Syntheses and structure-activity relationships of nonnatural β-C-nucleoside 5′-triphosphates bearing an aromatic nucleobase with phenolic hydroxy groups: Inhibitory activities against DNA polymerases

Article abstract of DOI:10.1021/jm020193w

Five nonnatural β-C-nucleoside 5′-triphosphates bearing a 3,4-dihydroxyphenyl (1TP), a 2-hydroxyphenyl (2TP), a 3-hydroxyphenyl (3TP), a 4-hydroxyphenyl (4TP), or a phenyl (5TP) group were synthesized, and their structure-activity relationships were exami

Full text of DOI:10.1021/jm020193w

5594
J . Med. Chem. 2002, 45, 5594-5603
Syn th eses a n d Str u ctu r e-Activity Rela tion sh ip s of Non n a tu r a l â-C-Nu cleosid e
5′-Tr ip h osp h a tes Bea r in g a n Ar om a tic Nu cleoba se w ith P h en olic Hyd r oxy
Gr ou p s: In h ibitor y Activities a ga in st DNA P olym er a ses
Saeko Aketani,† Kentaro Tanaka,† Kaneyoshi Yamamoto,‡ Akira Ishihama,*,‡ Honghua Cao,§ Atsushi Tengeiji,†
Shuichi Hiraoka,† Motoo Shiro,| and Mitsuhiko Shionoya*,†
Department of Chemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-0033, J apan, National Institute of Genetics, Yata, Mishima-shi, Shizuoka 411-8540, J apan,
The Graduate University for Advanced Studies, Myodaiji, Okazaki 444-8585, J apan, and Rigaku Corporation,
3-9-12 Matsubaracho, Akishima, Tokyo 196-8666, J apan
Received May 6, 2002
Five nonnatural â-C-nucleoside 5′-triphosphates bearing a 3,4-dihydroxyphenyl (1TP ), a
2-hydroxyphenyl (2TP ), a 3-hydroxyphenyl (3TP ), a 4-hydroxyphenyl (4TP ), or a phenyl (5TP )
group were synthesized, and their structure-activity relationships were examined for a series
of DNA polymerase reactions in vitro under typical polymerase chain reaction conditions. We
found that the 5′-triphosphates (1TP -5TP ) are not incorporated into DNA strands but inhibit
the DNA polymerase reactions in the presence of natural nucleoside 5′-triphosphates (dNTPs).
1TP having two phenolic hydroxy groups at the nucleobase moiety showed the most potent
inhibitory effect against DNA synthesis by Ex Taq polymerase (IC50 ) 30 µM). The competition
assay indicated that 1TP and dNTPs are most likely to affect DNA polymerase reactions
competitively. This finding may raise the appealing possibility that artificial nucleoside 5′-
triphosphates having phenolic hydroxy groups could exhibit potent inhibitory activity against
DNA-directed enzymatic reactions.
Ch a r t 1. Nucleosides (1-5) and Their 5′-Triphosphates
(1TP -5TP ) Used in This Study
In tr od u ction
DNA is a biopolymer that stores genetic information,
and transcription and replication of the information
involve hydrogen-bond-based recognition between two
pairs of complementary nucleobases (A-T and G-C).
To expand the genetic alphabet or to address a question
on whether the four-letter alphabet is the only solution
for the genetic code, unnatural base pairs that would
be mediated by alternative hydrogen bonding1 or by
hydrophobic packing interactions2,3 have been exploited
so far. Natural nucleoside 5′-triphosphates act as sub-
strates for DNA polymerases and then are incorporated
into DNA strands with extremely high accuracy in a
template-directed manner. In this context, nonnatural
nucleoside 5′-triphosphates with some specific functional
groups could possibly have an inhibitory effect on DNA
and RNA syntheses, and therefore, they have long been
recognized as promising candidates for antiviral and
antineoplastic agents.4-7 In addition, some unnatural
base pairs based on altered hydrogen bonding1b,c and
hydrophobic interactions2,3 have been enzymatically
incorporated into DNA strands leading to the chain
elongation.
We have recently envisioned metal coordination as
an alternative driving force for base pairing and re-
ported a few examples of artificial â-C-nucleosides
bearing a phenylenediamine,8 a catechol,9 a 2-amino-
phenol,9a,10 and pyridine11 as the nucleobase.12-15 In this
* To whom correspondence should be addressed. Phone and fax:
+81-3-5841-8061. E-mail: shionoya@chem.s.u-tokyo.ac.jp.
† The University of Tokyo.
study, the in vitro activity of the synthetic catechol-
bearing nucleoside 5′-triphosphate 1TP (Chart 1), which
could potentially form either two-point hydrogen bond-
ing or metal complexes, was examined for DNA poly-
‡ National Institute of Genetics.
§ The Graduate University for Advanced Studies.
| Rigaku Corporation.
10.1021/jm020193w CCC: $22.00 © 2002 American Chemical Society
Published on Web 11/09/2002

Nonnatural Nucleoside Triphosphates
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 25 5595
Sch em e 1a
Sch em e 2a
a
Reagents: (a) Cp2HfCl2-AgClO4, CH2Cl2, -78 to -75 °C; (b)
aqueous NH3, MeOH; (c) (1) POCl3 in trimethyl phosphate, proton
sponge, then tributylammonium pyrophosphate, tributylamine;
(2) NaClO4.
a
Reagents: (a) (1) SnCl4, CH2Cl2, -20 °C; (2) K2CO3, MeOH;
(b) (1) POCl3 in trimethyl phosphate, proton sponge, then tri-
butylammonium pyrophosphate, tributylamine, (2) NaClO4; (c) H2,
Pd-C, H2O.
Sch em e 3a
merase reactions under typical polymerase chain reac-
tion (PCR) conditions. As a result, we found that 1TP
is not incorporated into DNA strands and that it inhibits
the DNA polymerase reaction in the presence of natural
nucleoside 5′-triphosphates (dNTPs, where N ) A, T,
G, or C). To investigate the structure-activity relation-
ship, we further synthesized a series of â-C-nucleosides
with one or two hydroxy groups at the 2-, 3-, or
4-position (2-4), and examined the effect of their 5′-
triphosphates (2TP -4TP ) on a series of DNA poly-
merase reactions.
Resu lts a n d Discu ssion
Syn th eses of Nu cleosid es a n d Th eir 5′-Tr ip h os-
p h a te Der iva tives. Five â-C-nucleosides bearing a 3,4-
dihydroxyphenyl (1), a 2-hydroxyphenyl (2), a 3-hydroxy-
phenyl (3), a 4-hydroxyphenyl (4), or a phenyl (5) group
and their 5′-triphosphate derivatives (1TP -5TP , re-
spectively) were prepared to examine their effect on
DNA polymerase reactions (Chart 1).
a
Reagents: (a) (1) THF, -78 °C, (2) Et3SiH, BF3‚OEt2, CH2Cl2,
-78 °C; (b) TBAF, THF; (c) (1) POCl3 in trimethyl phosphate,
proton sponge, then tributylammonium pyrophosphate, tributyl-
amine, (2) NaClO4; (d) H2, Pd-C, MeOH-H2O.
The synthetic routes for the 5′-triphosphates are
depicted in Schemes 1-5. A 3,4-dihydroxyphenyl de-
rivative 1TP was prepared from O-protected catechol-
bearing nucleoside 8 (Scheme 1).9a The Friedel-Crafts
approach via electrophilic aromatic substitution was
chosen to build up the carbon skeleton of the nucleoside
8, where SnCl4 was used as a Lewis acid. The coupling
reaction of O-protected catechol 7 and 3,5-protected
methylglycoside 6 afforded the base-coupled nucleosides
as a mixture of R- and â-anomers (R/â ) 1:2) in 32%
yield. The removal of ethyl carbonate groups with
K2CO3 in MeOH followed by purification by column
chromatography afforded the desired â-C-nucleoside 8
in 14% yield. Its 5′-triphosphate derivative 9 was then
prepared by treatment with POCl3 in trimethyl phos-
phate followed by the addition of tributylammonium
pyrophosphate and tributylamine in DMF.16 After pu-
rification by reversed-phase HPLC, the triethylammo-
nium counterions were exchanged with sodium ions
using the modified ion exchange method.17 The protec-
tive benzyl groups were removed by catalytic hydroge-
nation to afford the desired 5′-triphosphate 1TP in 40%
overall yield from 8.
dation from triacetylated deoxyribose 10 to 12.18 The
coupling reaction preferentially took place at the R-posi-
tion of the hydroxy group of phenol 11, and consequently
the â-anomer was isolated as the main product in 34%
yield.19 Subsequent removal of acetyl groups afforded
2, which was then converted to its 5′-triphosphate 2TP
in 11% yield by the same method as that for 1TP .
The 3-hydroxyphenyl derivative 3TP was prepared
via nucleoside 16 (Scheme 3). The coupling reaction
between lithiated 14 and lactone 13 furnished a hy-
droxyketone, which was subsequently reduced by
Et3SiH-BF3‚Et2O to provide â-C-glycoside 15 selec-
tively in 35% yield. The following desilylation afforded
â-C-nucleoside 16 and was further converted to its 5′-
triphosphate 3TP in 11% yield.
