An improved method for synthesizing cyclic bis(3′-5′)diguanylic acid (c-di-GMP)

Article abstract of DOI:10.1246/bcsj.77.2089

This paper describes a new method for synthesizing biologically important cyclic bis(3′-5′)diguanylic acid (c-di-GMP) in a higher yield than that previously reported to be available by our synthetic method. In the new synthesis, the following two means, i

Full text of DOI:10.1246/bcsj.77.2089

Ó 2004 The Chemical Society of Japan
Bull. Chem. Soc. Jpn., 77, 2089–2093 (2004) 2089
An Improved Method for Synthesizing Cyclic Bis(30–50)diguanylic
Acid (c-di-GMP)
ꢀ
Mamoru Hyodo and Yoshihiro Hayakawa
Graduate School of Information Science/Human Informatics and CREST JST, Nagoya University,
Chikusa, Nagoya 464-8601
Received June 1, 2004; E-mail: yoshi@is.nagoya-u.ac.jp
This paper describes a new method for synthesizing biologically important cyclic bis(30–50)diguanylic acid (c-di-
GMP) in a higher yield than that previously reported to be available by our synthetic method. In the new synthesis, the
following two means, in place of those used in the previously reported synthesis, are employed as main strategies to
obtain an increase in product yield. One is the use of di-tert-butylsilanediyl protection for 30- and 50-hydroxy groups
of guanosine; this method allows regioselective production of a 20-O-(tert-butyldimethylsilyl)guanosine derivative that
is a key intermediate for the synthesis. The other is the use of a dimethylformamidine group as a protector for the 2-NH2
function of the guanine base, which can be easily introduced and results in an excellent yield.
Cyclic bis(30–50)diguanylic acid (c-di-GMP) (8 in Scheme 1)
is a naturally-occurring nucleotide that is now attracting great
attention in many research fields. This substance demonstrates
various important properties, such as regulation of cellulose
synthesis in the bacterium Acetobacter xylinum,1,2 acceleration
of DNA synthesis and retarding of cell division in Molt4 cells,3
and elevation of an expression of the CD4 receptor and cell
cycle arrest in Jurkat cells.4 The cell-growth retarding activi-
ties of c-di-GMP have suggested that this compound may have
anti-cancer activity. Further, on the basis of studies on activity
dmf
HO
O
O
G
G
G
O
O
O
a, b, c
d
e, f
(t-C H ) Si
(t-C H ) Si
4 9 2
4
9 2
HO
OH
O
OTBDMS
O
OTBDMS
1
2
dmf
G
DMTrO
dmf
G
HO
O
dmf
O
DMTrO
G
O
O
g
h, i, j
k, l, m
O
P
OTBDMS
O
OTBDMS
P
HO
OTBDMS
NCCH CH O
OCH CH=CH
2 2
2
2
NCCH CH O
N(i-C H )
2
2
3
7 2
3
5
4
dmf
HO
G
O
−
+
NCCH CH O
O
O
O
O
O
NH
HO
2
2
4
P
P
dmf
TBDMSO
O
O
G
G
O
O
O
OTBDMS
O
O
n, o
p, q
P
dmf
NCCH CH O
2
2
G
O
O
O
dmf
G
G
O
O
O
OTBDMS
O
O
O
OH
P
P
+
−
NCCH CH O
NH
O
2
2
4
O
O
OTBDMS
P
OCH
7
8
NCCH CH O
CH=CH
2
2
2
2
6
Scheme 1. (a) (t-C4H9)2Si(OTf)2, 0 ꢁC, 45 min; (b) imidazole, 25 ꢁC, 30 min; (c) TBDMSCl, 60 ꢁC, 2 h; (d) (CH3)2NCH(OCH3)2,
50 ꢁC, 5 h; (e) HF pyridine, pyridine, CH2Cl2, 0 ꢁC, 2 h; (f) DMTrCl, pyridine, 25 ꢁC, 12 h; (g) NCCH2CH2OP[N(i-C3H7)2]Cl,
ꢂ
ꢁ
ꢁ
2,4,6-collidine, N-methylimidazole, THF, 25 C, 1 h; (h) allyl alcohol, IMP, MS 3A, CH3CN, 25 C, 30 min; (i) a 6.7% BPO/
ꢁ
ꢁ
toluene solution, 25 C, 5 min; (j) a 20% Cl2CHCOOH/CH2Cl2 solution, 25 C, 10 min; (k) the phosphoroamidite 4, IMP, MS
3A, CH3CN, 25 ꢁC, 30 min; (l) a 6.7% BPO/toluene solution, 25 ꢁC, 5 min; (m) a 20% Cl2CHCOOH/CH2Cl2 solution, 25 ꢁC, 10
min; (n) NaI, acetone, reflux, 2 h; (o) TPSCl, N-methylimidazole, 25 ꢁC, 36 h; (p) conc. aq. NH3–CH3OH (1:1 v/v), 50 ꢁC, 12 h;
ꢁ
(q) (C2H5)3N 3HF, 25 C, 12 h.
