Studies on steric and electronic control of 2'-3' phosphoryl migration in 2'-phosphorylated uridine derivatives and its application to the synthesis of 2'-phosphorylated oligouridylates

Article abstract of DOI:10.1021/jo952073k

For the synthesis of 2'-phosphorylated oligouridylates by use of new phosphoramidite building units, several masked phosphoryl groups have been examined as 2'-phosphate precursors, which should not be migrated to the 3' position when the 3' hydroxy protecting group must be removed to introduce a phosphoramidite residue into the 3'-position. As a consequence, bis(2-cyano-1,1-dimethylethoxy)thiophosphoryl (BCMETP) was found to be the most suitable 2'-phosphate precursor. This thiophosphoryl group could be introduced into the 2'-hydroxyl of 3',5'-silylated uridine derivative 7 by phosphitylation with bis(2-cyano-1,1-dimethylethoxy)(diethylamino)phosphine followed by sulfurization. Treatment of the 2'-thiophosphorylated product 15 with (HF)(x)·Py in THF gave exclusively the 3',5'-unprotected uridine derivative 16a. Compound 16a was converted to the phosphoramidite unit 22 via a two-step reaction. This building block was used for the solution phase synthesis of U(2'-p)pU (29) and U(2'-ps)pU (30). Both the 2-cyano-1,1-dimethylethyl and 2-cyanoethyl groups were effectively removed from the fully protected derivative 25 by treatment with DBU in the presence of N,O-bis(trimethylsilyl)acetamide (BSA). The resulting 2'-thiophosphoryl group was successfully converted to a phosphoryl group by iodine treatment to give U(2'-p)pU (29). U(2'-ps)pU (30) was also synthesized by a modified procedure without the iodine treatment. Reaction of 29 with a new biotinylating reagent in aqueous solution in the presence of MgCl₂ gave a biotin-labeled product 35 having a pyrophosphate bridge at the 2' position. Reaction of 30 with monobromobimane gave the 2'-S-alkylated product 33 in aqueous solution. Application of the phosphoramidite unit 22 to the solid phase synthesis using aminopropyl CPG gel gave successfully [U(2'-p)p](n)U (n = 1, 3, 5). It was found that stability of the succinate linker between the CPG and oligouridylates was unaffected by the treatment with DBU when BSA was present. Several enzymatic properties of the synthetic 2'-phosphorylated and 2'-thiophosphorylated oligouridylates are also described.

Full text of DOI:10.1021/jo952073k

J . Org. Chem. 1996, 61, 4087-4100
4087
Stu d ies on Ster ic a n d Electr on ic Con tr ol of 2′-3′ P h osp h or yl
Migr a tion in 2′-P h osp h or yla ted Ur id in e Der iva tives a n d Its
Ap p lica tion to th e Syn th esis of 2′-P h osp h or yla ted Oligou r id yla tes
Mitsuo Sekine,* Hiroyuki Tsuruoka, Shin Iimura, Hiroshi Kusuoku, and Takeshi Wada
Department of Life Science, Tokyo Institute of Technology, Nagatsuta, Midoriku, Yokohama 226, J apan
Kiyotaka Furusawa
Research Institute for Polymers and Textiles, Yatabe Higashi 1-1-4, Tsukuba, Ibaraki 305, J apan
Received November 27, 1995X
For the synthesis of 2′-phosphorylated oligouridylates by use of new phosphoramidite building units,
several masked phosphoryl groups have been examined as 2′-phosphate precursors, which should
not be migrated to the 3′ position when the 3′ hydroxy protecting group must be removed to introduce
a phosphoramidite residue into the 3′-position. As a consequence, bis(2-cyano-1,1-dimethylethoxy)-
thiophosphoryl (BCMETP) was found to be the most suitable 2′-phosphate precursor. This
thiophosphoryl group could be introduced into the 2′-hydroxyl of 3′,5′-silylated uridine derivative
7 by phosphitylation with bis(2-cyano-1,1-dimethylethoxy)(diethylamino)phosphine followed by
sulfurization. Treatment of the 2′-thiophosphorylated product 15 with (HF)x‚Py in THF gave
exclusively the 3′,5′-unprotected uridine derivative 16a . Compound 16a was converted to the
phosphoramidite unit 22 via a two-step reaction. This building block was used for the solution
phase synthesis of U(2′-p)pU (29) and U(2′-ps)pU (30). Both the 2-cyano-1,1-dimethylethyl and
2-cyanoethyl groups were effectively removed from the fully protected derivative 25 by treatment
with DBU in the presence of N,O-bis(trimethylsilyl)acetamide (BSA). The resulting 2′-thiophos-
phoryl group was successfully converted to a phosphoryl group by iodine treatment to give U(2′-
p)pU (29). U(2′-ps)pU (30) was also synthesized by a modified procedure without the iodine
treatment. Reaction of 29 with a new biotinylating reagent in aqueous solution in the presence of
MgCl2 gave a biotin-labeled product 35 having a pyrophosphate bridge at the 2′ position. Reaction
of 30 with monobromobimane gave the 2′-S-alkylated product 33 in aqueous solution. Application
of the phosphoramidite unit 22 to the solid phase synthesis using aminopropyl CPG gel gave
successfully [U(2′-p)p]nU (n ) 1, 3, 5). It was found that stability of the succinate linker between
the CPG and oligouridylates was unaffected by the treatment with DBU when BSA was present.
Several enzymatic properties of the synthetic 2′-phosphorylated and 2′-thiophosphorylated oligou-
ridylates are also described.
Sch em e 1
In tr od u ction
In 1981, Konarska et al. discovered an unprecedented
2′-phosphomonoester, 3′-5′ phosphodiester linkage at the
ligation site of a circular RNA which was formed by
intramolecular cyclization of a linear RNA fragment Ω73
with wheat germ extracts having a unique RNA ligase
capability.1,2 A similar structure was also found in a
circular RNA formed by ligation of a linearized viroid
RNA by use of wheat germ or Chlamidomonas extracts.3
Abelson et al. reported that the 38th G at the splice
junction of the spliced yeast pre-tRNALeu3 is phosphory-
lated at the 2′-position.4 Later, such 2′-phosphomo-
noester, 3′-5′ phosphodiester linkages have been gener-
ally found in a number of spliced pre-tRNAs (Scheme
1).5,6
The 2′-phosphate group is removed by a tRNA specific
phosphatase with the help of NAD to give mature
tRNAs.7 Phizicky has recently clarified the mechanism
of this dephosphorylation, where the 2′-phosphate group
attacks the anomeric carbon with elimination of the
nicotinamide residue to produce an ADP-ribosylated 2′-
phosphorylated tRNA intermediate which, in turn, docom-
poses to the normal tRNA and a 1′′,2′′-cyclic phosphate
derivative of ADP-ribose.8 Although the biological mean-
ing and role of the 2′-phosphate group in the structure-
X Abstract published in Advance ACS Abstracts, May 1, 1996.
(1) Konarska, M.; Filipowicz, W.; Domdey, H.; Gross, H. J . Nature
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S0022-3263(95)02073-1 CCC: $12.00 © 1996 American Chemical Society

