Synthesis of 4-Thiouridine, 6-Thioinosine, and 6-Thioguanosine 3′,5′-O-Bisphosphates as Donor Molecules for RNA Ligation and Their Application to the Synthesis of Photoactivatable TMG-Capped U1 snRNA Fragments

Article abstract of DOI:10.1021/jo991432z

4-Thiouridine, 6-thioguanosine, and 6-thioinosine 3′,5′-bisphosphates (9, 20, and 28) were synthesized in good yields by considerably improved methods. In the former two compounds, uridine and 2-N-phenylacetylguanosine were converted via transient O-trimethylsilylation to the corresponding 4- and 6-O-benzenesulfonyl intermediates (2 and 13), which, in turn, were allowed to react with 2-cyanoethanethiol in the presence of N-methylpyrrolidine to give 4-thiouridine (3) and 2-N-phenylacetyl-6-thioguanosine derivatives (14), respectively. In situ dimethoxytritylation of these thionucleoside derivatives gave the 5′-masked products 4 and 15 in high overall yields from 1 and 11. 6-S-(2-Cyanoethyl)-5′-O-(4,4′-dimethoxytrityl)-6-thioinosine (23) was synthesized via substitution of the 5′-O-tritylated 6-chloropurine riboside derivative 22 with 2-cyanoethanethiol. These S-(2-cyanoethyl)thionucleosides were converted to the 2′-O-(tert-butyldimethylsilyl)ribonucleoside 3′-phosphoramidite derivatives 7, 18, and 26 or 3′,5′-bisphosphate derivatives 8, 19, and 27. Treatment of 8, 19, and 27 with DBU gave thionucleoside 3′,5′-bisphosphate derivatives 9, 20, and 28, which were found to be substrates of T4 RNA ligase. These thionucleoside 3′,5′-bisphosphates were examined as donors for ligation with m₃^2,2,7 G⁵′ pppAmUmA, i.e., the 5′-terminal tetranucleotide fragment of U1 snRNA. The 4-thiouridine 3′,5′-bisphosphate derivative 9 was found to serve as the most active substrate of T4 RNA ligase with a reaction efficiency of 96%.

Full text of DOI:10.1021/jo991432z

5104
J . Org. Chem. 2000, 65, 5104-5113
Syn th esis of 4-Th iou r id in e, 6-Th ioin osin e, a n d 6-Th iogu a n osin e
3′,5′-O-Bisp h osp h a tes a s Don or Molecu les for RNA Liga tion a n d
Th eir Ap p lica tion to th e Syn th esis of P h otoa ctiva ta ble
TMG-Ca p p ed U1 sn RNA F r a gm en ts
Michinori Kadokura, Takeshi Wada, Kohji Seio, and Mitsuo Sekine*
Department of Life Science, Tokyo Institute of Technology,
Nagatsuta, Midoriku, Yokohama 226-8501, J apan
msekine@bio.titech.ac.jp
Received September 13, 1999 (Revised Manuscript Received May 31, 2000)
4-Thiouridine, 6-thioguanosine, and 6-thioinosine 3′,5′-bisphosphates (9, 20, and 28) were synthe-
sized in good yields by considerably improved methods. In the former two compounds, uridine and
2-N-phenylacetylguanosine were converted via transient O-trimethylsilylation to the corresponding
4- and 6-O-benzenesulfonyl intermediates (2 and 13), which, in turn, were allowed to react with
2-cyanoethanethiol in the presence of N-methylpyrrolidine to give 4-thiouridine (3) and 2-N-
phenylacetyl-6-thioguanosine derivatives (14), respectively. In situ dimethoxytritylation of these
thionucleoside derivatives gave the 5′-masked products 4 and 15 in high overall yields from 1 and
11. 6-S-(2-Cyanoethyl)-5′-O-(4,4′-dimethoxytrityl)-6-thioinosine (23) was synthesized via substitution
of the 5′-O-tritylated 6-chloropurine riboside derivative 22 with 2-cyanoethanethiol. These S-(2-
cyanoethyl)thionucleosides were converted to the 2′-O-(tert-butyldimethylsilyl)ribonucleoside 3′-
phosphoramidite derivatives 7, 18, and 26 or 3′,5′-bisphosphate derivatives 8, 19, and 27. Treatment
of 8, 19, and 27 with DBU gave thionucleoside 3′,5′-bisphosphate derivatives 9, 20, and 28, which
were found to be substrates of T4 RNA ligase. These thionucleoside 3′,5′-bisphosphates were
G5′pppAmUmA, i.e., the 5′-terminal tetranucleotide
2,2,7
examined as donors for ligation with m3
fragment of U1 snRNA. The 4-thiouridine 3′,5′-bisphosphate derivative 9 was found to serve as
the most active substrate of T4 RNA ligase with a reaction efficiency of 96%.
In tr od u ction
sible to construct the plausible 3D models of functional
molecules such as hammerhead ribozymes,3 U snRNA,4
and rRNA.5 In addition, it was also reported that oligo-
nucleotides incorporating thionucleotides were available
for photocrosslinking assay of interaction between pro-
teins and nucleic acids.6 These thionucleotides as cross-
linking reagents are apparently powerful tools not only
for studies of interaction of RNA-RNA3-5 but also for
determination of specific structural elements in recogni-
tion domains of RNA-protein complexes.6
Lu¨hrmann and co-workers have recently reported that
the 5′-terminal 2,2,7-trimethylguanosine (TMG)-cap struc-
ture of U1 snRNA plays an important role as a signal to
transport U1 snRNA from the cytoplasm to the nucleus.7
They also isolated and identified a transport factor
Thio-substituted nucleotides are useful for various
purposes in molecular biology. Particularly, 4-thiouridine,
6-thioinosine, and 6-thioguanosine have been incorpo-
rated into oligonucleotides and utilized as functional
nucleosides for postsynthetic modification.1 The inherent
photo-cross-linking ability of thionucleoside-containing
oligonucleotides has been widely used to study three-
dimensional interaction between RNA-RNA or RNA-
proteins at the atomic level.2 In the photochemical
activation of 4-thiopyrimidine or 6-thiopurine nucleotides
by long wavelength UV light (330-350 nm), there is no
detrimental effect on the common nucletides A, G, C, and
U, such as is observed in photocrosslinking using short
wavelength UV (250-280 nm). Since the distance be-
tween the photoactivatable thiocarbonyl function and the
target molecule is nearly zero, the recognition site in
nucleotide-nucleotide interaction can be definitively de-
termined. Therefore, such detailed studies made it pos-
(3) (a) Woisard, A.; Favre, A. J . Am. Chem. Soc. 1992, 114, 10072-
10074. (b) Woisard, A.; Fourrey, J . L.; Favre, A. J . Mol. Biol. 1994,
239, 366-370. (c) Dos Santos, V. D.; Vianna, A.; Fourrey, J . L.; Favre,
A. Nucleic Acids Res. 1993, 21, 201-207. (d) Dos Santos, V. D.; Fourrey,
J . L.; Favre, A. Biochem. Biophys. Res. Commun. 1993, 190, 377-385.
(e) Wang, L.; Ruffner, D. E. Nucleic Acids Res. 1997, 25, 4355-4361.
(4) (a) Sontheimer, E. J .; Steitz, J . A. Science 1993, 262, 1989-1996.
(b) Kim, C. H.; Abelson, J . RNA 1996, 2, 995-1010. (c) Yu, Y.; Steitz,
J . A. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 6030-6035.
(5) (a) Dokudovskaya, S.; Dontsova, O.; Shpanchenko, O.; Bogdanov,
A.; Brimacombe, R. RNA 1996, 2, 146-152. (b) Baranov, P. V.; Gurvich,
O. L.; Bogdanov, A. A.; Brimacombe, R.; Dontsova, O. A. RNA 1998,
4, 658-668.
(6) (a) Nikiforov, T. T.; Connolly, B. A. Nucleic Acids Res. 1992, 20,
1209-1214. (b) McGregor, A.; Rao, M. V.; Duckworth, G.; Stockley, P.
G.; Connolly, B. A. Nucleic Acids Res. 1996, 24, 3173-3180. (c) Ping,
Y. H.; Liu, Y.; Wang, X.; Neenhold, H. R.; Rana, T. M. RNA 1997, 3,
850-860.
(7) Fischer, U.; Sumpter, V.; Sekine, M.; Satoh, T.; Lu¨hrmann, R.
EMBO J . 1993, 12, 573-583.
(1) (a) Coleman, R. S.; Siedlcki, J . M. J . Am. Chem. Soc. 1992, 114,
9229-9230. (b) Coleman, R. S.; Kesicki, E. A.; Arthur, J . C.; Cotham,
W. E. Bioorg. Med. Chem. Lett. 1994, 4, 1869-1872. (c) Coleman, R.
S.; Kesicki, E. A. J . Am. Chem. Soc. 1994, 116, 11636-11642.
(2) (a) Ito, A.; Robb, F. T.; Peak, J . G.; Peak, J . M. Photochem.
Photobiol. 1988, 47, 231-240. (b) Fourrey, J . L.; Gasche, J .; Fontaine,
C.; Guittet, E.; Favre, A. J . Chem. Soc., Chem. Commun. 1989, 1334-
1336. (c) Clivio, P.; Fourrey, J . L.; Gasche, J . J . Am. Chem. Soc. 1991,
113, 5481-5483. (d) Clivio, P.; Fourrey, J . L.; Gasche, J . Tetrahedron
Lett. 1992, 33, 1615-1618. (e) Clivio, P.; Fourrey, J . L.; Szabo, T.;
Stawinski, J . J . Org. Chem. 1994, 59, 7273-7283. (f) Saintome, C.;
Clivio, P.; Favre, A.; Fourrey, J . L.; Riche, C. J . Am. Chem. Soc. 1996,
118, 8142-8143.
10.1021/jo991432z CCC: $19.00 © 2000 American Chemical Society
Published on Web 07/22/2000

Donor Molecules for RNA Ligation
J . Org. Chem., Vol. 65, No. 17, 2000 5105
protein named Snurportin 1, which has proved to bind
to the TMG-cap structure of U1 snRNA.8 In their work,
m32,2,7 G5′pppAmUmApdCp-Hx-Biotin (Hx ) hexane-1,6-
diyl group), which was derivatized from the 5′-terminal
fragment of U1 snRNA synthesized by us,9 was utilized
for affinity column chromatography to isolate such a
TMG-binding protein. At the next stage of the continuing
studies on the mechanism of U snRNA transport, it is
highly desirable to determine the binding site of the TMG
cap in Snurportin 1. Therefore, it is of great importance
to incorporate a thionucleoside into the 5′-terminal region
of U1 snRNA.
ligation. The first-step ligation provides oligonucleotides
incorporating thionucleoside at the 3′-terminal site. The
oligonucleotides would be useful not only as the substrate
of the second-step enzymatic ligation but also as the
substrate of photo-cross-linking reactions.
