Chemical properties of 4,5-di(ethoxycarbonyl)-1,3-dioxolan-2-yl (DECDO) as a hydroxyl protecting group of the 2′-hydroxyl function in ribonucleosides

Article abstract of DOI:10.1002/jhet.5570440208

(Chemical Equation Presented) We describe basic chemical properties of 4,5-di(ethoxycarbonyl)-1,3-dioxolan-2-yl (DECDO) in view of its use as a protecting group for the 2′-hydroxyl function of ribonucleosides. The DECDO group is found to be compatible wit

Full text of DOI:10.1002/jhet.5570440208

Mar-Apr 2007
329
Chemical Properties of 4,5-Di(ethoxycarbonyl)-1,3-dioxolan-2-yl
(DECDO) as a Hydroxyl Protecting Group of the 2'-Hydroxyl
Function in Ribonucleosides
Boleslaw Karwowski†,a,c,*, Kohji Seiob,c, Mitsuo Sekine,a,c
aDepartment of Life Science, Tokyo Institute of Technology; b Frontier Collaborative Research Center,Tokyo
Institute of Technology; cCREST, JST (Japan Science and Technology Agency)
Received April 28, 2006
O
O
NH
O
NH
O
N
N
O
O
HO
DMTO
HO
OH
HO
O
O
CONH2
CONH2
O
We describe basic chemical properties of 4,5-di(ethoxycarbonyl)-1,3-dioxolan-2-yl (DECDO) in view of
its use as a protecting group for the 2'-hydroxyl function of ribonucleosides. The DECDO group is found to
be compatible with the DMTr strategy for the currently-used oligonucleotide synthesis. Post-synthetic
treatment with ammonia results in the conversion of this protecting group into the 4,5-dicarbamoyl-1,3-
dioxolan-2-yl (DCBDO) group which is unexpectedly more stable in aqueous acidic solution.
J. Heterocyclic Chem., 44, 329 (2007).
OEt
INTRODUCTION
O
O
HO
OH
a
Although a number of hydroxyl protecting groups have
been explored, [1-8] only a few have survived in the field of
nucleoside and nucleotide chemistry. This is due to the strict
conditions which are required for their chemical trans-
formation. For the synthesis of target materials, the
protecting group should fulfill the following requirements:
1) The reagent should be easy to prepare; 2) no new chiral
centers should be generated; 3) the protecting group should
be stable during the desired reaction, 4) removal of the
protecting group must be highly selective without any
damage to the other functional groups.
S
S
H
COOEt
H
COOEt
S
S
EtOOC
H
EtOOC
H
D-8
D-4
OEt
HO
OH
O
O
a
R
R
EtOOC
H
EtOOC
H
R
R
H
COOEt
H
COOEt
L-8
L-4
Scheme 1 Synthesis of 2-Ethoxy-1,3-dioxolane-4,5-dicarboxylic
acid diethyl ester and 2-Ethoxy-1,3-dioxolane-4,5-dicarboxylic acid
diamide. a) Toluene, CAS, 95 °C, 95%.
The methoxymethylidene group is well-known as the
protecting group of ribonucleoside cis-2',3'-diol
derivatives [9]. Viewed from a different perspective this
protection structure can be considered as an OH
protecting group in methanol by the 1,3-dioxolan-2-yl
group. This idea inspired us to investigate a new method
for the protection of the 2'-hydroxyl group of
ribonucleosides by exploiting the orthoester structure. In
the literature, there are only a few examples of the
orthoester-type protecting groups for RNA synthesis, [10-
14]. In addition they have several drawbacks such as low
yields of the precursors and the necessity to use an
expensive co-reagent. [14].
RESULTS AND DISCUSSION
Recently, orthoester type protecting groups, removable
by a two-step procedure, such as 3-methoxy-1,5-dicarbo-
methoxypentan-3-yl [12] or di(2-acetoxyethoxy)-methyl
[13-14] have been developed and applied to oligoribo-
nucleotide synthesis. The stability of the ester function
can be regulated by changing their electron donating/
withdrawing properties. It is expected that during the
synthetic cycle of oligoribonucleotides, the ester functions
of the protecting groups will be converted into the amide,
which are more labile. This idea has prompted us to study
new cyclic-type orthoester protecting groups. To verify
In this paper, we report the chemical properties of 4,5-
di(ethoxycarbonyl)-1,3-dioxolan-2-yl (DECDO) as
a
possible protecting group for the 2'-hydroxyl function of
ribonucleoside derivatives.

330
B. Karwowski, K. Seio, M.Sekine
Vol 44
5: R= -CH(OMe)2
O
O
O
6: R=
Method A: 2 or 3 /CAS/CH2Cl2
or
O
NH
O
NH
O
COOEt
N
N
O
Method B: 2 or 3 /CAS/TPSO/CH2Cl2
O
H
O
O
O
D-7: R=
Si
COOEt
Si
or
O
Method C: 4 /CAS/toluene
O
O
H
O
OH
O
O
R
Si
Si
D - DECDO
H
O
O
COOEt
H
TSPO
O
CAS
2
3
D-4
L-4
OEt
OEt
OEt
L-7: R=
(CH3O)3CH
O
Si
O
O
O
O
O
O
COOEt
COOEt
H
EtOOC
H
HO3S
H
H
COOEt
L - DECDO
EtOOC
OH
Method A: rt, overnight, 30% (5); Method B: rt, overnight, 86% (5), 90% (6); Method C: reflux, 2 h, 80% (D-7), 80% (L-7)
Scheme 2 Synthesis of 2'-O-dialkoxymethyluridine derivatives.
this hypothesis we have first studied the orthoester
exchange reaction of 3',5'-O-(1,1,3,3-tetraisopropyldisil-
oxane-1,3-diyl)-uridine (1) [15] with simple orthoester
compounds since the introduction of a protecting group
into the 2'-hydroxyl group of ribonucleosides under mild
conditions is of primary importance. Our attention turned
to trimethoxymethane (2) and 2-methoxy-1,3-dioxolane
(3) as well as a set of enantiomers, D- and L-diethyl 2-
ethoxy-1,3-dioxolane-4,5-dicarboxylate (D-4 and L-4)
[16-17] as reagents for the introduction of the orthoester
functions into the 2'-hydroxyl group of 1 (see Scheme 1).
These compounds are commercially available or easily
accessible and do not generate new chiral centres.
