Chemical Synthesis and Properties of Conformationally Fixed Diuridine Monophosphates as Building Blocks of the RNA Turn Motif

Article abstract of DOI:10.1021/jo971797o

Two intramolecularly cyclized diuridine monophosphates having an amide and a carbamate linker have been synthesized for fixation of the U-turn structure found in various tRNAs and hammerhead ribozymes. The structural analysis of these cyclic dimers using

Full text of DOI:10.1021/jo971797o

J . Org. Chem. 1998, 63, 1429-1443
1429
Ch em ica l Syn th esis a n d P r op er ties of Con for m a tion a lly F ixed
Diu r id in e Mon op h osp h a tes a s Bu ild in g Block s of th e RNA Tu r n
Motif
Kohji Seio, Takeshi Wada, Kensaku Sakamoto,† Shigeyuki Yokoyama,† and Mitsuo Sekine*
Department of Life Science, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology,
Nagatsuta, Midoriku, J apan, and Department of Biophysics and Biochemistry, Faculty of Science,
The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, J apan
Received September 29, 1997
Two intramolecularly cyclized diuridine monophosphates having an amide and a carbamate linker
have been synthesized for fixation of the U-turn structure found in various tRNAs and hammerhead
ribozymes. The structural analysis of these cyclic dimers using CD, 1H NMR, and 31P NMR
spectroscopy shows that they have rigid, unstacked conformations. Detailed computer simulations
of their 3D structures based on molecular mechanics calculations suggest that a two-state
equilibrium between a stretch and a turn conformation exists in favor of the former. The simulation
also suggested that the diuridine monophosphate containing the carbamate-linker is better as a
mimic of the U-turn structure than the amide-linked dimer. The enzymatic properties of these
cyclized dimers are also described.
In tr od u ction
to the conformational study of the â-turn motif5 in the
field of peptide chemistry, so far there have been no
applications in the field of nucleic acid chemistry.
The sharply bent structure called “U-turn” has been
commonly found in the anticodon loop of tRNAs, and its
structural properties have been studied in relation to
their biological role.1 A number of papers have reported
conformational studies of anticodon first-letter ribo-
nucleosides that are located at the bent point of the
U-turn.2 The latest discovery of a similar U-turn struc-
ture at the active site of the crystal structure of ham-
merhead ribozymes3 indicates a new biological role of this
motif. Since the importance of nucleotide sequences of
this active site for the ribozyme activity has already been
suggested by base mutation experiments,4 the stereo-
chemical requirement of this region, especially, the
U-turn, should be clarified next. An effective way to
study the conformational significance of the turn struc-
ture is to synthesize sterically fixed U-turn mimics.
Although such a strategy has been successfully applied
We have previously reported6 the chemical synthesis
of Upmnm5U, which corresponds to the 33-34th nucleo-
tide dimer sequence, i.e., the minimum U-turn unit, of
minor tRNAArg of E. coli,7a to study conformational
properties of the U-turn region. It was suggested that
hydrogen bonding between the 2′-hydroxyl group of
uridine (33rd) and the amino group of 5-[(methylamino)-
methyl]uridine (34th) might be a possible interresidual
interaction to stabilize the “rigid” C3′-endo conformation
of the anticodon first letter, mnm5U, which allows precise
recognition of A and G in the codon third letter of
mRNA.7b This possibility is discussed on the assumption
that the 3D structural conformation of the U-turn is
identical with that of tRNAPhe 8
. It is also likely that such
an interresidual interaction plays an additional role in
stabilization of the sharp bend of the U-turn9 as well as
predicts the C3′-endo sugar pucker in the first letter.
Therefore, our interest was focused on this cyclic struc-
ture via an interresidual hydrogen bonding, and we
† The University of Tokyo.
(1) Guenther, R. H.; Hardin, C. C.; Sierzputowska-Gracz, H.; Dao,
V.; Agris, P. F. Biochemistry 1992, 31, 11004. (b) Dao, V.; Guenther,
R. H.; Agris, P. F. Ibid. 1992, 31, 11012. (c) Auffinger, P.; Louis-May,
S.; Westhof, E. J . Am. Chem. Soc. 1995, 117, 6720. (c) Rould, M. A.;
Perona, J . J .; So¨ll, D.; Steitz, T. A. Science 1989, 246, 1135. (d)
Sundaralingam, M.; Mizuno, H.; Stout, C. D.; Rao, S. T.; Liebman, M.;
Yathindra, N. Nucl. Acids Res. 1976, 3, 2471. (e) Kim, S. H.; Sussman,
J . L. Nature 1976, 260, 645.
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Matsuda, A.; Ueda, T.; Yamaizumi, Z.; Kuchino, Y.; Nishimura, S.;
Miyazawa, T. Pure Appl. Chem. 1989, 61, 573. (b) Sierzputowska-
Gracz, H.; Sochacka, E.; Malkiewicz, A.; Kuo, K.; Gehrke, C. W.; Agris,
P. F. J . Am. Chem. Soc. 1987, 109, 7171. (c) Kasai, H.; Nishimura,
S.; Vorbru¨ggen, H.; Iitaka, Y. FEBS Lett. 1979, 2, 270. (d) Watanabe,
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Chrusciel, R. A.; Kahn, M. Biochem. Biophys. Res. Commun. 1992, 187,
999. (b) Nakanishi, H.; Chrusciel, R. A.; Shen, R.; Bertenshaw, S.;
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references therein.
(6) Sekine, M.; Seio, K.; Satoh, T.; Sakamoto, K.; Yokoyama, S.
Nucleosides Nucleotides 1993, 12, 305.
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Yamaizumi, Z.; Nishimura, S.; Miyazawa, T.; Yokoyama, S. Eur. J .
Biochem. 1993, 216, 369. (b) Sakamoto, K.; Kawai, G.; Watanabe, S.;
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S0022-3263(97)01797-0 CCC: $15.00 © 1998 American Chemical Society
Published on Web 02/20/1998

1430 J . Org. Chem., Vol. 63, No. 5, 1998
Seio et al.
F igu r e 1. Left: intraresidual hydrogen bonding between the U(33) and mnm5s2U(34) proposed by Hillen et al. Right: structures
of the amide-linked and carbamate-linked cyclized dinucleoside monophosphates (1 and 2).
Sch em e 1
designed two U-turn mimics 1 and 2 in which the
hydrogen bond is replaced by an amidomethyl and a
carbamate linkage, respectively, as depicted in Figure 1.
A preliminary result of the synthesis of 1 has already
been reported briefly by us.10
These two cyclic diuridylates are expected to be useful
not only as starting materials for preparation of the
minimum synthetic units of RNA or DNA oligonucleo-
tides having a U-turn but also as the first mimics of the
RNA turn chemically fixed by introducing a cyclic struc-
ture. In this paper, we report the detailed studies of the
chemical synthesis and conformational analysis of 1 and
2.
methyl)-2′,3′-O-isopropylideneuridine (7), which is the
common 3′-terminal nucleoside unit in the synthesis of
both 1 and 2 (Scheme 1). To introduce the aminomethyl
group to position 5 of 2′,3′-O-isopropylideneuridine, we
employed 5-(chloromethyl)-2′, 3′-O-isopropylideneuridine
(4)11 as an intermediate. Compound 4 was readily
obtained from 5-(hydroxymethyl)-2′,3′-O-isopropylideneu-
ridine (3)12 using trimethylsilyl chloride as a chlorinating
agent. Compound 4 was used in situ for the next reaction
without further purification because the chloromethyl
group of 4 was quite labile during chromatography,
producing a considerable amount of starting material 3.
Direct amination of 4 with aqueous NH3 was unsuccessful
because of further N-alkylation of the desired product and
competitive hydrolysis of the starting material. Shiau
et al. reported the successful chemical synthesis of
5-(aminomethyl)-2′-deoxyuridine by using the correspond-
Resu lts a n d Discu ssion
Syn t h esis of 5-(Am in om et h yl)u r id in e Der iva -
t ives. First, we examined the synthesis of 5-(amino-
(11) Sekine, M.; Peshakova, L. S.; Hata, T. J . Org. Chem. 1987, 52,
5060.
(12) Scheit, K. H. Chem. Ber. 1966, 99, 4.
(10) Sekine, M.; Seio, K.; Wada, T.; Sakamoto, K.; Yokoyama, S. J .
Am. Chem. Soc. 1994, 116, 4469.

Synthesis and Properties of Diuridine Monophosphates
Sch em e 2
J . Org. Chem., Vol. 63, No. 5, 1998 1431
ing 5-(azidomethyl)-2′-deoxyuridine derivative as an
intermediate.13 Therefore, the 5-(azidomethyl)uridine
derivative 5 was prepared in an overall yield of 50% from
3 by treatment of 4 with NaN3. Compound 5 was also
synthesized in 53% yield by the azidation of the N-methyl
quaternary salt 6 of 2′, 3′-O-isopropylidene-5-[(piperidin-
3-yl)methyl]uridine (9), which was prepared by the
Mannich reaction of 2′,3′-O-isopropylideneuridine (8).14
We have tested a number of methods to reduce the
azide group into an amino group.15 Consequently, the
best result was obtained by employing Letsinger’s
method15b using 1.6 equiv of triphenylphosphine as a
reducing agent. This reduction gave 7 in 71% yield.
Syn th esis of Am id e-Lin k ed Diu r id in e Mon op h os-
p h a te 1. For the preparation of a 5′-terminal uridine
unit of cyclic dinucleoside monophosphate 1, we required
2′-O-[(alkoxycarbonyl)methyl]uridine derivatives as key
intermediates. A procedure for the chemical synthesis
of such 2′-O-[(alkoxycarbonyl)methyl]ated uridine deriva-
tives by use of suitably protected riboses as starting
materials has been reported by Haner et al.16 Since this
method involved a multistep reaction, we searched for a
more straightforward procedure from a uridine deriva-
tive. Treatment of a 3′,5′-O-silylated uridine derivative
10 with NaH in THF followed by alkylation using ethyl
bromoacetate resulted in formation of a considerable
amount of N3-alkylated product, and the desired O-
alkylated product was obtained in 46% yield. To avoid
the N3-alkylation, we employed the triphenylmethane-
sulfenyl (TrS) group17 as a uridine imide protective group.
Compound 10 was converted to the N-protected uridine
derivative 11 (Scheme 2) by phase-transfer catalysis with
triphenylmethanesulfenyl chloride according to the pro-
cedure described previously.18 Treatment of 11 with NaH
followed by the successive alkylation with tert-butyl
bromoacetate gave the 2′-O-[(butoxycarbonyl)methyl]-
uridine derivative 12 in 78% yield. The 1,1,3,3-tetra-
isopropyldisiloxane-1,3-diyl (TIPDS) group of 12 was
removed by the use of TBAF in the presence of 2.2 equiv
of acetic acid19,20 to give the diol 13 in 98% yield. The
usual dimethoxytritylation of 13 gave the 5′-masked
product 14 in 88% yield. The TrS group of 14 was then
removed to give compound 15 in 70% yield according to
the procedure reported previously using 2.5 equiv of
tributyltin hydride in the presence of AIBN as an
initiator.18 The tert-butyl ester group of 15 thus obtained
was hydrolyzed in 0.2 M KOH-pyridine, and the result-
ing intermediate 16 was allowed to react with 2.9 equiv
of tert-butyldimethylsilyl chloride in the presence of 2.9
equiv of imidazole to give the 5′-terminal nucleoside unit
17 in an overall 78% yield from 15.
The 3′-O-silylated uridine derivative 17 was converted
to the succinamido ester 18 by the reaction with N-
hydroxysuccinimide in the presence of N-ethyl-N′-[2-
(dimethylamino)propyl]carbodiimide hydrochloride. The
successive aminolysis of 18 with 1.2 equiv of 7 gave
compound 19 in 93% yield, as shown in Scheme 3.
Phosphorylation of 19 with cyclohexylammonium S,S-
diphenyl phosphorodithioate in the presence of iso-
durenedisulfonyl dichloride (DDS)21 and 1H-tetrazole
resulted in formation of compound 20 in 82% yield. One
of the two phenylthio groups was removed from 20 under
weakly basic conditions (NEt3-H2O-pyridine), and the
TBDMS group was subsequently removed by treatment
with TBAF. In situ cyclization of the intermediate 21
using DDS in the presence of 1H-tetrazole gave the fully
protected cyclic diuridine monophosphate 22 in 50% yield
via a two-step reaction from 20.
(13) (a) Shiau, G. T.; Schinazi, R. F.; Chen, M. S.; Prusoff, W. H. J .
Med. Chem. 1980, 23, 127. (b) Kampf, A.; Pillar, C. J .; Woodford, W.
J .; Mertes, M. P. J . Med. Chem. 1976, 19, 909. (c) Edelman, M. S.;
Barfknecht, R. L.; Huet-Rose, R.; Boguslawski, S.; Mertes, M. P. J .
Med. Chem. 1977, 20, 669. (d) Hampton, A.; Kappler, F.; Chawla, R.
R. J . Med. Chem. 1979, 22, 621.
(14) (a) J ones, S. S.; Reese, C. B.; Ubasawa, A. Synthesis 1982, 259.
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877.
(15) (a) Hata, T,; Yamamoto, I.; Sekine, M. Chem. Lett. 1976, 601.
(b) Mungall, W. S.; Greene, G. L.; Heanner, G. A.; Letsinger, R. L. J .
Org. Chem. 1975, 40, 1659. (c) Samano, M. C.; Robins, M. J .
Tetrahedron Lett. 1991, 6293.
Finally, compound 22 thus obtained was deprotected
by treatment with 20 equiv of bis(tributyltin) oxide22 to
(16) Keller, T. H.; Haner, R. Helv. Chim. Acta 1993, 76, 884.
(17) (a) Branchaud, B. P. J . Org. Chem. 1983, 48, 3531. (b)
Branchaud, B. P. Ibid. 1983, 48, 3538. (c) Takaku, H.; Imai, K.; Nagai,
M. Chem. Lett. 1988, 857. (d) Netscher, T.; Weller, T. Tetrahedron
1991, 47, 8145.
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(19) Inoue, H. University of Hokkaido, unpublished work.
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J . Med. Chem. 1991, 34, 2917.
(21) Sekine, M.; Matsuzaki, J .; Hata, T. Tetrahedron 1985, 41, 5279.

