Replacement of oxygen with sulfur on the furanose ring of cyclic dinucleotides enhances the immunostimulatory effectviaSTING activation

Article abstract of DOI:10.1039/d1md00114k

Cyclic dinucleotides (CDNs) are secondary messengers composed of two purine nucleotides linkedviatwo phosphodiester linkages: c-di-GMP, c-di-AMP, 3′,3′-cGAMP, and 2′,3′-cGAMP. CDNs activate the stimulator of interferon genes (STING) and trigger immune res

Full text of DOI:10.1039/d1md00114k

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Replacement of oxygen with sulfur on the
furanose ring of cyclic dinucleotides enhances the
Cite this: DOI: 10.1039/d1md00114k
immunostimulatory effect via STING activation†
*
Noriko Saito–Tarashima,
Mao Kinoshita, Yosuke Igata,
*
Yuta Kashiwabara and Noriaki Minakawa
Cyclic dinucleotides (CDNs) are secondary messengers composed of two purine nucleotides linked via two
phosphodiester linkages: c-di-GMP, c-di-AMP, 3′,3′-cGAMP, and 2′,3′-cGAMP. CDNs activate the stimulator
of interferon genes (STING) and trigger immune responses in mammalian species. CDNs are thus
fascinating molecules as drug candidates, and chemically stable CDN analogues that act as STING agonists
are highly desired at present. We herein report the practical synthesis of 4′-thiomodified c-di-AMP
analogues, which have sulfur atoms at the 4′-position on the furanose ring instead of oxygen atoms, using
simple phosphoramidite chemistry. The resulting 4′-thiomodified c-di-AMP analogues acted as potent
STING agonists with long-term activity. Our results show that replacing O4′ on CDNs with sulfur can lead
to enhanced immunostimulatory effects via STING activation.
Received 30th March 2021,
Accepted 4th June 2021
DOI: 10.1039/d1md00114k
rsc.li/medchem
Cyclic dinucleotides (CDNs) have been recently recognized
secondary messengers found in a variety of organisms.1
Bacteria produce molecules in which two purine nucleotides
are linked via two 3′–5′ phosphodiester linkages to give
symmetrical cyclophane structures: cyclic-di-GMP (c-di-GMP),2
cyclic-di-AMP (c-di-AMP),3 and 3′,3′-cyclic-GMP-AMP (3′,3′-
cGAMP)4 (Fig. 1). These bacterial CDNs play a critical role in
some bacterial signaling networks involved in quorum
sensing, control of biofilm formation, and cell wall
homeostasis via binding to the related protein receptors and/
or riboswitches.5
Recently, the asymmetrical cyclic dipurine molecule: 2′,3′-
cGAMP, in which guanosine and adenosine monophosphates
are linked via one 3′–5′ linkage and another 2′–5′ linkage, was
also discovered in mammalian cells.6–8 2′,3′-cGAMP triggers
immune responses in mammalian species. The cyclic GMP–
AMP synthase (cGAS), which can identify the cytosolic DNA
resulting from pathogen invasion, cyclizes ATP and GTP to
produce 2′,3′-cGAMP.9,10 The produced 2′,3′-cGAMP then acts
as a secondary messenger that triggers the stimulator of
interferon genes (STING) pathway,11 resulting in the
production of type I interferons (IFNs.)12–14 Importantly, all
known bacterial CDNs (c-di-GMP, c-di-AMP, and 3′,3′-cGAMP)
also stimulate the STING.15–19
Pharmacologic activation of STING signaling has shown
promise in diverse clinically impactful applications,
including broad-acting antiviral treatments, vaccine
adjuvants, and immunogenic tumor clearance. Unfortunately,
however, CDNs may be too chemically unstable for research
and clinical use as 1) they are susceptible to
phosphodiesterase (PDE)-mediated degradation, and 2) their
hydrophilicity renders them impermeable to cell membranes.
As these poor druglikenesses of CDNs, some non-nucleotide
STING agonists have been developed.20–22 However, synthetic
CDN analogues with more favorable properties are urgently
needed to elucidate the biological significance of CDNs.
