Synthesis and properties of new nucleotide analogues possessing squaramide moieties as new phosphate isosters

Article abstract of DOI:10.1002/ejoc.200500520

New analogues of 2′-deoxynucleotides and ribonucleotides incorporating a unique squaramide structure were synthesized. Because of the strong acidity of this moiety (pK_a = 2.3), these nucleotide analogues exist in a monoanionic form, which can b

Full text of DOI:10.1002/ejoc.200500520

FULL PAPER
DOI: 10.1002/ejoc.200500520
Synthesis and Properties of New Nucleotide Analogues Possessing Squaramide
Moieties as New Phosphate Isosters
Kohji Seio,[a,c] Takuhei Miyashita,[b,c] Kousuke Sato,[b] and Mitsuo Sekine*[b,c]
Dedicated to Professor Wojciech J. Stec on the occasion of his 65th birthday
Keywords: Squaric acid / Acidity / Nucleotide analogues / Cyclic nucleotide analogues
New analogues of 2Ј-deoxynucleotides and ribonucleotides
incorporating a unique squaramide structure were synthe-
sized. Because of the strong acidity of this moiety (pKa = 2.3),
these nucleotide analogues exist in a monoanionic form,
we also synthesized 3Ј,5Ј-cyclic nucleotide analogues from
the 3Ј,5Ј-diazidonucleoside derivatives. NMR analysis re-
vealed that their ribose puckering was of an N-type form,
identical to that in cAMP and cGMP. Because of the unique
which can be regarded as an electronic isoster of 5Ј-nucleo- structural, electronic, and conformational properties of squar-
tides under physiological conditions. The synthesis of the nu-
cleotide analogues was achieved through the condensation
of 5Ј- or 3Ј-aminonucleosides with dimethyl squarate, whilst
the selective removal of the methyl group was effectively ac-
complished by treatment with sodium bromide. In addition,
amide-type nucleotide analogues, these analogues should be
quite interesting as potential biologically active compounds
such as antiviral and anticancer agents.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2005)
Introduction
Squaric acid (1) is a bibasic acid with a cyclobutene-3,4-
dione structure (Scheme 1). The unique properties of
squaric acid derivatives (2a) are characterized by the highly
polarized carbonyl group; this is attributable to the stability
of the resonance structure (2b),[1] which is aromatic accord-
ing to the Hückel rule (number of π electrons = 4n + 2: n =
0 for 2b). Such a highly polarized electronic structure, to-
gether with the strong acidity of squaric acid (pKa1 = 0.54;
pKa2 = 2.2),[2] makes such derivatives useful in medicinal
chemistry as isoelectronic isosters of carboxylic acids[3] and
sulfonic acids.[4] Recently, we have proposed similarities in
the electronic structures of squaric acid and phosphoric
acid, and have reported the synthesis of artificial DNAs
containing 3Ј–5Ј and 2Ј–5Ј squaryl dimide linkages, which
displayed an unusual bent structure and G–T mismatch sta-
bilization, respectively.[5,6] The potential of squaric acid as
a phosphate mimic has also been proposed by Seto and co-
workers[7] in the development of protein tyrosine phospha-
tase inhibitors.
Scheme 1. Structures of squaric acid derivatives.
In this paper we report the synthesis of new 5Ј- and 3Ј-
nucleotide analogues utilizing the properties of squaric acid
described above for the replacement of the phosphoric acid
residues of natural nucleotides with squaric acid skeletons.
In view of the chemical instability of squaric acid esters
under hydrolytic conditions, we designed squaramide deriv-
atives of nucleosides 3–9, as shown in Scheme 2. Although
Glusenkamp and co-workers[8] have reported the synthesis
of an 5Ј-adenosine squaramide derivative by condensation
of the corresponding 5Ј-aminonucleoside and diethyl squar-
ate, the removal of the ethyl group and the synthesis of
other nucleotide derivatives have not been reported. Be-
cause of the importance of nucleotides – 5Ј,3Ј-, and 5Ј,3Ј-
cyclic nucleotides, for example – in living systems, many
nucleotide and cyclic nucleotide analogues[9] of phospho-
rus-modified compounds have been developed as biochemi-
cal tools and drugs.[10] There has, however, been little study
[a] Frontier Collaborative Research Center, Tokyo Institute of
Technology
[b] Department of Life Science Tokyo Institute of Technology
[c] CREST, Japan Science and Technology Agency (JST)
4259 Nagatsuta, Midori-ku, Yokohama, 226-8501 Japan
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K. Seio, T. Miyashita, K. Sato, M. Sekine
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of analogues not containing phosphorus atoms, especially forms under physiological conditions, the results suggest the
on analogue design involving carbon acids.[11] A new class potential of the squaramide moiety as a phosphate mimic.
of nucleotide analogues, such as that reported in this paper,
should therefore be useful as biochemical and molecular
biological tools and for pharmaceutical applications.
Synthesis of 5Ј-Squaramides of Deoxynucleosides and of
Thymidine 3Ј-Squaramide
First of all, to establish a general procedure for the syn-
thesis of 5Ј-nucleotide analogues 3, the synthesis of the 5Ј-
thymidylate analogue was carried out as shown in
Scheme 3. The squaryl group was introduced onto the
amino group of 5Ј-amino-5Ј-deoxythymidine (11a)[12] by
condensation with dimethyl squarate[13,14] in the presence
of
a catalytic amount of N,N-diisopropylethylamine
(DIEA), as shown in Scheme 3. The reaction was complete
within 15 min and the target compound 12a was obtained
in 87% yield.
Next, removal of the methyl group was examined. Be-
cause the esters of squaric acid are fairly stable to aqueous
acids we first tested the deprotection under mild alkaline
conditions. The methyl group could be removed at room
temperature within 4 h to give 3 in 57% yield. Although the
hydrolysis of a small portion of the squaramide moiety was
also observed during the reaction, the target material could
be separated from the side-products by reversed-phase col-
umn chromatography.
The synthesis of 2Ј-deoxycytidine derivative 4 is also
shown in Scheme 3. The 5Ј-amino derivative[15] 11b was
condensed with dimethyl squarate to give the methyl squar-
ate derivative 12b in 66% yield. The methyl ester of 12b was
cleaved by treatment with aqueous NaOH to give the de-
sired compound 4 in 77% yield.
Next, as an example of the 3Ј-nucleotide analogue, thym-
idine 3Ј-squaramide (5) was synthesized as shown in
Scheme 4. Treatment of 5Ј-O-DMTr-3Ј-amino-3Ј-deoxythym-
idine (13)[16] with dimethyl squarate gave the squaramide
derivative 14 in 76% yield. Subsequently, the DMTr group
of 14 was removed by treatment with 80% acetic acid to
give the 5Ј-hydroxy derivative 15 in 60% yield. Finally, com-
Scheme 2. Structures of squaramide-type nucleotide analogues.
Results and Discussion
pKa of N-Methylsquaramide
Although the acidity of squaric acid has been well char-
acterized,[2] there has been no reported quantitative study
of the pKa values of squaric acid derivatives such as squaryl
monoamides. In order to confirm the similarity between
squaramide compounds and phosphates, the pKa value of
N-methylsquaramide (10) was measured by alkaline ti-
tration. As shown in Figure 1, compound 10 proved to be
a monobasic acid and its pKa was determined to be 2.3.
