A facile synthesis of cyclic bis(3′→5′)diguanylic acid

Article abstract of DOI:10.1016/S0040-4020(03)01045-7

This paper describes a new method for synthesizing biologically important cyclic bis(3′→5′)diguanylic acid (cGpGp) in a higher yield than that of the existing synthetic method. In the new synthesis, the following two means, in place of those used in the existing synthesis are employed as main strategies to cause the increase in product yield. One of these distinctive strategies in the new synthesis is that the phosphoramidite method is used for the preparation of a key synthetic intermediate of a linear guanylyl(3′→5′)guanylic acid derivative. This method allowed higher-yield formation of the intermediate than that by the triester method used in the existing synthesis. The second distinctive strategy used in the new synthesis is that allyloxycarbonyl and allyl groups are used for the protection of two guanine bases and two internucleotide bonds, respectively. These four allylic protectors can be removed all at once by the organopalladium-catalyzed reaction under neutral conditions. Thus, deprotection of the protected cGpGp precursor was achieved in the present synthesis in a shorter step and under milder conditions than the deprotection achieved in the existing synthesis, which uses diphenylacetyl and o-chlorophenyl groups as protectors for two guanine bases and two internucleotide bonds, respectively, whose full removal requires two different procedures including rather harsh basic treatment. As a result, technical loss and decomposition of the target product in the new synthesis is remarkably reduced.

Full text of DOI:10.1016/S0040-4020(03)01045-7

Tetrahedron 59 (2003) 6465–6471
A facile synthesis of cyclic bis(30!50)diguanylic acid
a
a
a
a
Yoshihiro Hayakawa,a,b Reiko Nagata, Akiyoshi Hirata, Mamoru Hyodo and Rie Kawai
,
*
aLaboratory of Bioorganic Chemistry, Graduate School of Human Informatics, Nagoya University, Chikusa, Nagoya 464-8601, Japan
bLaboratory of Bioorganic Chemistry, Graduate School of Information Science, Nagoya University, Chikusa, Nagoya 464-8601, Japan
Received 19 May 2003; revised 30 June 2003; accepted 3 July 2003
Abstract—This paper describes a new method for synthesizing biologically important cyclic bis(30!50)diguanylic acid (cGpGp) in a higher
yield than that of the existing synthetic method. In the new synthesis, the following two means, in place of those used in the existing synthesis
are employed as main strategies to cause the increase in product yield. One of these distinctive strateg0ies i0n the new synthesis is that the
phosphoramidite method is used for the preparation of a key synthetic intermediate of a linear guanylyl(3 !5 )guanylic acid derivative. This
method allowed higher-yield formation of the intermediate than that by the triester method used in the existing synthesis. The second
distinctive strategy used in the new synthesis is that allyloxycarbonyl and allyl groups are used for the protection of two guanine bases and
two internucleotide bonds, respectively. These four allylic protectors can be removed all at once by the organopalladium-catalyzed reaction
under neutral conditions. Thus, deprotection of the protected cGpGp precursor was achieved in the present synthesis in a shorter step and
under milder conditions than the deprotection achieved in the existing synthesis, which uses diphenylacetyl and o-chlorophenyl groups as
protectors for two guanine bases and two internucleotide bonds, respectively, whose full removal requires two different procedures including
rather harsh basic treatment. As a result, technical loss and decomposition of the target product in the new synthesis is remarkably reduced.
q 2003 Elsevier Ltd. All rights reserved.
1. Introduction
compound and consequently decreases the product yield.4
This paper reports a new method for the synthesis of cGpGp,
by which these drawbacks in the existing synthesis are
resolved.5
Cyclic bis(30!50)diguanylic acid (cGpGp) (5) is a naturally-
occurring substance with biologically important properties
such as (i) regulation of cellulose synthesis in the bacterium
Acetobacter xylinum1,2 and (ii) elevation of an expression
of the CD4 receptor and cell cycle arrest in Jurkat cells.3
Accordingly, studies aimed at the effective use of cGpGp
are actively being carried out in various research fields such
as biology, pharmacy, and clinical research. In order to
perform these studies, a sufficient supply of cGpGp and its
artificial analogs is crucial. Therefore, development of
chemical methods capable of synthesizing both cGpGp and
its artificial analogs is strongly demanded. Thus far, van
Boom and his co-workers has reported one example of such
a chemical synthesis.1,2 However, this synthesis seems to
have some problems with regard to the product yield. F0or
example, the reported yield of a linear guanylyl(30!5 )-
guanylic acid derivative (an important key intermediate),
which is prepared by means of the triester method, is not
satisfactorily high. Further, there is some apprehension that
removal of the diphenylacetyl protectors from guanine
bases, which is achieved by heating (508C) with conc.
