2′-Fluoro-c-di-GMP as an oral vaccine adjuvant

Article abstract of DOI:10.1039/c9ra08310c

Bis-(3′-5′)-cyclic dimeric 2′-deoxy-2′-fluoroguanosine monophosphate (2′-F-c-di-GMP) was synthesized through the modified H-phosphonate chemistry. Oral immunization of C57BL/6 mice with Helicobacter pylori cell-free sonicate extract adjuvanted with 2′-F-c

Full text of DOI:10.1039/c9ra08310c

RSC Advances
View Article Online
View Journal | View Issue
PAPER
20-Fluoro-c-di-GMP as an oral vaccine adjuvant†
a
Jia Li,a Rhonda Kuo Lee,b Wangxue Chenbc and Hongbin Yan
*
Cite this: RSC Adv., 2019, 9, 41481
Bis-(30–50)-cyclic dimeric 20-deoxy-20-fluoroguanosine monophosphate (20-F-c-di-GMP) was synthesized
through the modified H-phosphonate chemistry. Oral immunization of C57BL/6 mice with Helicobacter
pylori cell-free sonicate extract adjuvanted with 20-F-c-di-GMP led to the production of antigen-specific
antibodies in feces and sera, and lowered bacterial counts in the stomach upon post-vaccination
infections in immunized mice. Similarly, oral vaccination of BALB/c mice with flagillin proteins from
Clostridium difficile and Listeria monocytogenes adjuvanted with 20-F-c-di-GMP led to production of
antigen-specific antibodies both systemically and mucosally. The adjuvanticity of 20-F-c-di-GMP is
associated with the enhanced induction of interferon g. These results demonstrated the excellent oral
adjuvanticity of 20-F-c-di-GMP.
Received 12th October 2019
Accepted 5th December 2019
DOI: 10.1039/c9ra08310c
rsc.li/rsc-advances
antibodies, and provide excellent protective immunity of
immunized mice against H. pylori challenges. In a similar
Introduction
In the last decade or so, 30,50-cyclic diguanylic acid (c-di-GMP)
has been recognized as a potent immunostimulator and
a useful mucosal adjuvant in a number of models.1,2 It was
previously demonstrated by us that intranasal administration of
c-di-GMP prior to bacterial challenges provides mice with
protection against Acinetobacter baumannii infection by che-
mokine induction and enhanced neutrophil recruitment.3
Furthermore, we showed that intranasal immunization of mice
with pneumococcal surface adhesion A (PsaA) adjuvanted with
c-di-GMP invoked strong antigen-specic serum immunoglob-
ulin G (IgG) and secretory IgA antibody responses, and the
nasopharyngeal Streptococcus pneumoniae colonization in
immunized mice was signicantly reduced.4 In the present
study, we wish to demonstrate the adjuvanticity of c-di-GMP
and its 20-uoro-analog (20-F-c-di-GMP) in oral immunization
of mice against Helicobacter pylori. In this respect, uorine
atoms are small and electronegative. Incorporation of uorine
at the 20-position of nucleosides is an effective approach to
modulate sugar puckers.5 Furthermore, introduction of uorine
into therapeutic agents has been well recognized as a useful
modication to modulate pharmacological properties.6–9
manner, productions of antigen-specic antibodies were also
demonstrated in mice immunized with agillin proteins from
Gram-positive bacterium Clostridium difficille and intracellular
pathogen Listeria monocytogenes.
Results and discussion
Synthesis of 20-F-c-di-GMP via the modied H-phosphonate
chemistry
We previously demonstrated the synthesis of c-di-GMP via the
modied H-phosphonate chemistry.10,11 In a similar manner, the
synthesis of 20-F-c-di-GMP started with protecting the exocyclic
amino residues of 20-deoxy-20-uoro-guanosine 1 with isobutyryl
group. The resulting N-isobutyryl-20-deoxy-20-uoro-guanosine
was then protected with dimethoxytrityl (DMTr) group at the 50-
OH position. Thus, 50-O-DMTr-2-N-isobutyryl-20-deoxy-20-uoro-
guanosine 2 was obtained in 79% overall yield in two steps
(steps (i) and (ii), Scheme 1). The building blocks H-phosphonate
triethylammonium salt 3 and 30-O-levulinyl (Lev) nucleoside 4
were prepared according to the process described in Scheme 1.
In the modied H-phosphonate approach the fully protected
linear dimer phosphorothioate triester 5 was prepared by
reacting H-phosphonate triethylammonium salt 3 and 30-O-Lev
nucleoside 4 in the presence of pivaloyl chloride followed by
oxidation with 1-phenylsulfanyl-pyrrolidine-2,5-dione 11. Aer
removal of 30-O-levulinyl group from the dimer 5 by the treat-
ment with hydrazine hydrate in pyridine–acetic acid solution,
the product was transformed into the corresponding linear
dimer H-phosphonate triethylammonium salt, followed by
removal of 50-O-dimethoxytrityl group. The resulting linear
dimer H-phosphonate 7 was then subjected to cyclization under
high dilution conditions to give the fully protected 20-deoxy-20-
uoro cyclic diguanylic acid 8 in 59% yield.
We report herein that oral immunization of C57BL/6 mice
with H. pylori cell-free sonicate extract (HPCE) adjuvanted with
20-F-c-di-GMP led to the production of antigen-specic
aDepartment of Chemistry, Brock University, 1812 Sir Isaac Brock Way, St. Catharines,
ON L2S 3A1, Canada. E-mail: tyan@brocku.ca
bHuman Health and Therapeutics Research Center, National Research Council of
Canada, 100 Sussex Dr, Ottawa, ON K1A 0R6, Canada
cDepartment of Biological Sciences, Brock University, 1812 Sir Isaac Brock Way, St.
