Synthesis of 3′,5′-cyclic diguanylic acid (cdiGMP) using 1-(4-chlorophenyl)-4-ethoxypiperidin-4-yl as a protecting group for 2′-hydroxy functions of ribonucleosides

Article abstract of DOI:10.1080/15257770601112762

We herein report a convenient synthesis of 3′,5′-cyclic diguanylic acid via the modified H-phosphonate approach. The 1-(4-chlorophenyl)-4-ethoxypiperidin-4-yl (Cpep) group was used as protecting group for the 2′-hydroxy functions of ribonucleosides. Complete unblocking of the fully protected 3′,5′-cyclic diguanylic acid gave cdiGMP as a homogeneous compound in an excellent yield. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/15257770601112762

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Synthesis of 3’,5’-cyclic diguanylic acid
(cdiGMP) Using 1-(4-chlorophenyl)-4-
ethoxypiperidin-4-yl as a Protecting
Group for 2’-hydroxy Functions of
Ribonucleosides
Hongbin Yan a & Aimé López Aguilar a
a Department of Chemistry , Brock University , St. Catharines,
Ontario, Canada
Published online: 24 Jan 2007.
To cite this article: Hongbin Yan & Aimé López Aguilar (2007) Synthesis of 3’,5’-cyclic diguanylic
acid (cdiGMP) Using 1-(4-chlorophenyl)-4-ethoxypiperidin-4-yl as a Protecting Group for 2’-hydroxy
Functions of Ribonucleosides, Nucleosides, Nucleotides and Nucleic Acids, 26:2, 189-204, DOI:
10.1080/15257770601112762
To link to this article: http://dx.doi.org/10.1080/15257770601112762
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Nucleosides, Nucleotides, and Nucleic Acids, 26:189–204, 2007
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770601112762
SYNTHESIS OF 3’,5’-CYCLIC DIGUANYLIC ACID (cdiGMP) USING
1-(4-CHLOROPHENYL)-4-ETHOXYPIPERIDIN-4-YL AS A PROTECTING
GROUP FOR 2’-HYDROXY FUNCTIONS OF RIBONUCLEOSIDES
Hongbin Yan and Aime´ Lo´ pez Aguilar
Brock University, St. Catharines, Ontario, Canada
Department of Chemistry,
2
We herein report a convenient synthesis of 3ꢁ,5ꢁ-cyclic diguanylic acid via the modified H-
2
phosphonate approach. The 1-(4-chlorophenyl)-4-ethoxypiperidin-4-yl (Cpep) group was used as
protecting group for the 2ꢁ-hydroxy functions of ribonucleosides. Complete unblocking of the fully
protected 3ꢁ,5ꢁ-cyclic diguanylic acid gave cdiGMP as a homogeneous compound in an excellent
yield.
Keywords Biofilm; bacterial signaling pathway; H-phosphonate; cyclic ribonucleotide;
Cpep
INTRODUCTION
Biofilms are assemblies of microorganisms closely associated with a
surface and enclosed in a matrix of materials, such as polysaccharides,
nucleic acids, and proteins. Compared with organisms in their planktonic
phenotype, those in the biofilm state are very closely associated with each
other; as a consequence, microorganisms existing in biofilms are remarkably
more resistant to selection pressure, such as antibiotic treatment. It is known
that in biofilm formation, a process called quorum sensing is involved.
Thus, as the densities of microorganism change, certain compounds are
released from bacterial cells to interact with other cells by attaching to
and activating specific cell receptors. When the level of these compounds
reaches a certain threshold, signal transduction leads to the activation of
genes that control a variety of functions, including biofilm development and
regulation. 3ꢁ,5ꢁ-Cyclic diguanylic acid (cdiGMP) is an important signalling
Received 25 July 2006; accepted 6 October 2006.
This research was supported by awards from Research Corporation and Canada Foundation for
Innovation. The authors thank Professor Colin B. Reese for generous gifts of nucleosides, Tim Jones
and Razvan Simionescu for mass spectrometric and NMR analysis, respectively.
Address correspondence to Hongbin Yan, Department of Chemistry, Brock University, 500
Glenridge Ave., St. Catharines, Ontario, Canada L2S 3A1. E-mail: tyan@brocku.ca
189