aqueous ammonia, causes decomposition of the target
2. Results and discussion
Scheme 1 outlines the synthesis of cGpGp achieved in our
laboratory. First, the building block 2 was prepared in a 95%
overall yield through (1) the imidazolium perchlorate
(IMP)-promoted reaction6 of the nucleoside phosphorami-
dite 17 and 2-cyanoethanol in acetonitrile in the presence of
molecular sieves (MS) 3A (30 min),8 (2) oxidation using a
2-butanone peroxide (BPO)/toluene solution (5 min),9 and
(3) detritylation with dichloroacetic acid in dichloro-
methane (5 min). Subsequently, the two building blocks 1
and 2 (using 1 equiv. each) were condensed by the
assistance of imidazolium perchlorate in acetonitrile
with MS 3A, and the resulting product was subjected to
oxidation with 2-butanone peroxide followed by detrityl-
ation with dichloroacetic acid to provide the guanylyl-
(30!50)guanosine 30-phosphate derivative 3 in a 94%
isolated yield. The structure of 3 was confirmed by the
1H–1H COSY NMR spectrum, the 31P–1H COSY NMR
spectrum, and the MALDI-TOF high resolution mass
spectrum.
Keywords: nucleotides; phosphoramidites; cyclisation.
*
Corresponding author. Address: Laboratory of Bioorganic Chemistry,
Graduate School of Information Science, Nagoya University, Chikusa,
Nagoya 464-8601, Japan. Tel.: þ81-52-789-4848; fax: þ81-52-789-
5646; e-mail: yoshi@info.human.nagoya-u.ac.jp
The compound 3 was exposed to a conc. aqueous NH3/
0040–4020/$ - see front matter q 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0040-4020(03)01045-7

6466
Y. Hayakawa et al. / Tetrahedron 59 (2003) 6465–6471
Scheme 1. Synthesis of cGpGp: (a) HOCH2CH2CN, IMP, MS 3A, CH3CN, 30 min; (b) a 6.7% BPO/toluene solution, 5 min; (c) Cl2CHCOOH, CH2Cl2, 08C,
5 min; (d) the phosphoramidite 1, IMP, MS 3A, CH3CN, 30 min; (e) a 6.7% BPO/toluene solution, 5 min; (f) Cl2CHCOOH, CH2Cl2, 08C, 5 min; (g) conc. aq.
NH3–CH3OH (1:10 v/v), 60 min; (h) TPSCl, N-methylimidazole, THF, 12 h; (i) Pd2(dba)3·CHCl3, P(C6H5)3, n-C4H9NHþ3 HCOO2, THF, 10 min;
(j) (C2H5)3N·3HF, 12 h.
methanol (1:10 v/v) solution (60 min) to remove the
cyanoethyl group, and the resulting product was converted
to the cyclic compound 4 by treatment with a mixture of
triisopropylbenzenesulfonyl chloride (TPSCl) and
N-methylimidazole10 in a 0.05 M THF solution (20 h).
According to chromatographic analysis, this cyclization
afforded two major products, 4a and 4b. These two products
were isolated by chromatography and subjected to structure
determination by spectral analyses including the 1H–1H
COSY NMR spectrum, the 31P–1H COSY NMR spectrum,
and the MALDI-TOF high resolution mass spectrum.