Catharines, ON L2S 3A1, Canada
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c9ra08310c
This journal is © The Royal Society of Chemistry 2019
RSC Adv., 2019, 9, 41481–41489 | 41481

View Article Online
RSC Advances
Paper
Scheme 1 Reagents and conditions: (i) (a) (CH3)3SiCl, (CH3)2CHCOCl, C5H5N; (b) aq. NH3; (ii) DMTrCl, C5H5N; (iii) (a) 10, (CH3)3CCOCl, C5H5N; (b)
H2O; (iv) (a) 12, C5H5N; (b) Cl2CHCOOH, pyrrole, CH2Cl2; (v) (a) (CH3)3CCOCl, C5H5N; (b) 11, C5H5N; (vi) NH2NH2$H2O, CH3COOH, C5H5N, H2O;
(vii) (a) 10, (CH3)3CCOCl, C5H5N; (b) H2O; (c) Cl2CHCOOH, pyrrole, CH2Cl2; (viii) (a) (PhO)2P(O)Cl, C5H5N; (b) 11, C5H5N; (ix) (a) 13, TMG; (b) aq.
NH3, 55 ꢁC.
Two steps were involved in the deprotection of the fully of the product was found by ESI to be 693.1 as [M ꢀ H]ꢀ which is
protected 20-uoro cyclic diguanylic acid 8 (step (ix), Scheme 1), in agreement with the calculated value (693.1) (Fig. S3†).
treatment with 2-nitrobenzaldoxime 13 in the present of
N,N,N0,N0-tetramethylguanidine (TMG) followed by amminol-
Intranasally administered 20-F-c-di-GMP induces strong
ysis. The fully unprotected bis-(30–50)-cyclic dimeric 20-deoxy-20-
antigen-specic antibody responses in the serum and at
uoroguanosine monophosphate 9 was characterized by 1H and
multiple mucosal sites
31P NMR (Fig. S1†). The reverse phase HPLC prole conrmed
We and others have previously shown that c-di-GMP is a potent
mucosal adjuvant when administered intranasally.4,12–14 In this
the homogeneity of the product (Fig. S2†). The molecular mass
41482 | RSC Adv., 2019, 9, 41481–41489
This journal is © The Royal Society of Chemistry 2019

View Article Online
Paper
RSC Advances
study, we rst determined if i.n. immunization of mice with 20- the golden standard of mucosal adjuvant which has undesirable
F-c-di-GMP can elicit antigen specic mucosal immune toxicity for human applications. We have previously shown that
responses at a comparable magnitude to those induced by the immunization with PsaA alone at this dose showed no effect on
parental c-di-GMP. As shown in Fig. 1, co-administration of the bacterial colonization.4 These results demonstrated that 20-F-
pneumococcal protein PsaA with 20-F-c-di-GMP induced higher c-di-GMP is a potent mucosal adjuvant when administered by
levels of PsaA-specic IgA in feces and vaginal wash, and serum intranasal route, and that 20-F-c-di-GMP induces a potent,
IgG2a than the co-administration with c-di-GMP whereas the protective immunity against i.n. challenge with S. pneumoniae
serum PsaA-specic IgA and IgG1 levels were comparable when co-administered with the PsaA antigen via i.n. route.
between the two adjuvants. As expected, sham-immunized mice Therefore, further exploration of this molecule as a potential
showed no specic antibody responses in the serum or mucosal mucosal adjuvant is warranted.
samples.
More importantly, we found that the mucosal immune
Oral immunization with 20-F-c-di-GMP-adjuvanted vaccine
induces strong antigen-specic antibody responses in the
serum and at multiple mucosal sites
responses induced by the i.n. immunization with 20-F-c-di-GMP
adjuvanted vaccine were protective against mucosal infections
in the well-established mouse S. pneumoniae colonization model
(Fig. 2) in that mice i.n. immunized with PsaA + 20-F-c-di-GMP
showed signicantly reduced colonization of S. pneumoniae
when compared to sham-immunized mice (P < 0.05). The
magnitude of this reduction was comparable to that attained in
mice immunized with PsaA adjuvanted with cholera toxin (CT),4
Despite the well-recognized socioeconomic and safety advantages
of oral immunization over the parenteral or i.n. immunization,
only a limited number of oral vaccines are currently approved for
human use.15 Oral vaccination is the most challenging vaccina-
tion method due to the administration route. Indeed, we found
Fig. 1 Induction of antigen-specific mucosal IgA responses by intranasal administration of 20-F-c-di-GMP. Groups of 5 BALB/c mice were
intranasally immunized with 2 mg PsaA admixed with 2.5 mg 20-F-c-di-GMP, 2.5 mg c-di-GMP or 10 mg c-di-GMP at day 0, 14 and 21. Additional
group of mice were immunized with phosphate-buffered saline (PBS) and served as sham-immunized group. The feces, vaginal washing and
blood samples were collected at day 28 and assayed by ELISA for PsaA-specific IgA and IgG isotypes (IgG1 and IgG2a) responses. *P < 0.05 vs.
PsaA + c-di-GMP groups.
This journal is © The Royal Society of Chemistry 2019
RSC Adv., 2019, 9, 41481–41489 | 41483

View Article Online
RSC Advances
Paper
Fig. 2 Reduced nasopharyngeal colonization by S. pneumoniae in
mice intranasally immunized with 20-F-c-di-GMP-adjuvanted vaccine.
Groups of 5 BALB/c mice were intranasally immunized with 2 mg PsaA
admixed with 2.5 mg 20-F-c-di-GMP or sham-immunized with PBS at
day 0, 14 and 21. The mice were intranasally challenged at day 35 with
5 ꢂ 106 CFU type 14 S. pneumoniae and the bacterial numbers in the
nasal cavity of challenged mice were determined 3 days later. *P < 0.05
vs. PBS group.
that oral administration of the parental c-di-GMP as a mucosal
adjuvant failed to induce reliable mucosal or systemic immune
responses (unpublished data). In this study, we therefore
assessed if oral administration of 20-F-c-di-GMP induces antigen-
specic mucosal immune responses. As shown in Fig. 3, oral co-
administration of both high and low doses of HPCE with 20-F-c-
di-GMP induced substantial amount of antigen-specic fecal
IgA and serum IgG2a responses, which were similar in the
magnitude to those induced by CT (Fig. 3A). As expected, sham-
immunized mice showed no specic antibody responses in the
serum or fecal samples. Similarly, oral co-administration of 20-F-
c-di-GMP with agellin antigens puried from L. monocytogenes
(50 mg) or C. difficile (30 mg) induced substantial amount of C.
difficile agellin-specic IgA and small amount of Listeria
agellin-specic IgA in feces as well as serum IgG1 and IgG2a
responses, as compared with sham-immunized mice (Fig. 3B).