190
H. Yan and A. L. Aguilar
molecule that regulates quorum sensing; its signaling function was first
revealed in a bacterium associated with grapes—Acetobacter xylinum,[1] and
it is emerging as an important bacterial second messenger in regulating
bacterial growth on surfaces (for reviews on the role of cdiGMP in biofilm
regulation, see ref.[2,3]). cdiGMP Concentration is believed to be regulated
by cdiGMP synthetase and phosphodiesterase. Proteins that contain these
synthetase and phosphodiesterase domains may contribute to the regulation
of biofilm formation by controlling cdiGMP concentration.[4] This effect
was demonstrated in Vibrio cholerae and other microorganisms.[5,6] It re-
cently was shown that cdiGMP can serve as an inhibitor for Staphylococcus
aureus cell-cell interactions and biofilm formation.[7,8] Additionally, the role
of cdiGMP in the virulence of Pseudomonas aeruginosa[9] and the response of
the transcriptional profile of Escherichia coli to high levels of cdiGMP[10] have
been reported. Thus, a convenient synthetic methodology of this compound
is of importance in order to further evaluate its roles in the regulation of
bacterial functions.
So far, three methods have been reported for the synthesis of
cdiGMP.[11–13] These procedures involved the use of tert-butyldimethyl
silyl (TBDMS) group[12,13] or tetrahydrapyranyl[11] for the protection of
the 2ꢁ-hydroxy functions. The use of TBDMS[14] suffers from a notable
disadvantage in that it can readily undergo base-catalyzed migration. The
deprotection of 2ꢁ-O-tetrahydrapyranyl-uridine requires relatively drastic
acidic conditions (pH 2.0, 16 hours at room temperature), under which
conditions ribonucleotides may undergo migration and degradation. Ad-
ditionally, the tetrahydrapyranyl group is chiral; therefore, its use leads to
diastereoisomeric mixtures of products.[15]
Other protecting groups such as TOM,[16] ACE,[17] and
cyanoethoxymethyl[18] groups (Figure 1) also are used for the protection
of the 2ꢁ-hydroxy functions. However, introduction of the TOM and
cyanoethoxymethyl groups at O-2ꢁ position of ribose is not regiospecific.
In addition to this, unblocking of cyanoethoxymethyl is relatively difficult.
The ACE protecting group requires the use of relatively expensive starting
materials.
FIGURE 1 Some protecting groups for the 2ꢁ-OH of ribonucleosides.

Synthesis of cdiGMP
RESULTS AND DISCUSSION
191
The 1-(4-chlorophenyl)-4-ethoxypiperidin-4-yl (Cpep) group has unique
hydrolytic properties which make it the potential protecting group of choice
in solid-phase RNA synthesis.[19] Its apparent advantages are as follows: (i)
only cheap starting materials are used in the preparation of the monomeric
building blocks; (ii) 2ꢁ-O-Cpep protection of 2ꢁ-hydroxy functions proceeds
readily and regiospecifically; (iii) the Cpep group cannot migrate and
remains intact until the final deblocking step; (iv) as Cpep-protected RNA
sequences are stable to base and resistant to ribonucleases, they can readily
be purified and stored; (v) removal of the Cpep protecting groups in the
final deblocking step proceeds readily under very mild acidic conditions
(such as pH 4.0 at 35–40◦C for 5 hours) that do not promote cleavage or
migration of the internucleotide linkages. This chemistry was successfully
used to assemble the 3ꢁ-terminal decamer of yeast tRNAAla in solution.[20]
For these reasons, the Cpep was chosen as the protecting group for the
2ꢁ-hydroxy functions in this study.
In conjunction with the Cpep group, an acid-sensitive protecting group
also is required for the 5ꢁ-hydroxy functions of ribonucleosides. The ad-
vantage of using the 9-phenyl-xanthen-9-yl (or the pixyl as in Figure 2) as
the protecting group for 5ꢁ-hydroxy functions versus the 4,4ꢁ-dimethoxytrityl
group was recently demonstrated.[21] Because the pixyl group is about 3
times more readily removable under acidic conditions compared with the
DMTr group, its use shortens the exposure of Cpep groups to acids. This fea-
ture consequently ensures that the Cpep groups remain intact during depro-
tection of the 5ꢁ-hydroxy protecting group using trifluoroacetic acid. Thus,
the pixyl group is completely deprotected using trifluoroacetic acid in the
presence of pyrrole in 10 seconds. It is worthwhile to note that 5ꢁ-pixylated
nucleosides and nucleotides are stable compounds and can be readily puri-
fied by column chromatography without notable loss of the pixyl groups.
The guanine base residues were “doubly” protected with O6-(2,5-
dichlorophenyl)- and N 2-phenyl acetyl groups (Figure 3). Protection of
the O6-carbonyl function is necessary to both increase the solubility of
guanosine and prevent possible side-reaction with phosphorylating agents,
such as diphenyl chlorophosphate, which is used in the cyclization reaction
FIGURE 2 DMTr and Pixyl groups.

