Chemical synthesis of a 5′-terminal TMG-capped triribonucleotide m32,2,7G5′ pppAmpUmpA of U1 RNA

Article abstract of DOI:10.1021/jo952263v

The 5′-terminal TMG-capped triribonucleotide, m₃^2,2,7G^5′pppAmpUmpA, has been synthesized by condensation of an appropriately protected triribonucleotide derivative of ppAmpUmpA with a new TMG-capping reagent. During this total synthesis, it was found that the regioselective 2′-O-methylation of 3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-N-(4- monomethoxytrityl)adenosine was achieved by use of MeI/Ag₂O without affecting the base moiety. A new route to 2-N,2-N-dimethylguanosine from guanosine via a three-step reaction has also been developed by reductive methylation using paraformaldehyde and sodium cyanoborohydride. These key intermediates were used as starting materials for the construction of a fully protected derivative of pAmpUmpA and a TMG-capping reagent of Im-pm₃^2,2,7G. The target TMG-capped tetramer, m₃^2,2,7G^5′ pppAmpUmpA, was synthesized by condensation of a partially protected triribonucleotide 5′-terminal diphosphate species, ppA^MMTrmpUmpA, with Im-pm₃^2,2,7G followed by treatment with 80% acetic acid. The structure of m₃^2,2,7G^5′ pppAmpUmpA was characterized by ¹H and ³¹P NMR spectroscopy as well as enzymatic assay using snake venom phosphodiesterase, calf intestinal phosphatase, and nuclease P1.

Full text of DOI:10.1021/jo952263v

4412
J . Org. Chem. 1996, 61, 4412-4422
Ch em ica l Syn th esis of a 5′-Ter m in a l TMG-Ca p p ed
Tr ir ibon u cleotid e m 32,2,7G5′p p p Am p Um p A of U1 RNA
Mitsuo Sekine,* Michinori Kadokura, Takahiko Satoh, Kohji Seio, and Takeshi Wada
Department of Life Science, Tokyo Institute of Technology, Nagatsuta, Midoriku, Yokohama 226, J apan
Utz Fischer, Vicki Sumpter, and Reinhard Lu¨hrmann
Institut fu¨r Molekularbiologie und Tumorforschung, Philipps-Universita¨t Marburg,
Emil-Mannkopff-Strasse 2, D-3550 Marburg, Germany
Received December 27, 1995X
The 5′-terminal TMG-capped triribonucleotide, m32,2,7G5′pppAmpUmpA, has been synthesized by
condensation of an appropriately protected triribonucleotide derivative of ppAmpUmpA with a new
TMG-capping reagent. During this total synthesis, it was found that the regioselective 2′-O-
methylation of 3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-N-(4-monomethoxytrityl)adenosine
was achieved by use of MeI/Ag2O without affecting the base moiety. A new route to 2-N,2-N-
dimethylguanosine from guanosine via a three-step reaction has also been developed by reductive
methylation using paraformaldehyde and sodium cyanoborohydride. These key intermediates were
used as starting materials for the construction of a fully protected derivative of pAmpUmpA and
a TMG-capping reagent of Im-pm32,2,7G. The target TMG-capped tetramer, m32,2,7G5′pppAmpUmpA,
was synthesized by condensation of a partially protected triribonucleotide 5′-terminal diphosphate
species, ppAMMTrmpUmpA, with Im-pm32,2,7G followed by treatment with 80% acetic acid. The
structure of m32,2,7G5′pppAmpUmpA was characterized by 1H and 31P NMR spectroscopy as well as
enzymatic assay using snake venom phosphodiesterase, calf intestinal phosphatase, and nuclease
P1.
In tr od u ction
transport and splicing of RNAs.6 Because of these exciting
results, much attention has been paid to the structure-
function relationships of U1 RNA having a TMG cap.7
Therefore, chemical approaches to the synthesis of such
unique terminal sequences should be developed to clarify
the biochemical meaning of the hypermethylated struc-
ture in more detail. In connection with this study, Hata
and his co-workers have reported extensive studies of the
chemical and enzymatic synthesis of MMG-capped oligo-
ribonucleotides.8 The minimum-sized TMG caps
m32,2,7G5′pppG and m32,2,7G5′pppA were first synthesized
by Darzynkiewicz et al.5a Later, Iwase et al. reported
these TMG caps in a different way.9 Quite recently,
Stepinski et al. have reported the synthesis of DMG- and
TMG-caps as well as MMG cap analogs.10 Lo¨nnberg
extensively studied the fluorescence and absorption
spectroscopic properties of these caps.11 However, these
previous syntheses involved a tedious route to a key
U1 RNA is a small nuclear RNA which was found in
the nuclei of eukaryotic cells1 and plays an important role
in splicing of pre-mRNAs.2 One unique feature of U1
RNA is the cap structure containing 2,2,7-trimethyl-
guanosine (TMG) at its 5′-end. Mattaj3 and Lu¨hrmann4
have recently reported the interesting finding that TMG-
capped RNAs can transport from the cytoplasm to the
nucleus but 7-monomethylguanosine (MMG)-capped RNAs
cannot. Their studies revealed that the nucleomembrane
transport of capped RNAs is strictly regulated by the
number of methyl groups on the guanosine base of the
5′-terminal cap structure. TMG-capped RNAs have also
proved to have potential translational activity compared
with MMG-capped RNAs.5 The 5′-terminal hypermethyl-
ated structure of U1 RNA is thought to be essential for
X Abstract published in Advance ACS Abstracts, J une 1, 1996.
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S0022-3263(95)02263-8 CCC: $12.00 © 1996 American Chemical Society

Synthesis of a 5′-Terminal TMG-Capped Triribonucleotide
J . Org. Chem., Vol. 61, No. 13, 1996 4413
regioisomer using ion exchange column chromatography
followed by careful recrystallization. In fact, several
indirect methods suitable for the large-scale synthesis
of 2′-O-methylated adenosine derivatives have recently
been employed although they required more reaction
steps.15
If the selective 2′-O-methylation could be achieved by
using more readily available 3′,5′,N-protected ribonucleo-
sides, a bothersome process to separate the 2′- and 3′-
O-methylated regioisomers could be avoided. To our
knowledge, however, all attempts to obtain 2′-O-methyl
derivatives from 3′,5′,N-protected ribonucleosides have
failed. For example, the reaction of a typical N-benzoyl-
ated adenosine derivative 116 with methyl iodide in the
presence of silver oxide in benzene gave predominantly
the 2′,N1-dimethylated product 2 over the desired prod-
uct.
F igu r e 1. The 5′-terminal structure of U1 RNA from Hela
In this study we found that, when the MMTr group
was used as the 6-N protecting group,17 the selective 2′-
O-methylation was successfully carried out without the
N1-methylation on the masked adenine moiety: Reaction
of 6-N-(monomethoxytrityl)-3′,5′-O-(1,1,3,3-tetraisopro-
pyldisiloxane-1,3-diyl)adenosine18 (3) with methyl iodide
as the solvent in the presence of silver oxide19 under
reflux for 4 h afforded selectively the 2′-O-methylated
product 4 in 83% yield. These conditions are essential
for the high yield synthesis of 4, since the yield of 4 was
reduced drastically to 27% due to the overmethylation
on the base residue and the competitive decomposition
of the methylated products when the reaction was
prolonged for 15 h.
Two appropriately protected 3′-phosphoramidite units
7 and 11 of Am and Um were synthesized as follows:
Removal of the silyl group from 4 by the action of KF/
Et4NBr/H2O20 in acetonitrile gave quantitatively the 3′,5′-
diol 5. The tritylation of 5 with dimethoxytrityl chloride
gave compound 6 in 91% yield. The phosphitylation of
6 with chloro(2-cyanoethoxy)(diisopropylamino)phosphine21
gave the amidite unit 7 in 85% yield. In a similar
manner, the N3-benzoyl-2′-O-methyluridine 3′-phosphor-
amidite 11 was synthesized in 79% yield from the 5′-O-
dimethoxytrityl ether derivative 10, which was prepared
via compound 9 from compound 8 as described previ-
ously.19,22
cells.
intermediate, 2-N,2-N-dimethylguanosine,12 and its con-
version into a TMG-capping reagent via a multi-step
reaction. If m32,2,7G5′pppAmpUmpA, the smallest accep-
tor in RNA ligation, could be chemically prepared, TMG-
capped U1 RNA fragments with appropriate sequences
could be enzymatically synthesized by ligation of the
TMG-capped trimer with 3′-downstream RNA fragments
(pCpψpψ.....) that can be easily synthesized by an RNA
synthesizer. It should be noted that the combination of
the purine nucleoside A at the 3′-end of m32,2,7G5′ppp-
AmpUmpA and the pyrimidine nucleoside C at the 5′-
end of the 3′-downstream RNA fragments is the best one
in RNA ligation according to the previous results reported
by Uhlenbeck and other groups.13
In this paper, we report a markedly improved method
for the synthesis of 2-N,2-N-dimethylguanosine as well
as a new procedure for the regioselective 2′-O-methyla-
tion of adenosine derivatives and an application of these
procedures to the chemical synthesis of m32,2,7G5′-
pppAmpUmpA.
