Synthesis and immunostimulatory properties of the phosphorothioate analogues of cdiGMP

Article abstract of DOI:10.1016/j.bmcl.2008.08.088

The synthesis of mono- and bisphosphorothioate analogues of 3′,5′-cyclic diguanylic acid (cdiGMP) via the modified H-phosphonate chemistry is reported. The immunostimulatory properties of these analogues were compared with those of cdiGMP.

Full text of DOI:10.1016/j.bmcl.2008.08.088

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5631–5634
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and immunostimulatory properties of the phosphorothioate
analogues of cdiGMP
a
b
b
Hongbin Yan a, , Xiaolu Wang , Rhonda KuoLee , Wangxue Chen
*
a Department of Chemistry, Brock University, 500 Glenridge Avenue, St. Catharines, Ont., Canada L2S 3A1
b Institute for Biological Sciences, National Research Council Canada, Ottawa, Ont., Canada K1A 0R6
a r t i c l e i n f o
a b s t r a c t
The synthesis of mono- and bisphosphorothioate analogues of 30,50-cyclic diguanylic acid (cdiGMP) via
the modified H-phosphonate chemistry is reported. The immunostimulatory properties of these ana-
logues were compared with those of cdiGMP.
Article history:
Received 24 July 2008
Revised 23 August 2008
Accepted 26 August 2008
Available online 29 August 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
cdiGMP
Vaccine adjuvant
Immunostimulation
Phosphorothioate
30,50-Cyclic diguanylic acid (cdiGMP, 1c) has recently been rec-
ognized as an important bacterial second messenger.1–3 It has also
been shown to possess extraordinary immunostimulatory proper-
ties and is therefore evaluated as a potential vaccine adjuvant can-
didate.4–7
G
X
O
O
O
P
O
HO
1a X = O, Y = S;
b X = Y = S;
c X = Y = O
O
O
Y
OH
P
O
O
We previously reported a convenient synthesis of cdiGMP.8 In
order to explore the structure–immunostimulation relationship
of cdiGMP, we synthesized the phosphorothioate analogues of
cdiGMP (Fig. 1), where either one (cdiGMP-S1 1a)9 or two
(cdiGMP-S2 1b) sulfur atoms replace the non-bridging oxygen at
the internucleotide linkages.
G
Figure 1. cdiGMP 1c and its phosphorothioate analogues.
A four-step deprotection protocol (Scheme 2) was used to give
the fully deprotected cdiGMP-S1 1a and cdiGMP-S2 1b in good
yields (70–75%).15 Removal of the S-(2-cyanoethyl)- group by
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) under anhydrous condi-
tions was followed by treatment with 2-nitrobenzaldoxime 12 and
ammonolysis in the presence of mercaptoethanol (Scheme 2, steps
i–iii). The resulting partially protected cyclic dimers 11 were then
further deprotected in a triethylammonium formate buffer that
contains methanol (Scheme 2, steps iv and v). The 1H and 31P
NMR spectra of 1a and 1b are shown in Figure 3. Resonance at
ca. 55 and ꢀ1 ppm in the 31P NMR spectra correspond to phosp-
horothioate and phosphate, respectively. It is noted that the two
sets of phosphorous signals in cdiGMP-S1 1a integrate equally (pa-
nel b) and that there is no signal at ca. 0 ppm in the cdiGMP-S2 1b
(panel d).
The 20- and 50-hydroxyls of guanosine were protected with the
1-(4-chlorophenyl)-4-ethoxypiperidin-4-yl (Cpep)10 and the 9-
phenyl-xanthen-9-yl (or the pixyl)11,12 groups, respectively
(Fig. 2). Guanine was ‘doubly’-protected at both O-6 and N-2, as
is shown in Figure 2. The modified H-phosphonate approach13,14
was used due to its flexibility in the preparation of both
phosphates and phosphorothioates.
The synthesis of the phosphorothioates via the modified H-
phosphonate approach is illustrated in Scheme 1. In situ treatment
of H-phosphonate diesters with a sulfur-transfer reagent S-(2-cya-
noethyl)phthalimide 9 gave phosphorothioate triester 4, which
was further transformed into linear dimer H-phosphonate 6. Cycli-
zation of this linear dimer H-phosphonate 6 took place under high
dilution conditions to furnish the fully protected cyclic dinucleo-
tide phosphorothioate trieters 7a and 7b in good yields (75–80%).
The fully deprotected cdiGMP-S1 1a and S2 1b were also ana-
lyzed by reverse phase HPLC on a Dionex Acclaim PA C18 column
(Fig. 4).
We then carried out preliminary evaluation of the immuno-
stimulatory properties of cdiGMP, cdiGMP-S1, and cdiGMP-S2. In
* Corresponding author. Tel.: +1 905 688 5550x3545; fax: +1 905 682 9020.
E-mail address: tyan@brocku.ca (H. Yan).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.08.088