192
H. Yan and A. L. Aguilar
FIGURE 3 “Double” protection of guanine base residue.
as an activator.[22] The O6-(2,5-dichlorophenyl) is readily removable by
treatment with 2-nitro-benzaldoximate followed by aqueous ammonia.
The introduction of Cpep group at the O-2ꢁ positions of ribose is
straightforward and involves only relatively inexpensive starting material.[17]
2-N -Phenylacetylguanosine 1[23] first reacted with the 1,1-dichloro-1,1,3,3-
tetraisopropyldisiloxane (the Markiewicz reagent)[24] to give the 3ꢁ,5ꢁ-O
protected guanosine 2. After the O6- of guanine residue was protected by
2,5-dichlorophenol,[22] the Cpep group was introduced by treatment with
the enolether 3 under acidic conditions. After the “Markiewicz protecting
group” was removed by treatment with tetraethylammonium fluoride, the
“doubly” protected guanosine 4 with its 2ꢁ-hydroxy function protected with
Cpep group was obtained in 75% overall yields from 1 (Scheme 1). This 2ꢁ-O-
Cpep protected guanosine 4 was then transformed into the two components
5 and 6 that are required for the assembly of dinucleotides (Scheme 1, steps
v, vi, and vii).
The modified H-phosphonate approach[25] which involves an in
situ treatment of H-phosphonate diesters with a sulfur transfer reagent
has been shown to be a valuable method towards the synthesis of both
oligonucleotide phosphates and phosphorothioates.[26] Triethylammonium
2ꢁ-O-[1-(4-chlorophenyl)-4-ethoxypiperidin-4-yl]-6-O-(2,5-dichlorophenyl)-
2-N -phenylacetyl-5ꢁ-O-(xanthen-9-yl)guanosine 3ꢁ H-phosphonate 7 was
prepared in 95% yield by treatment of 2ꢁ-O-[1-(4-chlorophenyl)-4-ethoxy-
piperidin-4-yl]-6-O-(2,5-dichlorophenyl)-2-N -phenylacetyl-5ꢁ-O-(xanthen-
9-yl)guanosine 5 with p-tolyl H-phosphonate 8 (Scheme 1, steps viii and
ix).[27]
The overall strategy for the synthesis of fully protected cdiGMP 13 is
represented in Scheme 2. Thus, coupling of the H-phosphonate 7 with 6
was activated by pivaloyl chloride. This was followed by treatment with 1-
phenylsulfanyl-pyrrolidine-2,5-dione 11[28] in situ to give the fully protected
linear diguanylic acid 9. Removal of the 3ꢁ-levulinyl group from 9 is ef-
fected by treatment with hydrazine hydrate in aqueous acetic acid–pyridine
solution. Phosphitylation of the product gives fully protected diguanylic
acid H-phosphonate 10. Upon removal of the 5ꢁ-pixyl group, cyclization

Synthesis of cdiGMP
193
SCHEME 1 Reagents and conditions i, (i-Pr)2Si(Cl)OSi(i-Pr)2(Cl), C5H5N; ii, 3, CF3COOH, CH2Cl2; iii,
2,5-dichlorophenol, C5H5N, N -methylpyrrolidine, mesitylenesulfonyl chloride; iv. Et4N+ + F−, CH3CN;
v, PxCl, C5H5N; vi, (Lev)2O, C5H5N; vii, CF3COOH, pyrrole, CH2Cl2; viii, 8, (CH3)3COCl, C5H5N; ix,
aq. work-up.
of H-phosphonate 12 was carried out under high dilution conditions to
furnish fully protected cyclic product 13 in 73% yield, using diphenyl
chlorophosphate as an activating agent.
The linear diguanylic acid H-phosphonate 12 was characterized by 31P
NMR (Figure 4). As expected, two sets of peaks were observed, each of which
gave an integral representing one phosphorus. The resonance at ca. 2.9
ppm represents the H-phosphonate phosphorus center and is split by the
proton that is attached to it with a coupling constant of 626.9 Hz.

194
H. Yan and A. L. Aguilar
SCHEME 2 Reagents and conditions i. (CH3)3COCl, C5H5N; ii, 11, C5H5N; iii, NH2NH2·H2O,
CH3COOH, H2O, C5H5N; iv, 8, (CH3)3COCl, C5H5N, 0◦C; v, CF3COOH, pyrrole, CH2Cl2; vi,
(PhO)2P(O)Cl, CH2Cl2, C5H5N, -40◦C; vii, 11, C5H5N.
FIGURE 4 31P NMR spectra of linear diguanylic acid H-phosphonate 10. a) 1H decoupled; b) 1H
undecoupled.

Synthesis of cdiGMP
195
31P NMR spectrum of the fully protected cdiGMP 13 revealed three
distinguishable peaks (25.46, 28.73, and 28.94 ppm). Presumably, they
represent the four diastereoisomers ([R, R], [R, S], [S, S], and [S, R]) due
to the chirality of the internucleotide phosphorothioate linkages.
Unblocking of cdiGMP 13 involves three steps. First, an oximate treat-
ment displaces the P-SPh groups as well as the O6-phenyl protecting groups
on guanine base residues (Scheme 3, step i). This is followed by ammonoly-
sis to give the partially, that is, Cpep-protected cdiGMP 14 (Scheme 3, step
ii). Its molecular weight in the form of M− was found by ESI-MS to be 1163.3
1
(calculated 1163.27). This intermediate also was characterized by H and
31P NMR. Since the internucleotide phosphorothioate linkages in the fully
protected compound 13 are now converted to phosphate diesters, only one
phosphorus peak was observed in the 31P NMR spectrum at 0.38 ppm.
Unblocking of the Cpep protecting groups (Scheme 3, step iii) was ef-
fected by treatment with triethylammonium formate buffer [pH 2.52]–DMA
(2:3 v/v) (apparent pH 4.0) at 40◦C for 5 hours to give fully unblocked
cdiGMP 15 in 95% yield. The fully unblocked cdiGMP 15 was characterized
1
1
by H and 31P NMR (Figure 5). H NMR spectroscopy recorded at 40◦C
(Figure 5a) revealed all the protons of cdiGMP 15. Formation of homoge-
1
neous product was indicated by the H and 31P NMR spectra. HR-MALDI
for [M H]− was found to be 689.1095 (calculated 689.0870). Reverse phase
HPLC analysis (Figure 6b) also confirmed the homogeneity of cdiGMP 15.
CONCLUSION
Using Cpep as the protecting group for 2ꢁ-hydroxy functions, homoge-
neous cdiGMP was successfully synthesized in good yield. This method can
be extended to the preparation of other cyclic nucleotide analogues that
are under investigation for their roles in bacterial biofilm regulation.
SCHEME 3 Reagents and conditions i. 16, (CH3)2NC( NH)N(CH3)2, CH3CN; ii, aq. NH3, 55◦C; iii,
(CH3)2NC(O)CH3, NEt3-HCOOH buffer (pH2.52), 40◦C, 5 hours.