A key step in the synthesis of 4TP was the coupling
reaction of organozinc derivative 19 with O-protected
deoxyribose 18 followed by epimerization at the C1′
position (Scheme 4). Although this coupling reaction
selectively produced R-anomer 20 (R/â ) 7:1), epimer-
ization with benzenesulfonic acid in the presence of a
small amount of water20 led to partial conversion from
20 to the desired â-anomer 21 in 39% yield (R/â ) 1:2.2).
The resulting â-anomer 21 was converted to 4TP by
deprotection and phosphorylation.
A 2-hydroxyphenyl derivative 2TP was prepared as
shown in Scheme 2. A hafnium-based Lewis acid in
combination with AgClO4 was used for the C-glycosi-

5596 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 25
Aketani et al.
Sch em e 4a
F igu r e 1. DNA amplification by Ex Taq in the presence of
1TP : (lane 1) dNTPs, 0.2 mM each; (lane 2) dNTPs, 0.2 mM
each + 0.02 mM 1TP ; (lane 3) dNTPs, 0.2 mM each + 0.04
mM 1TP ; (lane 4) dNTPs, 0.2 mM each + 0.08 mM 1TP .
a
Reagents: (a) THF, reflux; (b) PhSO3H, H2O, H2SO4, toluene,
reflux; (c) NaOMe, MeOH; (d) (1) POCl3 in trimethyl phosphate,
proton sponge, then tributylammonium pyrophosphate, tributyl-
amine, (2) NaClO4; (e) H2, Pd-C, MeOH-H2O.
Sch em e 5a
a
Reagents: (a) (1) POCl3 in trimethyl phosphate, proton sponge,
then tributylammonium pyrophosphate, tributylamine, (2) Na-
ClO4.
The phenyldeoxynucleoside 5′-triphosphate 5TP was
derived from nucleoside 5, which was prepared accord-
ing to a previous report by Kool et al. (Scheme 5).21
The anomeric configuration of the C1′ position of each
free nucleoside was determined to be â by X-ray
analyses (see Supporting Information). The â-anomeric
F igu r e 2. Effect of 1TP on DNA polymerase reactions.
DNA strands. We then examined the influence of these
â-C-nucleoside 5′-triphosphates on DNA syntheses in
the presence of four regular substrates.
1
structure was also confirmed by comparison of the H
NMR splitting patterns for H1′ of each free nucleoside
with previously reported data.20,22 In all the cases, the
The artificial â-C-nucleoside 5′-triphophates 1TP -
4TP with one or two hydroxy groups showed inhibitory
activities against DNA polymerases when added to PCR
mixtures (Figures 2-5). In all the cases with inhibitory
activities, the formation of a full-length product was
reduced by the addition of â-C-nucleoside 5′-triphos-
phates 1TP -4TP in a dose-dependent manner, whereas
the quantity of unused primers was increased (for
instance, see Figure 1, lanes 2-4). No products shorter
than 180 bases were obtained, indicating that there is
no specific attenuation position of DNA chain elongation
along the template.
First, we measured the inhibitory activity of each â-C-
nucleoside 5′-triphophate on DNA syntheses catalyzed
by the five different species of DNA polymerases (Ex
Taq, LA-Taq, Pfu, Pyrobest, and Z-Taq). With all the
DNA polymerases examined, DNA syntheses were
inhibited by 1TP with two hydroxy groups (Figure 2).
The level of inhibition was, however, independent of
each DNA polymerase. For determination of the inhibi-
tory activities, the assay was repeated at least twice for
each enzyme, and the results were evaluated quanti-
tatively as summarized in Table 1. The concentrations
of 1TP required to exhibit 50% inhibition (IC50) ranged
from 0.03 (Ex Taq) to 0.73 mM (LA-Taq). 1TP acted as
1
5′-triphosphates 1TP -5TP were identified by H and
31P NMR, mass spectrometry, and elemental analyses
(see Experimental Section).
Effect of th e â-C-Nu cleosid e 5′-Tr ip h osp h a tes on
DNA P olym er a se Rea ction s. The effect of five â-C-
nucleoside 5′-triphosphates (1TP -5TP ) on DNA syn-
theses was evaluated using a series of DNA polymerases
(Ex Taq, LA-Taq, Z-Taq, Pfu, and Pyrobest). The phenyl-
bearing nucleoside 5′-triphosphate (5TP ) without aro-
matic hydroxy groups was used as the reference com-
pound for those (1TP -4TP ) with one or two hydroxy
groups. To increase the detection level, PCR was
employed throughout this study. The PCR was per-
formed using plasmid DNA (0.5 ng) as template and a
pair of primers (30-nucleotide-long lacUV501 and 28-
nucleotide-long lacUV502). Under these PCR conditions,
a DNA product of 180 bases in length is produced (for
instance, see Figure 1, lane 1). The amount of each PCR
product was determined by 8% acrylamide gel electro-
phoresis. When one of the â-C-nucleoside 5′-triphos-
phates 1TP -5TP was used as a substrate in place of
natural nucleoside 5′-triphosphates, virtually no DNA
synthesis was detected, and this result indicates that
these artificial 5′-triphosphates are not incorporated into

Nonnatural Nucleoside Triphosphates
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 25 5597
Ta ble 1. Comparative Inhibitory Activities of Synthetic
5′-Triphosphates (1TP -5TP ) on DNA Polymerases
a
IC50 (mM)
DNA polymerases
compd
Ex Taq
Pfu
LA-Taq
Z-Taq
Pyrobest
1TP
2TP
3TP
4TP
5TP
0.03
1.7
1.9
0.2
0.7
2.4
3.1
0.3
3.1
6.2
0.5
1.4
2.7
0.8
0.9
0.9
1.6
3.7
3.7
1.5
>16
>16
>16
>16
>16
a
IC50: concentrations of substrates to exhibit 50% inhibition
(mM).
F igu r e 4. Effect of 3TP on DNA polymerase reactions.
F igu r e 3. Effect of 2TP on DNA polymerase reactions.
the most efficient inhibitor against Ex Taq DNA poly-
merase. The second strongest inhibitory effect was
observed with Pfu, followed by Z-Taq, Pyrobest, and LA-
Taq in this order. The IC50 for LA-Taq with the lowest
inhibitory effect was 30 times larger than that for Ex
Taq with the highest inhibitory effect. The DNA am-
plification by Ex Taq was reduced to undetectable level
when 80 µM 1TP was added, while the other four
polymerases retained more than 70% activity at the
same concentrations.
To clarify the role of hydroxy groups of each substrate
in the inhibitory activity, the effect of 2TP , which has
one hydroxy group at the 2-position, on PCR was first
examined (Figure 3). PCR was carried out using the five
DNA polymerases in the presence of various concentra-
tions of 2TP and a fixed concentration (0.2 mM) of
regular nucleoside 5′-triphosphates (Figure 3). For all
five polymerases used, the inhibition by 2TP was less
than that by 1TP . The IC50 of 2TP ranged from 0.8 (Pfu)
to 3.1 mM (Z-Taq) as shown in Table 1. 2TP inhibited
DNA synthesis by Pfu most efficiently. The second
strongest inhibitory effect was observed with Pyrobest
followed by Ex Taq, LA-Taq, and Z-Taq in this order.
The IC50 for Z-Taq with the lowest inhibitory effect was
4 times larger than that for Ex Taq with the highest
inhibitory effect. The amplification activity by Pfu was
reduced to less than 5% when 1 mM 2TP was added,
while the other four polymerases retained more than
80% activity.
F igu r e 5. Effect of 4TP on DNA polymerase reactions.
inhibitory effect was observed with Ex Taq followed by
Pyrobest, LA-Taq, and Z-Taq in this order. The IC50 for
Z-Taq with the lowest inhibitory effect was 7 times
larger than that for Ex Taq with the highest inhibitory
effect. The amplification activity by Pfu was reduced to
approximately 5% when 1 mM 3TP was added, while
the other four polymerases retained more than 80%
activity.
DNA syntheses by the five DNA polymerases were
also examined in the presence of various concentrations
of 4TP (0-8.0 mM) with one hydroxy group at the
4-position (Figure 5). The IC50 of 4TP ranged from 0.9
(Pfu) to 3.7 mM (Z-Taq) as shown in Table 1. Again,
4TP acted as the most efficient inhibitor against DNA
synthesis by Pfu. The second strongest inhibitory effect
was observed with Pyrobest and Ex Taq followed by LA-
Taq and Z-Taq. The IC50 for LA-Taq and Z-Taq with the
lowest inhibitory effect was 4 times larger than that for
Pfu with the highest inhibitory effect. The DNA ampli-
fication by Pfu was reduced to approximately 5% when
1 mM 4TP was added, while the other four polymerases
retained more than 80% activity. On the basis of these
results with 1TP and three derivatives (2TP -4TP , with
one hydroxy group at different positions (Figures 3-5)),
we concluded that overall the inhibitory activity was
strongest with 1TP among the â-C-nucleoside 5′-tri-
phosphates tested in this study. The three â-C-nucleo-
side 5′-triphosphates with one hydroxy group are weaker
inhibitors compared with 1TP which has two hydroxy
groups at the 3- and 4-positions.