The ester exchange reactions of 1 with 2 and 3 were
studied in detail. As a result, the 2'-O-blocked products 5
and 6 were obtained in only ca. 30% yield each by the
ester exchange reaction in the presence of camphor-
sulfonic acid. However, it was found that the addition of
4-(tert-butyldimethylsilyloxy)pentene-2-one (TPSO) [14]
as a scavenger of methanol, formed as a byproduct during
the reaction, increased the yield of their synthesis up to
ca. 90%, as shown in Scheme 2. D-4 and L-4 were
synthesized in high yields by the reaction of D- and L-
tartaric acids, and D-8 and L-8 with triethyl orthoformate
(as shown in Scheme 1) to allow the synthesis of the 2'-O-
orthoester modified product 7.
O
NH
N
O
O
O
a
a
3
Si
5
6
O
O
O
Si
OMe
O
MeO
O
NH
O
NH
N
N
b
O
O
O
DMTO
O
Si
O
O
O
HO
O
O
O
Si
O
O
10
11
N
O
NH
O
O
NH
O
D-7
or
N
a
O
O
O
DMTO
b
Si
L-7
O
O
O
O
HO
O
O
Si
COOEt
COOEt
COOEt
O
O
COOEt
D-12 or L-12
D-13 or L-13
Scheme 3 a) THF, TBAF, RT, 30 min, 95% (10), 95% (D-12), 98% (L-12); b) DMT-Cl, pyridine, rt, 4h, 64% (11), 2 h, 94% (D-13), 2h, 96% (L-13) .

Mar-Apr 2007
Chemical Properties of 4,5-Di(ethoxycarbonyl)-1,3-dioxolan-2-yl (DECDO)
331
New protecting groups, D- and L-4,5-(diethoxycar-
bonyl)-2-ethoxy-1,3-dioxolan-2-yl (D- and L-DECDO),
which can be introduced to nucleosides using D-4 and L-
4, have been designed as follows (Scheme 2).
L-12. Similarly, 5'-dimethoxytritylation of these products
generated the 5'-O-protected compounds D-13 and L-13
without any loss of the DECDO group. These results
show that the DECDO group proved to be stable during
these transformations.
We expected that the stability of the DECDO group
could be changed after treatment with ammonia (Scheme
4, step c). The new created protecting group "4,5-dicar-
bamoyl-1,3-dioxolan-2-yl" (DCBDO) should be more
acid-labile. This lability originates from the superior
electron-donating ability of the carbamoyl group over the
ethoxycarbonyl group. The differences in the electron-
donating ability between the ethoxycarbonyl and
carbamoyl groups were estimated from the Hamett
constants σm in a series of substituted benzoic acids. The
values of the ethoxycarbonyl and carbamoyl groups were
reported to be 0.37 and 0.28, respectively [18]. Therefore,
we expected that the conversion of the DECDO group
into the DCBDO would allow a more facile acid
deprotection of the DECDO group at the final
deprotection step of the RNA synthesis. In addition, the
D- or L-DECDO group gave a single product when
introducted into the 2'-hydroxyl group of 1 because of the
inherent C2 symmetric property.
Stability of orthoester-type protecting groups. Since
the standard protocol for oligoribonucleotide synthesis
requires an acidic promoter for the activation of the
phosphoramidite building blocks, we checked the stability
of 11 and D-13 and L-13 under weak acidic conditions of
1H-tetrazole in CDCl3-DMSO (5:1 v/v). As a result,
compound 11 was found to gradually decompose after
120 min, while the DECDO group of D-13 or L-13
proved to be sufficiently stable.
To test the stability of 5'-O-DMTr-2'-O-[4,5-
di(ethoxycarbonyl)-1,3-dioxolan-2-yl]uridine D-13 and L-
13, we chose various acids at various concentrations. It
was found that our protecting group was stable during the
removal of the DMTr group by 1% DCA in CH2Cl2, as
evidenced by the 1H NMR data.
It proved possible to selectively remove the DMTr
group from the 5'-O position by treatment with 1% of
All attempts to obtain the uridine derivatives D-7 by
reaction of 1 with D-4, under conditions similar to those
described in the synthesis of 5 and 6, failed. The strategy
in which we used TPSO, as an ethanol scavenger, did not
give successful results. It is likely that the carbonium
cation derived from D-7 by an acid-catalyzed reaction
was destabilized because of the presence of two electron-
withdrawing ethoxycarbonyl groups at the 4 and 5
positions. However, it was found that the use of an
enhanced temperature (refluxing in toluene) together with
azeotropic removal of the once-generated ethanol resulted
in a satisfactory yield (80%) of the desired product D-7.
L-7 was obtained in similar yields by the use of L-4.
Synthesis of related compounds. In order to check the
stability of the orthoester-type functional groups, several
uridine derivatives were functionalized as shown in
Scheme 3.
Selective removal of the TIPS group from 5 and 6 was
carried out by treatment with Bu4NF in THF. However, it
was difficult to obtain 9 from 5. Contrary to this result,
the 3',5'-free product 10 was isolated with a yield of 95%
with respect to 6. The isolation of 10 by chromatograpy
required the addition of 1% pyridine to the eluent,
otherwise it decomposed. Moreover, when the reaction of
10 with DMTrCl in pyridine was carried out, the
simultaneous loss of the 1,3-dioxolan-2-yl group from the
product 11 was observed to a degree of ca. 50% after 10
h. This is due to a rather weak acidity of pyridinium
hydrochloride formed during the reaction.
Figure 1 Chart A: RP HPLC trace of the mixture 15 and 16 synthesized
using the monomers 14 (solvent system I), Chart B: RP HPLC profile of
the mixture obtained by treatment of 16 with 25% CD3COOD in D2O
(Solvent system II).
DCA in CH2Cl2, therefore we examined the properties of
the DECDO group at the dimer level to see if this group
could be used as the 2'-protecting group. Therefore, the
phosphoramidite derivative 14 with the isomeric ratio of
65:35, was synthesized at a yield of 60% by the reaction
of L-13 with the phosphitylating reagent
(Cl(iPr2N)P(OCH2CH2CN)) (Scheme 4).
We also
checked the use of compound 14 for the creation of the
internucleotidic phosphate bond by the liquid-phase
synthesis of the dinucleotide UpU.
During the formation of the internucleotide bond, we
did not find any unexpected signals in the 31P NMR
spectra. The crude product was analyzed by RP HPLC
(ammonium acetate buffer). We found that the product 15
had low water solubility but was easily dissolved in
CH3CN-water (10:90 v/v). The situation dramatically
changed when compound 15 was converted by ammonia
(28% in water) solution in methanol (1:1 v/v) to
compound 16, due to β-cyanoethyl removal and due to
Alternatively, desilylation of D-7 and L-7 gave high
yields of the 3',5'-O-free uridine derivatives i.e. D-12 and

332
B. Karwowski, K. Seio, M.Sekine
Vol 44
conversion of diester function to diamide one. This
compound had good solubility in water so that the
retention time (Rt = 35min) of 16 in RP-HPLC became
shorter than that of 15 (Rt = 37min) (Fig.1).
clarify these unexpected results, we studied the stability of
simpler substrates L-4 and L-17 under acidic conditions.