In this paper, we report improved methods for the
chemical synthesis of 4-thiouridine, 6-thioinosine and
6-thioguanosine 3′,5′-diphosphates psNp and T4 RNA
ligase mediated site-specific incorporation of thio-
nucleosides into the 3′-terminal site of a 5′-terminal U1
snRNA tetramer, m3 2,2,7G5′pppAmUmA, containing the
unique TMG-cap structure.
Incorporation of thionucleotides into oligonucleotides
by chemical1a,3a,5b,10 and enzymatic4a,11 methods has been
extensively studied. In the chemical method, various
oligonucleotides having thio-substituted nucleosides have
been prepared by automated synthesis by use of phos-
phoramidite units12 in which the thiol function was
appropriately protected to avoid side reactions during the
3′-phosphitylation and the oxidation by iodine after each
coupling cycle.
Resu lts a n d Discu ssion
Syn th esis of 4-Th iou r id in e 3′,5′-O-Bisp h osp h a te
(8). For the synthesis of 4-thiouridine, 6-thioguanosine
and 6-thioinosine derivatives, the 2-cyanoethyl13 or
pivaloxymethyl10d group has been used as the protecting
group of these thiocarbonyl functions. These protecting
groups were removed under basic conditions.10
On the other hand, the previously known enzymatic
methods required some thionucleoside derivatives. Thio-
nucleoside 5′-triphosphates have been used as substrates
of DNA-dependent RNA polymerases to obtain RNA
transcripts incorporating thionucleosides at the 3′-
terminal site or at random sites.11 The dinucleoside
monophosphate derivative 4sUG was also used as a
primer in RNA extension reaction using T7 RNA poly-
merase to introduce 4sU into an RNA at the 5′-terminal
site.4a In this case, the 5′-terminal site of the 4sUG-RNA
fragment thus obtained was phosphorylated enzymati-
cally and subjected to RNA ligation with another RNA′
to obtain RNA′-4sUG-RNA.4a However, this method is not
generally applicable to any site-specifically modified
oligonucleotides because of the requirement of the re-
stricted sequence of 4sUG.
Another enzyme widely used for RNA ligation is T4
RNA ligase. It joins the terminal 5′-phosphate of donor
oligonucleotides with the 3′-terminal hydroxyl group of
acceptor oligonucleotides. If thionucleoside 3′,5′-bis-
phosphates (psNp) are available as the donor substrates
for RNA ligation, it will be possible to obtain any site-
specifically modified RNA sequence using a two-step
In general, S-(2-cyanoethyl)thionucleosides have been
prepared by multistep reactions involving protection-
deprotection manipulation of the hydroxyl groups of the
ribose residues and substitution of appropriate leaving
groups at the C-4 (for pyrimidine nucleosides) or C-6 (for
purine nucleosides) position with 2-cyanoethanethiol.
In this study, we explored a more straightforward
method for the synthesis of 4-S-(2-cyanoethyl)-5′-O-(4,4′-
dimethoxytrityl)-4-thiouridine (4) in situ from uridine (1)
without extensive purification. A trisilylated species of
1, obtained by transient silylation with hexamethyldi-
silazane, was allowed to react with 2,4,6-triisopropyl-
benzenesulfonyl chloride in CH2Cl2-0.2 M Na2CO3 in the
presence of a catalytic amount of Bu4NBr.14 This phase-
transfer reaction gave the 4-O-triisopropylbenzenesul-
fonyl derivative (2),15 which was treated with 2-cyano-
ethanethiol in the presence of N-methylpyrrolidine. It
turned out that this process did not affect the fragile
trimethylsilyl groups so that the fully protected S-(2-
cyanoethyl) ether derivative could be easily extracted and
converted by treatment with trifluoroacetic acid to give
the product 3. Further in situ 5′-dimethoxytritylation of
3 gave the 5′-masked product 4 in 63% overall yield from
1. The same compound has been obtained in a lower yield
starting from uridine via a 6-step reaction, each step of
which required column chromatography except for the
first reaction.10j It should be emphasized that our route
to 4 requires only one-time separation at the last stage.
The 2′-O-silylation of 4 with tert-butyldimethylsilyl chlo-
ride in the presence of imidazole gave the 2′,5′-protected
thionucleoside 5 in 40% yield. The 3′-phosphitylation of
5 with chloro(2-cyanoethoxy)(diisopropylamino)phosphine
gave the amidite unit 7 in 79% yield (Scheme 1).
(8) Huber, J .; Cronshagen, U.; Kadokura, M.; Marshallsay, C.; Wada,
T.; Sekine, M.; Lu¨hrmann, R. EMBO J . 1998, 17, 4114-4126.
(9) Sekine, M.; Kadokura, M.; Satoh, T.; Seio, K.; Wada, T.; Fischer,
U.; Sumper, V.; Lu¨hrmann, R. J . Org. Chem. 1996, 61, 4412-4422.
(10) (a) Rappaport, H. P. Nucleic Acids Res. 1988, 16, 7253-7267.
(b) Connolly, B. A.; Newman, P. C. Nucleic. Acids Res. 1989, 17, 4957-
4974. (c) Christopherson, M. S.; Broom, A. D. Nucleic Acids Res. 1991,
19, 5719-5724. (d) Clivio, P.; Fourrey, J .-L.; Gasche, J .; Audic, A.;
Favre, A. Perrin, C.; Woisard, A. Tetrahedron Lett. 1992, 33, 65-68.
(e) Rao, T. S.; J ayaraman, K.; Durland, R. H.; Revankar, G. R.
Tetrahedron Lett. 1992, 33, 7651-7654. (f) Waters, T. R.; Connolly, B.
A. Nucleosides Nucleotides 1992, 11, 1561-1574. (g) Xu, Y.-Z.; Zheng,
Q.; Swann, P. F. J . Org. Chem. 1992, 57, 3839-3854. (h) Waters, T.
R.; Connolly, B. A. Nucleosides Nucleotides 1992, 11, 985-988. (i)
Clivio, P.; Fourrey, J .-L.; Favre, A. J . Chem. Soc., Perkin Trans. 1 1993,
2585-2590. (j) Adams, C. J .; Murray, J . B.; Arnold, J . R. P.; Stockley,
P. G. Tetrahedron Lett. 1994, 35, 765-768. (k) Adams, C. J .; Farrow,
M. A.; Murray, J . B.; Kelly, S. M.; Price, N. C.; Stockley, P. G.
Tetrahedron Lett. 1995, 36, 4637-4640. (l) Adams, C. J .; Murray, J .
B.; Farrow, M. A.; Arnold, J . R. P.; Stockley, P. G. Tetrahedron Lett.
1995, 36, 5421-5424.
The 3′,5′-bisphosphorylated compound 8 was synthe-
sized as follows. When the 5′-detritylation of 5 was
carried out by treatment with 1% TFA in CH2Cl2, the
(13) (a) Coleman, R. S.; Siedlecki, J . M. Tetrahedron Lett. 1991, 32,
3033-3034. (b) Christopherson, M. S.; Broom, A. D. Nucleic Acids Res.
1991, 19, 5719-5724.
(11) (a) Sheng, N.; Mougey, E. B.; Kelly, S.; Dennis, D. Biochemistry
1993, 32, 2248-2253. (b) Bartholomew, B.; Braun, B. R.; Kassaventis,
G. A.; Geiduschek, E. P. J . Biol. Chem. 1994, 269, 18090-18095. (c)
Dubreuil, L. Y.; Expert-Bezancon, A.; Favre, A. Nucleic Acids Res. 1991,
19, 3653-3660.
(12) Matteucci, M. D.; Caruthers, M. H. J . Am. Chem. Soc. 1981,
103, 3185-3191.
(14) Sekine, M. J . Org. Chem. 1989, 54, 2321-2326.
(15) (a) Daskalov, H. P.; Sekine, M.; Hata, T. Tetrahedron Lett. 1980,
21, 3899-3903. (b) Daskalov, H. P.; Sekine, M.; Hata, T. Bull. Chem.
Soc. J pn. 1981, 54, 3076-3083. (c) Sekine, M.; Matsuzaki, J .; Satoh,
M.; Hata, T. J . Org. Chem. 1982, 47, 571-573. (d) Kamimura, T.;
Masegi, T.; Sekine, M.; Hata, T. Tetrahedron Lett. 1984, 25, 4241-
4244.

5106 J . Org. Chem., Vol. 65, No. 17, 2000
Kadokura et al.
Sch em e 1a
a
Key: (a) hexamethyldisilazane, CH3CN, reflux; (b) ArSO2Cl, aq Na2CO3 or NEt3-DMAP; (c) HSCH2CH2CN, N-methylpyrrolidine,
CH2Cl2; (d) TFA, MeOH; (e) 4,4′-dimethoxytrityl chloride, pyridine; (f) TBDMSCl, imidazole; (g) 3% DCA in CH2Cl2; (h) 1H-tetrazole,
bis(2-cyanoethoxy)(N,N-diisopropylamino)phosphine, CH3CN; (i) tert-butyl hydroperoxide in hexane; (j) chloro(2-cyanoethoxy)(N,N-
diisopropylamino)phosphine, collidine, N-methylimidazole.
2′-3′ silyl migration occurred to give the desired 2′-O-
TBDMS derivative contaminated with a small amount
(ca. 5%) of its 3′-O-TBDMS isomer. However, it was found
that the use of 3% DCA gave only the 2′-O-silylated
uridine derivative 6 in a nearly quantitative yield. The
3′,5′-bisphosphitylation of the product 6 with bis (2-
cyanoethoxy)(diisopropylamino)phosphine in the pres-
ence of 1H-tetrazole followed by the oxidation gave the
bisphosphorylated compound 8 in 89% yield.
It is well-known that the cyanoethyl group can be
removed easily by DBU treatment from NCCH2CH2OP-
(O)(OR)2 but cannot from NCCH2CH2OP(O)(OR)(O-).16
Therefore, we employed our previous effective method of
the simultaneous removal of two 2-cyanoethyl groups
from (NCCH2CH2O)2P(O)(OR) by the combined use of
DBU and bis(trimethylsilyl)acetamide (BSA) for depro-
tection of all the 2-cyanoethyl groups in 8.
This method gave the completely decyanoethylated
product within 1.5 h. After the extraction, the 2′-O-
TBDMS group was removed by treatment with Et3N‚3HF
to give p4sUp (9) which was isolated in 67% yield by
anion-exchange column chromatography using DEAE
Sephadex A-25.