Comparison of the pKa of 10 with that of methyl phosphate
(pKa Ͻ 1) revealed that the acidity of the former was
weaker than that of the latter, but it should be noted that
the above pKa did indicate the existence of 10 in
monoanionic form under physiological conditions. Because
phosphate monoesters also exist in their monoanionic
Figure 1. Alkaline titration of squaramide.
Scheme 3. Synthesis of deoxynucleoside 5Ј-phosphate analogues.
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Squaramide Moieties as Phosphate Isosters in Nucleotides
FULL PAPER
pound 15 was converted to thymidine 3Ј-squaramide (5) in
75% yield by alkaline hydrolysis at 45 °C.
Scheme 4. Synthesis of thymidine 3Ј-phosphate analogues.
Synthesis of Ribonucleoside 5Ј-Squaramides
Scheme 5. Synthesis of ribonucleoside 5Ј-phosphate analogues.
In addition to the deoxynucleotide analogues described
above, we also tried to synthesize the 5Ј-ribonucleotide ana-
logues 6–8 (Scheme 2). Because ribonucleosides have higher
polarities than deoxynucleosides, we employed the 4,4Ј-di-
methoxytrityl (DMTr) group for protection of the exocyclic
amino groups of cytosine and adenine, to increase the lipo-
philicity of these materials. Introduction of the lipophilic
DMTr group allowed the purification of synthetic interme-
diates by silica gel chromatography; the reaction scheme is
shown in Scheme 5. As an example, N4-(dimethoxytrityl)-
Other 5Ј-ribonucleoside derivatives 6 and 8 were synthe-
sized by essentially the same procedure. In the case of the
uridine derivative 6, the base-unprotected derivative 20[12]
could be used because of the less polar nature of uridine.
Analogues of cAMP and cGMP with Squardiamide
Linkages
Finally, we tried to synthesize cAMP and cGMP ana-
cytidine (16) was converted into the 5Ј-azide derivative 18 logues incorporating squaryl diamide linkages. The key in-
by treatment with PPh3-LiN3/CBr4.[17] The azide group of termediates, the 3Ј,5Ј-diazido-3Ј,5Ј-dideoxynucleosides 28
18 was converted into the amino derivative 21 by treatment and 29, were synthesized by the procedure reported by Got-
with PPh3. Substitution of dimethyl squarate with the tikh,[19] with a slight modification. In our modified pro-
amino group of 21 gave the squaramide derivative 24 in cedure, we employed bis(trifluoromethylsulfonyl) derivative
68% yield.
26 in place of the di(methylsulfonyl) derivative reported in
For the synthesis of the ribonucleotide analogues, we the original procedure. The use of the triflic acid moiety as
searched for reaction conditions suitable for the removal of a leaving group enabled unfavorable olefin formation to be
both the DMTr group and the methyl group in order to reduced and the yield of 27 improved. Compound 27 was
simplify the reaction scheme. Previously, several researchers further converted into the diazidonucleosides 28 and 29 by
had reported removal of methyl groups from methyl phos- the original procedure.[19] The base moieties of 28 and 29
phate derivatives by use of sodium iodide under anhydrous were protected with DMTr groups to give the DMTr deriva-
conditions.[18] Because of the isoelectronic properties of tives 30 and 31, and the azide compounds were converted
phosphoric acid and squaric acid, we expected that this pro- into the amino derivatives 32 and 33 by use of tri-
cedure should also be effective for removal of methyl groups phenylphosphane. In spite of the highly polar natures of the
from the squaric acid derivatives. Consequently, we found diamino structures of 32 and 33, they could be purified by
that the methyl group of 24 was removed by treatment with silica gel column chromatography, because of the lipophil-
NaI in hexan-2-one at 120 °C to give the sodium salt of the icity of the DMTr group. Subsequent condensation of 32
desired uridine 5Ј-squaramide 9 in 57% yield. It should be and 33 with dimethyl squarate gave the DMTr derivatives
noted that the DMTr group could also be removed during of cAMP (34) and cGMP (35) analogues, which could be
this procedure, probably due to the instability of this pro- converted into the desired products, 9a and 9b, by removal
tecting group at such high temperature.
of the DMTr groups (Scheme 6).
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K. Seio, T. Miyashita, K. Sato, M. Sekine
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Conclusions
We have synthesized new analogues of 2Ј-deoxynucleo-
tides and ribonucleotides incorporating a unique squar-
amide structure. Because of the strong acidity (pKa = 2.3)
of squaramide, these nucleotide analogues exist in
monoanionic forms, which should act under physiological
conditions as electronic isosters of 5Ј-nucleotides. The syn-
thesis of the nucleotide analogues 3–8 was achieved by the
condensation of 5Ј- or 3Ј-aminonucleosides with dimethyl
squarate. The selective removal of the methyl group was
effectively carried out by treatment with sodium iodide. We
also synthesized 3Ј,5Ј-cyclic nucleotide analogues 9a and 9b,
starting from the 3Ј,5Ј-diazidonucleoside derivatives. NMR
analysis of 9b showed its ribose puckering to have the N-
type conformation, which is identical to that in cAMP and
cGMP. Because of these unique structural, electronic, and
conformational properties of squaramide-type nucleotide
analogues, these analogues are quite interesting as potential
biologically active compounds such as antiviral and anti-
cancer agents. Biological studies of the compounds synthe-
sized in this study are in progress and will be reported else-
where.
Experimental Section
General Remarks: 1H and 13C spectra were obtained on a Varian
Unity INOVA instrument at 500 and 126 MHz, respectively. The
chemical shifts were measured from tetramethylsilane in CDCl3 (δ
= 0 ppm), DMF in [D7]DMF (δ = 2.76 ppm), DMSO in [D6]-
DMSO (δ = 2.49 ppm), and DDS in D2O (δ = 0 ppm) for 1H
NMR, and CDCl3 (δ = 77.0 ppm), [D7]DMF (δ = 162.5 ppm), [D6]-
Scheme 6. Synthesis of cyclic nucleotide analogues.
DMSO (δ = 39.7 ppm) and dioxane in D2O (δ = 67.4 ppm) for 13
C
Conformation Properties of the Cyclic Nucleotide Analogue
9b
NMR spectroscopy. Column chromatography was performed with
Wako silica gel C-200. Amino-modified silica gel was purchased
from Fuji-silycia Co., Ltd. Recycle HPLC was performed on a
JALGEL GS-310 column with use of CH3CN as a solvent. TLC
was performed with Merck silica gel 60 (F254) plates. ESI mass
spectra were measured on a MarinerTM instrument.
