New approach for the synthesis of c-di-GMP and its analogues

Article abstract of DOI:10.1055/s-2006-950361

The synthesis of cyclic bis(3′-5′)diguanylic acid (c-di-GMP) by using a new flexible approach is reported. The flexibility of the method is exemplified by the synthesis of base-modified analogues that will find applications in the elucidation of c-di-GMP

Full text of DOI:10.1055/s-2006-950361

4230
PAPER
New Approach for the Synthesis of c-di-GMP and Its Analogues
S
ynthesis of c-di-G
i
MP an
c
d
A
nalogue
o
s
las Amiot, Karine Heintz, Bernd Giese*
Organic Chemistry Institute, University of Basel, St Johanns-Ring 19, 4056 Basel, Switzerland
Fax +41(61)2671105; E-mail: Bernd.Giese@unibas.ch
Received 31 July 2006; revised 4 September 2006
little data is available on c-di-GMP targets.1,4 In order to
study the biochemistry of c-di-GMP in more detail we
have started a research program dedicated to the synthesis
of c-di-GMP and its analogues. Because the literature pro-
cedures start from protected guanosines and are therefore
limited to the synthesis of c-di-GMP,6,12–15 we have de-
signed a new and flexible approach towards c-di-GMP
and its base derivatives.
Abstract: The synthesis of cyclic bis(3¢-5¢)diguanylic acid (c-di-
GMP) by using a new flexible approach is reported. The flexibility
of the method is exemplified by the synthesis of base-modified an-
alogues that will find applications in the elucidation of c-di-GMP
biological modes of action.
Key words: c-di-GMP, nucleotide, cyclization, nucleobases, phos-
photriester methodology
We have decided to develop a new approach where a cy-
clic sugar backbone 7 was synthesized first and the base
was introduced at the latest stage. The synthesis started
from readily available furanose building blocks 2 and
316,17 (Scheme 1). The phosphotriester methodology6,18
was used to produce dimer 5, which was deprotected with
ceric ammonium nitrate (CAN) in methanol19 (5 → 6). In
the next step, cyclization was achieved using 2-chlorophe-
nyl phosphorodichloridate in dilute pyridine solution.
Two fractions with notably different Rf values on TLC,
which contain, due to the chiral phosphates, three stereo-
isomers of 7, were isolated. The highest yields were ob-
tained after an hour of reaction time, any prolonged
reaction resulted in cleavage of phosphate linkages. The
cyclic sugar building block 7 was produced in yields of
33% over three steps, and the synthesis could be repro-
duced in a multi-gram scale.
Cyclic bis(3¢-5¢)diguanylic acid (c-di-GMP) 1 (Figure 1)
has been recently identified as a universal bacterial sec-
ondary messenger.1–5 It was first related to the regulation
of the cellulose synthesis in the bacterium Acetobacter
xylinum6 but it became clear later that c-di-GMP is in-
volved in several biological events including regulation of
biofilm formation in Vibrio cholerae7 and Yersinia pes-
tis,8 and the transition from motility to sessility of Escher-
ichia coli and Salmonella typhimurium.9 It is involved in
the inhibition of Staphylococcus aureus cell–cell interac-
tions and biofilm formation, as well as in the reduction of
the virulence of the biofilm-forming strains of the same
bacterium in a mouse model of mastitis infection.10 The
biological activity might be even wider since reports have
pointed out that this compound may have anticancer activ-
ity.11
In this paper we describe the synthesis of c-di-GMP using
the higher-Rf fraction. However, the lower-Rf fraction
gives equally good results and leads in the same way to c-
di-GMP. In the next step, the cyclic acetals were cleaved
and replaced by acetates20 that are required for the base in-
troduction reactions (Scheme 2). Modified Vorbrüggen
reaction conditions21,22 were used to obtain fully protected
c-di-GMP 9. In the first step, the guanine building block
O
N
NH
O
O–
O
N
N
NH2
P
OH
O
O
O
O
O
O
OH
P
N
N
8
21 was silylated. In the second step, the acetylated cyclic
O
H2N
N
O
precursor reacted with this activated nucleophile in pres-
ence of large excess of TMSOTf. The highest yields were
obtained if 7.5 equivalents of the Lewis acid were added
and the reaction time did not exceed 30 minutes. Any
longer reaction time resulted in degradation of the com-
pound, most probably due to nucleophilic attack on the
aryl protected phosphate linkage that results in phosphate
bond cleavage. The problem of the 9 versus 7 selectivity
encountered in the guanine base introduction was ad-
dressed by using the bulky p-nitrophenylethyl (Npe) pro-
tecting group at the O-6 position and high reaction
temperature as previously reported.21,23 The solvent of the
reaction was very important and toluene gave the highest
yields of the protected c-di-GMP 9. The o-chlorophenyl
as well as the Npe groups were removed using syn-pyri-
HN
1
Figure 1 c-di-GMP
Thus, c-di-GMP represents an excellent platform for drug
design in medicinal chemistry and especially in the field
of antibiotics where compounds with new modes of action
are required. However, the mechanisms of c-di-GMP de-
pendent signaling remain unknown, mainly because very
SYNTHESIS 2006, No. 24, pp 4230–4236
Advanced online publication: 02.11.2006
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2
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DOI: 10.1055/s-2006-950361; Art ID: T11906SS
© Georg Thieme Verlag Stuttgart · New York

PAPER
Synthesis of c-di-GMP and Analogues
4231
TBDMSO
O
TBDMSO
HO
O
O
O
O
i
O
P
O
O
O
O
OH
O
68%
OH
O
O
N
N
3
O
O
2
O
Cl
N
N
N
P
N
O
5
O
O
OH
O
HO
Cl
Cl
O
O
O
O
O
O
P
O
4
P
O
O
O
O
O
O
ii
O
iii
O
75%
O
64%
O
O
Cl
O
O
P
O
O
O
OH
O
6
7
Cl
Scheme 1 Synthesis of cyclic sugar backbone 5. Reaction conditions: (i) 4, THF, 4 Å molecular sieves; (ii) CAN, MeOH; (iii) 2-chlorophenyl
phosphorodichloridate, pyridine.
dine-2-carbaldoxime in the presence of N,N,N,N-tetra- regulation system. It was shown that the GGDEF domain,
methylguanidine.6 Therefore the use of DBU23 for the also called domain of unknown function 1 (DUF1), is in
deprotection of the Npe group was not necessary.
fact a diguanylate cyclase domain (DGC).2 The GGDEF
domain catalyzes the biosynthesis of c-di-GMP starting
from two GTP molecules. This synthetic compound was
also used to crystallize PleD and to get the crystal struc-
ture of a c-di-GMP binding protein.24
Finally, the acetyl and isobutyryl protecting groups were
removed by treatment with aqueous ammonium hydrox-
ide for two days at 50 °C.6 Pure c-di-GMP 1 was obtained
after purification using gel filtration chromatography and
HPLC.
