Alternate synthesis to D-glycero-β-D-manno-heptose 1,7-biphosphate

Article abstract of DOI:10.1016/j.carres.2017.08.011

D-glycero-β-D-manno-heptose 1,7-biphosphate (HBP) is an enzymatic intermediate in the biosynthesis of the heptose component of lipopolysaccharide (LPS), and was recently revealed to be a pathogen-associated molecular pattern (PAMP) that allows detection o

Full text of DOI:10.1016/j.carres.2017.08.011

Accepted Manuscript
Alternate synthesis to d-glycero-β-d-manno-heptose 1,7-biphosphate
Janelle Sauvageau, Milan Bhasin, Cynthia X. Guo, Itunuoluwa A. Adekoya, Scott D.
Gray-Owen, Stefan Oscarson, Lorenzo Guazzelli, Andrew Cox
PII:
S0008-6215(17)30430-5
10.1016/j.carres.2017.08.011
CAR 7436
DOI:
Reference:
To appear in: Carbohydrate Research
Received Date: 16 June 2017
Revised Date: 27 July 2017
Accepted Date: 22 August 2017
Please cite this article as: J. Sauvageau, M. Bhasin, C.X. Guo, I.A. Adekoya, S.D. Gray-Owen, S.
Oscarson, L. Guazzelli, A. Cox, Alternate synthesis to d-glycero-β-d-manno-heptose 1,7-biphosphate,
Carbohydrate Research (2017), doi: 10.1016/j.carres.2017.08.011.
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Alternate synthesis to D-glycero-β-D-manno-heptose 1,7-biphosphate
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Janelle Sauvageau,a* Milan Bhasin,a Cynthia X. Guo,b Itunuoluwa A. Adekoya,b Scott D. Gray-
Owen,b Stefan Oscarson,c Lorenzo Guazzelli,c1 Andrew Coxa
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a Vaccine Program, Human Health Therapeutics Portfolio, National Research Council, Ottawa,
Ontario, Canada, K1A 0R6
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b Department of Molecular Genetics, University of Toronto, Toronto, Canada, M5S 1A8
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c Centre for Synthesis and Chemical Biology, University College Dublin, Belfield, Dublin 4,
Ireland
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* Corresponding author: Janelle.Sauvageau@nrc-cnrc.gc.ca
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1 Present address: University of Pisa, Department of Pharmacy, Via Bonanno 6, 56126, Pisa,
Italy
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Abstract:
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D
-glycero-β-
D
-manno-heptose 1,7-biphosphate (HBP) is an enzymatic intermediate in the
biosynthesis of the heptose component of lipopolysaccharide (LPS), and was recently revealed to
be a pathogen-associated molecular pattern (PAMP) that allows detection of Gram-negative
bacteria by the mammalian immune system. Cellular detection of HBP depends upon its
stimulation of a cascade that leads to the phosphorylation and assembly of the TRAF-interacting
with forkhead-associated domain protein A (TIFA), which activates the transcription factor NF-
κB. In this note, an alternate chemical synthesis of HBP is described and its biological activity is
established, providing pure material for further assessing and exploiting the biological activity of
this compound.
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Keywords:
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D-glycero-β-D-manno-heptose 1,7-biphosphate (HBP), nuclear factor-kappa B (NF-κB),
pathogen-associated molecular pattern (PAMP), Wittig reaction, Traf-interacting with forkhead-
associated domain protein A (TIFA)
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1. Introduction
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Bacterial-liberated D-glycero-β-D-manno-heptose 1,7-biphosphate (HBP) was revealed to
function as a pathogen-associated molecular pattern (PAMP) by Gaudet et al.1 Once transported
into the mammalian cell cytoplasm, it promotes the alpha kinase 1 (ALPK1)-dependent
phosphorylation and oligomerization of the TRAF-interacting forkhead-associated domain-
containing protein A (TIFA), which leads to nuclear translocation of the transcription factor
nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB).1,2,3,4 Given the broad
function of NF-κB in immune activation, this discovery may be exploited for a broad variety of
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activities, ranging from the therapeutic stimulation of HIV from latency3 to immune modulation
for treatment of inflammatory diseases or cancer. A pure and plentiful supply of HBP would
facilitate these developments, leading to this development of a robust synthesis of HBP for
further biological testing.