Syn t h esis of 6-Th iogu a n osin e 3′,5′-O-Bisp h os-
p h a te (20). 6-S-(2-Cyanoethyl)-2-N-phenylacetyl-6-thio-
guanosine (14) was synthesized by a combined use of the
transient 2′,3′,5′-tri-O-TMS protection method and the
direct displacement of a 6-O-sulfonylated species with
2-cyanoethanethiol from a practical point of view: The
hydroxyl groups of 2-N-phenylacetylguanosine (11) were
transiently protected by treatment with HMDS. After the
2′,3′,5′-O-trisilylated compound 12 was sulfonylated at
the 6-O position with mesitylenesulfonyl chloride (MsCl)
in the presence of DMAP and Et3N, reaction of the
resulting 6-O-sulfonylated species 13 with 2-cyano-
ethanethiol in the presence of N-methylpyrrolidine gave
14 in 91% overall yield from 11. The 5′-tritylation of 14
gave the 5′-protected derivative 15 in 86% yield. The 2′-
O-silylation of 15 was carried out in a manner similar to
that described in the synthesis of 5 to give the desired
product 16 in 28% yield. The usual phosphitylation of
16 gave the amidite unit 18 in 77% yield. On the other
hand, the 3′,5′-bisphosphoylated compound 19 was ob-
tained in 78% yield in a manner similar to that described
in the synthesis of 8. In the case of p6sGp (20), decyano-
ethylation and desilylation were done under conditions
similar to those described above, and finally the 2-N-
phenylacetyl group was removed by treatment with NH3.
Thus, 6-thioguanosine 3′,5′-O-bisphosphate (20) was
obtained from 19 in 59% yield.
Syn th esis of 6-Th ioin osin e 3′,5′-O-Bisp h osp h a te
(28). In the previous synthesis of 6-thioinosine, introduc-
tion of the thiocarbonyl group into the C-6 position has
been performed by reaction of inosine derivatives with
Lawesson’s reagent or by displacement of a 6-triazolyl-
purine riboside derivative with hydrogen sulfide.10i To
prepare building units for incorporation of such thio-
nucleosides into oligonucleotides, the thiocarbonyl group
was further protected via S-alkylation with pivaloxy-
methyl chloride or cyanoethyl bromide under basic condi-
tions. Thus, these synthetic routes could not directly give
6-S-protected thioinosine derivatives. Therefore, we tried
to introduce an S-(2-cyanoethyl) function into the C-6
position via a 6-O-sulfonylinosine derivative. When 6-O-
sulfonylation of inosine derivatives by using 2,4,6-triiso-
propylbenzensufonyl chloride (TPS) was carried out, N1-
sulfonylation occurred simultaneously. The yields of the
(16) Sekine, M.; Tsuruoka, H.; Iimura, S.; Wada, T. Natural Products
Lett. 1994, 5, 41-46.

Donor Molecules for RNA Ligation
J . Org. Chem., Vol. 65, No. 17, 2000 5107
Sch em e 2a
a
Key: (a) 4,4′-dimethoxytrithyl chloride, pyridine; (b) HSCH2CH2CN, N-methylpyrrolidine, CH2Cl2; (c) TBDMSCl, imidazole, DMF;
(d) 3% DCA in CH2Cl2; (e) 1H-tetrazole, bis(2-cyanoethoxy)(N,N-diisopropylamino)phosphine, CH3CN; (f) tert-butyl hydroperoxide in hexane;
(g) chloro(2-cyanoethoxy)(N,N-diisopropylamino)phosphine, collidine, N-methylimidazole.
Sch em e 3
N1- and 6-O-TPS inosine derivatives were 56% and 31%,
respectively. Thus, direct introduction of the 2-cyano-
ethylthio group into the C-6 position via 6-O-sulfonylation
resulted in poorer yields of the desired product. There-
fore, we chose 6-chloropurine riboside (21) as a starting
material. After 5′-O-dimethoxytrityl-6-chloropurine ri-
boside (22) was synthesized in 94% yield from 21,
reaction of 22 with 2-cyanoethanethiol in the presence
of N-methylpyrrolidine produced the 6-S-(2-cyanoethyl)-
5′-O-dimethoxytrityl-6-thioinosine (23) in 96% yield. In
a manner similar to those described for uridine and
guanosine, the synthetic unit 26 and 3′,5′-bisphosphoyl-
ated 6-thioinosine derivative 27 were synthesized in 76
and 70% yields, respectively (Scheme 2).
reaction in the presence of GTP. This procedure initially
produces RNA oligomers starting from 4sUG at the 5′-
terminal site. Therefore, an additional enzyme reaction
is necessary to join the RNA oligomers with 5′-upstream
RNA fragments using T4 DNA ligase in the presence of
DNA templates. This method always requires guanylic
acid 3′-downstream from 4sU because of the enzyme’s
inherent specificity.
Cross-linking experiments using U1 snRNA fragments
incorporating 4sU are highly useful to clarify TMG-
proteins interaction in connection with the splicing
mechanism.17 Our target sequence, the 5′-terminal TMG-
capped RNA trimer (m32,2,7G5′pppAmUmA) of U1 snRNA,
does not have such a G nucleotide. Therefore, the above
strategy using 4sUpG as a primer cannot be applied to
the modification of the 5′-terminal sequence of U1
snRNA. Therefore, we used p4sUp for the site-specific
incorporation of 4sU into the 5′-terminal 10mer of U1
snRNA using T4 RNA ligase.
Site-Sp ecific In cor p or a tion of 4-Th ion u cleotid e
3′,5′-Bisp h osp h a te w ith T4 RNA Liga se. Oligonucleo-
tides containing 4sU have been prepared by use of 4sUTP
and appropriate polymerases such as T7 RNA polymerase
in the presence of DNA templates and primers. Since
4sUTP is incorporated four to five times more slowly than
UTP in this system, 4sU is randomly incorporated at all
the available positions confronting all the adenosines on
the templates.4c
The enzymatic synthesis of the U1 snRNA oligo-
mers containing 4sU, 6sI and 6sG, respectively, were
illustrated in Scheme 4. The U1 snRNA tetramer
(m32,2,7G5′pppAmUmA) was synthesized according to the
For the site-specific introduction of 4sU into RNA
oligomers at a definite position, a dinucleotide 4sUG has
often been used as the primer in T7 RNA polymerase
(17) Will, C. L.; Lu¨hrmann, R. Curr. Opin. Cell Biol. 1997, 9, 320-
328.

5108 J . Org. Chem., Vol. 65, No. 17, 2000
Kadokura et al.
Sch em e 4
F igu r e 2. UV spectra of the peaks 31a , 31b, and 31c shown
in Figure 1.
(pH 8.3) at 8 °C for 48 h. The conditions to incorporate
the other nucleotides are described in the Experimental
Section. The 3′-terminal phosphate group of the ligation
products, thus obtained, were removed by treatment with
calf intestinal alkaline phosphatase and the dephos-
phorylated pentamers were analyzed and purified by
anion exchange HPLC (Figure 1). The ligation efficiency
of p4sUp toward m32,2,7G5′pppAmUmA was 94% at a level
comparable to those of the reference materials pUp (88%)
or pCp (96%). In contrast, the ligation efficiency of a
purine nucleotide donors such as p6sIp and p6sGp were
relatively low (27% and 10%, respectively), as shown in
Table 1. The isolation yields of the ligation products are
also described in the same table. The structure of the
purified ligation products were characterized by the UV
spectra and the composition analysis after enzymatic
digestion with nuclease P1. The UV spectra of these
products showed λmax peaks at 330 nm which supported
the presence of thionucleotides (Figure 2). Furthermore,
the respective HPLC profiles cleary showed the appear-
ance of m32,2,7G5′pppAm, pUm, pA detected at 254 nm and
a thio-substituted nucleotide detected at 330 nm after
the nuclease P1 digestion (Figure 3). These observations
made us confirm the successful incorporation of the thio-
substituted nucleotides.
F igu r e 1. Anion-exchange HPLC profiles of the reaction
mixtures obtained by the RNA ligations as shown in entries 1
(A), 2 (B), and 4 (C) of Table 1. The HPLC conditions described
as method A (see the Experimental Section) were used for the
analysis for A and B. Those described as method B were used
for C.
Ta ble 1. Efficien cy of Th ion u cleosid e 3′5′-Bisp h osp a tes
9, 20, a n d 28 a s Don or s in Liga tion w ith m 32,2,7G5′
p p p Am Um A 30 by T4 RNA Liga se
ligation producta isolated yield
entry
donor
acceptor
(%)
(%)
1
2
3
4
5
6
p4sUp 9
p6sIp 20
p6sGp 28
p6sGp 28
pCp
30
30
30
30
30
30
31a (94)
31b (27)
31c (10)
31c (23)
31d (96)
31e (88)
81
19
6
7
74
60
pUp
a
The yield is estimated by HPLC analysis. All the reactions
were carried out for 48 h except for entry 4 (9 days).
Con clu sion
procedure reported previously by us9 and used as an
acceptor for the T4 RNA ligase reaction.18 For example,
the ligation reaction of m32,2,7G5′pppAmUmA (0.2 mM)
with p4sUp (2 mM) was performed using T4 RNA ligase
(175 units to donor 1 µmol) in the presence of 8 mM ATP,
20 mM MgCl2, and 5 mM DTT in 50 mM Tris-HCl buffer
In this study, we have established the hitherto most
practical route to synthesize thio-substituted nucleosides
and their derivatives. 4-S-(2-Cyanoethyl)-5′-O-(4,4′-
dimethoxytrityl)-4-thiouridine 4, a key intermediate for
the synthesis of 4-thiouridine 3′, 5′-bisphosphate, was
synthesized in a five-step reaction scheme which required
only one-time column chromatography separation at the
last stage. Similar reaction scheme, namely the transient
protection and 6-O-selective sulfonylation followed by the
direct displacement of the sulfonyloxy group with 2-
(18) (a) Uhlenbeck, O. C.; Cameron, V. Nucleic Acids Res. 1977, 4,
85-98. (b) England, T. E.; Uhlenbeck, O. C. Nucleic Acids Res. 1978,
17, 2069-2076. (c) Iwase, R.; Maeda, M.; Fujii, T.; Sekine, M.; Hata,
T. Nucleic Acids Res. 1992, 20, 1643-1648.

Donor Molecules for RNA Ligation
J . Org. Chem., Vol. 65, No. 17, 2000 5109
F igu r e 3. Anion-exchange HPLC profiles after nuclease P1 treatment of 31a (A), 31b (B), and 31c (C).