To increase enzymatic stability, the nonbridging oxygen of
phosphodiester linkages have been replaced with sulfur in
order to create phosphorothioate linkages. The resulting
compound 2′,3′-cGsAsMP is reported to be more resistant to
PDE-mediated degradation than natural 2′,3′-cGAMP,
prolonging its systemic half-life while maintaining a high
affinity for STING.23 Another phosphorothioate CDN
analogue made of two AMP moieties cyclized via 2′–5′ and 3′–
5′ phosphothioester bonds, known as ML-RR-S2-CDA,
MIW815, or ADU-S100, shows better IFN-β responses than
2′,3′-cGAMP.24 In addition, CDN analogues that lack
negatively charged phosphodiester linkages have been
developed to improve stability against PDE-mediated
degradation.25–29 However, they could not stimulate the
STING and it was difficult to develop stable CDN analogues
that have STING agonist activity.
Graduate School of Pharmaceutical Science, Tokushima University, Shomachi 1-78-
1, Tokushima 770-8505, Japan. E-mail: noriko.tarashima@tokushima-u.ac.jp;
Fax: +81 88 633 7288; Tel: +81 88 633 9539
We previously developed a series of 4′-thionucleic acids
with sulfur atoms at the 4′-position on the furanose ring
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d1md00114k
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Fig. 1 Structures of naturally occurring and 4′-thiomodified CDNs.
instead of oxygen atoms.30,31 The 4′-thionucleic acids
exhibited biological equivalency with natural DNA/RNA
nucleotides.32 Capitalizing on these favorable properties, we
reported the synthesis of c-di-4′-thioAMP (1) (Fig. 1).33 The 4′-
thiomodified analogue 1 exhibited enhanced resistance to
PDE-mediated degradation compared with natural c-di-AMP.
We also found that 1 acts as an artificial ligand for the
bacterial c-di-AMP riboswitch, ydaO. However, how the
replacement of oxygen with sulfur on the furanose ring of c-
di-AMP affects the immunomodulating effect via binding to
STING remains unclear, as our previous synthetic strategy for
1 had many drawbacks, including the need for repeated
purification using high-performance liquid chromatography
(HPLC).
We herein report the practical synthetic procedure of CDN
analogues using phosphoramidite chemistry. Using this
chemistry, we prepared 4′-thiomodified c-di-AMP analogues 1
and 2, and demonstrated that replacing oxygen with sulfur
on the furanose ring enhances the immunostimulatory effect
via STING activation.
Phosphoramidite chemistry has been widely utilized in
the synthesis of naturally occurring and chemically modified
oligonucleotides.34 Most synthetic nucleosides, including 4′-
thionucleosides, accept the reaction conditions of the
phosphoramidite oligonucleotide synthesis.30,31 Therefore,
adopting a synthetic approach to generating CDN analogues
based on phosphoramidite chemistry should expand the
structural diversity of CDN analogues.
Thus far, a number of synthetic approaches for CDNs have
been reported.35 In most cases, the CDNs and their
analogues are prepared by dimerizing nucleoside/nucleotide
monomers and then subjecting the resulting dimer
molecules to the cyclocondensation reaction. However, of the
many synthetic reports of CDNs, there have been few using
phosphoramidite chemistry in both dimerization and
cyclocondensation reactions to construct the CDN
skeleton.36–38 In these reports, the major challenge has been
the application of 3′,5′-hydroxyl-protecting groups that can
chemoselectively be removed prior to performing the
cyclocondensation of its synthetic precursor, and tedious
procedures are required to prepare appropriately and
specially protected nucleoside monomers. Therefore, we first
phosphoramidite chemistry with an easily prepared 3′,5′-
unprotected nucleoside monomer.
The coupling reaction between a commercially available
N6-benzoyl (Bz)-adenosine phosphoramidite derivative 3 (ref.
