Synthesis of 2'-modified cyclic bis(3'5') diadenylic acids (c-di-AMPs) and their promotion of cell division in a freshwater green alga

Article abstract of DOI:10.1246/cl.2012.1723

Three 2'-modified cyclic bis(3'5') diadenylic acids (c-di-AMPs) were synthesized from commercially available adenosine phosphoramidites and the effects of c-di-AMP derivatives on the cell division of Chlamydomonas reinhardtii, a kind of freshwater green algae, were investigated. This structureactivity relationship study suggests that the di(2'-O-methyl)-c-di-AMP showed the highest activity and that 2'-substituents influenced the cell division of C. reinhardtii.

Full text of DOI:10.1246/cl.2012.1723

doi:10.1246/cl.2012.1723
Published on the web December 8, 2012
1723
Synthesis of 2¤-Modified Cyclic Bis(3¤-5¤)diadenylic Acids (c-di-AMPs)
and Their Promotion of Cell Division in a Freshwater Green Alga
Takafumi Tezuka,*1,# Noritaka Suzuki,1,# Keigo Ishida,1 Kin-ichi Oyama,2 Setsuyuki Aoki,1 and Masaki Tsukamoto*1
1Graduate School of Information Science, Nagoya University, Chikusa-ku, Nagoya, Aichi 464-8601
2Chemical Instrumentation Facility, Research Center for Materials Science, Nagoya University,
Chikusa-ku, Nagoya, Aichi 464-8602
(Received October 3, 2012; CL-121020; E-mail: tsukamoto@is.nagoya-u.ac.jp)
Three 2¤-modified cyclic bis(3¤-5¤)diadenylic acids (c-di-
AMPs) were synthesized from commercially available adeno-
sine phosphoramidites and the effects of c-di-AMP derivatives
on the cell division of Chlamydomonas reinhardtii, a kind of
freshwater green algae, were investigated. This structure-activity
relationship study suggests that the di(2¤-O-methyl)-c-di-AMP
showed the highest activity and that 2¤-substituents influenced
the cell division of C. reinhardtii.
nucleotides and nucleic acids are correlated with phosphodies-
terase stability,9,10 cell permeability,11,12 and nucleic acid duplex
stability,13 which in turn influence biological activities. How-
ever, there are only a few reports on the synthesis of c-di-AMPs
with 2¤ modification.14-21 For example, cyclic deoxydiadenylic
acid (1a) (Chart 1) was synthesized by phosphotriester14-17 and
H-phosphonate18 methods. In addition, previously published
methods for synthesis of c-di-AMP such as homopolymerization
of adenosine 3¤-monophosphate,22 construction of a cyclic sugar
backbone followed by introduction of the protected adenine,23
and the phosphotriester method24,25 cannot be applied for
synthesis of various 2¤-modified analogs due to the low yield
of the final product and/or multistep reactions. Thus, we
synthesized the three c-di-AMP analogs shown in Chart 1 by a
combination of the phosphoramidite and phosphotriester ap-
proaches based on methods previously published by us20 and by
Hayakawa.26
The synthesis starts from the commercially available
adenosine cyanoethyl (CE) phosphoramidites 2 with the sub-
stituent R (H, OCH3, and F) at the 2¤-position as shown in
Scheme 1. First, the amidites 2 were converted into the 3¤-
phosphotriesters 3 by a three-step procedure consisting of
condensation with allyl alcohol in the presence of imidazolium
perchlorate (IMP),27 oxidation of the resulting phosphite triester
by tert-butyl hydroperoxide,28,29 and removal of the 5¤-O-p,p¤-
dimethoxytrityl (DMTr) group under acidic conditions. The
5¤-hydroxy-free nucleotides 3 were then elongated with the
amidites 2 by the above three-step procedure to afford the
protected linear dimers 4. The overall yields of 4 (71-76%)
from the beginning of synthesis were almost the same as in
the synthesis of c-di-AMP [R = tert-butyldimethylsilyloxy
(OTBDMS), 79%].20
c-di-AMP (Chart 1)1,2 is a recently identified second-
messenger molecule in bacteria, and it has great potential in
many research fields. This molecule performs numerous im-
portant functions in bacteria, such as monitoring DNA integrity
during sporulation and playing an essential role in cell wall
homeostasis in Bacillus subtilis,3,4 and controlling cell size and
envelope stress in Staphylococcus aureus.5 Although the
existence of endogenous c-di-AMP in animals has not been
disclosed, c-di-AMP secreted by intracellular Listeria mono-
cytogenes has been shown to activate a host type I interferon
response in cultured cells.6 Moreover, mucosally administered
c-di-AMP exerted strong adjuvant activities in mice.7
To date, the investigations of the physiological action of
c-di-AMP have been performed using bacteria1-6 and animals.6,7
However, as far as we know, there has been no report of an
investigation in planta. Therefore, we examined the potential
physiological actions by c-di-AMP in Chlamydomonas rein-
hardtii, a kind of freshwater green algae frequently used as a
model alga,8 and found that c-di-AMP promotes the cell division
of C. reinhardtii. Moreover, we synthesized various
c-di-AMP analogs to investigate the structure-activity relation-
ship related to this phenomenon.
Among various analogs, 2¤-modified c-di-AMPs are inter-
esting based on reports that the substituents at the 2¤ positions of
Subsequently, the allyl groups on the 3¤-terminal phosphates
of 4 were deprotected by sodium iodide. However, the aqueous
workup using a triethylammonium hydrogen carbonate solution,
followed by extraction with dichloromethane, was unable to
collect the linear dinucleotide 3¤-phosphodiesters 5 as triethyl-
ammonium salts due to their hydrophilic properties. Fortunately,
the sodium salts 5 were precipitated quantitatively as white
powder from the reaction mixture and were characterized by
FAB-MS and NMR measurements. This is quite in contrast with
the more hydrophobic 2¤-O-TBDMS analog (R = OTBDMS),
which can be isolated only by aqueous workup.20
NH2
N
O–
O
N
O
O
P
R
N
5'
N
O
4'
1'
3' 2'
O
N
O
R
N
N
O
–O
P
O
N
The sodium salts 5 thus obtained were cyclized by a mixture
of 2,4,6-triisopropylbenzenesulfonyl chloride and N-methyl-
imidazole in THF under high-dilution conditions (substrate
concentration: ca. 6 mM) to give the fully protected c-di-AMP
analogs 6.20,26,30 Yields of this cyclization were 46% and 43%
for 6b and 6c, respectively. The precise yield of 6a could not be
R = OH: c-di-AMP
R = H: 1a
R = OCH3: 1b
R = F: 1c
NH2
Chart 1.
Chem. Lett. 2012, 41, 1723-1725
© 2012 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/

