Synthesis of zwitterionic 1,1′-glycosylphosphodiester: A partial structure of galactosamine-modified francisella lipid A

Article abstract of DOI:10.1021/ol501639c

Synthesis of a "double glycosidic" phosphodiester comprising anomeric centers of two 2-amino-2-deoxy-sugars is reported. The carbohydrate epitope of Francisella lipid A modified with α-d-galactosamine at the anomerically linked phosphate has been stereose

Full text of DOI:10.1021/ol501639c

Letter
pubs.acs.org/OrgLett
Synthesis of Zwitterionic 1,1′-Glycosylphosphodiester: A Partial
Structure of Galactosamine-Modified Francisella Lipid A
David Baum, Paul Kosma, and Alla Zamyatina*
Department of Chemistry, University of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, Austria
S
* Supporting Information
ABSTRACT: Synthesis of a “double glycosidic” phosphodiester comprising anomeric centers of two 2-amino-2-deoxy-sugars is
reported. The carbohydrate epitope of Francisella lipid A modified with α-D-galactosamine at the anomerically linked phosphate
has been stereoselectively prepared and coupled to maleimide-activated bovine serum albumin via an amide-linked thiol-
terminated spacer group. H-Phosphonate and phosphoramidite approaches have been explored for the coupling of 4,6-DTBS-2-
azido-protected GalN lactol and peracetylated spacer-equipped reducing βGlcN(1→6)GlcN disaccharide via phosphodiester
linkage. Deprotection conditions preserving the integrity of the labile glycosidic zwitterionic phosphodiester were elaborated.
Francisella is a highly infectious Gram-negative intracellular
zoonotic bacterium that can cause tularemia, an extremely
contagious lethal pulmonary disease in mammals.1 F. tularensis,
which is a causative agent of tularemia in humans, provokes
special anxiety due to its classification as a bioterrorism agent.2
Lipopolysaccharide (LPS) is one of the principal virulence
factors of Gram-negative bacteria, whereas the lipid A portion of
LPS is primarily responsible for eliciting the innate immune
response through Toll-like Receptor 4 (TLR4)−myeloid
differentiation-2 (MD-2) complex. The major lipid A of
Francisella has an unusual tetraacylated structure, which lacks
the 4′-phosphate and a 3′-acyl chain and contains an α-D-GalN
residue at the glycosidically connected 1-phosphate (Figure
1).3−6 The four subspecies of genus F. tularensis, highly infectious
4′-phosphate,10−12 though F. tularensis LPS was shown to
provide protection against LVS infection in mice.13 The
biological significance of GalN modification of Francisella lipid
A has not yet been fully clarified, though it is associated with
enhanced bacterial virulence. F. novicida mutants which are
deficient in GalN modification have been shown to have
attenuated pathogenicity in mice and are capable of stimulating
the innate immune system.14
One of the most studied Francisella strains, F. novicida, was
shown to possess a truncated lipopolysaccharide form composed
of 90% of the α-D-GalN-modified lipid A portion, which is
deprived of the core sugars and polymeric O-antigen.7,15 The
diglucosamine backbone of lipid A modified by α-D-GalN at the
1-phosphate is, in this instance, the exposed and, possibly, the
antigen-presenting part of LPS. A novel system of LPS
remodelling enzymes involving a bifunctional Kdo-hydrolase
has been implicated in the synthesis of the partially truncated
Francisella LPS structures.16−18 To assess the antigenic potential
of the GalN modification, a lipid A - based epitope βGlcN(1→6)-
αGlcN(1 →P←1)-αGalN which is conserved in all Francisella
strains, has been synthetically prepared and coupled to
maleimide-activated bovine serum albumin (BSA) via thiol-
terminated spacer group.
The majority of naturally occurring glycosidically - linked
phosphodiesters contain phosphoester bonds connecting one
anomeric and one nonanomeric hydroxyl group. The formation
of this type of phosphodiesters is generally carried out in
conjunction with the well-established phosphotriester, phos-
phoramidite or H-phosphonate methodologies.19−23 Herein we
report on the first synthesis of a 1,1′-glycosylphosphodiester
wherein the anomeric centers of two 2-amino-2-deoxy-sugars
Figure 1. Structure of GalN-modified lipid A from Francisella and a
related synthetic neoglycoconjugate.
type A strain (Schu S4, tularensis), less virulent type B strain
(holartica), attenuated type B strain, designated as live vaccine
strain (LVS), F. mediasiatica, and a nonvirulent laboratory strain
F. novicida share a similar lipid A structure having a GalN
modification at the 1-phosphate.3,7−9 Francisella LPS was shown
to be not recognized by TLR4/MD-2 complex which is
attributed to the hypoacylated structure of its lipid A lacking a
Received: June 6, 2014
© XXXX American Chemical Society
A
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Letter
(αGlcN and αGalN as in Francisella lipid A) are involved. The
assembly of such “double glycosidic” phosphodiester represents
a formidable synthetic challenge with respect to the requirements
