Synthesis and properties of oligodeoxynucleotides possessing N-phosphoryl groups

Article abstract of DOI:10.1002/ejoc.200200294

This paper deals with the synthesis and properties of oligodeoxynucleotides of a Dickerson-Drew sequence, d(CGCGAATTCGCG), in which one of the four cytosine bases has been replaced with an N-phosphorylated or N-diethylphosphorylated cytosine base in order to study the effect of the N-phosphoryl or N-diethoxyphosphoryl group on their structural property and hybridization affinity. The CD spectra of these self-complementary duplexes showed the typical B DNA-type shape with very minor changes. In contrast, it was found from T_m experiments that N-phosphoryl groups destabilized the DNA duplexes significantly through steric hindrance and electronic repulsion. ( Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2003).

Full text of DOI:10.1002/ejoc.200200294

FULL PAPER
Synthesis and Properties of Oligodeoxynucleotides Possessing
N-Phosphoryl Groups
Tomohisa Moriguchi,[a] Takeshi Wada,[a] and Mitsuo Sekine*[a]
Keywords: Circular dichroism / DNA structures / Duplex stability / Oligonucleotides
This paper deals with the synthesis and properties of
oligodeoxynucleotides of Dickerson−Drew sequence,
d(CGCGAATTCGCG), in which one of the four cytosine
bases has been replaced with an N-phosphorylated or N-di-
ethylphosphorylated cytosine base in order to study the ef-
spectra of these self-complementary duplexes showed the
a
typical B DNA-type shape with very minor changes. In con-
trast, it was found from Tm experiments that N-phosphoryl
groups destabilized the DNA duplexes significantly through
steric hindrance and electronic repulsion.
fect of the N-phosphoryl or N-diethoxyphosphoryl group on ( Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
their structural property and hybridization affinity. The CD
Germany, 2003)
Introduction
phorylated deoxycytidine 3Ј-phosphoramidite unit was re-
quired as a building block.
A number of nucleoside phosphate derivatives exist in liv-
Here we report the synthesis and hybridization properties
ing cells, playing essential roles in biological reactions as of oligodeoxynucleotides incorporating N-phosphorylated
structural elements of DNA and RNA, phosphate donors deoxynucleoside derivatives.
in transphosphorylation, and second messengers. In con-
trast, N-phosphorylated nucleosides have not been disco-
Results and Discussion
vered, except for the 6-N-phosphorylated adenine derivative
agrocin 84.[1Ϫ5] In agrocin 84, N-phosphorylation is re-
quired for permeation of agrocin 84 into the target cells.[3]
Therefore, if in nature the nucleobase can be phosphoryl-
ated by some biological process, there is a possibility that
the presence of a phosphoryl residue attached to the amino
group has a certain biological meaning. We have recently
reported the synthesis of N-phosphorylated ribonucleos-
ides, phosphorylation being at the exo-amino groups of ad-
enosine, cytidine, and guanosine.[6]
Thermal Stability of N-Phosphorylated Ribonucleoside
Derivatives
It is well known that phosphate monoesters are hy-
drolyzed under thermal conditions, via metaphosphate
intermediates.[9Ϫ20] This dephosphorylation is accelerated
by neighboring group participation by proximal phosphate
esters, or by addition of divalent metal cations.[21] It seems
that the N-phosphorylated ribonucleosides are also hy-
drolyzed by simple heating, to give the parent correspond-
ing nucleosides. We therefore studied the thermal stabilities
of N-phosphorylated ribonucleosides 1Ϫ5 (Figure 1). Com-
pounds 1Ϫ5 were dissolved in 0.1  ammonium acetate
buffer (pH ϭ 7.0), and these solutions were heated at 90
°C. The products were analyzed by reversed-phase HPLC;
the results are summarized in Table 1. The hydrolysis of the
N-phosphorylated nucleoside derivatives 1Ϫ3 occurred ex-
tremely rapidly and was complete within 10 min in the cases
of all three nucleoside derivatives. The rates of hydrolysis of
these N-phosphorylated nucleoside derivatives 1Ϫ3 could
not be determined because of the extremely fast hydrolysis,
so the rate of the hydrolysis at 70 °C was measured in the
same solutions. No obvious difference in reaction rate be-
tween the nucleosides was observed, the hydrolysis reaction
appearing to proceed through metaphosphate intermediates
and the rates of formation of the metaphosphate intermedi-
ates therefore being very similar in these N-phosphorylated
It is known that 4-N-acetylcytidine (ac4C), found as a
minor modified nucleoside in tRNA, has an electron-with-
drawing acetyl group on its amino moiety.[7] We have also
reported that 4-N-acetyldeoxycytidine can form a stable
base pair with deoxyguanosine upon incorporation into
DNA in place of deoxycytidine.[8] It might therefore be ex-
pected that N-phosphorylated deoxycytidine derivatives
should have hybridization affinities similar to that of N-
acyldeoxycytidine when incorporated into DNA. We had
previously reported that N-phosphorylated cytidine deriva-
tives are fairly stable in comparison with N-phosphorylated
adenosine and guanosine derivatives. To synthesize oligo-
deoxynucleotides incorporating N-phosphorylated deoxycy-
tidine derivatives, an appropriately protected N-phos-
[a]
Department of Life Science, Tokyo Institute of Technology,
Nagatsuta, Midoriku, Yokohama 226-8501, Japan
Fax: (internat.) ϩ 81-45/924-5772
E-mail: msekine@bio.titech.ac.jp
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 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/ejoc.200200294
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Oligodeoxynucleotides Possessing N-Phosphoryl Groups
FULL PAPER
Figure 1. N-Phosphorylated ribonucleoside derivatives
Table 1. The hydrolysis of N-phosphorylated ribonucleosides
Scheme 1. (i) (7a) (a) HMDS, CH3CN, pyridine reflux, 1 h; (b)
(iPr)2NP(OCH2CH2CN)2, 1H-tetrazole, CH3CN, room temp., 1 h;
(c) tBuOOH, room temp., 15 min; (7b) (a) TMSCl, Et3N, CH2Cl2,
room temp., 30 min; (b) (EtO)2PCl, CH2Cl2, room temp., 30 min;
90 °C
t1/2
70 °C
tcomp
tcomp
t1/2
k [10Ϫ4 sϪ1
]
(c)
tBuOOH,
CH2Cl2,
room
temp.,
15 min;
(ii)
ClP(OCH2CH2CN)(NiPr2), Et3N, CH2Cl2, room temp., 30 min
(8a), 1 h (8b); CE ϭ 2-cyanoethyl
1
2
3
4
5
Ϫ
Ϫ
Ϫ
18 h
15 h
Ͻ 10 min
Ͻ 10 min
Ͻ 10 min
70 h
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
60 min
60 min
60 min
Ϫ
9.8[a]
9.8[a]
11[a]
(Scheme 1). The subsequent oxidation of the trivalent phos-
phite intermediate with tert-butyl hydroperoxide gave the
N-phosphorylated deoxycytidine derivative 7a in 37% yield.
