Chemical synthesis of the innate immune modulator – bacterial D-glycero-β-D-manno-heptose-1,7-bisphosphate (HBP)

Article abstract of DOI:10.1016/j.tetlet.2017.06.014

The bacterial metabolite and potent innate immune modulator D-glycero-β-D-manno-heptose-1,7-bisphosphate (HBP) and its α-configured counterpart D-glycero-α-D-manno-heptose-1,7-bisphosphate were synthesized via stereoselective anomeric phosphorylation of the peracetylated D,D-heptose 7-dibenzylphosphate by exploiting different nucleophilicity of equatorial and axial lactols in the D-manno-series. We also report a novel approach for anomeric phosphorylation using modified Mitsunobu reaction conditions and provide the first full structural characterization of HBP. The first chemical synthesis of HBP offers access to an anomerically pure structurally defined probe for biological studies and to a lead compound operating as a powerful stimulator of intracellular signaling for possible therapeutic immunomodulation.

Full text of DOI:10.1016/j.tetlet.2017.06.014

Tetrahedron Letters 58 (2017) 2826–2829
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Chemical synthesis of the innate immune modulator – bacterial
D-glycero-b-D-manno-heptose-1,7-bisphosphate (HBP)
⇑
Alessio Borio, Andreas Hofinger, Paul Kosma, Alla Zamyatina
Department of Chemistry, University of Natural Recourses and Life Sciences, Muthgasse 18, 1190 Vienna, Austria
a r t i c l e i n f o
a b s t r a c t
Article history:
The bacterial metabolite and potent innate immune modulator
D
-glycero-b-
-manno-heptose-1,7-bisphosphate were
synthesized via stereoselective anomeric phosphorylation of the peracetylated -heptose 7-diben-
zylphosphate by exploiting different nucleophilicity of equatorial and axial lactols in the -manno-series.
D-manno-heptose-1,7-bispho-
Received 24 April 2017
Revised 5 June 2017
Accepted 5 June 2017
Available online 13 June 2017
sphate (HBP) and its
a-configured counterpart D-glycero-a-D
D,D
D
We also report a novel approach for anomeric phosphorylation using modified Mitsunobu reaction con-
ditions and provide the first full structural characterization of HBP. The first chemical synthesis of HBP
offers access to an anomerically pure structurally defined probe for biological studies and to a lead
Keywords:
Carbohydrates
Anomeric phosphate
Phosphorylation
Mitsunobu reaction
Lipopolysaccharide
compound operating as
immunomodulation.
a
powerful stimulator of intracellular signaling for possible therapeutic
Ó 2017 Elsevier Ltd. All rights reserved.
Introduction
pyroptosis. The downstream signaling induced by NLRP3
inflammasome is associated with a number of acute and chronic
inflammatory disorders such as sepsis syndrome, diabetes,
atherosclerosis and Alzheimer disease related pathology.6–8 It has
been recently shown that NLRP3 inflammasome can be assembled
The innate immune response is the ‘‘first line of defense”
against microbial challenge and is engaged to recognize highly
conserved pathogen motifs, the so-called Pathogen Associated
Molecular Patterns (PAMPs) which are typically represented by
microbial surface antigens. For example, lipopolysaccharide (LPS),
a major constituent of the outer membrane of the cell-wall of
Gram-negative bacteria, can potently stimulate the mammalian
innate immune system via interaction with a transmembrane
Toll-like Receptor 4 (TLR4) complex which results in the triggering
of different intracellular signaling pathways leading to production
of pro-inflammatory mediators (cytokines and interleukins).1–3
In addition, a number of severe inflammatory effects are
induced by LPS-mediated activation of an atypical inflammasome,
a protein complex that is assembled in the cytosol of macrophages
in response to extracellular stimuli. Inflammasome promotes the
processing and secretion of inflammatory cytokines of the inter-
leukin-1 (IL-1) family which are critical for anti-bacterial
defense.4,5 The assembly and activation of the nucleotide oligomer-
ization domain-like (NOD) receptor family pyrin domain-contain-
ing protein 3 (NLRP3) inflammasome constitutes a principal
mechanism by which persistent inflammatory stress leads to
induction of the IL-1b pathway and programmed cell death by
and efficiently activated by TNF-
acting protein with a forkhead-associated domain (TIFA).9
Recent reports disclosed that TIFA-dependent NF B activation
resulting in the expression of cytokines is induced by the
Gram-negative bacterial metabolite -glycero-b- -manno-heptose
1,7-bisphosphate (HBP).10,11 Glycero-manno-heptose,
seven
a receptor-associated factor-inter-
j
D
D
a
carbon sugar of purely nonself origin, is an integral component of
the inner core region of LPS.12 Heptoses can be also sometimes
found in LPS outer core regions, in O-antigens and as constituents
of capsular polysaccharides and S-layer glycoproteins.13 The
a
-anomer of HBP,
D
-glycero-
a
-
D
-manno-heptose 1,7-bisphosphate,
-glycero- -manno-
is formed as an intermediate in the GDP-
D
a-D
heptose pathway during S-layer glycoprotein and capsular
polysaccharide biosynthesis, whereas its counterpart having an
anomeric phosphate group in the b-configuration,
D
-glycero-b-
D-
manno-heptose 1,7-bisphosphate, is generated during the ADP-
L-
glycero-b-D-manno-heptose biosynthetic pathway involved in the
assembly of LPS.14–16
HBP is currently available only in limited quantities as a hetero-
geneous enzymatic preparation which is poorly characterized.17 In
order to provide well-defined and pure material for biological
studies and to ascertain
immuno-modulator for potential pharmaceutical application, we
a reliable approach to HBP as an
⇑
Corresponding author.
E-mail address: alla.zamyatina@boku.ac.at (A. Zamyatina).
http://dx.doi.org/10.1016/j.tetlet.2017.06.014
0040-4039/Ó 2017 Elsevier Ltd. All rights reserved.