39) and a cognate 3′,5′-unprotected nucleoside monomer 4
(ref. 40) with the aid of a generally used promoter for
phosphoramidite coupling, 1H-tetrazole, in acetonitrile with
3 Å molecular sieves (MS) and subsequent oxidation of an
internucleotide phosphite(III) linkage with tert-butyl
hydroperoxide (TBHP) afforded
a complicated mixture
including the desired linear dimer 5 (Table 1, entry 1 and
Scheme 1). However, it was difficult to remove impurities
even by performing the subsequent detritylation reaction to
yield 6. We therefore sought a suitable promoter for efficient
phosphoramidite coupling between 3 and 3′,5′-unprotected
monomer 4 to give nucleotide dimer 5. The reaction using
other tetrazole-type coupling reagents, such as a 5-(ethylthio)-
1H-tetrazole41
and
5-(benzylthio)-1H-tetrazole,42
with
subsequent oxidation afforded the pure nucleotide dimer 5
in moderate yields (2 steps) (entries 2 and 3), with the side
reactions suppressed due to the enhanced reaction
efficiencies compared with that using 1H-tetrazole. To further
improve the reaction efficiency, we also explored the use of
the acid/azole complex promoters reported by Hayakawa
et al.43 Whereas the use of imidazolium triflate decreased the
reaction yield and elongated the coupling time (entry 4), a
similar reaction using N-phenylimidazolium triflate
(N-PhIMT) with subsequent oxidation showed excellent
activity, and the desired nucleotide dimer 5 was isolated in
97% yield (2 steps) (entry 5).
After detritylation of 5, the resulting 3′,5′-unprotected
dimer 6 was treated with strictly 1.1 mol equivalent of
2-cyanoethyl tetraisopropylphosphoramidite and N-PhIMT in
acetonitrile with 3 Å MS (Scheme 1). However, no obvious
reaction progress was observed on thin-layer chromatography
(TLC). When the solvent system was changed to the
combination of an acidic solvent CH2Cl2 with 4 Å MS (a
suitable drying agent for CH2Cl2), the phosphitylation of 6
smoothly progressed to form 7. The subsequent
intramolecular cyclocondensation of 7 was then performed
by adding two molar equivalents of N-PhIMT to the initial
phosphitylation reaction mixture. The cyclocondensation
reaction of 7 was allowed to proceed within a few minutes by
screened
conditions
for
preparing
CDNs
using
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Table 1 Formation of nucleotide dimer 5
Entry
Reagents
Coupling time (3 → 5)
Yieldsa
1
2
3
4
5
1H-Tetrazole
24 h
9 h
7 h
24 h
1.5 h
60%b
68%
62%
23%
97%
5-(Ethylthio)-1H-tetrazole
5-(Benzylthio)-1H-tetrazole
Imidazolium triflate
N-Phenylimidazolium triflate (N-PhIMT)
a
2 steps. b Including inseparable impurities. ABz = N6-benzoyladenine.
Scheme 1 Synthesis of c-di-AMP, c-di-4′-thioAMP (1) and 2 using phosphoramidite chemistry. ABz = N6-benzoyladenine.
stepwise phosphoramidite coupling without undesired
polymerization. Subsequent oxidation with TBHP gave a fully
protected c-di-AMP derivative 8 (ref. 44) in 3 steps 63% yield
based on the linear dimer 6. Finally, full-deprotection of 8
was performed in a manner similar to that reported by
Gaffney et al.45 The Bz and cyanoethyl (CE) protecting groups
on 8 were thus removed by treatment with saturated
ammonia in methanol at room temperature for 24 h. After
concentration to dryness, the residue was dissolved in
minimal volume of pyridine and treated with Et3N·3HF at 65
°C for 2.5 h. The final product, c-di-AMP, was then obtained
as a triethylammonium salt.
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Since simple phosphoramidite chemistry for CDN
synthesis had now been optimized, we next prepared 4′-
thiomodified CDN analogues. To synthesize c-di-4′-thioAMP
(1), the 4′-thioadenosine phosphoramidite derivative 9 (ref.