The phosphoramidite unit 8a was obtained in 82% yield by
the standard method.
0.12[b]
0.18[b]
60 h
Ϫ
[a]
[b]
70 °C. 90 °C.
Next, the diethyl ester derivative 8b was synthesized by
an alternative strategy. Firstly, the 3Ј-OH and amino groups
were trimethylsilylated by treatment with chlorotrimethylsil-
ane in pyridine. Phosphitylation of the amino group was
performed in pyridine through the use of a highly reactive
reagent, diethyl phosphorochloridite. When tert-butyl
hydroperoxide was used as the oxidizing agent in the follow-
ing oxidation step, however, the N-phosphoryl group was
found to be hydrolyzed completely. Since it had been found
that pyridine promoted the decomposition of the phosphite
intermediate, CH2Cl2 was used as a solvent and triethyl-
amine was additionally used as a base at the N-phosphityl-
ation. As the 3Ј-O-trimethylsilyl group was relatively stable
during the usual workup, the resulting mixture was further
treated with MeOH/H2O/pyridine (8:1:1, v/v/v) to remove
the trimethylsilyl group. The N-phosphorylated derivative
nucleosides 1Ϫ3. In contrast, the diethyl ester derivatives 4
and 5 were relatively stable and so were hydrolyzed much
more slowly than the N-phosphoryl derivatives 1Ϫ3, the
half-life times of the hydrolysis being 15 and 18 h for the
adenosine and cytidine derivatives 4 and 5, respectively. It
is obvious that there is a difference in the mechanisms of
hydrolysis of non-esterified and esterified N-phosphoramid-
ate derivatives. In the case of the diethyl ester derivatives,
the hydrolysis proceeded without the formation of the inter-
mediate metaphosphate.
Synthesis of Deoxycytidine Phosphoramidite Units
Containing N-Phosphoryl Groups
To synthesize oligodeoxynucleotides containing N-phos- 7b was thus obtained in 74% yield. The 3Ј-O-phosphityl-
phorylated deoxycytidine, the phosphoramidite derivative ation of 7b by the standard method gave 8b in 52% yield.
of N-phosphorylated deoxycytidine was prepared. The
The synthesis of deoxyadenosine derivatives containing
phosphorylation of the amino group of deoxycytidine was an N-diethoxyphosphoryl group by the same procedure
carried out by a method similar to that described previously as described in the case of deoxycytidine derivatives was
in the synthesis of N-phosphorylated ribonucleosides. 5Ј- attempted. Phosphorylation by the phosphoramidite re-
O-(4,4Ј-Dimethoxytrityl)-2Ј-deoxycytidine (6) was heated at agent indeed gave the corresponding N-diethoxyphosphoryl
reflux with hexamethyldisilazane in MeCN/pyridine for derivative, but this N-(phosphoryl)deoxyadenosine deriva-
30 min, and the resulting O-silylated species was allowed to tive was found to be unstable and decomposed during puri-
react with bis(2-cyanoethyl) N,N-diisopropylphosphoram- fication by silica gel column chromatography. It was also
idite in the presence of 1H-tetrazole as a promote found that the N-[bis(2-cyanoethoxy)phosphoryl]deoxy-
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T. Moriguchi, T. Wada, M. Sekine
FULL PAPER
adenosine derivative decomposed during chromatographic
separation. In addition, an non-esterified species of N-
(phosphoryl)deoxyadenosine exhibited instability in aque-
ous solution. These modified phosphorylated deoxyadenos-
ine building blocks were therefore not introduced into olig-
odeoxynucleotides.
Synthesis and Properties of dC*pT Dimers Containing N-
Phosphorylated Deoxycytidine Derivatives
To study the chemical properties of the N-phosphoryl
group in oligodeoxynucleotides, the deoxycytidyl(5Ј-3Ј)thy-
midine dimers d[C*pT] (11a) and d[C**pT] (11b), contain-
ing N-phosphoryl and N-diethoxyphosphoryl groups, were
synthesized by the liquid-phase method. Deoxycytidinyl
phosphoramidite units 8a and 8b were condensed with
3Ј-O-acetylthymidine (9) in the presence of 1H-tetrazole to
give the fully protected dimers 10a and 10b in satisfactory
yields Scheme 2). Deprotection of the fully protected dimers
10a and 10b was performed by the standard procedure to
give d[C*pT] 11a and d[C**pT] 11b, where * refers to the
phosphoryl or diethoxyphosphoryl group.
Some chemical properties of 11a and 11b were studied by
1
use of H NMR and CD spectroscopy. We have previously
reported that the N-phosphoryl moieties of N-phosphoryl-
ated ribonucleosides acted as electron-withdrawing groups.
It turned out that the 5-H and 6-H protons of the N-phos-
phorylated deoxycytidine derivatives are shifted to lower
magnetic field than those of cytidine. It has also been re-
ported that low magnetic field shifts of these base protons
were observed when electron-withdrawing groups such as
acetyl and benzoyl groups were introduced onto the amino
function of the cytosine moiety. In the case of d(C*pT) di-
mer 11a, it was also observed that the protons of the base
moiety were shifted to low magnetic field at the same level.
Next, in order to study the effect of the N-phosphoryl
groups on the structure, the CD spectra of these dimers
were measured at pH ϭ 7.0 at 0, 20 and 60 °C. The CD
Scheme 2. (i) (a) 1H-tetrazole, CH3CN, room temp., 30 min; (b)
tBuOOH, CH3CN, room temp., 15 min. (ii) (a) concd. NH3/pyri-
dine (3:1), room temp., 12 h, (b) 80% AcOH, room temp., 30 min;
CE ϭ 2-cyanoethyl
is weakened at 0 °C, due to steric hindrance around the N-
phosphorylated site in the case of 11a and 11b, and that the
contribution of the base-base stacking interaction to CD
disappears at 60 °C.