A. Borio et al. / Tetrahedron Letters 58 (2017) 2826–2829
2827
have developed a facile chemical synthesis of
D-glycero-b-D-manno-
heptose 1,7-bisphosphate 1 and its
a-configured counterpart 2
(Fig. 1).
The synthesis of anomeric phosphates is challenging with
respect to the requirements for strict anomeric stereocontrol and
the intrinsic instability of the intermediate glycosyl phosphotri-
esters, representing perfect leaving groups, which renders the
option of anomeric phosphorylation under glycosylation condi-
tions unreliable. Two major types of phosphorus compounds could
serve as intermediates in the synthesis of anomeric phosphates,
namely P(III)-based reagents such as phosphoramidites or P(V)-
derived chloroanhydrides of protected phosphoric acid. Upon
application of reagents of these types, the stereoselectivity of
anomeric phosphitylation/phosphorylation reflects the initial
a/b
ratio in the starting hemiacetal, unless the anomeric ratio is
manipulated in situ. The advantage of the phosphoramidite proce-
dure lies in the mildness of the phosphitylation conditions, though
an additional oxidation step is needed to produce a P(V)-phos-
phate. The reaction of a nucleophilic component with a phospho-
ramidite is catalyzed by 1H-tetrazole which generates an
extremely unstable P-N tetrazolide species that instantaneously
reacts with nucleophiles (e.g. alcohols, lactols) making the slow
process of in-situ anomerisation unsuitable. The merit of the P(V)
phosphotriester methodology involves bypassing of the additional
oxidation step along with relative stability and lower reactivity of
protected chlorophosphates which could allow for in-situ anomeri-
sation. Harsher reaction conditions and a necessity to use a strong
base count as drawbacks.
In protected synthetic heptose hemiacetal intermediates having
manno-configuration, the anomeric ratio is strongly shifted in favor
of the a-lactol such that the synthesis of the b-configured glycosyl
phosphate requires either in-situ anomerisation or inversion of
configuration. We considered the latter option particularly attrac-
tive and turned our attention to modified Mitsunobu reaction con-
ditions which entail application of phosphoric acid derivatives
instead of the classic carboxylic acid. Though such conditions were
successfully applied for the synthesis of sugar phosphates, only
primary hydroxyl groups were examined.18–21
Scheme 1. Synthesis of protected D,D-heptose 7-phosphate 10.
chromatography on silica gel in 53% and 43% yield, respectively.
To avoid acetyl migration, heptopyranoside 8 was immediately
subjected to further reaction with N,N-diisopropyl-bisbenzyl phos-
phoramidite in the presence of 1H-tetrazole followed by in-situ
oxidation with meta-chloroperbenzoic (m-CPBA) acid at ꢀ60 °C
to provide protected 7-O-phosphate 9 in 65% yield. Thiophenyl gly-
coside 9 was anomerically deprotected with N-bromosuccinimide
in aqueous acetone to furnish lactol 10 (a/b = 9:1).
To explore the stereoselectivity and efficiency of the anomeric
phosphorylation under Mitsunobu reaction conditions, we applied
peracetylated mannose hemiacetal 11 as a model compound.
When the transformation was performed under ‘‘classic condi-
tions” using diethyl azodicarboxylate (DEAD) and PPh3 in THF at
reduced temperature (ꢀ30 to 0 °C) with dibenzylphosphate as an
Our synthesis commenced with the preparation of peracety-
lated thiophenyl glycoside 4 by the Lewis acid catalysed reaction
of the D,D-heptose peracetate 3 with thiophenol which gave a mix-
ture of anomeric products (92%,
graphic separation of the
a
/b = 12:1) followed by chromato-
a
-thioglycoside (Scheme 1).
4
Deacetylation with aqueous methanolic Et3N provided 5 in quanti-
tative yield. Next, the primary OH group in position 7 was regios-
electively silylated by reaction with triisopropylsilyl chloride in
pyridine in the presence of 4,40-dimethylaminopyridine (DMAP)
to give 6 which was peracetylated to furnish 7. Cleavage of the TIPS
protecting group was performed under mild conditions with a
diluted solution of HF in pyridine at ꢀ20 °C to suppress acetate
group migration. The reaction was stopped prior to appearance
of the migration by-products (30 min); the target primary
alcohol 8 and unreacted 7 were separated by flash column
Fig. 1. Structure of
D
-glycero-b-
D
-manno-heptose 1,7-bisphosphate 1 (HBP) and its
Scheme 2. Anomeric phosphorylation under modified Mitsunobu reaction
a-configured counterpart 2.
conditions.