The effect of 3TP , which has one hydroxy group at
the 3-position, on PCR using the five DNA polymerases
was then examined in the presence of various concen-
trations of 3TP (0-10.0 mM; Figure 4). The IC50 of 3TP
ranged from 0.9 (Pfu) to 6.2 mM (Z-Taq) as shown in
Table 1. As in the case of 2TP , the second strongest

5598 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 25
Aketani et al.
F igu r e 7. Comparative amplification activities for Z-Taq
polymerase in the presence of 1TP and/or excess concentration
of natural nucleoside 5′-triphosphates: 1TP , dNTPs 0.2 mM
each + 1TP 1 mM; 1TP + T, dNTPs 0.2 mM each + 1TP 1mM
+ dTTP 1 mM; 1TP + G, dNTPs 0.2 mM each + 1TP 1 mM
+ dGTP 1 mM; 1TP + C, dNTPs 0.2 mM each + 1TP 1 mM
+ dCTP 1 mM; 1TP + A, dNTPs 0.2 mM each + 1TP 1 mM
+ dATP 1 mM; T, dNTPs 0.2 mM each + dTTP 1 mM; G,
dNTPs 0.2 mM each + dGTP 1 mM; C, dNTPs 0.2 mM each +
dCTP 1 mM; A, dNTPs 0.2 mM each + dATP 1 mM.
F igu r e 6. Effect of 5TP on DNA polymerase reactions.
As a control, we also examined the effect of 5TP
without hydroxy groups on DNA syntheses using the
five DNA polymerases. As shown in Figure 6, however,
the inhibition was not observed with 5TP at least up
to 16 mM. Owing to the solubility problem, we failed to
test the effect of 5TP at concentrations higher than 16
mM.
These results suggest that the hydroxy groups are
essential for the inhibitory activities. The two hydroxy
groups at the 3,4-positions of 1TP are most likely to play
a significant role in the inhibitory effect.23 The two
hydroxy groups may form two-point hydrogen bonding
with some functional groups around the catalytic cavity
of each DNA polymerase. In the cases of three 5′-
triphosphates with one hydroxy group (2TP -4TP ),
however, the extent of inhibition was not correlated with
the position of hydroxy group.
merases. The competitive inhibition of DNA synthesis
indicates the possibility that each DNA polymerase
forms a complex with nonnatural 5′-triphosphates (1TP -
4TP ), resulting in a reduction of the enzyme activity.
The primary sequence of Taq polymerase used in this
study is 38% identical to that of Eschericia coli DNA
polymerase I, an enzyme that has served as a model
for the study of DNA replication using enzymes of the
DNA Pol I family. This sequence homology implies that
1TP -4TP inhibit DNA polymerases other than those
used in this study. It is also noteworthy that the
nonnatural 5′-triphosphate 1TP shows the inhibitory
effect on RNA synthesis by E. coli RNA polymerase core
enzyme (M. Shionoya et al., unpublished results).
The present results indicate that the â-C-nucleoside
5′-triphophates 1TP -5TP are not incorporated into
DNA chains but are possibly recognized by the DNA
polymerases, resulting in the inhibition of polymeriza-
tion of the regular nucleoside 5′-triphosphate substrates.
If this is the case, the inhibition could be overcome by
the addition of a large excess of regular substrates. To
test this possibility, PCR was carried out in the presence
of a higher concentration (1.2 mM) of dATP, dTTP,
dGTP, or dCTP, where the concentrations of the other
three dNTPs were fixed at 0.2 mM. The DNA amplifica-
tion by Z-Taq in the presence of 1 mM 1TP significantly
increased by the addition of 1 mM dTTP, dGTP, or dCTP
(Figure 7). The most remarkable recovery was found
with dTTP (48%) followed by dGTP (33%) and dCTP
(14%), whereas the addition of dATP failed to recover
the DNA synthesis activity by Z-Taq but rather reduced
the amplification activity. The addition of 1 mM dATP,
dTTP, dGTP, or dCTP in the absence of 1TP showed
essentially the same activity as that under the standard
PCR condition. Since the level of recovery differed
between the nucleotides, the competition activity of the
â-C-nucleoside 5′-triphophate 1TP appears to depend
on the base species. Their competitive activity may arise
from the difference in the degree of structural analogy
of the nucleobase moieties.
Con clu sion
The effect of five â-C-nucleoside 5′-triphosphates
bearing a 3,4-dihydroxyphenyl (1TP ), a 2-hydroxy-
phenyl (2TP ), a 3-hydroxyphenyl (3TP ), a 4-hydroxy-
phenyl (4TP ), or a phenyl (5TP ) group on DNA poly-
merase reactions was investigated. It was found that
1TP with two hydroxy groups is a potent inhibitor
against DNA syntheses by the five different DNA
polymerases. A DNA amplification study using excess
dNTPs indicated that 1TP and dNTPs are recognized
by these DNA polymerases but the level of inhibition
differed among the five polymerases, presumably re-
flecting minor differences in their catalytic centers.
Although blood samples are known as PCR inhibitors,24
to our best knowledge, this is the first example of a
chelate-type â-C-nucleoside 5′-triphosphate that shows
potent inhibitory activity against DNA polymerases.25
This finding may raise the appealing possibility that
artificial nucleoside derivatives having phenolic hydroxy
groups could exhibit potent inhibitory activity against
DNA-directed enzymatic reactions. We will report else-
where the template effect of the chelate-type â-C-
nucleoside incorporated inside the DNA strands on DNA
polymerase reactions.
In addition, it should be noted that nonnatural 5′-
triphosphates 1TP -4TP interact with the DNA poly-
merase complexes, resulting in the shutting-off of
further elongation of the DNA strand by DNA poly-

Nonnatural Nucleoside Triphosphates
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 25 5599
COCH3), 2.15 (3H, s, COCH3). 13C NMR (CDCl3): δ 170.65,
170.60, 155.2, 129.4, 127.4, 123.3, 120.0, 117.1, 83.4, 81.6, 76.0,
63.7, 39.4, 20.9, 20.6. ESI-TOF mass, m/z: found, 317.0985;
calcd, [M + Na]+ 317.1001.
Exp er im en ta l Section
All reactions except for hydrogenation were carried out in
oven-dried glassware under argon atmosphere. Trimethyl
phosphate and tributylamine were distilled from BaO and
CaH2, respectively, under reduced pressure. Phosphorus oxy-
chloride was distilled under atmospheric pressure. All other
reagents were commercially available and used without fur-
ther purification. All reactions were monitored by thin-layer
chromatography (TLC) using Merck silica gel 60, F-254.
Column chromatography was conducted using Wakogel C-300
(silica gel, Wako). Purification via reversed-phase HPLC was
performed with a TOSO TSK gel ODS-80Ts column (21.5 mm
i.d. × 30 cm).
1-Hyd r oxy-2-(2′-d eoxy-â-D-r ibofu r a n osyl)ben zen e (2).
To a stirred solution of 12 (110 mg, 0.38 mmol) in MeOH (4
mL) was added dropwise 28% NH3 aqueous solution (1 mL).
The reaction mixture was stirred for 5 h at room temperature.
The solvent was removed in vacuo, and the residue was
chromatographed on silica gel with n-hexane-AcOEt-MeOH
(25:5:1). Compound 2 was obtained in 76% yield as a colorless
solid. Its configuration was confirmed by X-ray analysis of a
single crystal obtained by recrystallization from CHCl3-AcOEt
(for detailed data, see Supporting Information). Mp 135.5-
136.5 °C. 1H NMR (D2O-CD3OD ) 1:1): δ 7.26 (1H, d, J )
7.6 Hz, aromatic), 7.08 (1H, t, J ) 7.6 Hz, aromatic), 6.82 (1H,
t, J ) 7.6 Hz, aromatic), 6.77 (d, 1H, J ) 7.9 Hz, aromatic),
5.30 (1H, dd, J ) 5.5, 10.7 Hz, H-1′), 4.30-4.38 (1H, m, H-3′),
3.92 (1H, ddd, J ) 2.4, 4.6, 4.6 Hz, H-4′), 3.75-3.83 (2H, m,
H-5′), 2.18 (1H, ddd, J ) 1.5, 5.5, 13.4 Hz, H-2′), 1.95 (1H,
ddd, J ) 5.5, 10.7, 13.4 Hz, H-2′). 13C NMR (D2O-CD3OD )
1:1): δ 158.6, 132.6, 131.5, 129.7, 123.4, 119.7, 91.5, 83.2, 77.2,
66.3, 45.5. Anal. Calcd for C11H14O4: C, 62.85; H, 6.71.
Found: C, 62.79; H, 6.71.