Figure 1 shows 31P–1H COSY NMR spectra of 4a and
4b. The 31P signals were observed at d 22.31 and 1.91 ppm
in the spectrum of 4a and at d 1.50 ppm in the spectrum of
4b. 0 In the 1H NMR spectrum of 4a, signals due to
C(5 )CH2OH and C(30)CHOH observed around d 3.6–3.9
and 4.2–4.3 ppm, respectively, in the spectrum of 3
disappeared. Instead, signals for four protons due to two
C(50)H2OP(O)O and those for two protons due to two
C(30)HOP(O)O were observed around d 4.6–4.7 and 5.3–
the yield of the cyclic product 4, as a mixture of 4a and 4b,
was 75%.
The fully protected compound 4 (a mixture of 4a and 4b)11
was deblocked by treatment with a catalytic amount of
Pd2[(C6H5CHvCH)2CO]3·CHCl3 in the presence of tri-
phenylphosphine and butylammonium formate in THF12
(10 min) (removal of all allylic protectors on guanine bases
and phosphate moieties), followed by0 exposure to
(C2H5)3N·3HF13 (12 h) (removal of 2 -O-tert-butyl-
dimethylsilyl protecting groups). The 31P NMR analysis
of the resulting product indicated that this two-step
deprotection formed cGpGp (5) in a 77% overall yield.
Reverse-phase chromatography afforded a pure sample of
the diammonium salt of 5 (see, Fig. 2). The structure of this
product was confirmed by the following spectral analyses.
The ESI-TOF high resolution mass spectrum (negative
mode) (Fig. 3) showed a molecular ion peak at m/z
689.0853, which is consistent with the molecular weight
of 689.0876 calculated for 5 [(C20H23N10O14P22 ) (M2H2)].
1
1
5.4 ppm, respectively; these H signals correlate with the
The H NMR spectrum measured at 508C14 in D2O was
31P signals. Similar phenomena were observed in the
31P–1H COSY NMR spectrum of 4b. These observations
supported that 4a and 4b are stereoisomers having the cyclic
bis(30!50)diguanylic acid structure. The ESI-TOF high
resolution mass spectra (positive mode) of 4a and 4b,
showing molecular ion peaks at m/z 1247.4496 for 4a and
1247.4426 for 4b [calcd for 4 (C52H77N10O18P2Siþ2 )
(MþHþ) 1247.4435], also supported this structure. Thus,
identical with that of cGpGp illustrated in the literature.1 In
the 31P NMR spectrum, a sole signal was observed at d
20.86 ppm, which is also consistent with that previously
reported (d 20.62 ppm).1 The UV spectrum showed an
absorption at lmax¼254 nm (1 23,700), identical with that
previously reported (lmax¼252 nm; 1 24,700).1 Further, the
13C NMR spectrum indicated only five signals at d 62.8,
70.9, 73.7, 80.8, and 90.6 ppm as those due to ribose

Y. Hayakawa et al. / Tetrahedron 59 (2003) 6465–6471
6467
Figure 1. The 31P–1H COSY NMR spectra of 4a (A) and 4b (B).
Figure 2. HPLC profile of purified 5. Conditions: COSMOSIL 5C18-AR-300 column (25£200 mm); buffer A, 1 mM ammonium acetate; buffer B, 80%
acetonitrile–1 mM ammonium acetate; gradient, A:B¼100:0 to A:B¼40:60 in 60 min; detection 254 nm, flow rate, 5.0 mL/min, temperature 408C.

6468
Y. Hayakawa et al. / Tetrahedron 59 (2003) 6465–6471
Figure 3. The ESI-TOF mass spectrum (negative mode) of 5 [calcd for C20H23N10O14P22 (M2H2) 689.0876].
moieties and this observation supported the symmetric
structure of the product.