4088 J . Org. Chem., Vol. 61, No. 12, 1996
Sekine et al.
Recently, Szostak and co-workers12 have reported
artificial RNA ribozymes capable of catalyzing RNA liga-
tion and ATP(-γS)-dependent (thio)phosphorylation. In-
terestingly, they found that these ribozymes thiophos-
phorylate their internal 2′-hydroxyls. More recently,
Szostak’s research group has reported reversetranscrip-
tion of RNA containing a 2′-thiophosphate at a unique
internal site with AMV and MMLV H- reverse tran-
scriptases, showing that these reverse transcriptases
pause before the 2′-thiophosphate.13
Sch em e 2
These intriguing findings about 2′-phosphorylated or
hitherto unknown 2′-thiophosphorylated RNAs led us to
explore the chemical synthesis of such RNAs. Previously,
we have reported the first method for the synthesis of
2′-phosphorylated oligoribonucleotides using the tert-
butyl group as the 2′-phosphate protecting group.14
In this paper, we report an improved method for the
synthesis of 2′-phosphorylated and 2′-thiophosphorylated
oligouridylates by use of highly effective steric and
electronic control of the 2′-3′ phosphoryl migration in
2′-phosphorylated ribonucleosides.
function relationships of tRNAs is of great interest, no
basic physicochemical studies have been reported regard-
ing 2′-phosphorylated RNAs (Scheme 2). The chemical
synthesis of this kind of RNAs is highly desired for such
studies.
Resu lts a n d Discu ssion
Str a tegies for th e Syn th esis of 2′-P h osp h or yla ted
RNAs. For the synthesis of 2′-phosphorylated RNAs,
there would be two main strategies: One is the approach
in which appropriately protected monomer units having
a 2′-masked phosphate group are used as starting
materials. In fact, we have employed this strategy in our
previous paper.14 This kind of method has also been
utilized by us for the synthesis of branched RNAs.15
The other involves the introduction of 2′-phosphate
groups into oligoribonucleotide blocks at a later stage. If
the protecting groups of the internucleotidic phosphates
and 2′-hydroxy groups can be selectively removed to
produce the unprotected 2′-hydroxyl group, one can
introduce a protected phosphoryl group at each 2′-
position in a manner similar to that used for the
synthesis of branched RNAs16 reported by Kierzek and
Caruthers,17 Chattopadhyaya,18 and Imbach.19 In fact,
quite recently, Kierzek20 has adopted this type of ap-
proach to synthesize N(2′-p)pN, which has been utilized
as a reference material for elucidation of the biochemical
course of NAD-promoted dephosphorylation of a 2′-
phosphorylated tRNA species,8 as mentioned in the
foregoing text.
On the other hand, much attention has recently been
paid to polyanion species such as cyclodextrin polysul-
fates9 and heteropolyacids,10 which have proved to have
potent anti-HIV activities. In connection with these
studies, oligoribonucleotides having 2′-phosphates at each
2′-position are of interest as a new type of polyanion
species.
In connection with recent studies in antisense chem-
istry,11 oligonucleotides having a 2′-phosphate group
would be used as a new tool for introduction of a variety
of functional groups such as fluoresence probes and biotin
by the use of the intrinsic property of a phosphomo-
noester type of 2′-phosphate group which can be selec-
tively modified.
(8) Culver, G. M.; McCraith, S. M.; Zillmann, M.; Kierzek, R.;
Michaud, M.; LaReau, R. D.; Turner, D. H.; Phizicky, E. M. Science
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(9) Mitsuya, H.; Looney, D. J .; Kuno, S.; Ueno, R.; Wong-Staal, F.;
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M.; de Clercq, E.; Yamamoto, N. Antiviral Res. 1989, 11, 233. For recent
papers see: Moriya, T.; Saito, K.; Kurita, H.; Matsumoto, K.; Otake,
T.; Mori, H.; Morimoto, M.; Ueba, N.; Kunita, N. J . Med. Chem. 1993,
36, 1674. Cartledge, J . D.; Midgley, J .; Youle, M.; Gazzard, B. G. J .
Antimicrob. Chemoth. 1994, 33, 1071. Otake, T.; Schols, D.; Witvrouw,
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Ueba, N.; de Clercq, E. Antiviral Chem. Chemoth. 1994, 5, 155 and
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(10) Inouye, Y.; Take, Y.; Yoshida, T.; Yamamoto, A.; Yamase, T.;
Nakamura, S. Chem. Pharm. Bull. 1990, 38, 285. Inouye, Y.; Tokutake,
Y.; Yoshida, T.; Yamamoto, A.; Yamase, T.; Nakamura, S. Chem.
Pharm. Bull. 1991, 39, 1638. Take, Y.; Tokutake, Y.; Inouye, Y.;
Yoshida, T.; Yamamoto, A.; Nakamura, S. Antiviral Res. 1991, 15, 113.
Yamamoto, N.; Schols, D.; de Clercq, E.; Debyser, Z.; Pauwels, R.;
Balzarini, J . Mol. Pharmacol. 1992, 42, 1109. Inouye, Y.; Tokutake,
Y.; Yoshida, T.; Seto, T.; Fujita, H.; Dan, K.; Yamamoto, A.; Nishiya,
S.; Yamase, T.; Nakamura, S. Antiviral Res. 1993, 20, 317. Yamase,
T. Polyoxometalates: From Plantonic Solids to Anti-retroviral Activity;
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The Netherlands, 1993; pp 335-356.
(11) For comprehensive reviews see: Wickstrom, E. Prospects for
Antisense Nucleic Acid Therapy of Cancer and Aids; Wiley-Liss: New
York, 1991. Murray, J . A. H. Antisense RNA and DNA; Wiley-Liss,
Inc.: New York, 1992. Crooke, S. T.; Lebleu, B. Antisense Research
and Applications; CRC Press: Boca Raton, FL, 1993. Agarwal, S.
Protocols for Oigonucleotides and Analogs; Humana Press: Totowa,
NJ , 1993. Sanghvi, Y. S.; Cook, P. D. Carbohydrate Modifications in
Antisense Research; American Chemical Society: Washington, DC,
1994. Knorre, D. G.; Vlassov, V. V.; Zarytova, V. F.; Lebedev, A. V.;
Federova, O. S. Design and Targeted Reactions of Oligonucleotide
Derivatives; CRC Press: Boca Raton, 1994. Crooke, S. T. Therapeutic
Applications of Oligonucleotides; Springer-Verlag: New York, 1995.
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1995, 23, 2811.
(14) Sekine, M.; Iimura, S.; Furusawa, K. J . Org. Chem. 1993, 58,
3204. Sekine, M.; Iimura, S.; Mori, T.; Tsuruoka, H.; Furusawa, K.
Nucleic Acids Symp. Ser. 1991, 25, 65.
(15) Sekine, M.; Hata, T. J . Am. Chem. Soc. 1985, 107, 5813. Sekine,
M.; Hata, T. Nucleic Acids Symp. Ser. 1985, 16, 185. Sekine, M.;
Heikkila¨, J .; Hata, T. Tetrahedron Lett. 1987, 28, 5691. Sekine, M.;
Heikkila¨, J .; Hata, T. Bull. Chem. Soc. J pn. 1991, 64, 588.
(16) For a recent comprehensive review see: Sekine, M. In Studies
in Natural Products Chemistry; Elsevier Sci.: New York, 1994; pp 283-
312. (b) Hayakawa, Y. J . Org. Synth. Chem. J pn. 1988, 46, 753.
(17) Kierzek, R.; Kopp, D. W.; Edmonds, M.; Caruthers, M. H.
Nucleic Acids Res. 1986, 14, 4751.
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A.; Remaund, G.; Chattopadhyaya, J . Tetrahedron 1987, 43, 4685.
Sund, C.; Fo¨ldesi, A.; Yamakage, A.; Agback, P.; Chattopadhyaya, J .
Tetrahedron 1991 47, 6305. Sund, C.; Agback, P.; Chattopadhyaya, J .
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28, 415. Huss, S.; Gosselin, G.; Imbach, J . L. J . Org. Chem. 1988, 53,
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Synthesis of 2′-Phosphorylated Oligouridylates
J . Org. Chem., Vol. 61, No. 12, 1996 4089
Sch em e 3
In our present study, we have focused our efforts on
the former strategy. In our approach, 3′,5′-unprotected
2′-monophosphorylated ribonucleoside derivatives are
required as key synthetic intermediates, but such species
are known to be extremely unstable to undergo easy
cyclization and/or migration of the phosphoryl group
owing to the neighboring group participation of the
proximal OH group.21-23 Regulation of the cyclization
and migration of such a phosphoryl group is a serious
problem, if this strategy is chosen. We have recently
found the first clue to a general solution to this long-
standing problem, namely, that treatment of a 2′-phos-
phorylated adenosine derivative 1 with (HF)x‚Py gave
exclusively a 3′,5′-unprotected product 2, without 2′-3′
migration of the 2′-(di-tert-butoxyphosphoryl) (BTBP)
group.14 On the basis of this result, a new class of RNAs,
i.e., 2′-O-phosphorylated oligoribonucleotides, A(2′-p)pU
and A(2′-p)pA(2′-p)pU, have been synthesized using the
BTBP group as the 2′-phosphate precursor (Scheme 3).14
However, deprotection of the two tert-butyl groups from
the BTBP group required rather drastic conditions (20%
trifluoroacetic acid in acetic acid at rt for 3-5 h) which
are apparently unsuitable for the synthesis of long
oligomers. In fact, attempts to synthesize [U(2′-p)p]6U
in the solid phase approach on a CPG gel by the use of a
newly prepared uridine phosphoramidite unit 4 having
the BTBP group at the 2′-position gave complex mixtures.
Another drawback in this method is the unsatisfactory
yield of 1. The reason why such a sluggish phosphity-
lation occurred is now rationalized in terms of Wa-
tanabe’s recent finding24 that 1H-tetrazole used as acti-
vator at the 2′-phosphitylation step partially decomposes
the resulting di-tert-butyl phosphite intermediate by
protonation with formation of a hydrogen phosphonate
derivative. Therefore, a more ideal strategy capable of
complete avoidance of the phosphoryl migration as well
as facile 2′-phosphitylation and deprotection should be
developed.
conditions used for removal of the tert-butyl group as well
as to avoid the protonation on the phosphorus by 1H-
tetrazole at the stage of phosphitylation, we have searched
for sterically hindered protecting groups which should
be removed under more suitable basic conditions. The
2-cyano-1,1-dimethylethyl (CME) group25 was first in-
troduced to nucleic acid chemistry as a sterically hindered
phosphate protecting group by van Boom, who showed
that the CME group could be removed by the action of
ammonia. It was expected that the presence of the two
electron-withdrawing cyano groups causes less protona-
tion on the phosphorus of a phosphite intermediate.
Quite recently, we have also reported an effective method
for simultaneous removal of the two CME groups from
bis(2-cyano-1,1-dimethylethyl) 5′-O-dimethoxytritylthy-
midine 3′-phosphate (5) by use of DBU in the presence
of N,O-bis(trimethylsilyl)acetamide (BSA), which gave
the deprotected material (6) after desilylation by addition
of water.26 Evans27 and Bartlett28 have reported similar
strategies for the simultaneous removal of phosphate-
protecting groups capable of â-elimination.
In consideration of these facts, a phosphitylating
reagent, bis(2-cyano-1,1-dimethylethoxy)(diethylamino)-
phosphine (CMEAP), was prepared in situ by the reaction
of dichloro(diethylamino)phosphine with 2-cyano-1,1-
dimethylethanol in an attempt to use this CME group
as the 2′-protecting group in our strategy mentioned
above. The purity of this reagent was about 60% (31P
NMR). Since the other byproducts were inert species
having a pentavalent phosphorus atom, this reagent
underwent smooth phosphitylation with nucleosides in
spite of its relatively low purity. Reaction of N3-benzoyl-
3′,5′-O-(di-tert-butylsilanediyl)uridine (7)29 with 1.2 equiv
of CMEAP in the presence of 1.8 equiv of 1H-tetrazole
in CH2Cl2 for 2 h followed by the oxidation of the
resulting phosphite intermediate 8 with tert-butyl hy-
(25) Marugg, J . E.; Dreef, C. E.; van der Marel, G. A.; van Boom, J .
H. Recl. Trav. Chim. Pays-Bas 1984, 103, 97.
(26) Sekine, M.; Tsuruoka, H.; Iimura, S.; Wada, T. Nat. Prod. Lett.
1994, 5, 41.
(27) Evans, D. A.; Gage, J . R.; Leighton, J . L. J . Org. Chem. 1992,
57, 1964.
Syn th esis of 2′-P h osp h or yla ted Oligou r id yla tes
by Use of th e BCMETP Gr ou p . To avoid the acidic
(21) Brown, D. M.; Magrath, D. I.; Todd, A. R. J . Chem. Soc. 1955,
4396.
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Res., Symp. Ser. 1987, 18, 269.
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Trans. 1 1992, 1879.
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P. A. J . Am. Chem. Soc. 1992, 114, 3535.
(29) The di-tert-butylsilanediyl group has been reported as a 3′,5′-
protecting group by Furusawa: Furusawa, K.; Ueno, K.; Katsura, T.
Chem. Lett. 1990, 97. Furusawa, K.; Katsura, T. Tetradedron Lett.
1985, 26, 887. Furusawa, K. Chem. Lett. 1989, 509. Frusawa, K. Chem.
Exp. 1991, 6, 763.

4090 J . Org. Chem., Vol. 61, No. 12, 1996
Sekine et al.
ion-mediated Si-O3′ bond cleavage, a slightly suppressed
phosphoryl migration was observed (Figure 1B) but the
yield of 10a ,b was considerably decreased to 46%.
Several efforts to isolate 10a free from 10b by silica gel
column chromatography have failed. In an attempt to
separate the two regioisomers 10a and 10b at the next
step, dimethoxytritylation of a mixture of 10a and 10b
was conducted (Scheme 4). However, it was impossible
to separate the new products 11a and 11b by chromato-
graphic techniques.
Therefore, we tried to suppress the 2′-3′ phosphoryl
migration by changing the electronic environment on the
phosphorus as electrophilic center to avoid intramolecu-
lar attack of the proximal 3′-OH group. It was thought
that displacement of the PdO bond with a PdS bond
would result in the suppression of the 2′-3′ phosphoryl
migration since sulfur is lesser electron negative than
oxygen so that the phosphorus should become a poorer
electrophilic center. It is also known that thiophosphoric
acid monoesters can be converted into phosphoric acid
monoesters by treatment with iodine in aqueous solu-
tion.33 Therefore, we synthesized 2′-phosphorylated oli-
goribonucleotides via an indirect route involving an
additional step of oxidative S f O conversion, using bis-
(2-cyano-1,1-dimethylethoxy)thiophosphoryl (BCMETP)
as the 2′-phosphoryl precursor.
However, it is unknown whether the CME group can
be completely removed from ROP(S)(OCME)2 (R ) nu-
cleoside residue) by treatment with DBU/BSA under
conditions similar to those used for removal of the CME
group from ROP(O)(OCME)2.26 Therefore, a model com-
pound 13 was synthesized from 12 to study detailed
conditions for complete removal of the two CME groups
to give compound 14.
It was found that both CME groups could be more
rapidly removed from 13 in 3 min than those of the
corresponding phosphoryl compound 5, which required
1.5 h as reported previously by us.26 This marked
difference in elimination of the CME group between 5
and 13 is explained by the difference in leaving ability
between the phosphoryl and thiophosphoryl residues of
the two silylated species, ROP(O)(OCME)(OSiMe3) and
ROP(S)(OCME)(OSiMe3), respectively. In general, di-
alkyl phosphorothioates have lesser pKa values by ap-
proximately 0.5 unit than the corresponding dialkyl
phosphates. Therefore, the (thiophosphoryl)oxy group
should be stronger as a leaving group than the phospho-
ryloxy group so that the CME group of 13 was eliminated
faster than that of 5. Since the oxygen has a stronger
electronegative atom than the sulfur, it seems that the
phosphoryloxy group should be more readily removable
than the (thiophosphoryl)oxy group. However, this is not
correct since a thiophosphate anion once generated can
be isomerized to a more stable tautomer in which an
anion charge is located at the sulfur atom. It is likely
that, because of this further stabilization, preferential
elimination of the thiophosphoryl residue takes place over
the phosphoryl residue.
F igu r e 1. 2′-3′ isomerization of the 2′-phosphoryl group of
9 and 15 upon desilylation by HF-pyridine. A: The 31P NMR
spectrum of the product obtained by desilylation of 9 with
(HF)x‚Py-Py in THF. B: The 31P NMR spectrum of the product
obtained by desilylation of 9 with (HF)x‚Py-Py in THF in the
presence of acetic acid. C: The 31P NMR spectrum of the
product obtained by desilylation of 15 with (HF)x‚Py-Py in
THF.
droperoxide30 gave the 2′-phosphorylated product 9.
Further treatment of 9 with (HF)x‚Py14 in THF at rt for
10 min gave a 95:5 mixture of 3′,5′-unprotected 2′-
phosphorylated species 10a and the 3′-regioisomer 10b
in an overall yield of 72% yield from 7. The 31P NMR
spectrum of the mixture is shown in Figure 1A.
Compared with the BTBP group, the bis(2-cyano-1,1-
dimethylethoxy)phosphoryl (BCMEP) group caused phos-
phoryl migration to a degree of 5% owing to addition of
two electron-withdrawing cyano groups to the BTBP
group at the â-positions. When removal of the di-tert-
butylsilanediyl (DTBS) group from 7 was carried out in
the presence of acetic acid31,32 as a proton source that
neutralizes the 3′-oxide ion generated upon the fluoride
Since simultaneous rapid removal of the CME groups
from 13 was achieved, sulfurization of intermediate 8
was examined under various conditions. Consequently,
34
treatment of 8 with S8 in CS2 gave the corresponding
(30) Engels J .; J a¨ger, A. Angew. Chem., Int. Ed. Engl. 1982, 21, 912.
Engels, J .; J a¨ger, A. Tetrahedron Lett. 1984, 25, 1437. Hayakawa, Y.;
Uchiyama, M.; Noyori, R. Tetrahedron Lett. 1986, 27, 4191.
(31) Inoue, H. University of Hokkaido, private communication.
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J . Med. Chem. 1991, 34, 2917.
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16, 3209.