Synthesis and Properties of Oligodeoxynucleotides
Containing N-Phosphorylated Deoxycytidine Derivatives
In order to study the thermal properties of the N-phos-
spectra of these dimers are shown in Figure 2. All three phoryl function, oligodeoxynucleotides containing N-
showed large positive Cotton effects around 280 nm and (phosphoryl)deoxycytidine were prepared. For this study,
large negative Cotton effects around 220 nm. The intensity the DickersonϪDrew sequence of the dodecadeoxynucleo-
of the positive Cotton effect at 0 °C in the case of 11a and tide[22] d(CGCGAATTCGCG)2 was chosen. The structures
11b was apparently lower than in that of d(CpT). However, of many variants of this sequence, containing either
with an increase in temperature to 60 °C, the CD spectra matched or severely mismatched base pairs, have been stud-
of all three derivatives became more similar to one another. ied extensively by X-ray crystallography, NMR spec-
These results suggest that the base-base stacking interaction troscopy, and Tm experiments. We therefore decided to use
Figure 2. CD spectra of d(C*pT) dimers observed at various temperatures: 1: 0 °C; 2: 20 °C; 3: 60 °C; A: d(CpT); B: d(C*pT) (11a); C:
d(C**pT) (11b)
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Oligodeoxynucleotides Possessing N-Phosphoryl Groups
FULL PAPER
this well-known dodecamer in this study, and so the N-
(phosphoryl)deoxycytidine was introduced into the four
sites of deoxycytidine. Since the N-phosphoryl group is un-
stable under thermal conditions, the deprotection procedure
had to be carried out at room temperature, so the easily
removable dimethylformamidine group was selected as the
protecting group for the guanine moiety. The synthesis of
the oligodeoxynucleotides containing the N-phosphorylated
deoxycytidine was performed by the solid-phase method on
an automated synthesizer. The coupling efficiencies at the
N-(phosphoryl)deoxycytidine were similar to those of other
canonical nucleoside derivatives, and the average coupling
yields and isolated yields of all oligodeoxynucleotides are
shown in Table 2. These modified oligodeoxynucleotides
were purified by use of SepϪPak cartridges with the
DMTr-on mode and isolated by HPLC after detritylation
by treatment with 2% TFA. The isolated yields of oligo-
deoxynucleotides 12Ϫ20 were 15Ϫ37%. The structures of
these modified oligodeoxynucleotides were confirmed by
MALDI-TOF mass spectrometry.
Figure 3. The CD spectra of oligodeoxynucleotides contain-
ing
d(CGCGAATTCGCG);
d(CGCGAATTCGCG) D: d(CGCGAATTCGCG)
N-phosphorylated
deoxycytidine
derivatives:
A:
C:
B:
d(CGCGAATTCGCG);
Table 2. The average yields and isolated yields of DNA 12mers
containing N-phosphorylated deoxycytidine derivatives
of DNA duplexes,[23Ϫ26] and similar results were observed
Sequences (5Ј-3Ј)
Average yield Isolated yield in our experiments. This large destabilization affect was ob-
(%)
(%)
viously attributable to the introduction of the N-diethoxy-
phosphoryl group. A change of the N-diethoxyphosphoryl
group to the N-phosphoryl group caused a more drastic
destabilization effect, as shown in Table 3. Since the N-
phosphoryl group retains negative charges, a significant
electrostatic effect destabilizing the duplex was also to be
expected. The decrease in the Tm value per modification by
the introduction of this negatively charged phosphoryl
group is approximately 3 °C.
12
13
14
15
16
17
18
19
20
d(CGCGAATTCGCG)
d(C*GCGAATTCGCG)
d(CGC*GAATTCGCG)
d(CGCGAATTC*GCG)
d(CGCGAATTCGC*G)
d(C**GCGAATTCGCG)
d(CGC**GAATTCGCG)
d(CGCGAATTC**GCG)
d(CGCGAATTCGC**G)
99.5
99.2
98.5
98.5
99.4
99.2
99.3
98.7
99.9
28
15
25
37
22
15
30
19
30
In order to elucidate the global structures of the oligo-
deoxynucleotides 12Ϫ20, their CD spectra were observed at
20 °C (Figure 3).
All the oligodeoxynucleotides showed typical CD spec-
tra, with positive Cotton effects around 280 nm and nega-
tive Cotton effects around 250 nm, indicating the formation
of B-type duplexes. A slight difference in the intensity of
the Cotton effect of the unmodified oligodeoxynucleotide
12 was observed. This suggests that the presence of N-phos-
phoryl groups attached to the amino group of 2Ј-deoxycyti-
dine residues might slightly affect the conformation of the
duplexes.
Next, the thermal stabilities of the modified duplexes
were investigated by the melting temperature. The UV melt-
ing curves of all the duplexes exhibited two-state profiles
with shapes similar to those of the unmodified duplexes
(Figure 4). The Tm values are listed in Table 3, and those of
Figure 4. Melting temperatures of oligodeoxynucleotides contain-
ing N-phosphorylated deoxycytidine derivatives
Conclusion
In order to elucidate the chemical properties of the N-
the DNA duplexes were dramatically lower than that of the phosphoryl and N-diethoxyphosphoryl group in DNA,
unmodified DNA duplex. After addition of the N-diethoxy- oligodeoxynucleotides containing N-phosphorylated deoxy-
phosphoryl group, the Tm value dropped by approximately cytidine derivatives were synthesized by the phosphoramid-
5 °C. In general, introduction of a steric hindered group on ite approach. Some chemical properties of N-phosphoryl
an amino group of a cytosine moiety causes destabilization groups included in oligodeoxynucleotides were revealed by
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T. Moriguchi, T. Wada, M. Sekine
FULL PAPER
volume, diluted with CHCl3, and washed three times with 5%
NaHCO3 (aq.), and the aqueous layer was back-extracted with
CHCl3. The organic layer and washings were combined, dried with
Na2SO4, and filtered, and the solvents were evaporated to dryness.