Compound L-17 was prepared according to Scheme 5.
Table 1
This result is promising for future studies; the change of
this hydrophilic property enables us to purify them easily.
Surprisingly, when compound 16 was treated with 25%
CD3COOD in D2O at room temperature overnight, the
DCBDO group was found to be resistant to this acidic
medium. The EPS mass spectrometric analysis of the
reaction mixture suggested that the DMTr group was
completely removed and UpU, UpUisp, U(dcbco)pU, and
U(dcbdo)pUisp were observed in the ratio of
16.5:5.0:3.0:1.8. It was found that the DCBDO group was
Partial charges calculated by the DFT method,
B88-PW91/6-31G** basis set.
Partial charges
Atom
Partial
Atom
charges
Ester form
L-4
Amide form
L-17
-0.499
-0.497
-0.365
-0.367
-0.384
O1
O2
O3
O4
O5
-0.437
-0.481
-0.399
-0.406
-0.389
O1
O2
O3
O4
O5
Ura
O
DMTO
O
Ura
O
O
O
DMTO
O
a
NC
COEt
COEt
L-13
O
P O
O
O
N
O
O
NC
O
COEt
COEt
P
O
14
15
O
O
Ura
O
Ura
O
DMTO
O
O
O
O
c
d
CONH2
CONH2
UpU
+
P
O
O
O
UpUisp
+
16
O
O
U(dcbco)pU
+
Ura
O
DMTrU(dabdo)pUisp
U(dcbdo)pUisp
Scheme 4 Synthesis of UpU by the 1H-tetrazole-mediated reaction of 2',3'-O-isopropylideneuridine with 1,3-dioxolane-4,5-dicarboxylic acid
diethyl ester. a) CH2Cl2, rt, (iPr)2NEt, chloro(2-cyanoethoxy)diisopropylaminophosphine, overnight, 60%; b) i) CHCl3, 2',3'-O-
isopropylideneuridine, 1H-tetrazole; ii) I2-H2O-Py; c) NH3aq; d) 25% of CD3COOD in D2O.
removed somewhat faster than the isopropylidene group.
The latter cannot be used for the current RNA synthesis
because strong acidic conditions are required for its
removal. These results differ from our initial expectations
that the DCBDO group, derived from the DECDO group
by ammonia treatment, would be more acid-labile. To
The stablity of L-4 and L-17 was tested in 0.1%
camphorsulfonic acid (CSA) in methanol. As a result, the
t1/2 and tcom of the decomposition of L-4 were 3 min and
60 min, respectively. L-17 was found to be very stable
for 24 h under the same conditions.
When the
concentration of CSA was increased to 0.2%, the
decomposition of L-17 was observed with t1/2 and tcom
being 6 min and 2 h, respectively.
O5
EtO
O4
O5
EtO
O4
H
H
Quantum Mechanics Calculation. To investigate the
partial charges on the oxygen and carbon atoms of L-4
and L-17 the density function theory (DFT) with B88-
PW91[19-20] was used (the 6-31G** basis set was
applied). The data presented in Table 1 show that the
partial charges on the three oxygens O3, O4, and O5 of the
amide derivative are less negative than the ester group.
These results suggest that at the initial step of
O3
O3
O
O
O
1 O
O
O
O
1 O
a
C
H
C
H
EtO
H2N
H
C
OEt
H
C
NH2
O
O
O2
O2
L-4
L-17
Scheme 5 Synthesis of L-2-ethoxy-1,3-dioxolane-4,5-carboxamide
L-17. a) CH3OH, NH3aq, rt, overnight, 100%.

Mar-Apr 2007
Chemical Properties of 4,5-Di(ethoxycarbonyl)-1,3-dioxolan-2-yl (DECDO)
333
ESI mass spectra were measured on MarinerTM. MALDI-TOF
mass spectra were measured on Voyager RP. UV spectra were
measured by a U-2000 spectrophotometer. TLC was performed
with Merck silica gel 60 (F254) plates. Molecular calculation was
made by CAChe WorkSystem Pro Version 6.1 2000-2003
Fujitsu Ltd. B88-PW91 (Becke '88; Perdew & Wang '91) with
the 6-31G** basic set.
deproctection the ester derivative is more susceptible to
protonation. It is also important to consider the
neighboring electron-rich atoms as possible protonation
sites. Since the oxygen of the carbamoyl group is more
electron-rich than the carbonyl oxygen of the ester group,
the protonation of the O3, O4, and O5 atoms would be
suppressed by the presence of more basic carbamoyl
oxygens. As a result, it can be expected that when the
oxygen of the carbamoyl group is protonated, this group
becomes more electron-withdrawing so that the electron
density of the two oxygens of the ring and O5 decreases.
Synthesis of 2'-O-[di(methoxy)methyl]-3',5'-O-(1,1,3,3-
tetraisopropyldisiloxane-1,3-diyl)uridine (5). In
a 30 ml
round-bottom flask, 3',5'-O-(1,1,3,3-tetraisopropyldisiloxane-
1,3-diyl)uridine (1) (1.0 g, 2.05 mmol) was dissolved in 10 ml of
CH2Cl2 to give a clear solution. Trimethyl orthoformate (4.0 g,
0.038 mmol) and camphorsulfonic acid (63 mg, 0.27 mmol)
were then added to the solution. The reaction mixture was stirred
for 20 min and then 4-(tert-butyldimethylsilyloxy)-3-pentene-2-
one (890 mg, 4.1 mmol) was added. The solution was stirred
overnight at room temperature. The reaction was monitored by
TLC (CHCl3-CH3OH, 9:1 v/v). The solvent was evaporated on
completion of the reaction. The crude product was purified by
flash chromatography on a column of silica gel with CHCl3-
MeOH (100:0-99:1 v/v) to give compound 5 as a white foam
(0.98 g, 86%); Rf (CHCl3-CH3OH, 9:1 v/v) 0.38; δΗ (500 MHz;
CDCl3; Me4Si) 0.94-1.10 (m, 24H), 3.39 (s, 3H), 3.43 (s, 3H),
3.97-3.9 (d, 1H, J 13.4 Hz), 4.2-4.28 (m, 4H), 5.56 (s, 1H), 5.68-
5.7 (d, 1H, J 8. 3 Hz), 5.82 (s, 1H), 7.89-7.91 (d, 2H, J 8.3 Hz),
9.92 (s, 1H); δC (126 MHz; DMSO-d6) 12.65, 12.96, 13.01,
13.37, 17.43, 17.48, 17.53, 17.75, 17.82, 17.82, 51.05, 51. 68,
60.31, 68.74, 76.233, 81.68, 89.95, 101.72, 113.53, 124.57,
140.35, 150.29, 150.73, 163.96; ESI-MS (ES+) m/z 583.331
(M+Na. C24H44N2O9Si2Na requires 583.775).