capability for use in preparation of artificial analogs of
cGpGp with modified internucleotide linkages such as
phosphorothioates,15 phosphoroselenoates,16 and borano-
phosphates.17
3. Conclusion
We have developed a new method for synthesis of cGpGp,
which has several advantages over the existing one.1,20In the
existing synthesis, the yield of a linear guanylyl(3 !50)-
guanylic acid intermediate, which is prepared by the triester
method using one building block slightly excessive to
another building block, is not quite satisfactorily high. By
contrast, in the present synthesis, the linear diguanylic acid
intermediate 3 is prepared in an excellent yield via the MS
3A-presence phosphoramidite method using the two build-
ing blocks 1 and 2 in stoichiometric amounts; the yield of 3
is ca. 20% higher than that of a similar intermediate in the
existing synthesis. Further, in the existing synthesis,
because diphenylacetyl groups are employed as the
protectors of guanine bases, whose subsequent deprotection
requires treatment with hot concentrated ammonia, there is
the risk that the deprotection will cause decomposition of
the target product.4 By contrast, the present synthesis does
not include such risk, because it uses allyloxycarbonyl
groups for the protection of guanine bases, and these groups
can be removed by treatment with an organopalladium
catalyst under mild, almost neutral conditions. Furthermore,
the existing synthesis uses o-chlorophenyl protectors for the
phosphate moieties, which are not deprotected under the
conditions for removal of the N-diphenylacetyl protectors.
Therefore, two steps of procedure are required for the
removal of these protectors from a fully protected cGpGp
derivative. On the other hand, because the present synthesis
uses the allyl groups for the protection of internucleotide
bonds, and these groups are removable together with the
N-allyloxycarbonyl protectors by the organopalladium
reaction, we can deblock the four allylic protecting groups
from the fully protected cGpGp 4 in one step. This
improvement may diminish loss of the desired product in
the deprotection process. In addition, the present
synthesis constructs the cyclic diguanylic acid structure
by the phosphoramidite method, and thus has the
4. Experimental
4.1. General
A UV spectrum was taken on a JASCO V-500 spectrometer.
NMR spectra were obtained on a JEOL JNM-a400 or ECA-
500 instrument. The 1H, 13C, and 31P NMR chemical shifts
are described as d values in ppm relative to (CH3)4Si (for 1H
and 13C) and 85% H3PO4, respectively. Measurement of
MALDI-TOF HRMS and ESI-TOF HRMS spectra was
carried out on Applied Biosystems Voyager MDE and
Mariner spectrometers, respectively. HPLC analysis was
carried out using a COSMOSIL 5C18-MS column (Nacalai
Tesque, ODS-5 mm, 4.6£250 mm) on a Waters 2695
Separations Module chromatograph with a Waters 2996
Photodiode Array detector. Preparative HPLC using a
COSMOSIL 5C18-AR-300 column (Nacalai Tesque,
¨
25£200 mm) was achieved with an AKTA explorer
(Amersham Biosciences). Nacalai Tesque silica gel 60
(neutrality, 75 mm) was used for column chromatography.
Unless otherwise stated, reactions were carried out at
ambient temperature. Reactions requiring anhydrous
conditions were carried out under an argon atmosphere in
flasks dried by heating at 4008C under reduced pressure
(1.0–3.0 mm Hg) or by treatment with 5% dichloro-
dimethylsilane/dichloromethane solution followed by
washing with anhydrous dichloromethane and then heating
at 1008C.
4.2. Material and solvents
The protected guanosine 30-phosphoramidite 1,7 imi-
dazolium perchlorate,6 and tris(dibenzylideneacetone)di-
palladium(0)-chloroform complex [Pd2(dba)3·CHCl3]18
were prepared by the reported methods. A 6.7% 2-butanone
peroxide/dimethyl phthalate–toluene solution was prepared

Y. Hayakawa et al. / Tetrahedron 59 (2003) 6465–6471
6469
by dilution of a commercially supplied 55% 2-butanone
peroxide/dimethyl phthalate solution (Kishida) with
toluene. Acetonitrile, DMF, and dichloromethane were
used distilled from CaH2. Diethyl ether, THF, and toluene
were used after drying by reflux over sodium-benzophenone
ketyl. Other organic solvents were used as commercially
supplied without any purification. Nacalai tesque silica gel
60 (neutrality, 75 mm) was used for column chromato-
graphy. Solid and amorphous organic substances were used
dried over P2O5 at 50–608C for 8–12 h under reduced
pressure (1.0–3.0 mm Hg). Powdery MS 3A and 4A were
used after drying the commercially supplied one (Nacalai
tesque) at 2008C for 12 h under reduced pressure
(1.0–3.0 mm Hg).
dissolved in dichloromethane (20 mL). To this solution was
added dichloroacetic acid (3.3 mL, 5.2 g, 40 mmol) at 08C.