ꢂ
Published on the web November 11, 2004; DOI 10.1246/bcsj.77.2089

2090 Bull. Chem. Soc. Jpn., 77, No. 11 (2004)
Synthesis of Cyclic Bis(30–50)diguanylic Acid
of a regulatory protein, termed the regulation of cell signaling
(RocS) protein, in the switch between the smooth and rugose
phenotypes of V. cholerae, it was proposed that c-di-GMP
may have an important function in regulating exopolysaccha-
ride production, biofilm formation, and other phenotypes.5
Thus, we were stimulated to carry out extensive investigations
of the biological properties of c-di-GMP. Such investigations
required a sufficient amount of the substance. It was necessary
to develop an efficient chemical method for synthesizing c-di-
GMP in order to meet this requirement. Thus far, there have
been two methods developed, one by van Boom et al.,2 the oth-
er by us.6 Based on a comparison between these two methods,
our method appears more advantageous because it affords the
target compound in fewer steps and in a higher yield. Our
method still includes at least two unfavorable processes, which
do not provide satisfactory yields. One process is the tert-bu-
tyldimethylsilyl (TBDMS) protection of the 20-hydroxy of
guanosine. This protection was carried out using the 20-O-
and 30-O-free material, and took place in a nonregioselective
manner to give nearly equal amounts of 20-O-TBDMS, 30-O-
TBDMS, and 20,30-bis-O-TBDMS products.7 Therefore, isola-
tion of the desired 20-O-TBDMS compound requires tedious
chromatographic purification, causing a loss of the product.
In this approach, the desired 20-O-TBDMS product was isolat-
ed in a yield of only ca. 30%. Another process is the introduc-
tion of allyloxycarbonyl and allyl protecting groups to N2- and
O6-functions of the guanine base, respectively.8 This double
protection requires multiple steps, and the desired compound
was provided in an overall yield of only ca. 50%.7 According-
ly, we investigated development of an improved method that
would eliminate these drawbacks. This objective was achieved
by means of the following two new strategies. The first prob-
lem was resolved by use of the di-tert-butylsilanediyl ribonu-
cleoside-30,50-di-O-protection method (Furusawa method)9
and selective deblocking of this protector from the 30,50-O-
di-tert-butylsilanediyl-20-O-TBDMS guanosine intermediate.10
The second drawback was removed by use of an N2-dimethyl-
formamidine (dmf)-protected/O6-unprotected guanosine de-
rivative11 as a building block.