Resu lts a n d Discu ssion
Syn th esis of a Dip h osp h or yla ted Tr ir ibon u cleo-
tid e Block . For the synthesis of a triribonucleotide
block, appropriately protected 2′-O-methylated adenosine
units have been prepared usually by several-step reac-
tions from 2′-O-methyladenosine, which has been pre-
pared by reaction of adenosine with diazomethane or
methyl iodide.14 These procedures involved the use of a
hazardous reagent and tedious separation from its 3′-
(14) (a) Broom, A. D.; Robins, R. K. J . Am. Chem. Soc. 1965, 87,
1145-1146. (b) Khwaja, T. A.; Robins, R. K. J . Am. Chem. Soc. 1966,
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Biochemistry 1968, 7, 1406-1411. (d) Gin, J . B.; Dekker, C. A.
Biochemistry 1968, 7, 1413-1420. (e) Robins, M. J .; Naik, S. R.
Biochim. Biophys. Acta 1971, 246, 341-343. (f) Robins, M. J .; Naik,
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J .; Lee, A. S. K.; Norris, F. A. Carbohydr. Res. 1975, 41, 304-307. (h)
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15, 2735-2740. (i) Yano, J .; Kan, L. S.; Ts′O, P. O. P. Biochim. Biophys.
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Ishido, Y. Bull. Chem. Soc. J pn. 1982, 55, 2949-2955.
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Stolarrski, R.; Wieczorek, Z.; Cai, A. L.; Rhoads, R. E. Nucleosides
Nucleotides 1995, 14, 717-721. (b) Chlebicka, L.; Wieczovek, Z.;
Stolarski, R.; Stepinski, J .; Darezynkiewicz, E.; Sugar, D. Nucleosides
Nucleotides 1995, 14, 771-775.
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4414 J . Org. Chem., Vol. 61, No. 13, 1996
Sekine et al.
Sch em e 1
Condensation of 11 with the 3′-terminal adenosine unit
12 in acetonitrile in the presence of 1H-tetrazole gave
the fully protected dimer 13 in 86% yield. Removal of
the DMTr group from 13 was carried out by the use of
1% trifluoroacetic acid in CH2Cl2 to give the 5′-hydroxyl
compound 14 in 97% yield. This product was further
condensed with 2.0 equiv of 7 in a similar manner to give
the fully protected trimer block 15 in 86% yield. The
selective deprotection (84%) of 15 with 1 M ZnBr2 in
CH2Cl2-iPrOH (85:15, v/v)18,20,23 followed by phosphor-
ylation of the resulting 5′-hydroxyl product 16 with
cyclohexylammonium S,S-diphenyl phosphorodithioate24
in the presence of isodurenedisulfonyl dichloride (DDS)25
and 1H-tetrazole afforded the 5′-phosphorylated species
17 in 84% yield.
Syn th esis of 2-N,2-N-Dim eth ylgu a n osin e (DMG).
For the synthesis of a TMG-capping reagent 32, we have
explored a convenient route to 2-N,2-N-dimethylgua-
nosine (28: DMG) from guanosine by only a three-step
reaction. Borch and Hassid first reported a convenient
method for conversion of primary amines to N,N-di-
methylamines by reductive methylation.27a Later, other
groups successfully applied this method to the synthesis
of other amino compounds.27b,c Therefore, we have
studied in detail this procedure for the synthesis of DMG.
Consequently, we found that reaction of the easily
obtained 2′,3′,5′-tri-O-acetylguanosine (21)28 with
paraformaldehyde in the presence of sodium cyanoboro-
hydride in acetic acid at 40 °C for 4 h gave exclusively
the N,N-dimethylated product 23 in 95% yield. Five
stepwise additions of 3 equiv each of sodium cyanoboro-
hydride and paraformaldehyde was required for optimi-
zation of the yield of 23. DMG was prepared by hydrol-
ysis of 23. This simple method for the synthesis of DMG
is a really remarkable improvement, since the previous
methods developed by a number of laboratories12 required
a series of reactions involving more than six steps. Quite
recently, Eritja and co-workers12g reported an improved
method for the synthesis of DMG using (4-nitrophenyl)-
ethyl as the O6 protecting group of guanosine but this
approach still required a six-step reaction. Therefore, the
present reductive methylation provided the hitherto
shortest route to DMG. The results of the 2-N,2-N-
dimethylation of 21 and 2′,3′,5′-O-tris(tert-butyldimeth-
ylsilyl)guanosine (24) under various conditions are sum-
marized in Table 1.
Treatment of 17 with pyridinium phosphinate26 in
pyridine gave quantitatively the partially hydrolyzed
product 18. This compound was easily extracted with
CH2Cl2 with the help of the liphophilic MMTr group and
allowed to react with monotributylammonium phosphate
in the presence of iodine. After removal of excess iodine,
the resulting 5′-terminal diphosphate derivative 19 was
in situ treated with concentrated ammonia-pyridine to
give the 6-N-monomethoxytritylated trimer block,
ppAmMMTrpUmpA (20), in 73% yield. This compound was
easily monitored by TLC analysis because of the presence
of the MMTr group. The purity of 20 was more than 95%.
(23) Ito, H.; Ike, Y.; Ikuta, S.; Itakura, K. Nucleic Acids Res. 1982,
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(28) Bredereck, H. Chem. Ber. 1947, 80, 401.

Synthesis of a 5′-Terminal TMG-Capped Triribonucleotide
Sch em e 2
J . Org. Chem., Vol. 61, No. 13, 1996 4415
Sch em e 3
Ta ble 1. Meth yla tion of O-P r otected Gu a n osin e Der iva tives 21 a n d 24
monomethylated
dimethylated
compd
(CH2O)n, equiv
NaBH3CN,equiv
temp, °C
time, h
product (yield, %)
product (yield, %)
21
21
21
21
21
24
24
10
10
10
3 × 2
3 × 5
10
5 × 5
5
5
5
20
40
60
40
40
40
40
50
8
7
12
8
20
10
22 (52)
23 (52)
23 (54)
23 (63)
23 (36)
23 (95)
26 (9)
3 × 2
3 × 5
10
5 × 5
22 (26)
26 (65)
The acetyl group was superior to the tert-butyldimeth-
ylsilyl group which would cause steric hindrance around
the amino group of 24. The use of similar conditions
(ZnCl2, NaCNBH3)27c for the reductive methylation of 21
resulted in a poorer yield of 23.
in competitive formation of other byproducts and did not
allow a large-scale synthesis of MMG.29 On the basis of
these criteria, our new route to MMG via the three-step
reaction is practical and superior to the previous photo-
mediated method.
When a lesser amount of sodium cyanoborohydride was
employed, a mixture of a monomethylated product 22 and
23 was obtained as shown in Table 1. The former could
be easily isolated. As far as the synthesis of 22 is
concerned, 5 equiv of sodium cyanoborohydride should
be used. Compound 22 was quantitatively converted to
MMG by deacetylation. Although the photoirradiated
one-step synthesis of MMG from guanosine by use of tert-
butyl peracetate was reported, this procedure resulted
Syn th esis of a TMG-Ca p p ed Tr ir ibon u cleotid e,
m 32,2,7G5′p p p Am p Um p A. In our previous paper, we
prepared a TMG-capping reagent from DMG via a five-
step reaction (2′,3′-O-methoxylmethylation, 5′-bis(phen-
ylthio)phosphorylation, demethoxymethylation, dephen-
(29) (a) Bridson, P. K.; Markiewicz, W.; Reese, C. B.; Synthesis 1980,
1025 (b) Kemal, A.; Reese, C. B. Synthesis 1980, 1025-1028. (c) Zady,
M. F.; Wong, J . L. J . Org. Chem. 1980, 45, 2373-2377. (d) Boryski, J .;
Ueda, T. Nucleosides Nucleotides 1985, 4, 595- 606.

4416 J . Org. Chem., Vol. 61, No. 13, 1996
Sekine et al.
Sch em e 4
ylthiolation, and N-methylation).8 Since this procedure
was inconvenient, we have explored a more straight-
forward route to a TMG-capping reagent 32 as follows:
Reaction of DMG with an excess amount of methyl iodide
in DMF at 40 °C for 4 h followed by the successive
treatment of the 7-methylated DMG derivative 29 with
phosphorus oxychloride in trimethyl phosphate30 gave the
5′-phosphorylated product (31: pm32,2,7G) in 65% yield.
Reaction of 31 with imidazole in the presence of tri-
phenylphosphine and 2,2′-dipyridyl disulfide31 in DMF
gave ImpTMG (32) as sodium salt in 73% yield (method
A). The TMG-capping reagent 32 was also prepared as
an inner salt in a reverse way by phosphorylation of DMG
followed by methylation of the product 30 with methyl
iodide (method B). Since this inner salt was soluble in
DMF, it was used in situ without salt exchange for the
triphosphate bond formation.