After stirring for 5 min, the reaction mixture was poured to
an aqueous NaHCO3-saturated solution (100 mL) and the
organic layer was separated. The aqueous layer was
extracted with dichloromethane (100 mL, 50 mL£2). The
combined organic solutions were dried and concentrated.
The resulting material was chromatographed on a column of
silica gel (40 g) using a 1:1 ethyl acetate–hexane mixture
and then a 1:20:20 methanol–ethyl acetate–hexane mixture
as eluent to afford 3 (1.95 g, 94% yield; a mixture of two
1
diastereomers) as a colorless amorphous solid: H NMR
(CDCl3) 20.36–0.03 (m, 12H), 20.68–0.77 (m, 18H), 1.78
(br s, 1H), 2.79–2.81 (m, 2H), 3.79–3.96 (m, 4H), 4.30–
4.36 (m, 4H), 4.48–4.71 (m, 14H), 4.99–5.50 (m, 14H),
5.75–6.19 (m, 8H), 7.26–8.67 (m, 4H); 31P NMR (CDCl3)
21.32, 21.24, 21.11, 21.05, 20.88, 20.81; HRMS
(MALDIþ) calcd for C55H83N11O19P2Siþ2 (MþHþ)
1318.4797, found 1318.5267.
4.2.1. N 2-(Allyloxycarbonyl)-O 6-(allyl)-20-O-(tert-butyl-
dimethylsilyl)guanosine 30-(allyl 2-cyanoethyl)phosphate
(2). A heterogeneous mixture of the phosphoramidite 1
(2.0 g, 2.0 mmol), 2-cyanoethanol (0.16 mL, 170 mg,
2.4 mmol), and powdery MS 3A (200 mg) in acetonitrile
(20 mL) was stirred at 258C for 30 min. To this was added
imidazolium perchlorate (670 mg, 4.0 mmol) and stirring
was continued for additional 30 min. To the resulting
mixture was added a 6.7% 2-butanone peroxide/dimethyl
phthalate–toluene solution (4.0 mL). The mixture was
stirred for 5 min. MS 3A was removed by passage through
a Celite 545 pad. The filtrate was diluted with ethyl acetate
(100 mL) and washed with an aqueous NaHCO3-saturated
solution followed by brine. The organic layer was separated,
dried over sodium sulfate, and concentrated to give a
residual material. This material was dissolved in dichloro-
methane (20 mL) and cooled at 08C. To the solution was
slowly added dichloroacetic acid (3.3 mL, 5.2 g, 40 mmol)
and then the mixture was stirred at the same temperature for
5 min. The reaction mixture was poured to an aqueous
NaHCO3-saturated solution (100 mL) and the organic layer
was separated. The aqueous layer was extracted with
dichloromethane (100 mL, 50 mL£2). The combined
organic solutions were dried and concentrated to give a
residual product. This crude material was subjected to
column chromatography on silica gel (40 g) eluted with a
1:1 mixture of ethyl acetate and hexane followed by a
1:10:10 mixture of methanol, ethyl acetate, and hexane to
afford 2 (1.38 g, 95% yield; a mixture of two diastereomers)
as a colorless amorphous solid: IR (CH2Cl2) 3420, 3048,
2305, 1757, 1607, 1524, 1462, 1412, 1294, 1190, 1038,
4.2.3. Intramolecular cyclization of 3 giving the pro-
tected cyclic bis(30!50)diguanylic acid 4. To a solution of
3 (660 mg, 0.5 mmol) in methanol (10 mL) was added at
258C a concentrated aqueous ammonia solution (1.0 mL)
and the resulting solution was stirred for 60 min. The
reaction mixture was concentrated in vacuo. The resulting
residue was dissolved in toluene (20 mL) and the solvent
was evaporated. This treatment was carried out twice more
to give a colorless amorphous solid. This material was
dissolved in THF (100 mL). Powdery MS 4A was added to
the solution and the resulting mixture was stirred at 258C for
30 min to remove methanol, ammonia, and water. The MS
4A was removed by filtration. To the filtrate were
successively added N-methylimidazole (0.08 mL, 82 mg,
1.0 mmol) and 2,4,6-triisopropylbenzenesulfonyl chloride
(300 mg, 1.0 mmol). The resulting solution was stirred at
258C for 20 h. An aliquot of the reaction mixture was
subjected to TLC analysis using a 1:10:10 mixture of
methanol, ethyl acetate, and hexane as an eluent to indicate
that 4a (Rf¼0.40) and 4b (Rf¼0.49) was formed as
major products. The whole reaction mixture was directly
concentrated. The resulting residual material was chro-
matographed on a silica gel (40 g) column using a 1:2
mixture of ethyl acetate and hexane and then a 1:30:30
mixture of methanol, ethyl acetate, and hexane to afford 4a
(270 mg) and 4b (200 mg) as both amorphous solids. The
total yield of 4 was 75%.