Synthesis of 2′-Phosphorylated Oligouridylates
J . Org. Chem., Vol. 61, No. 12, 1996 4091
Sch em e 4
35
Sch em e 5
thiophosphorylated species 15. Et2NC(S)SS(S)NEt2
was less effective as the sulfurization reagent.
In situ desilylation of 15 thus formed was carried out.
As expected, treatment of 15 with (HF)x‚Py in THF
afforded exclusively the 2′-thiophosphorylated product
16a , as shown in Figure 1C. The overall yield of 16a
1
from 7 was 69%. The H, 13C, and 31P NMR spectra of
16a suggested that this compound was essentially free
from its 3′-regioisomer 16b.
To evaluate the importance of both the steric and
electronic effects of substituents, attached to the phos-
phorus, on the suppression of phosphoryl migration, a
2′-thiophosphorylated uridine derivative 17 having a less
hindered 2-cyanoethyl group than the CME group was
synthesized and treated similarly with (HF)x‚Py in THF.
Consequently, TLC analysis suggested that the 2′-thio-
phosphorylated product 18a was predominantly formed
over 18b when the reaction was completed, but very rapid
2′-3′ phosphoryl migration occurred even during extrac-
tion to give ultimately a nearly 1:1 mixture of 18a and
18b. These results clearly indicated that both the steric
and electronic effects are very essential in the present
study.
Next, the relative stability of the P(O)(OCME)2 (in
10a ), and P(S)(OCME)2 (in 16a ), P(O)(O-t-Bu)2 (in 19a )
groups attached to the 2′-hydroxyl of N3-benzoyluridine
was systematically examined. To obtain substrate 19a
having a P(O)(O-t-Bu)2 group, compound 7 was allowed
to react with di-tert-butyl phosphonate in the presence
of tris(2,4,6-tribromophenoxy)dichlorophosphorane (BD-
CP)36 in pyridine followed by oxidation with t-BuOOH.
This reaction gave 2′-phosphorylated derivative 20. Com-
pound 20 was in situ desilylated in a manner similar to
that described for 16a to give 19a as a crystalline
material in 58% yield (Scheme 5).
The 2′-phosphorylated uridine derivatives 10a , 16a ,
and 19a were kept in pyridine and in 80% acetic acid at
25 °C. These solutions were analyzed by 31P NMR to
monitor the phosphoryl migration. These results are
shown in Figure 2. In pyridine, an equilibrium between
10a and 10b was reached in ca. 50 days. About 0.7% of
phosphoryl migration occurred in 4 h, the time of which
is usually necessary for completion of the 5′-O-dimethox-
ytritylation in pyridine. This result is not acceptable.
(36) Hotoda, H.; Wada, T.; Sekine, M.; Hata, T. Tetrahedron Lett.
1987, 28, 1681. Wada, T.; Hotoda, H.; Sekine, M.; Hata, T. Tetrahedron
Lett. 1988, 29, 4143. Matsuzaki, J .; Hotoda, H.; Sekine, M.; Hata, T.
Nucleosides Nucleotides 1989, 8, 367. Wada, T.; Kato, R.; Hata, T. J .
Org. Chem. 1991, 56, 1243. Ohuchi, S.; Singh, R. K.; Wada, T.; Hata,
T. Chem. Lett. 1992, 1505.
(35) Vu, H.; Hirschbein, B. L. Tetrahedron Lett. 1991, 32, 3005.

4092 J . Org. Chem., Vol. 61, No. 12, 1996
Sekine et al.
F igu r e 2. A: Stability of 2′-(thio)phosphorylated uridine derivatives 10a , 16a , and 19a in pyridine. Each sample (0.06 mmol)
was dissolved in Py-Py-d5 (9:1, v/v, 0.6 mL), and the solution was kept at 25 °C: solid dot, 10a and 10b; open dot, 16a and 16b;
open square, 19a and 19b; solid line, the remaining amount of the starting material 10a , 16a , or 19a ; dotted line, the amount of
the isomerized product 10b and 16b. B: Stability of 2′-(thio)phosphorylated uridine derivatives 10a , 16a , and 19a in 80% acetic
acid. Each sample (0.06 mmol) was dissolved in acetic acid-D2O (4:1, v/v, 0.6 mL), and the solution was kept at 25 °C: solid dot,
10a and 10b; open dot, 16a and 16b; open square, 19a and 19b; solid line, the remaining amount of the starting material 10a ,
16a , or 19a ; dotted line, the amount of the isomerized product 10b, 16b, or 19b.
Compound 11a protected with the DMTr group at the
5′-position was similarly migrated to the regioisomer 11b.
There was almost no difference in the rate of the
isomerization between 10a and 11a . On the other hand,
the P(S)(OCME)2 group in 16a was so slowly migrated
to the 3′-position that an equilibrium mixture of 16a and
16b was not obtained even after 60 days. During the
dimethoxytritylation of 16a , phosphoryl migration was
expected to occur to a degree of 0.1%. This figure is
negligible. Actually, the 31P NMR spectrum of the 5′-O-
dimethoxytritylated product (21) obtained from 16a
showed a single resonance signal at 50.82 ppm. The
P(O)(O-t-Bu)2 group in 19a was extremely resistant to
the migration, which occurred to a degree of only 5% even
in 30 days.
elimination of the two CME groups and one CE group.
The silyl ester 26 was hydrolyzed by addition with water
to give a free acid 27. Treatment of 27 with I2 in
pyridine-water (9:1, v/v) at rt for 40 h resulted in
quantitative S f O conversion38 to give compound 28. It
was confirmed that the S f O conversion proceeded very
cleanly as evidenced by 31P NMR (Figure 3), which
suggests that quantitative reactions occurred at all three
steps. Thus, the BCMETP group could be readily con-
verted into a thiophosphoryl or a phosphoryl group under
mild conditions which are compatible with those used in
the current oligoribonucleotide synthesis.
Finally, successive treatments of 28 with concd NH3
(rt, 18 h) and 80% acetic acid (rt, 15 min) gave the desired
product 29, which was isolated in 72% yield (Scheme 6).
Compound 16a was the most stable in 80% acetic acid
among these three compounds, and more than 80% of 16a
remained unchanged even after 1 month. In contrast to
this, the tert-butyl group in 19a rapidly decomposed. This
difference is due simply to the destabilizing effect of the
carbonium cation by the electron-withdrawing 2-cyano-
ethyl group.
A phosphoramidite unit 22 was synthesized in an
overall yield of 78% from 16a via compound 21 by a two-
step reaction. The steric hindrance around the 2′-
thiophosphoryl residue of 16a did not affect phosphity-
lation of 21. A phosphotriester unit 23 was obtained in
85% yield by phosphorylation with cyclohexylammonium
S,S-diphenyl phosphorodithioate in the presence of DDS.37
During these reactions, no 2′-3′ phosphoryl migration
was detected. Condensation of 1.3 equiv of 22 with
N3,2′,3′-O-tribenzoyluridine (24) in the presence of 1H-
tetrazole in acetonitrile at rt followed by oxidation with
t-BuOOH gave the protected dimer 25 in 90% yield.
Treatment of 25 with 0.1 M DBU (3 equiv) and BSA (23
equiv)26 in pyridine at rt for 3 min resulted in quantita-
tive formation of the silyl ester 26 with simultaneous
The 2′-thiophosphorylated derivative 30 was also syn-
thesized in 80% yield from 25 by the following proce-
dure: (1) 1% trifluoroacetic acid in CH2Cl2 at rt for 15
min for removal of the DMTr group; (2) 0.1 M DBU-
BSA in pyridine at rt for 10 min; (3) concd NH3-pyridine
(9:1, v/v) at rt for 18 h. It was observed that compound
30 is gradually air-oxidized to give 31. The compound
30 isolated by paper chromatography always contained
an appreciable amount of 31. However, when compound
30 was isolated by HPLC and preserved at -30 °C, it
was stable for several months. It is expected that
compounds like 31 having a S-S linkage could be used
for physicochemical studies of DNA (or RNA)-DNA (or
RNA) cross-linking which can be released by reductive
cleavage using DTT or NaBH4. In fact, we confirmed that
reaction of 31 with NaBH4 gave quantitatively 30.
Since thiophosphate monoesters allow facile S-alkyla-
tion with alkyl halides without affecting the base residues
of nucleotides, the presence of such a reactive functional
group would be also useful for introduction of a variety
of reporter groups to RNA fragments via the 2′-position.
(37) Sekine, M.; Matsauzaki, J .; Hata, T. Tetrahedron 1985, 5279.
(38) Burgers, P. M. J .; Eckstein, F. Biochemistry 1979, 18, 450.

Synthesis of 2′-Phosphorylated Oligouridylates
J . Org. Chem., Vol. 61, No. 12, 1996 4093
F igu r e 3. 31P NMR spectra (Py-Py-d5, 9:1, v/v) of the mixtures obtained at each stage by a three-step reaction for conversion
of 25 to 28.
Sch em e 6
For example, a fluorescent group of bimane39 could be
easily introduced to the 2′-thiophosphoryl group via a
P-S bond by treatment of 30 with monobromobimane
32 in aqueous acetonitrile to give the product 33 in 60%
yield. The presence of this fluorescence group was
confirmed by a fluorescence detector attached to HPLC.
We have recently developed a new method for selective
biotin labeling of oligonucleotides having a 5′-monophos-
phate group by use of 6-(N-biotinylamino)hexyl phospho-
rimidazolidate 34.40 This reagent allows facile pyrophos-
phate bond formation with such a terminal phosphate
group in an aqueous medium without affecting the base
moieties and hydroxyl functions. The 2′-phosphorylated
(39) Cosstick, R.; McLaughlin, L. W.; Eckstein, F. Nucleic Acids Res.
1984, 12, 1791.
(40) Sekine, M. et al. Manuscript in preparation.