1432 J . Org. Chem., Vol. 63, No. 5, 1998
Seio et al.
Sch em e 3
Sch em e 4
remove the phenylthio group followed by hydrolysis of
the isopropylidene group under acidic conditions (60%
formic acid at rt) to give the desired amide-linked cyclic
diuridine monophosphate 1 in 61% yield. The chemical
structure of 1 was confirmed by 1H, 31P, 13C, 1H-1H
COSY, and NOESY NMR spectroscopic analysis.
carbamate-linked cyclic diuridine monophosphate 2 as
shown in Scheme 4. As the 5′-terminal nucleotide unit
of 2 we employed a 2′-O-carbonate ester derivative 23,
which has a reactive p-nitrophenyl carbonate function.
Reaction of compound 10 with 1.2 equiv of p-nitrophenyl
chloroformate23 in toluene in the presence of pyridine
gave 23 in 96% yield.
Syn th esis of Ca r ba m a te-Lin k ed Diu r id in e Mon o-
p h osp h a te. Next, we examined the synthesis of the
Condensation of 23 with 7 afforded the protected
dinucleoside 24 in 89% yield. The remaining 5′-hydroxyl
(22) Sekine, M.; Tanimura, H.; Hata, T. Tetrahedron Lett. 1985, 26,
4621.
(23) Letsinger, R. L.; Ogilvie, K. K. J . Org. Chem. 1967, 32, 296.

Synthesis and Properties of Diuridine Monophosphates
J . Org. Chem., Vol. 63, No. 5, 1998 1433
F igu r e 2. 1H NMR saturation transfer spectra of 27 measured at 270 MHz. Ha and Hb mean that the protons are attached to the
5′- and 3′-terminal nucleotide residues, respectively. The spectra recorded on saturating the 1′-Hb signal (top) or 1′-Ha (center)
are shown with the spectrum without irradiation (bottom).
group of 24 was protected by acetylation with acetic
anhydride in pyridine to give the fully protected diuridine
carbamate derivative 25 in a nearly quantitative yield.
Then, the TIPDS group of 25 was removed by TBAF to
give the diol 26 in 96% yield. Although 26 showed a
single spot on TLC after purification by silica gel column
chromatography, its 1H NMR spectrum showed the
presence of two components. It is well-known that
carbonyl-containing groups, such as acetyl, benzoyl, and
carbonate groups, attached to one of the two hydroxyl
groups of the cis-diol function in ribonucleosides, tend to
migrate easily to the neighboring hydroxyl group.24
Therefore, it should be carefully checked if the carbamate
group of 26 migrated from the 2′-hydroxyl group to the
proximal 3′-hydroxyl group in the 5′-terminal nucleoside
upon desilylation, giving rise to a mixture of the 2′- and
3′-carbamate derivatives. As discussed below, however,
unequivocal saturation transfers both from the 1′-Ha to
the 1′′-Ha signal and from the 1′-Hb to the 1′′-Hb signal.
These observations clearly proved that these two isomers
are in slow equilibrium with each other within an NMR
time scale.
One of the two phenylthio groups and the acetyl group
of 27 were removed under alkaline conditions (0.2 M
KOH-pyridine, 1:1, v/v), and the resulting intermediate
28 was cyclized by using DDS and 1H-tetrazole to give
the fully protected cyclic diuridine monophosphate 29 in
49% yield. Finally, compound 29 was deprotected using
the same procedure as employed in the synthesis of 1 to
give the carbamate-linked diuridine monophosphate 2 in
58% yield.
The 1H and 31P NMR spectra of 2 also showed splitting
patterns similar to those of compounds 26 and 27 as
1
noted above. Therefore, 31P and H NMR spectra were
1
further analysis using H NMR disclosed that the two
measured at various temperatures. In the 31P NMR
spectra, the two signals approached closer with an
increase of the temperature. Finally, they converged to
a single peak at the position of 1.14 ppm at 80 °C as
shown in Figure 3.
isomers observed are not a pair of regioisomers but an
equilibrium mixture of isomers that are probably rota-
mers around the OdC-N-C bond of the carbamate
linkage. The 5′-hydroxyl group of 26 was further blocked
with the 4,4′-dimethoxytrityl group, and the 3′-hydroxyl
group was phosphorylated by using PSS-DDS-1H-tetra-
zole to give the fully protected diuridine monophosphate
27 in 88% yield via two steps from 26. The 1H NMR
spectrum of 27 showed an isomeric pattern similar to that
of 26 as described in the foregoing.
These phenomena also supported the idea that the two
components in the final product are a set of equilibrium
isomers. A similar conclusion was obtained from analysis
of the 1H NMR signals, especially of the 6-H proton
signals of the 3′-terminal uridine. Although they were
apart from each other (7.38 and 7.89 ppm) at 25 °C, they
were melted into the baseline as the temperature was
raised to 70 °C.
To ascertain the presence of the rotamers, saturation
1
transfer H NMR experiments of 27 were carried out.25
These analyses as depicted in Figure 2 showed the
P r op er t ies of Cyclized Diu r id in e Mon op h os-
p h a tes tow a r d Nu clea ses 1 a n d 2. We examined the
sensitivity of the cyclized diuridine monophosphates
toward three enzymes of snake venom phosphodiesterase
(24) Reese, C. B.; Trentham, D. R. Tetrahedron Lett. 1965, 2467.
(25) (a) Neuhaus, D.; Williamson, M. In The Nuclear Overhauser
Effect in Structural and Conformational Analysis; VCH Publishers:
New York, 1989. (b) Campbell, I. D.; Dobson, C. M.; Ratcliffe, R. G.;
Williams, R. J . P. J . Magn. Reson. 1978, 29, 397.

1434 J . Org. Chem., Vol. 63, No. 5, 1998
Seio et al.
F igu r e 3. 31P NMR spectra of 27 in CDCl3 at various temperatures.
Ta ble 1. Nu clea ses Sen sitivity of 1 a n d 2a
should be noted that the observed conformational rigidity
of 1 and 2 is suitable for our purpose to introduce stable
turn structures into RNA or DNA oligomers and to
control their 3D structure. There are two different
aspects in the spectra of 1 and 2. One is the positive
Cotton effect around 200 nm of 2 that can be attributed
to the carbamate linkage, and the other is the larger
negative Cotton effect around 240 nm of 2 than that of
1. The difference in Cotton effect around 240 nm could
be due to the spatial geometry of the 5′- and 3′-terminal
bases because the Cotton effect around 240 nm is
commonly used as a sensitive indicator to discriminate
the A- and B-type DNA duplexes.27 Therefore, these CD
spectra reflect the conformational difference between 1
and 2 which could be the origin of the different stability
of these cyclized diuridine monophosphates toward NP1.
Con for m a tion a l Stu d ies Usin g 31P NMR An a lysis.
Various X-ray crystallographic studies have revealed
that, in the usual A-type RNA double helixes, the P-O
ester bonds (ú, R) of all ribonucleotide residues are in the
energetically most favorable conformation (g-, g-).28 To
stabilize the RNA turn structure, the conformation of the
two P-O ester bonds must be perturbed from normal (g-,
g-) to (g-, t), (t, g-), (g+, t), or (t, g+) [These four kinds of
conformers having the t and g+ (or g-) conformations are
represented as gt/tg in this paper]. Such gt/tg conformers
have been found, for example, in the crystal structure of
E. coli tRNAPhe at the position of U(33)pG(34) with the
dihedral angles of (g-, t). To determine the dihedral
angles around the P-O ester bond using NMR spectros-
copy, however, the Karplus equation, which has been
widely used in conformational studies of nucleic acids,
could not be used because of the absence of the corre-
sponding three-bond coupling constants, i.e., O-P-O-
C. On the other hand, Gorenstein et al. have reported
the experimental and theoretical evidence of the correla-
tion between the dihedral angle and 31P NMR chemical
shift of the O-P-O-C bond and its application to the
conformational analysis of oligonucleotides, DNA double
strands, and DNA-protein complexes.29 According to
their work, the 31P NMR chemical shifts of the inter-
SVP
CSP
NP1
1
2
stable
stable
stable
stable
digested
stable
a
Key: SVP, snake venom phosphodiesterase; CSP, calf spleen
phosphodiesterase; NP1, nuclease P1. The reaction conditions are
described in the Experimental Section.
(SVP), calf spleen phosphodiesterase (CSP), and nuclease
P1 (NP1). These results are summarized in Table 1.
The amide-linked dimer 1 was not digested by SVP and
CSP at all even after prolonged reaction time (4 days).
In contrast, this dimer was digested by NP1 completely
after 18 h to give a new single peak on HPLC that was
expected to be the ring-opened product 30. With regard
to these results, some research groups have reported the
interesting observation that some of the chemically
synthesized and naturally occurring cyclized nucleoside
diphosphates have similar tolerance and sensitivity
toward enzymes as described here.26 More interestingly,
there was a marked difference in sensitivity to NP1
between 1 and 2. As shown in Table 1, the carbamate-
linked dimer 2 was resistant to SVP, CSP, and even to
NP1 that could cleave the phosphodiester linkage of 1.
Since the structural difference between 1 and 2 is only
one methylene group, the observed difference in their
sensitivities in the NP1 digestion assay probably resulted
from a subtle conformational difference between 1 and
2. Therefore, we next studied the conformational prop-
erty of these cyclic diuridine monophosphates.
Con for m a tion a l Stu d ies Usin g Cir cu la r Dich r o-
ism An a lysis. In Figure 4 the CD spectra of 1, 2, and
uridylyl(3′-5′)uridine (UpU) at various temperatures are
shown.
The most significant were the peaks around 270 nm,
which reflect the orientation of the base moiety of 1, 2,
and UpU. UpU exhibited a striking temperature depen-
dence of the positive Cotton effect around 270 nm
changing its peak intensity, while little temperature
dependence was observed in both 1 and 2. These results
could be undoubtedly explained in terms of the relative
conformational rigidity of 1 and 2 compared to UpU. It
(27) Moore, D. S.; Wagner, T. E. Biopolymers 1974, 13, 977.
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1988, 110, 1691-1694. (b) Alagona, G.; Ghio, C.; Kollman, P. A. J .
Am. Chem. Soc. 1985, 107, 2229.
(29) Gorenstein, D. G. Chem. Rev. (Washington, D.C.) 1994, 94, 1315
and references therein.
(26) (a) Ikehara, M.; Tezuka, T. (b) Uesugi, S.; Yasumoto, M.;
Ikehara, M.; Fang, K. N.; Ts’O, O. P. J . Am. Chem. Soc. 1994, 116,
5480. (c) Liuzzy, M.; Weinfield, M.; Peterson, M. C. J . Biol. Chem.
1989, 264, 6355.