The residue was placed on a silica gel column. Column chromatog-
raphy with CH2Cl2 containing 0.5% pyridine, with a gradient of
methanol (3Ϫ3.5%), gave 7a (452 mg, 32%) as a colorless foam. 1H
NMR (CDCl3): δ ϭ 2.21Ϫ2.31 (m, 1 H, 2"-H), 2.44Ϫ2.53 (m, 1
H, 2Ј-H), 2.75 (t, J ϭ 6.3 Hz, 4 H, CH2CN), 3.42 (dd, J5ЈЈ,4Ј ϭ 3.3,
J5ЈЈ,5Ј ϭ 10.6 Hz, 1 H, 5"-H), 3.49 (dd, J5Ј,4Ј ϭ 3.0, J5Ј,5ЈЈ ϭ 10.6 Hz,
1 H, 5Ј-H), 3.80 (s, 6 H, OCH3 of DMTr), 4.03Ϫ4.06 (m, 1 H, 4Ј-
H), 4.19Ϫ4.28 (m, 4 H, 2 ϫ POCH2), 4.52Ϫ4.57 (m, 1 H, 3Ј-H),
Table 3. The Tm values of oligodeoxynucleotides containing N-
phosphorylated deoxycytidine derivatives
5.68 (d, J5,6 ϭ 7.9 Hz, 1 H, 5-H), 6.24 (dd, J1Ј,2Ј ϭ 6.3, J1Ј,2ЈЈ
ϭ
5.9 Hz, 1 H, 1Ј-H), 6.80Ϫ6.87 (d, J ϭ 8.9 Hz, 4 H, o-H of DMTr),
7.13Ϫ7.40 (m, 9 H, ArH), 7.84 (dd, 1 H, J6,5 ϭ 7.9, J ϭ 2.0 Hz,
the spectroscopic experiments. The CD spectra showed
slight changes in the global structure of modified forms of
DNA from that of the canonical B-type DNA structure.
Tm experiments, however, showed that drastic destabiliz-
ation occurred on the introduction of the phosphoryl
groups. This destabilization seems to be caused by the two
factors: steric effects and the electrostatic repulsion of the
phosphoryl groups.
3
6-H) ppm. 13C NMR (CDCl3): δ ϭ 19.6 (d, JPOCC ϭ 6.4 Hz,
2
CH2CN), 41.3 (2Ј-C), 55.2 (OMe of DMTr), 61.2 (d, JPOC
ϭ
3.7 Hz, POCH2), 62.8 (5Ј-C), 71.0 (3Ј-C), 85.6, 86.2, 86.9 (1Ј-C,
4Ј-C and tert-C of DMTr), 101.4, 101.7 (5-C), 113.2, 116.8 (CN),
125.2Ϫ130.0, 135.2, 135.3 (1,1Ј-C of DMTr), 140.7 (1"-C of
DMTr), 144.2 (6-C), 148.5, 158.6 (2-C), 160.2, 160.3 (4,4Ј-C of
DMTr) ppm. 31P NMR (CDCl3):
δ
ϭ
5.56 ppm.
C36H38N5O9P·1.5H2O (742.7): calcd. C 58.22, H 5.56, N 9.43;
found calcd. C 57.80, H 5.75, N 9.79.
Experimental Section
2Ј-Deoxy-N4-diethoxyphosphoryl-5Ј-O-(4,4Ј-dimethoxytrityl)-
cytidine (7b): Compound 6 (530 mg, 1.0 mmol) was dried by re-
peated coevaporation with dry pyridine and dissolved in dry
CH2Cl2 (10 mL). Trimethylsilyl chloride (381 µL, 3.0 mmol) and
triethylamine (694 µL, 5.0 mmol) were added, and the resulting
mixture was stirred at room temp. for 30 min. Diethyl phosphoro-
chloridite (289 µL, 2.0 mmol) was added. After the mixture had
then been stirred at room temp. for 30 min, tert-butyl hydroper-
oxide (containing 20% di-tert-butyl peroxide, 626 µL, 5.0 mmol)
was added. The solution was stirred at room temp. for 15 min. The
mixture was diluted with CHCl3 and washed three times with 5%
NaHCO3 (aq.), and the aqueous layer was back-extracted with
CHCl3. The organic layer and washings were combined, dried with
Na2SO4, filtered, and concentrated to dryness. The residue was
placed on a silica gel column. Column chromatography with
CH2Cl2 containing 1% pyridine, with a gradient of methanol
(2Ϫ3%), gave 7b (494 mg, 74%) as a colorless foam. 1H NMR
(CDCl3): δ ϭ 1.32 (t, J ϭ 7.3 Hz, 3 H, CH3 of Et), 2.20Ϫ2.30 (m,
1 H, 2"-H), 2.43Ϫ2.53 (m, 1 H, 2"-H), 3.41 (dd, J5ЈЈ,4Ј ϭ 3.6,
J5ЈЈ,5Ј ϭ 10.9 Hz, 1 H, 5"-H), 3.47 (dd, J5Ј,4Ј ϭ 3.3, J5Ј,5ЈЈ ϭ 10.9 Hz,
1 H, 5Ј-H), 3.80 (s, 6 H, OCH3 of DMTr), 4.02Ϫ4.13 (m, 5 H, 4Ј-
General Remarks: CH2Cl2 and MeCN were distilled from CaH2
after being heated at reflux for several hours, and were stored over
˚
molecular sieves (4 A). Pyridine was distilled after being heated at
reflux in the presence of p-toluenesulfonyl chloride for several
hours, redistilled from CaH2, and stored over molecular sieves (4
˚
A). tert-Butyl hydroperoxide (containing 20% di-tert-butyl per-
oxide) was purchased from Merck & Co. Inc. Bis(2-cyanoethoxy)(-
diisopropylamino)phosphane was synthesized by a previously re-
ported method.[27] 1H NMR spectra were obtained at 270 MHz
with a JEOL-EX-270 spectrometer with tetramethylsilane as an in-
ternal standard in CDCl3 and with sodium 3-(trimethylsilyl)pro-
panesulfonate (DSS) as an external standard in D2O. 13C NMR
spectra were obtained at 67.8 MHz with a JEOL-EX-270 spec-
trometer with tetramethylsilane as an internal standard and with
DSS as an external standard in D2O. 31P NMR spectra were ob-
tained at 109.25 MHz with a JEOL-EX-270 spectrometer, with 85%
H3PO4 as an external standard. UV spectra were recorded with a
Hitachi 220A spectrophotometer. Thin layer chromatography was
performed on precoated glass plates of Kieselgel 60 F254 (Merck,
no. 5715). Silica gel column chromatography was carried out on
Wakogel C-200. Reversed-phase column chromatography was per-
formed with Waters µBondapak C18. MALDI-TOF mass spectra
were obtained with a VoyagerTM spectrometer (PerSeptive Biosys-
tems). CE ϭ 2-cyanoethyl.