Wako Pure Chemical Industries, Ltd. and a minipump for a
cyanoethanethiol, was also effective to synthesize 6-thio-
guanosine derivatives. On the other hand, 6-S-(2-cyano-
ethyl)-6-thioinosine derivative 23 was obtained in a
higher yield when commercially available 6-chloropurine
riboside was used as a starting material. Three kinds of
pNp donors 9, 20, and 28 (Scheme 3) required for RNA
ligation were successfully synthesized. These donors have
proved to be useful for incorporation of thionucleotides
onto the 3′-terminal site of the TMG-capped RNA tet-
ramer by using T4 RNA ligase. The RNA oligomers
modified with a thionucleotide will be useful as a power-
ful tool for elucidation of RNA-RNA or RNA-protein
interaction by photocrosslinking methods. Especially, the
TMG capped RNA fragment 31a incorporating 4sU would
be used for determination of the TMG-cap binding site
of Snurportin 1.8 These studies are now under investiga-
tion. Moreover, our preliminary result showed that an
E. coli rRNA mimic 25mer (R-sarcin/ricin domain), which
has 6sG incorporated by use of p6sGp 20, proved to cross-
link to the binding site of another RNA fragment (thio-
strepton domain) of the same rRNA molecule upon UV
irradiation.19 This fact also demonstrates a potential
utility of 20 in studies related to molecular biology.
goldfish bowl was conveniently used to attain sufficient
pressure for rapid chromatographic separation. TLC was
performed on precoated TLC plates of silica gel 60 F-254
(Merck). Anion-exchange HPLC was performed on a Gen-Pak
Fax column (4.6 × 100 mm, waters) for the oligonucleotides
or Whatman Partisil 10 SAX WCS analytical column (4.6 ×
250 mm) for the enzymatic digestion. The analytical conditions
used were a 10-63% (method A) or 1-30% (method B) linear
gradient of solvent A (1.0 M NaCl in 25 mM NaH2PO4, pH
6.0) in solvent B (25 mM NaH2PO4, pH 6.0) for 30 min for the
Gen-Pak Fax column and 0-30% linear gradient of solvent C
(20% CH3CN in 0.5 M KH2PO4) in solvent D (20% CH3CN in
0.005 M KH2PO4) for 30 min for Whatman Partisil 10 SAX
WCS analytical column. The flow rate was 1.0 mL/min and
the column temperature was 50 °C. Ribonucleosides were
purchased from Yamasa Co., Ltd. Pyridine was distilled two
times from p-toluenesulfonyl chloride and from calcium hy-
dride and stored over molecular sieves 4A. Elemental analyses
were performed by the Microanalytical Laboratory, Tokyo
Institute of Technology, at Nagatsuta.
4-S-(2-Cya n oet h yl)-5′-O-(4,4′-d im et h oxyt r it yl)-4-t h io-
u r id in e (4). To a suspension of uridine 1 (3.66 g, 15 mmol) in
dry acetonitrile (150 mL) was added hexamethyldisilazane (16
mL, 75 mmol). After being refluxed for 2 h, the mixture was
cooled and ethanol (20 mL) was added. The solvent was
removed under reduced pressure and coevaporated with dry
toluene. The residue was dissolved in CH2Cl2 (300 mL). To
the solution were added 2,4,6-triisopropylbenzenesulfonyl
chloride (5.91 g, 19.5 mmol) and tetrabutylammonium bromide
(193 mg, 0.6 mmol). To the mixture was added 0.2 M Na2CO3
(600 mL), and the mixture was stirred vigorously at room
temperature for 16 h. The organic layer was washed three
times with water, dried over Na2SO4, filtered, and evaporated
under reduced pressure. The residue was dissolved in CH2Cl2
(150 mL), and 2-cyanoethanethiol (3.0 mL, 33.8 mmol) and
N-methylpyrrolidine (4.78 mL, 47 mmol) were added. After
being stirred at room temperature for 30 min, the mixture was
Exp er im en ta l Section
1H and 13C NMR spectra were measured at 270 and 67.8
MHz, respectively, with TMS as the internal reference. 31P
NMR spectra were measured at 109.4 MHz with 80% phos-
phoric acid as the external reference. Column chromatography
was performed with silica gel C-200 and C-300 purchased from
(19) Morishita, R.; Matsumoto, H.; Madin, K.; Sawasaki, T.; Uch-
iumi, T.; Sekine, M.; Endo, Y. Nucleic Acids Symposium Ser. 1998,
39, 157-158.

5110 J . Org. Chem., Vol. 65, No. 17, 2000
Kadokura et al.
diluted with CH2Cl2 and washed three times with 1 M
KH2PO4. The organic layer was collected, dried over Na2SO4,
filtered and evaporated under reduced pressure. To the residue
was added 2% trifluoroacetic acid in methanol (150 mL). After
being stirred at room temperature for 30 min, the reaction was
quenched by adding pyridine (50 mL). The solution was
evaporated under reduced pressure, and the residue was
diluted with water-pyridine (400 mL, 3:1,v/v). The aqueous
solution was washed three times with Et2O. Every time the
ethereal layer was back-extracted with water-pyridine (300
mL, 2:1, v/v) which was put in another separatory funnel. After
extraction was performed, the two aqueous layers were
combined and evaporated under reduced pressure. The residue
was rendered anhydrous by coevaporation three times with
dry pyridine and dissolved in dry pyridine (150 mL). To the
solution was added 4, 4′-dimethoxytrityl chloride (5.08 g, 15
mmol). After being stirred at room temperature for 20 h, the
mixture was diluted with CH2Cl2 (250 mL). The organic
solution was washed three times with sat. NaHCO3, dried over
Na2SO4, filtered and evaporated under reduced pressure. The
residue was chromatographed on a column of silica gel (180
g) eluted with CH2Cl2-MeOH (98.5:1.5-97.5: 2.5, v/v) to give
4 (5.81 g, 63%): 1H NMR (270 MHz, CDCl3) δ 2.91 (2 H, m),
3.21 (1 H, br), 3.35-3.47 (4 H, m), 3.80 (6 H, s), 4.38-4.39 (3
H, m), 5.40 (1 H, br), 5.78 (1 H, d, J ) 2.97 Hz), 6.04 (1 H, d,
J ) 6.83 Hz), 6.80-6.84 (4 H, m), 7.16-7.29 (9 H, m), 8.02 (1
H, d, J ) 7.26 Hz); 13C NMR (67.8 MHz, CDCl3) δ 18.06, 25.27,
55.15, 61.80, 70.03, 75.98, 84.49, 86.88, 92.51, 113.17, 118.04,
125.18, 127.01. 140.83, 144.13, 154.61, 158.56, 175.79. Anal.
Calcd for C33H33N3O7S‚H2O: C, 60.82; H, 5.72; N, 6.45; S, 4.92.
Found: C, 60.51; H, 5.09; N, 6.43; S, 6.67.
4-S-(2-Cya n oeth yl)-2′-O-(ter t-bu tyld im eth ylsilyl)-5′-O-
(4,4′-d im eth oxytr ityl)-4-th iou r id in e (5). Compound 4 (5.81
g, 9.43 mmol) was rendered anhydrous by coevaporation three
times each with dry pyridine and dry toluene and dissolved
in dry DMF (94 mL). To the solution were added imidazole
(963 mg, 14.2 mmol) and TBDMSCl (1.7 g, 11.3 mmol). The
mixture was stirred at room temperature for 36 h. The solvent
was removed under reduced pressure and the residue was
dissolved in CH2Cl2 (250 mL). The solution was washed three
times with sat. NaHCO3. The organic phase was collected,
dried over Na2SO4, filtered, and evaporated under reduced
pressure. The residue was chromatographed on a column of
silica gel (150 g) eluted with hexanes-EtOAc (7:3, v/v) to give
5 (2.72 g, 40%): 1H NMR (270 MHz, CDCl3) δ 0.26, 0.38 (6 H,
s), 0.98 (9 H, s), 2.91 (2 H, t, J ) 6.6 Hz), 3.40 (2 H, m), 3.61
(2 H, m, 5′-H), 4.13 (1 H, d, J ) 8.25 Hz), 4.34 (1 H, d, J )
4.29 Hz), 4.44 (1 H, m) 5.78 (1 H, d, J ) 7.26 Hz), 5.83 (1 H,
s), 6.88 (4 H, d, J ) 8.58 Hz) 7.18-7.45 (9 H, m), 8.39 (1 H, d,
J 5,6 ) 6.93 Hz); 13C NMR (67.8 MHz, CDCl3) δ -5.51, -4.45,
17.95, 18.17, 25.18, 25.75, 55.13, 60.81, 68.59, 76.37, 82.95,
86.92, 90.85, 103.18, 113.19, 118.04, 125.16, 127.03, 127.91,
128.07, 128.90, 130.01, 130.05, 134.95, 135.26, 141.01, 144.17,
153.46, 158.58, 175.27. Anal. Calcd for C39H47N3O7SSi H2O:
C, 62.63; H, 6.60; N, 5.62; S, 4.29. Found: C, 62.31; H, 6.25;
N, 5.52; S, 4.77.
(4 H, m), 7.16-7.45 (9 H, m), 8.34 8.40 (1 H, d, J ) 7.26 Hz);
13C NMR (67.8 MHz, CDCl3) δ -5.37, -4.63, -4.58 (Si (CH3)2),
17.77, 17.95, 19.88, 19.98, 20.07, 20.18, 21.15, 24.19, 24.28,
24.40, 24.51, 24.98, 25.59, 42.68, 42.81, 43.00, 54.95, 57.40,
57.54, 57.68, 57.85, 60.45, 60.63, 74.68, 75.31, 86.99, 86.70,
86.83, 91.30, 91.59, 103.02, 112.92, 117, 14, 117.23, 118,
124.99, 126.86, 127.62, 127.91, 128.10, 128.18, 128.72, 130.06,
134.75, 134.90, 135.08, 137.48, 140.90, 143.85, 144.04, 153.37,
153.42, 158.44, 174.61, 174.81; 31P NMR (67.8 MHz, CDCl3) δ
149.29, 151.44 (1:1). Anal. Calcd for C48H64N5O8SSiP: C, 61.98;
H, 6.93; N, 7.53; S, 3.45. Found: C, 61.87; H, 7.18; N, 7.38; S,
2.71.