Although these results are of prime importance, the eluci-
dation of the biochemistry of c-di-GMP remains to be
achieved. We believe that base-modified analogues can be
useful in addressing this problem. Following our new ap-
proach, we have synthesized three new derivatives of c-
di-GMP and proved that our synthesis can be employed to
synthesize this new class of compounds.
Using this completely new approach, the bacterial second-
ary messenger was synthesized in an efficient and up-scal-
able manner. One batch of c-di-GMP was used to study
the activity of PleD, a response regulator that plays an im-
portant role in the Caulobacter crescentus cell cycle, and
that has been shown to be part of the biofilm formation
ONpe
Cl
Cl
N
N
O
O
O
O
P
P
N
O
O
N
N
O
OAc
O
O
O
O
O
O
H
i
O
O
H
O
O
O
O
O
O
O
OAc
56%
N
N
P
N
N
P
O
O
O
O
N
7
9
Cl
Cl
ONpe
O
NO2
N
NH
O
O-
O
O
P
N
N
NH2
OH
O
O
N
ii
84%
N
O
O
O
P
-O
O
OH
H2N
N
N
N
N
N
H
H
O
HN
N
8
1
O
Scheme 2 Synthesis of c-di-GMP. Reaction conditions: (i) AcOH, Ac2O, H2SO4; 8, N,O-Bis(trimethylsilyl)acetamide (BSA), ClCH2CH2Cl
(DCE), 80 °C; TMSOTf, toluene, 80 °C; (ii) syn-pyridine-2-carbaldoxime, N,N,N,N-tetramethylguanidine; NH4OH, 50 °C.
Synthesis 2006, No. 24, 4230–4236 © Thieme Stuttgart · New York

4232
N. Amiot et al.
PAPER
Sigma-Aldrich Chemical Company Inc. TEAC buffer was prepared
by bubbling CO2 through a 0.25 M Et3N solution in H2O until pH
7.0.
The base-modified c-di-GMP 11a–c could be synthesized
from building block under similar conditions
7
(Scheme 3) as for the synthesis of 1. Base introduction
works well for thymidine and theophylline with yields of
51% and 47% to provide 11b and 11c, respectively. How-
ever, care had to be taken during the introduction of the
adenine since degradation products were observed.
Phosphorylating Agent 4 (1 M in THF)
To a solution of HOBt (13.78 g, 0.1 mol, 2.03 equiv) and pyridine
(8.33 mL, 0.1 mol, 2.07 equiv) in THF (25 mL) was added a solu-
tion of 2-chlorophenyl phosphorodichloridate (8.07 mL, 0.05 mol,
1 equiv) and THF (7 mL) under N2 at r.t. After a white suspension
had crashed out from the clear solution, the stirring was continued
for 18 h. The solution was filtered under N2 providing a 1 M stock
solution of phosphorylating agent that can be kept for several weeks
in the freezer.
Cl
Cl
O
O
O
O
P
B
P
OAc
O
O
O
O
O
O
O
O
i
(5¢-O-tert-Butyldimethylsilyl-1¢,2¢-bis-O-isopropylidene-D-ribo-
furanosyl)-(3¢-5¢)-(1¢,2¢-bis-O-isopropylidene-D-ribofuranosyl)-
2-chlorophenyl Phosphate (5)
O
O
O
OAc
O
O
O
B
P
O
O
P
O
O
O
O
a 28%
b 51%
c 47%
5¢-O-tert-Butyldimethylsilyl-1¢,2¢-bis(O-isopropylidene)-D-ribo-
furanoside (2; 2.0 g, 6.6 mmol, 1.0 equiv) was dissolved in THF (60
mL) and 4 Å molecular sieves were added. The solution was then
stirred for 3 h at r.t. under N2. Phosphorylating agent 4 (7.9 mL of 1
M stock solution, 7.9 mol, 1.2 equiv) was then added at r.t. under
N2. The mixture was stirred for 15 min after which time only zero-
mobility product was detected on TLC. 1¢,2¢-Bis(O-isopropy-
lidene)-D-ribofuranoside (3; 1.88 g, 9.9 mmol, 1.5 equiv) in THF
(30 mL) and 4 Å molecular sieves were added quickly and the mix-
ture was stirred for 1 h. The mixture was then filtered through Celite
and washed with CH2Cl2 (200 mL). The organic phase was washed
with 0.1 M TEAC buffer (200 mL) and then brine (200 mL). The
organic phase was then dried (MgSO4), filtered and the solvent was
removed under reduced pressure. Flash chromatography using a
gradient from hexane (100%) to hexane–EtOAc (50:50) provided 5
(2.98 g, 68%, mixture of 2 diastereoisomers) as a colorless oil;
Rf 0.61 (hexane–EtOAc, 50:50).
11a,b,c
Cl
7
Cl
ii a 81%
b 80%
O
NHi-Bu
c 76%
N
NH
N
N
O
O–
O
N
N
O
P
B
H
H
OH
O
O
10a
10b
N
O
O
N
O
O
O
OH
B
P
N
O
O
N
H
12a,b,c
B: Adenine, Thymine, Theophylline
10c
Scheme 3 Synthesis of analogues of c-di-GMP. Reaction condi-
tions: (i) AcOH, Ac2O, H2SO4; 10a, 10b or 10c, BSA, DCE, 80 °C,
respectively; TMSOTf, toluene, 80 °C; (ii) syn-pyridine-2-carb-
aldoxime, N,N,N,N-tetramethylguanidine; NH4OH, 50 °C.
Diastereoisomer 1
1H NMR (500.0 MHz, DMSO-d6): d = 7.58 (d, J = 7.9 Hz, 1 H,
Harom), 7.44 (d, J = 11.7 Hz, 1 H, Harom), 7.38 (m, 1 H, Harom), 7.26
(m, 1 H, Harom), 5.75 (d, J = 0.3 Hz, 1 H, H-1¢a), 5.67 (d, J = 3.6 Hz,
1 H, H-1¢b), 5.32 (d, J = 6.8 Hz, 1 H, H-3¢b), 4.72 (t, J = 4.3 Hz, 1
H, H-2¢a), 4.63 (m, 1 H, H-3¢a), 4.47 (m, 1 H, H-2¢b), 4.43 (m, 1 H,
H-5¢b), 4.16 (sext, J = 5.8 Hz, 1 H, H-5¢b), 4.01 (m, 1 H, H-4¢a),
3.95 (m, 1 H, H-4¢b), 3.83 (m, 1 H, H-5¢a), 3.75 (m, 1 H, H-3¢b),
3.71 (m, 1 H, H-5¢a), 1.43 (s, 6 H, CH3a, CH3b), 1.26 (s, 6 H, CH3a,
CH3b), 0.82 (s, 9 H, CH3), 0.015 (s, 3 H, CH3), 0.008 (s, 3 H, CH3).