H and 2 ϫ POCH2), 4.51Ϫ4.56 (m, 1 H, 3Ј-H), 5.67 (d, J5,6
ϭ
7.9 Hz, 1 H, 5-H), 6.26 (dd, J1Ј,2Ј ϭ 6.3, J1Ј,2ЈЈ ϭ 5.9 Hz, 1 H, 1Ј-
H), 6.84 (d, J ϭ 8.9 Hz, 4 H, o-H of DMTr), 7.19Ϫ7.43 (m, 9 H,
ArH), 7.77 (dd, 1 H, J6,5 ϭ 7.9, J ϭ 1.7 Hz, 6-H) ppm. 13C NMR
3
(CDCl3): δ ϭ 16.1 (d, JPOCC ϭ 7.3 Hz, CH3 of Et), 41.4 (2Ј-C),
N4-Bis(2-cyanoethoxy)phosphoryl-2Ј-deoxy-5Ј-O-(4,4Ј-dimethoxy-
trityl)cytidine (7a): 2Ј-Deoxy-5Ј-O-(4,4Ј-dimethoxytrityl)cytidine
(6) (1.06 g, 2.0 mmol) was dissolved in dry pyridine (10 mL)/dry
MeCN (10 mL). Hexamethyldisilazane (1.27 mL, 6.0 mmol) was
added to this solution, and the mixture was heated at reflux for
1 h. The reaction mixture was allowed to cool to room temp. and
concentrated to give a colorless foam. The resulting foam was dis-
solved in dry MeCN (20 mL), and bis(2-cyanoethoxy)(diisopropyl-
55.2 (OCH3 of DMTr), 62.8 (d, 2JPOC ϭ 7.3 Hz, CH2 of Et and 5Ј-
C), 71.0 (3Ј-C), 85.5, 86.1, 86.8 (1Ј-C, 4Ј-C and tert-C of DMTr),
101.7 (5-C), 113.2 (3,3Ј,5,5Ј-C of DMTr), 127.0, 128.0, 128.1, 130.0
(2,6-C of DMTr), 135.2, 135.4 (1,1Ј-C of DMTr), 140.2 (6-C), 144.3
(1"-C of DMTr), 149.2 (2-C), 158.6, 159.5 (4,4Ј-C of DMTr) ppm.
31P NMR (CDCl3): δ ϭ 6.34 ppm. C34H40N3O9P (665.7): calcd. C
61.35, H 6.06, N 6.31; found calcd. C 61.01, H 6.30, N 6.11.
amino)phosphane (814 mg, 3.0 mmol) and 1H-tetrazole (280 mg, N4-Bis(2-cyanoethoxy)phosphoryl-2Ј-deoxy؊5Ј-O-(4,4Ј-dimethoxy-
4.0 mmol) were added. After the mixture had been stirred at room
temp. for 1 h, tert-butyl hydroperoxide (containing 20% di-tert-bu-
tyl peroxide, 1.25 µL, 10.0 mmol) was added, and the mixture was
trityl)cytidin-3Ј-yl
(8a): Compound 7a (451 mg, 0.63 mmol) was dried by repeated co-
evaporation with dry pyridine and then dry toluene, and was then
2-Cyanoethyl-N,N-diisopropylphosphoramidite
stirred at room temp. for 15 min. It was concentrated to a small dissolved in dry CH2Cl2 (6 mL). Chloro(2-cyanoethoxy)(diisoprop-
2264  2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.eurjoc.org
Eur. J. Org. Chem. 2003, 2260Ϫ2267

Oligodeoxynucleotides Possessing N-Phosphoryl Groups
FULL PAPER
ylamino)phosphane (211 µL, 0.95 mmol) and N,N-diisopropyl-
(882.9): calcd. C 57.88, H 6.15, N 10.49; found calcd. C 57.15, H
ethylamine (220 µL, 1.26 mmol) were added, and the mixture was 5.98, N 10.25.
stirred at room temp. for 30 min. The mixture was diluted with
N4-Bis(2-cyanoethoxy)phosphoryl-2Ј-deoxy-5Ј-O-(4,4Ј-dimeth-
CHCl3 and washed three times with 5% NaHCO3 (aq.), and the
aqueous layer was back-extracted with CHCl3. The organic layer
and washings were combined, dried with Na2SO4, filtered, and con-
centrated to dryness under reduced pressure. The residue was
placed on a silica gel column. Column chromatography with
CH2Cl2 containing 1% pyridine, with a gradient of methanol
(2Ϫ3%), gave 8a (287 mg, 50%) as a colorless foam. 1H NMR
(CDCl3): δ ϭ 1.15Ϫ1.19 (m, 12 H, CH3 of iPr), 2.28Ϫ2.37 (m, 1
H, 2"-H), 2.46, 2.62 (2 t, J ϭ 6.3 Hz, 2 H, CH2CN), 2.52Ϫ2.65 (m,
1 H, 2Ј-H), 2.72Ϫ2.77 (2 t, J ϭ 6.3 Hz, 4 H, 2 ϫ CH2CN),
3.40Ϫ3.83 (m, 6 H, POCH2, 5Ј-H, 5"-H and 2 ϫ CH of iPr), 3.80,
3.80 (2 s, 6 H, OCH3 of DMTr), 4.10Ϫ4.27 [m, 5 H, 2 ϫ
P(O)OCH2, 4Ј-H], 4.67 (m, 1 H, 3Ј-H), 5.66Ϫ5.70 (m, 1 H, 5-H),
6.23Ϫ6.27 (m, 1 H, 1Ј-H), 8.83Ϫ8.87 (2 d, J ϭ 8.9 Hz, 4 H, o-H
of DMTr), 7.15Ϫ7.41 (m, 10 H, ArH), 7.84Ϫ7.92 (2 dd, J6,5 ϭ 7.9,
J ϭ 1.7 Hz,1 H, 6-H) ppm. 13C NMR (CDCl3): δ ϭ 19.5 [d,
3JPOCC ϭ 7.3 Hz, β-C of P(O)OCE], 20.1, 20.3 (d, 3JPOCC ϭ 7.3 Hz,
β-C of POCE), 24.3Ϫ24.5 (CH3 of iPr), 40.2Ϫ40.4 (2Ј-C), 43.0,
oxytrityl)-P-(2-cyanoethyl)cytidylyl-(3ЈǞ5Ј)-3Ј-O-acetylthymidine
(10a): 3Ј-O-Acetylthymidine (9) (28 mg, 0.10 mmol) and compound
8a (110 mg, 0.12 mmol) were dried by repeated coevaporation with
dry pyridine and then dry toluene, and were then dissolved in dry
MeCN (1 mL). 1H-Tetrazole (14 mg, 0.20 mmol) was added to the
mixture of 9 and 8a in dry MeCN. After the mixture had been
stirred at room temp. for 30 min, tert-butyl hydroperoxide (contain-