196
H. Yan and A. L. Aguilar
FIGURE 5 a) 1H (recorded at 40◦C) and b) 31P NMR spectra of the fully-deprotected cdiGMP 15 in D2O.
Experimental
1H and 31P NMR spectra were measured at 300 and 90 MHz on a
Bruker Avance (at the Department of Chemistry, Brock University) 300
spectrometer, respectively, unless stated otherwise; tetramethylsilane was
used as an internal standard, and J values are given in Hz. Reverse phase
high-performance liquid chromatography (HPLC) was carried out on a
4.6 × 150 mm Acclaim PA C18 3µ column: the column was eluted with
triethylammonium acetate (TEAA) buffer (0.1 M, pH7.0)—acetonitrile
mixtures [programme A: linear gradient of TEAA buffer-acetonitrile (100 :
FIGURE 6 Reverse phase HPLC profile of 14 (profile a, program A: linear gradient of 0.1 M linear
gradient of triethylammonium acetate [TEAA] buffer-acetonitrile (100 : 0 v/v to 80 : 20 v/v) over 10
minutes and then isocratic elution) and 15 (profile b, programme B: TEAA buffer–acetonitrile (100: 00
v/v to 98 : 2 v/v) over 10 minutes and then isocratic elution).

Synthesis of cdiGMP
197
0 v/v to 80 : 20 v/v) over 10 minutes and then isocratic elution; programme
B: linear gradient of TEAA buffer–acetonitrile (100 : 00 v/v to 98 : 2
v/v) over 10 minutes and then isocratic elution]. Merck silica gel 60 F254
TLC plates were developed in solvent system: [dichloromethane–methanol
(95:5 v/v)]. Merck Kieselgel H (Art 7729 and 9385) was used for short
column chromatography. Pyridine, 1-methylpyrrolidine, and acetonitrile
were dried by heating with calcium hydride, under reflux, and then
distilled; N ,N ,N ꢁN ꢁ-tetramethylguanidine was dried by distillation over cal-
cium hydride under reduced pressure; dichloromethane was dried over
phosphorous pentoxide and distilled; and toluene was dried by heating,
under reflux, with sodium and benzophenone, and then distilled. 1,1-
Dichloro-1,1,3,3-tetraisopropyldisiloxane, 9-phenyl-xanthene-9-ol, and 2-N -
phenylacetylguanosine were purchased from Rasayan, Inc. and were used
without further purification.
2ꢁ-O-[1-(4-Chlorophenyl)-4-ethoxypiperidin-4-yl]-6-O-(2,5-dichlorophenyl)-2-
N-phenylacetylguanosine 4
2ꢁ-O-[1-(4-Chlorophenyl)-4-ethoxypiperidin-4-yl]-2-N -phenylacetyl-3ꢁ,5ꢁ-
O-(1,1,3,3-tetraisopropyldisiloxyl)guanosine 2[19] (6.800 g, 7.713 mmol)
was azeotroped with dry pyridine (2 × 10 ml) and then redissolved in
pyridine (30 ml) and cooled (ice-water bath). Mesitylenesulfonyl chloride
(2.700 g, 12.34 mmol) and N -methylpyrrolidine (8.2 ml, 78.9 mmol)
followed after 15 minutes, 2,5-dichlorophenol (4.10 g, 25.19 mmol)
were added. The reaction mixture was then allowed to warm up to
room temperature. After 2 hours, the products were partitioned between
dichloromethane (150 ml) and saturated aqueous sodium hydrogen
carbonate (2 × 100 ml). The layers were separated and the aqueous layers
were back-extracted with dichloromethane (2 × 50 ml). The combined
dried (MgSO4) organic layers were concentrated under reduced pressure.
The residue was taken up with acetonitrile (40 ml) and a solution of
tetraethylammonium fluoride in acetonitrile (40 ml, 1 M, pH 8.0) was
added. After 30 minutes, the products were evaporated under reduced
pressure and the residue was partitioned between dichloromethane (200
ml) and saturated aqueous sodium hydrogen carbonate (100 ml). The
layers were separated and the aqueous layer was back-extracted with
dichloromethane (2 × 50 ml). The combined organic layers were dried
(MgSO4) and concentrated under reduced pressure. The residue was
purified by short column chromatography on silica gel. The appropriate
fractions, which were eluted with dichloromethane-methanol (98:2 v/v)
were pooled and evaporated under reduced pressure to give the title
compound as a colorless froth (5.500 g, 90.9%). Rf: 0.35. ESI-MS found
1
−
[M H]− = 781.1. 12C37 H3635Cl314N616O7 requires: 781.1711.