These results demonstrated that 20-F-c-di-GMP enhances
mucosal immune responses to microbial antigens when
administered via the oral route, and indicate that 20-F-c-di-GMP
can be used in oral vaccines as a potent mucosal adjuvant.
Oral immunization of mice with 20-F-c-di-GMP-adjuvanted
HPCE signicantly reduces gastric colonization by H. pylori
Fig. 3 Induction of antigen-specific mucosal IgA responses by oral
administration of 20-F-c-di-GMP. Groups of 5 C56BL/6 mice were
orally immunized with varying amount of H. pylori cell free sonicate
extract (HPCE) (A) or flagillin proteins from C. difficile and L. mono-
cytogenes (B) admixed with either 100 mg 20-F-c-di-GMP or 10 mg
cholera toxin (CT, positive control) at day 0, 14 and 21. Additional
group of mice were immunized with PBS and served as sham-
immunized group. Feces and blood samples were collected at day 28
and assayed by ELISA for antigen-specific IgA and IgG isotypes (IgG1
and IgG2a) responses.
We next determined if the mucosal immune responses induced
by the 20-F-c-di-GMP adjuvanted H. pylori oral vaccine protect
against H. pylori challenge in a mouse model of H. pylori
infection.16 As shown in Fig. 4, quantitative bacteriology showed
that oral immunization of mice with both low (250 mg) and high
(500 mg) doses of H. pylori HPCE + 20-F-c-di-GMP vaccines
signicantly reduced the bacterial burdens in the gastric
mucosa at 4 weeks post-challenge when compared to sham-
immunized mice (P < 0.001). Moreover, the magnitude of this
reduction was comparable to that attained in mice immunized
with HPCE adjuvanted with CT. As anticipated, immunization
of mice with HPCE alone failed to reduce the bacterial coloni-
zation. These results suggest that 20-F-c-di-GMP is capable of
inducing protective mucosal immunity against mucosal path-
ogens upon oral immunization with specic antigen.
Oral immunization with 20-F-c-di-GMP-adjuvanted vaccine
induces antigen-specic Th1/Th17 cytokine responses
Previous studies by others have implied that Th1/Th17 immune
responses may play an important role in host defence against H.
pylori infection.17 We next examined if oral administration of 20-
41484 | RSC Adv., 2019, 9, 41481–41489
This journal is © The Royal Society of Chemistry 2019

View Article Online
Paper
RSC Advances
F-c-di-GMP + HPCE induced antigen-specic Th1/Th17 cytokine
responses. Compared to sham-immunized mice, the spleno-
cytes from 20-F-c-di-GMP immunized mice produced substan-
tially higher amount of IFN-g, IL-2 and IL-17 in response to
HPCE stimulation (Fig. 5). The amount of IFN-g produced by
the splenocytes from 20-F-c-di-GMP immunized mice was even
higher than that produced by the cells from CT-immunized
mice although the latter mice produced higher amount of IL-2
and IL-17 than the former mice. These results indicate that
the protection against H. pylori infection induced by oral
immunization with 20-F-c-di-GMP in mice is associated with the
production of antigen-specic IFN-g.
Fig.
4 Reduced gastric colonization by H. pylori in mice orally
immunized with 20-F-c-di-GMP-adjuvanted vaccine. Groups of 5
C56BL/6 mice were orally immunized with varying amount of H. pylori
cell free sonicate extract (HPCE) admixed with either 100 mg 20-F-c-di-
GMP or 10 mg cholera toxin (CT, positive control) at day 0, 14 and 21.
Additional group of mice were immunized with PBS and served as
sham-immunized group. The mice were orally challenged 3ꢂ
between day 35 and 42 with 108 CFU H. pylori SS1 and the bacterial
numbers in the gastric mucosa of challenged mice were determine 4
weeks later. ***P < 0.001 vs. immunized groups.
Experimental
Synthesis of compounds
20-Fluoro-50-O-dimethoxytrityl-2-N-isobutyl-20-deoxyguanosine 2.
20-Fluoro-20-deoxyguanosine 1 (1.00 g, 3.51 mmol) was dried in
vacuo at 60 ꢁC for 5 h followed by addition of dry pyridine (10.0 ml).
Aer the solution was cooled (ice-water bath), chloro-
trimethylsilane (2.23 ml, 17.6 mmol) was added and the reaction
mixture was stirred for 30 min. The mixture was then evaporated to
ca. half of the original volume. To the residue, dry pyridine (5.0 ml)
was added and mixture was cooled (ice-water bath) followed by
dropwise addition of isobutyric anhydride (3.02 ml, 18.2 mmol).
Aer 3 h, the reaction was quenched by addition of water (3.0 ml)
followed aer 15 min aqueous ammonium hydroxide (30–33%,
10.0 ml). The products were rst evaporated under reduced pres-
ꢁ
sure while the temperature was kept below 10 C. Aer the bulk
ammonia was removed, the products were evaporated to dryness
under reduced pressure while the temperature was kept below
35 ꢁC. The residue was co-evaporated with dry pyridine (2 ꢂ 10 ml)
and then dissolved in dry pyridine (15.0 ml) followed by addition of
4,40-dimethoxytrityl chloride (1.15 g, 3.39 mmol). Water (2.0 ml) was
added aer 30 min to quench the reaction. The products were
concentrated under reduced pressure and the residue was dis-
solved in dichloromethane (25 ml) and extracted with saturated
aqueous sodium hydrogen carbonate (15 ml). The layers were
separated, and the aqueous layer was back extracted with
dichloromethane (2 ꢂ 5 ml). The combined organic layers were
dried (MgSO4) and concentrated with a rotary evaporator. The
residue was puried by column chromatography on silica gel. The
appropriate fractions, which were eluted with dichloromethane–
methanol (98 : 2 v/v) containing 0.5% triethylamine, were concen-
trated under reduced pressure to give the title compound as a col-
ourless glass (1.83 g, 79%). dH[(CD3)2SO]: 12.15 (1H, s, NH, ex),
11.65 (1H, s, NH, ex), 8.13 (1H, s, H-8), 7.34–7.19 (9H, m, Ar–H),
6.83–6.78 (4H, m, Ar–H), 6.21 (1H, d, J ¼ 18.7, H-10), 5.67 (1H, d, J ¼
7.2, 30-OH, ex), 5.42 (1H, dd, J ¼ 52.9 and 4.6, H-20), 4.67–4.53 (1H,
m, H-30), 4.12 (1H, m, H-40), 3.71 (3H, s, –OCH3), 3.72 (3H, s,
–OCH3), 2.76 (1H, CH, m), 1.12 (3H, d, J ¼ 7.0, CH3), 1.11 (3H, d, J ¼
7.0, CH3). Rf: 0.46 (dichloromethane–methanol 95 : 5, v/v).