ing 20% di-tert-butyl peroxide, 63 µL, 0.50 mmol) was added and
the mixture was stirred at room temp. for 15 min. It was then di-
luted with CHCl3 and washed three times with 5% NaHCO3 (aq.),
and the aqueous layer was back-extracted with CHCl3. The organic
layer and washings were combined and dried with Na2SO4, filtered,
and concentrated to dryness. The residue was placed on a silica
gel column. Column chromatography with CH2Cl2 containing 1%
pyridine, with a gradient of methanol (1Ϫ2%), gave 10a (104 mg,
1
93%) as a colorless foam. H NMR (CDCl3): δ ϭ 1.90 (s, 3 H, 5-
Me of Th), 2.08, 2.09 (2 s, 3 H, Ac), 2.26Ϫ2.44 (m, 4 H, 2"-H of
C and T, CH2CN of CE), 2.65Ϫ2.78 (m, 6 H, 2Ј-H of C and T, 2
ϫ CH2CN of CE), 3.47 (m, 2 H, 5"-H of C and T), 3.80 (s, 6 H,
OCH3 of DMTr), 4.13Ϫ4.35 (m, 10 H, 4Ј-H, 5Ј-H of C and T, 3
2
43.2 (d, JPNC ϭ 2.4 Hz, CH of iPr), 55.1 (OCH3 of DMTr), 57.9,
2
2
58.1 (d, JPOC ϭ 4.9 Hz, POCH2), 61.1 [d, JPOC ϭ 4.9 Hz,
2
P(O)OCH2], 62.1 (d, JPOC ϭ 7.3 Hz, 5Ј-C), 71.8Ϫ72.5 (3Ј-C),
ϫ POCH2), 5.16Ϫ5.25 (m, 2 H, 3Ј-H of C and T), 5.78 (d, J5,6
ϭ
85.1Ϫ86.8 (1Ј-C, 4Ј-C and tert-C of DMTr), 101.7, 102.0 (5-C),
113.1 (3,3Ј5,5Ј-C of DMTr), 116.7 [CN of P(O)OCE], 117.4, 117.6
(CN of POCE), 125.1Ϫ130.0 (2,6-C of DMTr), 134.9Ϫ135.2 (1,1Ј-
C of DMTr), 140.4 (1"-C of DMTr), 144.0 (6-C), 148.1, 158.5 (2-
C), 160.1 (4,4Ј-C of DMTr) ppm. 31P NMR (CDCl3): δ ϭ 6.15,
6.17, 149.48, 149.69 ppm. C45H55N7O10P2·H2O (932.9): calcd. C
57.88, H 6.15, N 10.49; found calcd. C 57.15, H 5.98, N 10.25.
7.9 Hz, 1 H, 5-H of C), 6.24Ϫ6.31 (m, 2 H, 1Ј-H of C and T), 6.85
(d, J ϭ 8.6 Hz, 4 H, ArH of DMTr), 7.22Ϫ7.36 (m, 10 H, ArH of
DMTr and 6-H of T), 7.69 (m, 1 H, 6-H of C), 9.40, 9.47 (2 s, NH
of T) ppm. 13C NMR (CDCl3): δ ϭ 12.4 (5-Me of T), 19.6 (d,
3
3JPOCC ϭ 4.7 Hz, β-C of CE), 36.6 (2Ј-C of T), 39.3 (d, JPOCC
ϭ
2.4 Hz, 2Ј-C of C), 55.2 (MeO of DMTr), 61.3Ϫ62.6 (5Ј-C of C
and T, α-C of CE), 67.6 (3Ј-C of T), 73.5 (3Ј-C of C). 82.3Ϫ85.2
(1Ј-C, 4Ј-C of C and T), 87.3 (tert-C of DMTr), 102.2 (5-C of C),
111.5, 111.7 (4-C of T), 113.1, 113.3 (3,3Ј-C of DMTr), 116.3,
116.4, 116.8 (CN), 127.2Ϫ130.0 (2,6-C of DMTr), 134.8, 134.9,
135.2 (1,1Ј-C of DMTr), 140.0 (6-C of C, 1"-C of DMTr, 2-C of
T), 150.4 (2-C of C), 158.7 (4,4Ј-C of DMTr), 160.1 (4-C of C),
163.65, 163.68 (6-C of T), 170.46, 170.53 (CϭO) ppm. 31P NMR
(CDCl3): δ ϭ Ϫ2.12, Ϫ2.07, 5.77 ppm. C51H56N8O17P2 (1115.0):
calcd. C 54.94, H 5.06, N 9.77; found calcd. C 56.34, H 5.20, N
9.77.
2Ј-Deoxy-N4-diethoxyphosphoryl-5Ј-O-(4,4Ј-dimethoxytrityl)-
cytidin-3Ј-yl 2-Cyanoethyl-N,N-diisopropylphosphoramidite (8b):
Compound 7b (239 mg, 0.36 mmol) was dried by repeated coeva-
poration with dry pyridine and then dry toluene, and was then
dissolved in dry CH2Cl2 (4 mL). Chloro(2-cyanoethoxy)(diiso-
propylamino)phosphane (120 µL, 0.54 mmol) and N,N-
diisopropylethylamine (125 µL, 0.72 mmol) were added, and the
mixture was stirred at room temp. for 1 h. The reaction mixture
was diluted with CHCl3 and washed three times with 5% NaHCO3
(aq.), and the aqueous layer was back-extracted with CHCl3. The
organic layer and washings were combined and dried with Na2SO4,
filtered, and concentrated to dryness under reduced pressure. The
residue was placed on a silica gel column. Column chromatography
with CH2Cl2 containing 1% pyridine, with a gradient of methanol
2Ј-Deoxy-N4-diethoxyphosphoryl-5Ј-O-(4,4Ј-dimethoxytrityl)-P-(2-
cyanoethyl)cytidylyl-(3ЈǞ5Ј)-3Ј-O-acetylthymidine (10b): 3Ј-O-Ace-
tylthymidine (9) (28 mg, 0.10 mmol) and compound 8b (110 mg,
0.12 mmol) were dried by repeated coevaporation with dry pyridine
(1Ϫ2%), gave 8b (158 mg, 51%) as a colorless foam. 1H NMR and then dry toluene, and were then dissolved in dry MeCN
(CDCl3): δ ϭ 1.15Ϫ1.19 (m, 12 H, CH3 of iPr), 1.33 (t, J ϭ 7.3 Hz, (1 mL). 1H-Tetrazole (14 mg, 0.20 mmol) was added to the mixture
3 H, CH3 of Et), 2.28Ϫ2.38 (m, 1 H, 2"-H), 2.55 (m, 2 H, CH2CN), of 9 and 3b in dry MeCN. After the mixture had been stirred at