4-S-(2-Cya n oet h yl)]-2′-O-(ter t-b u t yld im et h ylsilyl)-4-
th iou r id in e (6). To a solution of compound 5 (150 mg, 0.2
mmol) in CH2Cl2 (9.8 mL) was added dichloroacetic acid (250
µL). After being stirred at room temperature for 2 min, the
mixture was washed three times with sat. NaHCO3. The
organic layer was collected, dried over Na2SO4, filtered, and
evaporated under reduced pressure. The residue was chro-
matographed on a column of silica gel (5 g) eluted with CHCl3-
MeOH (99:1-98:2, v/v) to give 6 (85 mg, 99%): 1H NMR (270
MHz, CDCl3) δ 0.14, 0.17 (6 H, s), 0.91 (9 H, s), 2.89 (2 H, t, J
) 6.60 Hz), 3.42 (2 H, m), 3.86 (1 H, m), 4.05 (1 H, m), 4.15-
4.22 (2 H, m), 4.64 (1 H, t, J ) 3.96 Hz), 5.53 (1 H, d, J ) 3.30
Hz), 6.27 (1 H, d, J ) 6.27 Hz), 7.91 (1 H, J ) 6.27 Hz); 13C
NMR (67.8 MHz, CDCl3) δ -5.53, -4.89, 17.74, 17.86, 25.00,
25.48, 60.18, 68.79, 75.24, 84.57, 92.40, 103.50, 117.90, 142.05,
153.64, 175.62. Anal. Calcd for C18H29N3O5SSi: C, 50.56; H,
6.84; N, 9.83; S, 7.50. Found: C, 50.42; H, 6.50; N, 9.85; S,
6.85.
Tetr a k is(2-cya n oeth yl) Ester of 4-S-(2-Cya n oeth yl)-2′-
O-(ter t-bu tyld im eth ylsilyl)-4-th iou r id in e 3′,5′-Bisp h os-
p h a te (8). Compound 6 (214 mg, 0.5 mmol) was rendered
anhydrous by coevaporation three times each with dry pyri-
dine, dry toluene and dry CH3CN and finally dissolved in dry
CH3CN (5 mL). To the solution were added bis(2-cyanoethyl)-
(N,N-diisopropyl)phosphoramidite (405 mg, 1.5 mmol) and 1H-
tetrazole (160 mg, 2.25 mmol). The mixture was stirred under
argon atmosphere at room temperature for 1 h. tert-Butyl
hydroperoxide (1.5 mL, 15 mmol) was added and the resulting
mixture was stirred at room temperature for 30 min. The
mixture was diluted with CH2Cl2, and the solution was washed
twice each with sat. NaHCO3 (50 mL) and water. The organic
layer was dried over Na2SO4 and evaporated under reduced
pressure. The residue was chromatographed on a column of
silica gel (10 g) eluted with CH2Cl2-MeOH (98:2, v/v) to give
8 (354 mg, 89%): 1H NMR (270 MHz, CDCl3) δ 0.17, 0.26 (6
H, s), 0.94 (9 H, s) 2.76-2.91 (10 H, m), 3.40 (2 H, t, J ) 7.30
Hz) 4.29-4.59 (12 H, m), 4.74 (1 H, m), 5.69 (1 H, s), 6.33 (1
H, d, J ) 6.93 Hz), 7.83 (1 H, d, J ) 7.26 Hz); 13C NMR (67.8
MHz, CDCl3) δ -5.33, -4.76, 17.95, 18.08, 19.57, 19.68, 25.16,
25.54, 62.63, 62.70, 62.75, 62.82, 65.36, 73.05, 74.07, 77.18,
79.14, 92.29, 103.65, 116.39, 115.51, 115.62, 118.08, 140.04,
153.26, 175.97; 31P NMR (67.8 MHz, CDCl3) δ -1.81, -1.59.
Anal. Calcd for C30H43N57O11SSiP2‚5H2O: C, 40.49; H, 6.00;
N, 11.02; S, 3.60. Found: C, 40.33; H, 5.01; N, 10.66; S, 4.43.
4-Th iou r id in e 3′,5′-Bisp h osp h a te (9). To a solution of
Compound 8 (37 mg, 0.05 mmol) in dry pyridine (5 mL) were
added DBU (45 mL, 0.3 mmol) and BSA (460 mL, 1.88 mmol).
After being stirred at room temperature for 1.5 h, the mixture
was diluted with water. The aqueous solution was washed
three times with Et2O. The aqueous phase was collected and
evaporated under reduced pressure. The residue was rendered
anhydrous by coevaporation three times each with dry pyridine
and dry toluene. To the anhydrous mixture was added tri-
ethylamine trihydrofluoride (403 mg, 2.5 mmol). After being
stirred at room temperature for 20 h, the mixture was added
dropwise to 2 M TEAB buffer (2 mL). The reaction mixture
was purified by DEAE Sephadex A-25 (1 M ammonium
4-S-(2-Cya n oeth yl)-2′-O-(ter t-bu tyld im eth ylsilyl)-5′-O-
(4,4′-d im eth oxytr ityl)-4-th iou r id in 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 5 (1.46
g, 2 mmol) was rendered anhydrous by coevaporation three
times each with dry pyridine, dry toluene, and dry THF and
dissolved in dry THF (6 mL). To the solution under argon
atmosphere were added N-methylimidazole (80 µL, 1 mmol),
collidine (1.59 mL, 12 mmol), and chloro(2-cyanoethoxy)(N,N-
diisopropylamino)phosphine (870 µL, 4 mmol). After being
stirred at room temperature for 1 h, the mixture was diluted
with CH2Cl2 (100 mL) and washed three times with sat.
NaHCO3. The organic layer 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 hexanes-EtOAc-pyridine (70:30:0.5, v/v/v) to give 7 (1.47
g, 79%): 1H NMR (270 MHz, CDCl3) δ 0.16, 0.29 (6H, s), 0.91-
1.19 (2H, m), CH3 of iPr) 2.41 2.58 (2 H, t, J ) 6.27 Hz), 2.90
(2 H, m) 3.31-3.86 (8, H, m), 3.80 (6 H, m) 4.33-4.38 (3 H,
m), 5.59 5.71 (1 H, d, J ) 7.26 Hz) 5.27 5.79 (1 H, s), 6.84
bicarbonate). The eluate was lyophilized to give 9 (915 A330
,
67%): 1H NMR (270 MHz, D2O) δ 3.95-3.96 (2H, m), 4.41-
4.45 (2 H, m), 4.54 (1 H, m), 6.33 (1 H, d, J ) 6.93 Hz), 7.83 (1
H, d, J ) 7.26 Hz); 13C NMR (67.8 MHz, D2O) δ -5.33, -4.76,
17.95, 18.08, 19.57, 19.68, 25.16, 25.54, 62.63, 62.70, 62.75,
62.82, 65.36, 73.05, 74.07, 77.18, 79.14, 92.29, 103.65, 116.39,

Donor Molecules for RNA Ligation
J . Org. Chem., Vol. 65, No. 17, 2000 5111
115.51, 115.62, 118.08, 140.04, 153.26, 175.97; 31P NMR (67.8
MHz, CDCl3) δ 4.42, 4.60; FAB calcd for C9H11N2Na4O11P2S
m/z 508.92, obsd, 508.92; UV (H2O) λmax 243 nm, 330.5 nm,
λmin 226.5 nm, 274.5 nm.
NaHCO3. The organic layer was dried over Na2SO4 and
evaporated under reduced pressure. The residue was chro-
matographed on a column of high mesh silica gel (WAKO-
C300) (100 g) eluted with hexanes-Et2O (45:55, v/v) to give
16 (493 mg, 28%). The 3′-silylated isomer was eluted with
hexanes-Et2O (40:60, v/v) to give 16′ (799 mg, 45%): Analytical
data for 16: 1H NMR (270 MHz, CDCl3) δ -0.21, -0.01 (6 H,
s), 0.84 (9 H, s), 3.10 (2 H, t, J ) 6.76 Hz), 3.24-3.32 (2 H, m),
3.53 (2 H, m), 3.76 (6 H, s), 3.77 (2 H, s), 4.23 (1 H, m), 4.33 (1
H, m), 5.05 (1 H, t, J ) 5.61 Hz), 5.88 (1 H, d, J ) 5.94 Hz),
6.80 (4 H, m), 7.11-7.53 (14 H, m), 8.05 (1 H, s); 13C NMR
(67.8 MHz, CDCl3) δ -5.23, -5.07, 17.72, 18.37, 25.03, 25.39,
43.90, 55.10, 63.43, 71.16, 75.01, 77.18, 84.21, 86.43, 88.09,
113.19, 118.42, 126.98, 127.17, 127.91, 128.70, 129.31, 129.90,
133.84, 135.49, 135.67, 141.60, 144.71, 149.43, 151.45, 158.54,
160.34, 168.61. Anal. Calcd for C48H54N6O7SSi: C, 64.99; H,
6.14; N, 9.47; S, 3.61. Found: C, 65.49; H, 6.43; N, 9.03; S,
3.88. Analytical data for 16′: 1H NMR (270 MHz, CDCl3) δ
0.00, 0.08 (6 H, s), 0.87 (9 H, s), 2.94 (2 H, t, J ) 6.9 Hz), 3.23
(1 H, d, J ) 10.2 Hz), 3.40 (1 H, d, J ) 8.6 Hz),3.50 (2 H, t, J
) 6.9 Hz), 3.69 (2 H, s), 3.75 (6 H, s), 4.17 (1 H, m), 4.48 (1 H,
m), 4.69 (1 H, m), 5.91 (1 H, d, J ) 5.6 Hz), 6.80 (4 H, m),
7.11-7.38 (14 H, m), 7.95 (1 H, s), 8.10 (1 H, s); 13C NMR (67.8
MHz, CDCl3) δ -4.85, -4.78, 18.06, 18.58, 24.82, 25.50, 25.72,
44.48, 55.20, 62.12, 72.59, 74.86, 85.55, 86.56, 89.76, 113.15,
118.29, 126.94, 127.49, 127.87, 128.03, 128.75, 128.97, 129.43,
129.94, 133.85, 135.51, 135.60, 141.62, 144.38, 149.18, 151.39,
148.54. Anal. Calcd for C48H54N6O7SSi‚5/2H2O: C, 61.85; H,
6.38; N, 9.02. Found: C, 61.33; H, 5.83; N, 8.70.