Fig. 5 Induction of antigen-specific Th1/Th17 cytokine responses by
oral administration of 20-F-c-di-GMP. Groups of 5 C56BL/6 mice were
orally immunized with varying amount of H. pylori cell free sonicate
extract (HPCE) admixed with either 100 mg 20-F-c-di-GMP or 10 mg
cholera toxin (CT, positive control) at day 0, 14 and 21. Additional
group of mice were immunized with PBS and served as sham-
immunized group. The spleens were collected at day 28 and single cell
suspension was prepared for the determination of antigen-specific
cytokine responses. The cells were stimulated by 10 mg mlꢀ1 HPCE
and cultured at 37 ꢁC in a 5% CO2 atmosphere for 72 hours. At the end
of the culture, the supernatant was collected and assayed for the levels
of IFN-g, IL-2 and IL-17 by Luminex. *P < 0.05 and ***P < 0.001 vs. PBS
group.
20-Fluoro-30-O-levulinyl-2-N-isobutyryl-20-deoxyguanosine 4.
20-Fluoro-50-O-dimethoxytrityl-2-N-isobutyryl-20-deoxyguanosine
2 (1.00 g, 1.52 mmol) was co-evaporated with dry toluene (2 ꢂ 5
ml) and then dissolved in dry pyridine (10.0 ml) followed by
addition of levulinic anhydride (0.65 g, 3.04 mmol). Aer 16 h,
This journal is © The Royal Society of Chemistry 2019
RSC Adv., 2019, 9, 41481–41489 | 41485

View Article Online
RSC Advances
Paper
water (3.0 ml) was added and the solution was concentrated 2.73 (1H, sep, J ¼ 7.0, CH), 1.10 (15H, m). dP[(CD3)2SO]: ꢀ0.72 (dd,
under reduced pressure. The residue was dissolved in JPH ¼ 589.5, JPC ¼ 10.4). dF[(CD3)2SO]: ꢀ203.15 (ddd, J ¼ 18.8, 24.7
dichloromethane (25 ml) and extracted with saturated aqueous and 54.2).
sodium hydrogen carbonate (15 ml). The layers were separated
DMTr-G0p(s0)G0-Lev 5. 20-Fluoro-30-O-levulinyl-2-N-isobutyryl-20-
and the aqueous layer was back extracted with dichloromethane deoxyguanosine 4 (0.44 g, 0.97 mmol) and 20-uoro-50-O-dime-
(2 ꢂ 5 ml). The combined organic layers were dried (MgSO4) thoxytrityl-2-N-isobutyryl-20-deoxyguanosine
30-H-phosphonate
and evaporated under reduced pressure. The residue was co- triethylammonium salt 3 (1.00 g, 1.22 mmol) were co-evaporated
evaporated with dry toluene (3 ꢂ 5 ml) and dissolved in dry with dry pyridine (2 ꢂ 5 ml) and then dissolved in dry pyridine
dichloromethane (10.0 ml). Distilled pyrrole (1.05 ml, 15.1 (5.0 ml) and cooled (ice-water bath). Pivaloyl chloride (0.31 ml, 2.53
mmol) followed by dichloroacetic acid (0.56 ml, 6.79 mmol) mmol) followed by 1-phenylsulfanyl-pyrrolidine-2,5-dione 11
were added. Aer 10 min, the products were extracted with (0.56 g, 2.20 mmol) were added aer 5 min. The reaction mixture
saturated aqueous sodium hydrogen carbonate (15 ml). The was stirred for 30 min at room temperature, and then water (0.2
layers were separated and the aqueous layer was back extracted ml) was added. Aer 10 min the products were concentrated under
with dichloromethane (2 ꢂ 5 ml). The combined organic layers reduced pressure. The residue was dissolved in dichloromethane
were dried (MgSO4) and concentrated under reduced pressure. (25 ml) and extracted with saturated aqueous sodium hydrogen
The residue was puried by column chromatography on silica carbonate (15 ml). The layers were separated and the aqueous layer
gel. The appropriate fractions, which were eluted with was back extracted with dichloromethane (2 ꢂ 5 ml). The
dichloromethane–methanol (97 : 3 v/v), were combined and combined organic layers were dried (MgSO4) and concentrated
concentrated under reduced pressure to give the title compound under reduced pressure. The residue was puried by column
as a colourless glass (0.51 g, 74%). ESI-MS found [M ꢀ H]ꢀ ¼ 452.1, chromatography on silica gel. The appropriate fractions, which
ꢀ
C19H23FN5O7 requires 452.158. dH[(CD3)2SO]: 12.14 (1H, s, NH, were eluted with dichloromethane–methanol (98 : 2 v/v) contain-
ex), 11.69 (1H, s, NH, ex), 8.31 (1H, s, H-8), 6.17 (1H, dd, J ¼ 14.9 ing 0.5% of triethylamine, were pooled and concentrated under
and 4.2 Hz, H-10), 5.69 (1H, ddd, J ¼ 51.0, 4.5 and 4.5, H-20), reduced pressure to give the title compound as a colourless glass
5.43–5.35 (2H, m, H-30 and 5-OH), 4.23 (1H, d, J ¼ 3.9 Hz, H-40), (1.35 g, 109%. The yield is over 100%, due to inseparable impu-
3.76–3.59 (2H, m, H-50, H-500), 2.82–2.75 (3H, m, CH2 and CH), rities, likely phthalimide). ESI-MS found [M ꢀ H]ꢀ ¼ 1263.7, C60-
2.62–2.57 (2H, m, CH2), 2.13 (3H, s, CH3), 1.12 (6H, d, J ¼ 6.8, H62F2N10O15PSꢀ requires 1263.38. dP[(CD3)2SO]: 23.79, 23.02. Rf:
2ꢂCH3). dF[(CD3)2SO]: ꢀ206.2 (ddd, J ¼ 51.1, 11.7 and 12.3). 0.39 (dichloromethane–methanol 95 : 5, v/v).