2.50Ϫ2.64 (m, 1 H, 2Ј-H), 3.30Ϫ3.88 (m, 6 H, POCH2, 5Ј-H, 5"- room temp. for 30 min, tert-butyl hydroperoxide (containing 20%
H and 2 ϫ CH of iPr), 3.80, 3.81 (2 s, 6 H, OCH3 of DMTr),
4.05Ϫ4.22 [m, 5 H, 2 ϫ P(O)OCH2, 4Ј-H], 4.69 (m, 1 H, 3Ј-H), 5.67 was continued at room temp. for an additional 15 min. The reaction
(m, 1 H, 5-H), 6.25 (m, 1 H, 1Ј-H), 8.83Ϫ8.88 (2 d, J ϭ 8.9 Hz,4 H, mixture was diluted with CHCl3 and washed three times with 5%
di-tert-butyl peroxide, 63 µL, 0.50 mmol) was added and stirring
o-H of DMTr), 7.17Ϫ7.45 (m, 10 H, ArH), 7.85Ϫ7.92 (2 dd, J6,5 ϭ NaHCO3 (aq.), and the aqueous layer was back-extracted with
7.9, J ϭ 1.7 Hz, 1 H, 6-H) ppm. 13C NMR (CDCl3): δ ϭ 16.0 (d, CHCl3. The organic layer and washings were combined, dried with
3JPOCC ϭ 7.3 Hz, CH3 of Et), 20.1Ϫ20.3 (β-C of CE), 24.35, 24.39 Na2SO4, filtered, and concentrated to dryness. The residue was
3
(d, JPNCC ϭ 7.3 Hz, CH3 of iPr), 40.3 (2Ј-C), 43.1, 43.2 (d, placed on a silica gel column. Column chromatography with
2JPNC ϭ 12.2 Hz, CH of iPr), 55.1 (OCH3 of DMTr), 62.3Ϫ62.9 CH2Cl2 containing 1% pyridine, with a gradient of methanol
(CH2 of Et, 5Ј-C and α-C of CE), 72.8, 73.1 (3Ј-C), 85.0Ϫ87.0 (1Ј-
C, 4Ј-C, tert-C of DMTr), 113.1, 113.2 (3,3Ј,5,5Ј-C of DMTr), 117.4 (CDCl3): δ ϭ 1.32 (t, J ϭ 6.9 Hz, 6 H, CH3 of Et), 1.91 (s, 3 H, 5-
(1Ϫ2%), gave 10b (94 mg, 84%) as a colorless foam. 1H NMR
(CN), 127.0Ϫ130.1 (2,6-C of DMTr), 134.8, 135.0, 135.2 (1Ј-C of
Me of T), 2.08, 2.09 (2 s, 3 H, Ac), 2.27Ϫ2.41 (m, 4 H, 2"-H of C
and T, 2 ϫ CH2CN), 2.65Ϫ2.78 (m, 2 H, 2Ј-H of C and T),
DMTr), 39.4, 139.6 (6-C), 143.9, 144.1 (1"-C of DMTr), 148.9 (2-
C), 158.5, 158.6 (4,4Ј-C of DMTr), 159.2 (4-C) ppm. 31P NMR 3.43Ϫ3.47 (m, 2 H, 5"-H of C and T), 3.79, 3.80 (2 s, 6 H, MeO
(CDCl3): δ ϭ 6.33, 149.37, 149.76 ppm. C45H55N7O10P2·H2O
of DMTr), 4.03Ϫ4.35 [m, 10 H, 4Ј-H, 5Ј-H of C and T, 3 ϫ
Eur. J. Org. Chem. 2003, 2260Ϫ2267
www.eurjoc.org
 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2265

T. Moriguchi, T. Wada, M. Sekine
FULL PAPER
P(O)OCH2], 5.18Ϫ5.29 (m, 2 H, 3Ј-H of C and T), 5.69 (d, J5,6
ϭ
and T), 114.1 (6-C of C), 134.0 (6-C of T), 154.2 (2-C of C), 168.9
7.9 Hz, 1 H, 5-H of C), 6.26Ϫ6.33 (m, 2 H, 1Ј-H of C and T), 6.86
(2 d, J ϭ 7.6 Hz, 4 H, ArH of DMTr), 7.15Ϫ7.35 (m, 10 H, ArH
of DMTr and 6-H of T), 7.62Ϫ7.66 (m, 1 H, 6-H of C), 9.42 (s, 1
H, NH of T) ppm. 13C NMR (CDCl3): δ ϭ 2.4 (5-Me of T), 16.1
(d, 3JPOCC ϭ 6.1 Hz, CH3 of Et), 19.5Ϫ19.6 (2 d, 3JPOCC ϭ 6.1 Hz,
β-C of CE), 36.6 (2Ј-C of T), 39.3 (d, 3JPOCC ϭ 5.0 Hz, 2Ј-C of C),
55.2 (MeO of DMTr), 62.4Ϫ62.9 (CH2 of Et, 5Ј-C of C and T, α-
(4-C of C) ppm. 31P NMR (D2O): δ ϭ Ϫ2.04, Ϫ2.02, 5.79 ppm.
Synthesis and Purification of Modified Oligodeoxynucleotides:
Automated DNA synthesis was performed with an Applied Biosys-
tems Model 392 DNA/RNA Synthesizer. Deoxynucleoside phos-
phoramidite units and dG-LCAA-CPG were purchased from
PerkinϪElmer Japan, Inc. Synthesis of oligodeoxynucleotides was
performed on a 1-µmol scale by use of standard β-cyanoethyl phos-
phoramidite chemistry. After the automated synthesis, the solid
supports containing the DMTr-on oligodeoxynucleotides were
transferred to a glass snap vial and treated with conc. NH3 at room
temp. for 12 h. The DMTr-on oligodeoxynucleotides were purified
by a Sep-Pak cartridge by washing with distilled water. The detri-
tylation was performed by treatment with 2% TFA/H2O on the
Sep-Pak cartridge, and the subsequent purification of the detrityl-
ated oligodeoxynucleotides was performed with a Shimadzu 6A
system on a µBondapak column (Waters, C18-100A, 7.8 ϫ
300 mm) by use of a linear gradient of 0Ϫ30% acetonitrile in 0.1
 NH4OAc (pH ϭ 7.0) for 30 min at a flow rate of 3.0 mL/min at
room temp. The oligodeoxynucleotides synthesized were analyzed
by MALDI-TOF mass spectrometry. Compound 16: calcd.
3725.36; found 3726.43. Compound 20: calcd. 3781.47, found
3782.96.