The highest yields were obtained with an equimolar ratio
of adenine building block 10a.25 Moreover, the purifica-
tion by silica gel chromatography had to be carried out
quickly after the work-up, otherwise the base would
cleave the chlorophenyl protected phosphate group. Final
deprotection was achieved following the same reaction
sequence used in the synthesis of c-di-GMP and gave 12a, 13C NMR (125.8 MHz, DMSO-d6): d = 145.9 (Carom), 130.5, 128.5,
126.6, 124.4, 121.3 (CHarom), 112.3 (Cq), 111.6 (Cq), 103.6 (C-1¢a),
103.4 (C-1¢b), 78.9 (C-2¢b), 78.3 (C-4¢a), 77.6 (C-4¢b), 77.3 (C-2¢a),
74.2 (C-3¢a), 70.4 (C-3¢b), 67.7 (C-5¢b), 60.7 (C-5¢a), 26.6 (CH3a),
26.6 (CH3b), 26.4 (CH3a), 26.4 (CH3b), 25.8 (CH3), 18.0 (Cq), –5.4
(CH3), –5.4 (CH3).
12 b and 12c with yields of about 80%.
In summary, we have successfully designed a new syn-
thetic pathway towards c-di-GMP. The synthesis of dif-
ferent based modified analogues has also shown that this
procedure is flexible and that starting from compound 7,
new molecules can be created. These compounds are ex-
pected to play an important role in the elucidation of the
biological mode of action of c-di-GMP and experiments
to address this question are currently under way.
Diastereoisomer 2
1H NMR (500.0 MHz, DMSO-d6): d = 7.58 (d, J = 7.9 Hz, 1 H,
Harom), 7.44 (d, J = 11.7 Hz, 1 H, Harom), 7.38 (m, 1 H, Harom), 7.26
(m, 1 H, Harom), 5.75 (d, J = 0.3 Hz, 1 H, H-1¢a), 5.62 (d, J = 3.6 Hz,
1 H, H-1¢b), 5.32 (d, J = 6.8 Hz, 1 H, OH-3¢b), 4.76 (t, J = 4.3 Hz,
1 H, H-2¢a), 4.59 (m, 1 H, H-3¢a), 4.46 (m, 1 H, H-2¢b), 4.43 (m, 1
H, H-5¢b), 4.16 (sext, J = 5.8 Hz, 1 H, H-5¢b,), 4.01 (m, 1 H, H-4¢a),
3.95 (m, 1 H, H-4¢b), 3.79 (m, 1 H, H-5¢a), 3.74 (m, 1 H, H-3¢b),
3.60 (m, 1 H, H-5¢a), 1.46 (s, 6 H, CH3a, CH3b), 1.29 (s, 6 H, CH3a,
CH3b), 0.81 (s, 9 H, CH3), –0.011 (s, 3 H, CH3), –0.015 (s, 3 H,
CH3).
Reactions were monitored by TLC on silica gel 60 F254 (Merck)
with detection under UV and subsequent charring with 15% H2SO4
in MeOH. Flash column chromatography was performed on silica
gel 60 (0.040–0.063 mm, Fluka). NMR spectra were recorded on
Bruker VRX 500 instrument. Chemical shits are relative to TMS
(0.00). When necessary, assignments were based on DEPT, COSY,
HMQC, HMBC, NOESY and TOCSY. MALDI-ToF spectra were
measured with an Applied Biosystems Voyager DE Pro using p-ni-
troaniline as matrix. Chemicals were purchased from Fluka AG and
13C NMR (125.8 MHz, DMSO-d6): d = 146.5, 146.0 (Carom), 130.6,
128.5, 126.7, 124.4, 121.35 (CHarom), 112.4 (Cq), 111.6 (Cq), 103.7
(C-1¢a), 103.6 (C-1¢b), 78.9 (C-2¢b), 78.4 (C-4¢a), 77.6 (C-4¢b), 77.3
(C-2¢a), 74.3 (C-3¢a), 70.5 (C-3¢b), 68.1 (C-5¢b), 60.7 (C-5¢a), 26.6
Synthesis 2006, No. 24, 4230–4236 © Thieme Stuttgart · New York

PAPER
Synthesis of c-di-GMP and Analogues
4233
(CH3a, CH3b), 26.5 (CH3a), 26.4 (CH3b), 25.8 (CH3), 18.0 (Cq),
–5.5 (CH3), –5.4 (CH3).
(m, 2 H, H-3¢a, H-2¢b), 4.85–4.81 (m, 1 H, H-3¢b), 4.74 (t, J = 4.2
Hz, 1 H, H-2¢b), 4.52–4.45 (m, 2 H, H-5¢a, H-5¢b), 4.34–4.24 (m, 4
H, H-5¢a, H-5¢b, H-4¢a, H-4¢b), 1.52, 1.43, 1.32, 1.27 (4 s, 12 H,
CH3a, CH3b, CH3a, CH3b).
13C NMR (125.8 MHz, DMSO-d6): d = 145.7, 145.6 (Carom), 130.8,
130.6, 128.8, 128.5, 127.0, 126.8, 121.4, 121.0 (CHarom), 112.8,
112.6 (Cq), 103.6 (C-1¢a), 103.5 (C-1¢b), 77.4 (C-2¢a), 77.3 (C-2¢b),
74.4 (C-4¢a), 74.3 (C-4¢b), 74.1(C-3¢a), 74.1 (C-3¢b), 65.2, 65.1 (C-
5¢a, C-5¢b), 26.6, 26.4, 26.3 (CH3).