Rf: 0.31 (dichloromethane–methanol 95 : 5, v/v).
DMTr-G0p(s0)G0-OH 6. DMTr-G0p(s0)G0-Lev 5 (1.20 g, 0.95 mmol)
20-Fluoro-50-O-dimethoxytrityl-2-N-isobutyryl-20-deoxyguanosine was dissolved in pyridine (10.0 ml) followed by addition of
30-H-phosphonate triethylammonium salt 3. Ammonium p-tolyl H- a mixture of hydrazine monohydrate (0.40 ml, 8.24 mmol), acetic
phosphonate (0.95 g, 5.03 mmol) was co-evaporated with triethyl- acid (4.8 ml), water (0.8 ml) and pyridine (18.0 ml). The reaction
amine (1.40 ml, 10.0 mmol) and methanol (8.0 ml) under reduced mixture was stirred at room temperature for 15 min followed by
pressure. To the residue was added 20-uoro-50-O-dimethoxytrityl- addition of pentane-2,4-dione (1.20 ml). Aer 10 min the products
2-N-isobutyryl-20-deoxyguanosine 2 (1.10 g, 1.67 mmol), and the were concentrated under reduced pressure. The residue was dis-
mixture was co-evaporated with dry pyridine (2 ꢂ 5 ml) and then solved in dichloromethane (25 ml) and extracted with saturated
dissolved in dry pyridine (25 ml). To the cooled (ice-water bath) aqueous sodium hydrogen carbonate (15 ml). The layers were
mixture was added dropwise pivaloyl chloride (0.68 ml, 5.55 mmol) separated and the aqueous layer was back extracted with
over 5 min. Aer 1 h water (5.0 ml) was added. Aer a further dichloromethane (2 ꢂ 5 ml). The combined organic layers were
period of 1 h at room temperature, the products were concentrated dried (MgSO4) and concentrated under reduced pressure. The
under reduced pressure and the residue was dissolved in residue was puried by column chromatography on silica gel. The
dichloromethane (25 ml) and extracted with saturated aqueous appropriate fractions, which were eluted with dichloromethane–
sodium hydrogen carbonate (15 ml). The layers were separated and methanol (95 : 5 v/v), were concentrated under reduced pressure to
the aqueous layer was back extracted with dichloromethane (2 ꢂ 5 give the title compound as a colourless solid (1.05 g, 95%). ESI-MS
ml). The combined organic layers were extracted with triethy- found for [M ꢀ H]ꢀ 1166.3, C55H57F2N10O13PSꢀ requires 1166.35.
lammonium phosphate buffer (20 ml, 0.5 M, pH 7.0) and the dP[(CD3)2SO]: 23.68, 22.96. Rf: 0.27 (dichloromethane–methanol
organic layer was back extracted by dichloromethane (15 ml). The 95 : 5, v/v).
combined organic layers were dried (MgSO4) and concentrated
HO-G0p(s0)G0p(H) 7. Ammonium p-tolyl H-phosphonate 10
under reduced pressure. The residue was puried by short column (0.57 g, 3.01 mmol) was co-evaporated with triethylamine
chromatography on silica gel. The appropriate fractions, which (0.84 ml, 6.03 mmol) and methanol (5.3 ml). To the residue was
were eluted with dichloromethane–methanol (90 : 10 v/v), were added DMTr-G0p(s0)G0-OH 6 (1.03 g, 0.88 mmol) and the mixture
pooled and concentrated under reduced pressure to give triethy- was co-evaporated with dry pyridine (2 ꢂ 5 ml) and then dis-
lammonium salt of the title compound as a colourless glass solved in more dry pyridine (10.0 ml). To this cooled (ice-water
(1.18 g, 86%). ESI-MS found Mꢀ ¼ 720.2, C35H36FN5O9Pꢀ requires bath) reaction mixture was added pivaloyl chloride (0.37 ml,
720.2. dH[(CD3)2SO]: 12.37 (1H, s, NH, ex), 11.84 (1H, s, NH, ex), 3.0 mmol) over a period of 5 min. Aer 30 min water (1.0 ml) was
8.13 (1H, s, H-8), 7.21–7.10 (9H, m, Ar), 6.78 (4H, dd, J ¼ 3.3 and added. The products were allowed to warm up to room temper-
9.0, Ar), 6.21 (1H, d, J ¼ 18.4, H-10), 5.79 (1H, d, J ¼ 54.0, H-20), 4.12 ature and stirring was continued for another 1 h. The products
(1H, br, H-30), 3.70 (6H, s), 3.07 (1H, m, H-40), 2.92 (6H, q, J ¼ 7.0), were concentrated under reduced pressure and the residue was
41486 | RSC Adv., 2019, 9, 41481–41489
This journal is © The Royal Society of Chemistry 2019

View Article Online
Paper
RSC Advances
dissolved in dichloromethane (25 ml) and extracted with satu- sealed and heated at 55 ꢁC for 15 h. Aer the mixture was
rated aqueous sodium hydrogen carbonate (15 ml). The layers cooled, the products were concentrated to dryness under
were separated and the aqueous layer was back extracted with reduced pressure and then co-evaporated with ethanol (2 ꢂ 2
dichloromethane (2 ꢂ 5 ml). The combined organic layers were ml). The residue was dissolved in methanol (1.0 ml) and
dried (MgSO4) and concentrated under reduced pressure. The precipitated from diethyl ether (30.0 ml). The product was ob-
residue was co-evaporated with dry toluene (3 ꢂ 10 ml) and then tained by centrifugation at 5000 rpm. This precipitation–
dissolved in dry dichloromethane (20 ml). Freshly distilled centrifugation process was repeated 3 more times. The residue
pyrrole (1.04 ml, 15.0 mmol) and dichloroacetic acid (0.86 ml, was dissolved in HPLC grade water (0.2 ml) followed by addition