Apart from the enzymatic biosynthesis of HBP,5 there has been two reports of its chemical
synthesis.6,7 There has, however, been much research regarding
D-glycero-D-heptose synthesis as
well as the phosphorylated heptoses. A well-established method involves elongating a
mannofuranose derivative using a Wittig methodology and performing an osmoylation to obtain
the C-5, C-6 new stereocenters.8,9,10,11,12,13 The isomerisation at C-6 from
L-glycero-D-manno-
heptose to
D-glycero-D-manno-heptose provides a possible avenue to the synthesis of HBP and
was recently reported in a 16-step synthesis with a 1.2% overall yield.6 Another publication
regarding the synthesis of HBP started from peracetylated
11-step synthesis and overall yield of 9.7%.7
D-glycero-D-manno-heptose, with an
As Inuke et al. and Borio et al. 6,7 discussed, β-anomers are often regarded as being challenging
to prepare, and reports of mannose phosphate β-anomers are sparse. Inuke et al.6 developed a
method utilizing dibenzylphosphate for this purpose, whereas another option for β-
phosphorylation are chloro-phosphates.7, 14, 15, 16, 17 In this note, an alternate synthesis to
glycero-β- -manno-heptose 1,7-biphosphate is described.
D-
D
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2. Results and discussion:
O
O
RO
OR
O
OR
OR
P
P
O
P
O
O
O
O
HO
PO
OH
O
OP
O
O
HO
HO
OH
PO
PO
O
HO
P
O
O O
O
O
OH
OP
1
2
3
HBP
Wittig
MeO
HO
HO
OH
O
O
O
O O
O
O O
OH
O
HO
OP
OP
D-mannose
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4
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Scheme 1: Retrosynthesis of HBP, 1.
The retrosynthesis of HBP is described in Scheme 1. Here, HBP could be generated after
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phosphorylation and deprotection of intermediate 2, this intermediate could then be made from
furanose 3 after deprotection. Intermediate 3, could be obtained from alkene 4 after subsequent
ester reduction, osmoylation and phosphorylation. Elongated alkene 4 could be obtained after a
Wittig reaction from aldehyde 5. Aldehyde 5 could be obtained from
D-mannose after protecting
group manipulation, deprotection of the acetonide on 5ˈand 6ˈand sodium periodate cleavage.
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Scheme 2. Synthesis of phosphorylated derivative 14. a. Acetone/FeCl3, 94% b. NaH, PMBCl,
DMF, 78% c. Acetic acid/water , 96% d. NaIO4, Acetone, Water e. Ph3PCHCOOMe, toluene,
60% over two steps f. DIBAL, DCM, 81% g. Tetrazole, Acetonitrile, Dibenzyl N,N-
dimethylphosphoramidite, then tBuOOH, 67% h. OsO4, NMMO, Dioxane, Water, 70%.
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Diacetonide 6 was formed from D
-mannose such as in Güzlek et al. and Brimacombe et al.10,11,12
A benzyl group was first used as anomeric protecting group. The following steps including the
5ˈ,6ˈ acetonide deprotection, oxidation, Wittig reaction and reduction of the ester went as
reported by Brimacombe et al.. The phosphorylation using diphenylphosphoryl chloride was
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also successful, however, hydrolysis of the phenyls in the oxidation step caused lesser yields.
Another avenue which involved the protection of the C-7 hydroxyl with a TDBPS protecting
group was thus attempted. This avenue was successful, however the challenge laid in
deprotecting the acetonide and the benzyl group. At first, different hydrogenation catalysts were
used such as Pd/C, Pd(OH)2, PdCl2 with or without acetic acid in presence of different solvents
such as MeOH, EtOH, Ethyl acetate to no avail. Unsuccessfully, an acidic cleavage was
attempted at different temperatures with different acids such as acetic acid, pTsOH, TFA and
different acid concentrations.