Anal. Calcd for C24H44ClO12PSi (667.17): C, 50.41; H, 6.65; O,
28.78. Found: C, 50.45; H, 6.62; O, 28.77.
(1¢,2¢-Bis-O-isopropylidene-D-ribofuranosyl)-(3¢-5¢)-(1¢,2¢-bis-
O-isopropylidene-D-ribofuranosyl)-2-chlorophenyl Phosphate
(6)
Compound 5 (2.97 g, 4.45 mol, 1 equiv) was dissolved in MeOH
(60 mL) and CAN (2.45 g, 4.47 mol, 1 equiv) was added and the
mixture was stirred for 18 h under N2. The mixture was diluted with
EtOAc (100 mL) and 0.25 M TEAC buffer (200 mL) was added.
The aqueous phase was then extracted with EtOAc (4 × 50 mL).
The combined organic phases were dried (MgSO4) and the solvent
was removed under reduced pressure. Purification was achieved by
flash chromatography over silica gel using a mixture of CH2Cl2–
MeOH (90:10) as eluent to afford 6 (1.87 g, 76%, mixture of 2 dia-
stereoisomers) as a colorless oil; Rf 0.23 (CH2Cl2–MeOH, 95:5).
31P NMR (202 MHz, DMSO-d6): d = –8.35, –8.52.
Anal. Calcd for C28H32Cl2O14P2 (724.06): C, 46.36; H, 4.45; O,
30.88. Found: C, 46.29; H, 4.51; O, 30.98.
Low-Rf Stereoisomer 7
Rf 0.49 (hexane–EtOAc, 30:70).
1H NMR (500 MHz, DMSO-d6): d = 7.60–7.28 (m, 8 H, Harom), 5.85
(d, J = 3.4 Hz, 2 H, H-1¢a, H-1¢b), 4.82–4.78 (m, 4 H, H-2¢a, H-2¢b,
H-3¢a, H-3¢b), 4.46–4.45 (m, 4 H, H-5¢a, H-5¢b), 4.25–4.22 (m, 2 H,
H-4¢a, H-4¢b), 1.42, 1.28 (2 s, 12 H, CH3).
13C NMR (125.8 MHz, DMSO-d6): d = 145.6 (Carom), 130.6, 128.9,
128.4, 121.3 (CHarom), 112.5, 112.8 (Cq), 103.7 (C-1¢), 77.3 (C-2¢),
74.6 (C-4¢), 73.5 (C-3¢), 65.7 (C-5¢), 26.5, 26.3 (CH3).
Diastereoisomer 1
1H NMR (500.0 MHz, DMSO-d6): d = 7.59 (m, 4 H, Harom), 5.77 (d,
J = 3.7 Hz, 1 H, H-1¢a), 5.68 (d, J = 3.5 Hz, 1 H, H-1¢b,), 5.32 (dd,
J = 2.6, 4.2 Hz, 1 H, OH-3¢b), 4.89 (m, 1 H, OH-5¢a), 4.71 (m, 1 H,
H-2¢a), 4.61 (m, 1 H, H-3¢a), 4.48 (br t, 1 H, H-2¢b), 4.44 (m, 1 H,
H-5¢b), 4.18 (m, 1 H, H-5¢b), 3.99 (m, 1 H, H-4¢a), 3.96 (m, 1 H, H-
4¢b), 3.79 (m, 1 H, H-3¢b), 3.69 (m, 1 H, H-5¢ a), 3.47 (m, 1 H, H-
5¢a), 1.44 (s, 6 H, CH3a, CH3b), 1.27 (s, 6 H, CH3a, CH3b).
31P NMR (202.5 MHz, DMSO-d6): d = –8.08.
Anal. Calcd for C28H32Cl2O14P2 (724.06): C, 46.36; H, 4.45; O,
30.88. Found: C, 46.27; H, 4.49; O, 30.95.
Diastereoisomer 2
Cyclic Bis(3¢-5¢)-(2¢-O-acetyl-2-N-isobutyryl-6-O-p-nitro-
phenylethylguanosine)-2-chlorophenyl Phosphate (9)
1H NMR (500.0 MHz, DMSO-d6): d = 7.59 (m, 4 H, Harom), 5.77 (d,
J = 3.7 Hz, 1 H, H-1¢a), 5.63 (d, J = 3.6 Hz, 1 H, H-1¢b), 5.32 (dd,
J = 2.6, 4.2 Hz, 1 H, OH-3¢b), 4.86 (m, 1 H, OH-5¢a), 4.75 (m, 1 H,
H-2¢a), 4.61 (m, 1 H, H-3¢a), 4.47 (br t, 1 H, H-2¢b), 4.44 (m, 1 H,
H-5¢b), 4.18 (m, 1 H, H-5¢b), 3.99 (m, 1 H, H-4¢a), 3.96 (m, 1 H, H-
4¢b), 3.77 (m, 1 H, H-3¢ b), 3.63 (m, 1 H, H-5¢a), 3.47 (m, 1 H, H-
5¢a), 1.44 (s, 6 H, CH3a, CH3b), 1.29 (s, 3 H, CH3a), 1.27 (s, 3 H,
CH3b).
Cyclic compound 7 (670 mg, 0.93 mmol, 1 equiv) was dissolved in
glacial AcOH (10 mL). Ac2O (1 mL, 10.6 mmol, 11 equiv) was add-
ed as well as H2SO4 (0.4 mL) and the mixture was stirred for 18 h
at r.t. under N2. The mixture was then poured into ice water (50 mL)
and the aqueous phase was extracted with CH2Cl2 (4 × 50 mL). The
combined organic phases were washed with aq sat. NaHCO3 (100
mL), dried (MgSO4) and the solvent was removed under reduced
pressure. The residue was dissolved in CH2Cl2 and the solution was
filtered through a short pad of silica gel. The acetylated product was
used in the next step without further purification. Guanosine build-
ing block 8 (820 mg, 2.22 mmol, 3 equiv) was suspended into DCE
(20 mL). BSA (1.08 mL, 4.48 mmol, 6 equiv) was added and the
mixture was heated at 80 °C for 16 h in a sealed flask. The excess
of BSA and DCE were removed by evaporation under reduced pres-
sure. The resulting residue was dissolved in toluene (20 mL).
TMSOTf (0.70 mL, 3.87 mmol, 5 equiv) was added together with
the previously obtained cyclic acetylated precursor (600 mg, 0.74
mmol, 1 equiv) dissolved in toluene (10 mL). The mixture was
stirred for 30 min at 80 °C in a sealed flask. The mixture was diluted
with EtOAc (60 mL) and the solution was washed with 0.25 M
TEAC buffer (100 mL). The aqueous layer was then extracted with
EtOAc (2 × 100 mL). The combined organic phases were dried
(MgSO4), filtered and the solvent was evaporated under reduced
pressure. The cyclic diguanosine product 9 was purified by flash
chromatography over silica gel column using a gradient from pure
CH2Cl2 to CH2Cl2–MeOH (95:5). Fully protected c-di-GMP 9 was
obtained as a white oily residue (779 mg, 66%); Rf 0.53 (CH2Cl2–
MeOH, 95:5).