10.4 mmol) were added. The reaction mixture was stirred for of 1-butanol (10 ꢂ volume). The mixture was vortexed and then
10 min and then extracted with saturated aqueous sodium frozen. Aer centrifugation at 10 000 rpm for 10 min, the
hydrogen carbonate (15 ml). The layers were separated and the supernatant was discarded. This 1-butanol extraction step was
aqueous layer was back extracted with dichloromethane (2 ꢂ 5 repeated two more times and the nal residue was dissolved in
ml). The combined organic layers were extracted with triethy- HPLC water (1.0 ml) and passed through an Amberlite (Na+
lammonium phosphate buffer (20 ml, pH 7.0, 0.5 M) and the form) cation exchange column (1 ꢂ 20 cm). The fractions con-
aqueous layer was back extracted by dichloromethane (15 ml). taining nucleic acid were freeze-dried to give the fully unpro-
The combined organic layers were dried (MgSO4) and concen- tected disodium salt of 20-uoro-c-di-GMP 9 as a colourless
trated under reduced pressure. The residue was puried by short sponge solid (16 mg, 44%). ESI-MS found [M ꢀ H]ꢀ ¼ 693.1,
ꢀ
column chromatography on silica gel. The appropriate fractions,
C
20H21F2N10O12P2 requires 693.07. dH[D2O, 600 MHz]: 7.84
eluted with dichloromethane–methanol (90 : 10 v/v), were and 7.83 (2H, H-8), 6.09 (1H, d, J ¼ 15.0, H-10), 6.07 (1H, d, J ¼
concentrated under reduced pressure to give triethylammonium 15.0, H-10), 5.48 (2H, two broad signals with JF–H ¼ 52.2, H-20),
salt of HO-G0p(s0)G0p(H) as a colourless glass (367 mg, 40%). ESI- 5.03 (2H, br, H-30), 4.38 (2H, d, br, J ¼ 7.9, H-40), 4.31 (2H, d, br, J
MS found [M ꢀ H]ꢀ ¼ 927.3, C34H39F2N10O13P2Sꢀ requires ¼ 12.1, H-50), 4.02 (2H, dd, J ¼ 12.1 and 4.3, H-500). dP[D2O, 242.9
927.187. dP[(CD3)2SO]: 23.46 and 21.82 (1 P), 0.32 and 0.17 (1 P, MHz]: ꢀ1.40.
dd, JPH ¼ 602.6 Hz, JPC ¼ 11.4 Hz).
Antigens and adjuvants. c-di-GMP was synthesized accord-
Preparation of fully protected 20-uoro-c-di-GMP 8. HO- ing to procedures reported previously.10 Cholera toxin (CT) was
G0p(s0)G0p(H) triethylammonium salt 7 (100 mg, 0.097 mmol) purchased from Sigma-Aldrich Canada Ltd (Oakville, Ontario,
was co-evaporated with dry pyridine (2 ꢂ 3 ml) and then dis- Canada). The recombinant pneumococcal surface adhesion A
solved in dry dichloromethane (5.0 ml). This solution was (PsaA) of S. pneumoniae and H. pylori cell-free sonicate extract
added to a solution of diphenyl chlorophosphate (0.41 ml, 1.98 (HPCE) were prepared as described in details previously.4,18 For
mmol) in dry pyridine (5.0 ml) dropwise over 20 min while the the preparation of agellins from L. monocytogenes and C.
temperature was kept at ꢀ40 ꢁC (dry ice-acetone bath). Aer difficile, bacterial strains were grown overnight at 37 ꢁC on agar
20 min 1-phenylsulfanyl-pyrrolidine-2,5-dione 11 (0.45 g, 1.76 plates. Cells were scraped from surface and resuspended in
mmol) was added and the reaction mixture was allowed to warm 10 mM Tris pH 7.4. Flagella were sheared from surface of
up to room temperature. Stirring was continued for another bacterial cells using a warring blender (4 ꢂ 1 min). Bacterial
30 min and then water (0.5 ml) was added. Aer 5 min the cells were removed by low speed centrifugation and then
products were concentrated under reduced pressure. The agellar laments were collected from supernatant by ultra-
residue was dissolved in dichloromethane (25 ml) and extracted centrifugation (100 000 ꢂ g, 1 hour). Flagellar samples were
with saturated aqueous sodium hydrogen carbonate (15 ml). analysed by SDS-PAGE.
The layers were separated and the aqueous layer was back
Mice. Six to eight-week-old female BALB/c or C57BL/6 mice
extracted with dichloromethane (2 ꢂ 5 ml). The combined were purchased from Charles Rivers Laboratories (St.
organic layers were dried (MgSO4) and concentrated under Constant, Quebec). The animals were housed under specic-
reduced pressure. The residue was puried by short column pathogen-free conditions in
a federally licensed small
chromatography on silica gel. The appropriate fractions, which animal containment level 2 facility, and given free access to
were eluted with dichloromethane–methanol (97 : 3 v/v), were sterile water and certied mouse chow. The animals were
combined and concentrated under reduced pressure to give the maintained and used in accordance with the recommenda-
fully protected 20-uoro-c-di-GMP 8 as a colourless glass (58 mg, tions of the Canadian Council on Animal Care Guide to the
+
59%). ESI-MS found [M + H]+ ¼ 1019.2, C40H43F2N10O12P2S2
Care and Use of Experimental Animals. All experimental
requires 1019.19. dP[(CD3)2SO]: 24.50, 22.98, 21.60. Rf: 0.42, 0.35 procedures were approved by the institutional animal care
(dichloromethane–methanol 95 : 5, v/v).
committee (Institute for Biological Sciences, National
20-Fluoro-c-di-GMP 9. Fully protected 20-uoro-c-di-GMP 8 Research Council Canada, Ottawa, Ontario).