198
H. Yan and A. L. Aguilar
δH[(CD3)2SO] include the following peaks: 0.68 (3 H, t, CH2CH3
(Cpep), J = 6.9), 1.64 (2 H, m, CH2 (Cpep)), 1.85 (2 H, m, CH2 (Cpep)),
2.73 (2 H, m, CH2 (Cpep)), 2.96 (1 H, m, CH2 (Cpep)), 3.07 (1 H, m, CH2
(Cpep)), 3.32 (4 H, m, CH2CH3 (Cpep)), 3.65 (1 H, m, H-5ꢁ), 3.72 (1 H,
m, H-5ꢁꢁ), 4.00 (1 H, br, H-4ꢁ), 4.19 (1 H, br, H-3ꢁ), 4.93 (1 H, dd, H-2ꢁ, J =
5.1 and 6.6), 5.12 (1 H, t, 5ꢁ-OH, J = 5.4, ex), 5.22 (1 H, d, 3ꢁ-OH, J = 4.8,
ex), 6.09 (1 H, d, H-1ꢁ, J = 6.6), 7.14–7.44 (7 H, m), 7.66 (1 H, d, J = 8.7),
7.76 (1 H, d, J = 2.4), 8.58 (1 H, dd, J = 5.7 and 1.5), 8.71 (1 H, s, H-8),
10.63 (1 H, s, ex, NH).
2ꢁ-O-[1-(4-Chlorophenyl)-4-ethoxypiperidin-4-yl]-6-O-(2,5-dichlorophenyl)-2-
N-phenylacetyl-5ꢁ-O-(xanthen-9-yl)guanosine 5
4 (5.40 g, 6.89 mmol) Was evaporated with dry pyridine (2 × 10 ml).
The residue was redissolved in anhydrous pyridine (40 ml) followed by
addition of 9-chloro-9-phenylxanthene[29] (2.50 g, 8.54 mmol). After 30
minutes, a mixture of methanol–N -methylmorpholine (4 ml, 1:1 v/v) was
added. After a further period of 10 minutes, the products were partitioned
between dichloromethane (100 ml) and saturated aqueous sodium hydro-
gen carbonate (80 ml). The layers were separated and the aqueous layer was
back-extracted with dichloromethane (2 × 10 ml). The combined organic
layers were dried (MgSO4) and concentrated under reduced pressure. The
residue was fractionated by short column chromatography on silica gel. The
appropriate fractions, which were eluted with dichloromethane–methanol
(98.5:1.5 v/v) were combined and concentrated under reduced pressure to
give 5 as a colorless froth (6.31 g, 88%). Rf: 0.45.
δH[(CD3)2SO] include the following peaks: 0.76 (3 H, t, CH2CH3
(Cpep), J = 6.9), 3.63 (1 H, d, CH2Ph, J = 15.0), 3.70 (1 H, d, CH2Ph,
J = 15.0), 4.12 (1 H, br, H-4ꢁ), 4.25 (1 H, br, H-3ꢁ), 5.01 (1 H, t, H-2ꢁ, J = 5.6),
5.23 (1 H, d, 3ꢁ-OH, J = 6.0, ex), 6.10 (1 H, d, H-1ꢁ, J = 6.0), 7.66 (1 H, d,
J = 8.7), 8.50 (1 H, s, H-8), 8.58 (2 H, dd, J = 5.8 and 1.6), 10.54 (1 H, s,
ex, NH).
2ꢁ-O-[1-(4-Chlorophenyl)-4-ethoxypiperidin-4-yl]-6-O-(2,5-di-chlorophenyl)-2-
N-phenylacetyl-5ꢁ-O-(xanthen-9-yl)guanosine 3ꢁ H-phosphonate,
triethylammonium salt 7
p-Tolyl H-phosphonate 8 (1.14 g, 6.02 mmol) was evaporated with
triethylamine (1.70 ml, 12.20 mmol) and methanol (10 ml). To the residue
was added 5 (2.080 g, 2.0 mmol). The mixture was azeotroped with dry
pyridine (2 × 5 ml). The residue was redissolved in anhydrous pyridine
(25 ml) and cooled to 0◦C (ice-water bath). Pivaloyl chloride (0.92 ml, 7.47
mmol) was added over a period of 5 minutes. After 1 hour, water (5 ml)
was added and the reactants were allowed to warm up to room temperature.
After 1 hour, the products were partitioned between dichloromethane (100