Sch em e 5
First, condensation of the pyridinium salt of 20 with
the inner salt 32, in situ prepared by method B, in DMF
afforded m32,2,7G5′pppAMMTrmpUmpA, which was easily
detected with the help of the MMTr group. The succes-
sive treatment of m32,2,7G5′pppAMMTrmpUmpA with 80%
acetic acid gave m32,2,7G5′pppAmpUmpA in an overall
yield of 40%. When the sodium salt of 32 obtained as a
white powder by method A was passed through a column
of Dowex 50 W X8 (pyridinium form), the resulting inner
salt was hardly soluble in DMF. It is not clear why the
different solubility was observed in two inner salt ma-
terials obtained via method A and directly from method
B. The capping reaction using the above insoluble inner
salt resulted in a considerably reduced yield of m32,2,7G5′-
pppAmpUmpA. It should be noted that the inner salt
32 in situ generated by method B was sufficiently soluble
in DMF so that the capping reaction could be carried out
in an almost homogeneous solution giving a reasonable
yield of m32,2,7G5′pppAmpUmpA.
mixture of 20 and the sodium salt 32 accelerated the
capping reaction to give m32,2,7G5′pppAmpUmpA in 32%
yield from 20 after acid treatment.
The isolation of m32,2,7G5′pppAmpUmpA was performed
by paper electrophoresis on a relatively large scale
followed by purification using reverse phase HPLC. The
paper electrophoresis using Whatman 3MM papers made
it possible to separate ca. 100 A260 unit of the TMG-
capped trimer in one experiment. The electrophoretic
chromatogram of the deprotected mixture derived from
the condensation of 20 with 32 is shown in Figure 2. This
electrophoresis was quite effective for separation of the
desired TMG-capped tetramer from the unreacted cap-
ping reagent and other byproducts, but the material
separated by paper electrophoresis contained a consider-
able amount of unreacted diphosphate 20, as evidenced
Sawai and co-workers32 have recently reported that the
diphosphate and triphosphate bridge formation could be
done in aqueous media. Therefore, we tried the capping
reaction of 20 in DMF containing some amount of water
which enabled us to use the sodium salt of 32 without
salt exchange. Consequently, addition of water to the
(30) Yoshikawa, M.; Kato, T.; Takenishi, T. Bull. Chem. Soc. J pn.
1969, 42, 3505-3508.
(31) (a) Mukaiyama, T.; Hashimoto, M. J . Am. Chem. Soc. 1972,
94, 8528-8532.
(32) Shimazu, M.; Shinozuka, K.; Sawai, H. Tetrahedron Lett. 1990,
31, 235-238.

Synthesis of a 5′-Terminal TMG-Capped Triribonucleotide
J . Org. Chem., Vol. 61, No. 13, 1996 4417
1:1 mixture of m32,2,7G and Am as evidenced by Figure
5D. All digestion products containing m32,2,7G were
clearly detected by a fluorescence detector at ex 260 nm,
em 400 nm as depicted in panels A2, B2, C2, and D2 of
Figure 5.
Con clu sion s
The present method provided an efficient route to the
5′-terminal U1 RNA tetramer fragment, which would be
used as a powerful tool for elucidation of the mechanism
of mRNA splicing as well as transportation of RNAs since
the TMG capped structure can be used as a minimum
substrate capable of the 3′-selective ligation with pCp and
other labeled pCpX derivatives. Our preliminary result
showed that the synthetic 5′-terminal oligoribonucleotide,
m32,2,7G5′pppAmpUmpA, could serve as a good substrate
in the 3′-end 32P labeling with 32pCp in the presence of
RNA ligase, as expected. Further application of
m32,2,7G5′pppAmpUmpA to the study of interaction be-
tween snRNPs-U1RNA is now in progress.
F igu r e 2. Paper electrophoresis of the mixture obtained by
the reaction of the pyridinium salt of 20 with 32 in situ
prepared by method B.
Exp er im en ta l Section
Melting points are uncorrected. 1H and 13C NMR spectra
were measured at 270 and 67.8 MHz, respectively, with TMS
as the internal standard. 31P NMR spectra were recoreded at
109.25 MHz in D2O with 80% H3PO4 as the external reference.
In the case of 20 and 34, the lyophilized materials were
dissolved in D2O for these NMR analyses. Paper chromatog-
raphy was performed by use of a descending technique with
Whatman 3MM papers and Toyo Roshi 51 papers using the
following solvent system: 2-propanol-concd aqueous am-
monia-water, 7:1:2, v/v/v. Column chromatography was
performed with silica gel C-200 purchased from Wako Co.,
Ltd., and a minipump for a goldfish bowl was conveniently
used to attain sufficient pressure for rapid chromatographic
separation. Reverse-phase column chromatography was per-
formed by the use of µBondapak C-18 silica gel (Prep S-500,
Waters). TLC was performed on precoated TLC plates of silica
gel 60 F-254 (Merck). Reverse-phase HPLC was performed
on a Waters Model A25 using a µBondasphere 5 µm C18-100
Å, 3.9 mm × 15 cm (Waters) with a linear gradient starting
from 0.1 M NH4OAc, pH 7.0, and applying CH3CN at a flow
rate of 1.0 mL/min for 30 min. Ribonucleosides were pur-
chased from Yamasa Co., Ltd. Pyridine was distilled two times
from p-toluenesulfonyl chloride and from calcium hydride and
then stored over molecular sieves 4A. Elemental analyses
were performed by the Microanalytical Laboratory, Tokyo
Institute of Technology, at Nagatsuta.
F igu r e 3. The HPLC profile of the material separated by
paper electrophoresis as shown in Figure 2. The main peak is
m32,2,7G5′pppAmpUmpA.
by reverse phase HPLC (Figure 3). Finally, m32,2,7G5′-
pppAmpUmpA was purified by reverse phase HPLC.
The structure of m32,2,7G5′pppAmpUmpA thus obtained
1
was confirmed by H NMR, which exhibited four sharp
resonance signals at the aromatic region and four sets
of 1′-H resonance doublet signals around 6 ppm, as shown
in Figure 4A. The 31P NMR spectrum of m32,2,7G5′-
pppAmpUmpA (Figure 4B) clearly shows three types of
phosphorus resonance signals. In the high field of -24.66
ppm, a typical resonance of the â-phosphate of the
triphosphate bridge appeared, while the R- and γ-phos-
phate resonance signals were seen at -13.35 and -13.18
ppm, respectively. The internucleotidic phosphate groups
showed two peaks at -3.12 and -2.96 ppm.
Characterization of the final product was also per-
formed by enzyme analysis using venom phosphodi-
esterase and calf intestinal phosphatase. The former is
known to digest RNA form the 3′-side as an exonuclease
to give 5′-nucleotides. The latter is an enzyme used for
dephosphophoylation of nucleotides. The successive treat-
ments of m32,2,7G5′pppAmpUmpA with two enzymes gave
a mixture of TMG, Am, Um, and A in a ratio of nearly
1:1:1:1 as shown in panels B1 and B2 of Figure 5B.
Digestion of the synthetic TMG-capped trimer with
nuclease P1, which has 3′-exonuclease and endonuclease
acitivities to RNA, gave an equimolar mixture of
m32,2,7G5′pppAm, pUm and pA as shown in panels C1 and
C2 of Figure 5C. The TMG cap m32,2,7GpppAm was
further digested with snake venom phosphodiesterase
followed by calf intestinal phosphatase to give a nearly
2′,3′,5′-Tr i-O-a cetyl-2-N,2-N-d im eth ylgu a n osin e (23).12f
To a solution of compound 21 (219 mg, 0.5 mmol) in acetic
acid (5 mL) were added paraformaldehyde (45 mg, 1.5 mmol)
and sodium cyanoborohydride (314 mg, 1.5 mmol). The
mixture was vigorously stirred at 40 °C. At 8-h intervals (four
times), paraformaldehyde (45 mg, 1.5 mmol), and sodium
cyanoborohydoride (314 mg, 1.5 mmol) were stepwise added
to the mixture. The solvent was removed under reduced
pressure, and the residue was dissolved in CH2Cl2-pyridine
(1:1, v/v). The solution was washed three times with saturated
NaHCO3. Every aqueous layer was back-extracted with
CH2Cl2-pyridine (2:1, v/v). After extraction was performed,
the organic phase was collected, dried over Na2SO4, filtered,
and evaporated under reduced pressure. The residue was
chromatographed on a column of silica gel (10 g) eluted with
CH2Cl2-MeOH (98.5:1.5-98.2:1.8, v/v) to give 23 (208 mg,
95%): 1H NMR (270 MHz, CDCl3) δ 2.08, 2.11, and 2.12 (9H,
s), 3.24 (6H, s), 4.25 (1H, dd, J ) 4.6 Hz, J ) 11.2 Hz), 4.37
(1H, m), 4.44 (1H, dd, J ) 3.3 Hz, J ) 11.2 Hz), 5.69 (1H, t, J
) 5.6 Hz), 5.90 (1H, d, J ) 4.1 Hz), 6.07 (1H, dd, J ) 4.1 Hz,
J ) 5.6 Hz), 7.59 (1H, s), 10.34 (1H, br).