1
756 cm21; H NMR (CD3OD) 20.27 (s, 3H), 20.03 (s,
3H), 0.74 (s, 9H), 2.94 (t, J¼6.0 Hz, 2H), 3.85–3.97 (m,
2H), 4.30–4.43 (m, 3H), 4.70 (m, 4H), 5.00 (m, 1H), 5.08–
5.10 (m, 2H), 5.24–5.52 (m, 7H), 6.00–6.20 (m, 4H), 8.39
(s, 1H); 31P NMR (CD3OD) 24.69, 24.54.
1
Compound 4a: H NMR (CDCl3) 20.31, 20.21, 20.03,
0.04 (4 s, 12H), 0.76 (s, 18H), 4.03–4.09 (m, 2H), 4.26–
4.34 (m, 2H), 4.54–4.69 (m, 8H), 4.74–4.86 (m, 4H), 4.97–
5.15 (m, 6H), 5.21–5.52 (m, 12H), 5.64–5.68 (m, 1H),
5.74–5.82 (m, 3H), 5.94–6.01 (m, 4H), 6.08–6.18 (m,
2H), 7.53 (s, 1H), 7.78 (s, 1H), 7.81 (s, 1H), 8.03 (s, 1H);
31P NMR (CDCl3) 22.31, 1.91; HRMS (ESIþ) calcd
for C52H77N10O18P2Siþ2 (MþHþ) 1247.4426, found
1247.4496.
4.2.2. The guanylyl(30!50)guanosine 30-phosphate 3. A
mixture of the phosphoramidite 1 (1.6 g, 1.6 mmol) and the
50-O-free nucleoside phosphate 2 (1.1 g, 1.6 mmol) in the
presence of powdery MS 3A (200 mg) in acetonitrile
(15 mL) was stirred at 258C for 30 min. The mixture was
added imidazolium perchlorate (540 mg, 3.2 mmol) and
stirred for additional 30 min. To this was added a 6.7%
2-butanone peroxide/dimethyl phthalate–toluene solution
(3.2 mL). After 5 min, the reaction mixture was passed
through a Celite 545 pad to remove MS 3A. The filtrate was
concentrated to afford a viscous oil. This material was
Compound 4b: 1H NMR (CDCl3) 20.22, 20.08 (2 s, 12H),
0.74 (s, 18H), 4.07 (m, 2H), 4.48–4.51 (m, 2H), 4.65–4.68
(m, 8H), 4.96 (q, J¼11 Hz, 2H), 5.03–5.13 (m, 4H), 5.19–
5.58 (m, 16H), 5.77 (d, J¼7.0 Hz, 1H), 5.93–5.97 (m, 4H),
6.11–6.17 (m, 2H), 7.78 (s, 2H), 7.85 (s, 2H); 31P NMR

6470
Y. Hayakawa et al. / Tetrahedron 59 (2003) 6465–6471
(CDCl3) 1.50; HRMS (ESIþ) calcd for C52H77N10O18P2Siþ2
G. A.; van Boom, J. H.; Benziman, M. Nature (London) 1987,
325, 279–281.
(MþHþ) 1247.4426, found 1247.4435.
2. Ross, P.; Mayer, R.; Weinhouse, H.; Amikam, D.; Huggirat,
Y.; Benziman, M.; de Vroom, E.; Fidder, A.; de Paus, P.;
Sliedregt, L. A. J. M.; van der Marel, G. A.; van Boom, J. H.