4094 J . Org. Chem., Vol. 61, No. 12, 1996
Sekine et al.
Sch em e 7
dimer 29 could be selectively biotin-labeled at the 2′-
phosphoryl group via a pyrophosphate linkage by treat-
ment with 34 in aqueous solution in the presence of
MgCl2 by using Sawai’s reaction41 to give the product 35
in good yield. These new modified oligoribonucleotides
would be utilized as RNA probes or antisense RNAs as
well as site specific modulators for termination of re-
versetransriptase-mediated DNA chain elongation.13
F igu r e 4. A: Stability of DMTr-U(2′- or 3′-Bz)-suc-AP-CPG
under the conditions prescribed for removal of the CE and
CME groups. Solid dot: after DMTr-U(2′- or 3′-O-Bz)-suc-AP-
CPG (38.3 µmol/g, 28 mg, 1 µmol) was suspended in a solution
of BSA (200 µL, 0.8 mmol) in pyridine (188 µL) at room
temperature for 30 min, DBU (12 µL, 80 µmol) was added.
Open dot: DMTr-U(2′- or 3′-Bz)-suc-AP-CPG (1 µmol) was
suspended in pyridine (388 µL), and DBU (12 µL, 80 µmol)
was added. B: Stability of DMTr-U(2′- or 3′-O-Bz)-suc-LCAA-
CPG under the conditions prescribed for removal of the CE
and CME groups. Solid square: after DMTr-U(2′- or 3′-Bz)-
suc-LCAA-CPG (30.1 µmol/g, 38 mg, 1 µmol) was suspended
in a solution of BSA (200 µL, 0.8 mmol) in pyridine (188 µL)
at room temperature for 30 min, DBU (12 µL, 80 µmol) was
added. Open square: DMTr-U(2′- or 3′-Bz)-suc-LCAA-CPG (1
µmol) was suspended in pyridine, (388 µL) and DBU (12 µL,
80 µmol) was added.
P olym er -Su p p or t ed Syn t h esis of [U(2′-p )p ]n U
(n ) 1, 3, 5, 7, a n d 9). Finally, we applied our new
method to the solid phase synthesis of oligouridylates
[U(2′-p)p]nU (n ) 1, 3, 5, 7, and 9). Pfleiderer42 and
Brown43 reported that part of the oligodeoxyribonucle-
otide chain was eliminated from the CPG gel because of
the intramolecular cyclization of the succinate linker
which was catalyzed by DBU as depicted in Scheme 7.
Accordingly, the succinate linker must have been hitherto
avoided when DBU was used as reagent for removal of
the (4-nitrophenyl)ethyl group or 2-cyanoethyl group.
Indeed, they have used a sarcosine linker in place of the
usual succinate to avoid such undesirable side reactions.
Ta ble 1. P r otocol of th e Solid -P h a se Syn th esis of
[U(2′-p )p ]n U
step
manipulation
1% TFA/CH2Cl2
Therefore, it was checked to see if the 2′-phosphory-
lated oligouridylates were eliminated from CPG gel upon
treatment with DBU/BSA as prescribed for removal of
the internal CE and 2′-CME groups when the succinate
linker was used. We tested two kinds of CPG gels, i.e.,
aminopropyl CPG gel (AP-CPG) and long-chain alky-
lamino CPG gel (LCAA-CPG), from which DMTr(2′- or
3′-Bz)U-suc-AP-CPG and DMTr(2′- or 3′-Bz)U-suc-LCAA-
CPG were prepared, respectively. To gauge the instabil-
ity of the succinate linker, the amounts of DMTr(2′- or
3′-Bz) released from these CPG gels were measured by
the DMTr cation assay. These results are shown in
Figure 4. As reported by Pfleiderer, DMTr-U(2′- or 3′-
Bz) was eliminated from both DMTr-U(2′- or 3′-Bz)-suc-
AP-CPG and DMTr-U(2′- or 3′-Bz)-suc-LCAA-CPG. In-
terestingly, we found that DMTr(2′- or 3′-Bz)U-suc-AP-
CPG was more stable than DMTr(2′- or 3′-Bz)U-suc-
LCAAP-CPG when they were treated with 0.2 M of DBU
in pyridine. Furthermore, elimination of DMTr(2′- or 3′-
Bz)U from both resins was largely avoided when BSA
was added as shown in Figure 4. Approximately 10% of
DMTr-U(2′- or 3′-Bz) was lost after 24 h in the case of
LCAA-CPG gel. In particular, it should be noted that
DMTr(2′- or 3′-Bz)U remained almost intact in the case
1. detritylation
2. wash
pyridine
3. dry up
4. condensation
CH3CN
10 min
20 min
U-unit (0.15 M, 30 equiv),
1H-tetrazole (1.5 M,
300 equiv)/CH3CN
pyridine
5. wash
6. oxidation
0.1 M I2/THF-Py-H2O
(10:10:1, v/v/v)
pyridine
Ac2O-Py (1:9, v/v), 0.1 M
DMAP
2 min
2 min
7. wash
8. capping
of DMTr(2′- or 3′-Bz)U-suc-AP-CPG even even after 100
h. From these results, we finally chose AP-CPG gel for
our study.
To obtain [U(2′-p)p]nU on AP-CPG gel, N3,2′-O-diben-
zoyl-5′-O-(4,4′-dimethoxytrityl)uridine was attached to
AP-CPG gel via a succinate linker in the usual manner.44
The chain elongation was performed as shown in Table
1. Since the steric effect of the bulky 2′-thiophosphoryl
group was expected, the uridine unit 22 was used at a
concentration of 0.15 M which is more than the usual
(0.1 M). The condensation was performed for 20 min.
First, the synthesis of oligouridylates U(2′-p)pU on CPG
gel was tried. The coupling yield was 97% which was
estimated by the DMTr cation assay (Scheme 8).
Removal of the CE and CME groups and the successive
S-O conversion were carried out on CPG gel to facilitate
(41) Shimizu, M.; Shinozuka, K.; Sawai, H. Tetrahedron Lett. 1990,
31, 235.
(42) Strengele, K.; Pfleiderer, W. Tetrahedron Lett. 1990, 31, 2549.
Resmini, M.; Pfleiderer, W. Bioorg. Med. Chem. Lett. 1994, 4, 1909.
(43) Brown, T.; Pritchard, C. E.; Turner, G.; Salisbury, S. A. J . Chem.
Soc., Chem. Commun. 1989, 891.
(44) Tanimura, H.; Maeda, M.; Fukazawa, T.; Sekine, M.; Hata, T.
Nucleic Acids Res. 1987, 17, 8135.

Synthesis of 2′-Phosphorylated Oligouridylates
J . Org. Chem., Vol. 61, No. 12, 1996 4095
Sch em e 8
F igu r e 5. 20% Polyacrylamide gel electrophoresis of
[U(2′-p)p]nU (n ) 3, 5, 7, and 9): lane A, xylene cyanole and
bromophenol blue; lane B, [[U(2′-p)p]3U; lane C, [U(2′-p)p]5U;
lane D, [U(2′-p)p]7U; lane E, [U(2′-p)p]9U.
F igu r e 6. HPLC profiles of the mixtures obtained by polymer-
supported synthesis of [U(2′-p)p]nU (n ) 3 (A), 5 (B), 7 (C),
and 9 (D)). In the case of A, reversed-phase HPLC was carried
out. In the case of B, ion-exchange HPLC using a SAX column
was carried out. In the case of C and D, ion-exchange HPLC
using a FAX column was performed. In panels A-D, the
ordinate is UV absorbance at 254 nm.
removal of the excess reagents and the eliminated
species. Treatment with concentrated ammonia was
performed to release the oligomer from CPG gel as well
as to remove the benzoyl and succinyl groups. At the
final stage, treatment of the released product with 80%
acetic acid gave U(2′-p)pU by HPLC.
The best conditions for removal of the CE and CME
groups and for the S-O conversion were examined in
detail by using the CPG gel having a dimer UpU
sequence. As a result, successive treatments of the gel
with 0.2 M of DBU and BSA in pyridine for 40 h and
with iodine in aqueous pyridine for 48 h gave satisfactory
results as suggested by HPLC analysis. The isolated
yield of U(2′-p)pU was 75%.
On the basis of these results, the synthesis of longer
oligouridylates [U(2′-p)p]nU (n ) 3, 5, 7, and 9) was
examined on CPG gel. The average coupling yield was
usually ca. 97% in each synthesis. These crude reaction
mixtures obtained after deprotection were analyzed by
polyacrylamide gel electrophoresis (PAGE). As seen in
Figure 5, it seemed that the oligomers [U(2′-p)p]nU moved
as main bands in PAGE keeping a constant distance
among them. To our surprise, however, more detailed
HPLC analysis (Figure 6A-D) of the crude materials by
reversed-phase or ion-exchange HPLC showed that some
byproducts were simultaneously formed with the desired
2′-phosphorylated oligouridylates. It was possible to use
reversed-phase C18 HPLC for isolation of 2′-phosphory-
lated tetrauridylate [U(2′-p)p]3U as described in the case
of U(2′-p)pU. In the case of [U(2′-p)p]5U, however, anion-
exchange HPLC using a SAX column was neccesary for
its better separation as shown in Figure 6B. In the case
of n ) 7 or 9, ion exchange HPLC using a FAX column
was performed because of the increasing anion charge.
As shown in Figure 6C and D, a cluster of multipeaks
around the expected retention time appeared so that it
was extremely difficult to isolate the desired 2′-phospho-
rylated products. As far as estimation of the purity of
2′-phosphorylated oligoribonucleotides is concerned, the
PAGE analysis is unsuitable. The essential factor of the
mobility of oligonucleotides in PAGE is not the number
of the anion charges but the size of the substrates.45
Indeed, Brownlee et al. have recently reported the
difficulty in separating oligoribonucleotides having a 5′-
(45) Martin, R. Gel Electrophoresis: Nucleic Acids; B10S Scientific
Pub. Ltd: Oxford, 1996.

4096 J . Org. Chem., Vol. 61, No. 12, 1996
Sekine et al.
terminal diphosphate from oligoribonucleotides having
a 5′-terminal monophosphate by PAGE.46
neutral conditions. Therefore, it is expected that the use
of our P(S)(OCME)2 group in phospholipids would result
in complete avoidance of the phosphoryl migration.
These results imply that thiophosphorylated species of
acyclic 1,2-diols do not migrate to the proximal hydroxyl
group compared with those of ribonucleoside derivatives
which have more rigid conformations due to the presence
of a five membered ring.
Now we have achieved an approach to the solid phase
synthesis of 2′-phosphorylated oligouridylates. However,
we have encountered certain limitations to the present
method because of the considerable increase in relative
amounts of byproducts which comigrate with the desired
oligomers in PAGE. The HPLC analysis indicates that
the present method is limited to the synthesis of 2′-
phosphorylated oligoribonucleotides up to the level of six
mers. This limitation is due essentially to the multiple
complexity with the increasing number of the BCMETP
group involved in the synthetic intermediate, although
at the dimer level the amount of such byproducts is not
significant.
The synthesis of oligoribonucleotides or oligodeoxyri-
bonucleotides having a 2′-phosphate group at the specific
positions in the sequence is also of interest. Such
phosphate-bearing oligonucleotides would be utilized in
antisense DNA/RNA chemistry as described before.
These one-point modified oligomers would be more easily
prepared by a combined use of the phosphoramidite unit
22 and the commercially available standard phosphora-
midite units,49 since the conditions prescribed for removal
of the CME and the subsequent S-O conversion are
compatible with the 2′-TBDMS group which is used in
the usual phosphoramidite units.
Further study on the chemical synthesis of 2′-(thio)-
phosphate-containing oligonucleotides using four kinds
of synthetic units is now in progress. Our preliminary
results suggest that all four phosphoramidite units can
be constructed in a similar manner and a partial struc-
ture of 2′-phosphorylated RNA species can be synthesized
by the present strategy.49 The chemical, physicochemical,
and biochemical properties of mono- and multi-2′-(thio)-
phosphorylated oligoribonucleotides will be reported in
the near future.
We also observed that, as the chain becomes long, the
retention time of ion exchange HPLC is considerably
prolonged. In contrast, these polyanion charged oligou-
ridylates were not retained on reversed-phase HPLC
columns. In the case of the synthesis of [U(2′-p)p]3U and
[U(2′-p)p]5U, these compounds were fortunately obtained
as the main products as shown in Figure 6A,B. The
isolated yields of [U(2′-p)p]3U and [U(2′-p)p]5U were 19%
and 12%, respectively, from the fully protected 2′-phos-
phorylated uridylates bound to CPG gel. The structure
of this highly negative-charged oligouridylates was con-
firmed by enzymatic assay as described in a later section.
Our finding strongly suggests that BSA reacted with
the amide group of the succinyl linker to give a O-SiMe3
derivative which lost the NH proton responsible for the
side reaction as depicted in Scheme 7. This also implies
that the more accessible succinate linker can be used
even in the usual oligonucleotide synthesis using (4-
nitrophenyl)ethyl groups if silylating agents such as BSA
are present.
En zym e Assa y of 2′-P h osp h or yla ted Oligou r id y-
la tes. U(2′-p)pU was digested with snake venom phos-
phodiesterase to give U(2′-p) and pU while this dimer
was resistant to nuclease P1 and spleen phosphodi-
esterase. In the enzymatic internucleotide bond cleavage
of U(2′-p)pU with snake venom phosphodiesterase, the
peak corresponding to uridine was observed as a minor
product. This reaction required a relative excess of this
enzyme compared with the usual internucleotidic bond
hydrolysis. Accordingly, it is likely that the appearance
of uridine is due to the phosphatase acitivity contami-
nated in snake venom phosphodiesterase. Treatment of
U(2′-p)pU with calf intestinal alkaline phosphatase
(CIAP) gave the usual UpU dimer, which was digestable
with nuclease P1 to give U and pU. These enzymatic
properties are consistent with those of pG(2′-p)pA re-
ported previously.4 In the case of [U(2′-p)p]3U and [U(2′-
p)p]5U, dephosphorylation also occurred upon treatment
with CIAP. The stability of the 2′-phosphoryl group to
CIAP increased with an increase in length of the oligo-
mer. Contrary to these results, the 2′-thiophosphorylated
uridine dimer U(2′-ps)pU 30 was found to be completely
resistant to CIAP.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Melting points were
determined and are uncorrected. 1H NMR spectra were
recorded at 270 and 500 MHz with Me4Si (for water-insoluble
materials) or DSS (for water-soluble materials) as the external
reference. 13C NMR spectra were measured at 67.8 MHz with
TMS as the internal standard. Paper chromatography 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 ammonia-water,
6:1:3, 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.
Reversed-phase column chromatography was performed 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). Reversed-phase HPLC was performed using a µBon-
dasphere C-18 column 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. Ion-exchange high-performance liquid
chromatography (IE HPLC) was carried out at a flow rate of
1mL/min at 50 °C on a Whatman PARTISIL 10 SAX WCS
Con clu sion
Apart from the synthesis of 2′-phosphorylated RNAs,
the present strategy would provide a new promising tool
for the regioselective phosphorylation (thiophosphoryla-
tion) of not only ribonucleosides but also the neighboring
OH-containing phosphate compounds such as and myo-
inositol 1,4,5-triphosphates and phospholipids where
similar problems of phosphoryl isomerization have been
encountered.47 Quite recently, Heeb48 reported that the
bis(2-cyanoethoxy)thiophosphoryl [P(S)(OCE)2] group re-
mained almost intact upon acid-catalyzed removal of the
isopropylidene group from 1,2-O-isopropylidene-3-O-[bis-
(2-cyanoethoxy)thiophosphoryl]glycerin. As mentioned
before, compound 18a having a P(S)(OCE)2 group is
rapidly isomerized to the 3′-regioisomer 18b even under
(46) Brownlee, G. G.; Foder, E.; Pritlove, D. C.; Ground, K. G.;
Dalluge, J . J . Nucleic Acids Res. 1995, 23, 2641.
(47) Watanabe, Y.; Ogasawara, T.; Nakahira, H.; Matsuki, T.; Ozaki,
S. Tetrahedron Lett. 1988, 29, 5259 and references cited therein.
(48) Heeb, N. V.; Nambiar, K. P. Tetrahedron Lett. 1993, 34, 6193.
(49) Sekine, M. et al. Manuscript in preparation.