CONCLUSION
In conclusion, the 1,3-dioxolan-2-yl and methoxymethyl
groups are weak as secondary alcohol protecting groups,
but can be used when mild acidic conditions are necessary.
Surprisingly, it was found that the DCBDO group was
more stable than the DECDO group under acidic
conditions. This result was unexpected, however the
skeleton of the DCBDO group would be useful for the
introduction of a wide variety of functional groups on the
amido nitrogen via an N-alkyl spacer. This is promising
because the DCBDO group becomes more acid-resistant
and thereby such functional groups can remain intact
during the standard protocol of RNA or DNA synthesis.
Being a typical protecting group for the hydroxyl function,
the DECDO and DCBDO groups can also be used as new
protecting groups of the orthoester-type in nucleic acid
chemistry as well as in carbohydrate chemistry. The
stability of the DECDO group changes during the synthesis
and increases when the ester groups are transformed into
the amide groups. The stability of the resulting DCBDO
group rises but is still able to be removed under moderate
pH conditions in alcohol/water solutions. Additionally, the
purification of the crude post-synthetic oligonucleotides
should be fast and easy. Future studies will be continue to
adopt this strategy for the solid-phase synthesis of RNA or
DNA containing specific functional groups.
Stability of 2'-O-[di(methoxy)methyl]-3',5'-O-(1,1,3,3-tetra-
isopropyldisiloxane-1,3-diyl)uridine (5). Compound 5 (10 mg,
0.018 mmol) and 1H-tetrazole (25 mg, 0.36 mmol) were
dissolved in CDCl3-DMSO-d6 (5:1 v/v, 0.6 ml) in an NMR tube.
The progress of the reaction was monitored by 1H NMR. During
the reaction, the disappearance of the signal of the anomeric
proton at 5.40 ppm (DMSO) from the 1,3-dioxolane system was
observed.
Synthesis of 2'-O-(1,3-dioxolan-2-yl)-3',5'-O-(1,1,3,3-tetra-
isopropyldisiloxane-1,3-diyl)uridine (6). In a 30 ml round-
bottom flask, 3',5'-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-
uridine (1) (4.0 g, 8.0 mmol) was dissolved in 20 ml of CH2Cl2
to give a clear solution. 2-Methoxy-1,3-dioxolane (5.2 g, 50
mmol) and camphorsulfonic acid (0.17 g, 0.73 mmol) were
added to the solution. The reaction mixture was stirred for 20
min and then 4-(tert-butyldimethylsilyloxy)-3-pentene-2-one
(2.2 g, 9.3 mmol) was added. The solution was stirred at room
temperature overnight. The reaction progress was monitored by
TLC (CHCl3-CH3OH). The solvent was evaporated on
completion of the reaction. The crude product was purified by
flash chromatography on a column of silica gel in the CHCl3 and
0-1% CH3OH in CHCl3 to give 6 as a white foam (1.03 g, 90%);
Rf (CHCl3-CH3OH, 9:1, v/v) 0.42; δΗ (500 MHz; CDCl3; Me4Si)
1.0-1.1 (m, 28H), 3.95-4.01 (m, 3H), 4.17-4.26 (m, 6H), 5.66-
5.69 (d, 1H, J 8.3), 5.75 (s, 1H), 7.87-6.89 (d, 1H, J 8.3), 8.79 (s,
1H); δC (126 MHz; CDCl3) 12.76, 13.09, 13,23, 13.71, 17.08,
17.19, 17.22, 17.27, 17.47, 17.55, 17.63, 17.72, 31.84, 59.49,
63.84, 63.87, 63.87, 68.18, 76.66, 82.07, 89.69, 101.79, 113.9,
139.48, 150.09, 163.39; ESI-MS (ES+) m/z 581.110 (M+Na.
C12H16N2O8Na requires 581.233).
EXPERIMENTAL
General methods. 1H, 13C and 31P NMR spectra were
obtained on a Varian Unit 500 apparatus at 500, 126, 201 MHz,
respectively. The chemical shifts were measured from
tetramethylsilane (0 ppm) or DMSO-d6 (2.49 ppm) for 1H NMR,
CDCl3 (77.0 ppm) or DMSO-d6 (39.7 ppm) for 13C NMR and
85% phosphoric acid (0 ppm) for 31P NMR. Column
chromatography was performed by use of Wako silica gel C-
200. Reverse-phase HPLC was performed using µBondapak C-
18 columns (Waters Co., Ltd., 7.8 x 300 mm) at a flow rate of
1.0 mL/min at 50 °C. Elution was performed with the following
solvent systems I and II for 50 min and 20 min, respectively at a
flow rate of 1.0 mL/min. Solvent system I: 0.1 M Ammonium
acetate (pH 7.0)-acetonitrile (100-0 to 70:70 v/v); solvent system
II: 0.1 M Ammonium acetate (pH 7.0)-acetonitrile (100:0 to
80:20 v/v).
Synthesis of 2'-O-(1,3-dioxolan-2-yl)uridine (10). A
solution of 2'-O-[1,3-dioxalone]-3',5'-O-(1,1,3,3-tetraisopropyl-

334
B. Karwowski, K. Seio, M.Sekine
Vol 44
disiloxane-1,3-diyl)uridine (6) (2.0 g, 3.8 mmol) in dry THF 20
ml was treated with tertbutylamonium fluoride (0.70 g, 2.7
mmol). The mixture was stirred at room temperature for 30 min
and the solvent removed under reduced pressure. The residue
was purified by column chromatography on a column of silica
gel with CHCl3- CH3OH (20:1 to 20:2.5 v/v) containing 1%
pirydine to give 10 as a white foam compound (1.0 g, 95%); Rf
(CHCl3-CH3OH, 9:1 v/v) 0.18; δΗ (500 MHz; DMSO; Me4Si)
3.54-3.61 (m, 2H), 3.83-3.9 (m, 4H), 3.97-3.99 (m, 1H), 4.05-
4.07 (m, 1H), 4.16-4.18 (t, 1H), 5.14-5.16 (t, 1H), 5.19-5.2 (d,
1H, J 5.37 Hz), 5.43-5.66 (d, 1H, J 8.06 Hz), 5.87-5.88 (d, 1H, J
6.10 Hz), 5.93 (s. 1H), 7.87-7.89 (d, 1H, J 8.06 Hz), 11,31 (s,
1H); δC (126 MHz; DMSO-d6) 61.54, 63.91, 64.35, 69.94,
75.86, 86.1, 86.50, 86.56, 102.58, 114.52, 114.6, 141.25, 151.37,
163.78; ESI-MS (ES+) m/z 338.128 (M+Na. C12H16N2O8Na
requires 339.080).