It was theorised that a PMB group would be easier to cleave than a benzyl, intermediate 7
(Scheme 2) was synthesised. The deprotection of the acetonide (yield = 96%) and periodate
cleavage generated satisfying yields, however, surprisingly ester 10 was obtained in only 40%
yield over two steps. This low yield was caused by partial addition of methanol on the C-5
carbon preferentially to form intermediate 11. NMR convincingly showed an additional methoxy
group without any traces of characteristic alkene signals at 6 ppm in the proton NMR spectra,
additionally a correlation in the HMBC spectra from H-5ˈ to one of the methoxy carbon was key
in determining that it was linked at C-5ˈ. Mass-spectrometry data also agreed with this
identification (m/z Calcd for C20H32NO8= 414.2 found=414.0). Using toluene, improved the
yield slightly over two steps (60%) and the product was reduced to form alcohol 12 (81% yield).
After the phosphorylation of alcohol 12 (67% yield), the osmoylation yielded the expected diol
14 in a 92% yield. A mixture of the
D-glycero-α- D-manno-heptofuranose and L-glycero-β-L-
gulo-heptofuranose was obtained (3:1) and further purified with 70% yield. The new
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stereocenters at C-5ˈ and C-6ˈ were assigned with confidence after comparing 14 with the
benzylated intermediate described by Güzlek.12
O
OBn
OBn
P
BnO
BnO
OBn
O
OBn
O
O
P
P
O
O
OH
AcO
j.
HO
AcO
i.
OAc
O
OAc
O
O
O O
AcO
AcO
AcO
AcO
OPMB
OAc
OH
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15
14
k.
NaO
BnO
ONa
OBn
O
P
P
O
O
O
HO
AcO
AcO
OH
OAc
l.
O
O
O
O
HO
O
O
HO
AcO
P
P
NaO
PhO
ONa
OPh
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Scheme 3. Synthesis of
D
-glycero-β-
D
-manno-heptose 1,7-biphosphate i. TFA/Water/DCM then
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Acetic anhydride/Pyridine in DMF, 85%, j. DIPEA, Ammonium acetate in DMF, quant. k.
DMAP, diphenyl phosphoryl chloride in DCM, 77% l. H2, PtO2 in MeOH then H2, Pd/C in
MeOH then Et3N/Water/MeOH, 78% then NaCl treatment and desalting.
A two step one pot deprotection of acetonide and PMB protecting group followed by acetylation
afforded peracetylated 15 in 85% yield over two steps (Scheme 3). The deacylation of the
anomeric acetate was performed under slightly basic conditions, and yielded to intermediate 16
in quantitative yields. Phosphorylation afforded anomer 17β in a 1:4 (α:β) ratio (77% yield).
Key in this step is to add DMAP sparingly as well as adding the diphenyl phosphoryl chloride
dropwise. Should one wish to obtain the alpha anomer preferentially, it is possible to add 5
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equivalents of DMAP and introduce the phosphoryl chloride at a quicker rate. Both compounds
16 and 17β NMR data were in accordance with previously published data.7 Global deprotection
of the purified β-anomer afforded HBP, 1 in a 78% yield and an overall yield of 8.2% with 15
steps. For a representative evaluation of HBP biological function, endotoxin free water was used
for the last purification step. It was also reported that 293T cells are refractory to LPS.1
The biological function of synthetic HBP was assessed based upon its ability to activate NF-κB
in human cells, using the non-activating β-D-mannose phosphate as a negative control. In a first
experiment, HBP was shown to activate an NF-κB-driven luciferase reporter construct in human
embryonic kidney-derived (HEK 293) cells (Figure 1). In another experiment, the inflammatory
cytokine IL-8 was produced by human colonic epithelial (HCT 116) cells exposed to HBP
(Figure 2). Both batches of HBP induced a similar response in both assays, highlighting the
reproducibility of our synthesis. Furthermore, the activity was comparable to that of our positive
control – bacterial supernatant from a genetic mutant of Neisseria meningitidis that naturally
expresses HBP (∆gmhB), and was greater than supernatant from a mutant of N. meningitidis that
does not produce HBP (∆hldA).