1-Ben zyloxy-3-[3′,5′-O-((1,1,3,3-tetr a isop r op yl)d isilox-
a n d iyl)-2′-d eoxy-â-D-r ibofu r a n osyl]ben zen e (15). To a
solution of 3-benzyloxylbromobenzene (262 mg, 1.0 mmol) in
anhydrous THF (2.5 mL) under argon atmosphere was added
n-BuLi (0.6 mL, 1.6 M solution in n-hexane, 0.96 mmol) at
-78 °C, and the reaction mixture was further stirred for 30
min at -78 °C. A solution of 13 (220 mg, 0.59 mmol) in
anhydrous THF (2.5 mL) was added to the mixture at -78
°C. After 1 h, the reaction mixture was quenched with
saturated NH4Cl aqueous solution and extracted with
CH2Cl2. The organic phase was washed with saturated NH4-
Cl aqueous solution, water, and brine, dried over anhydrous
MgSO4, and concentrated in vacuo. The resulting yellowish
oil was used for the next reaction without further purification.
A solution of the crude oil in CH2Cl2 (2.5 mL) was treated
with Et3SiH (3 equiv) and BF3‚OEt2 (3 equiv) at -78 °C under
an argon atmosphere. The resulting solution was stirred for 6
h at -78 °C, and the reaction was quenched with saturated
NaHCO3 aqueous solution. The resulting mixture was ex-
tracted with CH2Cl2, and the organic phase was then washed
with saturated NaHCO3 aqueous solution, water, and brine,
dried over anhydrous MgSO4, and concentrated in vacuo. The
crude product was purified by silica gel column chromatogra-
phy with n-hexane-AcOEt (20:1), and the desired product 15
was obtained as a colorless oil in 35% yield. 1H NMR (CDCl3):
δ 7.43 (2H, d, J ) 7.0 Hz, CH2C6H5), 7.38 (2H, t, J ) 7.0 Hz,
CH2C6H5), 7.32 (1H, d, J ) 7.0 Hz, C6H4), 7.24 (1H, t, J ) 7.9
Hz, CH2C6H5), 6.98 (1H, s, C6H4), 6.93 (1H, d, J ) 7.6 Hz,
C6H4), 6.86 (1H, dd, J ) 2.4, 8.2 Hz, C6H4), 5.01-5.04 (1H, m,
H-1′), 5.05 (2H, s, CH2C6H5), 4.52 (1H, dt, J ) 4.5, 7.6 Hz,
H-3′), 4.14 (1H, dd, J ) 3.0, 11.0 Hz, H-5′), 3.84-3.96 (2H, m,
H-4′ and H-5′), 2.35 (1H, ddd, J ) 4.5, 6.7, 12.8 Hz, H-2′), 2.07
(1H, dt, J ) 7.9, 12.8 Hz, H-2′), 0.92-1.15 (28H, m, 4 ×
CH(CH3)2). 13C NMR (CDCl3): δ 158.9, 143.8, 137.0, 129.5,
128.6, 127.9, 127.6, 118.5, 113.7, 112.4, 86.5, 78.9, 73.5, 69.9,
63.8, 43.2, 17.6, 17.5, 17.44, 17.3, 17.16, 17.12, 17.02, 13.54,
13.44, 13.06, 12.59. HRMS (ESI-TOF) calcd for C30H46O5Si2Na
565.2781, found 565.2772.
1-Ben zyloxy-3-(2′-deoxy-â-D-r ibofu r an osyl)ben zen e (16).
To a stirred mixture of 15 (227 mg, 0.42 mmol) in anhydrous
THF (8.4 mL) was added dropwise n-Bu4NF (1.3 mL, 1.0 M
solution in anhydrous THF, 1.3 mmol). The resulting mixture
was stirred for 2 h at room temperature, and saturated
NaHCO3 and 10% NaOH aqueous solutions were added to the
reaction mixture for quenching. The mixture was extracted
with Et2O, and the organic extract was washed with saturated
NaHCO3 and 10% NaOH aqueous solutions, water, and brine.
The combined organic layer was dried over anhydrous MgSO4
and concentrated in vacuo. The crude product was purified by
silica gel column chromatography with CH2Cl2-MeOH (19:
1H and 13C NMR spectra were recorded on a Bruker DRX500
1
spectrometer (500 MHz for H, 125 MHz for 13C), and 31P NMR
spectra were recorded on a J EOL AL270B (109 MHz). Chemi-
cal shifts (δ) are reported in ppm, and coupling constants (J )
are reported in hertz. Mass spectra were recorded on an ESI-
TOF mass spectrometer (LCT, Micromass) or a FAB mass
spectrometer (J MS 700P, J EOL).
1,2-Dib e n zyloxy-4-(2′-d e oxy-â-D-r ib ofu r a n osyl)b e n -
zen e (8). To a stirred solution of ribose 6 (595 mg, 2.0 mmol)
in CH2Cl2 (1 mL) was added 7 (591 mg, 2.0 mmol). The mixture
was cooled to -20 °C, and then SnCl4 (600 µL, 2.0 mmol) was
added dropwise to the reaction mixture over 10 min. The
mixture was further stirred for 15 min before the reaction was
quenched with water (4 mL). The reaction mixture was
extracted with CH2Cl2 and washed with 1.0 M HCl aqueous
solution and brine, dried over anhydrous MgSO4, and concen-
trated in vacuo. The residue was purified by silica gel column
chromatograph with n-hexane-AcOEt (6:1). The title com-
pound was obtained as a mixture of R- and â-anomers (357
mg).
Without separation, K2CO3 (50 mg) was added to the
mixture (357 mg) in MeOH (5 mL), and the reaction mixture
was then stirred for 3 h at room temperature. The mixture
was extracted with CH2Cl2, and the organic phase was washed
with water, dried over anhydrous MgSO4, and evaporated. The
crude product was chromatographed on silica gel with n-hex-
ane-AcOEt (1:1). The title compound was obtained in 14%
yield as a colorless solid. Its configuration was confirmed by
X-ray analysis of a single crystal obtained by recrystallization
from CHCl3-AcOEt (for detailed data, see Supporting Infor-
1
mation). Mp 105.0-106.0 °C. H NMR (CDCl3): δ 6.73-7.25
(13H, m, aromatic), 5.08 (4H, s, 2 × CH2C6H5), 4.99 (1H, dd,
J ) 5.6, 9.8 Hz, H-1′), 4.28-4.34 (1H, m, H-3′), 3.88 (1H, ddd,
J ) 4.4, 4.4, 7.3 Hz, H-4′), 3.69 (1H, dd, J ) 4.1, 10.1 Hz, H-5′),
3.62 (1H, dd, J ) 6.6, 11.6 Hz, H-5′), 2.08 (1H, ddd, J ) 1.9,
5.5, 13.3 Hz, H-2′), 1.90 (1H, ddd, J ) 6.1, 10.0, 13.2 Hz, H-2′),
1.82 (1H, d, J ) 6.3 Hz, OH), 1.75 (1H, d, J ) 6.1 Hz, OH).
Anal. Calcd for C25H26O5: C, 73.87; H, 6.45. Found: C, 73.70;
H, 6.36.
1-Hyd r oxy-2-(3′,5′-d i-O-a cetyl-2′-d eoxy-â-D-r ibofu r a n o-
syl)ben zen e (12). To a stirred mixture of Cp2HfCl2 (2.4 g, 6.3
mmol), AgClO4 (2.6 g, 13 mmol), phenol 11 (400 mg, 4.2 mmol),
and powdered 4 Å molecular sieves (1.4 g) in CH2Cl2 (20 mL)
was added 1,3,5-tri-O-acetyl-2-deoxyribofuranose 10 (546 mg,
2.1 mmol) in CH2Cl2 (30 mL) at -78 °C. The temperature was
gradually raised to -40 °C over 2 h, and after the completion
of initial O-glycosidation, the temperature was further raised
to -5 °C over 2 h to convert to C-glycoside 12. The reaction
was quenched with saturated NaHCO3 aqueous solution, and
then the mixture was extracted with CH2Cl2. The organic
phase was washed with saturated NaHCO3 aqueous solution
and brine, dried over anhydrous MgSO4, and concentrated in
vacuo. The crude product was purified by silica gel column
chromatograph with n-hexane-AcOEt (30:1). The title com-
1
pound 12 was obtained in 34% yield as a pale-yellow oil. H
NMR (CDCl3): δ 7.60 (1H, s, C6H4OH), 7.19 (1H, t, J ) 8.0
Hz, aromatic), 7.05 (1H, d, J ) 7.6 Hz, aromatic), 6.87 (1H, d,
J ) 8.1 Hz, aromatic), 6.85 (1H, t, J ) 7.4 Hz, aromatic), 5.19-
5.23 (2H, m, H-1′ and H-3′), 4.42 (1H, dd, J ) 3.3, 12.0 Hz,
H-5′), 4.34 (1H, dd, J ) 3.2, 12.0 Hz, H-5′), 4.24 (1H, ddd, J )
3.2, 3.2, 5.7 Hz, H-4′), 2.30-2.34 (2H, m, H-2′), 2.18 (3H, s,

5600 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 25
Aketani et al.