2′,3′,5′-Tr i-O-a cetyl-2-N-m eth ylgu a n osin e (22).12f To a
solution of compound 21 (4.09 mg, 10 mmol) in acetic acid (100

4418 J . Org. Chem., Vol. 61, No. 13, 1996
Sekine et al.
F igu r e 4. Panel A: 1H NMR spectrum of m32,2,7G5′pppAmpUmpA in D2O. Panel B: 31P NMR spectrum of m32,2,7G5′pppAmpUmpA
in D2O.
F igu r e 5. Panel A: The HPLC profiles of purified m32,2,7G5′pppAmpUmpA. Panel B: The HPLC profiles of the mixture of m32,2,7G
(TMG), Am, Um, and A which was obtained after treatment of m32,2,7G5′pppAmpUmpA with SVPD and AP. Panel C: The HPLC
profiles of the mixture of m 2,2,7G5′pppAm, pUm and pA which was obtained after treatment of m32,2,7G5′pppAmpUmpA with nuclease
3
P1. Panel D: The HPLC profiles of the mixture of TMG and Am which was obtained after treatment of m32,2,7G5′pppAm with
SVPD and AP.
mL) were added paraformaldehyde (310 mg, 30 mmol) and
sodium cyanoborohydride (1.13 g, 30 mmol). The mixture was
vigorously stirred at 40 °C. After 8 h paraformaldehyde (310
mg, 10 mmol) and sodium cyanoborohydoride (1.13 g, 10 mmol)
were added to the mixture. The solvent was removed under
reduced pressure, and the residue was dissolved in CH2Cl2-
pyridine (1:1, v/v). The solution was washed three times with
saturated NaHCO3. Every aqueous layer was back-extracted
with CH2Cl2-pyridine (2:1, v/v). After extraction was per-
formed, the organic phase was collected, dried over Na2SO4,
filtered, and evaporated under reduced pressure. The residue
was chromatographed on a column of silica gel (50 g) eluted
with CH2Cl2-MeOH (98.5:1.5-98.2:1.8, v/v) to give 23 (1.58
g, 36%) and CH2Cl2-MeOH (97:3, v/v) to give 22 (1.10 g,
26%): 1H NMR (270 MHz, CDCl3) δ 2.08, 2.120, and 2.123 (9H,
s), 3.03 (3H, d, J ) 4.6 Hz), 4.29 (1H, dd, J ) 5.3 Hz, J )
11.7 Hz), 5.84 (1H, m), 5.93 (1H, d, J ) 4.0 Hz), 6.05 (1H, dd,
J ) 4.0 Hz, J ) 5.6 Hz), 7.60 (1H, s), 12.00 (1H, br).
2′,3′,5′-O-Tr is(ter t-bu tyld im eth ylsilyl)-2-N,2-N-d im eth -
ylgu a n osin e (26). To a solution of compound 24 (313 mg,
0.5 mmol) in acetic acid (5 mL) were added paraformaldehyde
(45 mg, 1.5 mmol) and sodium cyanoborohydride (314 mg, 1.5
mmol). The mixture was vigorously stirred at 40 °C. At 8-h
intervals (four times), paraformaldehyde (45 mg, 1.5 mmol)
and sodium cyanoborohydoride (314 mg, 1.5 mmol) were
stepwise added to the mixture. The solvent was removed
under reduced pressure, and the residue was dissolved in
CH2Cl2. The solution was washed three times with saturated
NaHCO3. Every aqueous layer was back-extracted with
CH2Cl2-pyridine (2:1, v/v). After extraction was performed,
the organic phase was collected, dried over Na2SO4, filtered,
and evaporated under reduced pressure. The residue was

Synthesis of a 5′-Terminal TMG-Capped Triribonucleotide
J . Org. Chem., Vol. 61, No. 13, 1996 4419
chromatographed on a column of silica gel (10 g) eluted with
CH2Cl2-MeOH (98.5:1.5-98.2:1.8, v/v) to give 26 (214 mg,
65%): 1H NMR (270 MHz, CDCl3) δ -0.17, -0.04, 0.09, 0.10.
0.11 (18H, s), 0.81, 0.92, 0.93 (27H, s), 3.24 (6H, s), 3.78 (1H,
dd, J ) 2.6 Hz, J ) 11.4 Hz), 3.92 (1H, dd, J ) 4.3 Hz, J )
11.4 Hz), 4.06 (1H, m), 4.28 (1H, t, J ) 4.0 Hz), 4.49 (1H, t, J ′
) 4.6 Hz), 5.88 (1H, d, J ) 5.0 Hz), 7.82 (1H, s), 11.30 (1H,
br); 13C NMR (67.8 MHz, CDCl3) δ -5.50, -5.46, -4.99, -4.76,
-4.53, 17.77, 17.99, 18.40, 25.59, 25.73, 25.97, 26.13, 38.26,
62.54, 71.83, 75.55, 84.71, 87.35, 116.39, 136.46, 151.55,
152.85, 159.21. Anal. Calcd for C30H59N5O5Si3.1/2H2O: C,
54.34; H, 9.12; N, 10.56. Found: C, 54.46; H, 9.10; N, 10.58.
2-N,2-N-Dim et h ylgu a n osin e (28).12f To a solution of
compound 22 (5.8 g, 13.3 mmol) in pyridine (400 mL) was
added 0.5 M NaOH (400 mL, 0.02 mol). After being stirred
at room temperature for 20 min, the mixture was applied to a
resin of Dowex 50WX8 (pyridinium form, 200 mL) and elution
was performed with pyridine-water (4000 mL, 9:1, v/v). The
eluate was evaporated under reduced pressure. Crystalliza-
tion of the residue from water (300 mL) gave 28 (3.04 g, 73%):
mp 218 °C (lit.12f 242 °C); 1H NMR (270 MHz, DMSO-d6-D2O,
at 70 °C) δ 3.06 (6H, s), 3.53 (1H, dd, J ) 4.6 Hz, J ) 11.6 Hz)
, 3.63 (1H, dd, J ) 4.0 Hz, J ) 11.6 Hz), 3.96 (1H, m), 4.14
(1H, t, J ) 4.6 Hz), 4.51 (1H, t, J ) 5.5 Hz), 5.72 (1H, d, J )
5.5 Hz), 7.84 (1H, s).
at room temperature for 2 h, the mixture was treated with
methanol (320 µL). The solvent was removed under reduced
pressure. The residue was dissolved in dry DMF (5 mL), and
32 was stored as a 0.01 mM solution in dry DMF at 0 °C. 32:
1H NMR (270 MHz, D2O) δ 3.07 (6H, s), 4.04 (3H, s), 4.04-
4.07 (2H, m), 4.19 (1H, m), 4.28 (1H, m), 4.42 (1H, m), 5.95
(1H, d, J ) 4.0 Hz), 6.92, 7.10, 7.77 (3H, s); 31P NMR (109.25
MHz, D2O) δ -7.27.
3′,5′-O-(1,1,3,3-Tetr a isop r op yld isiloxa n e-1,3-d iyl)-2′-O-
m et h yl-6-N-(m on om et h oxyt r it yl)a d en osin e (4). Com-
pound 3 (7.82 g, 10 mmol) was dissolved in methyl iodide (94
mL, 1.5 mol), and silver oxide (11.5 g, 50 mmol) was added.
The mixture was refluxed for 4 h. The resulting precipitate
was filtered and washed with CH2Cl2. The filtrate and the
washing were combined and evaporated under reduced pres-
sure. The residue was chromatographed on a column of silica
gel (400 g) eluted with hexane-EtOAc (5:1, v/v) to give 4 (6.57
g, 83%): 1H NMR (270 MHz, CDCl3) δ 1.06 (14 H, m), 3.66 (3
H, s), 3.78 (3 H, s), 3.94 (1H, dd, J ) 13.4 Hz), 4.01-4.15 (2
H, m), 4.14 (1H, dd, J ) 13.4 Hz), 4.85 (1 H, m), 5.94 (1H, s),
6.79 (2 H, d, J ) 8.6 Hz), 6.91 (1 H, s), 7.23-7.36 (14 H, m),
7.99 (1 H, s), 8.01 (1 H, s); 13C NMR (67.5 MHz, CDCl3) δ 12.49,
12.81, 12.90, 13.37, 14.14, 16.86, 17.00, 17.05, 17.15, 17.24,
17.27, 17.40, 55.15, 59.52, 59.75, 69.71, 70.91, 81.13, 83.40,
88.43, 113.06, 121.42, 126.76, 127.78, 128.86, 130.17, 137.20,
138.49, 145.14, 148.00, 152.24, 153.98, 158.24. Anal. Calcd
for C42H57N5O6Si2: C, 64.34; H, 7.33; N, 8.93. Found: C, 64.21;
H, 7.16; N, 8.45.