Synthesis and Properties of Diuridine Monophosphates
J . Org. Chem., Vol. 63, No. 5, 1998 1435
F igu r e 5. Temperature dependence of the 31P NMR chemical
shifts of 1, 2, and UmpU in 10 mM sodium cacodylate (pH
7.0).
Ta ble 2. 1H-1H Cou p lin g Con sta n ts of 1 Deter m in ed by
400 MHz NMRa
J (Hz)
5′-terminal
3′-terminal
protons
nucleotide residue
nucleotide residue
1′,2′
2′,3′
3′,4′
4′,5′
4′,5′′
4.6
4.3
2.8
3.8
2.8
4.3
n.d.
n.d.
n.d.
n.d.
a
The coupling constants that were not determined because of
the peak overlap are described as n.d.
in the case of both 1 and 2, the lines of 31P chemical shifts
drawn in Figure 5 show parallel shifts to lower field by
0.8 ppm for 1 and 0.9 ppm for 2 compared to that of the
UmpU upon heating from 26 to 80 °C. These low-field
shifts suggest that the fractional population of the gt/tg
conformers around the P-O ester bonds are increased
both in the amide- and carbamate-linked dimers relative
to UmpU. The amplitude of the chemical sift variation
∆δ ) δ(80 °C) - δ(26 °C) was expected to reflect the
conformational rigidity of these compounds. The order
∆δ (UmpU) ) 0.69 ppm > ∆δ (amide) ) 0.54 ppm > ∆δ
(carbamate) ) 0.43 ppm suggested that the carbamate-
linked dimer is conformationally most rigid among them,
the rigidity of the amide-linked dimer ranks second, and
the UmpU is the most flexible. This order is in good
agreement with the structural consideration based on the
number of ring members and the result of CD studies
described above.
F igu r e 4. CD spectra of amide-linked 1 (a), carbamate-linked
2 (b) and UpU (c) in 10 mM sodium phosphate (pH 7.0).
nucleotidic phosphates are shifted upfield (∼-1 ppm)
when they are in the normal (g-, g-) state and shifted
downfield (∼+1 ppm) when the fractional population of
the gt/tg conformer increases. Temperature dependence
of the 31P NMR chemical shifts of 1, 2, and 2′-O-
methyluridylyl(3′-5′)uridine (UmpU) as a reference com-
pound is shown in Figure 5. The chemical shifts of 2
plotted in Figure 5 were obtained by averaging those of
the two rotamers described above.
1
Con for m a tion An a lysis of 1 a n d 2 Usin g H NMR
1
a n d NOESY Sp ectr a . The H-1H coupling constants
of compound 1 were obtained by using 400 MHz NMR.
These values are listed in Table 2.
The 31P NMR chemical shifts of UmpU varied linearly
from -0.33 to +1.07 ppm with increasing temperature
(from 26 to 80 °C). This observation can be interpreted
as the result of the conformational transition of the P-O
bonds from the energetically stable (g-,g-) to the
unstable gt/tg. Although a similar trend was observed
The ribose conformation of the 5′- and 3′-terminal
nucleotides was analyzed using the J 1′,2′ and J 3′,4′ accord-
ing to the formula P(C2′-endo) ) J 1′,2′/(J 1′,2′ + J 3′,4′)
30 where
P(C2′-endo) represents the population of the C2′-endo
(30) Davies, D. B.; Danyluk, S. S. Biochemistry 1974, 13, 4417.

1436 J . Org. Chem., Vol. 63, No. 5, 1998
Seio et al.
F igu r e 6. Low-energy structures of 1 calculated by fixing the 5′-terminal and 3′-terminal ribose conformations in C3′-endo and
C3′-endo (a), C3′-endo and C2′-endo (b), C2′-endo and C3′-endo (c), and C2′-endo and C2′-endo (d), respectively.
conformation. The P(C2′-endo) calculated was 62% for
the 5′-terminal nucleotide.
the unstacked conformation in which the distance be-
tween the 5′- and 3′-nucleotide residues is relatively long.
The corresponding coupling constants of compound 2
except for the J 1′,2′ of both the 5′- and 3′-terminal residues
of the major equilibrium isomer were not determined
unambiguously because of the broadening and overlap-
ping of the signals induced by the slow exchange. The
J 1′,2′ values of the 5′- and 3′-terminal residues were 8.0
and 5.1 Hz, respectively. These relatively larger values
indicate that the conformation of the riboses of compound
2 is stabilized in the C2′-endo conformation.
3D Mod el Bu ild in g Usin g Molecu la r Mech a n ics
Ca lcu la tion s. The detailed 3D structure models of 1
and 2 were constructed by the molecular mechanics
calculation using MacroModel program version 4.5.31 The
all-atom AMBER*32 force field was used with the con-
stant dielectric constant ꢀ of 1.0 and with the implicit
solvent model, GB/SA.33 The conformation search was
done by using four independent series of simulations in
To study the conformation of compound 1 and 2, their
NOESY spectra were measured at 500 MHz. All cross
peaks observed, however, were attributed to those of
interresidual interactions, and no quantitative informa-
tion about the spatial geometry between the 3′-terminal
and 5′-terminal nucleotide units was obtained from the
NOESY spectra for either 1 or 2. These results suggest
(31) Mohamadi, F.; Richards, N. G. J .; Guida, W. C.; Liskamp, R.;
Caufield, C.; Chang, T.; Hendrickson, T.; Still, W. C. J . Comput. Chem.
1990, 11, 440.
(32) (a) Weiner, S. J .; Kollman, P. A.; Nguyen, D. T.; Case, D. A. J .
Comput. Chem. 1986, 7, 230. (b) Weiner, S. J .; Kollman, P. A.; Case,
D. A.; Singh, U. C.; Ghio, C.; Alagona, G.; Profeta, S., J r.; Weiner, P.
J . Am. Chem. Soc. 1984, 106, 765. (c) McDonald, D. Q.; Still, W. C.
Tetrahedron Lett. 1992, 33, 7743.

Synthesis and Properties of Diuridine Monophosphates
J . Org. Chem., Vol. 63, No. 5, 1998 1437
F igu r e 7. Low-energy structures of the trans rotamer of carbamate-linked dimer 2 calculated by fixing the 5′-terminal and
3′-terminal ribose conformations in C3′-endo and C3′-endo (a), C3′-endo and C2′-endo (b), C2′-endo and C3′-endo (c), and C2′-
endo and C2′-endo (d), respectively.
which the 5′- and 3′-terminal ribose conformations were
fixed to C3′-endo-C3′-endo, C3′-endo-C2′-endo, C2′-
endo-C3′-endo, and C2′-endo-C2′-endo each during the
simulations. In each series, 5000 conformers were
generated by using the MCMM option program,32 and the
low-energy conformers (within 12 kJ /mol from the lowest
energy structures) were sampled in each simulation.
Shown in Figure 6 are the lowest energy structures of
amide-linked dimer 1. Two classes of structures, which
were highly correlated with conformation of the 5′-
upstream ribose, were obtained. When the 5′-terminal
ribose was fixed in the C3′-endo conformation, the
simulations gave the “turn” conformations, as shown in
Figure 6a (-882.38 kJ /mol) and 6b (-875.71 kJ /mol), in
which the relative direction of the glycosyl bonds of the
5′- and 3′-terminal nucleotides was reversed and the
distance between the two nucleotide residues was short.
While the other two series of simulations obtained by
fixing the 5′-terminal ribose conformation in the C2′-endo
conformation gave the “stretch” conformations, as shown
in Figure 6c (-902.83 kJ /mol) and d (-893.80 kJ /mol),34
in which the 5′- and 3′-terminal nucleotide units were
oriented almost in the same direction and the distance
between them was long. The phosphate backbone con-
formations (ú, R) were (g+, g+), (g+, t), (t, g+), and (g-, t)
for the structures depicted as Figure 6a-d, respectively.
Those conformational properties are summarized as the
dominance of the gt/tg conformations that are in agree-
ment with those deduced from the 31P NMR study
1
described above. The NOESY and H NMR spectra of 1
described in the preceding section indicated that 1 has a
predominant conformation in which the ribose conforma-
tion was C2′-endo and the distance between the 5′- and
3′-terminal nucleotide units was long. The structures
depicted in Figure 6c,d satisfy these two properties.
Therefore, the solution structure of 1 should be pictured
as an equilibrium mixture of the “turn” (Figure 6a,b) and
“stretch” (Figure 6c,d) conformers in favor of the latter.
Although the turn conformation is expected to be minor
from this computer simulation, the two-state equilibrium
between the turn and stretch conformations of 1 suggests
the relative stability of the turn structure in this cyclized
(34) The energy values were calculated for the structures shown in
the figures after removing the constraints of the ribose. Even when
the structures were energy reminimized without the constraints, the
structural differences were small, rms < 0.3 Å, between the structures
before and after the reminimization procedure.
(33) Still, W. C.; Tempczyk, A.; Hawley, R. C.; Hendrickson, T. J .
Am. Chem. Soc. 1990, 112, 6127.