Synthesis of 2′-Phosphorylated Oligouridylates
J . Org. Chem., Vol. 61, No. 12, 1996 4097
analytical column (4.6 × 250 mm) or a Waters Gen-Pak FAX
column (4.6 × 100 mm). Analysis by SAX-HPLC was per-
formed using a linear gradient of 0-100% solution B (20% CH3-
CN in 0.005 M KH2PO4) in solution A (20% CH3CN in 0.005
M KH2PO4) for 20 min followed by maintaining the volume in
100% solution B for 20 min. In the case of FAX-HPLC
analysis, elution buffer by using 10-77% linear gradient of
solution C (1 M NaCl, 25 mM phosphate buffer, pH 6.0) in
solution D (25 mM phosphate buffer, pH 6.0) for 50 min was
employed. Uridine was purchased from Yamasa Co., Ltd.
Pyridine was distilled two times from p-toluenesulfonyl chlo-
ride and from calcium hydride and then stored over molecular
sieves 4A. Elemental analyses were performed by the Mi-
croanalytical Laboratory, Tokyo Institute of Technology, at
Nagatsuta.
N,N-diethylphosphoramidite (1.1 mL, 4.5 mmol). After the
mixture was stirred at room temperature for 3 h, tert-butyl
hydroperoxide (about 80% solution in t-BuOO-t-Bu, 1.5 mL,
1.5 mmol) was added. The solution was stirred at room
temperature for 15 min, diluted with CH2Cl2, washed twice
with 5% NaHCO3 and with water, dried over Na2SO4, filtered,
and evaporated under reduced pressure. The residue contain-
ing compound 20 was dissolved in THF (4 mL), and a mixture
of (HF)x‚Py (0.519 mL)-Py (2.9 mL) was added. The mixture
was stirred at room temperature for 10 min and then parti-
tioned between CH2Cl2 and 5% NaHCO3. The organic phase
was collected, washed with 5% NaHCO3 and water, dried over
Na2SO4, filtered, and evaporated under reduced pressure. The
residue was chromatographed on a column of silica gel (12 g)
with CH2Cl2-MeOH containing 0.5% pyridine to give 19a (172
1
2-Ch lor o-1,1-d im et h ylet h a n ol. 3-Chloro-2-methylpro-
pene (315 mL, 3 mol) was cooled at 0 °C, and 80% H2SO4 (230
mL), which was prepared in advance by addition of concen-
trated H2SO4 (168 mL) to water (62 mL), was added. The
mixture was stirred at room temperature for 2.5 h. The orange
viscous solution was diluted with water (1.3 L) cooled at 0 °C.
The mixture was steam distilled to give a mixture containing
the product, which was extracted with ether (500 mL × 3).
The extract was dried over Na2SO4, filtered, and concentrated
under reduced pressure. Distillation gave the title compound
mg, 56%): mp 116 °C dec from CH2Cl2-toluene (9:1, v/v); H
NMR (270 MHz, CDCl3, TMS) δ 1.47(18H, m, CH3 of t-Bu),
3.71 (1H, dd, J 4′H-5′′H ) 2.3 Hz, J 5′H-5′′H ) 12.2 Hz, 5′′-H), 3.95
(1H, dd, J 4′H-5′′H ) 2.3 Hz, J 5′H-5′′H ) 12.2 Hz, 5′-H), 4.20 (1H,
m, 4′-H), 4.46 (1H, dd, J 2′H-3′H ) 3.5 Hz, J 3′H-4′H ) 5.0 Hz, 3′-
H), 4.92 (1H, m, J 1′H-2′H ) 5.9 Hz, J 2′H-3′H ) 3.5 Hz, 2′-H), 5.87
(1H, d, J 5H-6H ) 7.9 Hz, 5-H), 5.95 (1H, d, J 1′H-2′H ) 5.9 Hz,
1′-H), 7.46-7.52 (2H, m, ArH), 7.62-7.67 (1H, m, ArH), 7.70
(1H, d, 6-H), 7.96-7.93 (2H, m, ArH); 13C NMR (67.8 MHz,
CDCl3) δ 29.49, 29.53, 29.58, 61.42, 70.01, 76.52, 76.98, 77.47,
78.20, 84.57, 84.69, 84.80, 84.92, 85.37, 87.62, 87.69, 87.78,
87.84, 102.50, 129.00, 130.37, 131.18, 135.02, 140.97, 149.20,
162.14, 168.37; 31P NMR (109 MHz, CDCl3, 85% H3PO4) δ
-8.97. Anal. Calcd for C24H33N2O10P‚1/2H2O: C,52.46; H,
6.24; N, 5.10. Found: C, 52.70; H, 6.17; N, 4.99.
Meth od B. BDCP36 (7.0 g, 6.4 mmol) was dissolved in dry
pyridine (40 mL), and di-t-butyl phosphonate (0.956 mL, 4.8
mmol) was added. The mixture was stirred at room temper-
ature for 15 min and then was added to N3-benzoyl-3′,5′-O-
(di-tert-butylsilanediyl)uridine (7) (1.95 g, 4.0 mmol) which was
rendered anhydrous by repeated coevaporation with dry
pyridine (20 mL × 5). The resulting solution was stirred at
room temperature for 1 h, and then tert-butyl hydroperoxide
(2 mL, 20 mmol) was added. After being stirred at room
temperature for an additional 1 h, the mixture was diluted
with CH2Cl2, washed with 5% NaHCO3 and water, dried over
Na2SO4, filtered, and evaporated under reduced pressure. The
residue was dissolved in THF (16 mL), and a mixture of (HF)-
x‚Py (2.1 mL)-Py (10.3 mL) was added. The mixture was
stirred at room temperature for 15 min. A workup similar to
that described above gave 19a (1.26 g, 58%).
2′-O-Bis[(2-cya n o-1,1-d im eth yleth oxy)p h osp h or yl]-N3-
ben zoylu r id in e (10a ). First, the phosphitylating reagent of
bis(2-cyano-1,1-dimethylethyl) N,N-diethylphosphoramidite was
prepared as follows: To a solution of (diethylamino)dichloro-
phosphine (4.13 mL, 26.3 mmol) in dry ether (18 mL) was
added dropwise a mixture of 2-cyano-1,1-dimethylethanol (5.12
mL, 50 mmol) and triethylamine (6.94 mL, 50 mmol) in dry
ether (12.5 mL) with cooling at 0 °C from -10 °C over a period
of 2 h. The solution was warmed to room temperature and
stirred for an additional 3.5 h. The white precipitate was
removed by filtration and washed with ether. The filtrate and
washing were combined and concentrated under reduced
pressure. The residue was extracted with ether and 5%
NaHCO3. The organic extract was dried over Na2SO4 and
filtrated. The solution was concentrated in vacuo. The purity
of the residue containing the phosphitylating reagent was
estimated by measurement of its 31P NMR spectrum (135.72
ppm in CDCl3), which showed that the purity varied usually
from 50-70%. Then, this residue was diluted by addition of
CH2Cl2 so as to obtain a 0.3-0.5 M solution of the reagent,
which was stocked in the presence of molecular sieves 3A in a
refrigerator at -20 °C. N3-Benzoyl-3′,5′-O-(di-tert-butylsi-
lanediyl)uridine (7) (488 mg, 1.0 mmol) and 1H-tetrazole (126
mg, 1.8 mmol) were rendered anhydrous by repeated coevapo-
ration with dry pyridine (5 mL × 5), dry toluene (5 mL × 2),
and CH2Cl2 (5 mL × 2) and dissolved in CH2Cl2 (7.5 mL). To
the mixture was added bis(2-cyano-1,1-dimethylethyl) N,N-
diethylphosphoramidite (purity 58%, 2.4 mL, 1.2 mmol). After
the mixture was stirred at room temperature for 2 h, tert-butyl
hydroperoxide (1 mL, 10 mmol) was added. The solution was
1
(145 g, 44%): bp 125-127 °C [lit.50 126.7 °C]; H NMR (270
MHz, CDCl3, TMS) δ 1.35 (6H, s, CH3), 2.28 (1H, s, OH), 3.54
(2H, s, CH2); 13C NMR (67.8 MHz, CDCl3) δ 26.29, 54.65, 70.01.
2-Cya n o-1,1-d im eth yleth a n ol. 2-Chloro-1,1-dimethyle-
thanol (31 mL, 0.3 mol) was dissolved in ethanol (688 mL) and
water (112 mL) and sodium cyanide (17.6 g, 0.36 mol). The
mixture was refluxed for 1 h. The brownish solution was
concentrated under reduced pressure and saturated NaCl (150
mL) and water (500 mL) were added. The residue was
extracted with ether (500 mL × 5). The ether extracts were
combined, dried over Na2SO4, filtered, and concentrated under
reduced pressure. The residue was distilled to give the title
compound (15.4 g, 51%): bp 118 °C/38 mmHg (lit.49 114 °C/30
mmHg); 1H NMR (270 MHz, CDCl3, TMS) δ 1.39 (6H, s, CH3),
2.33 (1H, s, OH), 2.52 (2H, s, CH2); 13C NMR (67.8 MHz,
CDCl3) δ 28.88, 32.65, 69.13, 117.70.
N3-Ben zoyl-3′,5′-O-(d i-ter t-bu tylsila n ed iyl)u r id in e (7).
N3-Benzoyluridine (1.04 g, 3 mmol) was rendered anhydrous
by repeated coevaporation with dry pyridine (10 mL × 3) and
toluene (10 mL × 3) and finally dissolved in dimethylforma-
mide (30 mL). To the mixture were added silver nitrate (1.33
g, 7.8 mmol) and di-tert-butyldichlorosilane (0.824 mL, 3.9
mmol). The resulting mixture was stirred vigorously at room
temperature for 30 min. The mixture was quenched at 0 °C
by addition of triethylamine (1.25 mL, 9 mmol), and the solvent
was removed under reduced pressure. The residue was
partitioned between CH2Cl2 and water. The organic phase was
collected, dried over Na2SO4, filtered, and concentrated under
reduced pressure. The residue was chromatographed on a
column of silica gel eluted with CH2Cl2-MeOH to give 7 (1.39
g, 95%): 1H NMR (270 MHz, CDCl3, TMS) δ 1.00-1.08 (18H,
m, CH3), 4.00-4.03 (2H, m, 5′-H and 5′′-H), 4.11-4.18 (1H,
m, 4′-H), 4.43 (1H, d, J 2′H-3′H ) 4.3 Hz, 3′-H), 4.47 (1H, d,
J 2′H-3′H ) 4.3 Hz, 2′-H), 5.61 (1H, s, 1′-H), 5.86 (1H, d, J 5H-6H
) 8.3 Hz, 5-H), 7.32 (1H, d, J 5H-6H ) 8.3 Hz, 6-H), 7.48-7.53
(2H, m, m-ArH), 7.64-7.69 (1H, m, p-ArH), 7.91-7.95 (2H,
m, o-ArH); 13C NMR (67.8 MHz, CDCl3) δ 20.18, 22.45, 27.01,
27.15, 66.94, 73.16, 74.50, 75.65, 93.96, 102.19, 129.06, 130.35,
131.03, 135.17, 140.81, 148.63, 161.87, 168.28. Anal. Calcd
for C17H26N2O6Si: C, 41.79; H, 5.36; N, 5.73. Found: C, 41.85;
H, 5.01; N, 5.42.
N 3-Be n zoyl-2′-O-(d i-t er t -b u t oxyp h osp h or yl)u r id in e
(19a ). Meth od A. A mixture of N3-benzoyl-3′,5′-O-(di-tert-
butylsilanediyl)uridine (7) (348 mg, 0.71 mmol) and 1H-
tetrazole (140 mg, 2.0 mmol) was rendered anhydrous by
repeated coevaporation with dry pyridine (5 mL × 5), dry
toluene (5 mL × 2), and CH2Cl2 (5 mL × 2) and was dissolved
in CH2Cl2 (10 mL). To the mixture was added di-tert-butyl
(50) Burgin, J .; Hearne, G.; Rust, F. Ind. Eng. Chem. 1941, 33, 385.
(51) Kjaer, A.; Boejensen, R. Acta Chem. Scand. 1958, 12, 1756.