1). The desired product 16 was obtained as a colorless solid in
83% yield. Its configuration was confirmed by X-ray analysis
of a single crystal obtained by recrystallization from CHCl3-
AcOEt (for detailed data, see Supporting Information). Mp
97.5-98.5 °C. 1H NMR (CDCl3-CD3OD ) 1:1): δ 7.41 (2H, d,
J ) 7.0 Hz, CH2C6H5), 7.37 (2H, t, J ) 7.0 Hz, CH2C6H5), 7.32
(1H, d, J ) 7.1 Hz, C6H4), 7.23 (1H, t, J ) 7.9 Hz, CH2C6H5),
6.98 (1H, s, C6H4), 6.90 (1H, d, J ) 7.8 Hz, C6H4), 6.88 (1H,
dd, J ) 2.7, 8.2 Hz, C6H4), 5.12 (1H, dd, J ) 5.7, 10.1 Hz, H-1′),
5.04 (2H, s, CH2C6H5), 4.33 (1H, dt, J ) 2.2, 6.3 Hz, H-3′),
3.97 (1H, ddd, J ) 4.9, 4.9, 3.0 Hz, H-4′), 3.68 (2H, d, J ) 5.0
Hz, H-5′), 2.21 (1H, ddd, J ) 1.9, 5.7, 13.3 Hz, H-2′), 1.95 (1H,
ddd, J ) 6.3, 10.2, 13.3 Hz, H-2′). 13C NMR (CDCl3): δ 158.9,
142.9, 136.9, 129.6, 128.6, 127.9, 127.5, 118.6, 113.8, 112.7,
87.3, 79.9, 73.4, 69.9, 63.3, 50.6, 43.5. Anal. Calcd for
of anhydrous MeOH (1 mL) and CHCl3 (0.5 mL) and treated
with 28% MeOH solution of NaOMe (80 µL). The reaction
mixture was stirred for 1.5 h at room temperature under an
argon atmosphere, and then the reaction was quenched with
solid NH4Cl. After filtration, the solvent was removed in vacuo.
The residue was extracted with AcOEt, and then the organic
phase was washed with saturated NaHCO3 aqueous solution
and brine, dried over anhydrous MgSO4, and concentrated in
vacuo. The crude product was purified by silica gel column
chromatograph with Et2O to Et2O-EtOH (75:1). The product
22 was obtained as a colorless solid in 76% yield. Its config-
uration was confirmed by X-ray analysis of a single crystal
obtained by recrystallization from CHCl3-AcOEt (for detailed
data, see Supporting Information). Mp 113.0-114.0 °C. 1H
NMR (CDCl3-CD3OD ) 1:1): δ 7.42 (2H, d, J ) 7.1 Hz,
CH2C6H5), 7.38 (2H, t, J ) 7.7 Hz, CH2C6H5), 7.32 (1H, d, J )
7.2 Hz, CH2C6H5), 7.27 (2H, d, J ) 8.7 Hz, C6H4), 6.96 (2H, d,
J ) 8.7 Hz, C6H4), 5.12 (1H, dd, J ) 5.6, 10.2 Hz, H-1′), 5.06
(2H, s, CH2C6H5), 4.39-4.43 (1H, m, H-3′), 3.98 (1H, ddd, J )
4.4, 4.4, 6.6 Hz, H-4′), 3.81 (1H, dd, J ) 3.8, 11.5 Hz, H-5′),
3.72 (1H, dd, J ) 4.3, 11.3 Hz, H-5′), 2.20 (1H, ddd, J ) 1.9,
5.6, 13.3 Hz, H-2′), 2.04 (1H, ddd, J ) 6.3, 10.2, 13.2 Hz, H-2′).
13C NMR (CDCl3-CD3OD ) 1:1): δ 158.4, 136.9, 133.1, 128.6,
127.9, 127.47, 127.41, 114.9, 87.1, 79.8, 73.8, 70.0, 63.3, 43.8.
Anal. Calcd for C18H20O4: C, 71.98; H, 6.71. Found: C, 71.66;
H, 6.73.
C
18H20O4: C, 71.98; H, 6.71. Found: C, 71.70; H, 6.65.
1-Ben zyloxy-4-(3′,5′-d i-O-tolu oyl-2′-d eoxy-r-D-r ibofu r a -
n osyl)ben zen e (20). To a solution of finely dried magnesium
turnings (500 mg, 21 mmol) and a few crystals of iodine in
anhydrous THF (1 mL) was added dropwise 4-benzyloxylbro-
mobenzene (5.0 g, 19 mmol) in THF (19 mL) under an argon
atmosphere. The reaction mixture was required to be heated
at about 40 °C for initiation. The concentration of the Grignard
reagent was determined to be 0.45 M by the acid-base
titration.
To finely dried ZnCl2 (497 mg, 3.7 mmol) was added the
Grignard reagent (13 mL, 0.45 M solution in anhydrous THF,
6.0 mmol) under an argon atmosphere. After heating for 3 h
at 80 °C, the reaction mixture was cooled to room temperature.
A solution of 1′-R-chrolo-3′,5′-di-O-toluoyl-2′-deoxyribose 18
(970 mg, 2.5 mmol) in anhydrous THF (15 mL) was introduced
into the reaction mixture at room temperature, which was
further heated at reflux for 5 h. The reaction was quenched
with distilled water, and then the solvent was removed in
vacuo. The residue was taken up into AcOEt, and the organic
layer was washed with saturated NaHCO3 aqueous solution
and brine, dried over anhydrous MgSO4, and concentrated in
vacuo. The crude product was purified by silica gel column
chromatograph with n-hexane-AcOEt (20:1). The major R-
anomer 20 and the minor â-anomer 21 were obtained as a pale-
yellow oil in 36% and 5% isolated yields, respectively.
1,2-Dib e n zyloxy-4-(2′-d e oxy-â-D-r ib ofu r a n osyl)b e n -
zen e 5′-Tr ip h osp h a te (9). Compound 8 (85 mg, 0.21 mmol)
and 1,8-bis(dimethylamino)naphthalene (proton sponge, 73
mg, 0.34 mmol) were dissolved in trimethyl phosphate (1.7
mL), and the mixture was cooled to 0 °C. After addition of
phosphorus oxychloride (23 µL, 0.25 mmol) dropwise, the
solution was stirred for 3 h at 0 °C. A solution of tributylamine
(330 µL, 1.4 mmol) and tributylammonium pyrophosphate (132
mg, 0.39 mmol) in dry DMF (2.3 mL) was added to the reaction
mixture, and the solution was stirred for 1 min and then
quenched with 1.0 M triethylammonium bicarbonate (TEAB,
33 mL, pH 8.5). After standing for 2.5 h at room temperature,
the reaction mixture was concentrated by lyophilization to
approximately 2 mL. Purification by RP-HPLC (30-46% CH3-
CN gradient over 50 min at a flow rate of 4 mL/min in 0.10 M
TEAB) yielded 9 as triethylammonium salts (285 mg) with a
retention time of 40 min. ESI-TOF mass, m/z: found, 644.90;
calcd, [M + 3H]- 645.07. The triethylammonium salts were
dissolved in water (1 mL), and then NaClO4 (255 mg, 2.1 mmol)
was added. After the mixture was stirred overnight, acetone
(15 mL) was added to the reaction mixture. The resulting
precipitate was collected by centrifugation, and the sodium
salts 9 were obtained as a colorless solid in 40% yield. 1H NMR
(D2O): δ 7.25-7.34 (10H, m, aromatic), 6.88-7.00 (3H, m,
aromatic), 5.09 (2H, s, CH2C6H5), 5.05 (1H, dd, J ) 5.4, 10.6
Hz, H-1′), 4.38-4.42 (1H, m, H-3′), 4.03-4.08 (1H, m, H-4′),
3.88-4.02 (2H, m, H-5′), 2.04 (1H, ddd, J ) 5.5, 5.9, 13.5 Hz,
H-2′), 1.93 (1H, ddd, J ) 5.8, 10.7, 13.6 Hz, H-2′). 31P NMR
(D2O): δ -10.3 (1P, d, J ) 18.3 Hz), -11.1 (1P, d, J ) 18.3
Hz), -22.9 (1P, t, J ) 18.3 Hz) (external reference, 20 mM
H3PO4 in D2O). Anal. Calcd for C25H26Na3O14P3‚3H2O: C,
39.18; H, 4.21. Found: C, 39.41; H, 4.19.