Synthesis of 5'-O-(4,4'-O-dimethoxytrityl)-2'-O-(1,3-
dioxolan-2-yl)uridine (11). 4,4'-dimethoxytrityl chloride (0.32
g, 0.92 mmol) was added to a solution of compound 10 (0.29 g,
0.92 mmol) in pyridine (5 ml). The reaction solution was stirred
at room temperature for 4 hours. The reaction mixture was
evaporated under reduced pressure to a 1/3 volume and the
residue was partitioned between CH2Cl2 and H2O (1:1 v/v, 15
ml). The organic layer was dried over MgSO4, filtered,
evaporated under reduced pressure and the crude product was
chromatographed on a column of silica gel with CHCl3 to give
the product 11 as a white foam (0.350g, 64%); Rf (CHCl3-
CH3OH, 9:1 v/v) 0.53; δΗ (500 MHz; CDCl3; Me4Si) 3.19-3.38
(d.m, 2H), 3.74 (s, 6H), 3.88-4.03 (m. 6H), 4.2-4.21 (m, 1H),
4.27-4.29 (m, 1H), 5.35-5.36 (m, 3H), 5.84-5.85 (d, 1H, J 4.64),
6.03 (s, 1H), 6.9-6.92 (d, 4H, J 8.79), 7.25-7.39 (t, m, 9H); 7.69-
7.71 (d, 1H, J 8.06), 11.43 (s,1H); δC (126 MHz; CDCl3) 55.76,
63.73, 63.89, 64.23, 69.613, 75.64, 79.89, 83.75, 86.66, 87.85,
102.307, 113.98, 114.56, 124.62, 127.53,128.41, 128.64, 130.49,
135.80, 136.06, 136.84, 141.24,145.36, 150.33, 151.15, 158.86,
163.73; ESI-MS (ES+) m/z 641.366 (M+Na. C33H34N2O10Na
requires 641.211).
6.09 (s, 1H). Compound L-4: δΗ (500 MHz; CDCl3; Me4Si) 1.2-
1.23 (t, 3H), 1.3-1.35 (m, 6H), 3.67-3.7 (m, 2H), 4.28-4.3 (m,
4H), 4.71-4.72 (d, 1H, J 4.40 Hz), 5.04-5.05 (d, 1H, J 4.40 Hz),
6.08 (s, 1H).
Synthesis of 2-ethoxy-1,3-dioxolane-4,5-dicarboxylic acid
diamide (17). L-(R,R)-2-Ethoxy-1,3-dioxolane-4,5-dicarboxylic
acid diethyl ester L-4 (5.0 g, 19 mmol) was dissolved in CH3OH
(10 ml) and a 50% solution of ammonia (28% in water) in
CH3OH (20 ml) was added with stirring at room temperature
overnight. The solvent and volatile substances were evaporated
under reduced pressure. The product as a white crystal was
obtained at a yield of 100%. Rf (CHCl3-CH3OH, 4:1 v/v) 0.30;
δΗ (500 MHz; DMSO; Me4Si) 1.10-1.13 (t, 3H), 3.59-3.65 (m,
2H), 4.43-4.44 (d, 1H, J 4.88 Hz), 4.63(d, 1H, J 4.88 Hz), 6.06
(s, 1H), 7.321 (s, 1H), 7.47-7.49 (d, 1H), 7.68 (s, 1H); δC (126
MHz; DMSO-d6) 15.53, 60.43, 77.01, 77.87, 116.95, 171.49,
171.63; ESI-MS (ES+) m/z 225.879 (M+Na. C7H12N2O5Na
requires 225.049).
Synthesis of 2'-O-[(4,5-di(ethoxycarbonyl)-1,3-dioxolan-
onic acid (7). The mixture was stirred in a single-neck round-
bottom flask equipped with a Dean-Stark receiver and a
condenser. The solution was refluxed (105-115 °C) until all the
ethanol had been azeotropically removed. The reaction was
monitored by TLC (CHCl3-CH3OH, 9:1 v/v). The mixture was
neutralized by addition of pyridine (1 ml) after cooling and
evaporated under reduced pressure. The residue was partitioned
between CHCl3 (50 ml) and water (50 ml). The CHCl3 solution
was collected and the aqueous layer was further extracted with
CHCl3 (50 ml). The organic extracts were combined, dried over
MgSO4, filtered and evaporated under reduced pressure. The
residue was chromatographed on a column of silica gel in CHCl3
to give the product D-7 or L-7.
Compound D-7 (561 mg, 80%); Rf (CHCl3-CH3OH, 9:1 v/v)
0.71; δΗ (500 MHz; CDCl3; Me4Si) 1.28-1.34 (m, 6H), 3.94-4.06
(m, 2H), 4.21-4.29 (m, 4H), 4.45-4.46 (d, 1H, J 4.64Hz), 4.74-
4.76 (d, 1H, J 5,37 Hz), 5.09-5.1 (d, 1H, J 5.13 Hz), 5.67-5.68
(d, 1H, J 7.79 Hz), 5.81 (s, 1H), 6.50 (s, 1H), 7.8-7.81 (d, 1H, J
8.06 Hz), 9.45 (s, 1H); ESI-MS (ES+) m/z 725.397 (M+Na.
C30H50N2O13Si2Na requires C30H50N2O13Si2Na requires 725.275).