1724
AdeBz
AdeBz
DMTrO
HO
O
O
O
1), 2)
3), 4)
a: 92%
b: 92%
c: 91%
a: 83%
b: 77%
c: 79%
R
O
P
2
O
R
P
CEO
OCH2CH=CH2
CEO
N(i-C3H7)2
3
AdeBz
AdeBz
HO
HO
O
O
5)
O
CEO
O
O
R
O
CEO
O
O
R
P
P
AdeBz
AdeBz
quant
O
O
Figure 1. Reversed-phase HPLC profiles of purified (a) 1a,
(b) 1b, and (c) 1c under the following conditions: column,
COSMOSIL 5C18-AR-II [4.6 (diameter) mm © 250 (height)
mm]; flow rate, 1.0 mL min¹1; detection, 254 nm; eluent and
gradient [A: 100 mM CH3COO¢NH4 in H2O, B: a 20:80 mixture
of H2O and CH3CN, 0-2 min A 100%, 2-32 min with a linear
gradient from A 100% to A 70%/B 30%, 32-50 min B 100%];
temperature, 40 °C.
O
CEO
O
R
O
CEO
O
ONa
R
P
P
OCH2CH=CH2
4
5
OCE
O
O
O
P
AdeBz
R
O
6)
7)
1
a: –
b: 46%
c: 43%
a: 43% from
5a
b: 90%
c: 91%
O
25
**
AdeBz
O
**
a: R = H
b: R = OCH3
c: R = F
O
R
P
*
*
CEO
O
20
15
10
5
6
Scheme 1. Reagents and conditions: 1) (i) allyl alcohol,
imidazolium perchlorate (IMP), molecular sieves 3 ¡ (MS
3 ¡), CH3CN, rt, (ii) t-C4H9OOH/toluene, rt; 2) CHCl2COOH,
CH2Cl2, 0 °C; 3) (i) 2, IMP, MS 3 ¡, CH3CN, rt, (ii) t-
C4H9OOH/toluene, rt; 4) CHCl2COOH, CH2Cl2, 0 °C; 5) NaI,
acetone, reflux; 6) 2,4,6-triisopropylbenzenesulfonyl chloride,
N-methylimidazole, THF, 28 °C; 7) concd aq. NH3-CH3OH (1:1
v/v), 50 °C.
0
control c-di-AMP 1a
1b
1c
Figure 2. The 2¤-substitution effects of c-di-AMP on the cell
division of Chlamydomonas reinhardtii. C. reinhardtii cells
were cultured in the presence and absence of c-di-AMP (10 ¯M),
1a (10 ¯M), 1b (10 ¯M), or 1c (10 ¯M) for 3 days under a light
condition of 17 ¯mol m¹2 s¹1 at 27 °C. Vertical bars represent the
means « S.E. (n = 50). The asterisks indicate a significant
difference in the cases of c-di-AMP, 1a, 1b, and 1c compared
with the control at P < 0.01 (**) and P < 0.05 (*) by Student’s
t-test.
calculated.31 However, the cyclization proceeded similarly, as
judged by the overall yield of 1a (vide infra). The two-step
yields (43-46%) of 6 from 4 are inferior to that of the 2¤-O-
TBDMS analog (57%) in c-di-AMP synthesis.20
Finally, the benzoyl (Bz) and cyanoethyl (CE) protecting
groups of 6 were removed by treatment with concentrated
aqueous ammonia and methanol to afford the target c-di-AMP
analogs 1 as triethylammonium salts. Figure 1 shows the HPLC
profiles of the final products obtained after purification by
reversed-phase preparative HPLC. The profiles indicate that the
purities were satisfactory. The scales and overall yields of the
synthesis as well as molecular ion peaks in ESI-MS analyses are
summarized [Table S1 in Supporting Information (SI)32]. The
observed molecular ion peaks are consistent with the calculated
values. Moreover, the fragmentation patterns of the analogs 1 in
the ESI tandem mass measurements are the same as those of
c-di-AMP (Figures S1-S4 in SI32), confirming that the final
products possess the cyclic dinucleotide structures.6,20 In
addition, the triethylammonium salt of 1a was converted into
the corresponding sodium salt by treatment with a cation-
of c-di-AMP, the optimum concentration was found to be 10 ¯M
(Figure S6 in SI32). The cell division (cell number) was
promoted to 19.04 © 108 cells mL¹1, or increased approximately
42%, by culture with 10 ¯M c-di-AMP, and this effect was
statistically significant (Figure 2). Other adenine nucleotides
such as cyclic adenosine 3¤,5¤-monophosphate (cAMP) and
adenosine 5¤-monophosphate (5¤-AMP) under the same condi-
tions showed lesser promotions with 17% and 14%, respectively
(Figure S7 in SI32).
The 2¤-substitution effects of c-di-AMP on the cell division
¹1
of C. reinhardtii (2.3 © 105 cells mL
at time zero) were
similarly investigated. Figure 2 shows that all of the analogs
enhanced the cell division. The cell numbers after culture
increased in the following order: 1b (increased by 45%), c-di-
AMP (41%), 1a (28%), and 1c (26%), which probably reflected
several factors of the c-di-AMP derivatives, such as phospho-
diesterase stability, cell permeability (or lipophilicity), affinity to
the receptor, and so on. The lipophilicities of the c-di-AMPs
decreased in the following order: 1b > 1c > 1a > c-di-AMP, as
1
exchange resin. The H and 31P NMR spectra of the obtained
sample are consistent with the reported data (see SI32).14
The biological activity of c-di-AMP was investigated as
follows. The culture of C. reinhardtii was started using 4 © 105
cells per plastic dish (2.0 © 105 cells mL¹1) at time zero. After
culturing for 3 days, the cell numbers of C. reinhardtii were
estimated. Based on the dose-response curve from 0 to 100 ¯M
Chem. Lett. 2012, 41, 1723-1725
© 2012 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/