1438 J . Org. Chem., Vol. 63, No. 5, 1998
Seio et al.
F igu r e 8. Low-energy structures of cis-carbamate-linked compound 2 calculated by fixing the 5′-terminal and 3′-terminal ribose
conformations in C3′-endo and C3′-endo (a), C3′-endo and C2′-endo (b), C2′-endo and C3′-endo (c), and C2′-endo and C2′-endo (d),
respectively.
dinucleoside monophosphate over that in the noncyclized
dinucleoside monophosphate because many more types
of conformations other than turn or stretch can be
assumed in the noncyclized usual dinucleoside mono-
phosphate.35 The conformation properties of the turn
structure (Figure 6a or b) are not completely identical
with those of the U-turn structure expected from crystal-
lographic study in terms of both the local conformation
(e.g., (ú, R) ) (g-, t) in the tRNA U-turn but (g+, g+) or
(g+, t) in the turn structure of 1) and the global confor-
mation. (vide infra)
Similar computer simulations were carried out for the
carbamate-linked dimer 2 using the same protocols and
the program. Shown in Figure 7 are the lowest energy
structures of 2 obtained from the calculation in which
the carbamate linkage, N-C(dO)-O-C, was fixed in the
trans conformation. The lowest energy values calculated
were -1009.17, -1023.30, -1023.31, and -1035.81 kJ /
mol for the structures in Figure 7a-d, respectively. The
turn structure emerged when the conformations of both
the 5′- and 3′-terminal riboses were fixed in the C3′-endo
conformation (Figure 7a) and the backbone conformation
was (ú, R) ) (g+, t). The other combinations of the 5′-
and 3′-terminal conformations showed the stretch con-
formation.
The lowest energy structures of 2 in which the car-
bamate linkage is fixed in the cis conformation are shown
in Figure 8. The energy values calculated were -1011.39,
-1002.96, -1026.73, and -1026.79 kJ /mol for the struc-
tures in Figure 8a-d, respectively. Although the simul-
taneous fixation of the 5′- and 3′-terminal ribose in C3′
endo and C2′-endo conformations, respectively (Figure
8b), gave the turn structure, the γ of the 3′ terminal
nucleotide was in the trans conformation, which is not
identical with that of the nucleotide unit in the U-turn
structure. All the other series of simulations uniformly
gave stretch conformations.
To see which is a better U-turn mimic, 1 or 2, compared
with the 3D structures of 1 and 2 to the U-turn structure,
the root mean square (rms) deviations between the
natural U-turn structure and the cyclized dinucleoside
monophosphates were calculated by the aid of the least-
squares superimposition (for the detailed protocol, see the
Experimental Section). Listed in Table 3 are the aver-
aged values of the rms deviations of the low-energy
structures (within 12 kJ /mol from the lowest energy
(35) Lee, C.-H.; Charney, E.; Tinoco, I., J r. Biochemistry 1979, 18,
5636.

Synthesis and Properties of Diuridine Monophosphates
J . Org. Chem., Vol. 63, No. 5, 1998 1439
Ta ble 3. Root Mea n Squ a r e Devia tion s (Å) of th e
Low -En er gy Str u ctu r es of th e Cyclized Dim er s
Ca lcu la ted fr om th e Str u ctu r es of Up U Ha vin g a U-Tu r n
Con for m a tion a
Con clu sion
In conclusion, we have shown that the introduction of
a cyclic structure, such as 1 and 2, is effective for fixation
of the conformation of dinucleoside monophosphates,
which would be useful as turn structure models of
functional RNAs. Between the amide-linked 1 and the
carbamate-linked 2, the latter is the better U-turn mimic
as expected from the computational simulation. The
rather large rms deviation of about 3.5 Å even in the best-
fitted conformation of the carbamate linked 2, however,
suggested the U-turn structure in the anticodon loop on
tRNA is stabilized not only by the interresidual hydrogen
bonding, which is the focus of our present work, but also
by other factors attributed to both the upstream and
downstream strands of the U(33)pN(34). Therefore, the
incorporation of the carbamate-linked dinucleoside mono-
phosphate 2 into the anticodon loop or hammerhead
ribozyme to explore the additive effect of the cyclic
structure on the U-turn structure stability and the
conformational changes of the U-turn region is an
intriguing subject of study. It will also be interesting to
incorporate cyclic structure 1, which is less suitable as a
U-turn mimic, into oligonucleotide duplexes because the
stretch conformer of 1 (Figure 6c or d) apparently serves
to extend the oligonucleotide chain toward both the 5′
and 3′ directions so that they can form bent duplexes.
Conversion of 1 and 2 into the corresponding phosphora-
midite units and further synthesis of oligonucleotides
having a U-turn and a bend are now in progress and will
be reported elsewhere.
compd 2
compd 1
trans
cis
5′:C3′-endo
3′:C3′-endo
5′:C3′-endo
3′:C2′-endo
5′:C2′-endo
3′:C3′-endo
5′:C2′-endo
3′:C2′-endo
4.15 (0.20)
3.87 (0.20)
4.22 (0.11)
4.26 (0.16)
4.40 (0.13)
4.45 (0.18)
4.53 (0.22)
5.05 (0.13)
4.60 (0.04)
3.86 (0.06)
5.13 (0.06)
4.30 (0.05)
a
The standard deviations are shown in parentheses.
Exp er im en ta l Section
Gen er a l Meth od s. 1H, 13C, and 31P NMR spectra were
recorded at 270, 68, and 109 MHz, respectively. The chemical
shifts were measured from tetramethylsilane or DSS for 1H
NMR spectra, CDCl3 (77 ppm) or DSS (0 ppm) for 13C NMR
spectra, and 85% phosphoric acid (0 ppm) for 31P NMR spectra.
The correct 1H-1H coupling constants were measured at 400
F igu r e 9. Superimposition of the structure of UpU (yellow)
constructed by diplacement of G(34) of U(33)pG(34) dimer unit
at the U-turn postition of tRNAPhe by uridine on the low-energy
structure of 2 without the carbamoylmethyl group having the
smallest rms deviation from the UpU described above.
MHz. For measurement of the temperature dependence of 31
P
NMR chemical shift, each sample (100 A260) was dissolved in
10 mM cacodylate-1 mM EDTA (500 µL)-D2O (100 µL) and
the pH was adjusted to 7.1 by addition of 1 M NaOH. CD
spectra were recorded on a spectrometer using a 0.5 cm cell.
TLC was performed by the use of silica gel 60-F-254 (0.25 mm).
Paper chromatography was performed by use of a descending
technique with 3M papers using the solvent system 2-pro-
panol-concentrated ammonia (25%)-water, 7:1:2, v/v/v. Col-
umn chromatography was performed with silica gel C-200, 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). Reversed-phase
HPLC was performed on a LC module 1 using a µBondasphere
C-18 column with a linear gradient starting from 0.1 M
NH4OAc, pH 7.0 and increasing CH3CN at a flow rate of 1.0
mL/min for 30 min. Uridine was purchased from Yamasa Co.,
Ltd. UpU and UmpU were synthesized according to the usual
phosphotriester method.36 Snake venom phosphodiesterase
and calf spleen phosphodiesterase were purchased from Boe-
hringer Mannheim Biochemica Co., Ltd., and Nuclease P1
from Seikagaku Kougyou Co., Ltd. Pyridine was distilled from
p-toluenesulfonyl chloride and then from calcium hydride and
stored over molecular sieves 4A. Elemental analyses were
performed by the Microanalytical Laboratory, Tokyo Institute
of Technology at Nagatsuta.
structures). Table 3 shows that the cis carbamate-linked
dimer of 2, in which the 5′- and 3′-terminal riboses is C3′-
endo and C2′-endo, respectively, has the smallest rms
deviation. The trans carbamate-linked dimer of 2, in
which the conformations of both 5′- and 3′-terminal
riboses are C3′-endo has a very similar rms deviation.
The analysis of the averaged rms deviation suggested
that the cis carbamate-linked conformer is a better
U-turn mimic. A single structure that has the smallest
rms deviation of 3.58 Å best fitting the natural U-turn
of all trans and cis conformers of 2 is seen in the eighth
lowest energy conformer (-1003.20 kJ /mol) of the trans
structure having the 3′-endo conformation for both 5′- and
3′-terminal riboses. The backbone conformation of the
lowest rms structure is (ú, R) ) (g-, t) with γ ) g+, which
is identical with that in the natural U-turn structure.
The superimposition of the lowest rms structure without
the carbamoylmethyl group on the UpU fitted to the
natural U-turn of tRNAPhe is shown in Figure 9, which
shows reasonable agreement between the 3D structures
of the turn structure of the carbamate linked dinucleoside
monophosphate and the tRNA U-turn.
(36) Honda, S.; Urakami, K.; Koura, K.; Terada, K.; Sato, Y.; Kohno,
K.; Sekine, M.; Hata, T. Tetrahedron 1984, 40, 153-163.