2
C of CE), 73.5 (3Ј-C of T), 79.0 (d, JPOC ϭ 6.1 Hz, 3Ј-C of C),
82.3Ϫ87.2 (1Ј-C, 4Ј-C of C and T, tert-C of DMTr), 101.8, 101.9,
102.2 (5-C of C), 111.6, 111.7 (4-C of T), 113.3 (3,3Ј,5,5Ј-C of
DMTr), 116.1, 116.2, 116.3 (CN), 127.2Ϫ130.0 (2,6-C of DMTr),
134.8, 135.1 (1,1Ј-C of DMTr), 139.3 (6-C of C), 143.88, 143.92
(1"-C of DMTr), 149.2 (2-C of T), 150.3 (2-C of C), 158.6, 158.7
(4,4Ј-C of DMTr), 159.3 (4-C of C), 163.5 (6-C of T), 170.4, 170.5
(CϭO) ppm. 31P NMR (CDCl3): δ ϭ 5.79, Ϫ2.02, Ϫ2.04 ppm.
2Ј-Deoxy-N4-(phosphoryl)cytidylyl-(3ЈǞ5Ј)thymidine (11a): The
fully protected d(CpT) dimer 10a was treated with NH3/pyridine
(20 mL, 3:1, v/v) at room temperature for 12 h. The mixture was
concentrated to a small volume, and the residue was dissolved in
dist. H2O. The solution was washed five times with diethyl ether.
The aqueous layer was concentrated to dryness under reduced
pressure, and the residue was treated with 80% aqueous AcOH at
room temperature for 30 min. The reaction mixture was concen-
trated and co-evaporated several times with dist. H2O. The residue
was dissolved in dist. H2O, and the solution was washed five times
with diethyl ether. The aqueous layer was concentrated to dryness
under reduced pressure. The residue was dissolved in a small
amount of water and placed on a gel filtration column (Sephadex
G-10, 300 ϫ 15 mm). Elution was performed with water. The eluent
was collected and lyophilized to give a crude product. This crude
product was further purified by C-18 reversed-phase column chro-
matography (80 ϫ 20 mm, H2O/MeCN, 100:0 to 85:15). The frac-
tions containing 11a were combined and lyophilized. The residue
was treated with cation-exchange resin (Dowex 50W ϫ 8, Naϩ
form) and lyophilized to give 11a (20 mg, 32%) as a white powder.
1H NMR (D2O): δ ϭ 1.88 (d, 3 H, J6-Me,5 ϭ 1.0 Hz, 6-Me of T),
Melting Experiments: Thermal denaturation studies of oligodeoxy-
nucleotides were carried out with a Hitachi U-2000 spectrophoto-
meter with 1-cm optical path length quartz cuvettes. The sample
temperature was increased from 0 to 80 °C at 1 °C/min with ab-
sorption readings at 260 nm taken every 0.5 °C. All samples were
prepared in a buffer containing 10 m sodium phosphate, pH ϭ
7.0, 150 m NaCl. The melting temperature (Tm) values were deter-
mined by the reading of the maximum point of the first derivative
of the melting profiles.
CD Spectra: The CD spectra of the d(CpT) dimers and oligodeoxy-
nucleotides containing N-phosphorylated deoxycytidine derivatives
were obtained with a JASCO J-500C spectrometer with 0.5-cm op-
tical path length quartz cuvettes. The d(CpT) dimers were dissolved
in 10 m sodium phosphate buffer (pH ϭ 7.0), and the final con-
centration was adjusted to about 10 µ. In the cases of the deoxy-
dodecamers, the solvent also contained 150 m NaCl, and the
sample concentration was 2 µ.
2.26Ϫ2.37 (m, 3 H, 2Ј-H of C, 2"-H of C and T), 3.72 (dd, J5ЈЈ,5Ј
ϭ
12.5, J5ЈЈ,4Ј ϭ 4.6 Hz, 1 H, 5"-H of C), 3.82 (dd, 1 H, J5Ј,5ЈЈ ϭ 12.5,
J5Ј,4Ј ϭ 3.3 Hz, 5Ј-H of C), 4.01Ϫ4.24 [m, 8 H, 4Ј-H of C and T,
5Ј-H and 5"-H of T, 2 ϫ P(O)OCH2], 4.52Ϫ4.57 (m, 1 H, 3Ј-H of
T), 4.78Ϫ4.87 (m, 1 H, 3Ј-H of C), 6.13 (t, 1 H, J1Ј,2Ј ϭ J1Ј,2ЈЈ
ϭ
6.3 Hz, 1Ј-H of T), 6.23 (m, 1 H, 1Ј-H of C), 6.29 (d, J5,6 ϭ 7.6 Hz,
1 H, 5-H of C), 7.67 (d, 1 H, J6,5-Me ϭ 1.3 Hz, 6-H of T), 7.91 (d,
J6,5 ϭ 7.6 Hz, 1 H, 6-H of C) ppm. 31P NMR (D2O): δ ϭ Ϫ2.12,
Ϫ2.07, 5.77 ppm.
Acknowledgments
This work was supported by a grant from ‘‘Research for the
Future’’ Program of the Japan Society for the Promotion of Science
(JSPS-RFTF97I00301) and a Grant-in-Aid for Scientific Research
from the Ministry of Education, Science, Sports and Culture,
Japan.
2Ј-Deoxy-N4-diethoxyphosphorylcytidylyl-(3ЈǞ5Ј)thymidine (11b):
A procedure similar to that described in the case of 11a gave 11b
(26 mg, 46%): 1H NMR (D2O): δ ϭ 1.31 (2 t, J ϭ 7.3 Hz, 6 H,
CH3 of Et), 1.86 (d, 3 H, J6-Me,5 ϭ 1.0 Hz, 6-Me of T), 2.24Ϫ2.37
(m, 3 H, 2Ј-H of C, 2"-H of C and T), 2.58Ϫ2.67 (m, 1 H, 2"-H of
T), 3.74 (dd, J5ЈЈ,5Ј ϭ 12.5, J5ЈЈ,4Ј ϭ 4.6 Hz, 1 H, 5"-H of C), 3.82
(dd, 1 H, J5Ј,5ЈЈ ϭ 12.5, J5Ј,4Ј ϭ 3.3 Hz, 5Ј-H of C), 4.02Ϫ4.24 [m,
8 H, 4Ј-H of C and T, 5Ј-H and 5"-H of T, 2 ϫ P(O)OCH2], 4.55
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2266
 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Oligodeoxynucleotides Possessing N-Phosphoryl Groups
FULL PAPER
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