2828
A. Borio et al. / Tetrahedron Letters 58 (2017) 2826–2829
Scheme 3. Stereoselective synthesis of D-b-D-heptose 1,7-bisphosphate 1 and D-a-D-heptose 1,7-bisphosphate 2.
acid component, only minor reaction progress could be detected
(Scheme 2). Using diisopropyl azodicarboxylate (DIAD) and pyri-
dine to accelerate deprotonation of the nucleophile (lactol 11)
improved the overall outcome resulting in full conversion, though
tosyl-1-phosphate from the relevant lactol can be manipulated by
the rate of addition of phosphorylating reagent.23,24 The higher
nucleophilicity of the b-manno-configured lactol ensures its higher
reaction rates with electrophiles (P-activated phosphates or phos-
phites). As soon as the faster reacting b-lactol is consumed by a
limited amount of the gradually added phosphorylating reagent,
the anomerisation process toward b-anomer supported by an
with unsatisfactory stereoselectivity 13/12 (a/b) = 4:1. The preva-
lence of the -phosphate 13 insinuated participation of a different
a
reaction mechanism, presumably, an acylation with the activated
dibenzylphosphate leading to phosphorylation without inversion
of configuration. Accordingly, to circumvent the latter event, we
avoided using pyridine and performed further trials using THF as
the reaction solvent.22 The best reaction outcome was achieved
by pre-forming the betaine (PPh3 was mixed with DIAD in THF
for 10 min) followed by addition of the nucleophilic component
lactol 11 and gradual addition (over 3 h) of dibenzylphosphate. Fol-
lowing the reaction progress by 1H and 31P NMR revealed that the
b-phosphate 12 was formed as a major product in the first 20 min
of reaction (ca. 40% overall conversion) followed by ensuing slow
excess of DMAP sets in. In this manner, different b-configured D-
manno-1-phosphates could be prepared in exceptionally high
yields.24–26 Along these lines, the in-situ anomerisation of lactol
10 in favor of the more nucleophilic b-configured anomer was
achieved by treatment with excess of DMAP. Slow continuous
addition of a diluted DCM solution of diphenyl phosphorochlori-
date (DPCP, 0.5 equiv/h) to a solution of 10 resulted in a preferen-
tial formation of a kinetically controlled product, b-heptosyl
phosphotriester 16 (a/b = 1:4), which was isolated in 51% yield
(Scheme 3). The somewhat lower yield compared to the
formation of the
a
-phosphate 13 indicating a switch from the Mit-
b-manno-phosphorylation outcome (85%) in our previous synthesis
sunobu reaction mechanism to other competing processes (appar-
ently, SN1 reaction or concurrent formation of an oxocarbenium
ion which is then trapped by the nucleophile dibenzylphosphate).
To gain deeper insight into the mechanistic backgrounds for the
poor stereoselective outcome, we applied the conditions described
above for phosphorylation of the secondary OH groups in GlcN
derivatives 14 and 15 which was anticipated to proceed with
inversion of configuration. The expected products could not be
detected, demonstrating the involvement of a different reaction