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Figure 1. Effect of synthetic or naturally-produced HBP preparations on HEK 293T cells
encoding an NF-κB-driven luciferase reporter gene.
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Figure 2. Inflammatory response of human colonic epithelial cells exposed to synthetic or
naturally-produced HBP preparations. EF H2O, endotoxin-free water (solvent); nd, not detected.
KO; TIFA knockout
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3. Conclusion
In conclusion, we describe herein a method to obtain D-glycero-β-D-manno-heptose 1,7-
biphosphate in 8.7% overall yield and 15 steps. In this synthesis, mannose is elongated using the
Wittig methodology where an alkene is formed. After reduction of the ester and
phosphorylation, oxidation of the alkene leads us to a protected furanose form of
D-glycero-D-
manno-heptose. HBP thus formed was tested for its activity and shown to activate NF-κB in
HEK 293 cells and induce the TIFA- and NF-κB-dependent production of IL-8 in HCT 116 cells.
This method is robust and will be useful to further determine how this molecule can be exploited
for a variety of biological applications.
4. Experimental
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4.1 General
All chemicals were purchased from Aldrich, Fisher Scientific or Alpha Aesar. They were used
without further purification. Proton and Carbon NMR spectra were measured on either a Varian
(1H, 500 MHz), Bruker (1H, 600 MHz, 13C 150 MHz, 31P 242 MHz) or Bruker (1H, 400 MHz, 13C
100 MHz, 31P 162 MHz) spectrometer reported with the solvent residual signal (CDCl3, 7.26
ppm for 1H and 77.36 ppm for 13C, D2O, 4.79 ppm for 1H and externally with dioxane (67.19
ppm) for 13C). Compounds were purified using a CombiFlash RF system and RediSep RF silica
columns. MS data were recorded on a SQ2 from waters and HRMS data were recorded on a
Ultima from Waters with the LC/MS Calibrant Mix from Agilent as internal standard.
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4.2 Biological Assays
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Synthetic compounds were resuspended in endotoxin-free water (EF H2O; Sigma, Oakville,
Ontario, Canada). HCT 116 cells were maintained in McCoy’s 5A medium and HEK 293T cells
were maintained in a DMEM (Dulbecco's Modified Eagle's medium) buffer. All media was
supplemented with 10% FBS and 1% glutamax. Neisseria meningitidis supernatants were
purified as previously described.1
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HEK 293T cells were transfected with a plasmid encoding an NF-κB-driven luciferase reporter.
After 24 hours, cells were stimulated for 20 minutes in permeabilization buffer (5 µg/mL
digitonin) in the presence of culture supernatant from N. meningitidis mutants with (∆gmhB) or
without (∆hldA) HBP, or with 20 µg/mL of synthetic compounds. Treatment was removed, and
cells were washed and incubated for 3.5 hours in complete medium Luciferase activity
determined using the Dual-Glo Luciferase Assay System (Promega) as previously described1 and
luminescence was measured using a luminometer (Cytation 5). Results are expressed as fold
increase relative to transfected, mock-treated cells. The results are means ± standard error mean
of technical triplicates.
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HCT 116 that were either wild type (WT) or deficient in TIFA protein expression (knockout,
KO)18 were stimulated for 20 minutes in permeabilization buffer (5 µg/mL digitonin) in the
presence of culture supernatant from N. meningitidis mutants with (∆gmhB) or without (∆hldA)
HBP, or with 10 µg/mL of synthetic compound. Treatment was removed, and cells were washed,
then incubated for 6 hours in complete media. Quantitative measurement of IL-8 levels in the
culture supernatant was then performed using an ELISA kit from BD Biosciences. The results
are means ± standard error mean of technical triplicates.
