 127.6, 127.7, 127.8, 127.9, 128.0, 128.1, 128.2,
134.4, 134.9, 135.4, 136.4, 137.1, 137.2, 137.3, 137.4, 137.7.
{4-[2-(ꢀ-D-Ribofuranos-1-yl)ethyl]imidazolyl}methyl 2,2-dimethyl-
propionate (8). NaH (60%, 161 mg, 4.0 mmol) in mineral oil was
added to THF (20 mL) while stirring to give a suspension. A
solution of (E,Z)-7 (1.33 g, 2.7 mmol) in THF (30 mL) was added
to the suspension, and the resulting mixture was stirred at rt for 40
min. A solution of chloromethyl pivaloate (607 mg, 4.0 mmol) in
THF (30 mL) was then added. After stirring for 2 h, H2O (0.5 mL)
was added followed by evaporation to a residue that was subse-
quently dissolved in EtOAc. The organic layer was washed with
water and brine, dried, and evaporated. The crude product was
purified by column chromatography on silica gel using 50% EtOAc
in hexane to give (E,Z)-{4-[(E,Z)-2-(2,3,5-tri-O-benzyl-ꢀ-D-ribo-
furanos-1-yl)vinyl]imidazolyl}methyl 2,2-dimethylpropionate [17,
1.49 g, 92%, E/Z ) 2/3] as a colorless oil. The 2:3 ratio of the E
and Z isomers of 17 was assigned based on the following 1H NMR
1
data: H NMR (CDCl3) δ 1.04 [s, 5.4H, C(CH3a)3], 1.17 [s, 3.6H,
C(CH3b)3], 3.52-3.64 (m, 2H), 3.81 (t, 0.4H, J ) 5.6 Hz), 3.86 (t,
0.6H, J ) 5.6 Hz), 3.94 (t, 0.4H, J ) 5.6 Hz), 4.01 (t, 0.6H, J )
5.6 Hz), 4.22-4.30 (m, 1H), 4.47-4.76 (m, 6.4H), 5.31 (dd, 0.6H,
J ) 9.6, 5.6 Hz), 5.57 (dd, 0.6H, J ) 12.4, 9.6 Hz, CHadCH-Im),
5.72 (q, 1.2H, J ) 11.5 Hz), 5.76 (s, 0.8H), 6.34 (dd, 0.4H, J )
15.3, 7.6 Hz, CHbdCH-Im), 6.45 (d, 0.6H, J ) 12.4 Hz,
CHdCHa-Im), 6.58 (d, 0.4H, J ) 15.3 Hz, CHdCHb-Im), 6.83
(s, 0.4H), 7.24-7.35 (m, 15.6H), 7.61 (s, 0.4H), 7.64 (s, 0.6H);
13C NMR (CDCl3) δ 27.2, 39.0, 67.7, 67.8, 70.3, 70.4, 71.9, 72.0,
72.1, 72.2, 73.4, 73.5, 77.4, 77.6, 78.1, 80.8, 81.1, 81.2, 81.8, 82.1,
108.9, 117.0, 118.7, 122.6, 123.9, 127.1, 127.1, 127.2, 127.3, 127.4,
127.5, 127.6, 127.7, 127.8, 127.9, 127.9, 128.8, 137.4, 137.5, 137.8,
137.9, 138.4, 139.7, 176.9 (COO, overlapped); HRMS calcd for
C37H42N2O6 (M+) 610.3040, found 610.3032. Next, a mixture of
(E,Z)-methyl 2,2-dimethylpropionate (165 mg, 0.27 mmol), 20%
Pd(OH)2-C (99 mg), and cyclohexene (0.82 mL, 8.1 mmol) in
EtOH (7 mL) was refluxed for 1 h. After filtration through Celite,
the filtrate was evaporated to give a residue, which was purified
by silica column chromatography [MeOH/EtOAc (1/9)] to give 8
(77 mg, 84%) as a colorless oil: 1H NMR (CD3OD) δ 1.15 (s, 9H),
1.72-1.86 (m, 1H), 1.86-2.00 (m, 1H), 2.55-2.77 (m, 2H), 3.56
(dd, 1H, J ) 11.4, 5.2 Hz), 3.64-3.80 (m, 4H), 3.92 (t, 1H, J )
5.2 Hz), 5.89 (s, 2H), 6.99 (s, 1H), 7.72 (s, 1H); 13C NMR (CD3OD)
δ 25.3, 27.3, 34.5, 39.8, 63.5, 69.1, 72.7, 76.1, 83.1, 85.3, 116.7,
138.7, 142.8, 178.0 (COO); HRMS (EIMS) calcd for C16H27N2O6
(M + H)+ 343.1867, found 343.1863.
{4-[2-(3,5-O-TIPDS-ꢀ-D-ribofuranos-1-yl)ethyl]imidazolyl}methyl
2,2-dimethylpropionate (9). 1,3-Dichloro-1,1,3,3-tetraisopropyldisi-
loxane (0.064 mL, 0.2 mmol) was added dropwise to a solution of
(30) Beattie, T. L.; Olive, J. E.; Collins, R. A. Proc. Natl. Acad. Sci. U.S.A.