31) and the corresponding 3′,5′-unprotected monomer 10
(ref. 32) were coupled in the presence of N-PhIMT, and
subsequent oxidation gave c-di-4′-thioadenosine dimer 11 in
93% yield (2 steps), much the same yield as in the
corresponding reaction to give 5. After detritylation of 11, the
resulting dimer 12 was subjected to the cyclocondensation
reaction using a diamidite reagent to give a fully protected c-
di-4′-thioAMP 14 in 66% yield (3 steps). These results
indicated that our phosphoramidite strategy for constructing
the CDN skeleton was useful, even for preparing chemically
modified CDN analogues. The Bz and CE group of 14 were
then deprotected with saturated ammonia in methanol at
room temperature. After concentration to dryness, the
residue was suspended in H2O, and the resulting white solid
was treated with Et3N·3HF at 65 °C in methanol. Finally,
purification of the reaction mixture using solid phase
extraction cartridges afforded the pure 1.
Next, we evaluated the immunostimulatory effect of 4′-
thiomodified CDN analogues 1 and 2 using HEK-293T-
derived interferon regulatory factor (IRF) and IFN-β reporter
cells (293T-Dual™ Null cells). Since HEK-293T cells are
known to have a non-functional STING pathway, we stably
transfected R232 hSTING, which is the most prevalent
isoform (occurrence of approximately 60% in the human
population),46 into 293T-Dual™ Null cells. As shown in
Fig. 2a, significant IFN-β induction was observed when
compound 1, which has two sulfur atoms on each furanose
ring, was simply added to the cell culture medium of 293
T-Dual R232 hSTING cells, and its induction degree was
much more potent than that of the parent compound c-di-
AMP. The asymmetrical 4′-thiomodified CDN analogue 2 also
showed a stronger immunostimulatory effect than c-di-AMP.
In addition, both the 4′-thiomodified CDN analogues 1 and 2
exhibited much more potent IFN-β inductions than c-(RpRp)-
di-Aps, which has two sulfur atoms instead of the two
nonbridging oxygens of the phosphodiester linkages.
Compounds 1 and 2 did not respond to the HEK-293T cells
lacking a functional STING pathway (293T-Dual™ Null cells),
indicating that they induced IFN-β responses as STING
agonists.
To evaluate how the replacement of oxygen with sulfur on
the furanose ring affected the activation of STING, we also
prepared asymmetrical analogue 2, in which an oxygen atom
on one of two furanose rings was substituted with a sulfur, in
a similar manner to that described for 1 while using 3 and 10
as starting materials. All CDN analogues were able to be
prepared without HPLC purification in this study.
To further compare the immunostimulatory effects, we
also evaluated the agonist activities of 1 and 2 to A230
hSTING, a non-synonymous variant of STING (Fig. 2b). A230
hSTING contains a single amino acid substitution G230A.
G230 is located in the flexible loop that forms a lid above the
Fig. 2 The evaluation of the INF-β induction by STING activation. The degree of INF-β induction by CDNs was measured in a) 293T-Dual™ Null
and 293T-Dual R232 hSTING cells and b) 293T-Dual A230 hSTING cells. CDNs (20 nmol mL−1) were added to the cells. INF-β induction was
measured by a luciferase reporter assay. Luminescence data are the mean relative light units
standard error of the mean (SEM) of at least 3
experiments (n = 3). *P < 0.05 and ****P < 0.0001 by Student's t-test.
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CDN binding pocket, and the G230A variant, A230 hSTING, is
able to respond to lower concentrations of CDNs.46 As a
result of simple addition to the cell culture medium of 293T-
Dual A230 hSTING, 4′-thiomodified CDN analogues 1 and 2
showed a superior induction of IFN-β to c-di-AMP and c-
(RpRp)-di-Aps. In addition, 1 and 2 both showed potent IRF3
induction as a result of STING activation (Fig. S1 in ESI†).