As described above, the 2Ј-deoxyribo- and ribonucleotide
analogues 3–8 and the cyclic nucleotide analogues 9a and
9b were successfully synthesized. The cyclic nucleotide ana-
logues differ from naturally occurring cyclic AMP and
GMP in that the oxygen atoms at the 5Ј- and the 3Ј-posi-
tions are replaced by nitrogen atoms and the phosphorus
atoms are replaced by a cyclobutene ring. It should there-
fore be interesting to elucidate the effects of these two modi-
fications on the sugar conformations of the cyclic nucleo-
N-Methylsquaramide (10): Diethyl squarate (1 g, 5.9 mmol) was dis-
solved in ethanol (30 mL). Methylamine (40% in methanol,
0.6 mL, 5.9 mmol) was added, and the resulting solution was
stirred for 15 min at ambient temperature. The solvents were re-
moved under reduced pressure, and the residue was dissolved in
NaOH (1 , 5.9 mL, 5.9 mmol). After 12 h, the solution was con-
centrated under reduced pressure and was then diluted with meth-
anol (10 mL). Dowex 50 W×8 (H+ form, 5.9 mmol) was added and
removed by filtration. The solvent was removed under reduced
pressure to give 10 (5.7 mmol, 97%). 1H NMR (270 MHz, [D6]-
DMSO): δ = 3.30 (s, 3 H), 4.59 (br., 1 H) ppm. 13C NMR
(67.8 MHz, [D6]DMSO): δ = 29.7, 182.0, 188.4 ppm. ESI-MS
calcd. for C5H5NO3 [M + H]+: 127.0269; found 127.0270.
tide analogues. The proton–proton coupling constants J1Ј2Ј
,
J2Ј3Ј, and J3Ј4Ј of the ribose moieties in cAMP and cGMP
have been reported to be 0.7–0.8 Hz, 5.2–5.7 Hz, and 9–
10 Hz, respectively.[20] The splitting pattern of these cyclic
nucleotides cAMP and cGMP is characteristic of N-type
sugar puckering. Significantly, the coupling constants J1Ј2Ј
,
J2Ј3Ј, and J3Ј4Ј in 9b were quite similar to those of the cyclic
nucleotides and proved to be 0 Hz, 5.4 Hz, and 9.0 Hz,
respectively. This splitting pattern is characteristic of the N-
type conformation. These results suggested that the change
of the two hydroxy groups on the ribose ring and the phos-
phorus atom for the squaramide-type structure produced
a ribose conformation identical to that in the 3Ј,5Ј-cyclic
nucleotide.
General Procedure 1. Reactions between 5Ј- or 3Ј-Aminonucleoside
and Dimethyl Squarate
5Ј-Amino-5Ј-N-(2-methoxy-3,4-dioxocyclobuten-1-yl)-5Ј-deoxy-
thymidine (12a): 5Ј-Amino-5Ј-deoxythymidine (11a, 300 mg,
1.2 mmol) was dissolved in methanol (4 mL), and N,N-diisopropy-
lethylamine (110 µL, 0.62 mmol) and 3,4-dimethoxy-3-cyclobutene-
1,2-dione (dimethyl squarate, 180 mg, 1.2 mmol) were added. The
resulting solution was stirred at ambient temperature for 15 min.
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FULL PAPER
The solvent was removed under reduced pressure, and the residue
was chromatographed on a column of silica gel with chloroform/
in acetic acid (80%, 2.0 mL). The reaction mixture was stirred at
ambient temperature for 3.5 h. The solvent was removed under re-
methanol (100:6, v/v) to give 12a (380 mg, 87 %). 1H NMR duced pressure, and the residue was coevaporated twice with water
(270 MHz, D2O): δ = 2.27–2.43 (m, 2 H), 3.46–3.87 (m, 3 H), 4.15,
(2×5 mL). The residue was chromatographed on a column of silica
4.18 (2×s, 3 H), 4.29, 4.42 (m, 1 H), 6.01–6.06 (t, 1 H), 7.20, 7.22 gel with chloroform/methanol (100:7, v/v) to give 15 (59 mg, 60%).
(2×s, 1 H) ppm. 13C NMR (67.8 MHz, D2O): δ = 12.4, 12.5, 40.1,
47.1, 61.1, 72.2, 72.4, 86.1, 86.3, 86.5, 111.8, 137.8, 138.0, 152.1,
166.1, 174.9, 178.2, 185.0, 185.3, 189.4 ppm. ESI-MS calcd. for
C15H17N3O7 [M + H]+: 351.1067; found 351.1216.
1H NMR (270 MHz, [D6]DMSO): δ = 1.70 (s, 3 H), 2.22–2.27 (m,
2 H), 3.55 (m, 2 H, m), 4.22 (s, 2 H), 4.84 (s, 3 H), 6.09–6.14 (m,
1 H), 7.65, 7.69 (2×s, 1 H), 8.45–8.48, 8.88–9.07 (br., 1 H), 11.21
(br., 1 H) ppm. 13C NMR (67.8 MHz, [D6]DMSO): δ = 12.5, 37.6,
44.6, 53.5, 60.7, 83.4, 84.4, 84.5, 109.3, 136.2, 150.3, 163.6, 171.6,
174.2, 177.3, 177.8, 182.3, 182.7, 187.0, 188.9 ppm. ESI-MS calcd.
for C15H18N3O7 [M + H]+: 352.1145; found 352.1158.
General Procedure 2. Synthesis of Nucleoside 5Ј-Squarate by Alka-
line Hydrolysis. 5Ј-Amino-5Ј-N-(2-hydroxy-3,4-dioxocyclobuten-1-
yl)-5Ј-deoxythymidine (3) from 12a: Compound 12a (100 mg,
0.28 mmol) was dissolved in methanol (2.0 mL), aqueous NaOH
(1 , 1.1 mL, 1.1 mmol) was added, and the resulting solution was
stirred at ambient temperature for 4 h. The reaction mixture was
neutralized by addition of Dowex 50W×8 (2 mL, free form). The
resin was removed by filtration, and the filtrate was concentrated
under reduced pressure. The residue was chromatographed on a
C18 reversed-phase column with CH3CN/H2O (100:5, v/v) to give
3Ј-Amino-3Ј-N-(2-hydroxy-3,4-dioxocyclobuten-1-yl)-3Ј-deoxy-
thymidine (5): Aqueous NaOH (1.0 , 1.1 mL) was added to a solu-
tion of compound 15 (100 mg, 0.28 mmol) in ethanol (2 mL), and
the solution was stirred at 45 °C for 14 h. The reaction mixture
was neutralized by addition of Dowex 50W×8 (2 mL), filtered, and
concentrated under reduced pressure. The residue was chromato-
graphed on a C18 reversed-phase column with CH3CN/H2O
(100:1, v/v) to give 5 (71 mg, 75%). 1H NMR (270 MHz, [D6]-
DMSO): δ = 1.70 (s, 3 H), 2.22–2.27 (m, 2 H), 3.55 (m, 2 H), 4.22
(s, 2 H), 6.09–6.14 (m, 1 H), 7.69 (s, 1 H), 8.46, 8.88 (br., 1 H),
11.21 (br., 1 H) ppm. 13C NMR (67.8 MHz, [D6]DMSO): δ = 12.5,
37.6, 44.6, 53.5, 83.7, 84.7, 109.5, 136.5, 150.3, 163.6, 182.3, 188.0,
198.3 ppm. ESI-MS calcd. for C14H16N3O7 [M + H]+: 338.0988;
found 338.0972.
1
3 (54 mg, 57%). H NMR (270 MHz, [D6]DMSO): δ = 1.76 (s, 3
H), 2.03–2.05 (m, 2 H), 3.59–3.79 (m, 3 H), 4.19 (m, 1 H), 6.09–
6.14 (t, 3J(H,H) = 6.8 Hz, 1 H), 7.19 (br., 1 H), 7.45 (s, 1 H), 11.26
(br., 1 H) ppm. 13C NMR (67.8 MHz, CD3OD): δ = 12.1, 44.5,
70.4, 83.7, 85.7, 109.6, 135.64, 150.3, 163.6, 181.2, 188.3 ppm. ESI-
MS calcd. for C14H16N3O7 [M + H]+: 338.0988; found 338.0998.