4098 J . Org. Chem., Vol. 61, No. 12, 1996
Sekine et al.
stirred at room temperature for 10 min, diluted with CH2Cl2,
washed twice with 5% NaHCO3 and with water, dried over
Na2SO4, filtered, and evaporated under reduced pressure. The
residue was dissolved in THF-AcOH (1:1, v/v, 4 mL), and a
mixture of (HF)x‚Py (0.52 mL)-Py (2.9 mL) was added. The
mixture was stirred at room temperature for 10 min, and then
an excess amount of pyridine (5 mL) was added. The mixture
was partitioned between CH2Cl2 and 5% NaHCO3. The
organic phase was collected, washed with 5% NaHCO3 and
water, dried over Na2SO4, filtered, and evaporated under
reduced pressure. The residue was chromatographed on a
column of silica gel (18 g) with CH2Cl2-MeOH containing 0.5%
pyridine to give 10a (purity 97%, 269 mg, 46%). When the
solvent for removal of the DTBS group was changed from
THF-AcOH to THF, 10a was obtained in 72% yield with a
purity of 95%. 10a : 1H NMR (270 MHz, CDCl3, TMS) δ 1.59-
1.69 (12H, m, CH3 of CME), 2.63-3.05 (4H, m, CH2 of CME),
3.87 (2H, s, 5′-H and 5′′-H), 4.25 (1H, d, J 3′H-4H ) 1.65 Hz,
4′-H), 4.58 (1H, dd, J 3′H-4′′H ) 1.65 Hz, J 2′H-3′H ) 4.62, 3′-H),
4.91 (1H, m, 2′-H), 5.91 (1H, d, J 5H-6H ) 8.25 Hz, 5-H), 6.27
(1H, d, J 1′H-2′H ) 6.93 Hz, 1′-H), 7.47-7.52 (2H, m, m-ArH),
7.62-7.68 (1H, m, p-ArH), 7.97-7.98 (2H, m, o-ArH), 8.06 (1H,
d, J 5H-6H ) 8.25 Hz, 6-H); 13C NMR (67.8 MHz, CDCl3) δ 26.99,
27.03, 27.15, 27.21, 27.30, 27.37, 31.25, 31.32, 31.43, 31.50,
61.24, 69.88, 69.92, 79.01, 79.08, 81.82, 81.92, 85.07, 87.03,
87.15, 102.59, 116.78, 116.93, 129.06, 130.48, 131.11, 135.17,
140.59, 149.33, 162.16, 168.70; 31P NMR (109 MHz, CDCl3,
85% H3PO4) δ -9.51. Anal. Calcd for C26H31N4O10P‚1/2H2O:
C, 52.09; H, 5.38; N, 9.34. Found: C, 51.95; H, 5.34; N, 9.17.
3′-O-[Bis(2-cyan o-1,1-dim eth yleth oxy)th ioph osph or yl]-
5′-O-(4,4′-d im eth oxytr ityl)th ym id in e (13). A mixture of 5′-
O-(4,4′-dimethoxytrityl)thymidine (545 mg, 1.0 mmol) and 1H-
tetrazole (158 mg, 2.25 mmol) was dried by repeated
coevaporation with dry pyridine (2 mL × 3), toluene (2 mL ×
2), and CH2Cl2 (2 mL × 1) and finally dissolved in CH2Cl2 (6
mL). To the solution was added a 0.5 M solution of bis(2-
cyano-1,1-dimethylethyl) N,N-diethylphosphoramidite in CH2-
Cl2 (purity 58%, 4 mL, 1.5 mmol) prepared above. After being
stirred for 50 min, the mixture was treated with elemental
sulfur (S8) (1.78 g, 6 mmol) for another 12 h. Then, it was
extracted by the use of CH2Cl2/H2O. The organic layer was
dried over Na2SO4, filtered, and evaporated under reduced
pressure. The crude material was purified by chromatography
on a silica gel column eluted with CH2Cl2-CH3OH containing
0.5% pyridine to give 13 (441 mg, 55%): 1H NMR (270 MHz,
CDCl3, TMS) δ 1.43-1.71 (15H, m, CH3 of CME and Th), 2.46-
2.73 (2H, m, 2′-H and 2′′-H), 2.79-3.07 (4H, m, CH2 of CME),
3.45 (2H, d, J 4′H-5′H ) 2.31 Hz, 5′-H and 5′′-H), 4.26 (1H, d,
J 4′H-5′H ) 2.31 Hz, 4′-H), 5.42 (1H, m, 3′-H), 6.30 (1H, m, 1′-
H), 6.83-6.88 (4H, m, ArH), 7.23-7.42 (9H, m, ArH), 7.57 (1H,
s, 6-H), 8.50 (1H, br, NH); 13C NMR (67.8 MHz, CDCl3, TMS)
δ 163.7, 158.65, 150.46, 144.04, 135.20, 135.04, 130.03, 128.12,
128.05, 127.96, 127.08, 116.53, 116.39, 113.26, 111.57, 97.08,
84.42, 84.33, 84.30, 82.41, 82.21, 79.12, 63.29, 55.17, 38.92,
38.85, 31.50, 31.43, 31.39, 31.34, 27.42, 27.39, 27.32, 27.13,
27.08, 11.61; 31P NMR (109 MHz, CDCl3, 85% H3PO4) δ 49.19;
Anal. Calcd for C41H47N4O9PS‚1/2H2O: C, 60.66; H, 5.94; N,
6.89. Found: C, 60.40; H, 6.20; N, 7.33.
mixture was partitioned between CH2Cl2 and 5% NaHCO3. The
organic phase was collected, washed with 5% NaHCO3 and
water, dried over Na2SO4, filtered, and evaporated under
reduced pressure. The residue was chromatographed on a
column of silica gel (40 g) with CH2Cl2-MeOH containing 0.5%
pyridine to give 16a (864 mg, 69%): 1H NMR (270 MHz,
CDCl3, TMS) δ 1.60-1.74 (12H, m, CH3 of CME), 2.63-2.97
(4H, m, CH2 of CME), 3.86 (2H, s, 5′-H and 5′′-H), 4.26 (1H, s,
4′-H), 4.58 (1H, d, J 2′H-3′H ) 4.9 Hz, 3′-H), 5.13 (1H, ddd, J 1′H-2′H
) 6.9 Hz, J 2′H-3′H ) 4.9 Hz, J 2′H-P ) 11.5 Hz, 2′-H), 5.92 (1H,
d, J 5H-6H ) 8.3 Hz, 5-H), 6.30 (1H, d, J 1′H-2′H ) 6.9 Hz, 1′-H),
7.47-7.52 (2H, m, m-ArH), 7.62-7.68 (1H, m, p-ArH), 7.96-
7.99 (2H, m, o-ArH), 8.10 (1H, d, J 5H-6H ) 8.3 Hz, 6-H); 13C
NMR (67.8 MHz, CDCl3) δ 26.88, 26.92, 27.35, 27.41, 27.48,
27.55, 28.36, 28.45, 31.16, 31.41, 31.47, 62.50, 71.45, 79.03,
79.10, 82.50, 82.62, 83.60, 83.72, 86.00, 86.11, 86.25, 103.22,
116.86, 117.27, 129.08, 130.64, 131.38, 135.04, 140.54, 149.54,
162.05, 168.55; 31P NMR (109 MHz, CDCl3, 85% H3PO4) δ
50.21. Anal. Calcd for C26H31N4O9PS‚1/2H2O: C, 50.73; H,
5.24; N, 9.10. Found: C, 50.71; H, 5.28; N, 9.01.
2′-O-[Bis(2-cyan o-1,1-dim eth yleth oxy)th ioph osph or yl]-
N3-ben zoyl-5′-O-(4,4′-d im eth oxytr ityl)u r id in e (21). Com-
pound 16a (2.1 g, 3.5 mmol) was rendered anhydrous by
repeated coevaporation with dry pyridine (20 mL × 3) and
finally dissolved in dry pyridine (35 mL). To the mixture was
added 4,4′-dimethoxytrityl chloride (1.78 g, 5.25 mmol), and
the solution was kept at room temperature for 4 h. Extraction
was performed with CH2Cl2 and 5% NaHCO3. The usual
workup followed by silica gel column chromatography eluted
with CH2Cl2-MeOH containing 0.5% pyridine gave 21 (2.94g,
93%); 1H NMR (270 MHz, CDCl3, TMS) δ 1.61-1.75 (12H, m,
CH2 of CME), 2.72-2.92 (4H, m, CH2 of CME), 3.41 (1H, dd,
J 4′H-5′′H ) 2.3 Hz, J 5′H-5′′H ) 11.2 Hz, 5′′-H), 3.52 (1H, dd,
J 4′H-5′H ) 2.3 Hz, J 5′H-5′′H ) 11.2 Hz, 5′-H), 4.25 (1H, d, J 4′H-5′′H
) 2.3 Hz, 4′-H), 4.80 (1H, dd, J 2′H-3′H ) 4.6 Hz, J 3′H-4′H ) 2.0
Hz, 3′-H), 5.31-5.41 (2H, m, J 1′H-2′H ) 6.3 Hz, J 2′H-3′H ) 4.6
Hz, J 5H-6H ) 8.2 Hz, 2′-H and 5-H), 6.30 (1H, d, J 1′H-2′H ) 6.3
Hz, 1′-H), 6.84-6.90 (4H, m, ArH), 7.14-7.67 12H, m, ArH),
7.93-8.02 (3H, m, ArH and 6-H); 13C NMR (67.8 MHz, CDCl3)
δ 26.99, 27.39, 27.44, 27.51, 27.57, 31.47, 31.54, 31.88, 31.95,
55.20, 63.14, 70.65, 79.59, 79.66, 82.79, 82.89, 83.77, 83.90,
84.48, 85.66, 85.81, 87.51, 102.62, 113.42, 116.75, 127.17,
128.01, 128.14, 128.97, 129.06, 130.03, 130.12, 130.57, 131.30,
134.70, 135.04, 140.16, 144.02, 149.27, 158.69, 158.72, 161.70,
168.50; 31P NMR (109 MHz, CDCl3, 85% H3PO4) δ 50.82. Anal.
Calcd for C47H49N4O11PS: C, 62.11; H, 5.43; N, 6.16. Found:
C, 61.61; H, 5.46; N, 5.86.
2′-O-[Bis(2-cyan o-1,1-dim eth yleth oxy)th ioph osph or yl]-
N3-ben zoyl-5′-O-(4,4′-d im eth oxytr ityl)u r id in e 3′-(2-Cya -
n oeth yl-N,N-d iisop r op ylp h osp h or a m id ite) (22). Com-
pound 21 (2.27 g, 2.5 mmol) was rendered anhydrous by
successive coevaporations with dry pyridine (20 mL × 3) and
dry toluene (20 mL × 2) and finally dissolved in dry CH2Cl2
(25 mL). To the mixture were added triethylamine (1.39 mL,
10 mmol) and 2-cyanoethoxy (N,N-diisopropylamino)phospho-
rochloridite (1.09 mL, 5.0 mmol). The resulting mixture was
stirred for 1 h and then extracted with CH2Cl2. The CH2Cl2
extract was washed with 5% NaHCO3 and water. The usual
workup followed by silica gel column chromatography eluted
with hexane-ethyl acetate containing 0.5% pyridine gave 22
(2.34 g, 84%): 1H NMR (270 MHz, CDCl3, TMS) δ 1.07-1.76
(26H, m, CH3 of CME and i-Pr), 2.40-3.05 (6H, m, CH2CN of
CE and CME), 3.48-3.97 (4H, m, CH of iPr, and 5′-H and 5′′-
H), 3.80 (6H, s, OCH3 of DMTr), 4.28-4.35 (1H, m, 4′-H), 4.75-
4.84 (3H, m, 3′-H), 5.34-5.5.51 (3H, m, POCH2 of CN, 2′-H),
6.27-6.46 (1H, m, 1′-H), 6.85-6.91 (4H, m, ArH), 7.16-7.68
(13H, m, 6-H and ArH), 7.96-8.04 (2H, m, ArH); 13C NMR
(67.8 MHz, CDCl3) δ 20.11, 20.36, 20.45, 24.49, 24.58, 24.71,
24.84, 26.97, 27.05, 27.33, 27.39, 27.51, 27.62, 27.68, 27.73,
31.48, 31.57, 31.63, 31.68, 43.13, 43.16, 43.36, 55.26, 63.31,
82.39, 82.46, 82.57, 83.56, 83.69, 84.46, 85.23, 85.30, 86.11,
87.69, 87.75, 102.66, 113.46, 116.50, 116.59, 117.07, 117.38,
117.83, 127.24, 128.07, 128.19, 129.06, 130.10, 130.15, 130.40,
130.66, 130.75, 130.80, 131.41, 134.81, 149.45, 158.78, 162.01,
168.55; 31P NMR (109 MHz, CDCl3, 85% H3PO4) δ 153.26,
151.29, 51.55, 51.21. Anal. Calcd for C56H66N6O12P2S: C,
2′-O-[Bis(2-cyan o-1,1-dim eth yleth oxy)th ioph osph or yl]-
N3-ben zoylu r id in e (16a ). N3-Benzoyl-3′,5′-O-(di-tert-butyl-
silanediyl)uridine (7) (1.02 g, 2.08 mmol) and 1H-tetrazole (328
mg, 4.68 mmol) were rendered anhydrous by repeated co-
evaporation with dry pyridine (5 mL × 5), dry toluene (5 mL
× 2), and CH2Cl2 (5 mL × 2) and dissolved in CH2Cl2 (7.5 mL).
To the mixture was added a 0.25 M solution of bis(2-cyano-
1,1-dimethylethyl) N,N-diethylphosphoramidite (1.2 mmol) in
CH2Cl2. After the mixture was stirred at room temperature
for 45 min, a 1 M solution of S8 in CS2 (20 mL) was added.
The solution was stirred at room temperature for 2 h and then
evaporated under reduced pressure. The residue was dissolved
in CH2Cl2 (300 mL). The precipitate was removed by filtration.
The filtrate was washed twice with 5% NaHCO3 and with
water, dried over Na2SO4, filtered, and evaporated under
reduced pressure. The residue was dissolved in THF (8 mL),
and a mixture of (HF)x‚Py (1.2 mL)-Py (5.4 mL) was added.
The mixture was stirred at room temperature for 10 min, and
then a excess amount of pyridine (10 mL) was added. The