6-S-(2-Cya n oeth yl)]-2′-O-(ter t-bu tyld im eth ylsilyl)-5′-O-
(4′,4′′-d im e t h o x y t r it y l)-2-N -p h e n y la c e t y l-6-t h io g u a -
n 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 (18). Compound 16 (177 mg, 0.2 mmol) was rendered
anhydrous by coevaporation three times each with dry pyri-
dine, dry toluene and dry THF, and finally dissolved in dry
THF (0.6 mL). To the solution were added N-methylimidazole
(8 ul, 0.1 mmol), collidine (159 µL, 1.2 mmol) and chloro(2-
cyanoethyl)(N,N-diisopropylamino)phosphine (87 µL, 0.4 mmol)
under argon atmosphere. After being stirred at room temper-
ature for 40 min, the mixture was diluted with CH2Cl2 (25
mL) and washed three times with sat. NaHCO3. The organic
layer 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 hexanes-EtOAc-
Et3N (70:30:1, v/v/v) to give 18 (217 mg, 77%): 1H NMR (270
MHz, CDCl3) δ -0.26, -0.05, -0.01 (6H, s), 0.76 (9 H, s), 0.95-
1.32 (12 H, m), 2.18 (2 H, m), 2.61 (2 H, m), 3.09-4.02 (16 H,
m), 4.24-4.38 (2 H, m), 5.01 5.14 (1 H, m), 5.84 5.99 (1 H,
d, J ) 7.26 Hz), 6.77-7.64 (14 H, m), 8.08 8.10 (1 H, s); 13C
NMR (67.8 MHz, CDCl3) δ -5.26, -4.83, -4.77, 17.70, 17.81,
18.39, 19.80, 19.91, 20.11, 20.18, 24.42, 24.53, 24.62, 25.11,
25.34, 25.43, 25.48, 29.15, 45.59, 42.79, 43.18, 43.36, 43.51,
43.79, 55.13, 58.73, 58.94, 63.04, 63.24, 72.04, 72.26, 75.72,
84.17, 84.64, 86.34, 86.67, 87.19, 88.05, 113.24, 117.11, 117.70,
118.55, 127.04, 127.82, 128.00, 128.49, 128.57, 128.81, 129.38,
129.83, 129.88, 129.99, 133.89, 134.14, 135.31, 135.42, 135.62,
135.87, 141.15, 141.89, 144.49, 144.85, 149.70, 151.41, 151.64,
158.56, 160.14, 160.25, 169.00; 31P NMR (67.8 MHz, CDCl3) δ
149.44, 151.84. Anal. Calcd for C57H71N8O8SSiP: C, 62.96; H,
6.58; N, 10.31. Found: C, 63.24; H, 6.70; N,10.14.
6-S -(2-Cya n oe t h yl)-2-N -p h e n yla ce t yl-6-t h iogu a n o-
sin e (14). To a suspension of compound 11 (803 mg, 2 mmol)
in dry acetonitrile was added hexamethyldisilazane (2.1 mL,
10 mmol). After being refluxed for 2 h, the mixture was cooled,
and ethanol (2 mL) was added. The solvent was removed under
reduced pressure, and the residue was coevaporated with dry
toluene and dissolved in CH2Cl2 (300 mL). To the mixture were
added mesitylenesulfonyl chloride (525 mg, 2.4 mmol), tri-
ethylamine (1.12 mL, 8 mmol), and 4-(dimethylamino)pyridine
(12 mg, 0.1 mmol). After being stirred at room temperature
for 1 h, the mixture which was cooled at 0 °C. To the mixture
was added N-methylpyrrolidine (2.08 mL, 20 mmol). After 30
min, 2-cyanoethanethiol (2.0 mL, 20 mmol) was added, and
the mixture was stirred at 0 °C for 30 min. The mixture was
diluted with CH2Cl2 and washed three times with 1 M KH2-
PO4. The organic layer was collected, dried over Na2SO4,
filtered and evaporated under reduced pressure. To the residue
was added 2% trifluoroacetic acid/methanol (50 mL). After
being stirred at room temperature for 20 min, the mixture was
quenched by addition of pyridine (20 mL). The organic solution
was evaporated under reduced pressure, and the residue was
chromatographed on a column of silica gel (30 g) eluted with
CH2Cl2-MeOH (96:4 v/v). After the eluent was evaporated
under reduced pressure, the residue was diluted with CH2-
Cl2-pyridine (2:1, v/v) and the solution was washed three
times with water. Every time the aqueous layer was back-
extracted three times with CH2Cl2-pyridine (1:1, v/v) which
was put in another separatory funnel. After the extraction,
the two organic layers were combined and evaporated under
reduced pressure to give compound 14 (943 mg, 91%): 1H NMR
(270 MHz, DMSO) δ 3.15 (2 H, t, J ) 7.26 Hz), 3.55 (2 H, t, J
) 6.92 Hz), 3.55-3.64 (2 H, m), 3.82 (2 H, s), 3.95 (1 H, m),
4.19 (1 H, m), 4.54 (1 H, dd), 4.99 (1 H, t), 5.17 (1 H, d, J )
4.29 Hz), 5.49 (1 H, d, J ) 5.61 Hz), 5.93 (1 H, d, J ) 5.28 Hz),
7.26-7.34 (5 H, m), 8.58 (1 H, s), 10.86 (1 H, bs); 13C NMR
(67.8 MHz, DMSO) δ 15.89, 22.28, 41.24, 59.25, 68.36, 71.95,
83.69, 85.19, 117.34, 124.55, 125.77, 126.29, 127.40, 133.68,
140.38, 147.80, 149.97, 156.66, 167.12. Anal. Calcd for
C
21H22N6O5S: C, 53.61; H, 4.71; N, 17.86; S, 6.81. Found: C,
52.62; H, 4.80; N, 17.34; S, 7.94.
6-S-(2-Cyan oeth yl)-5′-O-(4,4′-dim eth oxytr ityl)-2-N-ph en -
yla cetyl-6-th iogu a n osin e (15). Compound 14 (613 mg, 1.3
mmol) was rendered anhydrous three times with dry pyridine
and dissolved in dry pyridine (13 mL). To the mixture was
added 4,4′-dimethoxytrityl chloride (485 mg, 1.43 mmol). After
being stirred at room temperature for 18 h, the mixture was
diluted with CH2Cl2. The CH2Cl2 solution was washed three
times with sat. NaHCO3. The organic layer was dried over
Na2SO4 and evaporated under reduced pressure. The residue
was chromatographed on a column of silica gel (20 g) eluted
with CH2Cl2-MeOH (100:0-99:1) to give 15 (863 mg, 86%):
1H NMR (270 MHz, CDCl3) δ 2.80 (2 H, t, J ) 7.25 Hz), 3.18,
3.35 (2 H, m), 3.53 (2 H, m), 3.75 (6 H, s), 3.82 (2 H, s), 4.39 (1
H, m), 4.48 (1 H, m), 4.96 (1 H, m), 5.91 (1 H, d, J ) 6.27 Hz),
6.67 (4 H, m), 7.06-7.43 (18 H, m), 8.14 (1 H, s); 13C NMR
(67.8 MHz, CDCl3) δ 18.71, 24.51, 40.05, 55.11, 63.78, 73.80,
86.54, 87.01, 92.06, 112.99, 118.17, 123.74, 125.23, 126.77,
127.69, 127.82, 128.14, 128.57, 128.95, 129.20, 129.40, 129.79,
129.85, 133.41, 135.26, 135.29, 141.24, 144.12, 148.30, 149.65,
150.89, 158.38, 158.42, 160.20, 169.16. Anal. Calcd for
6-S-(2-Cya n oeth yl)-2′-O-(ter t-bu tyld im eth ylsilyl)-2-N-
p h en yla cetyl-6-th iogu a n osin e (17). To a solution of com-
pound 16 (0.2 mmol, 177 mg) in CH2Cl2 was added dichloro-
acetic acid (250 µL). After being stirred at room temperature
for 2 min, the mixture was washed three times with sat.
NaHCO3. The organic layer was collected, dried over Na2SO4,
filtered, and evaporated under reduced pressure. The residue
was chromatographed on a column of silica gel (5 g) eluted
with CHCl3-MeOH (99:1-98:2, v/v) to give 17 (106 mg, 91%):
1H NMR (270 MHz, CDCl3) δ -0.35, -0.13 (6 H, s), 0.81 (9 H,
s), 2.99 (2 H, t, J ) 6.93 Hz), 3.50 (2 H, m), 3.71-3.94 (2 H,
m), 3.84 (2 H, s), 4.30 (1 H, s), 4.36 (1 H, d, J ) 4.95 Hz). 5.96
(1 H, dd, J ) 6.93 Hz, J ) 4.95 Hz), 5.74 (1 H, d, J ) 7.26 Hz),
7.32-7.45 (5 H, m), 7.89 (1 H, s), 7.91 (1 H, bs); 13C NMR (67.8
MHz, CDCl3) δ -5.39, -5.35, 17.67, 18.33, 24.85, 25.38, 44.78,
C
42H40N6O7S: C, 65.27; H, 5.22; N, 10.87; S, 4.51. Found: C,
65.70; H, 5.44; N, 10.12; S, 4.21.
6-S-(2-Cya n oeth yl)-2′-O-(ter t-bu tyld im eth ylsilyl)-5′-O-
(4,4′-d im e t h oxyt r it yl)-2-N -p h e n yla ce t yl-6-t h iogu a n o-
sin e (16). A mixture of compound 15 (1.55 g, 2 mmol) and
imidazole (200 mg, 3 mmol) was rendered anhydrous by
coevaporation three times each with dry pyridine and dry
toluene. The mixture was dissolved in dry DMF (20 mL) and
TBDMSCl (360 mg, 2.4 mmol) was added. After being stirred
at room temperature for 16 h, the mixture was diluted with
EtOAc. The EtOAc solution was washed three times with satd

5112 J . Org. Chem., Vol. 65, No. 17, 2000
Kadokura et al.
62.55, 71.97, 74.46, 77.18, 86.69, 90.42, 118.22, 127.57, 129.06,
129.31, 129.38, 133.68, 142.61, 148.48, 150.99, 160.99, 168.48.
Anal. Calcd for C27H36N6O5SSi: C, 55.46; H, 6.20; N, 14.37; S,
5.48. Found: C, 55.22; H, 6.17; N, 14.31; S, 5.30.
6-S-(2-Cya n oet h yl)-5′-O-(4,4′-d im et h oxyt r it yl)-6-t h io-
in osin e (23). To a solution of 22 (1.29 g, 2 mmol) in CH2Cl2
(40 mL) were added N-methylpyrrolidine (1.8 mL, 20 mmol)
and 2-cyanoethanethiol (2.04 mL, 20 mmol). After being stirred
at room temperature for 2 h, the mixture was diluted with
CH2Cl2 (150 mL). The CH2Cl2 solution was washed three times
with 1 M KH2PO4 buffer. The organic phase was dried over
Na2SO4 and evaporated under reduced pressure. The residue
was chromatographed on a column of silica gel (60 g) eluted
with CH2Cl2-MeOH (99:1-98:2) to give 23 (2.47 g, 96%): 1H
NMR (270 MHz, CDCl3) δ 2.91 (2 H, t, J ) 7.26), 3.31 (1 H,
dd, J ) 3.30 Hz, J ) 10.56 Hz), 3.45 (1 H, dd, J ) 3.30 Hz, J
) 10.56 Hz), 3.61 (2 H, t, J ) 7.26), 3.76 (6 H, s), 4.40-4.46 (2
H, m), 4.83 (1 H, t, J ) 5.28 Hz), 6.02 (1 H, d, J ) 5.61), 6.72-
6.75 (4 H, m), 7.16-7.28 (9 H, m), 8.24 (1 H, s), 8.66 (1 H, s);
13C NMR (67.8 MHz, CDCl3) δ 18.42, 21.30, 24.28, 53.55, 55.06,
63.24, 71.63, 75.15, 85.09, 86.47, 89.70, 113.01, 117.97, 123.92,
125.12, 126.79, 127.71, 127.87, 128.05, 128.86, 129.83, 131.41,
135.31, 141.85, 144.21, 147.82, 148.81, 151.39, 158.38, 159.10.