5Ј-Amino-5Ј-N-(2-methoxy-3,4-dioxocyclobuten-1-yl)-2Ј,5Ј-dideoxy-
cytidine (12b): Compound 11b (100 mg, 0.44 mmol) was converted
into the title compound 12b (120 mg, 66%) as described in General
N4-(4,4Ј-Dimethoxytrityl)cytidine (16): Cytidine (973 mg, 4 mmol)
was dissolved in pyridine (20 mL), and chlorotrimethylsilane
(3.8 mL, 30 mmol) was added. After 1 h, DMTrCl (1.6 g,
4.8 mmol) was added, and the resulting solution was stirred for 8 h.
The reaction was quenched by addition of aq. NaHCO3, and the
mixture was diluted with ethyl acetate (60 mL). The organic layer
was washed twice with saturated aqueous NaCl (30 mL), dried with
MgSO4, filtered, and concentrated under reduced pressure. The res-
idue was chromatographed on a column of silica gel with chloro-
form/methanol (100:3, v/v) to give 16 (1.7 g, 79 %). 1H NMR
(500 MHz, CDCl3): δ = 3.70–3.80 (m, 2 H), 3.77 (s, 6 H), 4.08 (m,
1
Procedure 1. H NMR (270 MHz, [D6]DMSO): δ = 1.89–2.12 (m,
2 H), 3.44–3.68 (m, 2 H), 3.80 (m, 1 H), 4.14 (m, 1 H), 4.26 (2×s,
3
3 H), 5.36 (br., 1 H), 5.72–5.74 (m, 1 H), 6.11–6.16 (t, JH,H
=
3
6.8 Hz, 1 H), 7.16, 7.23 (br., 2 H), 7.51 (d, JH,H = 7.3 Hz, 1 H),
8.76, 8.96 (br., 1 H) ppm. 13C NMR (67.8 MHz, [D6]DMSO): δ =
45.8, 46.2, 60.0, 60.2, 70.8, 79.2, 84.6, 84.8, 94.2, 140.8, 154.8,
165.3, 172.0, 172.5, 176.9, 177.5, 182.1, 182.3, 188.9, 189.3 ppm.
ESI-MS calcd. for C14H17N4O6 [M + H]+: 337.1148; found
337.1078.
3
3
1 H), 4.27 (t, JH,H = 4.9 Hz, 1 H), 4.37 (t, JH,H = 4.6 Hz, 1 H),
5Ј-Amino-5Ј-N-(2-hydroxy-3,4-dioxocyclobuten-1-yl)-2Ј,5Ј-dideoxy-
cytidine (4): Compound 12b (100 mg, 0.3 mmol) was converted into
the title compound 4 (74 mg, 77%) as described in General Pro-
cedure 2. 1H NMR (270 MHz, D2O): δ = 2.13–2.35 (m, 2 H), 3.56–
3.81 (m, 2 H), 3.90–3.97 (m, 1 H), 4.14–4.33 (m, 1 H), 5.90 (d,
3
3
5.11 (d, JH,H = 7.6 Hz, 1 H), 5.57 (d, JH,H = 4.2 Hz, 1 H), 6.80
(d, JH,H = 8.1 Hz, 4 H), 7.09–7.29 (m, 9 H), 7.47 (d, JH,H
3
3
=
7.6 Hz, 1 H) ppm. 13C NMR (125.7 MHz, CDCl3): δ = 50.5, 55.1,
55.1, 63.0, 70.8, 72.3, 73.7, 77.2, 87.7, 91.2, 113.1, 121.6, 123.9,
126.8, 127.8, 128.6, 129.9, 130.0, 136.8, 139.9, 145.0, 147.0, 149.2,
151.6, 154.3, 158.2, 158.2 ppm. ESI-MS calcd. for C30H32N3O7 [M
+ H]+: 546.2240; found 546.9822.
3
3JH,H = 7.6 Hz, 1 H), 6.03, 6.07 (2×t, JH,H = 6.3 Hz, 6.7 Hz, 1
H), 7.50 (d, 3JH,H = 7.6 Hz, 1 H) ppm. 13C NMR (67.8 MHz, [D6]-
DMSO): δ = 41.0, 41.2, 47.3, 51.5, 51.7, 61.9, 73.2, 87.3, 87.5, 88.3,
88.6, 98.6, 98.9, 133.2, 143.5, 143.9, 153.3, 158.9, 159.1, 167.8,
175.7,184.0, 188.8, 190.4, 197.4, 205.4 ppm. ESI-MS calcd. for
C13H14N4O6Na [M + Na]+: 345.0811; found 345.0835.
N6-(4,4Ј-Dimethoxytrityl)adenosine (17): Adenosine (1.1 g, 4 mmol)
1
was converted into 17 (1.4 g, 61%) as described for 16. H NMR
(500 MHz,CDCl3): δ = 3.58–3.67 (m, 1 H), 3.71 (s, 6 H), 3.80 (d,
3JH,H = 12.4 Hz, 1 H), 4.15 (brs, 2 H), 4.83 (brs, 1 H), 5.19 (br., 1
3Ј-Amino-5Ј-O-(4,4Ј-dimethoxytrityl)-3Ј-N-(2-methoxy-3,4-dioxocy-
clobuten-1-yl)-3Ј-deoxythymidine (14): 3Ј-Amino-5Ј-O-(4,4Ј-di-
methoxytrityl)-3Ј-deoxythymidine (13, 200 mg, 0.37 mmol) was
converted into the title compound (180 mg, 76%) as described in
3
H), 5.57 (d, JH,H = 7.8 Hz, 1 H), 6.71–6.74 (m, 4 H), 7.14–7.26
( m , 9 H ) , 7 . 5 7 ( s, 1 H ) , 7 . 9 4 ( s, 1 H ) p p m . 1 3 C N MR
(125.7 MHz,CDCl3): δ = 55.1, 63.0, 70.8, 72.3, 73.7, 87.7, 91.3,
113.1, 121.7, 125.2, 126.8, 127.8, 128.2, 128.7, 129.0, 130.0, 130.0,
136.95, 139.9, 145.0, 147.1, 151.7, 154.3, 158.3 ppm. ESI-MS calcd.
for C31H32N5O6 [M + H]+: 570.2353; found 570.2294.
1
General Procedure 1. H NMR (270 MHz, CDCl3): δ = 1.70 (s, 3
H), 2.22–2.27 (m, 2 H), 3.79 (s, 6 H), 4.22 (s, 2 H), 4.84 (s, 3 H),
6.09–6.14 (m, 1 H), 6.83–6.86 (m, 4 H), 7.62–7.65 (m, 10 H), 7.69
(2 × s, 1 H), 8.45–8.48, 8.88, 9.07 (m, 1 H) ppm. 13C NMR
(67.8 MHz, CDCl3): δ = 11.6, 55.3, 56.6, 63.6, 79.4,84.5, 86.2, 87.2,
113.0, 127.3, 127.9, 128.1, 130.0,134.6, 143.7, 151.8, 158.7,163.0,
170.4, 178.1, 181.1, 190.6 ppm. ESI-MS calcd. for C36H36N3O9 [M
+ H]+: 654.2452; found 654.2488.