mechanism in the anomeric b-manno-phosphorylation under mod-
ified Mitsunobu reaction conditions. Because of unsatisfactory
of ADP-D-glycero-b-D
-manno-heptose24 could be explained by a
remote influence of the bulky 7-O-dibenzylphosphate group which
shields the anomeric position, hindering access from the b-face.
Also the separation of 16 from the residual a-anomer 17 was less
efficient due to a smaller difference in Rf values inflicted by the
7-phospate substituent. By using the phosphoramidite approach
for the phosphitylation of 10, the equilibrium could be
shifted toward formation of the thermodynamically preferred
a-phosphite, which, after oxidation with m-CPBA and separation
by column chromatography, furnished -heptosyl phosphotriester
a
19 as a major product. Phenyl protecting groups at phosphorus in
diphosphates 16 and 17 were readily cleaved by catalytic
hydrogenation in the presence of PtO2, followed by complete
stereoselectivity 13/12 (a/b) = 1:1, the Mitsunobu reaction condi-
tions were no longer pursued.
Thus, our stereoselective synthesis of
tose 1,7-bisphosphate 1 was based on our previous finding
disclosing that the anomeric ratio in the synthesis of D-manno-hep-
D
-glycero-b-
D
-manno-hep-
deacetylation with MeOH/H2O/Et3N (7:3:1) which afforded
heptose 1,7-bisphosphate 1 and -heptose 1,7-bisphosphate
2. The assignment of the anomeric configuration in the
D-b-D-
D-a-D
Table 1
1H and 13C NMR data for HBP 1 and 2.
Proton/carbon
H-1 ppm/3J Hz
C-1 ppm/2J Hz
H-2 ppm/3J Hz
C-2 ppm/2J Hz
H-3 ppm/3J Hz
C-3 ppm/2J Hz
H-4 ppm/3J Hz
C-4 ppm/2J Hz
H-5 ppm/3J Hz
C-5 ppm/2J Hz
H-6 ppm/3J Hz
C-6 ppm/2J Hz
H-7a ppm/3J Hz
C-7 ppm/2J Hz
H-7b ppm
3.92 (m)
1
5.05 (d)
3.91 (m)
3.60(dd)
3J2,3 = 3.2
3J3,4 = 9.8
72.84
3.67 (t)
3.44 (dd)
4.11 (dt)
3J7b,6 = 7.4
3J7a,6 = 3.5
70.89
4.02 (ddd)
2J7a,7b = 11.1
3J7a,P = 6.6
65.41
1H NMR
3J1,P = 8.5
3J4,5 = 9.8
3J5,6 = 3.1
1
95.54
70.57
67.06
76.73
13C NMR
2
2J1,P = 4.4
5.36 (dd)
3J1,2 = 1.9
3J1,P = 8.0
96.36
3J2,P = 7.7
3.93 (m)
3J6,P = 6.6
4.14 (dt)
3J7a,6 = 7.6
2J7,P = 5.5
4.05 (ddd)
2J7a,7b = 10.9
3J7a,P = 6.5
66.25
3.85 (dd)
3J2,3 = 3.3
3J3,4 = 9.5
70.79
3.79 (t)
3.88 (dd)
3.93 (m)
1H NMR
3J4,5 = 9.8
3J5,6 = 2.9
3J5,6
71.45
3J6,P = 7.7
=
3J7b,6 = 3.3
2
71.02
67.62
74.00
13C NMR
2J1,P = 5.2
3J2,P = 8.8
2J7,P = 4.4

A. Borio et al. / Tetrahedron Letters 58 (2017) 2826–2829
2829
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In conclusion, we describe the first chemical synthesis of the
innate immune modulator,
D-b-D-heptose 1,7-bisphosphate
(HBP), which was efficiently prepared by application of stereose-
lective b-manno-phosphorylation and isolated in anomerically
pure form. We show that anomeric phosphorylation under modi-
fied Mitsunobu reaction conditions proceeds with only partial
inversion of configuration and undergoes different reaction mech-
anism which needs to be further investigated. Biological studies
applying D-b-D- and D-a-D-heptose 1,7-bisphosphates 1 and 2 will
be published elsewhere.
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