(50 mg, 0.049 mmol) was co-evaporated with dry toluene (2 ꢂ 2
Intranasal or oral immunization in mice
ml) and then dissolved in dry acetonitrile (3.0 ml), followed by
addition of 2-nitrobenzaldoxime 13 (0.12 g, 0.72 mmol) and For intranasal immunization, mice were lightly anesthetized
N,N,N0,N0-tetramethylguanidine (86 ml, 0.69 mmol). Aer 16 h under isouorane. On day 0, 14 and 21, groups of mice (n ¼ 5)
the products were evaporated to dryness under reduced pres- were inoculated with 50 ml of various vaccine preparations or
sure and the residue was taken up in concentrated aqueous controls as detailed in Results section. For oral immuniza-
ammonium hydroxide (33%, 2.0 ml). The reaction mixture was tion, various vaccine preparations or controls in 0.5 ml
This journal is © The Royal Society of Chemistry 2019
RSC Adv., 2019, 9, 41481–41489 | 41487

View Article Online
RSC Advances
Paper
volume were administered by gavage via an 18-gauge feeding chocolate agar. Inoculated mice were sacriced 3 days later and
needle. the nasal cavity was lavaged with 0.5 ml lavage uid and
At day 28, blood and mucosal samples (fecal pellets or/and aliquots (100 ml) of 10-fold serial dilutions of the lavage uids
vaginal wash) were collected as described elsewhere.19 In were cultured, in duplicates, on chocolate agar plates to quan-
some experiments, nasal wash samples were also collected at tify the number of viable organisms.
the end of experiment. For both the vaginal and nasal wash, the
volume of the lavage uid recovered from each mouse was
recorded. Since the recovery of lavage volumes among different
mice in our study were very similar (about 90%), the small
H. pylori and oral H. pylori infection
The mouse-adapted H. pylori SS1 that was established by Lee
et al.20 was used as the challenge strain. Bacteria were grown on
brain–heart infusion (BHI) broth supplemented with 5% horse
serum under a microaerophilic atmosphere created by a Cam-
variation among individual samples was not adjusted. All
ꢁ
samples were stored at ꢀ20 C until assay.
ꢁ
pyGen generator (Oxoid Ltd, Hampshire, England) at 37 C for
Enzyme-linked immunosorbent assay (ELISA) for
measurement of antigen-specic antibodies in the serum and
mucosal samples
48 h. The bacterial cells were harvested by centrifugation at
12 000 ꢂ g for 10 min at 4 ꢁC, washed with PBS three times and
then suspended in PBS.
The mice were inoculated orally with 1 ꢂ 108 CFU of freshly
harvested H. pylori in 500 ml BHI broth by using a 18-gauge
feeding needle as described previously.16 The inoculations were
repeated twice (a total of three inoculations) over a period of 5
days. Mice were killed by an overdose of carbon dioxide 4 weeks
aer the last inoculation, and the serum and stomachs of the
animals were collected for analysis. In some experiments,
spleens were removed for splenocyte culture and determination
of cytokine responses.
Levels of antigen-specic antibodies in serum and mucosal
samples were measured by an enzyme-linked immunosorbent
assay (ELISA) modied from a previously described procedure.16
Briey, 96-well microplates (Thermo Electron Corporation,
Milford, MA) were coated with puried PsaA (5 mg mlꢀ1, 100 ml
per well), puried agellins (5 mg mlꢀ1, 50 ml per well), or HPCE
(20 mg mlꢀ1; 50 ml per well) in 0.015 M sodium carbonate and
ꢁ
0.035 M sodium bicarbonate buffer (pH 9.6) at 4 C overnight.
All the subsequent incubations were carried out at room
temperature. The wells were blocked by incubation with 5%
bovine serum in PBS for 1 h, and then rinsed three times with
PBS-0.05% Tween 20. Duplicates of 100 ml pre-diluted samples
were added to the wells. Sample dilutions were as follows: 1 : 2
for fecal extracts and nasal washes, 1 : 2000 for serum IgG1,
1 : 500 for serum IgG2a and 1 : 50 for serum IgA. Aer the plates
were incubated for 3 h, alkaline phosphatase-conjugated goat
antibodies specic for mouse IgA, IgG1 and IgG2a (all from
Caltag Laboratories, Burlingame, CA) were added and incu-
bated for 1 h. Colour reactions were developed by the addition
of p-nitrophenyl phosphate (pNPP) substrate (KPL, Inc., Gai-
thersburg, MD), and optical density was measured at 405 nm
with an automated ELISA plate reader (Multiskan Ascent,
Thermo Labsystems, Vantaa, Finland). Pooled samples
collected from mice that had been intranasally immunized with
Assessment of H. pylori infection
The presence of H. pylori infection in individual mice was
determined by quantitative bacterial culture as described
previously.16 Briey, the mouse stomach was opened longitu-
dinally along the greater curvature and gently washed three
times in PBS to remove the stomach contents. The stomach
tissue was then homogenized in 5 ml saline with Teon
pestles and glass tubes. The homogenates were serially diluted
in saline, and 100 ml of the dilutions were plated onto GSSA
(Glaxo Selective Supplement Antibiotics)-supplemented agar
plates. The plates were incubated for 96 h at 37 ꢁC under
microaerophilic conditions, and the number of colonies were
counted and expressed as CFU per stomach. With this
method, log10 2.7 CFU per stomach represented the limit of
detection.
¨
the relevant vaccines or from the naıve mice were used as
positive or negative controls respectively.