Synthesis of cdiGMP
199
ml) and saturated aqueous sodium hydrogen carbonate (2 × 80 ml). The
combined aqueous layers were back extracted with dichloromethane (2 ×
20 ml). The combined organic layers were further washed with triethylam-
monium phosphate buffer (0.5 M, pH 7.0, 80 ml). The layers were separated
and the aqueous layer was back extracted with dichloromethane (20 ml).
The combined dried (MgSO4) organic layers were concentrated under
reduced pressure. The residue was fractionated by short column chro-
matography on silica gel. The appropriate fractions, which were eluted with
dichloromethane–methanol (93:7 v/v) were combined and concentrated
under reduced pressure to give 7 as a colorless froth (2.20 g, 91%). ESI-MS
1
16
found M−=1101.22. 12C56 H4935Cl314N6
O
10
31P− requires: 1101.2313.
δH[(CD3)2SO] include the following peaks: 0.76 (3 H, t, CH2CH3
(Cpep), J = 6.9), 1.11 (9 H, t, NH+(CH2CH3)3, J = 2.7), 4.35 (1 H, br,
H-4ꢁ), 4.75 (1 H, dd, H-3ꢁ, J = 8.4 and J 3.9), 5.17 (1 H, dd, H-2ꢁ, J = 7.2
and 4.2), 5.79 (0.5 H, s, PH), 6.13 (1 H, d, H-1ꢁ, J = 7.2), 6.85 (2 H, d, J =
9.0), 6.97 (1 H, t, J = 7.5), 7.15–7.32 (17 H, m), 7.40 (1 H, dd, J = 6.5 and
2.4), 7.64 (1 H, d, J = 8.7), 7.74 (0.5 H, s, PH), 7.75 (1 H, s), 8.45 (1 H, s,
H-8), 10.71 (1 H, s, ex, NH).
δP[(CD3)2SO]: 0.18 (1J P-H = 564.9)
2ꢁ-O-[1-(4-Chlorophenyl)-4-ethoxypiperidin-4-yl]-6-O-(2,5-dichlorophenyl)-3ꢁ-
O-levulinyl-2-N-phenylacetyl-guanosine 6
Compound 5 (1.50 g, 1.442 mmol) was dried by azeotroping with dry
pyridine (2 × 5 ml), and then redissolved in dry pyridine (10 ml). Levunilic
anhydride (0.628 g, 2.932 mmol) was then added, and the reaction was
allowed to proceed for 15 hours. Methanol (2 ml) was added. After another
10 minutes, the products were concentrated to dryness under reduced
pressure. The residue was co-evaporated with toluene (3 × 5 ml), and then
redissolved in dichloromethane (10 ml). Pyrrole (1.00 ml, 14.41 mmol)
followed by trifluoroacetic acid (0.67 ml, 9.02 mmol) were added. After
30 seconds, N -methylmorpholine (1.00 ml, 9.09 mmol) was added. The
products were poured into saturated aqueous sodium hydrogen carbonate
(15 ml). The layers were separated and the aqueous layer was back-extracted
with dichloromethane (20 ml). The organic layers were then combined,
dried (MgSO4), and concentrated. The residue was purified by short
column chromatography. The appropriate fractions, which were eluted with
dichloromethane–methanol (98:2 v/v) were combined and concentrated
under reduced pressure to give 6 as a colorless froth (1.143 g, 90%).
Rf: 0.43. ESI-MS found [M-H]− = 879.2.
C
1H4235Cl314N616O9 requires:
42
12
879.207.
δH[(CD3)2SO] include the following peaks: 0.61 (3 H, t, CH2CH3
(Cpep), J = 6.9), 2.95 (2 H, m, CH2 (Cpep)), 3.19 (2 H, m, CH2 (Cpep)),
4.14 (1 H, H-4ꢁ), 5.26 (1 H, H-2ꢁ), 5.29 (1 H, H-3ꢁ), 5.34 (1 H, ex, 5ꢁ-OH),