5Ј-Azido-5Ј-deoxy-N4-(4,4Ј-dimethoxytrityl)cytidine (18): Com-
pound 16 (3.0 g, 5.5 mmol) was dissolved in DMF (6 mL), tri-
phenylphosphane (2.9 g, 11 mmol), lithium azide (1.4 g,
27.5 mmol), and tetrabromomethane (3.6 g, 11 mmol) were added,
and the resulting solution was stirred for 1 h. Water (3 mL) and
then ethyl acetate (10 mL) were added. The aqueous layer was re-
moved, and the organic layer was washed five times with water
3Ј-Amino-3Ј-N-(2-methoxy-3,4-dioxocyclobuten-1-yl)-3Ј-deoxy-
thymidine (15): Compound 14 (180 mg, 0.28 mmol) was dissolved
Eur. J. Org. Chem. 2005, 5163–5170
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
5167

K. Seio, T. Miyashita, K. Sato, M. Sekine
FULL PAPER
3
(10 mL). The organic layer was dried with Na2SO4, filtered, and
concentrated under reduced pressure. The residue was chromato-
graphed on a column of silica gel with chloroform/methanol (100:4,
v/v) to give 18 (1.2 g, 38%). 1H NMR (500 MHz, CDCl3): δ =
3.50–3.78 (m, 2 H), 3.79 (s, 6 H), 4.06–4.08 (m, 1 H), 4.14–4.16 (m,
5.68 (m, 1 H), 5.90–5.91 (d, JH,H = 4.6 Hz, 1 H), 7.73–7.75 (d,
3JH,H = 8.1 Hz, 1 H, 6-H), 8.27, 8.93 (br., 1 H, NH), 11.37 (br., 1
H, NH) ppm. 13C NMR(125.7 MHz, [D7]DMF):δ = 46.5, 47.0,
60.5, 60.6, 71.6, 73.7, 73.8, 83.5, 83.8, 89.8, 90.1, 102.6, 102.6,
141.8, 151.6, 163.8, 173.4, 173.9, 178.2, 178.8, 183.5, 183.8, 189.9,
190.4 ppm. ESI-MS calcd. for C14H16N3O8 [M + H]+: 354.0937;
found 354.0939
3
1 H), 4.19–4.22 (m, 1 H), 5.15 (d, JH,H = 7.8 Hz, 1 H), 5.75–5.76
3
(d, 1 H, JH,H = 4.2 Hz), 6.82–6.84 (m, 4 H), 7.12–7.50 (m, 13
H) ppm. 13C NMR (67.8 MHz, [D6]DMSO): δ = 12.5, 37.6, 44.6,
53.5, 60.7, 83.4, 84.4, 84.5, 109.3, 136.2, 150.3, 163.6, 171.6, 174.2,
177.3, 177.8, 182.3, 182.7, 187.0, 188.9 ppm. ESI-MS calcd. for
C15H18N3O7 [M + H]+: 352.1145; found 352.1158.
5Ј-Amino-5Ј-N-(2-methoxy-3,4-dioxocyclobuten-1-yl)-5Ј-deoxy-4-N-
(4,4Ј-dimethoxytrityl)cytidine (24): Compound 21 (544 mg,
1.0 mmol) was condensed with dimethyl squarate for 1 h as de-
scribed in General Procedure 1 to give 24 (448 mg, 68%). 1H NMR
(500 MHz, [D6]DMSO,75 °C): δ = 3.59–3.74 (m, 2 H, 5Ј-, 5ЈЈ-H),
3.74 (s, 6 H, OMe of DMTr), 3.82–3.88 (m, 2 H, 4Ј-H, 3Ј-H), 3.96
(m, 1 H, 2Ј-H), 4.23 (s, 3 H, OMe of squaric acid), 4.86 (br., 1 H,
5Ј-Azido-5Ј-deoxy-N6-(4,4Ј-dimethoxytrityl)adenosine (19): Com-
pound 17 (8.5 g, 15 mmol) was converted into 19 (4.4 g, 50%) as
1
described for 18. H NMR (500 MHz, CDCl3): δ = 3.48–3.66 (m,
3
3
2 H), 3.76 (s, 6 H), 4.28–4.29 (m, 2 H), 4.47–4.49 (t, JH,H
=
OH), 5.06 (br., 1 H, OH), 5.63, 5.64 (d, JH,H = 4.9 Hz, 1 H, 1Ј-
3
4.88 Hz, 1 H), 5.89–5.90 (d, JH,H = 5.13, 1 H), 6.77–6.79 (m, 5
H), 7.01–7.97 (m, 8 H), 8.00 (s, 1 H), 8.03 (s, 1 H) ppm. 13C NMR
(125.7 MHz, CDCl3): δ = 52.4, 55.4, 71.0, 71.7, 72.8, 75.2, 75.4,
77.5, 84.3, 90.2, 113.4, 121.2, 127.1, 128.1, 129.0, 130.3, 137.4,
138.2, 145.3, 147.9, 152.0, 154.5, 158.5 ppm. ESI-MS calcd. for
C31H31N8O5 [M + H]+: 595.2417; found 595.2466.
H), 6.83–7.28 (14 H, m, 6-H, DMTr), 7.55 (1 H, br., NH) ppm.
13C NMR (125.7 MHz, [D6]DMSO, 75 °C): δ = 45.7, 54.9, 59.4,
59.7, 69.4, 70.5, 72.7, 81.8, 90.1, 112.8, 126.1, 127.3, 128.3, 129.2,
129.6, 136.7, 144.9, 153.9, 157.6, 169.9, 172.5, 182.5, 189.0 ppm.
ESI-MS calcd. for C35H35N4O9 [M + H]+: 655.24; found 655.3326.
5Ј-Amino-5Ј-N-(2-methoxy-3,4-dioxocyclobuten-1-yl)-5Ј-deoxy-6-N-
(4,4Ј-dimethoxytrityl)adenosine (25): Compound 22 (853 mg,
1.5 mmol) was condensed with dimethyl squarate for 3 h as de-
scribed in General Procedure 1 to give 25 (319 mg, 31%). 1H NMR
([D6]DMSO, 75 °C): δ = 3.70–3.77 (m, 8 H, 5ЈH, 5ЈЈH, Me of
DMTr), 4.05–4.08 (m, 1 H, 4Ј-H), 4.19 (m, 4 H, 3Ј-H, OMe), 5.08–
5.09 (m, 1 H, 2Ј-H), 5.29 (br., 1 H, OH), 5.30 (br., 1 H, OH), 5.80–
5Ј-Amino-5Ј-deoxy-N4-(4,4Ј-dimethoxytrityl)cytidine (21): Com-
pound 18 (1.1 g, 2.0 mmol) was dissolved in pyridine (20 mL), tri-
phenylphosphane (1.0 g, 3.9 mmol) was added, and the resulting
solution was stirred for 6 h. Concentrated ammonia (10 mL) was
added, and the solution was stirred for 12 h. The solvents were
removed under reduced pressure, and the residue was chromato-
graphed on an amino-modified silica gel column with chloroform/
methanol (100:3, v/v) to give 21 (627 mg, 69 %). 1H NMR
3
5.88 (d, JH,H = 5.86 Hz, 1 H, 1Ј-H), 6.83–6.89 (m, 4 H, DMTr),
7.12–7.31 (m, 11 H, DMTr), 7.96 (br., 1 H, NH) 8.24 (s, 1 H, 2-
H), 8.29 (s, 1 H, 8-H), 8.92 (br., 1 H, NH) ppm. 13C NMR
(125.7 MHz, [D7]DMF): δ = 55.5, 60.3, 70.9, 72.1, 73.9, 84.7, 113.4,
113.7, 127.1, 127.9, 128.2, 128.4, 129.3, 129.7, 130.6, 138.3, 141.1,
141.2, 141.5, 146.3, 149.0, 152.1, 154.9, 159.1, 173.7, 178.4, 183.8,
190.0 ppm. ESI-MS calcd. for C36H35N6O8 [M + H]+: 679.2516;
found 679.2541.