1440 J . Org. Chem., Vol. 63, No. 5, 1998
Seio et al.
5-(Azid om et h yl)-2′,3′-O-isop r op ylid en eu r id in e
(5).
(250 µL, 4.3 mmol) were added. The mixture was stirred at
room temperature for 40 min. The solution was diluted with
CH2Cl2 and washed with saturated NaHCO3. The organic
layer was dried over Na2SO4, filtered, and evaporated to
dryness under reduced pressure. The residue was chromato-
graphed on a column of silica gel with CH2Cl2-MeOH (100:
1-100:1.5, v/v) to give 13 (1.23 g, 98%): 1H NMR (270 MHz,
CDCl3) δ 1.68 (9H, s, t-Bu), 4.00 (1H, d, 5′-Ha, J gem ) 11.9 Hz),
5-(Hydroxymethyl)-2′,3′-O-isopropylideneuridine (3) (4.5 g,
14.3 mmol) was dissolved in dioxane (140 mL). Trimethylsilyl
chloride (9.07 mL, 71.5 mmol) was added, and the mixture was
stirred at 60 °C for 2.5 h. Solvent was removed under reduced
pressure, and the residue was dissolved in DMF (140 mL).
Sodium azide (5.58 g, 85.8 mmol) was added, and the mixture
was stirred at 60 °C for 3 h. The resulting precipitate was
filtered, and the filtrate was concentrated. The concentrate
was extracted five times with CH2Cl2-pyridine (4:1, v/v), and
the organic layer was concentrated under reduced pressure.
The residue was chromatographed on a column of silica gel
(CH2Cl2-MeOH, 100:1.5, v/v) gave 5 (2.41 g, 50%): 1H NMR
(270 MHz, CDCl3) δ 1.37 (3H, s, CH3), 1.59 (3H, s, CH3), 3.83
(1H, d, 5′-Ha, J gem ) 12.0 Hz), 3.95 (1H, dd, 5′-Hb, J gem ) 12.0
Hz, J 4′,5′ ) 2.4 Hz), 4.14 (1H, s, CH2N3), 4.81 (1H, dd, 4′-H,
J 3′,4′ ) 3.0 Hz, J 4′,5′ ) 2.4 Hz), 4.96 (1H, dd, 3′-H, J 3′,4′ ) 3.0
4.15 (1H, d, 5′-Hb, J gem ) 11.9 Hz), 4.19 (1H, d, CH2CO, J gem
17.1 Hz), 4.21 (2H, m, 3′-H and 4′-H), 4.45 (1H, d, CH2CO,
J gem ) 17.1 Hz), 4.55 (1H, br, 2′-H), 5.46 (1H, d, 1′-H, J 1′,2′
)
)
2.3 Hz), 5.72 (1H, d, 5-H, J 5,6 ) 8.3 Hz), 7.41-7.66 (15H, m,
ArH), 7.70 (1H, d, 6-H, J 5,6 ) 8.3 Hz); 13C NMR (67.8 MHz,
CDCl3) δ 28.07, 60.94, 67.80, 68.84, 74.95, 83.22, 84.10, 84.57,
91.84, 100.77, 127.33, 127.46, 127.92, 130.75, 132.02, 138.94,
142.35, 152.04, 162.82, 171.21.
Anal.
Calcd for
C34H36N2O8S‚1/2H2O: C, 63.64; H, 5.81; N, 4.36. Found: C,
63.35; H, 5.86; N, 4.36.
Hz, J 2′,3′ ) 6.3 Hz), 5.02 (1H, dd, 2′-H, J 2′,3′ ) 6.3 Hz, J 1′,2′
)
2.6 Hz), 5.65 (1H, d, 1′-H, J ) 2.6 Hz), 7.51 (1H, s, 6-H), 9.05
(1H, br, NH); 13C NMR (67.8 MHz, CDCl3) δ 25.21, 27.19,
46.99, 62.55, 80.31, 83.79, 86.94, 95.60, 109.38, 114.47, 140.89,
2 ′-O -[(t e r t -B u t o x y c a r b o n y l )m e t h y l ]-5 ′-O -(4 ,4 ′-
d i m e t h o x y t r i t y l)-N 3 -(t r i p h e n y lm e t h a n e s u lfe n y l)-
u r id in e (14). Compound 13 (1.23 g, 1.94 mmol) was rendered
anhydrous by repeated coevaporation with dry pyridine and
finally dissolved in dry pyridine (20 mL). Dimethoxytrityl
chloride (723 mg, 2.10 mmol) was added, and the solution was
stirred for 5 h. The mixture was partitioned with CH2Cl2-
H2O and extracted with saturated NaHCO3. The organic layer
was dried over Na2SO4, filtered, and evaporated under reduced
pressure. The residue was coevaporated three times with
toluene and chromatographed on a column of silica gel with
CH2Cl2-toluene (1:4, v/v) to give 14 (1.68 g, 88%): 1H NMR
(270 MHz, CDCl3) δ 1.53 (9H, s, t-Bu), 3.82 (7H, m, 5′-Ha,
OCH3), 4.05 (1H, m, 4′-H), 4.16 and 4.32 (2H each, d, CH2CO,
J gem ) 18.0 Hz), 4.29 (2H, m, 2′-H and 3′-H), 5.16 (1H, d, 5-H,
J 5,6 ) 8.3 Hz), 5.38 (1H, s, 1′-H), 6.85-7.52 (23H, m, ArH),
7.82 (1H, d, 6-H, J 5,6 ) 8.3 Hz); 13C NMR (67.8 MHz, CDCl3)
δ 28.02, 55.17, 60.59, 67.35, 68.25, 74.93, 82.95, 83.07, 84.80,
86.78, 89.76, 100.49, 113.19, 123.65, 127.03, 127.26, 127.39,
127.91, 128.05, 129.99, 130.14, 130.71, 135.13, 135.35, 135.87,
137.47, 142.46, 144.49, 149.78, 151.82, 158.58, 158.63, 162.82,
171.05. Anal. Calcd for C55H54N2O10S: C, 70.65; H, 5.82; N,
2.99. Found: C, 70.74; H, 5.82; N, 2.85.
2′-O-[(ter t-Bu toxyca r bon yl)m eth yl]-5′-O-(4,4′-d im eth -
oxytr ityl)u r id in e (15). Compound 14 (464 mg, 0.50 mmol)
was dissolved in toluene (5 mL) and heated to 120 °C.
Tributyltin hydride (340 µL, 1.3 mmol) was added and the
mixture stirred for 5 min in the presence of azabisisobuty-
ronitrile (2 mg). The mixture was cooled with water and
chromatographed on a column of silica gel with CH2Cl2-MeOH
(100:1, v/v) to give 15 (379 mg, 78%): 1H NMR (270 MHz,
CDCl3) δ 1.49 (9H, s, t-Bu), 3.54 (2H, m, 5′-H), 3.80 (6H, s,
OCH3), 3.95 (1H, d, 2′-H J ) 4.9 Hz), 4.25 and 4.37 (2H each,
d, CH2CO, J gem ) 17.0 Hz), 4.15 (2H, m, 4′-H, 3′-OH), 4.46
(1H, dd, 3′-H), 5.26 (1H, d, 5-H, J 5,6 ) 8.2 Hz), 5.87 (1H, s,
1′-H), 6.82-7.40 (13H, m, ArH), 8.03 (1H, d, 6-H, J 5,6 ) 8.2
Hz), 8.56 (1H, br, N3-H): 13C NMR (67.8 MHz, CDCl3) δ 28.03,
55.23, 61.13, 68.00, 68.57, 83.15, 83.36, 85.27, 86.97, 88.43,
101.89, 113.30, 128.00, 128.12, 130.06, 130.19, 135.09, 135.35,
139.95, 144.42, 149.97, 158.65, 158.71, 162.93, 170.67. Anal.
Calcd for C36H40N2O10‚2H2O: C, 62.06; H, 6.36; N, 4.01.
Found: C, 62.24; H, 5.81; N, 4.00.
150.03, 162.46; IR (KBr) 2100 cm-1
.
Anal. Calcd for
C13H17N5O5‚H2O: C, 46.02; H, 5.05; N, 20.63. Found: C, 46.09;
H, 4.97; N, 19.81.
5-(Am in om et h yl)-2′,3′-O-isop r op ylid en eu r id in e (7).
Compound 5 (860 mg, 2.54 mmol) was rendered anhydrous
by three coevaporations with dry pyridine and dissolved in dry
pyridine (15 mL). Triphenylphosphine (1.07 g, 4.06 mmol) was
added, and the resulting mixture was stirred at ambient
temperature for 3 h. Aqueous ammonia (15 mL, 25%) was
added, and this solution was stirred for an additional 3 h. The
precipitate was filtered, and water was added to the filtrate.
This mixture was washed two times with CH2Cl2, and the
aqueous layer was concentrated under reduced pressure. The
residue was charged on a C18 reversed-phase column. Elution
with water gave 7 (567 mg, 71%). The presence of the amino
group could be readily confirmed by the color identification
test using the ninhydrin reagent, which showed a yellow spot
on TLC: 1H NMR (270 MHz, D2O) δ 1.40 (3H, s, CH3), 1.60
(3H, s, CH3), 3.75 (1H, d, 5′-Ha, J gem ) 12.5 Hz), 3.86 (1H, d,
5′-Hb, J gem ) 12.5 Hz), 3.94 (1H, s, CH2N), 4.40 (1H, m, 4′-H),
4.89 (1H, dd, 3′-H, J 3′,4′ ) 3.3 Hz, J 2′,3′ ) 6.6 Hz), 5.08 (1H, dd,
2′-H, J 2′,3′ ) 6.6 Hz, J 1′,2′ ) 2.6 Hz), 5.86 (1H, d, 1′-H, J ) 2.6
Hz), 8.01 (1H, s, 6-H); 13C NMR (67.8 MHz, D2O) δ 27.06, 28.79,
40.25, 64.11, 83.10, 86.86, 89.06, 95.90, 111.90, 117.51, 143.92,
158.54, 173.72. Anal. Calcd for C13H19N3O5‚3/2H2O: C, 45.88;
H, 6.22; N, 12.34. Found: C, 45.37; H, 6.15; N, 12.18.
2′-O-[(t er t -Bu t oxyca r b on yl)m e t h yl]-3′,5′-O-(1,1,3,3-
t e t r a i s o p r o p y ld i s i lo x a n e -1,3-d i y l)-N 3 -(t r i p h e n y l-
m eth a n esu lfen yl)u r id in e (12). Compound 11 (1.88 g, 2.5
mmol) was rendered anhydrous by repeated coevaporation
three times with dry pyridine followed by three coevaporations
with dry toluene. The residue was dissolved in dry THF (25
mL). Sodium hydride (400 mg, 10 mmol) was added, and the
mixture was stirred under argon. After 30 min, tert-butyl
bromoacetate (1.6 mL, 10 mmol) was added, and the resulting
mixture was stirred for 18 h. The mixture was quenched by
addition of 200 mL of phosphate buffer (pH 7.0). After
extraction with CH2Cl2, the organic layer was concentrated
under reduced pressure. The residue was chromatographed
on a column of silica gel with CH2Cl2-hexane (2:1, v/v) to give
12 (1.72 g, 78%): 1H NMR (270 MHz, CDCl3) δ 1.03-1.09
(27H, m, i-Pr), 3.94 (1H, br, 5′-Ha), 3.96 (1H, m, 5′-Hb), 4.03-
4.22 (5H, m, CH2CO, 2′-H, 3′-H, 4′-H), 5.35 (1H, s, 1′-H), 5.48
(1H, d, 5-H, J ) 8.1 Hz), 7.20-7.49 (15H, m, ArH), 7.63 (1H,
d, 6-H, J ) 8.1 Hz); 13C NMR (67.8 MHz, CDCl3) δ 12.29, 12.89,
12.94, 13.70, 16.82, 17.07, 17.25, 17.41, 28.11, 59.30, 74.89,
81.46, 81.55, 82.37, 89.51, 100.23, 100.54, 127.21, 127.39,
127.91, 130.01, 130.75, 137.45, 142.46, 142.61, 151.57, 163.07,
169.69. Anal. Calcd for C46H62N2O9SSi2: C, 63.13; H, 7.14;
N, 3.20. Found: C, 63.34; H, 7.05; N, 2.89.
3′-O-(ter t-Bu tyld im eth ylsilyl)-2′-O-(ca r boxym eth yl)-5′-
O-(4,4′-d im eth oxytr ityl)u r id in e (17). Compound 15 (226
mg, 0.34 mmol) was dissolved in 0.2 M KOH-pyridine (5 mL:5
mL). After being stirred for 1.5 h, the mixture was neutralized
with Dowex 50W × 8 (pyridinium form) and filtered. The
filtrate was concentrated and partitioned between ether and
H2O. The aqueous layer was washed twice with ether,
evaporated under reduced pressure, and rendered anhydrous
by repeated coevaporation with dry pyridine. The residue was
further coevaporated with dry toluene and finally dissolved
in DMF (1 mL). tert-Butyldimethylsilyl chloride (150 mg, 1.0
mmol) and imidazole (68 mg, 1.0 mmol) were added. The
solution was stirred at room temperature for 60 h and then
at 40 °C for 30 min. The mixture was diluted with CH2Cl2-
pyridine (2:1, v/v) and washed three times with saturated
2′-O-[(t er t -Bu t oxyca r b on yl)m e t h yl]-N 3-(t r ip h e n yl-
m eth a n esu lfen yl)u r id in e (13). Compound 12 (1.72 g, 2.0
mmol) was dissolved in THF (20 mL), and tetrabutylammo-
nium fluoride monohydrate (1.36 g, 4.3 mmol) and acetic acid