Compound L-7 (562 mg, 80%); Rf (CHCl3-CH3OH, 9:1 v/v)
0.71; δΗ (500 MHz; CDCl3; Me4Si) 0.93-1.1 (m, 28H), 1.29-1.35
(m, 6H), 3.95-3.98 (m, 2H), 4.15-4.33 (m, 4H), 4.39-4.41 (d,
1H, J 3.35 Hz), 4.78-4.79 (d, 1H, J 5.13 Hz), 5.02-5.21 (d, 1H, J
5.13 Hz), 5.64 (s, 1H), 5.65-5.67 (d, 1H, J 8.06 Hz), 6.52 (s,
1H), 7.8-7.81 (d, 1H, J 8.30 Hz), 8.90 (s, 1H). ESI-MS (ES+)
m/z 725.400 (M+Na. C30H50N2O13Si2Na requires 725.275).
Synthesis of 2'-O-[4,5-di(ethoxycarbonyl)-1,3-dioxolan-2-
yl]uridine (12). Tetrabutylamonium fluoride (0.5 mmol) was
added to a solution of compound D-7 or L-7, (1.0 mmol) in dry
THF (10 ml). The mixture was stirred at room temperature for
60 min, and then the solvent was removed under reduced
pressure. The residue was purified by column chromatography
on a column of silica gel with CHCl3-CH3OH (20:1 to 20:2.5
v/v) containing 1% pyridine.
Stability of 2'-O-(1,3-dioxolan-2-yl)uridine (10). Compound
10 (16 mg, 0.05 mmol) and 1H-tetrazole (148 mg, 2.1 mmol)
was dissolved in CDCl3-DMSO-d6 (5:1 v/v, 1.168 g) in an NMR
1
tube. The progress of the reaction was monitored by H NMR.
The disappearance of the signal of the anomeric proton 5.93
ppm was observed during the reaction.
Synthesis of 2-ethoxy-1,3-dioxolane-4,5-dicarboxylic acid
diethyl ester (4). To a solution of diethyl D- or L-tartarate (D-8
or L-8) (9.8 g, 48 mmol) in toluene (50 ml) were added triethyl
orthoformate (19.4 g, 0.131 mol) and camphorsulfonic acid (20
mg, 0.086 mmol). Triethyl orthoformate (19.4 g, 0.131 mol)
and camphorsulfonic acid (20 mg, 0.086 mmol) was added to a
solution of diethyl D- or L-tartarate (D-8 or L-8) (9.8 g, 48
mmol) in toluene (50 ml). The mixture was stirred vigorously in
a single-neck round-bottom flask equipped with a Dean-Stark
receiver and a condenser. The solution was refluxed (95°C) until
all ethanol had been azeotropically removed. The reaction
progress was monitored by TLC (CHCl3-CH3OH, 9:1 v/v). The
mixture was neutralized by addition of pyridine (1 ml) after
cooling and evaporated under reduced pressure. Purification was
carried out by flash column chromatography on a column of
silica gel with CHCl3 to give the product 4 as an oil (12.2 g,
95%); Compound D-4: δΗ (500 MHz; CDCl3; Me4Si) 1.19-1.29
(t, 3H), 1.3-1.35 (m, 6H), 3.66-3.70 (m, 2H), 4.27-4.32 (m, 4H),
4.726-4.734 (d, 1H, J 4.15Hz), 5.05-5.06 (d, 1H, J 4.15 Hz),
Compound D-12 (437 mg, 95%); Rf (CHCl3-CH3OH, 9:1
v/v) 0.21; δΗ (500 MHz; CDCl3; Me4Si) 1.30-1.35 (m, 6H), 3.0-
3.04 (b, 2H), 3.82-3.97 (m, 2H), 4.06-4.08 (m, 1H), 4.26-4.31
(m, 4H), 4.45-4.47 (m, 1H), 4.73-4.74 (d, 1H, J 3.91 Hz), 4.75-
4.78 (m, 1H), 4.97-4.98 (d, 1H, J 3.91 Hz), 5.7-5.72 (d, 1H, J
8.06 Hz), 5.85-5.86 (d, 1H, J 4.64 Hz), 6.32 (s, 1H), 7.69-7.71
(d, 1H, J 8.06 Hz), 8.61-8.63 (m, 1H); δC (12662-14.34, 17.02,
17.17-17.7, 59.6, 62.29, 62.36, 67.57, 75.74, 76.57, 76.97,

Mar-Apr 2007
Chemical Properties of 4,5-Di(ethoxycarbonyl)-1,3-dioxolan-2-yl (DECDO)
335
89.62, 101.79, 116.49, 140.13, 150.07, 164.15, 168.68, 168.93;
ESI-MS (ES+) m/z 483.229 (M+Na. C18H24N2O12Na requires
483.122).
4H), 7.24-7.75 (m, 8H), 7.28-7.42 (m, 2H), 7.77-7.81 (dd, 1H J
8.30); δC (126 MHz; CDCl3) 13.11, 13.35, 14.30, 14.34, 17.35,
17.37, 20.53, 20.58, 23.09, 23.18, 24.7, 24.75, 24.81, 24.87,
43.38, 43.61, 45.50, 45.55, 55.44, 59.16, 59.29, 62.42, 62.48,
75.46, 75.9, 76.67, 76.97, 83.15, 83.19, 87.32, 87.56, 102.58,
113.48, 115.94, 118.28, 123.98, 127.36, 128.17, 128.17-128.54,
130.43, 130.48, 135.2, 135.41, 135.45, 136.29, 140.38, 144.36,
149.9, 150.61, 158.92, 163.69, 168.46, 168.59, δP (201 MHz;
CDCl3; 85% H3PO4) 152.157, 151.205; ESI-MS (ES+) m/z
985.589 (M+Na. C48H59N4O15PNa requires 985.361).
Compound L-12 (451 mg, 98%); Rf (CHCl3-CH3OH, 9:1 v/v)
0.21; δΗ (500 MHz; CDCl3; Me4Si) 1.29-1.34 (m, 6H), 3.25 (b,
signals 2H), 3.7-3.75 (m, 2H), 4.02-4.02 (m, 1H), 4.24-4.29 (m,
4H), 4.3-4.45 (m, 1H), 4.799-4.808 (d, 1H, J 4.15 Hz), 5.041-
5.049 (d, 1H, J 4.15 Hz), 5.60-5.62 (d, 1H, J 8.06 Hz), 6.01-6.02
(d, 1H, J 5.62 Hz), 6.27 (s, 1H), 7.98-7.99 (d, 1H, J 8.06 Hz); δC
(16 168.16; ESI-MS (ES+) m/z 483.235 (M+Na. C18H24N2O12Na
requires 483.122).