13C NMR (125.8 MHz, DMSO-d6): d = 146.5, 146.0 (Carom), 130.6,
128.6, 126.61, 121.41 (CHarom), 112.2 (Cq), 111.6 (Cq), 103.6 (C-
1¢a), 103.4 (C-1¢b), 79.0 (C-2¢b), 78.6 (C-4¢a), 77.6 (C-4¢b), 77.4 (C-
2¢a), 74.6 (C-3¢a), 70.4 (C-3¢b), 67.9 (C-5¢b), 59.1 (C-5¢a), 26.6
(CH3a, CH3b), 26.4 (CH3a, CH3b).
Anal. Calcd for C22H30Cl2O12P (552.90): C, 47.79; H, 5.47; O,
34.72. Found: C, 47.62; H, 5.67; O, 34.70.
Cyclic Bis(3¢-5¢)-(1¢,2¢-bis-O-isopropylidene-D-ribofuranosyl)-
2-chlorophenyl Phosphate (7)
Dimer 6 (1.80 g, 3.26 mmol, 1 equiv) was co-evaporated with pyri-
dine (2 × 10 mL) and dissolved in pyridine (900 mL). 2-Chlorophe-
nyl phosphorodichloridate (0.79 mL, 4.89 mmol, 1.5 equiv) was
added and the mixture was stirred at r.t. under N2 for 1 h. The sol-
vent was evaporated, the residue taken up in CH2Cl2 (200 mL) and
washed with 0.25 M TEAC buffer (200 mL). The organic phase was
dried (MgSO4) and the solvent was removed under reduced pres-
sure. Flash chromatography over silica gel was used for the purifi-
cation with a gradient from hexane (100%) to hexane–EtOAc
(50:50). Two fractions containing the different possible stereoiso-
mers of 7 were isolated. The first fraction of higher Rf on silica gel
TLC plate was isolated as a white oily residue (683 mg, 29%)
whereas the lower-Rf fraction was isolated as a colorless oil (826
mg, 35%).
1H NMR (500 MHz, CDCl3): d = 8.78, 8.70 (2 s, 2 H, NH), 8.14 (m,
4 H, Harom, Npe), 7.85, 7.76 (2 s, 2 H, H-8a, H-8b), 7.52 (m, 4 H,
Harom, Npe), 7.50–7.00 (m, 8 H, Harom, chlorophenyl), 6.33 (m, 1 H,
H-3¢a), 6.19 (dd, J = 4.6, 5.6 Hz, 1 H, H-2¢a), 6.15 (dd, J = 4.0, 5.2
Hz, 1 H, H-2¢b), 6.02 (d, J = 4.4 Hz, 1 H, H-1¢b,), 5.99 (m, 1 H, H-
3¢b), 5.86 (d, J = 3.6 Hz, 1 H, H-1¢a,), 4.98 (ddd, J = 6.5, 7.8, 10.7
Hz, 1 H, H-5¢b), 4.91–4.80 (m, 4 H, OCH2, Npe), 4.70 (m, 1 H, H-
4¢b), 4.65–4.49 (m, 3 H, H-5¢a, H-4¢a, H-5¢a), 4.45 (ddd, J = 3.6,
High-Rf Stereoisomer 7
Rf 0.71 (hexane–EtOAc, 30:70).
1H NMR (500 MHz, DMSO-d6): d = 7.73 (m, 8 H, Harom), 5.88 (d,
J = 3.5 Hz, 1 H, H-1¢a), 5.75 (d, J = 3.6 Hz, 1 H, H-1¢b), 4.93–4.87
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N. Amiot et al.
PAPER
5.0, 10.7 Hz, 1 H, H-5¢b), 3.34–3.30 (m, 4 H, CH2C, NPe), 2.92 (br
d, 2 H, CH, isobutyryl), 1.99, 1.94 (2 s, 6 H, CH3, acetyl), 1.21–1.15
(m, 12 H, CH3, isobutyryl).
as a colorless oily residue (29 mg, 28%); Rf 0.46 (CH2Cl2–MeOH,
95:5).
1H NMR (500 MHz, DMSO-d6): d = 10.74, (s, 1 H, NHa), 10.70 (s,
1 H, NHb), 8.74 (s, 1 H, H-8a), 8.68 (s, 1 H, H-2a), 8.66 (s, 1 H, H-
8b), 8.60 (s, 1 H, H-2b), 7.64 (m, 1 H, Haromb), 7.49 (m, 1 H, Haroma),
7.45 (m, 1 H, Haromb), 7.38 (m, 1 H, Haroma), 7.32 (m, 1 H, Haromb),
7.31 (m, 1 H, Haromb), 7.20 (m, 1 H, Haroma), 7.15 (m, 1 H, Haroma),
6.44 (d, J = 6.4 Hz, 1 H, H-1¢a,), 6.38–6.44 (m, 1 H, H-1¢b), 6.36 (m,
1 H, H-2¢a), 6.17–6.44 (d, J = 5.2 Hz, 1 H, H-2¢b), 5.86 (m, 1 H, H-
3¢b), 5.74 (m, 1 H, H-3¢a), 4.87 (m, 1 H, H-4¢b), 4.80 (m, 1 H, H-
4¢a), 4.60 (m, 2 H, H-5¢a, H-5¢b), 4.50 (m, 2 H, H-5¢a, H-5¢b), 2.94
(q, J = 6.9 Hz, 2 H, isobutyryl), 1.95 (s, 3 H, CH3, acetyl a), 1.94 (s,
3 H, CH3, acetyl b), 1.13 (d, J = 6.9 Hz, 12 H, CH3, isobutyryl).
13C NMR (125.8 MHz, DMSO-d6): d = 175.3 (C=O, isobutyryl),
169.2 (C=O, acetyl a), 169.1 (C=O, acetyl b), 151.9 (C-6a), 151.8
(C-6b), 151.6 (C-2a), 151.3 (C-2b), 150.2 (2 s, C-5), 145.6 (2 s, C-
4), 145.5 (2 s, Carom), 143.3 (C-8a), 143.2 (C-8b), 130.8, 130.6,
128.8, 128.2, 127.3, 126.8 (CHarom), 124.3 (2 s, Carom), 121.7, 120.8
(CHarom), 85.7 (C-1¢a), 85.1 (C-1¢b), 80.5 (2 s, C-4¢), 78.2 (2 s, C-
3¢), 71.4 (C-2¢a), 70.8 (C-2¢b), 65.5 (C-5¢a), 65.4 (C-5¢b), 39.0 (CH,
isobutyryl), 20.2 (CH3, acetyl), 19.3 (CH3, isobutyryl).