1995, 92, 4686–4690.
J. Org. Chem. Vol. 74, No. 6, 2009 2355

Araki et al.
8 (35 mg, 0.1 mmol) in pyridine (1.5 mL) at 0 °C. The resulting
mixture was stirred for 1 h at the same temperature and then at rt
for 2 h. After evaporation, the crude product was purified by column
chromatography [EtOAc/hexane (4/1)] to yield 9 (60 mg, quant)
as a colorless oil: 1H NMR (CDCl3) δ 0.95-1.10 (br s, 28H), 1.17
(s, 9H), 1.78-2.07 (m, 2H), 2.60-2.77 (m, 2H), 3.77-3.91 (m,
4H), 4.01 (dd, 1H, J ) 11.3, 3.3 Hz), 4.21 (t, 1H, J ) 6.6 Hz),
5.77 (s, 2H), 6.81 (s, 1H), 7.60 (s, 1H); 13C NMR (CDCl3) δ 13.2,
13.6, 13.8, 17.40, 17.46, 17.49, 17.6, 17.7, 17.9, 24.6, 27.1, 33.4,
39.0, 63.1, 67.8, 72.6, 74.6, 77.2, 82.0, 83.5, 115.2, 137.1, 142.4,
176.9; HRMS (EIMS) calcd for C28H52N2O7Si2 (M+) 584.3310,
found 584.3317.
2.44-2.85 (m, 2H), 2.63 (t, 2H, J ) 6.0 Hz), 3.16 (dd, 1H, J )
10.1, 4.7 Hz), 3.29 (dd, 1H, J ) 10.1, 3.4 Hz), 3.62-3.84 (m, 2H),
3.67 (t, 1H, J ) 5.4 Hz), 3.78 (s, 6H), 3.94 (m, 2H), 4.09 (t, 1H,
J ) 5.4 Hz), 5.76 (s, 2H), 6.81 (d, 4H, J ) 8.3 Hz), 7.14-7.48 (m,
10H), 7.62 (s, 1H); 13C NMR (CDCl3) δ 19.5, 24.7, 27.2, 30.0,
33.8, 39.0, 55.3, 64.2, 65.1, 67.7, 71.7, 79.8, 82.8, 83.5, 85.9, 112.7,
115.4, 117.2, 126.3, 127.4, 127.8, 129.7, 135.5, 135.6, 137.1, 141.9,
144.4, 157.8, 176.9; IR (film, cm-1) 1735 (CO-O), 2250 (CN);
HRMS (FABMS: NBA) calcd for C40H48N3O8 (M + H)+ 698.3441,
found 698.3445.
[4-(2-{5-O-DMT-2-O-(2-cyanoethyl)-3-O-[(2-cyanoethyl)-(N,N-
diisopropylamino)phosphoramidyl]-ꢀ-D-ribofuranos-1-yl}ethyl)imi-
dazolyl]methyl 2,2-dimethylpropionate (1). 2-Cyanoethyl N,N,N′,N′-
tetraisopropylphosphodiamidite (0.04 mL, 0.121 mmol) and 4,5-
DCI (9.0 mg, 0.073 mmol) were added to a solution of compound
12 (42.2 mg, 0.061 mmol) in dry dichloroethane (0.7 mL) under
an argon atmosphere. The resulting mixture was stirred at 40 °C
for 20 min and then evaporated. The residual oil was subjected to
NH/silica gel chromatography using a solvent system (30% and
then 50% EtOAc in hexane) to give 1 (39.1 mg, 72%) as a white
{4-[2-(2-O-CE-3,5-O-TIPDS-ꢀ-D-ribofuranos-1-yl)ethyl]imidazo-
lyl}methyl 2,2-dimethylpropionate (10). Acrylonitrile (0.27 mL, 4.0
mmol) and cesium carbonate (67.3 mg, 0.203 mmol) were added
to a solution of 9 (118 mg, 0.2 mmol) in t-butanol (1.0 mL). After
vigorously stirring at rt for 6 h, the mixture was filtered through
Celite. The filtrate was evaporated to a residue that was purified
by column chromatography with EtOAc/hexane (50:50 to 100:0,
v/v) as eluent to give 10 (124.6 mg, 97%) as an oil: IR (film, cm-1
)
1
1
1735 (CO-O), 2250 (CN); H NMR (CDCl3) δ 0.86-1.08 (m,
28Η), 1.12 (s, 9H), 1.71-1.96 (m, 2H), 2.48-2.64 (m, 4H),
3.52-4.17 (m, 8H), 5.70 (s, 2H), 6.75 (s, 1H), 7.51 (s,1H); 13C
NMR (CDCl3) δ 13.0, 13.2, 13.5, 13.9, 17.3, 17.5, 17.6, 17.67,
17.73, 17.8, 19.6, 24.5, 27.1, 34.1, 38.9, 60.5, 65.6, 67.7, 72.1, 80.0,
82.6, 83.2, 115.3, 117.4, 137.1, 142.0, 176.8; HRMS (FABMS:
NBA) calcd for C31H56N3O7Si2 (M + H)+ 638.3656, found
638.3658.