Synthesis of dinucleotide UpU in solution. (232 mg, 0.24
mmol), 2',3'-O-isopropylideneuridine (76 mg, 0.28 mmol) and
1H-tetrazole (40 mg, 0.57 mmol) were dried together in vacuum
at 40 °C for 6 h. This mixture was dissolved in dry CH2Cl2 (5
ml). The reaction was monitored by 31P NMR (CDCl3). At the
beginning of the reaction, the 31P signals (500 MHz; CDCl3;
85% H3PO4) of 14 were observed at 152.16 and 151.21 ppm in
the ratio of 65:35. After 10 min these peaks disappeared and two
other signals at 140.29 and 140.04 ppm derived from the
tervalent phosphorus (III) intermediate appeared in the ratio
49:51. These PIII derivatives were oxydized by a 1% solution of
I2 in H2O-pyridine (1:9 v/v, 2 ml). After 5 min, the mixture was
quenched by the addition of sat. Na2S2O3 (3 ml). The PV species
were formed as shown by 31P NMR which showed the signals
derived from 15 were observed at -1.56 and -1.92 ppm at the
ratio of 50:50. The diasteromeric product 15 was isolated from
the reaction mixture by extraction from the aqueous suspension
by CH2Cl2 (25 ml). The organic layer was dried over MgSO4,
filtered and evaporated under reduced pressure. The residue was
chromatographed on a column of silica gel. Compound 15 thus
obtained was dissolved in methanol and 28% ammonia (1:1 v/v,
10 ml) for 2 h. Then the mixture was evaporated under reduced
pressure. The 31P NMR spectrum of the residue involving 16
showed a signal at 0.067 ppm. After the solvent was evaporated
under reduced pressure, the residue was treated with 25%
CD3COOD in D2O (2 ml). After the solution was evaporated
under reduced pressure, the residue containing a mixture of
UpU, UpUisp, U(dcbco)pU, and U(dcbdo)pUisp was analyzed
by 31P NMR (D2O) that showed only a signal at -0.299 ppm and
also analyzed by ESI mass spectrography.
Compound (15) δΗ (500 MHz; CDCl3; Me4Si) 1.24-1.38 (m.
9H), 1.53-1.68 (s, 3H), 3.43.3.49 (m, 2H), 3.79 (s, 6H), 4.14-
4.43 (m, 11H), 4.7-4.72 (t, 1H, J 4.64 Hz), 4.83-4.96 (m, 2H),
4.91-4.94 (d, 1H, J 4.88 Hz), 5.00-5.13 (m, 1H), 5.29-5.32 (d.d,
1H), 5.51-5.64 (dm, 1H), 5.60-5.62 (d, 1H, J 8.06), 5.69-5.70
(d, 1H, J 8.06), 6.08-6.12 (dd, 1H, J 6.10, 6.84), 6.26-6.32 (ds,
1H), 6.84-6.86 (d, 4H, J 8.79 Hz), 7.8-7.10 (d, 1H, 8.30 Hz),
7.20-7.37 (m, 9H), 7.54-7.62 (dd, 1H, J 8.30, 8.06), 9.03 (b,
1H), 9.27-9.37 (db. 1H); δC (126 MHz; CDCl3) 14.27, 14.29,
19.65, 19.71, 25.42, 27.27, 55.48, 62.6, 62.57, 62.74, 62.88,
63.13, 63.18, 75.81, 75, 86, 76.69, 77.58, 80.97, 84.55, 85.85,
85.99, 86.44, 87.64, 94.8, 95.54, 102.88, 102.94, 103.00, 103.17,
113.56, 114.7, 114.73, 116.19, 116.35, 116.81, 117.21, 127.46,
128.27, 128.51, 130.42, 130.45, 130.49, 135.07, 135.193,
135.25, 140.293, 142.74, 144.24, 150.44, 150.48, 150.93,
151.17, 158.97, 163.53, 163.66, 163.8, 163.95, 168.2, 168.39,
168.491, 168.55; δP (201 MHz; CDCl3; 85% H3PO4) -1.557, -
1.924; ESI-MS (ES+) m/z 1182.800 (M+Na. C54H60N5O22PNa
requires 1184.337).
Synthesis of 5'-O-(4,4'-dimethoxytrityl)-2'-O-[4,5-di-
(ethoxycarbonyl)-1,3-dioxolan-2-yl]uridine (13). 4,4'-di-
methoxytrityl chloride (508 mg, 1.5 mmol) was added to a
solution of compound D-12 or L-12 (1.0 mmol) in pyridine (35
ml). The mixture was stirred at room temperature for 2 h. The
reaction was monitored by TLC (CHCl3-CH3OH, 9:1 v/v). The
mixture was evaporated under reduced pressure to a 1/3 volume
and the residue was taken up by CH2Cl2-H2O (1:1 v/v, 100 ml).
The organic layer was dried over MgSO4, filtered and
evaporated under reduced pressure. The residue was chroma-
tographed on a column of silica gel with CHCl3 to give the
product D-13 or L-13.
Compound D-13 (717 mg, 94%); Rf (CHCl3-CH3OH, 9:1 v/v)
0.63; δΗ (500 MHz; CDCl3; Me4Si) 1.29-1.33 (m, 6H), 3.0 (b.d,
1H), 3.52-3.53 (m, 2H), 3.793-3.796 (d, 6H, J 1.47 Hz), 4.01-
4.02 (m, 1H), 4.24-4.3 (m, 4H), 4.53-4.55 (m, 2H), 4.77-4.78 (d,
1H, J 4.15 Hz), 5.05-5.06 (d, 1H, J 4.40 Hz), 5.31-5.32 (d, 1H, J
9.77 Hz), 6.05 (d, 1H, J 1.47), 6.44 (s, 1H), 6.83-6.86 (d, 4H,
9.03 Hz), 7.23-7.31 (m, 8H), 3.38-3.74 (m, 2H), 7.92-7.93 (d,
1H, J 8.30), 9.08 (b, 1H); δC (126, 61.50, 62.43, 62.75, 68.53,
75.86, 76.52, 83.33, 86.99, 88.05, 102.05, 113.27, 113.29,
116.43, 123.75, 127.11, 127.99, 128.15, 130.11, 130.19, 135.13,
135.35, 136.01, 140.17, 144.39, 149.79, 150.05, 158.66, 158.66,
158.69, 163.26, 168.26, 169.38; ESI-MS (ES+) m/z 785.46463
(M+Na. C39H42N2O14Na requires 785.253).