3
(500 MHz, CDCl3): δ = 2.86–2.90 (dd, JH,H = 4.9 Hz, 5.4 Hz, 1
H), 2.98–3.01 (dd, J = 3.9 Hz, 3.2 Hz, 1 H), 3.78 (s, 6 H), 4.00–
4.03 (m, 2 H), 4.15 (t, 3JH,H = 4.0 Hz, 1 H), 5.09 (d, 3JH,H = 7.6 Hz,
3
1 H), 5.67 (s, JH,H = 2.9 Hz, 1 H), 6.81–6.83, (m, 5 H), 7.11–7.69
(m, 14 H) ppm. 13C NMR (125.7 MHz, [D6]DMSO): δ = 43.4, 55.1,
69.5, 70.5, 73.4, 79.3, 84.7, 88.9, 96.7, 112.9, 113.6, 126.2, 126.5,
127.6, 128.6, 129.6, 129.7, 129.7, 130.1, 132.1, 132.1, 132.2, 133.8,
137.1, 140.6, 145.3, 154.4, 157.6, 163.4 ppm. ESI-MS calcd. for
C30H33N4O6 [M + H]+: 545.2400; found 545.2419.
5Ј-Amino-5Ј-N-(2-hydroxy-3,4-dioxocyclobuten-1-yl)-5Ј-deoxy-
cytidine Sodium Salt (7): Compound 24 (178 mg, 0.27 mmol) and
NaI (49 mg, 0.32 mmol) were dissolved in hexan-2-one. The reac-
tion mixture was stirred at 120 °C for 19 h. The solvent was re-
moved under reduced pressure, and the resulting residue was chro-
matographed on a C-18 column with water/CH3CN (5:95, v/v) to
give 7 (52 mg, 57%). 1H NMR (500 MHz, D2O): δ = 4.27–4.28,
5Ј-Amino-5Ј-deoxy-N6-(4,4Ј-dimethoxytrityl)adenosine (22): Com-
pound 19 (3.4 g, 5.7 mmol) was dissolved in pyridine (10 mL), tri-
phenylphosphane (3.0 g, 11.5 mmol) was added, and the resulting
solution was stirred for 4 h. Concentrated ammonia (10 mL) was
added, and the solution was stirred for 14 h. The mixture was di-
luted with ethyl acetate (100 mL) and was then washed with water
(100 mL) and twice with saturated NaCl (100 mL). The organic
layer was dried with MgSO4, filtered, and concentrated under re-
duced pressure. The residue was chromatographed on an amino-
modified silica gel column with chloroform/methanol (100:5) to
3
4.30, 4.31–4.40, 4.43, (m, 2 H), 4.61–4.63 (dd, JH,H = 4.9 Hz,
3
11.2 Hz, 1 H), 4.72 (dd, JH,H = 3.9 Hz, 4.6 Hz, 1 H), 5.13–5.14
3
3
(m, 1 H), 6.3 (d, JH,H = 3.7 Hz, 1 H), 6.4 (d, JH,H = 7.6 Hz, 1
H), 7.99–8.00 (d, 1 H, 3JH,H = 7.6 Hz) ppm. 13C NMR (125.7 MHz,
D2O): δ = 44.2, 69.7, 73.3, 81.9, 90.1, 96.1, 141.2, 157.2, 165.8,
181.5, 188.1, 195.0 ppm. ESI-MS calcd. for C13H15N4O7
[M + H]+: 339.09407; found 340.2517.
1
give 22 (2.7 g, 83%). H NMR (500 MHz, CDCl3): δ = 2.85–2.99
(m, 2 H), 3.74 (s, 6 H), 4.09–4.12 (m, 1 H), 4.29–4.27 (m, 1 H),
3
4.58–4.61 (t, 3JH,H = 5.25 Hz, 1 H), 5.80–5.81 (d, JH,H = 5.37 Hz,
5Ј-Amino-5Ј-N-(2-hydroxy-3,4-dioxocyclobuten-1-yl)-5Ј-deoxy-
uridine Sodium Salt (6): Compound 23 (106 mg, 0.3 mmol) was
treated with NaI in hexan-2-one for 13 h as described for 7 to give
6 (67 mg, 59%). 1H NMR (500 MHz, D2O): δ = 3.92–3.96 (dd,
3JH,H = 5.6, 14.6 Hz, 1 H), 4.07–4.10 (dd, 3JH,H = 3.7 Hz, 14.6 Hz,
1 H), 6.75–6.78 (m, 4 H), 7.17–7.31 (m, 9 H), 7.91(s, 1 H), 7.95 (s,
1 H) ppm. 13C NMR (125.7 MHz, CDCl3): δ = 45.4, 55.1, 70.6,
71.5, 74.4, 85.9, 89.8, 113.0, 121.2, 126.8, 127.8, 128.7, 130.0, 137.2,
138.7, 145.1, 147.9, 151.9, 154.2, 158.2 ppm. ESI-MS calcd. for
C31H33N6O5 [M + H]+: 569.2512; found 569.2557.
3
1 H), 4.26–4.29 (dd, JH,H = 5.7 Hz, 9.6 Hz, 1 H), 4.36–4.38 (t,
3
5Ј-Amino-5Ј-N-(2-methoxy-3,4-dioxocyclobuten-1-yl)-5Ј-deoxy-
uridine (23): Compound 20 (1.2 g, 5.0 mmol) was condensed with
dimethyl squarate for 1 h as described in General Procedure 1 to
give 23 (695 mg, 39%). 1H NMR (500 MHz, [D7]DMF): δ = 3.70–
3JH,H = 5.7 Hz, 1 H), 4.51–4.53 (t, JH,H = 4.4 Hz, 5.1 Hz, 1 H),
3
3
5.92 (d, JH,H = 3.7 Hz, 1 H), 5.92–5.94 (d, JH,H = 8.0 Hz, 1 H),
7.69–7.71 (d, JH,H = 8.0 Hz, 1 H) ppm. 13C NMR (125.7 MHz,
3
D2O): δ = 47.2, 72.6, 75.8, 85.2, 92.8, 105.2, 144.8, 154.2, 168.7,
3.77 (m, 2 H, 5Ј-H, 5ЈЈ-H), 3.93–3.95 (m, 1 H, 4Ј-H), 4.03–4.04 (m, 184.4, 190.8, 197.9, 205.7 ppm. ESI-MS calcd. for C13H14N3O8 [M
1 H, 3Ј-H), 4.04–4.06 (m, 1 H, 3Ј-H), 4.33–4.35 (m, 3 H), 5.63–
+ H]+: 340.0781; found 340.0802.