in a 78% yield. The 31P NMR signals observed at ꢀ 148.9 and
150.0 supported the structure of 4 (a 68:32 mixture of two dia-
stereomers). The compound 4 was treated with allyl alcohol
(1.2 molar amount) in the presence of imidazolium perchlorate
(IMP)13 (2.0 mol. amt.) and molecular sieves 3A (MS 3A)14 in
ꢁ
acetonitrile (25 C, 30 min), followed treatment with 2-buta-
none peroxide (BPO) (2.0 mol. amt.) in toluene (25 ꢁC, 5
min),15 and then, from the resulting product, the DMTr pro-
tecting group was removed by exposure to a 20% solution of
dichloroacetic acid in dichloromethane to furnish the phospho-
triester 5 (an 86% overall yield from 3). The structure of 5 was
confirmed by the 31P NMR spectrum that showed signals at ꢀ
ꢃ2:3, and by the ESI-TOF mass spectrum that indicated a mo-
lecular peak at m=z 626.2628 [calcd for (C25H41N7O8Si)þ:
1
626.2518]. The H NMR data were also consistent with 5 as
a mixture of diastereomers. The compound 5 was reacted with
the phosphoramiditeꢁ4 by the aid of IMP in acetonitrile con-
taining MS 3Aꢁ(25 C, 30 min), and then oxidized by BPO
in toluene (25 C, 5 min) to give the diguanylic acid ester,
which led to 6 (an 82% overall yield from 5) by detritylation
using a 20% dichloroacetic acid/dichloromethane solution
(25 ꢁC, 10 min). The 31P NMR signals that appear at ꢀ
ꢃ1:78, ꢃ1:72, ꢃ1:45, ꢃ1:32, ꢃ1:05, and ꢃ0:83 and an ESI-
TOF-mass peak that appears at m=z 1193.5944 [calcd for
(C47H75N14O15P2Si2)þ: 1193.4545] supported the structure
of the product. From the 30-terminal phosphotriester moiety
of 6, the allyl group was removed by the action of sodium io-
dide in refluxing acetone (2 h), and the resulting product was
converted to the fully protected cyclic dinucleotide 7 by treat-
ment with 2,4,6-triisopropylbenzenesulfonyl chloride (TPSCl)
ꢁ
in the presence of N-methylimidazole in THF (25 C, 36 h).16
In this process, 7 was obtained in an 85% yield. This product
showed a molecular peak at m=z 1135.4455 [calcd for
(C44H69N14O14P2Si2)þ: 1135.4126] in the ESI-TOF mass
spectrum and a signal at ꢀ 0.75 in the 31P NMR spectrum.
These data were consistent with 7. Finally, 7 was exposed to
a 1:1 mixture of concentrated aqueous ammonia and methanol
ꢁ
(50 C, 12 h)17 to remove the dmf and cyanoethyl protecting
ꢁ
groups and then treated with (C2H5)3N 3HF18 (25 C, 12 h)
ꢂ
to deblock the TBDMS protector. The resulting crude product
was purified by reverse-phase column chromatography to give
c-di-GMP (8) as the diammonium salt in an 89% isolated
Results and Discussion
1
Scheme 1 illustrates a newly developed synthetic pathway
to c-di-GMP. Successive single-flask treatment of guanosine
yield. The H NMR, 31P NMR, UV, and ESI-TOF mass spec-
tral data of this product were identical to those of the authentic
sample of 8.6
ꢁ
in DMF with di-tert-butylsilanediyl di(triflate) at 0 C for 45
ꢁ
min, with imidazole at 25 C for 30 min, and with TBDMS
We have thus developed a novel synthesis of c-di-GMP,
which has eliminated two major drawbacks of our previous
method. One improvement is perfectly regioselective produc-
tion of a 20-O-TBDMS-protected guanosine derivative ob-
tained using the Furusawa method for the 30,50-di-O-protection
of guanosine. The other improvement is an easy and high-
yielding preparation of the N2-protected guanosine achieved
using the dmf group as the protector. These modifications
greatly improved the yield of the target product. Actually,
when the synthesis using the same amount of the starting gua-
nosine is carried out by the present new method and by our
previously reported method, the former method gives the tar-
get product in an amount approximately ten-fold larger than
does the latter method.
chloride at 60 ꢁC for 2 h gave 1 in a 70% isolated yield.12 Sub-
sequently, the 2-amino function of the guanine base was pro-
tected byꢁthe reaction of 1 and (CH3)2NCH(OCH3)2 in meth-
anol (50 C, 5 h) to provide 2 in a 98% isolated yield. The
product 2 was converted to the 30-O-free guanosine 3 in an
86% overall yield through removal of the di-tert-butylsilane-
diyl protector by exposure to a mixture of HF pyridine com-
ꢂ
plex and pyridine (4.0:7.4 molar ratio) in dichloromethane (0
ꢁC, 2 h), and subsequent dimethoxytritylation using p,p0-di-
methoxytrityl chloride (DMTrCl) in pyridine (25 ꢁC, 12 h).
Condensation of 3 with NCCH2CH2OP[N(i-C3H7)2]Cl in the
presence of 2,4,6-collidine and N-methylimidazole in THF
(25 ꢁC, 1 h) afforded the phosphoramidite 4 with >90% purity

M. Hyodo et al.
Bull. Chem. Soc. Jpn., 77, No. 11 (2004) 2091
found m=z 593.3330.