13C NMR (125.8 MHz, CDCl3): d = 175.9 (C=O, isobutyryl), 169.4,
169.2 (C=O, acetyl), 160.8 (C=O-4), 152.4, 152.3, 152.1, 151.9 (C-
2, C-6), 146.9, 146.8, 146.0, 145.9, 145.8, 145.7 (Carom), 141.1 (C-
8), 130.9, 130.7, 130.1, 130.0, 128.1, 127.9, 126.9, 126.6, 125.5,
125.0, 123.8, 123.7, 121.4, 120.9 (CHarom), 118.9, 118.6 (C-5), 88.0,
87.0 (C-1¢), 81.2, 79.0 (C-4¢), 75.6, 74.6 (C-3¢), 72.4, 72.2 (C-2¢),
66.1, 65.9 (C-5¢), 35.8, 35.0 (CH, isobutyryl), 20.4, 20.3, 19.4, 19.3
(CH3, acetyl, isobutyryl).
31P NMR (202.5 MHz, CDCl3): d = –5.8, –9.5.
Anal. Calcd for C60H60Cl2N12O22P2 (1432.28): C, 50.25; H, 4.22; O,
24.55. Found: C, 50.14; H, 4.29; O, 24.64.
c-di-GMP 1
Fully protected c-di-GMP 9 (430 mg, 0.30 mmol) in pyridine (5
mL) and syn-pyridine-2-carbaldoxime (1.5 g, 12.3 mmol, 40 equiv)
were mixed with N,N,N,N-tetramethylguanidine (1.35 mL, 10.7
mmol, 35 equiv). The mixture was stirred for 16 h at r.t. Aq 14 M
NH4OH (100 mL) was then added and the mixture was stirred for 2
days at 50 °C in a sealed flask. The solution was evaporated to 1/10
of its volume and then washed with CH2Cl2 (2 × 20 mL). A first pu-
rification step was achieved using size-exclusion chromatography
with Sephadex G15 and nanopure H2O as eluent. A final purifica-
tion by reverse-phase HPLC chromatography yielded pure c-di-
GMP 1 (173 mg, 84%, 99.99% pure).
1H NMR (500 MHz, D2O): d = 7.90 (s, 2 H, H-8), 5.84 (d, J = 1.3
Hz, 2 H, H-1¢), 4.74 (dd, J = 8.5, 5.0 Hz, 2 H, H-3¢), 4.59 (dd,
J = 5.0 Hz, 2 H, H-2¢), 4.27 (dd, J = 8.5 Hz, 2 H, H-4¢), 4.21, 3.96
(2 m, 4 H, H-5¢).
13C NMR (125.8 MHz, D2O): d = 158.8, 153.8 (C-4, C-6), 150.8 (C-
2), 137.1 (C-8), 116.3 (C-5), 89.2 (C-1¢), 79.8 (C-4¢), 73.3 (C-2¢),
70.5 (C-3¢), 62.2 (C-5¢).
MS (MALDI-ToF): m/z calcd for C44H46Cl2N10O18P2 (1102.13);
found: 1125.13 [M + Na]+.
c-di-AMP 12a
Fully protected c-di-AMP 11a (29 mg, 0.03 mmol, 1 equiv) in pyri-
dine (2 mL) and syn-pyridine-2-carbaldoxime (118 mg, 0.97 mmol,
40 equiv) was mixed with N,N,N,N-tetramethylguanidine (110 mL,
0.87 mmol, 35 equiv). The mixture was stirred for 16 h at r.t. Aq 14
M NH4OH (30 mL) was then added and the mixture was stirred for
2 days at 50 °C in a sealed flask. The solution was evaporated to
1/10 of its volume and then washed with CH2Cl2 (2 × 10 mL). A
first purification step was achieved using gel filtration chromatog-
raphy with Sephadex G10 and a mixture of MeOH–nanopure H2O
(1:1) as eluent. A final purification by reverse-phase HPLC chroma-
tography yielded pure c-di-AMP 12a (14 mg, 81%).
HRMS-ESI: m/z [M – H]– calcd for C20H23N10O14P2: 689.0870;
1H NMR (500 MHz, DMSO-d6): d = 8.45 (s, 2 H, H-8), 8.14 (s, 2
H, H-2), 7.32 (s, 4 H, NH2), 5.88 (d, J = 4.4 Hz, 2 H, H-1¢), 4.93 (m,
2 H, H-2¢), 4.68 (t, J = 5.3 Hz, 2 H, H-3¢), 4.22 (dd, J = 5.3, 9.7 Hz,
2 H, H-4¢), 4.00–3.98 (m, 4 H, H-5¢).
found: 689.0871.
Cyclic Bis(3¢-5¢)-(2¢-O-acetyl-6-N-isobutyryladenosine)-2-chlo-
rophenyl Phosphate (11a)
Cylic compound 7 (70 mg, 0.10 mmol, 1 equiv) was dissolved in
glacial AcOH (2 mL). Ac2O (0.2 mL, 2.13 mmol, 21 equiv) was
added as well as H2SO4 (42 mL) and the mixture was stirred for 18
h at r.t. under N2. The mixture was then poured into ice water (10
mL) and the aqueous phase was extracted with CH2Cl2 (4 × 10 mL).
The organic phase was washed with aq sat. NaHCO3 (20 mL), dried
(MgSO4) and the solvent was removed under reduced pressure. The
residue was dissolved in CH2Cl2 and the solution was filtered
through a short pad of silica gel. The acetylated residue was used in
the next step without further purification. Adenine building block
10a (35 mg, 0.19 mmol, 2 equiv) was suspended in DCE (2.5 mL).