2′-O-Meth yl-6-N-(m on om eth oxytr ityl)aden osin e (5). To
a solution of 4 (6.0 g, 7.65 mmol) in acetonitrile-water (77
mL, 99:1, v/v) were added KF (3.6 g, 61 mmol) and Et4NBr
(12.86 g, 61 mmol). The mixture was stirred at room temper-
ature for 6 h. The solvent was removed under reduced
pressure, and the residue was partitioned between CH2Cl2 (200
mL) and water (200 mL) in a separatory funnel A. The
aqueous layer was transferred to another separatory funnel
B and extracted with CH2Cl2.(200 mL), and then the aqueous
layer was discarded. The CH2Cl2 layer in the first separatory
funnel A was washed five times with water (200 mL), which
was transferred to separatory funnel B, extracted with the
same CH2Cl2, and discarded each time. The two CH2Cl2 layers
were combined, dried over Na2SO4, filtered, and evaporated
under reduced pressure. The residue was chromatographed
on a column of silica gel (200 g) eluted with CH2Cl2-MeOH
(100:0-98.5:1.5, v/v) to give 5 (4.0 g, 97%): 1H NMR (270 MHz,
CDCl3) δ 3.36 (3H, s), 3.69 (1H, dd, J ) 12.2 Hz), 3.78 (3H, s),
3.91 (1H, dd, J ) 12.2 Hz), 4.33 (1H, br s), 4.56 (1H, m), 4.71
(1H, dd, J ) 7.6 Hz, J ) 4.6 Hz), 5.83 (1H, d, J ) 7.6 Hz), 6.80
(2H, d, J ) 8.9), 7.06 (1H, s), 7.23-7.36 (14H, m), 7.78 (1H, s),
8.01 (1H, s); 13C NMR (67.5 MHz, CDCl3) δ 55.11, 58.64, 63.16,
70.46, 71.03, 82.46, 88.23, 89.51, 113.08, 122.34, 126.85,
127.82, 128.77, 130.12, 136.84, 140.13, 144.83, 147.21, 151.61,
154.50, 158.27. Anal. Calcd for C31H31N5O5‚3/4H2O: C, 65.64;
H , 5.77; N, 12.35. Found: C, 65.82; H, 5.63; N, 12.00.
2′-O-Met h yl-5′-O-(d im et h oxyt r it yl)-6-N-(m on om et h -
oxytr ityl)a d en osin e (6). Compound 5 (1.107 g, 2.0 mmol)
was rendered anhydrous by repeated coevaporations with dry
pyridine and dissolved in dry pyridine (20 mL). Dimethoxy-
trityl chloride (813 mg, 2.4 mmol) was added, and the mixture
was stirred at room temperature for 12 h. The mixture was
diluted with CH2Cl2, and the CH2Cl2 solution was washed two
times each with saturated NaHCO3 (150 mL) and with water.
The organic phase was collected, dried over Na2SO4, filtered,
and evaporated under reduced pressure. The residue was
chromatographed on a column of silica gel (100 g) eluted with
CH2Cl2-MeOH (100:0-99:1, v/v) to give 6 (1.67 g, 98%): 1H
NMR (270 MHz, CDCl3) δ 3.38 (1H, dd, J ) 4.4 Hz, J ) 11.6
Hz), 3.49 (1H, dd, J ) 3.3 Hz, J ) 11.6 Hz), 3.52 (3 H, s), 3.76
(9 H, s), 4.08 (1 H, m), 4.30-4.35 (2 H, m), 6.01 (1 H, d, J )
3.6 Hz), 6.69 (2 H, d, J ) 8.9), 6.71 (2 H, d, J ) 8.9 Hz), 6.81 (1
H, s), 7.09-7.33 (21 H, m), 7.87 (1 H, s), 7.90 (1 H, s); 13C NMR
(67.5 MHz, CDCl3) δ 21.37, 55.10, 58.71, 62.98, 69.69, 70.89,
83.06, 83.67, 86.33, 86.45, 113.03, 113.08, 121.19, 125.19,
126.74, 126.83, 127.76, 128.07, 128.12, 128.81, 128.93, 129.96,
130.03, 130.12, 135.53, 135.67, 137.11, 138.20, 144.42, 145.10,
148.37, 152.27, 153.98, 158.18, 158.44. Anal Calcd for
2-N,2-N,7-Tr im eth ylgu a n osin e 5′-O-P h osp h a te (31). To
a suspension of 28 (311 mg, 1 mmol) in dry DMF (17 mL) was
added methyl iodine (1.25 mL, 20 mmol). After the mixture
was stirred at 40 °C for 4 h, the solvent was removed under
reduced pressure. Compound 29 was precipitated from ether
(100 mL) and was dissolved in trimethyl phosphate (6 mL).
The resulting solution was stirred at 0 °C for 10 min, and
phosphorus oxychloride (280 µL, 1.3 mmol) was added. After
being stirred at 0 °C for 4 h, the mixture was diluted with
water and washed three times with CH2Cl2. The aqueous
phase was evaporated under reduced pressure. Compound 31
was purified by the following methods: Meth od A. The
residue was applied to a column of Dowex 50WX8 (H+ form,
60 mL) and eluted with water to give 31 (249 mg, 61%).
Meth od B. The residue was chromatographed on Whatman
3MM papers developed with butanol-acetic acid-H2O (5:2:3,
v/v/v). The band of Rf 0.7 was cut and eluted with water. The
eluate was condensed and applied to a column of Dowex1X8
-
(HCO3 form, 60 mL). Elution with water followed by lyo-
philization gave 31 (260 mg, 65%): 1H NMR (270 MHz, D2O)
δ 3.02 (6H, s), 3.92 (3H, s), 3.92-4.08 (2H, m), 4.21 (1H, m),
4.29 (1H, m), 4.53 (1H, m), 5.93 (1H, d, J ) 3.3 Hz), 8.94 (1H,
s); 13C NMR (67.8 MHz, D2O) δ 31.35, 36.91, 38.65, 64.36,
70.04, 75.29, 84.72, 90.51, 107.80, 137.37, 150.45, 154.90,
156.29; 31P NMR (109.25 MHz, D2O) δ 0.65: FAB Calcd for
m/ z 404.0971. Observed for m/ z 404.0964.
5′-O -(I m i d o ly lp h o s p h o )-2-N ,2-N ,7-t r i m e t h y lg u a -
n osin e (32). Meth od A. A mixture of 31 (81 mg, 0.2 mmol)
and imidazole (136 mg, 2 mmol) was rendered anhydrous by
successive coevaporations with dry pyridine, dry toluene, and
dry DMF, and finally suspended in dry DMF (2 mL). To the
mixture was added triethylamine (200 µL), tri-n-octylamine
(100 µL), triphenylphosphine (157 mg, 0.6 mmol), and 2,2′-
dipyridyl disulfide (132 mg, 0.6 mmol), and the mixture was
stirred at room temperature for 2 h. Precipitation of the
mixture from acetone-ether-triethylamine (61 mL, 40:20:1,
v/v/v) gave 32 (65 mg, 73%). Meth od B. Compound 28 (311
mg, 1 mmol) was suspended in trimethyl phosphate (6 mL),
and the mixture was stirred at 0 °C for 10 min. Phosphorus
oxychloride (280 µL, 3 mmol) was added to the resulting
solution. After being stirred at 0 °C for 4 h, the mixture was
diluted with water and washed three times with CH2Cl2. The
aqueous phase was evaporated under reduced pressure. The
residue was chromatographed on Whatman 3MM papers
developed with butanol-acetic acid-H2O (5:2:3, v/v/v) to give
30 (350 mg, 90%). Part of this compound (200 mg, 0.5 mmol)
was suspended in DMF (10 mL), and methyl iodide (625 µL,
10 mmol) was added. After the mixture was stirred at 40 °C
for 16 h, the solvent was removed under reduced pressure.
The residue was dissolved in dry DMF, and 1,1′-carbonyldi-
imidazole (811 mg, 5 mmol) was added. After being stirred

4420 J . Org. Chem., Vol. 61, No. 13, 1996
Sekine et al.
C52H49N5O7; C, 72.97; H, 5.77; N, 8.18. Found: C, 73.53; H,
5.61; N, 7.79.