Synthesis and Properties of Diuridine Monophosphates
J . Org. Chem., Vol. 63, No. 5, 1998 1441
NaHCO3. The organic layer was dried over Na2SO4, filtered,
and evaporated under reduced pressure. The residue was
chromatographed on a column of silica gel with CH2Cl2-MeOH
(100:5, v/v) to give 17 (379 mg, 78%): 1H NMR (270 MHz,
CDCl3) δ 0.67 and 0.66 (6H each, s, CH3), 0.79 (9H, s, t-Bu),
3.34 (1H, d, 5′-H, J gem ) 9.2 Hz), 3.79 (7H, m, OCH3, 5′-H),
4.06 (1H, d, 2′-H J ) 2.7 Hz), 4.18 (1H, m, 4′-H), 4.28 (12H,
m, CH2CO, 3′-H), 4.44 (1H, d, CH2CO, J gem ) 16.2 Hz), 6.10
(1H, s, 1′-H), 5.29 (1H, d, 5-H, J 5,6 ) 7.9 Hz), 6.82-7.36 (13H,
m, ArH), 8.20 (1H, d, 6-H, J 5,6 ) 7.9 Hz), 10.29 (1H, br, N3-H);
13C NMR (67.8 MHz, CDCl3) δ -5.16, -4.56, 17.92, 25.55,
55.20, 68.07, 69.36, 82.80, 82.97, 102.18, 113.15, 113.21,
125.23, 127.21, 127.91, 128.16, 128.34, 128.97, 130.24, 134.90,
134.99, 140.54, 143.92, 150.82, 158.72, 164.26, 173.40. Anal.
Calcd for C38H45N2O10Si‚1/2H2O: C, 62.71; H, 6.51; N, 3.85.
Found: C, 62.68; H, 6.71; N, 3.84.
evaporation under reduced pressure. The residue was dis-
solved in THF (6 mL), and tetrabutylammonium fluoride
monohydrochloride (62 mg, 0.24 mmol) was added. The
solution was stirred for 3 h and evaporated to dryness under
reduced pressure. The residue was dissolved in H2O-pyridine
(6 mL, 1:1, v/v) and chromatographed on a Dowex 50W × 8
column (pyridinium form, 16 mL) with H2O-pyridine (100 mL,
1:1, v/v). The fraction was rendered anhydrous by repeated
coevaporation with dry pyridine, and the residue was dissolved
in dry pyridine (5 mL). DDS (94 mg, 0.29 mmol) was added.
The mixture was stirred at room temperature for 1.5 h and
diluted with CH2Cl2. The solution was washed three times
with saturated NaHCO3. The organic layer was dried over
Na2SO4, filtered, and evaporated under reduced pressure. The
residue was coevaporated with toluene and chromatographed
on a column of silica gel with CH2Cl2-MeOH (100:2.5, v/v) to
give 22 (51 mg, 50%): 1H NMR (270 MHz, CDCl3) δ 1.33 and
1.58 (3H each, s, isopropylidene), 3.22 and 3.57 (1H each, m,
5′-Ha), 3.80 (6H, s, OCH3), 4.01-4.10 (3H, m, CH2N, 4′-Ha),
4.12-4.29 (3H, m, 2′-Ha, CH2CO), 4.41-4.50 (3H, m, 4′-Hb,
5′-Hb), 4.53 (1H, dd, 3′-Hb, J 2′,3′ ) 6.3 Hz, J 3′,4′ ) 3.0 Hz), 4.62
(1H, dd, 2′-Hb, J 2′,3′ ) 6.3 Hz, J 1′,2′ ) 2.3 Hz), 4.92 (1H, m, 3′-
Ha), 5.30 (1H, d, 5-Ha, J gem ) 8.2 Hz), 5.97 (1H, d, 1′-Hb, J 1′,2′
) 2.3 Hz), 6.09 (1H, d, 1′-Ha, J 1′,2′ ) 5.6 Hz), 6.82-7.64 (19H,
m, ArH, 6-Hb), 7.67 (1H, d, 6-Ha, J ) 8.2 Hz), 8.89, 9.07 (1H,
1H, br, br, N3-H); 13C NMR (67.8 MHz, CDCl3) δ 25.30, 27.08,
55.28, 61.89, 67.80,70.14, 79.32, 81.42, 81.74, 84.31, 84.48,
85.46, 86.15, 91.45, 103.38, 111.30, 113.41, 114.45, 124.17,
124.28, 127.48, 128.12, 129.79, 130.21, 134.50, 134.57, 134.86,
134.93, 138.81, 143.65, 150.21, 150.80, 158.90, 162.50, 162.75,
168.54; 31P NMR (109 MHz, CDCl3) δ 25.5, 24.7 ppm. Anal.
Calcd for C51H52N5O16SP‚2H2O: C, 56.19; H, 5.18; N, 6.42.
Found: C, 56.21; H, 5.18; N, 6.67.
Am id e-Lin k ed Diu r id in e Dim er 19. Compound 17 (222
mg, 0.28 mmol) and N-hydroxysuccinimide (38 mg, 0.34 mmol)
were dissolved in DMF (2 mL). N-Ethyl-N′-(dimethylami-
no)carbodiimide hydrochloride (63 mg, 0.34 mmol) was added,
and the mixture was stirred for 6 h. Compound 7 (107 mg,
0.34 mol) and triethylamine (47 µL, 0.34 mmol) were added,
and the resulting solution was stirred for another 2 h. The
mixture was partitioned between CH2Cl2 and H2O. The
CH2Cl2 extract was washed three times with saturated NaH-
CO3 and five times with water. The organic layer was dried
over Na2SO4 and filtered. The filtrate was evaporated to
dryness under reduced pressure and chromatographed on a
column of silica gel with CH2Cl2-MeOH (100:2, v/v) to give
19 (312 mg, 93%): 1H NMR (270 MHz, CDCl3) δ -0.12 and
0.02 (3H each, s, CH3), 0.77 (9H, s, t-Bu), 1.34 and 1.57 (3H
each, s, isopropylidene), 3.32 (1H, d, 5′-Ha1, J gem ) 9.2 Hz),
3.72-3.85 (9H, m, 2′-Ha, 5′-Hb1, OCH3, 5′-Ha2), 3.95 (1H, d, 5′-
H
b2, J gem ) 12.2 Hz), 4.01-4.16 (3H, m, 4′-Ha, CH2N), 4.19 (2H,
Am id e-Lin k ed Diu r id in e Mon op h osp h a t e 1. Com-
pound 22 (39 mg, 36.5 µmol) was dissolved in pyridine (2 mL)
and bis(tributyltin) oxide (370 µL, 0.7 mmol). After 3 h,
trimethylsilyl chloride (140 µL, 1.1 mmol) was added, and the
mixture was stirred for 5 min. The solution was diluted with
H2O-pyridine (1:1) and washed three times with hexane. The
aqueous layer was extracted with CH2Cl2, and the extract was
dried over Na2SO4 and filtered. The filtrate was evaporated,
and the remaining pyridine was completely removed by
repeated coevaporation with H2O. The residue was dissolved
in 60% formic acid (10 mL) and stirred at room temperature
for 20 h. The solution was evaporated under reduced pressure,
and the residue was chromatographed on paper (Whatman
s, CH2CO), 4.28 (1H, m, 3′-Ha), 4.89 (2H, br, 2′-Hb, 3′-Hb), 5.30
(1H, d, 5-Ha, J 5,6 ) 7.9 Hz), 5.80 (1H, s, 1′-Hb), 5.95 (1H, d,
1′-Ha, J 1′,2′ ) 1.2 Hz), 6.82-7.35 (14H, m, ArH, 6-Hb), 7.55 (1H,
t, NHCO), 8.05 (1H, d, 6-Ha, J 5,6 ) 7.9 Hz), 9.60 and 9.90 (1H
each, br, N3-H). Anal. Calcd for C51H63N5O15Si‚H2O: C, 59.35;
H, 6.15; N, 6.78. Found: C, 59.59; H, 6.34; N, 6.67.
P h osp h or yla ted Diu r id in e Dim er 20. Compound 19
(128 mg, 0.13 mmol), cyclohexylammonium S,S-diphenyl phos-
phorodithioate (99 mg, 0.26 mmol), and 1H-tetrazole (36 mg,
0.52 mmol) was rendered anhydrous by repeated coevaporation
with dry pyridine and dissolved in dry pyridine (2 mL). DDS
(129 mg, 0.39 mmol) was added. The mixture was stirred at
room temperature for 30 min and partitioned between CH2Cl2
and H2O. The organic layer was washed three times with
saturated NaHCO3, dried over Na2SO4, and filtered. The
filtrate was evaporated under reduced pressure. The residue
was coevaporated with toluene and chromatographed on a
column of silica gel with CH2Cl2-MeOH to give 20 (135 mg,
82%): 1H NMR (270 MHz, CDCl3) δ -0.13 and 0.01 (3H each,
s, CH3), 0.76 (9H, s, t-Bu), 1.34 and 1.57 (3H each, s,
isopropylidene), 3.32 and 3.73 (1H each, d, 5′-Ha, J gem ) 9.2
Hz), 3.79 (6H, s, OCH3), 3.83 (1H, dd, 2′-Ha, J 2′,3′ ) 4.5 Hz,
J 1′,2′ ) 1.7 Hz), 3.97-4.30 (6H, m, CH2N, CH2CO, 4′-Ha, 3′-
Ha), 4.35 (1H, m, 4′-Hb), 4.43 (2H, m, 5′-Hb), 4.75 (1H, dd, 3′-
Hb, J 2′,3′ ) 6.3 Hz, J 3′,4′ ) 3.3 Hz), 4.92 (1H, d, 2′-Hb, J 2′,3′ ) 6.3
Hz, J 1′,2′ ) 2.3 Hz), 5.29 (1H, d, 5-Ha, J gem ) 8.2 Hz), 5.77 (1H,
d, 1′-Hb, J 1′,2′ ) 2.3 Hz), 5.94 (1H, d, 1′-Ha, J 1′,2′ ) 1.7 Hz),
6.82-7.56 (24H, m, ArH, 6-Hb), 8.04 (1H, d, 6-Ha, J 5,6 ) 8.2
Hz), 9.32 and 9.65 (1H each, br, N3-H); 13C NMR (67.8 MHz,
CDCl3) δ -5.21, -4.49, 17.81, 25.09, 25.48, 27.05, 35.83, 55.22,
60.63, 62.46, 69.51, 70.12, 80.88, 82.82, 83.72, 85.39, 87.10,
87.78, 88.12, 94.59, 102.46, 109.76, 113.19, 113.23, 113.59,
125.25, 127.28, 127.94, 128.18, 128.30, 128.99, 130.21, 134.81,
134.90, 139.30, 141.04, 143.85, 150.19, 150.60, 158.76, 163.46,
163.56, 170.55; 31P NMR (109 MHz, CDCl3) δ 50.7 ppm. Anal.
Calcd for C67H72N5O16SiS2P‚2H2O: C, 65.14; H, 5.22; N, 2.26.
Found: C, 65.30; H, 5.45; N, 2.69.
i
3MM) with PrOH-concentrated ammonia (25%)-H2O (7:1:2,
v/v/v) to give 1 (438 A260, 61%): 1H NMR (270 MHz, D2O) δ
3.81 (1H, dd, 5′-Ha, J gem ) 13.2 Hz, J 4′,5′′ ) 3.8 Hz), 3.93 (1H,
dd, 5′′-Ha, J gem ) 13.2 Hz, J 4′,5′′ ) 2.8 Hz), 4.2-4.3 (8H, m,
CH2N, 4′-Ha, 2′-Hb, 3′-Hb, 4′-Hb, 5′-Hb), 4.35 (3H, m, 4′-Ha,
CH2CO), 4.40 (1H, t, 2′-Ha, J 1′,2′ ) J 2′,3′ ) 4.9 Hz), 4.78 (1H,
ddd, 3′-Ha, J 2′,3′ ) 4.3 Hz, J 3′,4′ ) 2.8 Hz, J 3′,P ) 7.8 Hz), 5.88
(1H, d, 5-Ha, J gem ) 7.9 Hz), 6.00 (1H, d, 1′-Hb, J 1′,2′ ) 3.9 Hz),
6.07 (1H, d, 1′-Ha, J 1′,2′ ) 4.6 Hz), 7.52 (1H, s, 6-Hb), 7.87 (1H,
d, 6-Ha, J 5,6 ) 7.9 Hz); 13C NMR (67.8 MHz, D2O) δ 35.24,
61.06, 65.87, 69.48, 70.26, 72.23, 74.86, 81.08, 83.59, 83.73,
84.53, 88.48, 89.07, 103.39, 112.26, 136.26, 142.31, 172.68; 31
P
NMR (109 MHz, D2O) δ 0.61 ppm.
2′-O-[(4-Nitr op h en oxy)ca r bon yl]-3′,5′-O-(1,1,3,3-tetr a -
isop r op yld isiloxa n e-1,3-d i-yl)u r id in e (23). Compound 10
(4.5 g, 9.3 mmol) was dissolved in dry toluene (100 mL).
4-Nitrophenyl chloroformate (2.2 g, 11.1 mmol) and pyridine
(840 µL, 11.1 mmol) were added, and the resulting mixture
was stirred at room temperature for 1 h. The solution was
dissolved with ether (300 mL) and washed three times with
saturated NaHCO3 (300 mL). The ether layer was dried over
Na2SO4, filtered, and evaporated to dryness under reduced
pressure. The residue was chromatographed on a column of
silica gel with CH2Cl2-MeOH (100:1, v/v) to give 23 (5.8 g,
96%): 1H NMR (270 MHz, CDCl3) δ 1.02-1.11 (27H, m, i-Pr),
4.00-4.14 (2H, m, 5′′-H, 4′-H), 4.27 (1H, d, 5′-H, J gem ) 13.5
Hz), 4.46 (1H, dd, 3′-Hb, J 2′,3′ ) 4.6 Hz, J 3′,4′ ) 9.2 Hz), 5.33
(1H, d, 2′-H, J 2′,3′ ) 4.6 Hz), 5.75 (1H, d, 5-H, J 5,6 ) 7.9 Hz),
5.96 (1H, s, 1′-H), 7.40 (2H, d, Ar-H, J ) 9.2 Hz), 7.76 (1H, d,
Th e F u lly P r otected Am id e-Lin k ed Diu r id in e Mon o-
p h osp h a te 22. Compound 20 (122 mg, 95 µmol) was dissolved
in triethylamine-pyridine-H2O (2 mL:2 mL:1.5 mL) and
stirred at 40 °C for 1 h. The solvent was removed by