Experimental
N2 -(Dimethylaminomethylene)-20 -O-(tert-butyldimethylsi-
lyl)-50-O-(p,p0-dimethoxytrityl)guanosine (3). A chilled solu-
tion of hydrogen fluoride–pyridine (2.0 mL, 100 mmol) in pyri-
dine (12 mL) was added to a solution of 2 (14.8 g, 25 mmol) in
dichloromethane (100 mL) at 0 ꢁC. The reaction mixture was stir-
red for 2 h. The reaction mixture was washed with water and with
an aqueous sodium hydrogencarbonate-saturated solution. The or-
ganic layer was evaporated under reduced pressure to give a resid-
ual material. This resulting product was dissolved in pyridine (50
mL) and to this was added dimethoxytrityl chloride (9.3 g, 27.5
mmol). The mixture was stirred for 12 h, then the reaction was
quenched by adding methanol (5 mL). Concentration of the result-
ing mixture gave an oily product. This material was dissolved in
ethyl acetate and washed with water, a sodium hydrogencarbonate
solution, and brine. The organic layer was dried and subjected to
silica gel (400 g) column chromatography to give 3 (16 g, 86%
yield) as an amorphous solid: 1H NMR (CDCl3) ꢀ ꢃ0:13 (s,
3H), 0.01 (s, 3H), 0.83 (s, 9H), 2.80 (d, J ¼ 3:7 Hz, 2H), 3.02
(s, 3H), 3.07 (s, 3H), 3.35 (dd, J ¼ 4:0, 10.5 Hz, 2H), 3.77 (s,
6H), 4.20 (q, J ¼ 3:5 Hz, 1H), 4.30 (q, J ¼ 3:5 Hz, 1H), 4.69
(t, J ¼ 5:5 Hz, 1H), 5.97 (d, J ¼ 6:0 Hz, 1H), 6.80–6.82 (m,
4H), 7.18–7.32 (m, 13H), 7.41–7.43 (m, 2H), 7.80 (s, 1H), 8.51
(s, 1H), 9.46 (s, 1H); HRMS (ESIþ) calcd for C40H50N6O7Siþ
(M + Hþ) m=z 755.3583, found m=z 755.3579.
N2 -(Dimethylaminomethylene)-20 -O-(tert-butyldimethylsi-
lyl)-50 -O-(p,p0 -dimethoxytrityl)guanosine 30 -[(2-Cyanoethyl)
N,N-Diisopropylaminophosphoramidite] (4). To a solution
of 3 (3.8 g, 5 mmol), 2,4,6-collidine (4.4 g, 4.8 mL, 36 mmol),
and N-methylimidazole (205 mg, 0.2 mL, 2.5 mmol) in THF
(25 mL) was added NCCH2CH2OP[N(i-C3Hꢁ7)2]Cl (3.0 g, 12.5
mmol) at 0 ꢁC. The mixture was stirred at 25 C for 1 h and then
diluted with ethyl acetate. The resulting mixture was washed with
an aqueous sodium hydrogencarbonate solution, then with brine,
and the organic layer was concentrated to give an oily material.
This crude product was dissolved in dichloromethane (20 mL)
and the resulting solution was added dropwise to hexane (1 L).
The resulting precipitate was collected by filtration and dried un-
der reduced pressure to give 5 in ca. 90% purity (3.7 g, 78% yield)
as a colorless powder: 1H NMR (CDCl3) ꢀ ꢃ0:13, ꢃ0:10, 0.02,
0.03 (4 s, 6H), 0.81, 0.82 (2 s, 9H), 1.16–1.30 (m, 12H), 2.24–
2.39 (m, 2H), 2.95, 3.07, 3.08, 3.09 (4 s, 6H), 3.24–3.28 (m,
1H), 3.53–3.64 (m, 4H), 3.78, 3.79, 3.80, 3.81 (4 s, 6H), 4.27–
4.39 (m, 2H), 4.67–4.74 (m, 1H), 5.97, 6.04 (2 d, J ¼ 6:4 Hz,
1H), 6.71–6.85 (m, 4H), 7.21–7.48 (m, 13H), 7.85, 7.88 (2 s,
1H), 8.50, 8.58 (2 s, 1H), 8.67, 8.69 (2 s, 1H); 31P NMR (CDCl3)
ꢀ 148.88, 150.02; HRMS (ESIþ) calcd for C49H68N8O8Siþ (M +
Hþ) m=z 955.4662, found m=z 955.4639.
General Procedures, Materials, and Solvents. Each UV
spectrum was measured on a JASCO V-500 spectrometer. NMR
spectra were taken on a JEOL JNM-ꢁ400 or ECA-500 instrument.