foam: Rf ) 0.73, 0.63 (EtOAc); H NMR (CDCl3) δ 1.11-1.18
(overlapping peaks, 23H), 1.94 (m, 1H), 2.08 (m, 1H), 2.28 (t, 0.8H,
J ) 6.2 Hz), 2.50-2.90 (m, 5.2H), 3.07 (dt, 1H, J ) 10.3, 3.4
Hz), 3.26 (dd, 0.6H, J ) 10.3, 3.4 Hz), 3.34 (dd, 0.4H, J ) 10.3,
3.4 Hz), 3.46-3.91 (m, 5H), 3.78 (s, 6H), 3.98 (m, 1H), 4.13 (m,
1H), 4.26 (m, 1H), 5.76 (s, 2H), 6.82 (m, 4H), 7.15-7.49 (m, 10H),
7.59 (s, 1H); 31P NMR (CDCl3) δ 149.2, 150.0; IR (film, cm-1
)
1735 (CO-O), 2250 (CN); HRMS (FABMS: TEOA+
NaCl)28 calcd for C49H65N5O9P (M + H)+ 898.4520, found
898.4515; calcd for C49H64N5O9PNa (M + Na)+ 920.4339, found
920.4336.
{4-[2-(2-O-CE-ꢀ-D-ribofuranos-1-yl)ethyl]imidazolyl}methyl 2,2-
dimethypropionate (11). Et3N·3HF (0.11 mL, 0.7 mmol) and Et3N
(0.05 mL, 0.35 mmol) were added to a solution of compound 10
(125 mg, 0.2 mmol) in THF (2.0 mL). The mixture was stirred at
rt for 1.5 h and was evaporated. The residue was subjected to
chromatography with CHCl3/MeOH (100:0 to 95:5, v/v) to give
11 (70 mg, 91%) as an oil: IR (film, cm-1) 1735 (CO-O), 2250
Acknowledgment. We thank Miss Ayumi Amenomori for
her technical support. We are grateful to Dr. K. Minoura and
Ms. M. Fujitake of Osaka University of Pharmaceutical Sciences
for NMR and MS measurements, respectively. This work was
supported in part by Grants-in-Aid for Scientific Research [No.
16590024 (to S.H.) and No. 17790024 (to L.A.)] from JSPS
and a Grant-in-Aid for “High Technology Research Center”
Project for Private Universities: matching fund subsidy from
MEXT (Ministry of Education, Sciences, Sports and Culture),
2007-2009, Japan. We also acknowledge the financial support
of Cancer Research UK.
1
(CN); H NMR (CDCl3) δ 1.18 (s, 9H), 1.93 (m, 2H), 2.57-2.80
(m, 2H), 2.66 (t, 2H), 3.60-3.98 (m, 7H), 4.20 (t, 1H, J ) 5.2
Hz), 5.78 (s, 2H), 6.83 (s, 1H), 7.61 (s, 1H); 13C NMR (CDCl3) δ
20.0, 24.9, 27.7, 34.0, 39.5, 62.5, 65.6, 68.3, 71.2, 80.9, 83.8, 84.6,
115.9, 117.9, 137.6, 142.5, 177.5; HRMS (FABMS: NBA) calcd
for C19H30N3O6 (M + H)+ 396.2135, found 396.2134.
{4-[2-(5-O-DMT-2-O-CE-ꢀ-D-ribofuranos-1-yl)ethyl]imidazolyl}-
methyl 2,2-dimethylpropionate (12). Compound 11 (30 mg, 0.08
mmol) was coevaporated three times with pyridine (1 mL) and
redissolved in dry pyridine (0.5 mL). DMTCl (41 mg, 0.12 mmol),
Et3N (0.02 mL, 0.115 mmol), and DMAP (0.2 mg, 0.002 mmol)
were added to the solution. After stirring at rt for 2.5 h, methanol
(0.5 mL) was added. The solvents were removed to give a residue
that was purified by column chromatography (NH/silica gel) using
a gradient solvent system [CHCl3/benzene (30:70, 50:50, 70:30 to
100:0)] as eluent to give compound 12 (44.3 mg, 83%) as white
foam: 1H NMR (CDCl3) δ 1.15 (s, 9H), 1.84-2.12 (m, 2H),
Supporting Information Available: General procedure and
the procedures for the synthesis of 4ꢀ, 5, 13, 14ab, 15, and 16.
Copies of NMR spectra (1H, 13C, and/or 31P) of all new
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
JO802556S
2356 J. Org. Chem. Vol. 74, No. 6, 2009