5632
H. Yan et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5631–5634
Cl
Cl
O
N
Cl
N
N
O
G
'' =
OEt
N
O
Px
N
N
Cpep
H
Figure 2. Protecting groups used in this study.
a
c
b
8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5
ppm
d 70
60
50
40
30
20
10
0
ppm
8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5
ppm
70
60
50
40
30
20
10
0
ppm
Figure 3. 1H and 31P NMR spectra of 1a (a and b) and 1b (c and d).
G"
PxO
G''
PxO
O
the first experiment, groups of five female 8-week-old C57BL/6
mice were intranasally administered with 0, 5, 10, and 69 g of
O
l
G"
PxO
O
O
O(Cpep)
O
O
O
O
H
O
O(Cpep)
cdiGMP in injectable phosphate buffered saline (PBS). The mice
were killed 24 h later and their lungs were lavaged with PBS sup-
plemented with 3 mM EDTA (1 ml, 5ꢁ). The total and differential
cell counts as well as a panel of 21 chemokines and cytokines were
measured. As can be seen in Figure 5, intranasal instillation of
cdiGMP induced a dose-dependent recruitment of inflammatory
cells into the bronchoalveolar spaces with the majority of recruited
P
P
O
2
HNEt3
G"
O
i, ii
RS
iii, iv
O(Cpep)
O
O
+
O
P
G"
HO
G"
O
RS
O
H
O(Cpep)
O
P
(Lev)O
O(Cpep)
O
O(Cpep)
(Lev)O
3
5 R = CNCH2CH2-
4
R = CNCH2CH2-
v
G"
O
HO
O
G
X
O
O
O
G"
O
R'S
O
P
O
O
O(Cpep)
P
O
(Cpep)O
G
P
vi, vii
(Cpep)O
G ''
O
O
G"
O
RS
i, ii, iii
iv, v
O
Y
O(Cpep)
O
O(Cpep)
O
O
1a or 1b
7
P
P
O
O
SR
O
O
H
O(Cpep)
O
O
7a; R = CNCH2CH2-, R' = Ph
b; R = R' = CNCH2CH2-
P
11a; X = O, Y = S
b; X = Y = S
O
6 R = CNCH2CH2-
O
O
NO
2
O
O
CN
N
SPh
Lev =
Me
O
P
H
O
N
S
NH4
O
O
10
O
8
9
12
N
OH
Scheme 1. Synthesis of fully protected cdiGMP-S1 and S2. (i) (CH3)3COCl, C5H5N;
(ii) 9, C5H5N; (iii) NH2NH2ꢂH2O, CH3COOH, H2O, C5H5N; (iv) 10, (CH3)3COCl, C5H5N,
0 °C; (v) CF3COOH, pyrrole, CH2Cl2; (vi) (PhO)2P(O)Cl, CH2Cl2, C5H5N, ꢀ40 °C; (vii) 8
or 9, C5H5N.
Scheme 2. Unblocking of fully protected cdiGMP-S1 and S2. (i) DBU, (CH3)3SiCl,
CH3CN; (ii) 12, DBU, CH3CN; (iii) aq NH3, HSCH2CH2OH, 55 °C; (iv) CH3OH, NEt3-
HCOOH buffer (pH 3.75), 40 °C, 4 h; (v) Amberlite IR-120, Na+ form.