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4.3 Synthesis
4.3.1 1,2,3,4,6-penta-O-acetyl-7-O-[bis(benzyloxy)phosphoryl]-
heptopyranose) (15).
D-glycero- D-manno-
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Compound 14 (120 mg, 190 µmol) was stirred with DCM (5 mL) and water (1 mL) at 0°C. TFA
(5 mL) was then added and the reaction stirred for 1 hour. After few minutes a pink shade
appears, this transforms slowly into a purple shade as the reaction warms up to RT and observed
to be to completion on TLC (Rf = 0.14, ethyl acetate:hexane, 1:1, v:v). Concentration in vacuo of
the reaction mixture and co-evaporation with toluene (6 times) gave a yellow oil. The mixture
was dissolved in anhydrous DMF (200 µL), then anhydrous pyridine (1 mL) and acetic
anhydride (1 mL) were added and stirred for 16 hours at RT. DCM was added and washed with
NaHCO3 (sat. aq.) until a neutral pH was reached and then washed with brine. The organic layer
was dried over Na2SO4 and concentrated in vacuo, flash chromatography afforded compound 15
(1:0.6, α:β) in 85% yield (110 mg, 162 µmol). 1H NMR (600 MHz, CDCl3): δ= 7.4-7.3 (m, 10H,
Bn), 6.03 (d, JH1ˈα,H2ˈα = 1.9Hz, 1H, H1ˈα), 5.80 (d, JH1ˈβ,H2ˈβ = 1.4Hz, 1H, H1ˈβ), 5.41 (m, 1H,
H2ˈβ), 5.33-5.26 (m, 2H, H4ˈα and H3ˈα), 5.23-5.17 (m, 3H, H4ˈ β, H6ˈβ, H2ˈα), 5.13 (m, 1H,
H6ˈα), 5.07-4.96 (m, 5H, CH2Bn, H3ˈβ), 4.34-4.16 (m, 2H, H7ˈα and β), 4.08 (JH5ˈα,H4ˈα = 9.0Hz,
JH5ˈα,H6ˈα = 4.1Hz, dd, 1H, H5ˈα), 3.79 (JH5ˈβ,H4ˈβ = 9.1Hz, JH5ˈβ,H6ˈβ = 4.1Hz, dd, 1H, H5ˈβ), 2.15-
1.94 (m, 15H, CH3). 13C NMR (150 MHz, CDCl3): δ= 170.33, 170.19, 170.10, 170.0, 169.9,
168.5, 168.3 (CH3C(O)), 136.1, 136.0, 135.9 (CCH2O, Bn), 128.9, 128.8 (CBn), 128.2, 128.1,
128.0 (CHBn), 90.3 (C1ˈα and β), 74.0 (C5ˈβ), 71.4 (d, JC6ˈα,P = 7.9 Hz, C6ˈα), 71.2 (C5ˈα), 71.1
(C6ˈβ), 70.4 (C3ˈβ), 69.7 and 69.6 (CH2Ph), 69.0 (C3ˈα), 68.3 (C2ˈα), 67.8 (C2ˈβ), 66.8 (C4ˈα),
66.3 (C4ˈβ), 65.0 (d, JC7,P = 5.2 Hz, C7ˈ), 21.1, 21.0, 20.9, 20.8 (CH3C(O)); ESI-MS: m/z Calcd
for C31H37NaO15P [M+Na]+, 703.17; found, 703.17.
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4.3.2 2,3,4,6-tetra-O-acetyl-7-O-[bis(benzyloxy)phosphoryl]-
heptopyranose (16).
D -glycero- D-manno-
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Compound 15 (50 mg, 78 µmol) was dissolved into DMF (5 mL) and to this was added
diisopropylethylamine (1 mL) and ammonium acetate (200 mg) under a nitrogen atmosphere.