5168
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© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2005, 5163–5170

Squaramide Moieties as Phosphate Isosters in Nucleotides
5Ј-Amino-5Ј-N-(2-hydroxy-3,4-dioxocyclobuten-1-yl)-5Ј-deoxy- the resulting solution was stirred at ambient temperature. After 1 h,
FULL PAPER
adenosine Sodium Salt (8): Compound 25 (102 mg, 0.15 mmol) was
treated with NaI in hexan-2-one for 15 h as described for 7 to give
8 (45 mg, 79%). 1H NMR (500 MHz, D2O): δ = 3.70–3.84 (m, 2
4,4Ј-dimethoxytrityl chloride (948 mg, 2.8 mmol) was added, and
the mixture was stirred at ambient temperature for 8.5 h. The reac-
tion was quenched by portionwise addition of saturated NaHCO3
(100 mL). After 1 h, the products were extracted twice with ethyl
H), 4.17–4.20 (m, 1 H), 4.42–4.44 (t, 3JH,H = 5.1 Hz, 5.4 Hz, 1 H),
3
4.77–4.79 (m, 1 H), 5.92 (d, JH,H = 5.1 Hz, 1 H), 8.07 (s, 1 H), acetate (20 mL), and the organic layer was dried with Na2SO4, fil-
8.13 (s, 1 H) ppm. 13C NMR (500 MHz, D2O): δ = 43.9, 70.2, 72.9,
83.1, 88.0, 118.7, 140.1, 148.5, 152.5, 155.2, 181.4, 187.7,
194.7 ppm. ESI-MS calcd. for C14H14N6O6 [M + H]+: 362.0975;
found 362.0668.
tered, and concentrated under reduced pressure. The residue was
chromatographed on a column of silica gel with chloroform/hexane
(5:1, v/v) to give crude 30. The crude material was dissolved in
pyridine (120 mL), triphenylphosphane (950 mg, 3.6 mmol) was
added, and the mixture was stirred at ambient temperature for 14 h.
Aqueous ammonia (28%, 12 mL) was added, and the solution was
stirred for 23 h. The solvents were removed under reduced pressure,
and the residue was dissolved in ethyl acetate (30 mL), washed
three times with saturated aqueous NaCl (50 mL), and concen-
trated under reduced pressure. The residues was chromatographed
on a silica gel column with chloroform/methanol/triethylamine
(100:10:1, v/v/v) to give 32 (32% from 28). 1H NMR (500 MHz,
CDCl3): δ = 2.97–3.11 (m, 2 H), 3.76–3.80 (m, 7 H), 3.88–3.91 (m,
1 H), 4.45–4.47 (m, 1 H), 5.89 (d, 3JH,H = 3.17 Hz, 1 H), 6.78–6.80
(m, 5 H), 7.21–7.33 (m, 8 H), 7.97 (s, 1 H), 8.01 (s, 1 H, 8-H) ppm.
13C NMR (125.7 MHz, CDCl3): δ = 43.3, 53.6, 55.0, 70.4, 75.1,
85.6, 90.8, 112.9, 121.2, 126.6, 127.7, 128.6, 129.9, 137.2, 138.4,
145.1, 147.7, 151.9, 154.0, 158.1 ppm. ESI-MS calcd. for
C31H34N7O4 [M + H]+: 568.2672; found 568.2672.
1,2-O-Isopropylidene-3,5-O-bis(trifluoromethylsulfonyl)-α-D-xylose
(26): 1,2-O-Isopropylidene-α--xylose (11.4 g, 60 mmol) was ren-
dered anhydrous by repeated coevaporation with toluene, and
CH2Cl2 (360 mL) and N,N-diisopropylethylamine (28 mL,
162 mmol) were added. The resulting mixture was cooled to –78 °C,
and trifluoromethanesulfonic anhydride (29 mL, 171 mmol) was
then added dropwise over 1 h. After the reaction was complete, the
organic solution was washed twice with water (100 mL), and then
with saturated NaHCO3. The organic layer was dried with Na2SO4,
filtered, and evaporated under reduced pressure. The residue was
chromatographed on a column of silica gel with hexane/ethyl ace-
tate (100:1, v/v) to give 26 (20 g, 75 %). 1H NMR (500 MHz,
CDCl3): δ = 1.34 (s, 3 H, CH3), 1.52 (s, 3 H,CH3), 4.59–4.79 (m, 3
3
H, 3-H, 4-H, 5-H), 5.24–5.25 (dd, JH,H = 2.2 Hz, 1 H, 2-H), 6.04
(d, 3JH,H = 3.7 Hz, 1-H) ppm. 13C NMR (125.7 MHz, CDCl3): δ =
26.1, 26.5, 70.4, 75.6, 83.0, 86.7, 104.7, 113.7, 117,0, 119.6,
119.7 ppm. C10F6O9S2·H2O: C 25.43, H 2.99, S 13.58, F 24.13;
found C 25.45, H 2.85, S 13.84, F 24.86.
N2-(4,4-Dimethoxytrityl)-3,5-diaminoguanosine (33): Compound 31
(418 mg, 0.66 mmol) was dissolved in dry pyridine (6 mL), and tri-
phenylphosphane (690 mg, 2.6 mmol) was added. After 24 h, aque-
ous ammonia (28%, 12 mL) was added, and the mixture was stirred
for 15 h. Water (20 mL) was added, and the material was extracted
three times with ethyl acetate (10 mL). The organic layers were
combined, dried with Na2SO4, filtered, and concentrated under re-
duced pressure. The residue was chromatographed on a column of
silica gel with chloroform/methanol (100:7, v/v) to give 33 (226 mg,
1,2-O-Isopropylidene-3,5-diazido-α-D-xylose (27): Compound 26
(454 mg, 1 mmol) was dissolved in DMF (1 mL), lithium azide
(245 mg, 5 mmol) was added, and the resulting mixture was stirred
at 40 °C for 4 h. The solution was diluted with diethyl ether
(10 mL) and was then washed with water (10 mL) and three times
with saturated aqueous NaCl (10 mL). The organic layer was dried
with MgSO4, filtered, and concentrated under reduced pressure.
The residue was chromatographed on a column of silica gel with
hexane/diethyl ether (100:3, v/v) to give 27 (84 mg, 42 %). The
structure was confirmed by comparison of the spectroscopic data
with those reported in the literature.[18]
1
59%). H NMR (500 MHz, [D6]DMSO): δ = 2.62–2.74 (m, 2 H),
3.33–3.34 (m, 1 H), 3.72–3.75 (m, 8 H), 5.17–5.18 (m, 2 H), 6.86–
6.87 (m, 5 H), 7.19–7.32 (m, 12 H), 7.56 (s, 1 H), 7.81 (s, 1 H), 8.32
(br., 1 H) ppm. 13C NMR (125.7 MHz, [D6]DMSO): δ = 43.7, 54.1,
55.0, 69.4, 73.6, 79.2, 84.9, 89.3, 112.9, 113.0, 117.7, 126.4, 127.7,
128.2, 129.7, 129.7, 136.9, 137.2, 145.2, 149.0, 150.8, 156.5,
157.6 ppm. ESI-MS calcd. for C31H34N7O5 [M + H]+: 584.2621;
found 584.2673.