168.66. Anal. Calcd for C38H36N2O9: C, 68.66; H, 5.46; N,
4.22. Found: C, 68.59; H, 5.67; N, 4.13.
2′-O-Met h yl-5′-O-(d im et h oxyt r it yl)-6-N-(m on om et h -
oxytr ityl)a d en osin e 3′-O-(2-cya n oeth yl N,N-d iisop r op yl-
p h osp h or a m id ite) (7). Compound 6 (428 mg, 0.5 mmol) was
rendered anhydrous by repeated coevaporations with dry
pyridine and then with dry toluene and dissolved in CH2Cl2
(5 mL). To the solution were added ethyldiisopropylamine
(0.357 mL, 1.2 mmol) and (2-cyanoethoxy)(N,N-diisopropyl-
amino)phosphine (0.163 mL, 0.75 mmol). The resulting mix-
ture was stirred under argon atmosphere at room temperature
for 1 h. The mixture was diluted with CH2Cl2, and the CH2Cl2
solution was washed two times each with saturated NaHCO3
(100 mL) and with water. The organic phase was collected,
dried over Na2SO4, filtered, and evaporated under reduced
pressure. The residue was chromatographed on a column of
silica gel (25 g) eluted with hexane-EtOAc-pyridine (70:30:
0.5, v/v/v) to give 7 (449 mg, 85%): 1H NMR (270 MHz, CDCl3)
δ 1.05-1.17 (12 H, d, J ) 6.6 Hz), 2.36 and 2.63 (2 H, t, J )
6.6 and 6.3 Hz), 3.45 and 3.46 (3 H, s), 3.28-3.65 (4 H, m),
3.76 and 3.77 (9 H, s), 3.84-3.94 (2 H, m), 4.31 and 4.36 (1H,
m), 4.55-4.68 (2 H, m), 6.06 (1H, m), 6.77-6.82 (6H, m), 6.92
(1H, bs), 7.13-7.42 (27H, m), 7.72, 7.96, 7.97, 7.98 (2 H, s,
2H, 8H); 13C NMR (67.5 MHz, CDCl3) δ 19.97, 20.07, 20.20,
20.29, 24.46, 24.57, 29.60, 43.00, 43.18, 55.11, 57.81, 58.10,
58.24, 58.71, 58.96, 62.61, 63.09, 70.48, 70.73, 70.87, 81.62,
81.94, 83.49, 86.31, 86.42, 86.51, 113.05, 117.32, 117.63,
121.33, 126.74, 126.81, 127.77, 128.09, 128.18, 128.81, 129.97,
130.05, 130.12, 135.49, 135.60, 137.16, 138.78, 144.35, 145.12,
148.64, 148.73, 152.26, 154.00, 158.20, 158.45; 31P NMR
(109.25 MHz, CDCl3) δ 151.63, 150.84 (40; 60). Anal. Calcd
for C61H66N7O8P; C, 69.36; H, 6.30; N, 9.29. Found: C, 69.21;
H, 6.42; N, 9.04.
N 3-Be n zoyl-2′-O-m e t h yl-5′-O-(d im e t h oxyt r it yl)u r i-
d in e 3′-O-(2-cya n oeth yl N,N-d iisop r op ylp h osp h or a m id -
ite) (11). Compound 10 (2.00 g, 3 mmol) was rendered
anhydrous by coevaporations three times each with dry
pyridine, dry toluene, and dry CH2Cl2, and finally dissolved
in dry CH2Cl2 (30 mL). To the solution were added diisopro-
pylethylamine (2.1 mL, 12 mmol) and (2-cyanoethoxy)(N,N-
diisopropylamino)phosphine (0.98 mL, 4.5 mmol). The mixture
was stirred at room temperature for 1 h and then diluted with
CH2Cl2. The CH2Cl2 solution was washed two times each with
saturated NaHCO3 (150 mL) and water. The organic phase
was collected, dried over Na2SO4, filtered, and evaporated
under reduced pressure. The residue was chromatographed
on a column of silica gel (60 g) eluted with hexane-EtOAc-
pyridine (70:30:1, v/v/v) to give 11 (2.22 g, 85%): 1H NMR (270
MHz, CDCl3) δ 1.21 (12H, m), 2.47 and 2.65 (2H, t, J ) 5.9
and 6.3 Hz), 3.55 and 3.56 (3H, s), 3.81 and 3.82 (6H, s), 3.45-
3.95 (6H, m), 4.00 (1H, m), 4.23 (1H, m), 4.57 and 4.76 (1H,
m), 5.27 and 5.32 (1H, d, J ) 8.3 Hz), 5.92 and 5.97 (1H, d, J
) 2.3 Hz), 6.83-6.90 (4H, m), 7.25-8.63 (14H, m), 8.17 and
8.26 (1H, d, J ) 8.3 Hz); 13C NMR (67.5 MHz, CDCl3) δ 20.20,
20.11, 20.13, 24.19, 24.30, 24.37, 24.46, 42.84, 43.04, 54.90,
57.38, 57.70, 57.86, 58.01, 58.13, 58.40, 60.00, 60.54, 68.93,
69.22, 69.44, 81.85, 82.43, 83.49, 86.63, 86.83, 87.12, 101.49,
101.60, 112.99, 117.50, 117.59, 123.43, 124.99, 126.95, 127.73,
128.01, 128.72, 128.82, 130.01, 130.31, 131.18, 134.61, 134.72,
134.81, 134.93, 135.67, 137.47, 139.69, 143.90,144.13, 148.95,
149.45, 158.49, 161.92, 161.99, 168.73; 31P NMR (109.25 MHz,
CDCl3) δ 151.10, 150.98 (43:57). Anal. Calcd for C47H53N4O10P
.1/4H2O: C, 64.92; H, 6.20; N, 6.45. Found: C, 65.05; H, 6.38;
N, 6.49.
N3-Ben zoyl-2′-O-m eth ylu r id in e (9). To a solution of 8
(1.81 g, 3 mmol) in dry THF (30 mL) were added acetic acid
(0.38 mL, 6.6 mmol) and Bu4NF.H2O (1.73 mg, 6.6 mmol). The
mixture was stirred at room temperature for 30 min. The
solvent was removed under reduced pressure, and the residue
was dissolved in CH2Cl2-pyridine (3:1, v/v). The solution was
washed two times each with saturated NaHCO3 and water.
The organic phase was collected, dried over Na2SO4, filtered,
and evaporated under reduced pressure. The residue was
dissolved in pyridine-water (300 mL, 1:2, v/v) and passed
through a Dowex 50WX8 resin (H+ form, 80 mL). Further
elution was performed with water (600 mL). The eluate was
evaporated under reduced pressure and chromatographed on
a column of silica gel (60 g) eluted with CH2Cl2-MeOH (100:
0-98.2:1.8, v/v) to give 9 (1.02 g, 94%): 1H NMR (270 MHz,
CDCl3) δ 3.58 (3H, s), 3.87-4.10 (4H, m), 4.34 (1H, dd, J )
5.6 Hz, J ) 7.6 Hz), 7.64-7.70 (2H, m), 7.51 (2H, m), 7.67
(1H, m), 7.93-8.00 (3H, m); 13C NMR (67.5 MHz, CDCl3) δ
58.65, 60.58, 68.00, 83.22, 84.55, 88.70, 102.09, 129.22, 130.49,
131.20, 135.31, 140.59, 149.20, 162.16, 168.55. Anal. Calcd
for C17H18N2O7: C, 56.35; H, 5.01; N, 7.73. Found: C, 56.35;
H, 4.57; N, 7.58.
Syn th esis of F u lly P r otected Dir ibon u cleotid e De-
r iva tive 13. A mixture of 12 (205 mg, 0.3 mmol) and 1H-
tetrazole (95 mg, 1.35 mmol) was rendered anhydrous by
coevaporations three times each with dry toluene and dry
pyridine and with dry acetonitrile. The mixture was mixed
with 11 (390 mg, 0.45 mmol) and dissolved in dry acetonitrile
(3 mL). After being stirred under argon atmosphere at room
temperature for 2.5 h, the mixture was treated with a solution
of iodine (343 mg, 1.35 mmol) in pyridine-water (3 mL, 98:2,
v/v). The resulting mixture was stirred at room temperature
for 40 min, and then saturated Na2SO3 was added to reduce
the excess iodine. The mixture was diluted with CH2Cl2, and
the CH2Cl2 solution was washed two times each with saturated
NaHCO3 (100 mL) and with water. The organic phase was
collected, dried over Na2SO4, filtered, and evaporated under
reduced pressure. The residue was chromatographed on a
column of silica gel (20 g) eluted with hexane-EtOAc-pyridine
(45:55:0.5, v/v/v) to give 13 (449 mg, 85%): 31P NMR (109.25
MHz, CDCl3) δ -1.92, -1.99 (61:39). Anal. Calcd for
C79H67N8O19P: C, 64.84; H, 4.62; N, 7.66. Found: C, 65.27;
H, 4.86; N, 6.87.