1,2-Dih yd r oxy-4-(2′-d eoxy-â-D-r ibofu r a n osyl)ben zen e
5′-Tr ip h osp h a te (1TP ). Compound 9 (31 mg, 0.042 mmol)
was dissolved in distilled water (4 mL), and Pd-C (36 mg, 10%
Pd) was added to the reaction mixture. The suspended mixture
was stirred vigorously for 4 h under an H2 atmosphere. After
filtration and removal of the solvent, 1TP was obtained
quantitatively as a colorless solid. 1H NMR (D2O): δ 7.01-
7.03 (1H, m, C6H4), 6.81-6.83 (3H, m, C6H4), 5.02 (1H, dd, J
) 6.6, 8.9 Hz, H-1′), 4.47-4.52 (1H, m, H-3′), 4.02-4.12 (3H,
m, H-4′ and H-5′), 2.07-2.14 (2H, m, H-2′). 31P NMR (D2O): δ
-9.28 (1P, d, J ) 21.3 Hz), -10.87 (1P, d, J ) 18.3 Hz), -22.68
(1P, dd, J ) 21.3, 18.3 Hz) (external reference, 20 mM H3PO4
in D2O). HRMS (FAB) calcd for C11H15O14P3Na 486.9572, found
486.9565.
1
20. H NMR (CDCl3): δ 7.96 (2H, d, J ) 8.2 Hz, C6H4CH3),
7.76 (2H, d, J ) 8.2 Hz, C6H4CH3), 7.42 (2H, d, J ) 7.5 Hz,
CH2C6H5), 7.34-7.38 (4H, m, aromatic), 7.30 (1H, t, J ) 7.3
Hz, CH2C6H5), 7.21 (2H, d, J ) 7.9 Hz, aromatic), 7.17 (2H, d,
J ) 8.0 Hz, aromatic), 6.96 (2H, d, J ) 8.7 Hz, C6H4), 5.58
(1H, ddd, J ) 3.5, 3.9, 7.1 Hz, H-3′), 5.29 (1H, dd, J ) 6.8, 6.8
Hz, H-1′), 5.04 (2H, s, CH2C6H5), 4.66 (1H, ddd, J ) 4.6, 4.6,
6.9 Hz, H-4′), 4.58 (1H, dd, J ) 5.1, 11.7 Hz, H-5′), 4.54 (1H,
dd, J ) 4.5, 11.5 Hz, H-5′), 2.89 (1H, ddd, J ) 6.8, 7.1, 13.6
Hz, H-2′), 2.38 (3H, s, C6H4CH3), 2.37 (3H, s, C6H4CH3), 2.27
(1H, ddd, J ) 4.4, 6.4, 13.6 Hz, H-2′).
1
21. H NMR (CDCl3): δ 7.98 (2H, d, J ) 8.1 Hz, C6H4CH3),
7.94 (2H, d, J ) 8.1 Hz, C6H4CH3), 7.21-7.42 (11H, m,
aromatic), 6.93 (2H, d, J ) 8.7 Hz, C6H4), 5.58-5.63 (1H, m,
H-3′), 5.20 (1H, dd, J ) 4.9, 10.2 Hz, H-1′), 5.05 (2H, s,
CH2C6H5), 4.62-4.68 (2H, m, H-5′), 4.50-4.55 (1H, m, H-4′),
2.4-2.5 (1H, m, H-2′), 2.43 (3H, s, C6H4CH3), 2.40 (3H, s,
C6H4CH3), 2.23 (1H, ddd, J ) 6.1, 11.0, 13.9 Hz, H-2′). HRMS
(ESI-TOF) calcd for C34H32O6Na 559.2099, found 559.2099.
1-Ben zyloxy-4-(3′,5′-d i-O-tolu oyl-2′-d eoxy-â-D-r ibofu r a -
n osyl)ben zen e (21). To a stirred solution of 20 (965 mg, 1.8
mmol) in toluene (40 mL) were added a catalytic amount of
benzenesulfonic acid (10%), two drops of concentrated H2SO4,
and seven drops of H2O. The reaction mixture was heated at
reflux for 2 h. The mixture was then poured into 5% NaHCO3
aqueous solution (100 mL) and extracted with AcOEt. The
organic layer was dried over anhydrous MgSO4 and concen-
trated in vacuo. The crude product was purified by silica gel
chromatography eluting with n-hexane-AcOEt (20:1). The
desired product 21 was obtained as a pale-yellow oil in 39%
yield.
1-Ben zyloxy-4-(2′-deoxy-â-D-r ibofu r an osyl)ben zen e (22).
Compound 21 (19 mg, 0.035 mmol) was dissolved in a mixture
1-Hyd r oxy-2-(2′-d eoxy-â-D-r ibofu r a n osyl)ben zen e 5′-
Tr ip h osp h a te (2TP ). Compound 2 (88 mg, 0.42 mmol) and

Nonnatural Nucleoside Triphosphates
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 25 5601
proton sponge (288 mg, 1.3 mmol) were dissolved in trimethyl
phosphate (1.5 mL), and the mixture was cooled to 0 °C. After
addition of phosphorus oxychloride (77 µL, 0.84 mmol) drop-
wise in trimethyl phosphate (1.5 mL), the solution was stirred
for 3 h at 0 °C. A solution of tributylamine (1.3 mL, 5.6 mmol)
and tributylammonium pyrophosphate (530 mg, 0.98 mmol)
in dry DMF (4.7 mL) was added to the reaction mixture, and
the solution was stirred for 1 min and then quenched with
1.0 M TEAB (70 mL, pH 8.5). After standing for 1 h at room
temperature, the reaction mixture was lyophilized to dryness.
Purification by RP-HPLC (10-25% CH3CN gradient over 1 h
at a flow rate of 5 mL/min in 0.10 M TEAB) yielded 2TP as
triethylammonium salts with a retention time of 26 min. ESI-
TOF mass, m/z 449.0 (M + 3H)-. The obtained salts were
dissolved in distilled water (1 mL), and NaClO4 (403 mg, 3.3
mmol) was added to the solution. After the mixture was stirred
for 3 h, acetone (20 mL) was added to the solution. The
resulting precipitate was collected by centrifugation, washed
with acetone, and dried in vacuo. The sodium salts 2TP were
2.09-2.15 (1H, m, H-2′), 2.00 (1H, ddd J ) 1.2, 5.5, 10.7 Hz,
H-2′). HRMS (FAB) calcd for C11H13O13P3Na3 514.9262, found
514.9252.
1-Ben zyloxy-4-(2′-d eoxy-â-D-r ibofu r a n osyl)ben zen e 5′-
Tr ip h osp h a te (23). Compound 22 (58 mg, 0.19 mmol) and
proton sponge (66 mg, 0.31 mmol) were dissolved in trimethyl
phosphate (2.0 mL) and cooled to 0 °C. After addition of
phosphorus oxychloride (21 µL, 0.23 mmol) in trimethyl
phosphate (1 mL) dropwise, the solution was stirred for 3 h
at 0 °C. A solution of tributylamine (310 µL, 1.3 mmol) and
tributylammonium pyrophosphate (122 mg, 0.22 mmol) in dry
DMF (2.0 mL) was added to the reaction mixture, and the
solution was stirred for 1 min and then quenched by addition
of 1.0 M TEAB (30 mL, pH 8.5). After standing for 1.5 h at
room temperature, the reaction mixture was lyophilized to
dryness. Purification by RP-HPLC (20-55% CH3CN gradient
over 60 min at a flow rate of 5 mL/min in 0.10 M TEAB)
yielded 23 as triethylammonium salts with a retention time
of 38 min. ESI-TOF mass, m/z 539.1 (M + 3H)-, 561.1 (M +
Na + 2H)-.
1
obtained as a colorless solid in 11% yield. H NMR (D2O): δ
7.33 (1H, d, J ) 7.7 Hz, C6H4), 7.10 (1H, t, J ) 7.8 Hz, C6H4),
6.86 (1H, t, J ) 7.5 Hz, C6H4), 6.76 (1H, d, J ) 8.1 Hz, C6H4),
5.32 (1H, dd, J ) 5.5, 10.4 Hz, H-1′), 4.41-4.45 (1H, m, H-3′),
4.03-4.06 (1H, m, H-4′), 3.93-4.02 (2H, m, H-5′), 2.14 (1H,
ddd, J ) 1.9, 5.5, 13.5 Hz, H-2′), 2.03 (1H, ddd, J ) 6.0, 11.0,
14.0 Hz, H-2′). 31P NMR (D2O): δ -10.1 (1P, d, J ) 15.2 Hz),
-11.0 (1P, d, J ) 18.3 Hz), -22.75 (1P, dd, J ) 15.3, 18.3 Hz)
(external reference, 20 mM H3PO4 in D2O). Anal. Calcd for
The salts thus obtained were dissolved in distilled water (1
mL), and NaClO4 (150 mg, 1.2 mmol) was added to the
solution. After the mixture was stirred for 2 h, acetone (20
mL) was added to the reaction mixture. The resulting precipi-
tate was collected by centrifugation, washed with acetone, and
dried in vacuo. The sodium salts 23 were obtained as a
1
colorless solid in 15% yield. H NMR (D2O): δ 7.36 (2H, d, J
) 7.5 Hz, CH2C6H5), 7.24-7.32 (5H, m, aromatic), 6.93 (2H,
d, J ) 8.4 Hz, aromatic), 5.02 (2H, s, CH2C6H5), 4.99 (1H, dd,
J ) 7.5, 8.7 Hz, H-1′), 4.42-4.45 (1H, m, H-3′), 4.00-4.04 (1H,
m, H-4′), 3.92-3.98 (2H, m, H-5′), 2.03-2.06 (2H, m, H-2′).