1442 J . Org. Chem., Vol. 63, No. 5, 1998
Seio et al.
6-H, J 5,6 ) 7.9 Hz), 8.28 (2H, d, Ar-H, J ) 9.2 Hz), 9.73 (1H,
br, 3-H); 13C NMR (67.8 MHz, CDCl3) δ 12.64, 12.80, 13.32,
16.64, 16.77, 16.89, 17.13, 17.16, 17.25, 17.33, 59.21, 64.62,
67.67, 80.10, 81.92, 87.87, 102.37, 121.64, 125.30, 138.90,
145.46, 149.92, 151.09, 155.35, 163.32. Anal. Calcd for
Calcd for C25H31N5O: C, 48.00; H, 5.00; N, 11.19. Found: C,
47.71; H, 5.25; N, 10.97.
P h osp h or yla ted Ca r ba m a te-Lin k ed Diu r id in e Dim er
27. Compound 26 (694 g, 1.11 mmol) was rendered anhydrous
by repeated coevaporation with dry pyridine and dissolved in
dry pyridine (10 mL). 4,4′-Dimethoxytrityl chloride (488 mg,
1.44 mmol) was added, and the resulting mixture was stirred
at room temperature. After 4 h, cyclohexylammonium S,S-
diphenyl phosphorodithioate (677 mg, 1.78 mmol), 1H-tetrazole
(310 mg, 4.43 mmol), and DDS (2.01 g, 6.66 mmol) were added.
The solution was stirred for 3.5 h and then extracted between
CH2Cl2-water (50 mL:50 mL). The organic layer was washed
three times with saturated NaHCO3 (50 mL), dried over
Na2SO4, filtered, and evaporated to dryness by repeated
coevaporation with toluene. The residue was chromato-
graphed on a column of silica gel with CH2Cl2-MeOH (100:
1.5, v/v) to give 27 (1.15 g, 88%). The fraction obtained was
an equilibrium mixture of the two rotamers at the carbamate
bond, which gave two distinguishable set of signals in NMR
spectra in CDCl3: 1H NMR (270 MHz, CDCl3) major isomer δ
1.20, 1.58 (3H, 3H, s, s, CH3), 2.01 (3H, s, CH3CO), 3.33 (2H,
m, 5′′-Ha), 3.77 (7H, m, OCH3, 5′-Ha), 4.08 (1H, br, 4′-Ha), 4.20-
4.50 (5H, m, 4′-Hb, 5′-Hb, 5′′-Hb, CH2N), 4.88 (1H, m, 3′-Hb),
5.11-5.31 (2H, m, 2′-Ha, 3′-Ha), 5.40 (1H, d, 5-Ha, J 5,6 ) 7.3
Hz), 5.45 (1H, m, 2′-Ha), 5.88 (1H, s, 1′-Hb), 6.17 (1H, d, 1′-Ha,
J 1′,2′ ) 7.3 Hz), 6.83-7.65 (15H, m, 6-Ha, 6-HbArH), 7.90 and
8.55 (1H each, br, 3-Ha and 3-Hb); 31P NMR (109 MHz, CDCl3)
δ 50.00; minor isomer δ 1.50 and 1.63 (3H each, s, CH3), 3,45
(1H, m, 5′-Ha), 5.00 (1H, m, 3′-Hb), 6.02 (1H, s, 1′-Hb), 6.41
(1H, d, 1′-Ha, J 1′,2′ ) 7.9 Hz), 9.05 and 9.69 (1H each, br, 3-Ha
and 3-Hb); 31P NMR (109 MHz, CDCl3) δ 50.18; 13C NMR (67.8
MHz, DMSO-d6) δ 20.72, 25.09, 26.85, 53.35, 55.17, 62.82,
64.31, 73.96, 81.53, 83.52, 84.26, 85.10, 85.73, 87.60, 96.05,
103.02, 110.71, 113.35, 114.68, 115.00, 125.52, 125.62, 125.71,
127.21, 128.03, 128.07, 128.12, 129.45, 129.52, 129.56, 129.72,
129.83, 130.01, 130.06, 130.10, 134.56, 135.78, 138.96, 139.12,
140.97, 143.52, 143.68, 149.72, 142.79, 150.91, 150.96, 154.50,
158.62, 158.71, 162.50, 163.45, 164.10, 170.57. Anal. Calcd
for C58H58N5O17PS2: C, 58.43; H, 4.90; N, 5.87. Found: C,
58.08; H, 4.94; N, 5.69.
C
28H41N3O4Si2: C, 51.60; H, 6.34; N, 6.44. Found: C, 51.54;
H, 6.28; N, 6.38.
Ca r ba m a te-Lin k ed Diu r id in e Dim er 24. A mixture of
23 (521 mg, 0.80 mmol) and 7 (275.7 mg, 0.88 mol) in DMF (3
mL) was stirred for 1 min and then partitioned between
CH2Cl2 (50 mL) and saturated NaHCO3 (50 mL). The aqueous
layer was extracted five times with CH2Cl2 (50 mL), and the
organic layer was collected, dried over Na2SO4, and filtered.
After being evaporated to dryness, the residue was chromato-
graphed on a column of silica gel with CH2Cl2-MeOH (100:
2.5, v/v) to give 24 (587 mg, 89%): 1H NMR (270 MHz, CDCl3)
δ 0.92-1.25 (27H, m, i-Pr), 1.36 and 1.58 (3H each, s, CH3),
3.64-3.99 (6H, m, 5′-Ha1, 5′-Hb1, 5′-Hb2, CH2N, 4′-Ha), 4.19 (1H,
d, 5′-Ha2, J gem ) 12.2 Hz), 4.29 (1H, m, 3′-Ha), 4.45 (1H, m,
4′-Hb), 4.87-4.94 (2H, m, 2′-Hb, 3′-Hb), 5.17 (1H, d, 2′-Ha, J 2′,3′
) 4.9 Hz), 5.69 (1H, d, 5-Ha, J 5,6 ) 7.9 Hz), 5.77 (2H, m, 1′-Ha,
1′-Hb), 5.87 (1H, t, OCONH, J ) 5.8 Hz), 7.66 (1H, d, 6-Ha,
J 5,6 ) 7.9 Hz), 7.85 (1H, s, 6-Hb), 9.20 (2H, br, 3-Hz, 3-Hb); 13
C
NMR (67.8 MHz, CDCl3) δ 12.53, 12.81, 13.35, 16.71, 16.79,
17.18, 17.22, 17.3, 17.36, 25.09, 26.99, 37.92, 59.55, 62.36,
67.75, 76.07, 80.90, 82.00, 85.45, 87.78, 88.46, 94.88, 102.21,
109.90, 113.55, 139.17, 140.75, 150.08, 150.24, 155.44, 163.56,
164.11. Anal. Calcd for C35H55N5O4Si2‚2H2O: C, 48.77; H,
6.90; N, 8.12. Found: C, 48.55; H, 7.02; N, 8.34.
Acetyla tion of 24. Compound 24 (1.15 g, 1.4 mmol) was
dissolved in pyridine (10 mL), and acetic anhydride (520 µL,
5.6 mmol) was added. The resulting solution was stirred at
room temperature for 12 h. The mixture was diluted with
CH2Cl2 (100 mL) and washed three times with saturated
NaHCO3 (100 mL). The organic layer was collected, dried over
Na2SO4, evaporated to dryness by repeated coevaporation with
toluene. The residue was chromatographed on a column of
silica gel with CH2Cl2-MeOH (100:1.8, v/v) to give 25 (1.2 g,
99%): 1H NMR (270 MHz, CDCl3) δ 0.92-1.25 (27H, m, i-Pr),
1.56, 1.34 (3H, 3H, s, s, CH3), 2.07 (3H, s, CH3CO), 3.92-4.30
(9H, m, 3′-Ha, 4′-Ha, 4′-Hb 5′-Ha, 5′-Hb, CH2N), 4.98 (1H, m,
3′-Hb), 4.97 (1H, d, 2′-Hb, J 2′,3′ ) 6.3 Hz), 5.20 (1H, d, 2′-Ha,
J 2′,3′ ) 5.3 Hz), 5.66 (1H, d, 5-Ha, J 5,6 ) 8.2 Hz), 5.76 (1H, s,
1′-Hb), 5.81 (2H, m, 1′-Ha, NHCO), 7.48 (1H, s 6-Hb), 7.61 (1H,
d, 6-Hb, J 5,6 ) 8.2 Hz), 9.48 and 9.73 (1H each, br, 3-Ha and
3-Hb); 13C NMR (67.8 MHz, CDCl3) δ 12.42, 12.64, 12.72, 13.26,
16.66, 17.06, 17.11, 17.16, 17.25, 20.65, 25.12, 26.94,
37.56, 59.53, 64.15, 67.64, 75.80, 81.10, 81.87, 84.35, 85.09,
88.57, 94.43, 102.00, 111.12, 114.36, 139.10, 141.11, 149.85,
149.97, 154.90, 163.50, 163.95, 170.48. Anal. Calcd for
Th e F u lly P r otected Ca r ba m a te-Lin k ed Diu r id in e
Mon op h osp h a te 29. Compound 27 (204 mg, 0.17 mmol) was
dissolved in 0.2 N KOH-pyridine (10 mL:10 mL) and stirred
for 40 min at room temperature. The mixture was passed
through a Dowex 50W × 8 column (pyridinium form, 10 mL),
and elution was performed with H2O-pyridine (1:1, v/v, 100
mL). Triethylamine (5 mL) was added, and the eluent was
evaporated to dryness under reduced pressure. The residue
was rendered anhydrous together with 1H-tetrazole (60 mg,
0.85 mmol) by repeated coevaporation with pyridine and finally
dissolved in dry pyridine (2 mL). To this solution was added
DDS (169 mg, 0.51 mmol), and the resulting mixture was
stirred at room temperature for 40 min. The solution was
diluted with ether (50 mL) and washed three times with
saturated NaHCO3 (50 mL). The organic layer was collected,
dried over Na2SO4, filtered, and evaporated to dryness by
repeated coevaporation with toluene. The residue was chro-
matographed on a column of silica gel with CH2Cl2:MeOH (100:
2.5, v/v) to give 29 (87 mg, 49%): 1H NMR (270 MHz, CDCl3)
δ 1.30 and 1.52 (3H each, s, CH3), 3.46 (2H, br, 5′-Ha), 3.78
and 3.79 (6H each, s, OCH3), 3.92-4.37 (5H, m, 5′-Hb, 4′-Hb,
CH2), 4.53-4.79 (2H, m, 2′-Hb, 4′-Ha), 4.83 (1H, m, 3′-Hb), 5.13
(1H, dd, 3′-Ha, J ) 4.1 Hz, J ) 9.2 Hz), 5.44-5.63 (2H, m,
2′-Ha, 5-H), 6.17 (1H, d, 1′-Ha, J 1′,2′ ) 8.2 Hz), 6.29 (1H, d, 1′-
Hb, J 1′,2′ ) 2.0 Hz), 6.83-7.72 (17H, m, ArH, 6-Ha, 6-Hb), 10.03
and 10.21 (1H each, br, 3-Ha and 3-Hb); 13C NMR (67.8 MHz,
CDCl3) δ 25.32, 25.45, 27.12, 38.44, 38.69, 55.24, 62.98, 68.11,
68.10, 78.08, 78.64, 82.91, 83.11, 84.10, 84.87, 85.39, 87.62,
87.75, 88.97, 89.09, 103.38, 111.52, 111.70, 113.39, 113.46,
114.99, 115.38, 123.72, 123.86, 127.22, 128.05, 128.09, 128.16,
128.75, 129.72, 129.99, 130.05, 130.84, 134.72, 134.86, 134.99,
135.06, 135.33, 135.42, 135.60, 137.43, 138.69, 143.65, 149.60,
149.88, 151.48, 151.73, 154.75, 154.93, 158.72, 158.78, 162.82,
162.96, 163.83; 31P NMR (109 MHz, D2O) δ 26.15, 20.48 ppm.
C
37H57N5O15Si2‚2H2O: C, 49.43; H, 6.84; N, 7.79. Found: C,
49.10; H, 6.77; N, 7.86.
Diol Der iva tive 26. Compound 25 (1.12 g, 1.29 mmol) was
dissolved in THF (12 mL), and tetrabutylammonium fluoride
monohydrate (900 mg, 2.84 mol) and acetic acid (160 µL, 2.84
mmol) were added. The resulting solution was stirred at room
temperature for 40 min. The mixture was diluted with
CH2Cl2-pyridine (60 mL:30 mL) and washed with saturated
NaHCO3 (100 mL). The aqueous layer was back-extracted
with CH2Cl2-pyridine (60 mL:30 mL). The conbined organic
layers were dried over Na2SO4, filtered, and evaporated by
repeated coevaporation with toluene. The residue was chro-
matographed on a column of silica gel with CH2Cl2-MeOH
(100:5.5, v/v) to give 26 (770 mg, 96%): 1H NMR (270 MHz,
CDCl3-5% CD3OD) δ 1.29 and 1.53 (3H, 3H, s, s, CH3), 1.97
(3H, s, CH3CdO), 3.76 (2H, m, 5′-Ha), 4.15-4.28 (6H, m, 3′-
Ha, 4′-Ha, 5′-Hb, CH2N), 4.74 (1H, m, 3′-Hb), 5.02 (2H, m, 2′-
Ha, 2′-Hb), 5.62 (1H, d, 5-Ha, J 5,6 ) 7.9 Hz), 5.72 (1H, d, 1′-Hb,
J 1′,2′ ) 1.7 Hz), 5.94 (1H, d, 1′-Ha, J 1′,2′ ) 5.9 Hz), 7.31 (1H, s,
6-Hb), 7.82 (1H, d, 6-Hb, J 5,6 ) 7.9 Hz), 7.90 and 8.55 (1H each,
br, 3-Ha and 3-Hb); 13C NMR (67.8 MHz, CD3OD) δ 20.75,
25.50, 27.40, 48.05, 62.35, 65.46, 70.92, 77.48, 82.84, 85.71,
86.52, 86.93, 88.84, 96.06, 102.98, 112.27, 115.40, 141.92,
142.71, 151.78, 152.25, 157.41, 165.31, 165.96, 172.43. Anal.