Synthesis of 2′-Phosphorylated Oligouridylates
J . Org. Chem., Vol. 61, No. 12, 1996 4099
60.64; H, 6.00; N, 7.57; S, 2.89. Found: C, 60.91; H, 6.38; N,
7.50; S, 2.63.
with dry pyridine (5 mL × 3), and BSA (0.167 mL, 0.675 mmol)
and a 0.1 M solution (0.9 mL, 0.09 mmol) of DBU in pyridine
were added. After being stirred at room temperature for 10
min, the mixture was quenched by addition of water (0.2 mL).
Iodine (38 mg, 0.3 mmol) was added, and the mixture was
stirred at room temperature for 40 h. The mixture was diluted
with pyridine-water (5 mL, 1:1, v/v), and the excess iodine
was removed by extraction with ether (5 mL × 7). The
aqueous phase was collected and evaporated under reduced
pressure. The residue was dissolved in concentrated ammonia
(45 mL)-pyridine (5 mL), and the mixture was stirred at room
temperature for 18 h. After the solvent was removed under
reduced pressure, the residue was coevaporated with water
(5 mL × 2) and treated with 80% acetic acid (5 mL) at room
temperature for 15 min. The solvent was removed under
reduced pressure, and the residue was partitioned between
water and ether. The aqueous layer was further washed with
ether (5 mL × 2) and concentrated under reduced pressure.
Paper chromatography using Whatman 3MM papers devel-
oped with 2-PrOH-concd NH3-H2O (6:1:3, v/v/v) gave 29
(421A260, 72%): Rf ) 0.19; UV(H2O) λmax 260 nm, λmin 229 nm;
S,S-Dip h en yl 2′-O-[Bis(2-cya n o-1,1-d im eth yleth oxy)-
th iop h osp h or yl]-N3-ben zoyl-5′-O-(4,4′-d im eth oxytr ityl)-
u r id in e 3′-P h osp h or od ith ioa te (23). A mixture of 21 (943
mg, 1.04 mmol), cyclohexylammonium S,S-diphenyl phospho-
rodithioate (633 mg, 1.66 mmol), and 1H-tetrazole (291 mg,
4.16 mmol) was rendered anhydrous by repeated coevaporation
with dry pyridine (5 mL × 7) and finally dissolved in dry
pyridine (10 mL). To the mixture was added isodurenedisul-
fonyl dichloride (1.10 g, 3.33 mmol). After being stirred at
room temperature for 2 h, the mixture was partitioned between
CH2Cl2-pyridine (99:1, v/v, 200 mL) and saturated NaHCO3
(200 mL). The organic phase was collected, washed with
saturated NaHCO3 and water, dried over Na2SO4, filtered, and
concentrated under reduced pressure. The residue was chro-
matographed on a column of silica gel with hexane-ethyl
acetate containing 0.5% pyridine to give 23 (1.03 g, 85%): 1H
NMR (270 MHz, CDCl3, TMS) δ 1.55-1.76 (12H, m, CH3 of
CME), 2.63-2.90 (4H, m, CH2 of CME), 3.31 (2H, s, 5′-H and
5′′-H), 3.81 (6H, s, CH3 of DMTr), 4.02 (1H, s, 4′-H), 5.42-
5.48 (3H, m, 2′-H, 3′-H, and 5′-H), 6.31 (1H, d, J 1′H-2′H ) 6.9
Hz, 1′-H), 6.86-6.89 (4H, m, ArH), 7.24-7.67 (18H, m, ArH),
7.90 (1H, d, J 5H-6H ) 8.6 Hz, 6-H), 8.02 (2H, d, J ) 7.59 Hz,
o-ArH); 13C NMR (67.8 Mz, CDCl3) δ 26.94, 26.97, 27.05, 27.10,
27.14, 27.19, 27.53, 27.60, 31.34, 31.43, 31.72, 31.79, 55.26,
63.00, 76.21, 76.78, 77.20, 83.97, 84.12, 84.67, 84.78, 87.98,
102.89, 113.53, 116.50, 116.60, 125.61, 125.73, 125.84, 127.35,
128.05, 128.27, 129.11, 129.52, 129.56, 129.65, 129.70, 129.78,
129.96, 130.05, 130.15, 130.76, 131.30, 134.55, 134.72, 135.13,
135.22, 135.45, 135.54, 140.00, 143.79, 149.38, 158.85, 161.87,
168.48; 31P NMR (109 MHz, CDCl3, 85% H3PO4) δ 51.20, 51.90.
Anal. Calcd for C59H58N4O12P2S2‚H2O: C, 59.94; H, 5.03; N,
4.73. Found: C, 59.77; H, 4.82; N, 4.74.
1H NMR (270 MHz, D2O, DSS, 40 °C) δ 3.82 (2H, d, J 4′H-5′′H
)
3.3 Hz, 5′-H and 5′′-H of Up), 4.08-4.27 (4H, m, 3′-H, 4′-H,
and 5′-H and 5′′-H of pU), 4.31-4.40 (2H, m, 2′-H of pU and
4′-H of Up), 4.73-4.78 (1H, m, 3′-H of Up), 4.83-4.90 (1H m,
2′-H of Up), 5.99 (1H, d, J 5H-6H ) 8.2 Hz, 5-H of Up), 5.94-
5.97 (2H, m, J 5H-6H ) 8.3 Hz, J 1′H-2′H ) 4.0 Hz, 5-H and 1′-H
of pU), 6.04 (1H, d, J 1′H-2′H ) 6.6 Hz, 1′-H of Up), 7.82 (1H, d,
J 5H-6H ) 8.2 Hz, 6-H of Up), 7.92 (1H, d, J 5H-6H ) 8.3 Hz, 6-H
of pU); 13C NMR (67.8 MHz, D2O, dioxane) δ 61.87, 65.84,
65.91, 70.37, 74.44, 74.75, 74.84, 74.97, 84.08, 84.13, 84.18,
85.08, 88.50, 88.55, 89.43, 103.37, 103.46, 142.42, 143.39,
152.23, 152.61, 166.95, 166.99; 31P NMR (109 MHz, D2O, 85%
H3PO4) δ 0.153, 0.180; MS (FAB-) calcd for C18H23O17N4P2 (M+
- H) 629.0547, found 629.0558.
F u lly P r otected 2′-P h osp h or yla ted Dim er 25. A mix-
ture of 2′,3′,5′-tri-O-benzoyluridine (24) (224 mg, 0.4 mmol)
and 1H-tetrazole (56 mg, 0.8 mmol) was rendered anhydrous
by repeated coevaporation with dry pyridine (5 mL × 5), dry
toluene (5 mL × 3), and dry acetonitrile (5 mL × 2) and finally
dissolved in acetonitrile (4 mL). To the mixture was added
the phosphoramidite unit 22 (592 mg, 0.52 mmol), and the
solution was stirred at room temperature for 3.5 h. tert-Butyl
hydroperoxide (0.3 mL, 2.4 mmol) was added, and stirring was
continued for an additional 50 min. The solvent was removed
under reduced pressure, and extraction was performed with
CH2Cl2-5% NaHCO3. The CH2Cl2 extract was washed with
5% NaHCO3 and water. The usual workup followed by silica
gel column chromatography eluted with CH2Cl2-MeOH con-
taining 0.5% pyridine gave 25 (570 mg, 90%): 1H NMR (270
MHz, CDCl3, TMS) δ 1.57-1.79 (12H, m, CH2 of CME), 2.67-
3.01 (6H, m, CH2 of CME and CE), 3.64-4.98 (2H, m, 5′-H
and 5′′-H of Up), 3.78-3.80 (6H, m, CH3 of DMTr), 4.23-4.56
(6H, m, 4′-H of Up, 4′-H and 5′-H and 5′′-H of pU, POCH2 of
CE), 5.37-5.44 (2H, m, 5-H and 3′-H of Up), 5.50-5.68 (2H,
m, 2′-H of Up and pU), 5.66-5.75 and 5.77-5.81 (1H, m, 3′-H
of diasteromeric pU), 5.88-5.94 (1H, m, 5-H of pU), 6.17 and
6.24 (1H, d, J 1′H-2′H ) 5.9 Hz, 1′-H of diastereomeric pU), 6.34-
6.39 (1H, m, 1′-H of Up), 6.86-6.90 (4H, m, m-ArH of DMTr),
7.27-7.63 (22H, m, 6-H of pU and p- and m-ArH), 7.83-7.99
(9H, m, 6-H of Up and o-ArH); 13C NMR (67.8 MHz, CDCl3) δ
19.52, 19.55, 19.63, 21.37, 26.79, 26.85, 27.17, 27.23, 31.54,
31.59, 31.65, 31.95, 32.04, 55.22, 62.93, 63.02, 63.07, 63.15,
67.23, 67.28, 70.35, 70.48, 73.35, 80.97, 81.08, 83.12, 83.23,
83.29, 83.33, 83.81, 83.92, 84.04, 85.39, 88.00, 88.03, 102.80,
103.29, 103.45, 113.39, 113.50, 116.42, 116.60, 116.68, 116.80,
116.86, 125.21, 127.33, 128.07, 128.14, 128.23, 128.36, 128.45,
128.50, 128.61, 128.95, 129.06, 129.09, 129.70, 129.74, 129.81,
130.01, 130.17, 130.28, 130.33, 130.46, 130.58, 130.69, 131.12,
131.29, 133.78, 134.41, 134.72, 135.06, 139.95, 140.11, 143.76,
149.24, 149.29, 158.83, 161.49, 161.60, 161.81, 165.19, 165.30,
165.48, 168.18, 168.30, 168.37; 31P NMR (109 MHz, CDCl3,
85% H3PO4) δ -2.12, -2.04, 51.14, 51.25. Anal. Calcd for
C80H75N7O22P2S‚3.5H2O: C, 58.46; H, 5.03; N, 5.97. Found:
C, 58.36; H, 4.88; N, 5.91.
Syn th esis of U(2′-p s)p U (30). Compound 25 (48 mg, 0.03
mmol) was dissolved in a 1% solution (2 mL) of trifluoroacetic
acid in CH2Cl2, and the mixture was stirred at room temper-
ature for 15 min. The mixture was quenched by addition of
MeOH-pyridine (5 mL, 1:1, v/v) and extracted with CH2Cl2.
The CH2Cl2 extract was washed successively with 5% NaHCO3
and water, dried over Na2SO4, filtered, and evaporated under
reduced pressure. The residue was rendered anhydrous by
repeated coevaporation with dry pyridine (5 mL × 3), and N,O-
bis(trimethylsilyl)acetamide (0.167 mL, 0.675 mmol) and a 0.1
M solution (0.9 mL, 0.09 mmol) of DBU in pyridine were added.
After being stirred at room temperature for 10 min, the
solution was evaporated under reduced pressure. The residue
was dissolved in concentrated ammonia (45 mL)-pyridine (5
mL), and the mixture was stirred at room temperature for 18
h. After the solvent was removed under reduced pressure, the
residue was coevaporated with water (5 mL × 2) and parti-
tioned between water and ether. The aqueous layer was
further washed with ether (5 mL × 2) and concentrated under
reduced pressure. Paper chromatography using Whatman
3MM papers developed with 2-propanol-concentrated am-
monia-water (6:1:3, v/v/v) gave 30, which was passed through
a column of Dowex 50 W × 8 (sodium salt form, 1.5 × 15 cm)
1
(472A260, 80%): UV(H2O) λmax 260 nm, λmin 229 nm; H NMR
(270 MHz, D2O, DSS) δ 3.83 (2H, d, J 4′H-5′H ) 3.3 Hz, 5′-H
and 5′′-H of Up), 4.19-4.28 (3H, m, 4′-H and 5′-H and 5′′-H of
pU), 4.36-4.43 (2H, m, 3′-H of pU and 4′-H of Up), 4.46-4.50
(1H, m, 2′-H of pU), 4.78-5.00 (2H, m, 2′-H and 3′-H of up),
5.88 (1H, d, J 5H-6H ) 7.6 Hz, 5-H of Up), 5.94-5.99 (2H, m,
1′-H and 5-H of pU), 6.10 (1H, d, J 1′H-2′H ) 7.3 Hz, 1′-H of
Up), 7.89 (1H, d, J 5H-6H ) 7.6 Hz, 6-H of Up), 7.96 (1H, d,
J 5H-6H ) 8.3 Hz, 6-H of pU); 13C NMR (67.8 MHz, D2O,
dioxane) δ 62.24, 66.03, 66.12, 70.46, 74.39, 75.77, 75.85, 84.49,
84.61, 85.48, 88.41, 88.48, 89.23, 103.21, 103.48, 142.48,
144.16, 152.99, 153.15, 167.43, 167.32; 31P NMR (109 MHz,
D2O, 85% H3PO4) δ 0.244, 46.14; MS (FAB-) calcd for
C18H22O16N4P2SNa (M+ - H) 667.0124, found 667.0134.
Compound 30 was gradually air-oxidized upon standing to
give 31: 1H NMR (270 MHz, CDCl3, TMS) δ 3.84-3.87 (4H,
m, 5′-H and 5′′-H of Up), 4.19-4.30 (6H, m, 4′-H, 5′-H, 5′-H of
pU), 4.39-4.45 (6H, m, 4′-H of Up and 2′-H, 3′-H of pU), 4.77-
Syn th esis of U(2′-p )p U (29). Compound 25 (48 mg, 0.03
mmol) was rendered anhydrous by repeated coevaporation