J. Biol. Chem. 1990, 265, 18933–18943.
4.2.4. Full deprotection of 4 giving cGpGp 5. To a
solution of 4 (a mixture of 4a and 4b) (50 mg, 0.04 mmol) in
THF (1.6 mL) were added triphenylphosphine (16 mg,
0.06 mmol), n-butylamine (49 mL, 36 mg, 0.48 mmol),
formic acid (18 mL, 22 mg, 0.48 mmol), and Pd2(dba)3·
CHCl3 (12 mg, 0.012 mmol). The resulting mixture was
stirred at 258C for 30 min. During this period, white solid
precipitated. To this mixture was added ethyl acetate
(10 mL). The precipitate was collected by filtration and
dried in vacuo. This solid material was mixed with
(C2H5)3N·3HF (0.3 mL) and the mixture was stirred at
258C for 12 h. An aliquot (30 mL) of the reaction mixture
was taken out and subjected to the following analysis. The
aliquot was dissolved in a mixture of a 0.1 M Na2HPO4
aqueous solution (40 mL) and D2O (500 mL). The resulting
solution was subjected to the 31P NMR measurement; the
yield of the desired product 5 was estimated by comparison
of intensity of the signal due to 5 (d 20.94 ppm) and that
due to Na2HPO4 (0.78 ppm). The yield of 5 indicated by this
analysis was 77%. The whole aliquot taken out was mixed
again with the reaction mixture. To the reaction mixture was
added a 1 mM ammonium formate buffer solution (1 mL).
The resulting mixture was vigorously stirred under heating
at 30–408C to precipitate a pale yellow solid. After removal
of the resulting precipitate, the aqueous solution was
subjected to preparative HPLC using a COSMOSIL 5C18-
AR-300 column [25 (diameter)£200 (height) mm]. Elution
was carried out by the following conditions [A¼a 1.0 mM
ammonium acetate buffer solution, B¼a 0.2 mM
ammonium acetate solution in a 20:80 mixture of H2O
and acetonitrile; gradient: 0–8 min A 100%, 8–55 min
linear gradient A 100% to A 40%/B 60%, 55–63 min B
100%; detection 254 nm; flow rate 10 mL/min] to give the
diammonium salt of 5 (12 mg, 40% yield). UV (a 50 mM
3. Steinberger, O.; Lapidot, Z.; Ben-Ishai, Z.; Amikam, D. FEBS
Lett. 1999, 444, 125–129.
4. In Refs. 1,2, there is no information about the yield of the
desired compound in the deprotection of the N-diphenylacetyl
protectors. Thus, we cannot know whether the deprotection
causes decomposition of the desired product in the existing
synthesis. However, according to experiments carried out by
us, cGpGp was decomposed to no small extent by treatment
with hot conc. aqueous ammonia to give undesired products.
The structure of the products was not determined because
analytical samples of them were not obtained though
chromatographic purification was attempted under several
conditions; we supposed that the products are those resulting
from cleavage of internucleotide linkage.
5. This paper eliminates another defect of the existing synthesis.
In the field of chemical synthesis, detailed experimental
protocol is very important for reexamination of the synthesis.
However, Refs. 1,2 do not describe in detail the protocol; this
is troublesome for further investigations. By contrast, this
paper provides detailed experimental procedures for all
reactions.
6. Hayakawa, Y.; Kawai, R.; Hirata, A.; Sugimoto, J.; Kataoka,
M.; Sakakura, A.; Hirose, M.; Noyori, R. J. Am. Chem. Soc.
2001, 123, 8165–8176.
7. Heidenhein, S. B.; Hayakawa, Y. Nucleosides Nucleotides
1999, 18, 1771–1787.
8. Hayakawa, Y.; Hirata, A.; Sugimoto, J.; Kawai, R.; Sakakura,
A.; Kataoka, M. Tetrahedron 2001, 57, 8823–8826.
9. Kataoka, M.; Hattori, A.; Okino, S.; Hyodo, M.; Asano, M.;
Kawai, R.; Hayakawa, Y. Org. Lett. 2001, 3, 815–818.