The 1H, 13C, and 31P NMR chemical shifts are described as ꢀ val-
ues in ppm relative to (CH3)4Si (for 1H and 13C) and 85% H3PO4,
respectively. ESI-TOF high resolution mass (HRMS) spectra were
obtained on Applied Biosystems Voyager MDE and Mariner spec-
trometers, respectively. HPLC analysis was carried out using a
COSMOSIL 5C18-MS column (Nacalai Tesque, ODS-5 mm,
4:6 ꢄ 250 mm) on a Waters 2695 Separations Module chromato-
graph with a Waters 2996 Photodiode Array detector. Preparative
HPLC was achieved using a COSMOSIL 5C18-AR-300 column
¨
(Nacalai Tesque, 25 ꢄ 200 mm) on an AKTA explorer (Amer-
sham Biosciences). Column chromatography was performed using
Nacalai Tesque silica gel 60 (neutrality, 75 mm). Unless otherwise
noted, synthetic reactions were carried out at ambient temperature.
The reactions requiring anhydrous conditions were achieved under
an argon atmosphere in flasks dried by heating at 400 ꢁC under
133–400 Pa, or by washing with a 5% solution of dichlorodi-
methylsilane in dichloromethane, followed by waꢁshing with anhy-
drous dichloromethane, and then heating at 100 C. Imidazolium
perchlorate,13 a 6.7% 2-butanone peroxide/dimethyl phthalate–
toluene solution,6,15 and NCCH2CH2OP[N(i-C3H7)2]Cl19 were
prepared by the reported methods. Diethyl ether, THF, and toluene
were used after drying by reflux over sodium–diphenyl ketyl. Ace-
tonitrile, DMF, and dichloromethane were distilled from calcium
hydride. Other organic reagents were used as commercially sup-
plied without any purification. Solid and amorphous organic sub-
ꢁ
stances were used after drying over P2O5 at 50–60 C for 8–12 h
under 133–400 Pa. Powdery molecular sieves (MS) 3A were em-
ployed after drꢁying the commercially supplied product (Nacalai
tesque) at 200 C for 12 h under 133–400 Pa.
20-O-(tert-Butyldimethylsilyl)-30,50-O-(di-tert-butylsilane-
diyl)guanosine (1). To a stirred suspension of guanosine (11.3 g,
40 mmol) in DMF (80 mL) was added di-tert-butylsilanediyl di-
ꢁ
(triflate) (19.4 g, 16.0 mL, 44 mmol) at 0 C over 15 min. After
stirring for 30 min at the same temperature, imidazole (13.6ꢁg,
200 mmol) was added. The resulting mixture was stirred at 0 C
for 5 min and then at room temperature for 25 min. To this was
added tert-butyldimethylchlorosilane (7.2 g, 48 mmol) and the re-
action mixture was heated at 60 ꢁC for 2 h. The occurring precip-
itate was collected by filtration, washed with cold methanol, and
dried under reduced pressure to give 1 (15.0 g, 70% yield) as a
colorless powder: 1H NMR (DMSO-d6) ꢀ 0.07 (s, 3H), 0.09 (s,
3H), 0.86 (s, 9H), 1.01 (s, 9H), 1.06 (s, 9H), 3.93–4.00 (m, 2H),
4.27–4.35 (m, 2H), 4.57 (d, J ¼ 5:6 Hz, 1H), 5.72 (s, 1H), 6.34
(br, 2H), 7.90 (s, 1H), 10.64 (s, 1H).
N2 -(Dimethylaminomethylene)-20 -O-(tert-butyldimethylsi-
lyl)guanosine 30-(Allyl 2-Cyanoethyl Phosphate) (5). A heter-
ogeneous mixture of the phosphoramidite 4 (3.82 g, 4.0 mmol),
allyl alcohol (0.33 mL, 279 mg, 4.8 mmol), and powdery MS
N2 -(Dimethylaminomethylene)-20 -O-(tert-butyldimethylsi-
lyl)-30,50-O-(di-tert-butylsilanediyl)guanosine (2). N,N-Dimeth-
ylformamide dimethyl acetal (11.9 g, 13.3 mL, 100 mmol) was
added with stirring to a suspension of 1 (14.3 g, 25 mmol) in
methanol (150 mL). The reaction was heated at 50 ꢁC for 5 h.