Syn th esis of 5′-Un p r otected Dir ibon u cleotid e Der iva -
tive 14. To a solution of 13 (283 mg, 0.19 mmol) in CH2Cl2
was added trifluoroacetic acid (95 µL). After being stirred at
room temperature for 20 min, the mixture was diluted with
CH2Cl2 and the CH2Cl2 solution was washed two times with
saturated NaHCO3 (150 mL) and with water. The organic
phase was collected, dried over Na2SO4, filtered, and evapo-
rated under reduced pressure. The residue was chromato-
graphed on a column of silica gel (25 g) eluted with CH2Cl2-
MeOH (100:0-98:2, v/v) to give 14 (214 mg, 97%): 1H NMR
(270 MHz, CDCl3) δ 2.71-2.79 (2H, m), 3.43 and 3.51 (3H, s),
3.76-3.82 (2H, m), 4.19-4.36 (4H, m), 4.58 (2H, m), 4.72 (1H,
m), 5.00-5.02 (1H, m), 5.75-5.85 (1H, m), 5.86-5.89 (1H, m),
6.08-6.12 (1H, m), 6.20 and 6.28 (1H, t, J ) 5.3 Hz), 6.58 (1H,
d, J ) 5.0 Hz), 7.34-8.03 (26H, m), 8.25 and 8.55 (1H, s), 8.67
and 8.69 (1H, s); 13C NMR (67.5 MHz, CDCl3) δ 19.39, 19.50,
19.60, 29.60, 53.39, 58.60, 59.73, 62.64, 62.72, 70.84, 71.02,
73.93, 74.25, 81.38, 81.51, 81.80, 83.58, 86.58, 86.74, 88.18,
102.37, 102.43, 116.48, 127.35, 128.09, 128.30, 128.54, 128.57,
128.79, 129.13, 129.38, 129.72, 129.76, 129.80, 130.42, 130.48,
131.21, 131.25, 133.19, 133.78, 133.93, 135.11, 140.45, 141.20,
143.22, 143.56, 149.31, 151.95, 152.56, 152.69, 152.83, 161.98,
N 3-Be n zoyl-2′-O-m e t h yl-5′-O-(d im e t h oxyt r it yl)u r i-
d in e (10). Compound 9 (0.63 g, 1.7 mmol) was rendered
anhydrous by repeated coevaporations with dry pyridine and
finally dissolved in dry pyridine (17 mL). To the solution was
added dimethoxytrityl chloride (0.65 g, 1.9 mmol). The
mixture was stirred at room temperature for 12 h. The
mixture was diluted with CH2Cl2, and the CH2Cl2 solution was
washed two times each with saturated NaHCO3 (150 mL) and
with water. The organic phase was collected, dried over
Na2SO4, filtered, and evaporated under reduced pressure. The
residue was chromatographed on a column of silica gel (50 g)
eluted with hexane-EtOAc-pyridine (65:35:1, v/v/v) to give
10 (1.12 g, 99%): 1H NMR (270 MHz, CDCl3) δ 3.56-3.63 (2H,
m), 3.61 (3H, s), 3.81 (6H, s), 3.85 (1H, m), 4.02 (1H, d, J )
8.3 Hz), 4.53 (1 H, br), 5.36 (1H, d, J ) 8.3 Hz), 5.94 (1H, s),
6.88 (4H, d, J ) 8.6 Hz), 7.14-7.44 (9H, m), 7.50 (2H, m), 7.66
(2H, m), 7.95 (2H, d, J ) 7.3 Hz), 8.22 (1H, d, J ) 8.3 Hz); 13
C
NMR (67.5 MHz, CDCl3) δ 55.22, 58.64, 60.81, 68.11, 83.24,
83.90, 87.12, 87.30, 101.94, 113.30, 125.25, 127.17, 128.01,
128.12, 128.99, 129.15, 130.08, 130.15, 130.49, 131.30, 134.95,
135.17, 135.22, 137.81, 139.73, 144.24, 149.09, 158.72, 161.99,

Synthesis of a 5′-Terminal TMG-Capped Triribonucleotide
J . Org. Chem., Vol. 61, No. 13, 1996 4421
165.05, 168.50, 168.59, 172.31, 172.36; 31P NMR (109.25 MHz,
for C104H91N14O27P3S2: C, 58.75; H, 4.31; N, 9.23; S, 3.02.
Found: C, 58.96; H, 4.56; N, 8.96; S, 3.83.
CDCl3)
δ
-1.74, -1.80 (50:44). Anal. Calcd for
C58H49N8O17P: C, 60.00; H, 4.25; N, 9.65. Found: C, 60.05;
H, 4.58; N, 9.30.
Syn th esis of 5′-P yr op h osp h or yla ted Tr ir ibon u cleotid e
Der iva tive 20. Compound 17 (63 mg, 0.03 mmol) was
rendered anhydrous by coevaporations three times with dry
pyridine and dissolved in a solution of 5 M pyridinium
phosphinate (0.30 mL)-triethylamine (0.105 mL, 0.75 mmol).
After being stirred at room temperature for 1 h, the mixture
was diluted with CH2Cl2 (25 mL). The CH2Cl2 solution was
washed 15 times with 0.5 M triethylammonium bicarbonate
(25 mL). Every aqueous layer was back-extracted with
CH2Cl2-pyridine (25 mL, 4:1, v/v) put in another separatory
funnel. After extraction was performed, the two CH2Cl2 layers
were combined, dried over Na2SO4, filtered, and evaporated
under reduced pressure. The residue was coevaporated with
six times with dry pyridine and then dissolved in a solution
of tributylammonium phosphate (1.2 mmol) in dry pyridine
(2 mL). The resulting solution was stirred at room tempera-
ture for 5 min, and then iodine (152 mg, 0.6 mmol) was added.
After being stirred for 2 h, the mixture was diluted with
pyridine-water (50 mL, 1:3, v/v) and washed six times with
ether (50 mL). Each ethereal extract was back-extracted with
pyridine-water (50 mL, 1:2, v/v) in another separatory funnel.
The aqueous laylers were combined, dried over Na2SO4,
filtered, and evaporated under reduced pressure. The residue
was treated with 25% ammonia-pyridine (50 mL, 9:1, v/v) at
room temperature for 60 h. The solution was diluted with
water, washed three times with ether, and evaporated under
reduced pressure. The residue was chromatographed on
Whatman 3MM papers developed with concd ammonia-2-
propanol-H2O, 7:1:2, v/v/v). The band of Rf 0.2 was cut and
eluted with water. Lyophilization of the eluate gave 20 (940
A257.5, 73%): 31P NMR (109.25 MHz, D2O) δ -9.49, -4.65 (5′-
pyrophosphate), -0.20 (internucleotidic phosphate); Rf 0.2. UV
(H2O) λmax 257.5 nm.
Syn th esis of th e 5′-ter m in a l r egion of U1RNA. Com-
pound 34 (0.022 mmol) obtained in the above experiment was
dissolved in water, and the solution was applied to a column
of Dowex 50WX8 (pyridinium form, 4 mL), and elution was
performed with pyridine-water (50 mL, 1:1, v/v). The eluate
was evaporated under reduced pressure. The residue was
lyophilized to give a white powder (pyridinium salt of 20). This
compound was dissolved in a 0.01 mM solution of TMG capping
unit 32 (2.2 mL, 0.022 mmol, the one obtained by method B)
in dry DMF . The mixture was stirred at room temperature
for 3 days, and 80% acetic acid (2 mL) was added in this
solution. After being stirred at room temperature for 2 h, the
mixture was diluted with water and washed three times with
ether (20 mL). The aqueous phase was evaporated under
reduced pressure. The residue was purified with paper
electrophoresis (0.2 M ammonium acetate buffer, 600 V, 2 h)
on four sheets of Whatman 3MM papers (14 cm × 46 cm). The
band of Mob. 0.7-0.8 (relative to ppG) was cut and eluted with
water. The eluate was lyophilized and dissolved in water.
Syn th esis of F u lly P r otected Tr ir ibon u cleotid e De-
r iva tive 15. A mixture of 14 (407 mg, 0.35 mmol) and 1H-
tetrazole (245 mg, 3.5 mmol) was rendered anhydrous by
coevaporations three times each with dry toluene and dry
pyridine and with dry acetonitrile. The mixture was mixed
with 7 (741 mg, 0.7 mmol) and dissolved in dry acetonitrile
(3.5 mL). After being stirred under argon atmosphere at room
temperature for 1 h, the mixture was treated with a solution
of iodine (533 mg, 2.1 mmol) in pyridine-water (3.5 mL, 98:2,
v/v). The resulting mixture was stirred at room temperature
for 30 min, and then saturated Na2SO3 was added to reduce
the excess iodine. The mixture was diluted with CH2Cl2, and
the CH2Cl2 solution was washed two times each with saturated
NaHCO3 (100 mL) and water. The organic phase was col-
lected, dried over Na2SO4, filtered, and evaporated under
reduced pressure. The residue was chromatographed on a
column of silica gel (38 g) eluted with hexane-EtOAc-pyridine
(10:90:0.5, v/v/v) to give 15 (639 mg, 86%): 31P NMR (109.25
MHz, CDCl3) δ -1.39, -1.47, -1.56, -1.77, -1.97, -2.08,
-2.27. Anal. Calcd for C113H100N14O26P2: C, 63.65; H, 4.73;
N, 9.22. Found: C, 63.36; H, 4.63; N, 8.99.