Intranasal challenge of mice with S. pneumoniae
Splenocyte culture and cytokine assay
Fresh inocula were prepared for each experiment from a frozen Spleens were aseptically removed and placed in 5 ml Dulbecco's
stock of S. pneumoniae (type 14). Stock vials of S. pneumoniae modied Eagle's medium (DMEM). Single cell suspensions
were thawed and the culture revived on chocolate agar, which were prepared by pressing the spleen through a 100 mm Nylon
was then used to inoculate Todd-Hewitt and Columbia broth cell strainer into a sterile Petri dish, using the rubber end of
supplemented with 1% glucose and 0.1% sodium bicarbonate. a 1 ml syringe plunger. Erythrocytes from spleens were lysed
The broth culture was incubated in a candle jar at 37 ꢁC for using an ACK lysis buffer. Cells were washed one time with
approximately 6 h. The broth culture in mid-logarithmic growth DMEM and resuspended at a concentration of 3 ꢂ 107 cells
phase was centrifuged at 12 000 ꢂ g for 15 min, cells were per ml in DMEM supplemented with 10% fetal bovine serum
resuspended in PBS and used immediately. Fourteen days aer (FBS). Cells were seeded in duplicate on a 96-well cell culture
the last immunization, mice were anesthetized and intranasally plate and stimulated with the mixed H. pylori antigens (10 mg
inoculated with approximately 107 colony-forming units (CFU) mlꢀ1 each) for 48 h at 37 ꢁC and 5% CO2. Culture supernatants
S. pneumoniae in 50 ml saline. The actual inoculum concentra- were collected and assayed for the concentrations of IFN-g, IL-2
tions were determined by plating 10-fold serial dilutions on and IL-17 using Luminex kits.21
41488 | RSC Adv., 2019, 9, 41481–41489
This journal is © The Royal Society of Chemistry 2019

View Article Online
Paper
RSC Advances
the agellin antigens used in the study and Mr Harris Greg for
Statistical analysis
technical assistance for the part of the study.
Data are presented as mean ꢃ standard deviation (SD) for
parametric data, unless otherwise indicated. Differences among
experimental groups were analyzed by Student's t-test or one-
way ANOVA followed by Bonferroni or Dunnett's multiple
pairwise comparison tests, when appropriated. Differences were
considered signicant at P < 0.05. All statistical analyses were
conducted using GraphPad Prism Version 5.0 (GraphPad So-
ware, San Diego, CA).
Notes and references
1 W. Chen, R. KuoLee and H. Yan, Vaccine, 2010, 28, 3080.
2 R. Libanova, P. D. Becker and C. A. Guzman, Microb.
Biotechnol., 2012, 5, 168.
3 L. Zhao, R. KuoLee, G. Harris, K. Tram, H. Yan and W. Chen,
Int. Immunopharmacol., 2011, 11, 1378.
4 H. Yan, R. KuoLee, K. Tram, H. Qiu, J. Zhang, G. B. Patel and
W. Chen, Biochem. Biophys. Res. Commun., 2009, 387, 581.
5 F. Guo, Q. Li and C. Zhou, Org. Biomol. Chem., 2017, 15, 9552.
Conclusions
The modied H-phosphonate chemistry was found to be suit-
able for the synthesis of 20-F-c-di-GMP. This uorinated c-di-
GMP analogue was shown to possess excellent adjuvanticity
both intranasally and orally. In this respect, i.n. immunization
of BALB/c mice with PsaA adjuvanted with 20-F-c-di-GMP led to
the induction of antigen-specic antibodies both systemically
and in the mucosa, and provided protective immunity against S.
pneumoniae infections. Similar results were seen in C56BL/6
mice orally immunized with H. pylori HPCE adjuvanted with
20-F-c-di-GMP. Furthermore, immunization of C56BL/6 mice
with agillin proteins from C. difficile and L. monocytogenes
adjuvanted with 20-F-c-di-GMP led to successful induction of
antigen-specic antibodies both systemically and mucosally.
Mechanistically, this work showed that the protection against
H. pylori infection induced by oral immunization with 20-F-c-di-
GMP as adjuvant is associated with the enhanced production of
antigen-specic IFN-g. This observation appears to be in
contrast to the cytokine induction when CT was used as an
adjuvant. In the latter case, production of IL-17 appeared to be
more profound, suggesting different mode of activation of the
host immune system by 20-F-c-di-GMP and CT as adjuvants.
¨
6 K. Muller, C. Faeh and F. Diederich, Science, 2007, 317, 1881.
7 E. P. Gillis, K. J. Eastman, M. D. Hill, D. J. Donnelly and
N. A. Meanwell, J. Med. Chem., 2015, 58, 8315.
8 P. Richardson, Expert Opin. Drug Discovery, 2016, 11, 983.
9 D. E. Yerien, S. Bonesi and A. Postigo, Org. Biomol. Chem.,
2016, 14, 8398.
10 H. Yan and A. L. Anguilar, Nucleosides, Nucleotides Nucleic
Acids, 2007, 26, 189.
11 H. Yan, X. Wang, R. KuoLee and W. Chen, Bioorg. Med.
Chem. Lett., 2008, 18, 5631.
12 P. M. Gray, G. Forrest, T. Wisniewski, G. Porter, D. C. Freed,
J. A. DeMartino, D. M. Zaller, Z. Guo, J. Leone, T. M. Fu and
K. A. Vora, Cell. Immunol., 2012, 278, 113.
13 T. Ebensen, K. Schulze, P. Riese, C. Link, M. Morr and
C. A. Guzman, Vaccine, 2007, 25, 1464.
14 D. L. Hu, K. Narita, M. Hyodo, Y. Hayakawa, A. Nakane and
D. K. R. Karaolis, Vaccine, 2009, 27, 4867.
´
15 A. Miquel-Clopes, E. G. Bentley, J. P. Stewart and
S. R. Carding, Clin. Exp. Immunol., 2019, 196, 205.
16 W. Chen, D. Shu and V. S. Chadwick, J. Gastroenterol.
Hepatol., 2001, 16, 377.
17 T. Larussa, I. Leone, E. Suraci, M. Imeneo and F. Luzza, J.
Immunol. Res., 2015, 981328.
18 W. Chen, D. Shu and V. S. Chadwick, J. Gastroenterol.
Hepatol., 2000, 15, 1000.
19 G. B. Patel, H. Zhou, A. Ponce and W. Chen, Vaccine, 2007,
25, 8622.
Conflicts of interest
Work described in this manuscript is partially disclosed in WO
patent 2015/074145A1 and US patent 10092644.
20 A. Lee, J. O'Rourke, M. C. De Ungria, B. Robertson,
G. Daskalopoulos and M. F. Dixon, Gastroenterologia, 1997,
112, 1386.
Acknowledgements
The authors thank Natural Sciences and Engineering Research
Council of Canada and National Research Council Canada for 21 G. Harris, R. KuoLee, H. H. Xu and H. Chen, Sci. Rep., 2019,
funding this work. We also thank Dr Susan Logan for providing
9, 6538.
This journal is © The Royal Society of Chemistry 2019
RSC Adv., 2019, 9, 41481–41489 | 41489