Cooling the reaction mixture afforded a colorless precipitate,
which was collected by filtration, washed with cold methanol,
and dried under reduced pressure to give 2 (14.5 g, 98% yield)
ꢁ
3A (200 mg) in acetonitrile (16 mL) was stirred at 25 C for 30
min. To this was added imidazolium perchlorate (1.35 mg, 8.0
mmol) and stirring was continued for an additional 30 min. To
the resulting mixture was added a 6.7% solution of 2-butanone
peroxide/dimethyl phthalate in toluene (8.0 mL). The mixture
was stirred for 5 min and then MS 3A were removed by passage
through a Celite 545 pad. The filtrate was diluted with ethyl ace-
tate (100 mL) and then washed with an aqueous sodium hydrogen
carbonate-saturated solution, followed by washing with brine. The
organic layer was concentrated to give a residue material. This
1
as a colorless powder: H NMR (CDCl3) ꢀ 0.14 (s, 6H), 0.93 (s,
9H), 1.05 (s, 9H), 1.07 (s, 9H), 3.13 (s, 3H), 3.34 (s, 3H), 4.00–
4.06 (m, 1H), 4.18–4.21 (m, 2H), 4.42 (d, J ¼ 3:6 Hz, 1H),
4.49–4.52 (m, 1H), 5.93 (s, 1H), 7.60 (s, 1H), 8.59 (s, 1H); HRMS
þ
(ESIþ) calcd for C27H49N6O5Si2 (M + Hþ) m=z 593.3298,

2092 Bull. Chem. Soc. Jpn., 77, No. 11 (2004)
Synthesis of Cyclic Bis(30–50)diguanylic Acid
product was dissolved in dichloromethane (30 mL) and then
cooled at 0 ꢁC. To the resulting solution was slowly added di-
chloroacetic acid (6.6 mL, 10.4 g, 80 mmol) at 0 ꢁC, and then
the mixture was stirred at the same temperature for 5 min. The re-
action mixture was poured into an aqueous sodium hydrogen car-
bonate-saturated solution (100 mL), and the organic layer was
separated. The aqueous layer was extracted with dichloromethane
(100 mL, 50 mL ꢄ 2). The combined organic extracts were dried
and concentrated to give a residual product. This crude material
was subjected to column chromatography on silica gel (100 g) us-
ing a 1:20 mixture of methanol and dichloromethane, followed by
a 1:10 mixture of methanol and dichloromethane as eluents, to
afford 5 (2.15 g, 86% yield; a mixture of two diastereomers) as
action mixture was extracted with ethyl acetate (50 mL ꢄ 3) and
the organic layer was concentrated. The resulting residual material
was subjected to column chromatography on silica gel (50 g). Elu-
tion with a 1:20 mixture of methanol and dichloromethane and
then a 1:10 mixture of methanol and dichloromethane afforded
7 (676 mg, 85% yield) as an amorphous solid: 1H NMR (CD3OD)
ꢀ ꢃ0:14 (s, 6H), 0.09 (s, 6H), 0.76 (s, 18H), 2.95 (t, J ¼ 6:0 Hz,
4H), 3.14 (s, 6H), 3.31 (s, 6H), 4.13–4.21 (m, 2H), 4.35–4.68 (m,
10H), 5.36, 5.38 (2 d, J ¼ 5:0 Hz, 2H), 5.31–5.44 (m, 2H), 5.91–
5.96 (m, 4H), 7.08 (s, 2H), 7.94 (s, 2H), 8.69 (s, 2H); 31P NMR
þ
(CD3OD) ꢀ 0.75; HRMS (ESIþ) calcd for C44H69N14O14P2Si2
(M + Hþ) m=z 1135.4126, found m=z 1135.4455.
Preparation of c-di-GMP (8) by Full Deprotection of 7. To
a suspension of 7 (116 mg, 0.1 mmol) in methanol (8 mL) was
added a concentrated aqueous ammonia solution (8 mL), and
1
a colorless amorphous solid: H NMR (CDCl3) ꢀ ꢃ0:274, ꢃ0:23
(2 s, 3H), ꢃ0:09 (s, 3H), 0.78, 0.79 (2 s, 9H), 2.80 (t, J ¼ 6:0
Hz, 2H), 3.12 (s, 3H), 3.19 (s, 3H), 3.76 (q, J ¼ 11:6 Hz, 1H),
3.88–3.91 (m, 1H), 4.27–4.35 (m, 2H), 4.46–4.48 (m, 1H),
4.63–4.67 (m, 2H), 4.97 (m, 1H), 5.08–5.17 (m, 1H), 5.31–5.37
(m, 1H), 5.41–5.45 (m, 1H), 5.69–5.72 (m, 1H), 5.95–6.03 (m,
1H), 7.63 (s, 1H), 8.40 (s, 1H), 9.37 (br, 1H); 31P NMR (CDCl3)
ꢀ ꢃ2:34; HRMS (ESIþ) calcd for C25H40N7O8PSiþ (M + Hþ)
m=z 626.2518, found m=z 626.2628.