The reaction was stirred for 16 hours at RT after which TLC monitoring showed that the reaction
was to completion (Rf = 0.8, ethyl acetate:hexane, 1:1, v:v). The reaction mixture was diluted
with DCM, washed with NaHCO3 (sat. aq.) and water. The DCM fraction was then dried with
Na2SO4, filtered and concentrated in vacuo. Column chromatography afforded title compound
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16 in quantitative yield (47 mg, 74 µmol). H NMR (400 MHz, CDCl3): δ= 7.4-7.3 (m, 10H,
Bn), 5.45 (JH3ˈ,H2ˈ = 3.5Hz, JH3ˈ,H4ˈ = 9. Hz, dd, 1H, H3ˈ), 5.26 (JH2ˈ,H1ˈ = 1.7Hz, JH2ˈ,H3ˈ = 3.5Hz,
dd, 1H, H2ˈ), 5.22 (JH4ˈ,H3ˈ = 9.8 Hz, JH4ˈ,H5ˈ = 9.8 Hz, dd (apt), 1H, H4ˈ), 5.12 (d, JH1ˈ,H2ˈ
=
1.4Hz, 1H, H1ˈ), 5.06-5.00 (m, 5H, CH2Bn and H6ˈ), 4.48 and 4.09 (m, 1H, H7ˈ), 4.29 (JH5ˈ,H6ˈ
=
7.4Hz, JH5ˈ,H4ˈ = 9.8Hz, dd, 1H, H5ˈ), 2.11 (s, 3H, CH3COOCH2ˈ), 2.03 (s, 3H, CH3COOCH4ˈ),
2.01 and 2.00 (s, 3H, CH3COOCH3ˈ and CH3COOCH6ˈ). 13C NMR (100 MHz, CDCl3) δ=
170.3 (x2), 170.1 (x2) (C(O)), 135.8 (x2), 135.7 (x2) (CBn), 129.1, 129.0, 128.9 (x2) (CHBn),
92.7 (C1ˈ), 73.1 (JC6’,P = 5.8 Hz, d, C6ˈ), 70.3 (C2ˈ), 70.3-70.0 (CH2Bn), 69.3 (C3ˈ), 68.7 (C4ˈ),
66.1 (C5ˈ), 65.7 (JC6’,P = 5.7 Hz, C7ˈ), 21.2-20.9 (CH3). 31P NMR (161 MHz, CDCl3) δ -1.0;
HRMS m/z Calcd for C29H35NaO14P [M+Na]+ 661.1662, found 661.1683.
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4.3.3 Diphenyl (2,3,4,6-tetra-O-acetyl-[7-O-(bis[benzyloxy]phosphoryl)-
D-glycero-β-D-manno-
heptopyranosyl) phosphate (17β).
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Compound 16 (29.3 mg, 43 µmol) was coevaporated using toluene and dried under vacuum
overnight. After 16 hours, 16 was dissolved in anhydrous DCM (1 mL) and DMAP (5 mg, 43
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µmol) was added. Phosphoryl chloride (90 µL, 430 µmol) was also coevaporated with toluene
and was dissolved into DCM (1 mL) and using a syringe pump, dropped at a rate of 0.5 mL/h
over 2 hours under N2, the reaction was to completion after 5 hours. DCM was then added and
the organic layer was washed with TEAB buffer (until a basic pH was reached), water and brine.
The organic layer was then dried with Na2SO4, filtered and concentrated under reduced pressure.