Syn th esis of 5′-Un p r otected Tr ir ibon u cleotid e Der iva -
tive 16. Compound 15 (640 mg, 0.3 mmol) was dissolved in a
0.5 M solution of ZnBr2 in CH2Cl2 -2-propanol (12 mL, 85:15,
v/v) at -5 °C. After being stirred at -5 °C for 2 h, the mixture
was diluted with CH2Cl2 and the CH2Cl2 solution was washed
three times with water. The organic phase was collected, dried
over Na2SO4, filtered, and evaporated under reduced pressure.
The residue was chromatographed on a column of silica gel
(28 g) eluted with CH2Cl2-MeOH (100:0-98:2, v/v) to give 16
(463 mg, 84%): 1H NMR (270 MHz, CDCl3) δ 2.74 (4Η, m),
3.32-3.51 (6H, m), 3.77 (3H, m), 4.20-4.43 (10H, m), 4.58 (2H,
m), 4.72 (1H, m), 4.78 (1H, m), 5.07 (1H, m), 5.29 (1H, m),
5.81-5.95 (3H, m), 6.15 (1H, m), 6.28 (1H, m), 6.56 (1H, m),
6.78-7.93 (28H, m), 8.48-8.69 (2H, m); 31P NMR (109.25 MHz,
CDCl3) δ -1.46, -1.56, -1.63, -1.85, -2.01. Anal. Calcd for
C92H82N14O24P2‚2H2O: C, 61.05; H, 4.52; N, 10.72. Found: C,
60.54; H, 5.46; N, 9.95.
Syn th esis of 5′-P h osp h or yla ted Tr ir ibon u cleotid e De-
r iva tive 17. A mixture of 16 (440 mg, 0.24 mmol), cyclohexyl-
ammonium S,S-diphenyl phosphorodithioate (108 mg, 0.29
mmol), and 1H-tetrazole (67 mg, 0.96 mmol) was rendered
anhydrous by coevaporations three times with dry pyridine
and finally dissolved in dry pyridine (2.4 mL). To the mixture
was added isodurenedisulfonyl dichloride (DDS, 159 mg, 0.48
mmol), and the mixture was stirred at room temperature for
30 min. The mixture was diluted with CH2Cl2, and the CH2Cl2
solution was washed two times with saturated NaHCO3 (50
mL) and with water. The organic phase was collected, dried
over Na2SO4, filtered, and evaporated under reduced pressure.
The residue was chromatographed on a column of silica gel
(28 g) eluted with CH2Cl2-MeOH (100:0-98:2, v/v) to give 17
(423 mg, 84%): 1H NMR (270 MHz, CDCl3) δ 2.56-2.75 (4H,
m), 3.36, 3.41, 3.42, 3.43, 3.46, 3.47, 3.49, 3.50 (6H, s), 3.75
(3H, m), 4.20-4.56 (10Η, m), 4.71 (1H, m), 4.82 (1H, m), 5,06
(1H, m), 5.25 (1H, m), 5.33 (1H, m), 5.75-5.87 (3H, m), 6.05
(2H, m), 6.14 (1H, m), 6.26 (1H, m), 6.57 (1H, d, J ) 5.3 Hz),
6.79, 6.95, 7.21-7.93 (51 H, m), 8.47, 8.50, 8.51, 8.57, 8.67,
8.68, 8.69, 8.693 (2H, s); 13C NMR (60.85 MHz, CDCl3) δ 19.34,
19.63, 53.34, 54.96, 58.58, 58.64, 58.78, 62.61, 64.71, 65.52,
66.17, 66.88, 70.73, 73.66, 73.82, 74.93, 80.23, 80.56, 81.22,
81.62, 86.22, 86.45, 88.64, 89.18, 102.59, 109.60, 111.66,
112.90, 116.50, 116.68, 116.75, 116.86, 121.26, 125.18, 125.30,
125.41, 126.63, 127.40, 127.64, 127.96, 128.19, 128.37, 128.61,
128.99, 129.18, 129.36, 129.60, 129.94, 130.28, 130.96, 131.00,
132.96, 133.62, 135.00, 135.08, 136.86, 139.05, 140.02, 140.25,
140.50, 140.58, 143.49, 143.65, 143.90, 144.87, 147.98, 148.34,
148.39, 148.93, 149.02, 151.81, 151.97, 152.11, 152.28, 152.63,
152.96, 153.91, 158.04, 161.62, 164.89, 165.07, 168.36, 168.41,
169.63, 172.09; 31P NMR (109.25 MHz, CDCl3) δ -1.63, -1.69,
-1.73, -1.93, -2.08, -2.16, -2.47, 52.08, 52.33. Anal. Calcd
Semipreparative reverse phase HPLC gave 34 (373 A258.5
,
40%): 1H NMR (270 MHz, D2O) δ 2.94 (6Η, s N(CH3)2), 3.41
(3H, s, 2′-OCH3 of Am or Um), 3.52 (3H, s, 2′-OCH3 of Am or
Um), 4.04 (3H, s, N7-CH3 of TMG), 3.89-4.83 (m, 2′,3′, 4′, 5′a
and 5′b-H of TMG, Am, Um, A), 5.62 (1H, d, J 5,6 ) 8.3 Hz, H-6
of Um), 8.02, 8.13, 8.24, 8.36 (4H, s, 2-H of Am or A, 8-H of
Am or A); 31P NMR (109.25 MHz, D2O) δ -24.66 (â-phosphate
of triphosphate, dd, J R,â
J â,γ ) 17.0 or 18.3 Hz), -13.35,
or
-13.18 (R,γ-phosphate of triphosphate bridge), -3.12, -2.96
(internucleotidic phosphate); UV (H2O) λmax 258.5 nm (hypo-
chromicity 13.3%) ꢀ258.5
) 42.4 × 103.
nm
En zym a tic Tr ea tm en t of m 32,2,7G5′p p p Am p Um p A (34)
w ith Sn a k e Ven om P h osp h od iester a se (SVP D) a n d Al-
k a lin e P h osp h a ta se (AP ). Compound 34 (2.0 A at its λ max
)
was dissolved in 0.05 M Tris-HCl (pH 8.0, 400 µL) containing
0.02 M ZnCl2 and 20 µL of SVPD (20 units, 1.0 unit /µL) in
glycerin-water (1:1, v/v) was added. The resulting mixture was
incubated at 37 °C for 4 h and heated at 90 °C for 2 min. A
20 µL volume of AP (20 unit, 1.0 unit/µL) was added in the
resulting mixture. After being incubated for 3 h, the mixture
was heated at 90 °C for 2 min. The mixture was analyzed by
reverse-phase HPLC.

4422 J . Org. Chem., Vol. 61, No. 13, 1996
Sekine et al.
En zym a tic Tr ea tm en t of m 32,2,7G5′p p p Am p Um p A (34)
w ith Nu clea se P 1. Compound 34 (2.0 A258.5) was dissolved
in 0.05 M AcOH-AcONa (pH 5.3, 400 µL) containing 0.1 mM
ZnCl2 and 6 µL of nuclease P1 (6 units, 1.0 unit/µL) in
glycerine-water (1:1, v/v) was added. The resulting mixture
was incubated at 50 °C for 5 h and then heated at 90 °C for 2
min. The mixture was analyzed by reverse-phase HPLC, and
the peak of m32,2,7G5′pppAm was collected. The fractions
collected were freeze-dried two times and used in the next
enzyme reaction.
was added 20 µL of AP (20 unit, 1.0 unit/µL). After being
incubated for 3 h, the mixture was heated at 90 °C for 2 min.
The mixture was analyzed by reverse-phase HPLC.
Ack n ow led gm en t. This work was supported by a
Grant-in-Aid for Scientific Research on Priority Areas
from the Ministry of Education, Science, Sports and
Culture, J apan.
En zym a tic Tr ea tm en t of m 32,2,7G5′p p p Am w ith Sn a k e
Ven om P h osp h od iester a se a n d Alk a lin e P h osp h a ta se.
m32,2,7G5′pppAm (1.0 A260) collected was dissolved in 0.05 M
Tris-HCl (pH 8.0, 200 µL) containing 0.02 M ZnCl2, and 10 µL
of SVPD (10 units, 1.0 unit/µL) in glycerine-water (1:1, v/v)
was added. The resulting mixture was incubated at 37 °C for
4 h and heated at 90 °C for 2 min. To the resulting mixture
Su p p or tin g In for m a tion Ava ila ble: Copies of 1H and 13C
NMR spectra (40 pages). This material is contained in
libraries on microfiche, immediately follows this article in the
microfilm version of the journal, can be ordered from the ACS;
see any current masthead page for ordering information.
J O952263V