31P NMR (D2O): δ -9.08 and -9.81 (1P, m), -11.0 (1P, d, J )
18.3 Hz), -22.28 and -23.03 (1P, m) (external reference, 20
mM H3PO4 in D2O). Anal. Calcd for C18H20Na3O13P3‚4H2O: C,
31.87; H, 4.16. Found: C, 31.35; H, 3.99.
C
11H14Na3O13P3‚3H2O: C, 23.17; H, 3.54. Found: C, 23.28; H,
3.62. HRMS (FAB) calcd for C11H15O13P3Na 470.9623, found
470.9627.
1-Ben zyloxy-3-(2′-d eoxy-â-D-r ibofu r a n osyl)ben zen e 5′-
Tr ip h osp h a te (17). Compound 16 (39 mg, 0.18 mmol) and
proton sponge (60 mg, 0.28 mmol) were dissolved in trimethyl
phosphate (1.4 mL) and cooled to 0 °C. After addition of
phosphorus oxychloride (19 µL, 0.21 mmol) dropwise in tri-
methyl phosphate (0.5 mL), the solution was stirred for 3 h at
0 °C. A solution of tributylamine (278 µL, 1.2 mmol) and
tributylammonium pyrophosphate (192 mg, 0.35 mmol) in dry
DMF (2 mL) was added to the reaction mixture, and the
solution was stirred for 1 min. Then the reaction was quenched
by addition of 1.0 M TEAB (30 mL, pH 8.5). After standing
for 30 min at room temperature, the reaction mixture was
lyophilized to dryness. Purification by RP-HPLC (0-50% CH3-
CN gradient over 50 min at a flow rate of 5 mL/min in 0.10 M
TEAB) yielded compound 17 with a retention time of 51 min.
ESI-TOF mass, m/z: found, 538.8; calcd, [M + 3H]- 539.0.
1-Hyd r oxy-4-(2′-d eoxy-â-D-r ibofu r a n osyl)ben zen e 5′-
Tr ip h osp h a te (4TP ). Compound 23 (25 mg, 0.028 mmol) was
dissolved in a mixture of distilled water (1 mL) and MeOH (1
mL), and then Pd-C (30 mg, 10% Pd) was added to the
reaction mixture. The suspended mixture was stirred over-
night under an H2 atmosphere. After filtration and removal
of the solvent, 4TP was obtained quantitatively as a colorless
1
solid. H NMR (D2O): δ 7.27 (2H, d, J ) 8.5 Hz, C6H4), 6.81
(2H, d, J ) 8.5 Hz, C6H4), 5.02 (1H, t, J ) 8.2 Hz, H-1′), 4.45-
4.49 (1H, m, H-3′), 4.04-4.08 (1H, m, H-4′), 3.94-4.03 (2H,
m, H-5′), 2.07-2.11 (2H, m, H-2′). 31P NMR (D2O): δ -9.11
(1P, d, J ) 18.3 Hz), -10.9 (1P, d, J ) 18.3 Hz), -22.4 (1P, t,
J ) 18.3 Hz) (external reference, 20 mM H3PO4 in D2O). FAB
mass, m/z: found, 470.9612, 492.9494, 514.9336; calcd, [M +
Na + 2H]- 470.9623, [M + 2Na + H]- 492.9443, [M + 3Na]-
514.9262.
The salts thus obtained were dissolved in distilled water (1
mL), and NaClO4 (161 mg, 1.3 mmol) was added to the
solution. After the mixture was stirred for 2 h, acetone (20
mL) was added to the reaction mixture. The resulting precipi-
tate was collected by centrifugation, washed with acetone, and
dried in vacuo. The sodium salts 17 were obtained as a
(2′-Deoxy-â-D-r ibofu r a n osyl)ben zen e 5′-Tr ip h osp h a te
(5TP ). Compound 5 was prepared via the procedure reported
by Kool et al.21 The â-configuration of 5 was confirmed by X-ray
analysis (see Supporting Information). Compound 5 (48 mg,
0.25 mmol) and proton sponge (84 mg, 0.39 mmol) were
dissolved in trimethyl phosphate (2.0 mL), and the mixture
was cooled to 0 °C. After addition of phosphorus oxychloride
(27 µL, 0.30 mmol) dropwise in trimethyl phosphate (1 mL),
the solution was stirred for 3 h at 0 °C. A solution of
tributylamine (390 µL, 1.6 mmol) and tributylammonium
pyrophosphate (268 mg, 0.49 mmol) in dry DMF (2.7 mL) was
added to the reaction mixture, and the solution was stirred
for 1 min. Then the reaction was quenched with the addition
of 1.0 M TEAB (39 mL, pH 8.5). After standing for 1 h at room
temperature, the reaction mixture was concentrated in vacuo
and chromatographed on a DEAE-Sephadex A25 with a
gradient of TEAB (0.10-1.0 M, pH 7.5). The appropriate
fractions were collected and concentrated in vacuo. Purification
by RP-HPLC (0-15% CH3CN gradient over 30 min and fixed
to 15% for an additional 15 min at a flow rate of 5 mL/min in
0.10 M TEAB) yielded 5TP as triethylammonium salts with
a retention time of 43 min. ESI-TOF mass, m/z: found, 433.0,
455.0; calcd, [M + 3H]- 433.0, [M + Na + 2H]- 455.0.
1
colorless solid in 11% yield. H NMR (D2O): δ 7.38 (2H, d, J
) 7.2 Hz, CH2C6H5), 7.33 (2H, t, J ) 7.2 Hz, CH2C6H5), 7.29
(1H, d, J ) 7.2 Hz, C6H4), 7.23 (1H, t, J ) 5.5 Hz, CH2C6H5),
6.96-7.02 (2H, m, C6H4), 6.86-6.90 (1H, m, C6H4), 5.07 (2H,
s, CH2C6H5), 5.03 (1H, dd, J ) 5.4, 10.5 Hz, H-1′), 4.41-4.47
(1H, m, H-3′), 4.05-4.12 (1H, m, H-4′), 3.95-4.05 (2H, m, H-5′),
2.13 (1H, dd, J ) 5.6, 13.6 Hz, H-2′), 1.96-2.04 (1H, m, H-2′).
31P NMR (D2O): δ -8.89 to -9.17 (1P, m), -10.8 (1P, d, J )
18.3 Hz), -22.06 to -22.38 (1P, m) (external reference, 20 mM
H3PO4 in D2O). Anal. Calcd for C18H19Na4O13P3‚4H2O: C,
30.70; H, 4.09. Found: C, 30.70; H, 4.09.
1-Hyd r oxy-3-(2′-d eoxy-â-D-r ib ofu r a n osyl)b en zen e 5′-
Tr ip h osp h a te (3TP ). To a stirred solution of 17 (18 mg, 0.092
mmol) in a mixture of distilled water (1 mL) and MeOH (1
mL), Pd-C (30 mg, Pd 10%) was added. The suspended
mixture was stirred overnight under an H2 atmosphere. After
filtration and removal of the solvent, 3TP was produced
quantitatively as a colorless solid. 1H NMR (D2O): δ 7.13-
7.18 (1H, m, C6H4), 6.82-6.94 (2H, m, C6H4), 6.68-6.72 (1H,
m, C6H4), 5.00 (1H, dd, J ) 4.9, 10.1 Hz, H-1′), 4.41 (1H, m,
H-3′), 4.02 (1H, m, H-4′), 4.00 (1H, m, H-5′), 3.88 (1H, m, H-5′),

5602 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 25
Aketani et al.
Biochemistry 1993, 32, 10489-10496. (c) Horlacher, J .; Hottiger,
M.; Podust, V. N.; Hu¨bscher, U.; Benner, S. A. Recognition by
Viral and Cellular DNA Polymerase of Nucleosides Bearing
Bases with Nonstandard Hydrogen Bonding Patterns. Proc.
Natl. Acad. Sci. U.S.A. 1995, 92, 6329-6333.
The obtained residue was dissolved in distilled water (1 mL),
and NaClO4 (170 mg, 1.4 mmol) was added to the solution.
After the mixture was stirred for 2 h, addition of acetone (20
mL) resulted in precipitation. The precipitate was collected
by centrifugation, washed with acetone, and dried in vacuo.