4100 J . Org. Chem., Vol. 61, No. 12, 1996
Sekine et al.
4.90 (4H, m, 3′-H of Up and 2′-H of pU), 5.93-6.00 (6H, m,
5-H of Up and 1′-H, 5-H of pU), 6.17 (2H, 2d, J 1′H-2′H ) 6.9
Hz, 1′-H of Up) 7.92-7.95 (4H, m, 6-H of Up, pU); 31P NMR
(109 MHz, D2O, 85% H3PO4) δ -0.15, 16.00.
(20 µL) of CSP (2 mg/µL). The incubation was done at 37 °C
for 23 h. After being heated at 100 °C for 1 min, the mixture
was analyzed by HPLC.
Tr ea tm en t w ith Sn a k e Ven om P h osp h od iester a se.
Compound 29 (0.5A260, 26 nmol) was dissolved in Tris-HCl
buffer (pH 7.5, 10 µL). To the mixture was added SVP (1 µL,
1 mg/µL). The incubation was done at 37 °C for 24 h. After
being heated at 100 °C for 1 min, the mixture was analyzed
by HPLC.
U(2′-p p -biotin )p U (35). Compound 29 (50A260, 2.55 mmol)
was dissolved in 2 M N-ethylmorpholine buffer (pH 7.0, 10
µL). To the mixture were added water (30 µL), 1 M MgCl2
(20 µL), and 0.25 M biotin-HA-pIm (40 µL). The mixture was
stirred at 37 °C for 40 h. Paper chromatography on Whatman
3MM papers developed with 2-propanol-concentrated am-
An At t em p t t o Syn t h esize [U(2′-p )p ]6U on P olym er
Su p p or t. A CPG gel having U unit 4 (1 µmol, 26.4 µmol/g)
as the 3′-terminus was used. Chain extension was performed
according to the protocol described in Table 1. After all
condensations were finished, a mixture of concentrated am-
monia-pyridine (10 mL, 9:1, v/v) was added. The mixture was
slowly rotated by a rotary evaporator at room temperature for
24 h, filtered, and washed with water. The filtrate and
washing were collected, and the solution was evaporated under
reduced pressure. The residue was treated with 20% trifluo-
roacetic acid in acetic acid (10 mL). Aliquots were taken after
3 and 5 h and analyzed by paper electrophoresis.
Solid P h a se Syn th esis of [U(2′-p )p ]n U (n ) 1, 3, 5, 7,
a n d 9). A CPG gel having a U unit (1 µmol, 26.4 µmol/g) as
the 3′-terminus was used. The synthetic unit 22 was used for
condensation. Chain extension was performed according to
the protocol described in Table 1. After all operations were
over, the CPG gel was evaporated with acetonitrile and
suspended in BSA-pyridine (1:1, v/v, 0.4 mL). The suspension
was slowly rotated in a vessel at room temperature for 1 h,
and then DBU (12 µL, 0.08 mmol) was added. Rotation was
continued at room temperature for 40 h. The CPG gel was
then filtered and washed with pyridine (1 mL × 3). The CPG
gel was treated with a 1 M solution of iodine in THF-
pyridine-water (0.5 mL, 10:10:1, v/v/v) at room temperature
for 48 h. Filtration was performed, and the CPG gel was
washed with pyridine (1 mL × 7). The CPG gel was collected
and transferred to a round flask. Concentrated ammonia-
pyridine (5 mL, 9:1, v/v) was added, and the suspension was
slowly rotated by a rotary evaporator for 24 h. The CPG gel
was removed by filtration and washed with water. The filtrate
and washings were combined and evaporated under reduced
pressure. The residue was dissolved in water and subjected
to polyacrylamide gel electrophoresis and HPLC. In the case
of n ) 1, 3, and 5, the main peaks were pooled and analyzed
by HPLC.
monia-water (6:1:3, v/v/v) gave title compound 32 (12A260
,
23%): UV(H2O) λmax 258 nm, λmin 228 nm; 1H NMR (270 MHz,
D2O, DSS) δ 1.27-1.73 (14H, m, CH2), 2.24 (2H, t, J ) 7.26
Hz, CH2C(O)), 2.77 (1H, d, J Ha-Hb ) 12.9 Hz, Ha of SCH2),
2.96 (1H, dd, J Ha-Hb ) 12.9 Hz, J Hb-CHN ) 4.6 Hz, Hb, of CH2S),
3.14-3.19 (2H, m, CH2NH), 3.30-3.35 (1H, m, SCH), 3.79-
3.86 (4H, m, POCH2 and 5′-H and 5′′-H of Up), 4.19-4.43 (7H,
m, 4′-H of Up, 2′-H, 3′-H, 4′-H, 5′-H, and 5′′-H of pU and
CHCHNH), 4.60 (1H, d, J 5H-6H ) 8.2 Hz, 5-H of Up), 5.94 (1H,
d, J 5H-6H ) 7.9 Hz, 5-H of pU), 6.00 (1H, d, J 1′-2′ )4.95 Hz,
1′-H of pU), 6.09 (1H, d, J 1′H-2′H ) 6.3 Hz, 1′-H of Up), 7.83
(1H, d, J 5H-6H ) 8.2 Hz, 6-H of Up), 7.91 (1H, d, J 5H-6H ) 7.9
Hz, 6-H of pU); 31P NMR (109 MHz, D2O, 85% H3PO4) δ
-0.014, 9.828 (d, J P-P ) 18.5 Hz), -11.45 (d, J P-P ) 18.5 Hz);
MS (FAB-) calcd for C34H51O22N7P3S (M+ - H) 1034.2021,
found 1034.2037.
U(2′-p s-bim a n e)p U (33). Compound 30 (59A260, 3 µmol),
which contained a small amount of 31, was dissolved in
ethanol (0.5 mL)-water (0.3 mL), and NaBH4 (1.1 mg, 0.03
mmol) was added. The mixture was stirred at room temper-
ature for 24 h, and acetone (0.5 mL) was added to decompose
the excess reagent. The solvent was removed under reduced
pressure, and the residue was dissolved in water (0.5 mL). To
the mixture was added a solution of monobromobimane (1.2
mg, 4.4 µmol). The solution was stirred for 16 h and then
chromatographed on Whatman 3MM papers developed with
2-propanol-concentrated ammonia-water (6:1:3, v/v/v) to give
33 (38A260, 64%): UV(H2O) λmax 258 nm, λmin 230 nm; 1H NMR
(270 MHz, D2O, DSS) δ 1.74 (3H, S, CH3 of bimane), 1.81 (3H,
s, CH3 of bimane), 2.39 (3H, s, CH3 of bimane), 2.88-2.95 (1H,
m, CH2 of bimane), 3.34-3.39 (1H, m, CH2 of bimane), 3.81
(2H, d, J ) 2.31 Hz, 5′-H and 5′′-H of Up), 4.15-4.36 (6H, m,
2′-H, 3′-H, 4′-H, and 5′-H and 5′′-H of pU, and 4′-H of Up),
4.75-4.90 (1H, m, 3′-H of Up), 4.91-5.01 (1H, m, 2′-H of Up),
5.80-5.87 (3H, m, 1′-H and 5-H of pU and 5-H of Up), 6.06
(1H, d, J 1′H-2′H ) 6.6 Hz, 1′-H of Up), 7.83-7.88 (2H, 2d, 6-H
of Up and pU); 31P NMR (109 MHz, D2O, 85% H3PO4) δ 0.00,
17.16; MS (FAB-) calcd for C28H33O18N6P2S (M+ - H) 835.1047,
found 835.1049.
En zym a tic Assa y of U(2′-p )p U. Tr ea tm en t w ith Ca lf
In testin a l Alk a lin e P h osp h a ta se F ollow ed by Tr ea tm en t
w ith Nu clea se P 1. Compound 29 (0.5A260, 26 nmol) was
dissolved in 50 mM Tris-HCl buffer (pH 8.5, 250 µL) contain-
ing 0.1 mM EDTA. To the mixture was added a solution (50
µL) of CIAP (1 unit/µL). The incubation was done at 50 °C
for 1 h. After being heated at 100 °C for 1 min, the mixture
was analyzed by HPLC. The main peak which corresponds
to UpU was pooled, lyophilized, and treated with a solution
(10 µL) of nuclease P1 (1 unit/µL) in 20 mM AcOH-AcONa
buffer (pH 5.3, 0.1 mL) at 50 °C for 1 h. After being heated at
100 °C for 1 min, the mixture was analyzed by HPLC.
Tr ea tm en t w ith Nu clea se P 1. Compound 29 (0.5A260, 26
nmol) was dissolved in 20 mM AcOH-AcONa buffer (pH 5.3,
0.25 mL). To the mixture was added a solution (25 µL) of
nuclease P1 (1 unit/µL). The incubation was done at 50 °C
for 40 h. After being heated at 100 °C for 1 min, the mixture
was analyzed by HPLC.
Ack n ow led gm en t. This work was supported by The
Naito Foundation, Toray Foundation, and a Grant-in-
Aid for Scientific Research on Priority Areas from the
Ministry of Education, Science and Culture, J apan.
1
Su p p or tin g In for m a tion Ava ila ble: Copies of H NMR
1
data for compounds 11, 18a ,b, 33, and 35; H and 13C NMR
data for compounds 5, 7, 8, 10a , 13, 15-23, 25, 29-31, 33,
and 35; 31P NMR data for compounds 5, 6, 15-20, 33, and 35;
FAB mass data of 29, 30, 33, and 35; 1H-1H COSY NMR data
of 25, 29, 33, and 35; Figure 7 (stability of 11a in pyridine),
Figures 8 and 9 (HPLC profiles and 31P NMR spectra of 29
and 30), Figures 10 and 11 (HPLC profiles of the mixtures
obtained by the reactions of 30 with 32 and 34, respectively),
Figure 12 (analysis of the polymer supported synthesis of U(2′-
p)pU under various conditions), and Figures 13-15 (enzymatic
analysis of [U(2′-p)p]nU, n ) 1, 3, and 5) (58 pages). This
material is contained in libraries on microfiche, immediately
follows this article in the microfilm version of the journal, and
can be ordered from the ACS; see any current masthead page
for ordering information.
Tr ea tm en t w ith Ca lf Sp leen P h osp h od iester a se. Com-
pound 29 (0.5A260, 26 nmol) was dissolved in 0.1 M NH4OAc
buffer (pH 5.7, 90 µL). To the mixture was added a solution
J O952073K