BSA (83 mL, 0.39 mmol, 4 equiv) was added and the mixture was
heated at 80 °C for 16 h in a sealed flask. The excess of BSA and
DCE were removed by evaporation under reduced pressure. The re-
sulting residue was dissolved in toluene (2 mL). TMSOTf (78 mL,
0.43 mmol, 5 equiv) was added together with the previously ob-
tained cyclic acetylated precursor dissolved in toluene (2 mL). The
mixture was stirred for 30 min at 80 °C in a sealed flask. The mix-
ture was diluted with EtOAc (20 mL) and the solution was washed
with 0.25 M TEAC buffer (20 mL). The aqueous layer was then ex-
tracted with EtOAc (2 × 10 mL). The combined organic phases were
dried (MgSO4), filtered and the solvent was evaporated under re-
duced pressure. The cyclic diadenosine product 11a was purified by
preparative TLC using a mixture of CH2Cl2–MeOH (95:5) as eluent
shortly after work-up. Fully protected c-di-AMP 11a was obtained
13C NMR (125.8 MHz, DMSO-d6): d = 156.1 (C-4), 152.7 (C-6),
149.6 (C-5), 139.7 (C-8), 119.2 (C-2), 87.0 (C-1¢), 81.1 (C-2¢), 73.5
(C-3¢), 71.3 (C-4¢), 63.9 (C-5¢).
HRMS-ESI: m/z [M – H]– calcd for C20H23N10O12P2: 657.0972;
found: 657.0967.
Cyclic Bis(3¢-5¢)-(2¢-O-acetylthymidine)-2-chlorophenyl Phos-
phate (11b)
Compound 7 (62 mg, 0.09 mmol, 1 equiv) was dissolved in glacial
AcOH (3 mL). Ac2O (0.3 mL, 3.2 mmol, 35 equiv) was added as
well as H2SO4 (14 mL) and the mixture was stirred for 18 h at r.t. un-
der N2. The mixture was then poured into ice water and the aqueous
phase was extracted with CH2Cl2 (4 × 10 mL). The organic phase
was washed with 0.1 M TEAC buffer (10 mL), dried (MgSO4) and
the solvent was removed under reduced pressure. The residue was
dissolved in CH2Cl2 and the solution was filtered through a short
pad of silica gel. The acetylated residue was used in the next step
without further purification. Thymine 10b (22.8 mg, 0.18 mmol, 2.1
equiv) was suspended into DCE (3 mL) and BSA (088 mL, 0.36
mmol, 4.2 equiv) was added. The mixture was heated at 80 °C for
16 h in a sealed flask. The excess of BSA and DCE were removed
by evaporation under reduced pressure. The resulting residue was
dissolved in toluene (3 mL). TMSOTf (117 mL, 0.68 mmol, 7.5
equiv) was added to the mixture together with the previously ob-
tained cyclic sugar dissolved in toluene (1.5 mL). The mixture was
Synthesis 2006, No. 24, 4230–4236 © Thieme Stuttgart · New York

PAPER
Synthesis of c-di-GMP and Analogues
4235
stirred for 30 min at 80 °C in a sealed flask. The mixture was diluted
with EtOAc (10 mL) and the solution was washed with 0.25 M
TEAC buffer (10 mL). The aqueous layer was then extracted with
EtOAc (2 × 10 mL). The combined organic phases were dried
(MgSO4), filtered and the solvent was evaporated under reduced
pressure. Flash chromatography (gradient of CH2Cl2 to CH2Cl2–
MeOH, 95:5) afforded a white oily residue (41.4 mg, 51%); Rf 0.41
(CH2Cl2–MeOH, 9:1).
was washed with 0.25 M TEAC buffer (10 mL). The aqueous layer
was then extracted with EtOAc (2 × 10 mL). The combined organic
phases were dried (MgSO4), filtered and the solvent was evaporated
under reduced pressure. Flash chromatography (gradient of CH2Cl2
to CH2Cl2–MeOH, 95:5) afforded 11c as a white residue (42 mg,
47%); Rf 0.32 (CH2Cl2–MeOH, 9:1).
1H NMR (500 MHz, DMSO-d6): d = 8.49 (s, 1 H, H-8a), 8.44 (s, 1
H, H-8b), 7.63 (d, J = 7.6 Hz, 1 H, Haroma), 7.56 (d, J = 7.9 Hz, 1 H,
1H NMR (500.0 MHz, DMSO-d6): d = 7.73 (m, 2 H, H-6), 7.63 (m,
2 H, Harom), 7.46 (m, 2 H, Harom), 7.43 (m, 2 H, Harom), 7.31 (m, 2 H,
H
aromb), 7.47 (m, 1 H, Haromb), 7.45 (m, 1 H, Haromb), 7.38 (m, 1 H,
Haroma), 7.32 (m, 1 H, Haromb), 7.26 (m, 1 H, Haroma), 7.24 (m, 1 H,
Haroma), 6.39 (d, J = 6.7 Hz, 2 H, H-1¢a, H-1¢b), 6.08 (m, 1 H, H-
H
arom), 5.92 (d, J = 5.9 Hz, 2 H, H-1¢), 5.63 (t, J = 5.9 Hz, 2 H, H-
2¢), 5.36 (m, 2 H, H-3¢a), 4.70 (m, 2 H, H-5¢), 4.38 (m, 4 H, H-4¢, H-
5¢), 1.89 (s, 6 H, CH3), 1.76 (s, 6 H, CH3, acetyl).
2¢b), 6.04 (t, J = 6.3 Hz, 1 H, H-2¢a,), 5.62 (m, 1 H, H-3¢b), 5.58 (m,
1 H, H-3¢a), 4.79 (m, 1 H, H-5¢a), 4.76 (m, 1 H, H-5¢b), 4.51 (m, 1
H, H-4¢b), 4.50 (m, 1 H, H-4¢a), 4.33 (m, 2 H, H-5¢a, H-5¢b), 3.43 (s,
3 H, H-4a), 3.41 (s, 3 H, H-4b), 3.24 (s, 3 H, H-2a), 3.23 (s, 3 H, H-
2b), 1.95 (s, 3 H, CH3, acetyl b), 1.93 (s, 3 H, CH3, acetyl a).
13C NMR (125.8 MHz, DMSO-d6): d = 169.1 (C=O, acetyl b), 168.8
(C=O, acetyl a), 154.1 (C=O-3a), 153.9 (C=O-3b), 150.7 (C=O-1a),
150.6 (C=O-1a), 149.7 (C-5b), 149.6 (C-5a), 145.7 (Caromb), 145.6
(Caroma), 143.6 (C-8b), 142.9 (C-8a), 130.8 (CHaroma), 130.7
(CHaromb), 128.8 (CHaromb), 128.0 (CHaroma), 127.3 (CHaromb),
126.7 (CHaroma), 124.6 (Caroma), 124.2 (Caromb), 121.7 (CHaroma),
120.7 (CHaromb), 105.6 (C-6b), 105.5 (C-6a), 86.8 (C-1¢b), 86.5 (C-
1¢a), 80.5 (C-4¢a), 80.0 (C-4¢b), 75.4 (C-3¢b), 74.4 (C-3¢a), 71.7 (C-
2¢a), 71.6 (C-2¢b), 65.5 (C-5¢a), 64.8 (C-5¢b), 29.7 (C-4), 27.9 (C-2),
20.2 (CH3, acetyl b), 20.1 (CH3, acetyl a).