Compound L-13 (732 mg, 96%); Rf (CHCl3-CH3OH, 9:1
v/v) 0.63; δΗ (500 MHz; CDCl3; Me4Si) 1.31-1.36 (m, 6H),
3.51-3.53 (m, 2H), 3.785-3.794 (d, 6 Hz, 4.64 Hz), 4.14-4.15 (t,
1H); 4.27-4.33 (m, 4H); 4.44-4.60 (m, 1H), 4.46-4.51 (m, 1H),
4.83-4.84 (d, 1H, J 3.91 Hz), 5.02-5.03 (d, 1H, 3.91 Hz), 5.29-
5.31 (d, 1H, 8.30 Hz), 6.02-6.03 (d, 1H, J 3.42 Hz), 6.38 (s, 1H),
6.8-6.85 (m, 4H,), 7.19-7.45 (m, 10H), 7.88-7.89 (d, 1H, J 8.06
Hz), 8.85 (s, 1H); C(126 170.33; ESI-MS (ES+) m/z 785.459
(M+Na. C39H42N2O14Na requires 785.253).
Synthesis of 5'-O-(4,4'-dimethoxytrityl)-2'-O-[4,5-di-
(ethoxycarbonyl)-1,3-dioxolan-2-yl]uridine-3'-O-(2-cyano-
ethyl-N,N-diisopropyl)phosphoramidite (14). Ethyldiisoprop-
ylamine (125 mg, 1.1 mmol) was added to a solution of L-13
(760 mg, 1.0 mmol) in CH2Cl2 (10 ml). The mixture was stirred
at room temperature for 5 min and then chloro(2-cyanoethoxy)-
diisopropylaminophosphine (0.28 g, 1.2 mmol) was added. After
being stirred at room temperature overnight, the mixture was
evaporated under reduced pressure. The residue was chroma-
tographed on a column of silica gel with CHCl3 containing 2%
triethylamine to give compound 14 as a white foam (0.57 g,
60%); Rf (CHCl3-CH3OH, 9:1 v/v) 0.53; δΗ (500 MHz; CDCl3;
Me4Si) 1.02-1.35 (d.m, 20H), 2.63-2.72 (m, 2H), 3.52-3.87 (m,
11H), 4.19-4.31 (m, 4H), 4.55-5.60 (m, 1H), 5.56-5.66 (m, 1H),
4.69-4.74 (dd, 1H, J 4.63 Hz, J 5.13 Hz), 5.00-5.04 (dd, 1H, J
4.64 Hz, J 5.13 Hz), 5.27-5.34 (dd, 1H, J 8.06, J 8.06 Hz), 6.02-
6.03 (d.d, 1H, J 2.44 Hz), 6.27-6.34 (d.s, 1H), 6.83-6.86 (m,
Compound (16) δΗ (500 MHz; CD3OD; Me4Si) 1.28 (s, 3H),
1.52 (s, 3H), 3.51-3.55 (m, 2H), 3.78 (s, 6H), 3.86-4.06 (dm,
2H), 4.22-4.28 (m, 1H), 4.29-4.30 (m, 1H), 4.64-4.67 (m, 3H),

336
B. Karwowski, K. Seio, M.Sekine
Vol 44
†
On leave from the Medical University of Lodz, Poland.
4.79-4.98 (m, 6H), 5.14-5.16 (d, 1H, J 8.06 Hz), 5.66-5.67 (d,
1H, J 8.06 Hz), 5.84-5.85 (d, 1H, J 2.93Hz), 5.98-5.99 (d, 1H, J
3.42 Hz), 6.52 (s, 1H), 6.87-6.89 (d, 4H, J 8.03), 7.23-7.33 (m,
8H), 7.41-7.42 (d, 2H), 7.66-7.71 (d, 1H, J 8.06 Hz); δC (126
MHz; CD3OD) 24.37, 24.42, 26.38, 35.77, 47.33, 47.33, 54.61,
61.96, 65.51, 71.9, 76.76, 77.13, 78.52, 81.09, 81.23, 82.46,
82.52, 84.37, 84.49, 85.42, 85.35, 85.41, 87.33, 87.99, 92.23,
92.53, 101.53, 101.87, 101.97, 113.15, 113.91, 114.06, 116.74,
117.04, 127.04, 127.86, 128.51, 130.41, 130.47, 135.269,
135.41, 140.76, 142.28, 142.39, 144.55, 150.89, 150.97, 159.15,
159.18, 164.76,164.95, 172.46; 173.13; δP (201 MHz; CD3OD;
85% H3PO4) 0.067; ESI-MS (ES+) m/z 1051.600 (M+H.
C47H52N6O20P requires 1051.297).
[1] T. W. Greene, P. G. M. Wuts in Protective Groups in Organic
Synthesis, (1999), 3rd Ed., John Wiley & Sons, New York.
[2] R. P. Iyer in Comprehensive Natural Products Chemistry, ed.
E. T. Kool, Elsevier Science B.V., Amsterdam, 7, 105-152 (1999).
[3] K. Jarowicki, P. Kocienski J. Chem. Soc., Perkin Trans. 1,
1999, 1589-1616.
[4] Sekine M., Hata T.; Current Organic Chemistry, 3, 25-66
(1999).
[5] S. Pitsch, Chimia, 55, 320-324 (2001).
[6] D. Orain J. Ellard M. Bradley, J. Comb. Chem., 4, 1-16
(2002).
[7] J. Robertson, P. M. Stafford Carbohydrates, 2003, 9-68.
[8] C. B. Reese Tetrahedron, 58, 8893-8920 (2002).
[9] L. P. D. Green, R. Ravindranathan, C. B. Reese, R. Saffihill
Tetrahedron, 26, 1031-1041 (1970).
UpU: ESI-MS (ES-) m/z 549.087 (M-H). C18H22N4O14P
requires 549.088.
[10] T. Hata, J. Azizian, Tetrahedron Lett., 10, 4443-4446 (1969).
[11] M. Sekine, T. Hata J. Am. Chem. Soc., 105, 2044-2049
(1983).
[12] S. Sandstrom, A. Kwiatkowski, J. Chattopadyaya, Acta
Chem. Scand. B., 39, 273-290 (1985).
UpUisp: ESI-MS (ES-) m/z 589.118 (M-H). C21H26N4O14P
requires 589.119.
U(dcbco)pU: ESI-MS (ES-) m/z 707.472 (M-H).
C23H28N6O18P requires 707.120.
U(dcbdo)pUisp: ESI-MS (ES-) m/z 747.536 (M-H).
C26H32N6O18P requires 747.152.
[13] S. A. Scaringe, F. E. Wincott, M. H. Caruthers, J. Am. Chem.
Soc., 120, 11820-11821 (1998).
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Acknowledgements. This work was supported by a Grant
from CREST of JST (Japan Science and Technology Agency).
This work was also supported by the COE21 project.
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Present and Correspondence Address: Medical University
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