1725
estimated by the retention times in the reversed-phase HPLC
analysis (Figure 1 and SI32).20 Thus, the lipophilicity is not the
only dominant factor for the biological activity: the other factors
might contribute as well. Consequently, the 2¤-substituents did
not interfere with the enhancement of cell division by c-di-AMP.
In summary, based on the practical synthesis of c-di-AMP
and its 2¤-modified analogs, we revealed that the c-di-AMPs
promote the cell division of C. reinhardtii, which is the first
such finding in planta. The three 2¤-modified analogs from the
commercially available adenosine phosphoramidites were syn-
thesized in overall yields of 28-32%. The key intermediates are
the linear dinucleotide 3¤-phosphodiester sodium salts, which
can be cyclized by the phosphotriester method to give fully
protected cyclic products. Our method provides a general route
for various 2¤-modified analogs regardless of the electronic
properties of 2¤-substituents. Among the analogs, di(2¤-O-
methyl)-c-di-AMP showed the highest activity (albeit compara-
ble to that of c-di-AMP), and all the 2¤-substituents positively
influenced the cell division. We believe that the structure-
activity relationship studies including the 2¤-substitution effects
will lead to an understanding of the signaling mechanism of
c-di-AMP.
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We are grateful to Drs. Takuya Matsuo and Masahiro Ishiura
(Center for Gene Research, Nagoya University) for providing
C. reinhardtii and assistance. This study was supported by a
Grant-in-Aid for Scientific Research (No. 22750148) from the
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© 2012 The Chemical Society of Japan
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