H. Yan et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5631–5634
5633
450
200
-50
160
a
b
WVL:260 nm
14.05 - 14.0
mAU
mAU
WVL:260 nm
1 - 12.82 - 12.805
100
50
min
min
-20
0.0
10.0
Retention Time[min]
20.0
0.0
10.0
Retention Time [min]
20.0
Figure 4. HPLC profiles of 1a (a) and 1b (b) (Programme: linear gradient of 0.1 M triethylammonium acetate buffer–acetonitrile (100:0–90:10 v/v) over 10 min and then
isocratic elution).
100
80
60
40
20
0
50
40
30
20
10
0
8.0
6.0
4.0
2.0
0.0
Figure 5. Dose-dependent recruitment of inflammatory cells into the bronchoal-
veolar spaces by cdiGMP.
Figure 7. Induction of pulmonary neutrophil recruitment by cdiGMP and its
analogues (cdiGMP-S1 and cdiGMP-S2).
cells being neutrophils (solid bars in the right panel). This effect
was particularly evident when 69 lg of cdiGMP was used.
Similarly, Luminex analysis of the lavage fluid showed a signif-
icant dose-dependent induction of key proinflammatory cytokines
and chemokines such as IL-12(p40), IL-1beta, IL-6, KC, MCP-1, MIP-
1beta, RANTES, and TNF-alpha (Fig. 6) whereas there was no
change in the levels of GM-CSF, IFN-gamma, IL-10, IL-13, IL-17,
IL-1alpha, IL-2, IL-3, IL-4, IL-5, IL-9, or VEGF. These data suggests
that cdiGMP is immunostimulatory at one of the mucosal sites
and has the potential to function as a mucosal adjuvant.
When the same experiments were carried out with cdiGMP-S1
and cdiGMP-S2, these two analogues appear to retain their immu-
nostimulatory function by induction of pulmonary neutrophil
recruitment (Fig. 7) and local proinflammatory cytokine/chemo-
kine responses (Fig. 7). However, the inflammatory responses elic-
ited by the two analogues, particularly the cdiGMP-S1, were
substantially milder than cdiGMP (Figs. 7 and 8), in that fewer neu-
trophils and lower amounts of chemokine KC, MIP-1beta, and RAN-
TES were induced in cdiGMP-S1-treated than in cdiGMP-treated
1000
cdiGMP-S1
cdiGMP-S2
100
10
1
cdiGMP
PBS
Figure 8. Induction of key proinflammatory cytokines and chemokines by cdiGMP
and its analogues (cdiGMP-S1 and cdiGMP-S2).
BAL 21-plex
1000.0
0 ug
mice. Although the induction of proinflammatory responses is a
prerequisite for an adjuvant to induce effective immune responses,
excessive tissue inflammation is also detrimental to the host. Thus,
it is possible that these analogues are superior to cdiGMP as muco-
sal adjuvants with fewer side effects. More detailed immunological
evaluations of these analogues are currently underway.
5 ug
10 ug
69 ug
100.0
10.0
1.0
Acknowledgments
This work is supported by Research Corporation, Canada Foun-
dation for Innovation, Natural Sciences and Engineering Research
Council of Canada, and National Research Council Canada (A-base).
The authors thank Tim Jones and Razvan Simionescu for mass
Figure 6. Dose-dependent induction of key proinflammatory cytokines and
chemokines by cdiGMP.

5634
H. Yan et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5631–5634
1 ml). The residue was dissolved in dry acetonitrile (2 ml) followed by
addition of trimethylsilyl chloride (20 l) and DBU (0.10 ml, 0.67 mmol).
After 30 min, the products were concentrated under reduced pressure and
the residue was redissolved in dry acetonitrile (2 ml) followed by addition of
2-nitrobenzaldoxime (92 mg, 0.55 mmol). The reaction was allowed to
proceed overnight at room temperature, and the products were evaporated
spectrometric and NMR analysis, respectively, and Mr. Greg Harris
for Luminex assay.
l
References and notes
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methanol (1.5 ml) and precipitated with
a mixture of ethyl acetate and
diethyl ether (30 ml, 1:3 v/v). The precipitate was collected by centrifugation
at 5000 RPM. This precipitation–centrifugation process was repeated two
more times. The dried residue was suspended in methanol (3.0 ml) followed
by addition of triethylammonium formate buffer (4.5 ml, pH 3.75, 0.5 M)
and incubated at 40 °C. After 6 h, the pH of the reaction mixture was
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an Amberlite ionic exchange column (Na+ form, 1ꢁ 10 cm). The appropriate
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12
14
16
C
1H23
N
O
31P232S2 requires: 721.04.
20
10 12
S2 7b (50 mg, 27
lmol) was dried by evaporation with dry pyridine (2ꢁ