Anal. Calcd for C34H33N5O6S: C, 63.84; H, 5.20; N, 10.95; S,
5.06. Found: C, 65.18; H, 5.39; N, 10.20; S, 4.64.
6-S-(2-Cya n oeth yl)-2′-O-(ter t-bu tyld im eth ylsilyl)-5′-O-
(4,4′-d im eth oxytr ityl)-6-th ioin osin e (24). A mixture of
compound 23 (1.29 g, 2 mmol) and imidazole (204 mg, 3 mmol)
was rendered anhydrous by coevaporation three times each
with dry pyridine and dry toluene, and finally dissolved in dry
DMF (20 mL). To the mixture was added TBDMSCl (361 mg,
2.4 mmol). After being stirred at room temperature for 24 h,
the mixture was diluted with ethyl acetate (150 mL), and the
solution was washed three times with sat. NaHCO3. The
organic phase was dried over Na2SO4 and evaporated under
reduced pressure. The residue was chromatographed on a
column of silica gel (WAKO C-300, 100 g) eluted with hex-
anes-Et2O (50:50, v/v) to give 24 (414 mg, 27%). The 3′-
silylated isomer (24′) was eluted with hexanes-Et2O (30:70-
20:80, v/v) to give 24′ (614 mg, 41%): Analytical data for 24:
1H NMR (270 MHz, CDCl3) δ -0.13, 0.02 (6 H, s), 0.86 (9 H,
s), 2.96 (2 H, t, J ) 7.09 Hz), 3.41 (1 H, dd, J ) 3.96 Hz, J )
10.56 Hz), 3.53 (1 H, dd, J ) 2.97 Hz, J ) 10.56 Hz), 3.63 (2
H, t, J ) 6.16), 3.80 (6 H, s), 4.28 (1 H, m), 4.38 (1 H, m), 5.01
(1 H, t, J ) 4.95 Hz), 6.09 (1 H, d, J ) 5.28 Hz), 6.82 (4 H, d,
J ) 8.91 Hz), 7.22-7.46 (9 H, m), 8.22 (1 H, s), 8.64 (1 H, s);
13C NMR (67.8 MHz, CDCl3) δ -5.30, -5.10, 17.70, 18.48,
24.28, 25.38, 55.02, 63.16, 71.34, 75.53, 84.08, 86.51, 88.23,
113.05, 117.95, 125.77, 127.73, 127.91, 129.88, 131.63, 135.38,
141.87, 144.37, 148.46, 151.75, 158.40, 158.83. Anal. Calcd for
Tetr a k is(2-cya n oeth yl) Ester of 6-S-(2-Cya n oeth yl)-2′-
O-(ter t-bu tyldim eth ylsilyl)-6-th iogu an osin e 3′,5′-Bisph os-
p h a tes (19). Compound 17 (58 mg, 0.1 mmol) was rendered
anhydrous by coevaporation three times each with dry pyri-
dine, dry toluene and dry CH3CN. The residue was dissolved
in dry CH3CN (1 mL). To the solution were added bis(2-
cyanoethyl) (N,N-diisopropyl) phosphoramidite (81 mg, 0.3
mmol) and 1H-tetrazole (32 mg, 0.45 mmol). The mixture was
stirred at room temperature for 1 h and then tert-butyl
hydroperoxide (0.3 mL, 3 mmol) was added. After 30 min, the
solution was diluted with CH2Cl2 and washed with sat.
NaHCO3. The organic layer was dried over Na2SO4, filtered,
and evaporated under reduced pressure. The residue was
chromatographed on a column of silica gel (3 g) eluted with
CH2Cl2-MeOH (97:3, v/v) to give 19 (75 mg, 78%): 1H NMR
(270 MHz, CDCl3) δ -0.02, -0.00 (6 H, s), 0.78 (9 H, s), 2.76-
2.81 (8 H, m), 3.04-3.12 (2 H, m), 3.46-3.76 (2 H, m), 3.84 (2
H, s), 4.29-4.38 (8 H, m), 4.50-4.58 (3 H, m), 5.18 (1 H, m),
5.40 (1 H, m), 5.81 (1 H, d, J ) 5.94 Hz), 7.29-7.41 (5 H, m),
7.92 (1 H, s), 9.35 (1 H, s); 13C NMR (67.8 MHz, CDCl3) δ
-5.37, -5.08, 17.76, 18.44, 19.34, 19.63, 24.89, 25.30, 25.54,
44.44, 62.28, 62.36, 62.61, 62.66, 66.92, 71.75, 77.18, 80.99,
89.33, 116.48, 116.55, 116.64, 118.53, 127.12, 128.68, 129.27,
134.65, 142.62, 149.00, 151.79, 160.50, 168.84; 31P NMR (109.4
MHz, CDCl3) δ -2.55, -1.98.
6-Th iogu a n osin e 3′,5′-Bisp h osp h a tes (20). To a solution
of compound 19 (75 mg, 0.078 mmol) in dry pyridine (7.8 mL)
were added DBU (70 µL, 0.47 mmol) and BSA (723 µL, 2.93
mmol). After being stirred at room temperature for 1.5 h, the
mixture was diluted with H2O. The aqueous solution was
washed three times with Et2O. The aqueous phase was
collected and evaporated under reduced pressure. The residue
was treated with 25% ammonia-EtOH (40 mL 3:1,v/v) at 55
°C for 3 h. After the solution was evaporated under reduced
pressure, the residue was diluted with water, washed three
times with CH2Cl2, and evaporated under reduced pressure.
The residue was rendered anhydrous by coevaporations three
times each with dry pyridine and dry toluene. To the anhy-
drous mixture was added triethylamine trihydrofluoride (629
mg, 3.9 mmol). After being stirred at room temperature for
16 h, the mixture was added dropwise to 2 M TEAB buffer (2
mL). The reaction mixture was purified by DEAE Sephadex
A-25 (1 M ammonium bicarbonate). The elution followed by
lyophilization gave 20 (1144 A338.5, 59%); 1H NMR (270 MHz,
D2O) δ 3.94 (2 H, m), 4.45 (1 H, m), 4.66 (2 H, m), 4.77 (1 H,
m), 5.93 (1 H, d, J ) 6.93 Hz), 8.28 (1 H, s); 13C NMR (67.8
MHz, D2O) δ 63.91, 73.81, 73.90, 84.42, 86.25, 129.01, 138.77,
148.30, 157.24, 176.69; 31P NMR (67.8 MHz, CDCl3) δ 4.70,
4.88; FAB calcd for C10H12N5Na4O10P2S m/z 547.94, obsd for
C
40H47N5O6SSi H2O: C, 62.23; H, 6.40; N, 9.07; S, 4.25.
Found: C, 61.95; H, 6.78; N, 9.80; S, 4.29. Analytical data for
24′: 1H NMR (270 MHz, CDCl3) δ 0.00, 0.16 (6 H, s), 0.89 (9
H, s), 2.93 (2 H, t, J ) 7.26 Hz), 3.14 (1 H, d, J ) 6.59 Hz)
3.25 (1 H, d,J ) 6.94 Hz), 3.50 (1 H, d, J ) 7.59 Hz), 3.61 (2
H, t, J ) 7.26 Hz), 3.78 (6 H, s), 4.19 (1 H, m), 4.59 (1 H, m),
4.74 (1 H, m), 6.03 (1 H, d, J ) 4.62 Hz), 6.80 (4 H, d, J ) 8.59
Hz), 7.22-7.46 (9 H, m), 8.24 (1 H, s), 8.68 (1 H, s); 13C NMR
(67.8 MHz, CDCl3) δ -4.75, -4.59, 18.11, 18.77, 24.54, 25.64,
25.78, 55.32, 62.76, 72.12, 74.69, 84.67, 86.70, 88.70, 89.41
113.27, 127.05, 127.97, 128.17, 130.07, 132.09, 135.59, 135.64,
142.22, 144.41, 148.63, 152.00, 158.68, 159.13. Anal. Calcd for
C
10H12N5Na4O10P2S m/z 547.95; UV (H2O) λmax 254 nm, 338.5
nm, λmin 240.5 nm, 288.5 nm.
6-Ch lor o-5′-O-(4,4′-d im et h oxyt r it yl)p u r in e R ib osid e
(22). Compound 21 (880 mg, 3.07 mmol) was rendered
anhydrous by coevaporation three times with dry pyridine. The
residue was dissolved in dry pyridine (30 mL), and DMTrCl
(1.25 g, 3.68 mmol) was added. After being stirred at room
temperature for 6 h, the mixture was diluted with CH2Cl2 and
washed three times with satd Na2CO3. The organic layer was
dried over Na2SO4 and evaporated under reduced pressure.
The residue was chromatographed on a column of silica gel
(30 g) eluted with CH2Cl2-MeOH (99.5:0.5-99:1) to give 22
(1.69 g, 94%): 1H NMR (270 MHz, CDCl3) δ 3.33 (1 H, dd, J )
3.30 Hz, J ) 10.89 Hz), 3.46 (1 H, dd, J ) 3.63 Hz, J ) 10.53
Hz), 3.77 (6 H, s), 4.42-4.48 (2 H, m), 4.88 (1 H, t, J ) 5.28
Hz), 6.05 (1 H, d, J ) 5.61 Hz), 6.74 (4 H, m), 7.15-7.31 (9 H,
m), 8.38 (1 H, s), 8.73 (1 H, s); 13C NMR (67.8 MHz, CDCl3) δ
55.17, 63.34, 72.09, 75.49, 85.63, 86.72, 90.50, 113.14, 123.94,
125.25, 126.95, 127.84, 127.91, 128.18, 128.99, 129.90, 135.31,
136.46, 143.90, 144.21, 149.24, 150.94, 151.63, 151.96, 158.54.
Anal. Calcd for C31H29N4O6SCl: C, 63.21; H, 4.96; N, 9.51; Cl,
6.02. Found: C, 64.44; H, 5.24; N, 9.08; Cl, 5.71.
C
40H47N5O6SSi‚1/2H2O: C, 62.97; H, 6.34; N, 9.18. Found: C,
62.91; H, 6.22; N, 8.93.