Synthesis and Properties of Diuridine Monophosphates
J . Org. Chem., Vol. 63, No. 5, 1998 1443
Anal. Calcd for C50H50N5O16PS‚7/2H2O: C, 54.45; H, 5.21; N,
6.35. Found: C, 54.49; H, 4.82; N, 6.18.
Com p u ta tion a l Meth od s. The four initial structures of
1 and 2 each were constructed by using the structure of uridine
or thymidine residues implemented in Macro Model ver. 4.5.
The ribose moieties were fixed in the conformation of the initial
structure by applying the constraints on all five endocyclic
torsions of the riboses. The four initial structures in which
the 5′-terminal ribose and 3′-terminal riboses were fixed in
C3′-endo or C2′-endo conformations were simulated indepen-
dently in the following calculation. The force field used was
the AMBER* by modifying the distance dependent dielectric
constant to the constant dielectric. The effect of the solvent
was included by using the GB/SA solvent model implemented
in the software. The default values for the atomic partial
charges were used in all the calculations. The initial struc-
tures were energy minimized, and the structures thus obtained
were used as starting structures for the next simulations. The
local minima were searched by the 500 step energy minimiza-
tion of 5000 structures generated by using the MCMM option.
The low-energy structure (within 20 kJ /mol from the lowest
energy structures) was energy reminimized until the rms
gradient became less than 0.01 kJ /mol. The low-energy
structures of 1 and 2 were compared with that of U-turn
structures as follows: First, the UpU structure having the
same conformation as those of 1 and 2 was made by eliminat-
ing the CH2C(O)NHCH2 group and C(O)NHCH2 group from 1
and 2, respectively. The UpU structure having the same
conformation as that of the U-turn of the tRNAPhe was
constructed by replacing the G(34) residue of U(33)pG(34)
dimer extracted from the X-ray crystallographic structure of
tRNAPhe (obtained from PDB Brookhaven National Laboratory)
with uracil. The UpU structures were superimposed with the
aid of the method of the least-squares fit, and the rms (Å)
values were calculated.
Ca r ba m a te-Lin k ed Diu r id in e Mon op h osp h a te 2. Com-
pound 29 (41 mg, 40 µmol) was dissolved in pyridine (2 mL).
Bis(tributyltin) oxide (410 µL, 0.80 mmol) was added, and the
resulting mixture was stirred for 3 h at ambient temperature.
Trimethylsilyl chloride was added, and then the solution was
diluted with H2O-pyridine (10 mL:10 mL). After being
washed three times with hexane (20 mL), the mixture was
extracted three times with CH2Cl2. The CH2Cl2 extracts were
collected, dried over Na2SO4, filtered, and coevaporated five
times with water to remove pyridine completely. The residue
was dissolved in 60% formic acid (10 mL), and the solution
was stirred for 20 h. The formic acid was removed from the
mixture by repeated coevaporation with water. The resulting
residue was chromatographed on Whatman 3MM paper with
i-PrOH-concentrated ammonia-H2O (7:1:2, v/v/v) to give 29
(459 A260, 58% assuming that the ꢀ value of 29 was same as
that of UpU): 1H NMR (270 MHz, D2O) of the major equilib-
rium isomer δ 3.81-3.86 (3H, m, 5′′-Hb and 5′-Ha), 3.92 (1H,
br, 5′-Hb), 4.05-4.10 (3H, m, 5′-Ha, CH2), 4.17-4.20 (2H, m,
2′-Hb and 4′-Hb), 4.27-4.39 (2H, m, 3′-Hb and 4′-Ha), 4.75 (1H,
m, 3′-Ha), 5.30 (1H, m, 2′-Ha), 5.85 (1H, d, 5-Ha, J 5,6 ) 7.9 Hz),
6.00-6.03 (2H, m, 1′-Ha and 1′-Hb, J 1′a,2′a ) 8.0 Hz, J 1′b,2′b
)
5.1 Hz), 7.80 (1H, d, 6-Ha, J 5,6 ) 7.9 Hz), 7.89 (1H, s, 6-Hb); 1H
NMR (270 MHz, D2O) of the minor equilibrium isomer δ 5.38
(m, 2′-Ha), 6.12 (d, 1′-Ha, J 1′,2′ ) 4.5 Hz), 7.38 (s, 6′-Hb); 13C
NMR (67.8 MHz, D2O) δ 30.13, 38.13, 60.30, 61.04, 63.97,
65.57, 68.69, 70.38, 71.73, 73.19, 73.54, 74.01, 74.48, 82.62,
84.09, 84.94, 85.46, 87.76, 88.06, 102.60, 112.02, 134.73,
139.17, 141.31, 151.35, 151.78, 156.15, 165.10, 165.87, 166.01;
31P NMR (109 MHz, D2O) δ 0.86, 0.32 ppm.
CD Sp ectr a . Dinucleoside monophosphate was dissolved
in 10 mM sodium phosphate pH 7.0 (2 mL), and the absorbance
was adjusted to 0.5A260nm. The spectra were accumulated 16
times and then averaged for smoothing.
400 MHz NMR Sp ectr a . Dinucleoside monophosphate
(100A260nm) was dissolved in 10 mM sodium phosphate pH 7.0
(600 µL) and then lyophylized three times with 99.8% D2O and
finally redissolved in 99.95% D2O (600 µL). The spectra were
recorded at 400 MHz, and the coupling constants were
simulated by using the “Deconvolusion” option compiled in the
spectrometer.
En zym a tic Digestion . The cyclized dinucleoside mono-
phosphate was dissolved in a buffer of 50 mM Tris-HCl pH
8.0 for SVP, 20 mM sodium acetate-0.1 mM ZnCl2 for NP1,
or 200 mM ammonium acetate pH 5.7 for CSP and incubated
at 35 °C. The reactions were analyzed by using reversed-phase
HPLC.
Ack n ow led gm en t. This work was partially sup-
ported by Toray Scientific Foundation and by a Grant-
in-Aid for Scientific Research from Ministry of Educa-
tion, Science, Culture, Sports and Culture, J apan.
Su p p or tin g In for m a tion Ava ila ble: The 1H, 13C, and 31P
NMR spectra of compounds 1-29, 1H-1H DQF COSY and
NOESY spectra of compounds 1 and 2, and HPLC profiles of
the enzymatic digestion of compounds 1 and 2 (47 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.
J O971797O