Flash chromatography using hexane: diethyl ether gave title compound 17β in 77% yield (30 mg,
1
34 µmol, α:β, 1:4). H NMR, (400 MHz, CDCl3) δ= 7.4-7.1 (m, 2OH, Bn and Ph), 5.67 (JH1ˈ,H2ˈ
= 1.7Hz, JH1ˈ,P = 7.5Hz, dd, 1H, H1ˈ), 5.40 (JH2ˈ,H1ˈ = 1.7Hz, JH2ˈ,H3ˈ = 3.1Hz, dd, 1H, H2ˈ), 5.31
(m, 1H, H6ˈ), 5.21 (JH4ˈ,H3ˈ = JH4ˈ,H5ˈ = 7.9Hz, dd (apt), 1H, H4ˈ), 5.08 (JH3ˈ,H2ˈ = 3.1Hz, JH3ˈ,H4ˈ
=
7.9Hz, dd, 1H, H3ˈ), 5.08-5.01 (m, 4H, CH2Bn), 3.89 (JH5ˈ,H6ˈ = 5.8Hz, JH5ˈ,H4ˈ = 7.3Hz, dd, 1H,
H5ˈ), 4.24 (m, 2H, H7ˈ), 2.06, 2.02, 2.00, 1.98 (CH3). 13C NMR (100MHz, CDCl3) δ= 170.0,
169.9, 169.8, 169.7 (C(O)CH3 ), 150.5, 150.4, 150.3, 150.2 (C, Ph and Bn), 136.0, 135.9, 135.9,
135.8 (CH, Ph and Bn), 130.14, 130.10 (CH, OBn), 128.8 (m, CH, OBn), 128.12 and 128.09
(CH, OPh), 126.0 and 128.9 (CH, OPh), 120.34 (m, CH, OPh), 94.84 (2J31P, C1ˈ = 4.6Hz, C1ˈ),
73.3 (C5ˈ), 70.7 (3J31P, C6ˈ, = 7.2Hz, C6ˈ), 69.5 (m, CH2Bn), 69.1 (C3ˈ), 67.2 (3J31P, C2ˈ, = 7.3Hz,
C2ˈ), 66.2 (C4ˈ), 64.9 (2J31P, C7ˈ = 5.7Hz, C7ˈ), 20.8, 20.7, 20.5 (CH3). 31P NMR (161 MHz,
CDCl3) δ -0.36 and -13.27. HRMS m/z Calcd for C41H44NaO17P2 [M+Na]+ 893.1951, found
893.1942.
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4.3.4 D-glycero-D-manno-heptopyranose 1β,7-biphosphate (1, HBP-β).
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Compound 17β (6 mg, 6.9 µmol) was stirred in anhydrous methanol at RT with PtO2 under
balloon pressure for 48 hours. When activated, the brown PtO2 turns to black. After filtration
over celite and concentration, the compound was again dissolved in anhydrous methanol and
Pd/C was added. After 48 hours of stirring under a H2 atmosphere, filtration over celite and
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concentration under reduced pressure, the compound was dissolved into methanol:endotoxin free
water:triethylamine (7:3:1, v:v:v) at pH 11 for 4 hours after which it was concentrated and freeze
dried over water. Purification using endotoxin free water on a desalting column (G-10) and
exchanging the triethylamine ions for sodium ions gave compound 1, HBP-β in 78% yield (2
mg, 5.4 µmol). NMR assignments are in agreement with literature data.5,6 HRMS m/z Calcd for
C7H16NaO13P2, 392.9964, found, 392.9941.
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Acknowledgements:
We acknowledge Ken Chan and The-Minh Tu for measuring HRMS on the synthesised
compounds. We would like to thank also Evgueni Vinogradov, Wei Zou and Dean Williams for
valuable discussions.
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Financial Support:
This work was supported by Canadian Institutes of Health Research (CIHR) operating grant
HOP-137697 to SGO. Financial support from Science Foundation Ireland (Grant 13/IA/1959 to
S. O.) and a Marie Curie-Intra-European Fellowship (FP7-PEOPLE-2011-IEF, project number
299710 to L. G.) is gratefully acknowledged.
Declaration of interest:
The authors, with the exception of Milan Basin, Itunuoluwa A. Adekoya and Cynthia X. Guo,
have filed patents concerning the therapeutic potential of HBP.
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Highlights:
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•
Alternate synthesis to
The synthesis starts from mannose and uses Wittig methodology and osmoylation as means to
obtain -glycero- -manno heptose
Cell-based assays validate that synthetic
Nf-κB through a TIFA-dependent pathway
D-glycero-D-manno-heptose 1,7-diphosphate
D
D
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D-glycero-D-manno-heptose 1,7-diphosphate stimulates