200
H. Yan and A. L. Aguilar
6.12 (1 H, d, H-1ꢁ, J = 7.5), 6.83 (1 H, d, J = 9.0), 7.16–7.31 (7 H, m), 7.43
(1 H, dd, J = 8.7 and 2.4), 7.66 (1 H, d, J = 8.7), 7.76 (1 H, d, J = 2.4), 8.74
(1 H, s, H-8), 10.68 (1 H, s, ex, NH).
Px-Gꢁꢁp(sꢁ)Gꢁꢁ-Lev∗ 9
Compounds 6 (0.463 g, 0.525 mmol) and 7 (0.750 g, 0.622 mmol)
were dried by azeotroping with dry pyridine (2 × 5 ml). The residue
was redissolved in anhydrous pyridine (5 ml) and cooled (ice-water bath).
Pivaloyl chloride (0.16 ml, 1.30 mmol) was added. After 5 minutes, 1-
phenylsulfanyl-pyrrolidine-2,5-dione 11 (0.310 g, 1.496 mmol) was added.
The reactants were then warmed up to room temperature. After 30 minutes,
water (0.2 ml) was added. After 5 minutes, the products were partitioned
between dichloromethane (25 ml) and saturated aqueous sodium hydrogen
carbonate (25 ml). The layers were separated and the aqueous layer was
back extracted with dichloromethane (2 × 5 ml). The combined organic
layers were dried (MgSO4) and concentrated under reduced pressure. The
residue was purified by short column chromatography on silica gel. The
appropriate fractions, which were eluted with dichloromethane–methanol
(98.5:1.5 v/v) were combined and concentrated under reduced pressure to
give 9 as a colorless froth (1.02 g, 93.5%). Rf: 0.52.
δP[(CD3)2SO]: 22.10, 22.73
Preparation of HO-Gꢁꢁp(sꢁ)Gꢁꢁp(H) 12
Px-Gꢁꢁp(sꢁ)Gꢁꢁ-Lev 9 (0.900 g, 0.433 mmol) was dissolved in pyridine (6
ml) followed by addition of a mixture of hydrazine monohydrate (0.20 ml,
4.12 mmol), acetic acid (2.4 ml), water (0.4 ml) and pyridine (9 ml) at
room temperature. After 15 minutes, pentan-2,4-dione (0.85 ml) was added.
Stirring was continued for 10 minutes and the products were partitioned
between dichloromethane (30 ml) and saturated aqueous sodium hydrogen
carbonate (3 × 30 ml). The layers were separated and the aqueous layers
were back-extracted with dichloromethane (2 × 50 ml). The combined
dried (MgSO4) organic layers were concentrated under reduced pressure.
The residue was dissolved in dichloromethane (1.0 ml) and added drop-wise
to diethyl ether (100 ml) under stirring. The precipitate was collected
by filtration to give a colorless solid (0.80 g). This material (abbrevi-
ated as Px-Gꢁꢁp(sꢁ)Gꢁꢁp(H) 10) was used for the next step without further
purification.
∗A system of abbreviations[30] is followed for protected oligonucleotides in which nucleoside
residues and internucleotide linkages are italicized if they are protected in some defined way. In
the present context, Gꢁꢁ represents guanine protected on O-6 with a 2,5-dichlorophenyl group and
N -2-protected with a phenylacetyl group; and p(sꢁ) represents an S-phenyl-protected phosphorothioate.

Synthesis of cdiGMP
201
p-Tolyl H-phosphonate 8 (0.222 g, 1.174 mmol) was evaporated with
triethylamine (0.50 ml, 3.59 mmol) and methanol (1.0 ml). To the residue
was added Px-Gꢁꢁp(sꢁ)Gꢁꢁ OH 10 (0.780 g, 0.394 mmol). The mixture was
azeotroped with dry pyridine (2 × 5 ml). The residue was redissolved in
anhydrous pyridine (8 ml) and cooled to 0◦C (ice-water bath). Pivaloyl
chloride (0.15 ml, 1.22 mmol) was added in one portion. After 30 minutes,
water (1.0 ml) was added. The reactants were stirred at room temperature
for 1 hour. The products were then partitioned between dichloromethane
(30 ml) and saturated aqueous sodium hydrogen carbonate (30 ml). The
combined aqueous layers were back-extracted with dichloromethane (2 ×
10 ml). The organic phases were combined, dried (MgSO4) and concen-
trated under reduced pressure, followed by co-evaporation with toluene
(3 × 10 ml). The residue was redissolved in dichloromethane (15 ml).
Pyrrole (0.41 ml, 5.91 mmol) followed by trifluoroacetic acid (0.23 ml, 3.10
mmol) were added. After 1 minute, N -methylmorpholine (0.48 ml) was
added. The products were then extracted with saturated aqueous sodium hy-
drogen carbonate (20 ml). The layers were separated and the aqueous layers
were back extracted with dichloromethane (2 × 10 ml). The combined or-
ganic layers were further washed with triethylammonium phosphate buffer
(0.5 M, pH 7.0, 20 ml). The layers were separated and the aqueous layer
was back-extracted with dichloromethane (10 ml). The combined organic
layers were dried (MgSO4) and concentrated under reduced pressure. The
residue was purified by short column chromatography on silica gel. The
appropriate fractions, which were eluted with dichloromethane–methanol
(92:8 v/v) were pooled and concentrated under reduced pressure to give
H-phosphonate 12 as a colorless froth (0.560 g, 70% for three steps).
12
14
16
ESI-MS found M− = 1781.2.
C
80
1H7735Cl6
N
O
31P232S− requires:
12 17
1781.2857.
δP[(CD3)2SO]: 2.89, 3.09 (1 P), 23.79, 25.05 (1 P, 1J P-H = 626.9).
Preparation of Fully Protected cdiGMP 13
HO-Gꢁꢁp(sꢁ)Gꢁꢁp(H) 12 (0.190 g, 0.100 mmol) was co-evaporated with dry
pyridine (2 × 3 ml) and dissolved in anhydrous dichloromethane (10 ml).
This solution was then added dropwise over a period of 20 minutes to a
cooled solution of diphenyl chlorophosphate (0.41 ml, 1.98 mmol) in dry
pyridine (10 ml) at −40◦C. After 20 minutes, 1-phenylsulfanyl-pyrrolidine-
2,5-dione 11 (0.453 g, 2.18 mmol) was added and the reactants were allowed
to warm up to room temperature over 30 minutes. Water (0.5 ml) was then
added and after a further period of 5 minutes, the products were partitioned
between dichloromethane (15 ml) and saturated aqueous sodium hydrogen
carbonate (2 × 10 ml). The layers were separated and the aqueous layers
were back extracted with dichloromethane (2×5 ml). The combined dried
(MgSO4) organic layers were concentrated under reduced pressure and the