There are three possible factors responsible for the
improvement in the immunostimulatory effects of
compounds 1 and 2: 1) enhancement of the stability against
PDE-mediated degradation, 2) improvement in the
membrane permeability, and 3) changes in the binding mode
to the STING arising from the replacement of oxygen with
sulfur on the furanose ring. Regarding the potential
involvement of factor 1), we previously reported that
compound 1 displayed resistance against PDE specific for
CDNs, YybT.33 Since the immunostimulatory effects of
compounds 1 and 2 actually increased over time from 24 to
72 h post-compound addition (Fig. 2 and S1†), the enhanced
stability of 1 and 2 arising from the replacement of O4′ with
sulfur might have led to an improvement in STING
activation. To determine the potential involvement of the
other two factors, evaluations of other 4′-thiomodified CDNs,
such as c-di-4′-thioGMP, are currently underway.
293T-Dual™ Null cells using Lipofectamine 2000 (Thermo
Fisher Scientific, Waltham, MA, USA) according to the
manufacturer's instructions. Stable clones were selected for
resistance to blasticidin (10 mg mL−1) and then further
screened using Western blotting.
Cell stimulation and the SEAP/luciferase assay
293T-Dual R232 hSTING or 293T-Dual A230 hSTING cells
were seeded in a 96-well plate (3 × 104 cells/100 μL per well)
in DMEM with 10% FBS, 100 μg mL−1 streptomycin, 100
units per mL penicillin, normocin (100 μg mL−1), hygromycin
(100 μg mL−1), zeocin (100 μg mL−1), and blasticidin (10 μg
mL−1). After incubation for 24 h, the cells were treated with
each CDN. After further incubation for the intended
duration, the supernatant was collected, and the IRF-3/INF-β
induction was analyzed based on luminescence using
QUANTI-Luc (InvivoGen) and a SEAP Reporter Assay kit
(InvivoGen) according to the manufacturer's instructions.
Author contributions
N. S. T. designed and performed the experiments, analyzed
data, and wrote the paper. M. K., Y. I., and Y. K. performed
experiments. N. M. supervised the research and proofread
the paper. All authors discussed the results and contributed
to the final manuscript.
In conclusion, we developed practical synthetic procedures
for CDN analogues using phosphoramidite chemistry. Using
this procedure, 4′-thiomodified c-di-AMP analogues (1 and 2)
were successfully prepared without the need to perform Conflicts of interest
tedious purification using HPLC. The resulting 4′-
The authors declare no competing financial interests in
association with the present study.
thiomodified CDN analogues 1 and 2 showed potent and
long-term immunostimulatory effects by activating STING.
Our results indicate that the replacement of oxygen with
sulfur on the furanose ring is a useful strategy for designing
bioisosteres of c-di-AMP.
Acknowledgements
This work was financially supported in part by JSPS KAKENHI
Grant Numbers 17K15481 (N.S.T.) and 19K16316 (N.S.T.), the
Takeda Science Foundation (N.S.T.), the Naito Foundation (N.
S.T.), and the Mochida Memorial Foundation for Medical and
Pharmaceutical Research (N.S.T.). Y. I. is grateful for a
scholarship from the Tokyo Biochemical Research Foundation
and the research program for the development of intelligent
Tokushima artificial exosome (iTEX) from Tokushima
University.
Methods
Synthesis of CDNs
The detailed synthetic procedure and full characterization
data are presented in the ESI.†
Immunostimulatory effects: cell culture and transfections
293T-Dual™ Null cells (Invivogen, San Diego, CA, USA),
derived from the HEK293T cell line by the stable integration
of a SEAP reporter construct (to monitor the activation of the
transcription factor IFN-stimulated response elements) and a
luciferase reporter construct (to monitor the expression of
IFN-β), were propagated in Dulbecco's modified Eagle's
medium (DMEM) supplemented with 10% fetal bovine serum
(FBS), 100 μg mL−1 streptomycin, 100 units per mL penicillin,
normocin (100 μg mL−1), hygromycin (100 μg mL−1), and
zeocin (100 μg mL−1) in a humidified atmosphere with 5%
CO2. Stable R232 hSTING- or A 230 hSTING-expressing cell
lines (293T-Dual R232 hSTING or 293T-Dual A230 hSTING)
were obtained by transfection of the expression vector
pUNO1-hSTING-WT or pUNO1-hSTING-A230 (Invivogen) into
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