10. Efimov, V. A.; Reverdatto, S. V.; Chakhmakhcheva, O. G.
Tetrahedron Lett. 1982, 23, 961–964.
1
solution of NH4OAc in H2O) lmax 254 nm (1 23,700); H
NMR (D2O) 4.04–4.06 (m, 2H), 4.38–4.44 (m, 4H), 5.08
(s, 2H), 5.33 (s, 2H), 6.12 (s, 2H), 8.25 (s, 2H); 13C NMR
(D2O) 62.8, 70.9, 73.7, 80.8, 90.6, 116.4, 136.9 (weak),
150.4 (weak), 154.1, 157.8; 31P NMR (D2O) 20.86; HRMS
(ESI2) calcd for C20H23N10O18P22 (M2H2) 689.0876,
found 689.0853.
11. Deprotection of the single material of 4a and 4b carried out in
a similar manner also provided 5 as a sole product. This result
also supported the assigned structure of 4a and 4b.
12. (a) Hayakawa, Y.; Kato, H.; Uchiyama, M.; Kajino, H.;
Noyori, R. J. Org. Chem. 1986, 51, 2400–2402. (b) Hayakawa,
Y.; Hirose, M.; Noyori, R. J. Org. Chem. 1993, 58,
5551–5555. (c) Hayakawa, Y.; Wakabayashi, S.; Kato, H.;
Noyori, R. J. Am. Chem. Soc. 1990, 112, 1691–1696.
13. (a) Gasparutto, D.; Livache, T.; Bazin, H.; Duplaa, A.-M.;
In a similar manner, a pure sample of 4a or 4b was fully
deprotected to give 5.
´
Guy, A.; Khorlin, A.; Molko, D.; Roget, A.; Teoule, R.
Nucleic Acids Res. 1992, 20, 5159–5166. (b) Westman, E.;
Acknowledgements
¨
Stromberg, R. Nucleic Acids Res. 1994, 22, 2430–2431.
(c) Pirrung, M. C.; Fallon, L.; Lever, D. C.; Shuey, S. W.
We acknowledge a part of financial supports by contri-
butions from Mitsui Chemicals, Inc., and a Grant-in-Aid for
Scientific Research [(No. 11101001) (Y. H.)] and a Grant-
in-Aids for JSPS Fellows [(No. 12003794) (R. K.)] from the
Ministry of Education, Science, Sports and Culture.
J. Org. Chem. 1996, 61, 2129–2136.
14. The 1H NMR spectrum listed in Ref. 1 was taken at 508C.
Therefore, this work also took the spectrum under the same
conditions in order to carry out exact comparison of both
spectra.
15. Representative works: (a) Hayakawa, Y.; Hirose, M.; Noyori,
R. Nucleosides Nucleotides 1994, 13, 1337–1345. (b) Iyer,
R. P.; Guo, M.-J.; Yu, D.; Agrawal, S. Tetrahedron Lett. 1998,
39, 2491–2494. (c) Boesen, T.; Kehler, J.; Dahl, O.
Nucleosides Nucleotides 1999, 18, 1241–1242.
References
1. Ross, P.; Weinhouse, H.; Aloni, Y.; Michaeli, D.; Weinberger-
Ohana, P.; Mayer, R.; Braun, S.; de Vroom, E.; van der Marel,
16. (a) Nemer, M. J.; Ogilvie, K. K. Tetrahedron Lett. 1980, 21,

Y. Hayakawa et al. / Tetrahedron 59 (2003) 6465–6471
6471
4149–4153. (b) Stec, W. J.; Zon, G.; Egan, W.; Stec, B. J. Am.
Chem. Soc. 1984, 106, 6077–6079.
18. Ukai, T.; Kawazura, H.; Ishii, Y.; Bonnet, J. J.; Ibers, J. A.
J. Organomet. Chem. 1974, 65, 253–266, This compound is
commercially available from Aldrich.
17. (a) Sood, A.; Shaw, B. R.; Spielvogel, B. F. J. Am. Chem. Soc.
1990, 112, 9000–9001. (b) Chen, Y.-Q.; Qu, F.-C.; Zhang,
Y.-B. Tetrahedron Lett. 1995, 36, 745–748.