202
H. Yan and A. L. Aguilar
residue was fractionated by short column chromatography on silica gel. The
appropriate fractions, which were eluted with dichloromethane–methanol
(98.5:1.5–98:2 v/v) were pooled and concentrated under reduced pressure
to give the fully protected cdiGMP 13 as a colorless froth (0.138 g, 73%). Rf:
0.51 and 0.45.
δP[(CD3)2SO]: 25.46, 28.73, 28.94.
Preparation of 2ꢁ-O-Cpep Cyclic bis(3ꢁ→5ꢁ)diguanylic Acid 14 by
Partial-Unblocking of Fully Protected Cyclic Diguanydic Acid 13
Fully-protected cdiGMP 13 (30 mg, 15.99 µmol) was azeotroped with dry
toluene (2 × 2 ml) and the residue was dissolved in dry acetonitrile (2 ml).
2-Nitrobenzaldoxime 16 (38.3 mg, 0.231 mmol) followed by N ,N ,N ꢁ,N ꢁ-
tetramethylguanidine (26 µl, 0.21 mmol) were added. The reactants were
stirred at room temperature for 16 hours and were then concentrated
under reduced pressure. To the residue was added concentrated aqueous
ammonia (33%, d 0.88, 2.0 ml) and the mixture was heated at 55◦C for
15 hours. After the products had been cooled, they were concentrated to
dryness followed by co-evaporation with ethanol (2× 2 ml) under reduced
pressure. The residue was dissolved in methanol (1 ml) and precipitated
with diethyl ether (30 ml). The precipitate was collected by centrifuga-
tion. This precipitation-centrifugation process was repeated for one more
time and the solid residue was dried in vacuo to give 2ꢁ-O-Cpep cyclic
bis(3ꢁ→5ꢁ)diguanylic acid 14 as a colorless solid (17.0 mg, 89.5%). ESI-MS
12
14
16
−
found M−=1163.3.
C
46
1H5535Cl2
N
O
31P2 requires: 1163.2711. Rt
12 16
(Programme A): 7.8 minutes.
δP[D2O]: −0.38.
δH[D2O, 600 MHz] include the following peaks: 0.67 (6 H, s, CH2CH3
(Cpep)), 3.45 (2 H, br), 3.99 (2 H, br, H-5ꢁ), 4.21 (2 H, br, H-5ꢁꢁ), 4.55 (2 H,
br, H-4ꢁ), 4.85 (2 H, br, H-2ꢁ), 5.00 (2 H, br, H-3ꢁ), 5.86 (2 H, br, H-1ꢁ), 6.79
(4 H, br, Cpep), 7.11 (2 H, s, br, Cpep), 7.12 (2 H, s, br, Cpep), 7.88 (2 H, s,
H-8).
Cyclic bis(3ꢁ→5ꢁ)diguanylic Acid 15 (cdiGMP)
Substrate 14 (10 mg, 8.60 µmol) was dissolved in dimethylacetamide
(1.8 ml) followed by addition of triethylammonium formate buffer (0.5
M, pH 2.52, 1.2 ml, prepared with sterile water). The reactants were then
sealed and heated at 40◦C. After 5 hours, the products were cooled, and
the pH was adjusted to ca. 7.0 with triethylamine. The products were
then extracted with chloroform (4 × 2.0 ml). The organic layers were
discarded. The aqueous layer was evaporated under reduced pressure at
room temperature to a volume of ca. 0.2 ml. To the residue was added

Synthesis of cdiGMP
203
n-butanol (2.0 ml). The mixture was vortexed vigorously and then chilled
at –78◦C (dry ice–acetone bath) for 10 minutes. Then it was centrifuged for
10 minutes. The supernatant was discarded and the pellet was redissolved
in sterile water (0.2 ml). The above precipitation–centrifugation process
was repeated two more times. The final pellet was then dried in vacuo.
This material was dissolved in sterile water (1.0 ml) and passed through an
Amberlite (IR120, Na+ form) cation-exchange column (0.5 × 2.0 cm). The
fractions which contained oligonucleotide were pooled and freeze-dried to
give the fully unblocked cdiGMP 15 as a colorless froth (6.0 mg, 94.9%). HR-
−
−
1
14
16
MALDI found M = 689.1095. 12C20 H23
N
10
O
14
31P2 requires 689.0870.
Rt (Programme B): 8.3 minutes.
δH[(CD3)2SO, 600 MHz 40◦C]: 4.08 (2 H, dd, H-5ꢁ, J 11.6 and 3.7), 4.38
(2 H, d, H-5ꢁꢁ, J 11.5), 4.44 (2 H, d, H-4ꢁ, J 8.3), 4.82 (2 H, d, H-2ꢁ, J 3.6),
5.04 (2 H, m, H-3ꢁ), 5.92 (2 H, s, H-1ꢁ), 8.11 (2 H, s, H-8).
δP[D2O, 242 MHz]: −1.55.
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