13C NMR (125.8 MHz, DMSO-d6): d = 169.1 (C=O, acetyl), 163.6
(C=O-4), 150.4 (C=O-2), 145.6 (Carom), 136.9 (C-6), 130.8, 128.8,
127.2 (CHarom), 124.4 (Carom), 121.6 (CHarom), 110.1 (C-5), 86.7 (C-
1¢), 79.1 (C-4¢), 73.5 (C-3¢), 70.6 (C-2¢), 65.5 (C-5¢), 20.2 (CH3,
acetyl), 12.0 (CH3).
MS (MALDI-ToF): m/z calcd for C36H36Cl2N4O18P2 (944.09);
found: 967.76 [M + Na]+.
c-di-TMP 12b
Compound 11b (29 mg, 0.03 mmol, 1 equiv) was dissolved in pyri-
dine (2 mL) and treated with syn-pyridine-2-carbaldoxime (150 mg,
1.23 mmol, 40 equiv) as well as N,N,N,N-tetramethylguanidine
(134 mL, 1.08 mmol, 35 equiv). The mixture was stirred for 16 h at
r.t.. The solvents were evaporated and the residue was taken up in
H2O (10 mL) and washed with CH2Cl2 (2 × 10 mL). The aqueous
phase was then evaporated under reduced pressure. A first purifica-
tion step was achieved using size-exclusion chromatography with
Sephadex G10 and a mixture of nanopure H2O–MeOH (1:1) as elu-
ent. A final purification by reverse-phase HPLC chromatography
yielded pure c-di-TMP 12b (23.2 mg, 80%).
1H NMR (500.0 MHz, DMSO-d6): d = 11.32 (s, 2 H, NH), 9.33 (s,
1 H, OH), 8.38 (s, 1 H, OH), 7.73 (s, 2 H, H-6), 5.75 (d, J = 5.1 Hz,
2 H, H-1¢), 4.47 (m, 2 H, H-3¢), 4.19 (t, J = 4.9 Hz, 2 H, H-2¢), 4.07
(m, 2 H, H-4¢), 3.95 (m, 2 H, H-5¢), 3.87 (m, 2 H, H-5¢), 1.77 (s, 6
H, CH3).
MS (MALDI-ToF): m/z calcd for C40H40Cl2N8O18P2 (1052.13);
found: 1075.17 [M + Na]+.
Cyclic Bis(3¢-5¢)-theophylline Monophosphate 12c
Compound 11c (22 mg, 0.02 mmol, 1 equiv) was dissolved in pyri-
dine (2 mL) and treated with syn-pyridine-2-carbaldoxime (150 mg,
0.84 mmol, 40 equiv) and N,N,N,N-tetramethylguanidine (92 mL,
0.73 mmol, 35 equiv). The mixture was stirred for 16 h at r.t. The
solvents were evaporated and the residue was taken up in H2O (10
mL) and washed with CH2Cl2 (2 × 10 mL). The aqueous phase was
then evaporated under reduced pressure. A first purification step
was achieved using size-exclusion chromatography with Sephadex
G10 and a mixture MeOH–nanopure H2O (1:1) as eluent. A final
purification by reverse-phase HPLC chromatography yielded pure
12c (11.8 mg, 76%).
13C NMR (125.8 MHz, DMSO-d6): d = 163.6 (C=O-4), 150.4
(C=O-2), 135.6 (C-6), 109.7 (C-5), 87.6 (C-1¢), 80.0 (C-4¢), 72.0 (C-
3¢), 71.7 (C-2¢), 62.9 (C-5¢), 11.9 (CH3).
HRMS-ESI: m/z [M – H]– calcd for C20H25N4O16P2: 639.0741;
found: 639.0735.
1H NMR (500 MHz, DMSO-d6): d = 8.51 (s, 1 H, H-8), 7.17 (s, 1
H, OH-2¢), 6.12 (d, J = 5.8 Hz, 1 H, H-1¢), 4.57 (m, 2 H, H-2¢, H-3¢),
4.18 (m, 1 H, H-4¢), 3.91 (m, 2 H, H-5¢), 3.43 (s, 3 H, H-4), 3.23 (s,
3 H, H-2).
13C NMR (125.8 MHz, DMSO-d6): d = 154.1 (C=O-1), 150.9
(C=O-3), 148.9 (C-5), 141.0 (C-8), 105.9 (C-6), 89.1 (C-1¢), 81.2
(C-4¢), 73.0 (C-3¢), 72.4 (C-2¢), 63.3 (C-5¢), 29.5 (C-4), 27.7 (C-2).
Cyclic Bis(3¢-5¢)-(2¢-O-acetyltheophylline)-2-chlorophenyl
Phosphate (11c)
Compound 7 (62 mg, 0.09 mmol, 1 equiv) was dissolved in glacial
AcOH (3 mL). Ac2O (0.3 mL, 3.2 mmol, 35 equiv) was added as
well as H2SO4 (14 mL) and the mixture was stirred for 18 h at r.t. un-
der N2. The mixture was then poured into ice water and the aqueous
phase was extracted with CH2Cl2 (4 × 10 mL). The combined or-
ganic phases were washed with 0.1 M TEAC buffer (10 mL), dried
(MgSO4) and the solvent was removed under reduced pressure. The
residue was dissolved in CH2Cl2 and the solution was filtered
through a short pad of silica gel. The acetylated residue was used in
the next step without further purification. Theophylline 10c (32.5
mg, 0.18 mmol, 2.1 equiv) was suspended into DCE (3 mL) and
BSA (88 mL, 0.36 mmol, 4.2 equiv) was added. The mixture was
heated at 80 °C for 16 h in a sealed flask. The excess of BSA and
DCE were removed by evaporation under reduced pressure. The re-
sulting residue was dissolved in toluene (3 mL). TMSOTf (117 mL,
0.64 mmol, 7.5 equiv) was added to the mixture together with the
previously obtained cyclic acetylated sugar dissolved in toluene
(1.5 mL). The mixture was stirred for 30 min at 80 °C in a sealed
flask. The mixture was diluted with EtOAc (10 mL) and the solution
HRMS-ESI: m/z [M – H]– calcd for C24H29N8O16P2: 747.1177;
found: 747.1171.
Acknowledgment
This work was supported by the Swiss National Science Founda-
tion.
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