ꢁ
the resulting mixture was stirred at 50 C for 12 h. The reaction
mixture was concentrated under reduced pressure, and the residue
was dried in vacuo. The resulting product was mixed with
(C2H5)3N 3HF (1.0 mL) and this mixture was stirred for 12 h.
ꢂ
To the reaction mixture was added a 1 M (= 1 mol dmꢃ3) ammo-
nium acetate buffer solution (10 mL). The mixture was vigorously
stirred at 30–40 ꢁC to precipitate a pale yellow solid. After remov-
al of the resulting precipitate, the aqueous solution was subjected
to preparative HPLC using a COSMOSIL 5C18-AR-300 column
[25 (diameter) ꢄ 200 (height) mm]. Elution was carried out under
these conditions: [A = a 1.0 mM ammonium acetate buffer solu-
tion, B = a 0.2 mM ammonium acetate solution in a 20:80 mix-
ture 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 di-
ammonium salt of 8 (67 mg, 89% yield): UV (a 50 mM solution
The Guanylyl(30–50)guanosine 30-Phosphate 6. A mixture of
the phosphoramidite 4 (1.7 g, 1.6 mmol) and the 50-O-free nucleo-
side phosphate 5 (980 mg, 1.6 mmol) in the presence of poꢁwdery
MS 3A (200 mg) in acetonitrile (10 mL) was stirred at 25 C for
30 min. The mixture was treated with imidazolium perchlorate
(540 mg, 3.2 mmol) and stirred for an additional 30 min. To this
mixture was added a 6.7% 2-butanone peroxide/dimethyl phtha-
late–toluene solution (3.2 mL). After 5 min, the reaction mixture
was passed through a Celite 545 pad to remove MS 3A. The fil-
trate was then concentrated to afford a viscous oil. This material
was dissolved in dichloromethane (20 mL). To this solution was
added dichloroacetic acid (3.3 mL, 5.2 g, 40 mmol) at 0 ꢁC. After
stirring for 5 min, the reaction mixture was poured into an aqueous
sodium hydrogen carbonate solution (100 mL) and the organic
layer was separated. The aqueous layer was extracted with di-
chloromethane (100 mL, 50 mL ꢄ 2). The organic solutions were
combined and concentrated. The resulting material was chromato-
graphed on a silica gel (50 g) column using a 1:20 methanol–di-
chloromethane mixture and then a 1:10 methanol–dichlorometh-
ane mixture as an eluent to afford 6 (1.53 g, 82% yield; a mixture
of four diastereomers) as a colorless amorphous solid: 1H NMR
(CDCl3) ꢀ ꢃ0:26 to 0.03 (m, 12H), 0.74–0.96 (m, 18H), 2.72–
2.80 (m, 4H), 3.09, 3.11, 3.18, 3.22 (4 s, 12H), 3.70–3.81 (m,
2H), 4.17–4.64 (m, 11H), 4.88–5.25 (m, 4H), 5.31–5.44 (m,
2H), 5.73–6.00 (m, 3H), 7.51–7.82 (m, 4H), 8.37 (s, 1H), 8.61
(s, 1H), 9.24 (br, 2H); 31P NMR (CDCl3) ꢀ ꢃ1:78, ꢃ1:72,
ꢃ1:45, ꢃ1:32, ꢃ1:05, ꢃ0:83; HRMS (ESIþ) calcd for
1
of NH4OAc in H2O) ꢂmax 254 nm (" 23700); 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) ꢀ ꢃ0:61; HRMS (ESIꢃ) calcd for
ꢃ
C20H23N10O18P2
689.0853. These spectral data were identical to those of the au-
thentic sample.6
(M ꢃ Hꢃ) m=z 689.0876, found m=z
We acknowledge partial financial support in the form of
contributions from Mitsui Chemicals.
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¨
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