The sodium salts 5TP were obtained as a colorless solid in
25% yield. 1H NMR (D2O): δ 7.31-7.36 (2H, m, C6H5), 7.28
(2H, t, J ) 7.6 Hz, C6H5), 7.22 (1H, t, J ) 7.3 Hz, C6H5), 5.03
(1H, dd, J ) 5.8, 10.0 Hz, H-1′), 4.43-4.47 (1H, m, H-3′), 4.20-
4.34 (1H, m), 4.02-4.12 (2H, m, H-5′), 3.89-3.99 (1H, m),
2.01-2.13 (2H, m, H-2′). 31P NMR (D2O): δ -5.55 (1P, d, J )
21.3 Hz), -10.6 (1P, d, J ) 18.3 Hz), -21.4 (1P, dd, J ) 18.3,
21.3 Hz) (external reference, 20 mM H3PO4 in D2O). Anal.
Calcd for C11H14Na3O12P3‚4H2O: C, 23.09; H, 3.88. Found: C,
23.12; H, 3.99.
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P CR Am p lifica tion Con d ition s. Five DNA polymerases
were examined for PCR amplification: TaKaRa Ex Taq
(TaKaRa Shuzo, J apan); native Pfu polymerase from Pyrococ-
cus furiosus (Stratagene, CA); TaKaRa Z-Taq (TaKaRa Shuzo);
TaKaRa LA-Taq (TaKaRa Shuzo); Pyrobest DNA polymerase
(TaKaRa Shuzo) from Pyrococcus sp. PCR amplifications were
carried out under the optimum conditions for each polymerase
as suggested by the manufactures: in 1 × Ex Taq buffer
containing 2 mM Mg2+ and a 0.2 mM concentration of each
deoxynucleoside 5′-triphosphate (dNTP) with Ex Taq; in native
Pfu buffer containig 2 mM Mg2+ and a 0.2 mM concentration
of each dNTP with Pfu; in 1 × Z-Taq buffer containing 3 mM
Mg2+ and a 0.2 mM concentration of each dNTP with Z-Taq;
in 1 × LA-Taq buffer containing 2.5 mM Mg2+ and a 0.2 mM
concentration of each dNTP with LA-Taq; in 1 × Pyrobest
buffer containing 2 mM Mg2+ and a 0.2 mM concentration of
each dNTP with Pyrobest. The standard reaction mixture
contained 0.5 µL (0.4 ng/µL) of DNA template pGMI401 (4263
nt), 5 µL (5 pmol) of each primer, lacUV501 (d(5′-GAAGATCT-
CAGCTGGCACGACAGGTTTC-3′), 28 nt), lacUV502 (d(5′-
CGATGCATAGCTGTTTCCTGTGTGAAATTG-3′), 30 nt), vari-
ous concentrations of synthetic 5′-triphosphates (1TP -5TP ),
0.5 µL (5 U/µL) of DNA polymerase in a final volume of 50 µL.
Reaction mixtures were incubated in a thermocycler (model
Gene Amp PCR system 9700; PE Applied Biosystems) for 5
min at 95 °C, followed by 30 cycles at 95 °C for 30 s, 50 °C for
30 s, and 72 °C for 60 s and then by a final extension at 72 °C
for 5 min in the case of Ex Taq, Pfu, and LA-Taq. Mixtures
were also incubated at 95 °C for 5 min, followed by 25 cycles
at 95 °C for 30 s, 50 °C for 30 s, and 72 °C for 30 s and then
by a final extension at 72 °C for 5 min in the cases of Z-Taq
and Pyrobest. The polymerase units were defined by the
supplier, in all cases reflecting conversion of 10 nmol of dNTP
into an acid-insoluble form in 30 min under defined reaction
conditions.
(5) Rose, K. M.; Bell, L. E.; J acob, S. T. Specific Inhibition of
Chromatin-Associated Poly(A) Synthesis in Vitro by Cordycepin
5′-Triphosphate. Nature 1977, 267, 178-180.
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Inhibiton of Mouse Myeloma DNA Polymerase R by 5′-Triphos-
phates of 1-â-D-Arabinofuranosylthymine and 1-â-D-Arabino-
furanosylcytosine. J . Biochem. 1978, 83, 1511-1515.
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Matthes, E. Inhibitory Activity of 3′-Fluoro-2′-deoxythymidine
and Related Nucleoside Analogues against Adenoviruses in
Vitro. Antiviral Res. 1997, 34, 113-119.
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for Alternative DNA Base Pairing through Metal Complexation.
J . Org. Chem. 1999, 64, 5002-5003. (b) Shionoya, M.; Tanaka,
K. Synthetic Incorporation of Metal Complexes into Nucleic
Acids and Peptides Directed toward Functionalized Molecules.
Bull. Chem. Soc. J pn. 2000, 73, 1945-1954.
(9) (a) Tanaka, K.; Tasaka, M.; Cao, H.; Shionoya, M. An Approach
to Metal-Assisted DNA Base Pairing: Novel â-C-Nucleosides
with a 2-Aminophenol or a Catechol as the Nucleobase. Eur. J .
Pharm. Sci. 2001, 13, 77-83. (b) Cao, H.; Tanaka, K.; Shionoya,
M. An Alternative Base Pairing of Catechol-Bearing Nucleosides
by Borate Formation. Chem. Pharm. Bull. 2000, 48, 1745-1748.
(10) (a) Tasaka, M.; Tanaka, K.; Shiro, M.; Shionoya, M. A Palladium-
Mediated DNA Base Pair of a â-C-Nucleoside Possesing a
2-Aminophenol as the Nucleobase. Supramol. Chem. 2001, 13,
671-675. (b) Tanaka, K.; Tasaka, M.; Cao, H.; Shionoya, M.
Toward Nano-Assembly of Metals through Engineered DNAs.
Supramol. Chem. 2002, 14, 255-261.
(11) Tanaka, K.; Yamada, Y.; Shionoya, M. Formation of Silver(I)-
Mediated DNA Duplex and Triplex through an Alternative Base
Pair of Pyridine Nucleobases. J . Am. Chem. Soc. 2002, 124,
8802-8803.
(12) Meggers, E.; Holland, P. L.; Tolman, W. B.; Romesberg, F. E.;
Schultz, P. G. A Novel Copper-Mediated DNA Base Pair. J . Am.
Chem. Soc. 2000, 122, 10714-10715.
(13) Weizman, H.; Tor, Y. 2,2′-Bipyridine Ligandoside: A Novel
Building Block for Modifying DNA with Intra-Duplex Metal
Complexes. J . Am. Chem. Soc. 2001, 123, 3375-3376.
(14) Atwell, S.; Meggers, E.; Spraggon, G.; Schultz, P. G. Structure
of a Copper-Mediated Base Pair in DNA. J . Am. Chem. Soc.
2001, 123, 12364-12367.
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Containing a Non-Hydrogen-Bonding and Non-Shape-Comple-
mentary Base Couple: Interstrand Stacking as the Stability
Determining Factor. Angew. Chem., Int. Ed. 2001, 40, 3012-
3014.
Aliquots of PCR products (8 µL each) were analyzed by
electrophoresis (8% acrylamide gel) and visualized by staining
with SYBR gold. Band intensity measurement was employed
with phosphorimager SI (Vista Fluorescence), and each data
point is shown as an average of two independent experiments.
Ack n ow led gm en t. We are grateful for Grants-in-
Aid for Scientific Research (B), No. 13554024, and
Priority Area, No. 13128202, from Ministry of Educa-
tion, Culture, Sports, Science and Technology, J apan,
to M.S. This work was also partially supported by a
grant from Hayashi Memorial Foundation for Female
Natural Scientists to S.A.
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in DNA. Nature 1999, 399, 704-707.
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bonucleotides to 5′-Triphosphates. J . Am. Chem. Soc. 1965, 87,
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Su p p or tin g In for m a tion Ava ila ble: The X-ray analytical
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available free of charge via the Internet at http://pubs.acs.org.
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Base Analogues. Synthesis, Structure, and Fluorescence in DNA.
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Refer en ces
(1) (a) Piccirilli, J . A.; Krauch, T.; Moroney, S. E.; Benner, S. A.
Enzymatic Incorporation of a New Base Pair into DNA and RNA
Extends the Genetic Alphabet. Nature 1990, 343, 33-37. (b)
Switzer, C. Y.; Moroney, S. E.; Benner, S. A. Enzymatic Recogni-
tion of the Base Pair between Isocytidine and Isoguanosine.
(22) All the isomers assigned as â showed H1′ resonances as a doublet
of doublets with coupling constants of J ) 5 and 10 Hz. The
coupling constants for H2′-H3′ were J ) 2 and 6 Hz, and these

Nonnatural Nucleoside Triphosphates
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 25 5603
values indicate that the ring conformation in solution is almost
the same as that in the solid state.
(23) Catechol was also an inhibitor against DNA synthesis by Ex Taq
with an IC50 of 0.06 mM.
(24) Al-Soud, W. A.; J o¨nsson, L. J .; Rådstro¨m, P. Identification and
Characterization of Immunoglobulin G in Blood as a Major
Inhibitor of Diagnostic PCR. J . Clin. Microbiol. 1995, 33, 596-
601.
(25) To explore the possibility of 1TP -4TP as therapeutic agents,
further study on the triphosphorylaton of hydrophobic 1-4 by
cellular enzymes should be required, since the hydrophilic 5′-
triphosphate derivatives are not spontaneously incorporated into
cells through the cellular membrane.
J M020193W