N2-(4,4-Dimethoxytrityl)-3,5-diazidoguanosine (31): Compound 29
(457 mg, 1.4 mmol) was rendered anhydrous by repeated coevapo-
ration with dry pyridine. The residue was dissolved in dry pyridine
(13 mL), and chlorotrimethylsilane (433 µL, 3.4 mmol) was added.
After 1 h, 4,4Ј-dimethoxytrityl chloride (555 mg, 1.6 mmol) was
added, and the resulting mixture was stirred for 24 h. The reaction
was quenched by addition of saturated NaHCO3 (13 mL). The ma-
terial was extracted twice with ethyl acetate (50 mL), and the or-
ganic layers were combined, dried with Na2SO4, filtered, and con-
centrated under reduced pressure. The residue was chromato-
graphed on an amino-modified silica gel column with chloroform/
methanol (100:5, v/v) to give 31 (510 mg, 48 %). 1H NMR
(500 MHz, CDCl3): δ = 3.22–3.39 (m, 2 H), 3.43 (s, 6 H), 3.91 (m,
2 H), 4.54 (m, 1 H), 5.53 (s, 1 H), 6.45–6.47 (m, 4 H), 6.79–7.01
(m, 9 H), 7.63 (s, 1 H), 7.68 (s, 1 H) ppm. 13C NMR (125.7 MHz,
CDCl3): δ = 52.0, 55.3, 62.0, 70.9, 75.8, 77.5, 81.2, 90.4, 90.4, 113.3,
121.2, 127.0, 128.0, 128.9, 130.2, 137.4, 137.4, 138.5, 145.3, 147.8,
152.2, 152.2, 154.4, 158.4 ppm. ESI-MS calcd. for C31H30N11O5 [M
+ H]+: 636.2431; found 636.2416.
N6-(4,4Ј-Dimethoxytrityl)-3Ј,5Ј-diamino-3Ј,5Ј-N-(3,4-dioxocyclobut-
ene-1,2-diyl)adenosine (34): N,N-Diisopropylethylamine (63 µL,
0.37 mmol) and dimethyl squarate (104 mg, 0.73 mmol) were added
to a solution of compound 32 (414 mg, 0.73 mmol) in methanol
(1 mL). The mixture was stirred at ambient temperature for 1 h,
the solvent was removed under reduced pressure, and the residue
was chromatographed on a column of silica gel with chloroform/
methanol (100:5, v/v) to give 34 (208 mg, 32 %). 1H NMR
(500 MHz, CDCl3): δ = 3.77–3.80 (m, 7 H), 3.99–4.01 (m, 1 H),
4.26–4.30 (m, 1 H), 4.37–4.39 (m, 1 H), 5.13–5.14 (m, 1 H), 5.82
3
(d, JH,H = 0.7 Hz, 1 H), 6.77–7.44 (m, 13 H), 7.78 (s, 1 H), 8.02
(s, 1 H) ppm. 13C NMR (125.7 MHz, [D7]DMF): δ = 51.8, 55.1,
62.5, 70.6, 75.9, 81.7, 86.4, 92.7, 113.1, 121.7, 126.6, 126.8, 127.7,
127.8, 128.2, 128.5, 128.7, 128.9, 130.0, 136.1, 137.3, 138.3, 144.9,
145.2, 147.9, 152.6, 154.2, 158.3, 158.3, 167.6, 168.5, 181.7,
182.7 ppm. ESI-MS calcd. for C35H32N7O6 [M + H]+: 646.2412;
found 646.2502.
N6-(4,4-Dimethoxytrityl)-3,5-diaminoadenosine (32): Compound 28
(738 mg, 2.3 mmol) was rendered anhydrous by repeated coevapo- 3Ј,5Ј-Diamino-3Ј,5Ј-N-(3,4-dioxocyclobutene-1,2-diyl)adenosine
ration with dry pyridine. Additional pyridine (24 mL) and chloro-
(9a): Compound 35 (147 mg, 0.23 mmol) was dissolved in trifluoro-
trimethylsilane (737 µL, 5.8 mmol) were added to the residue, and
acetic acid/dichloromethane (1% v/v, 3 mL), and the solution was
Eur. J. Org. Chem. 2005, 5163–5170
© 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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K. Seio, T. Miyashita, K. Sato, M. Sekine
FULL PAPER
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stirred at ambient temperature for 7 h. The product was extracted
three times with water (20 mL), and the aqueous solution was con-
centrated under reduced pressure. The solution was neutralized
with dimethylaminomethyl-polystyrene, filtered, and concentrated
under reduced pressure. The residue was purified by gel-filtration
chromatography. The fraction containing the desired product was
concentrated, and the residue was triturated with diethyl ether to
give 9a (33 mg, 42%). 1H NMR (500 MHz, [D6]DMSO): δ = 3.70–
3.72 (m, 2 H), 4.07–4.09 (m, 1 H), 4.11–4.22 (m, 1 H), 4.75–4.77
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1 H), 7.34 (br., 2 H), 8.15 (s, 1 H), 8.29 (s, 1 H), 8.64–8.70 (br., 2
H) ppm. 13C NMR (125.7 MHz, [D7]DMF): δ = 48.7, 63.8, 76.1,
82.0, 92.0, 120.3, 140.1, 149.9, 153.6, 157.2, 169.0, 169.5, 183.0,
183.7 ppm. ESI-MS calcd. for H14N7O4 [M + H]+: 344.1107; found
346.1106.
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(9b): N,N-Diisopropylethylamine (27.4 µL, 0.16 mmol) and di-
methyl squarate (46 mg, 0.32 mmol) were added to a solution of
compound 33 (188 mg, 0.32 mmol) in methanol (3 mL). The mix-
ture was stirred at ambient temperature for 2 h, the solvent was
removed under reduced pressure, and the residue was chromato-
graphed on a column of silica gel with chloroform/methanol (100:5,
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at ambient temperature for 18 h. The product was extracted three
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dimethylaminomethyl-polystyrene, filtered, and concentrated under
reduced pressure. The residue was purified by gel-filtration
chromatography. The fraction containing the desired product was
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3
D2O): δ = 3.87 (dd, JH,H = 2.4 Hz, 12 Hz, 1 H, 5Ј-H), 4.35–4.39
3
(m, 1 H, 4Ј-H), 4.57 (dd, JH,H = 5.4 Hz, 9.0 Hz, 1 H, 3Ј-H), 4.86
3
(d, JH,H = 5.4 Hz, 1 H, 2Ј-H), 5.97 (s, 1 H, 1Ј-H), 7.93 (s, 1 H, 8- [18] a) J. M. Delfino, C. J. Stankovic, S. L. Schreiber, F. M. Rich-
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81.8, 91.7, 118.0, 136.4, 151.2, 154.6, 157.3, 168.6, 169.2, 182.6,
183.3 ppm. ESI-MS calcd. for C14H14N7O5 [M + H]+: 360.1056;
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Published Online: October 19, 2005
5170
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Eur. J. Org. Chem. 2005, 5163–5170