6-S-(2-Cyan oeth yl)-2′-O-(ter t-bu tyldim eth ylsilyl)-6-th io-
in 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 (26). Compound 24 (60 mg, 0.08 mmol) was rendered
anhydrous by coevaporation three times each with dry pyri-
dine, dry toluene and dry THF, and finally dissolved in dry
THF (0.24 mL). To the solution were added N-methylimidazole
(3 µL, 0.04 mmol), collidine (64 µL, 0.48 mmol) and chloro(2-
cyanoethoxy) (N,N-diisopropylamino)phosphine (35 µL, 0.16
mmol) under argon atmosphere. The resulting mixture was
stirred at room temperature for 40 min. The mixture was
diluted with CH2Cl2 (25 mL) and washed three times with satd
NaHCO3. The organic layer was collected, dried over Na2SO4,
filtered, and evaporated under reduced pressure. The residue
was chromatographed on a column of silica gel (5 g) eluted
with hexanes-EtOAc-Et3N (80:20:0.5, v/v/v) to give 26 (76

Donor Molecules for RNA Ligation
J . Org. Chem., Vol. 65, No. 17, 2000 5113
mg, 76%): 1H NMR (270 MHz, CDCl3) δ -0.22 -0.21 , -0.04
-0.02 (6 H, s), 0.75 (9 H, s), 1.03-1.25 (9 H, m), 2.29 2.65
(2 H, t, J ) 6.27 Hz), 2.95 (2 H, t, J ) 7.26), 3.29-3.96 (14 H,
m), 4.35-4.43 (2 H, m), 5.05 (1 H, m), 6.02 6.08 (1 H, d, J )
6.26 Hz), 6.79-7.48 (13 H, m), 8.21 8.24 (1 H, s), 8.60 8.24
(1 H, s); 13C NMR (67.8 MHz, CDCl3) δ -5.17, -4.67, 17.81,
17.86, 18.67, 19.98, 20.09, 20.36, 20.45, 22.54, 24.44, 24.58,
24.67, 24.76, 25.43, 25.54, 29.22, 31.70, 42.81, 42.98, 43.25,
43.45, 53.73, 55.20, 57.40, 57.72, 58.87, 63.09, 63.25, 72.54,
72.76, 73.21, 73.35, 74.48, 75.22, 77.20, 83.81, 84.22, 86.56,
86.72, 88.11, 88.39, 89.02, 113.12, 113.17, 117.23, 117.54,
118.13, 126.92, 127.30, 127.85, 127.89, 128.03, 128.16, 130.01,
130.06, 130.10, 131.84, 131.91, 135.42, 135.47, 135.62, 135.67,
1142.19, 142.30, 144.40, 144.53, 148.75, 151.84, 158.51, 158.71;
31P NMR (109.4 MHz, CDCl3) δ 149.69, 151.64. Anal. Calcd
for C49H64N7O7SSiP: C, 61.68; H, 6.76; N, 10.28; S, 3.36.
Found: C, 61.73; H, 7.14; N, 9.67; S, 3.64.
6-Th ioin osin e 3′,5′-Bisp h osp h a te (28). To a solution of
compound 27 (83 mg, 0.1 mmol) in dry pyridine (10 mL) were
added DBU (90 µL, 0.6 mmol) and BSA (930 µL, 3.75 mmol).
After being stirred at room temperature for 1.5 h, the mixture
was diluted with H2O. The aqueous solution was washed three
times with Et2O. The aqueous layer was collected and evapo-
rated under reduced pressure. The residue was rendered
anhydrous by coevaporation three times each with dry pyridine
and dry toluene. To the anhydrous mixture was added tri-
ethylamine trihydrofluoride (806 mg, 5 mmol). After being
stirred at room temperature for 16 h, the mixture was added
dropwise to 2 M TEAB buffer (2 mL). The reaction mixture
was purified by DEAE Sephadex A-25 (1 M ammonium
bicarbonate). The eluate was lyophilized to give 28 (1467 A311
,
75%): 1H NMR (270 MHz, D2O) δ 4.00 (2 H, m), 4.49 (1 H, m),
4.70-4.78 (2 H, m), 6.12 (1 H, d, J ) 6.27 Hz), 8.29 (1 H, s),
8.67 (1 H, s); 13C NMR (67.8 MHz, D2O) 63.80 (J
) 3.66
(COP)
Hz), 73.55 (J (COP) ) 3.66 Hz), 74.62, 84.57 (J (CCOP) ) 6.54 Hz),
87.33, 134.93, 142.15, 144.56, 146.23, 175.09; 31P NMR (109.4
MHz, CDCl3) δ 3.77, 4.30; FAB calcd for C10H11N4Na4O10P2S
m/z 532.93, obsd 532.93; UV (H2O) λmax 228.5 nm, 311 nm, λmin
213 nm, 256 nm.
6-S-(2-Cyan oeth yl)-2′-O-(ter t-bu tyldim eth ylsilyl)-6-th io-
in osin e (25). To a solution of compound 24 (151 mg, 0.2 mmol)
in CH2Cl2 (9.75 mL) was added dichloroacetic acid (250 µL).
After being stirred at room temperature for 2 min, the mixture
was washed three times with sat. NaHCO3. The organic layer
was collected, dried over Na2SO4, filtered, and evaporated
under reduced pressure. The residue was chromatographed
on a column of silica gel (3 g) eluted with CHCl3-MeOH (99.5:
0.5-99:1, v/v) to give 25 (85 mg, 92%): 1H NMR (270 MHz,
CDCl3) δ -0.18, -0.42 (6 H, s), 0.79 (9 H, s), 2.95 (2 H, t, J )
7.26 Hz), 3.63 (2 H, m), 3.78, 3.96 (2 H, m), 4.34-4.36 (2 H,
m), 5.08 (1 H, dd, J ) 7.26 Hz, J ) 4.95 Hz), 5.81 (1 H, d, J )
7.26 Hz), 8.01 (1 H, s), 8.71 (1 H, s); 13C NMR (67.8 MHz,
CDCl3) δ -5.59, -5.48, 17.59, 18.44, 24.33, 25.29, 62.95, 72.38,
74.39, 87.26, 90.91, 117.81, 132.70, 143.22, 147.21, 151.11,
160.49. Anal. Calcd for C19H29N5O4SSi‚1/2H2O: C, 49.54; H,
6.56; N, 15.20; S, 6.96. Found: C, 49.45; H, 6.11; N, 14.89; S,
6.84.
Liga tion of m 32,2,7G5′p p p Am Um A w ith Th ion u cleosid e
3′,5′-Bisp h osp h a tes Usin g T4 RNA Liga se. Ligation accep-
tor 30 (1.7A259) and donor pNp [N ) 4sU (6.8 A330), 6sI (7.84
A
330), 6sG (9.92 A330), C (3.64 A259), or U (4.0 A259)] were
dissolved in 50 mM Tris-HCl (pH 8.0, 200 µL) containing 20
mM ZnCl2, 5 mM DTT and 8 mM ATP. T4 RNA ligase (70
units, 10 unit/µL) in glycerin-water (1:1,v/v) was added, and
the resulting mixture was incubated at 8 °C. After 48 h, the
reaction mixture was heated at 100 °C for 3 min and cooled to
room temperature. Calf intestinal alkaline phosphatase (50
unit, 1 unit/µL) was added, and the resulting solution was incu-
bated at 37 °C. After being incubated for 2 h, the mixture was
heated at 90 °C for 2 min. The mixture was chromatographed
on anion exchange HPLC, and the peak of m32,2,7G5′pppAm-
UmAN was collected. The fractions collected were lyophilized,
and the contaminated salts were removed by gel filtration
using Sephadex G-15 (17 mm × 100 cm). The eluent was
lyophilized to give the desired ligated product. The yields of
the ligated products 31a -e are listed in Table 1.
Tetr a k is(2-cya n oeth yl) Ester of 6-S-(2-Cya n oeth yl)-2′-
O-(ter t-bu tyld im eth ylsilyl)-6-th ioin osin e 3′,5′-Bisp h os-
p h a te (27). Compound 25 (138 mg, 0.3 mmol), which was
rendered anhydrous three times each with dry pyridine, dry
toluene and dry CH3CN, was dissolved in dry CH3CN (5 mL).
To the solution were added bis(2-cyanoethoxy) (N,N-diisopro-
pyl)phosphoramidite (244 mg, 0.9 mmol) and 1H-tetrazole (95
mg, 1.35 mmol). The mixture was stirred at room temperature
for 1 h and tert-butyl hydroperoxide (0.9 mL, 9 mmol) was
added. After 30 min, the solution was diluted with CH2Cl2 (50
mL) and washed with sat. NaHCO3. The organic layer was
evaporated under reduced pressure. The residue was chro-
matographed on a column of silica gel (10 g) eluted with
CH2Cl2-MeOH (97:3, v/v) to give 27 (174 mg, 70%): 1H NMR
(270 MHz, CDCl3) δ -0.07, 0.06 (6 H, s), 0.83 (9 H, s), 2.76-
2.85 (4 H, m), 3.62 (2 H, m), 4.28-4.41 (8 H, m), 4.48-4.61 (3
H, m), 5.11-5.15 (2 H, m), 6.03 (1 H, d, J ) 4.29), 8.28 (1 H,
s), 8.73 (1 H, s); 13C NMR (67.8 MHz, CDCl3) δ -5.39, -5.19,
17.69, 18.46, 19.41, 19.52, 24.19, 25.23, 25.43, 62.50, 62.57,
62.68, 62.73, 65.99, 73.32, 75.22, 77.20, 80.36, 88.84, 116.46,
116.60, 118.06, 131.79, 141.98, 148.12, 151.77, 159.03; 31P
NMR (109.4 MHz, CDCl3) δ -1.85, -1.94. Anal. Calcd for
C31H43N9O10SSiP2‚2H2O: C, 43.30; H, 5.51; N, 14.66; S, 3.73.
Found: C, 43.73; H, 5.33; N, 13.17; S, 5.09.
En zym a tic Digestion of m 32,2,7G5′p p p Am Um AN (31a ,
31b, 31c) w ith Nu clea se P 1. The ligation products (31a :0.76,
31b:0.18, 31c:0.30 A259) was dissolved in 20 mM AcOH-
AcONa (pH 5.3, 100 µL) containing 0.1 mM ZnCl2. Nuclease
P1 (4 units, 1 unit/µL) in glycerin-water (1:1, v/v) was added
and the resulting mixture was incubated at 50 °C. After being
incubated for 5 h, the mixture was heated at 100 °C for 3 min.
The mixture was analyzed by anion-exchange HPLC. The
HPLC profiles are shown in Figure 3.
Ack n ow led gm en t. This work was supported by a
Grant from the “Research for the Future” Program of
the J apan Society for the Promotion of Science (J SPS-
RFTF97I00301) and a Grant-in-Aid for Scientific Re-
search from the Ministry of Education, Science, Sports
and Culture, J apan. We thank Dr. Sei-ichiro Higashiya
for his measurement of FAB mass.
J O991432Z