for the anomeric stereocontrol and intrinsic instability of the 2-
amino-2-deoxy-glycosylphosphate derivatives under a variety of
chemical conditions.
excess of pyridine (over SalPCl) which afforded glycosyl-H-
phosphonate 5 (α/β = 4:1, according to the 1H NMR spectra of
the crude product), isolated with a higher proportion of the α−
anomer (α/β = 5:1) as the ammonium salt in 78% yield.
Thereafter, the β−(1→6)-linked diglucosamine 12, equipped
with a 6-thioacetylhexanoylamino spacer group at C-2′, as well as
its 2′-hexanoylamino counterpart 11 as a model compound, were
prepared (Scheme 2). To this end, the βGlcN(1→6)GlcN
Two classes of P(III) phosphorus compounds could serve as
intermediates in the synthesis of double anomeric phospho-
diesters, namely phosphoramidite (three-coordinated) or H-
phosphonate (tetra-coordinated). Both possess an electrophilic
phosphorus center capable of easily reacting with various
nucleophiles. The advantage of the phosphoramidite procedure
lies in the mildness of the phosphitylation and oxidation
conditions, whereas the instability of the intermediary glycosyl
phosphoramidites and glycosyl phosphites, which represent
perfect glycosyl donors,24 count as drawbacks. The merits of the
H-phosphonate methodology include substantial stability of the
intermediate glycosidic H-phosphonate monoesters as well as
the absence of protecting groups at phosphorus. However, the
oxidation of the H-phosphonate phosphodiesters into P(V)
species often requires harsh conditions which can eventually lead
to side-reactions. Herein we explored the applicability of both
methods toward the synthesis of a Francisella lipid A - related
epitope containing αGlcN(1→P←1)αGalN fragment.
Scheme 2. Preparation of (1→6) Diglucosamine Lactol
The synthesis of αGalN-H-phosphonate 5 commenced with
regioselective introduction of the 4,6-O-di-tert-butylsilylene
(DTBS) group into triol 125 to furnish compound 2 (Scheme
1). The DTBS group was expected to enhance the α/β ratio in
Scheme 1. Synthesis of the Glycosyl-H-phosphonate
disaccharide was assembled by a TMSOTf-assisted glycosylation
of the 6-OH acceptor 7a28 with peracetylated trichloroacetimi-
date donor 6.29 The acceptor was applied as a 4:1 mixture of
positional isomers, 4-O-acetyl-6-OH compound 7a and 4-OH-6-
O-acetyl compound 7b, which inevitably arise due to the known
propensity of the 4-O-acetyl group in glucopyranoses to undergo
(4→6) migration in a variety of chemical conditions.30 Owing to
the diminished reactivity of 7b compared with 7a in a (1→6)
glycosylation, the target (1→6) disaccharide 8 could be
efficiently prepared and readily isolated in 63% yield. Reductive
cleavage of the 2′N-Troc protecting group with Zn in hexanoic
acid followed by addition of N,N′-diisopropylcarbodiimide
(DIC) afforded amide 9. To install the thiol-terminated spacer,
the 2′N-Troc group was reduced by Zn in acetic acid, the
resulting amine was in situ acylated with 6-thioacetyl-hexanoic
acid/DIC which afforded compound 10. Anomeric deprotection
by isomerization of allyl- to propenyl- group, followed by
hydrolysis with aqueous I2 provided anomeric lactols 11 (α/β =
2.4:1) and 12 (isolated with a high preponderance of α-anomer,
α/β = 10:1).
To explore the stereoselectivity and efficiency of phosphity-
lation with GalN-H-phosphonate 5, a pivaloyl chloride (PivCl)-
mediated coupling of 5 to the thiol-free model compound 11 was
first performed (Scheme 3). A diluted solution of PivCl had to be
gradually added to the reaction mixture to avoid a formation of P-
acyl byproducts and possible over-reaction of the intermediate
H-phosphonate phosphodiester with an excess of PivCl.31
The H-phosphonate coupling was monitored by 31P NMR
(162 MHz, CDCl3) spectroscopy which indicated gradual
the anomeric lactol 4, since this cyclic protection has been shown
to exert a remote α-directing effect in the glycosylation reactions
involving 4,6-O-DTBS-protected galactose donors.26 Next, the
3-OH group in compound 2 was protected by reaction with
TBDMS-chloride in DMF in the presence of imidazole to give 3.
N-Bromo-succinimide-mediated hydrolysis of the thioethyl
glycoside provided the anomeric lactol 4 as a mixture of anomers
(α/β = 2(3):1). Since the stereoselectivity of the ensuing
phosphitylation with P(III) reagents generally depends on the α/
β ratio in the starting hemiacetal, we attempted to increase the
proportion of the α-configured lactol in 4 by in situ
anomerisation. Experiments in the NMR tube confirmed that
lactol 4 could be enriched in the α-anomer under acidic
conditions. Therefore, a solution of 4 in chloroform was treated
with triethylammonium formate - formic acid buffer (pH 5) for
12 h, followed by the traceless removal of buffer by coevaporation
with toluene. The reaction of 4 (α/β = 4:1) with 2-chloro-1,3,2-
benzodioxaphosphorin-4-one (salicyl-chlorophosphite,
SalPCl)27 was performed in DCM in the presence of a 2-fold
disappearance of the H-phosphonate 5 (δ: 1.06 ppm, JP−H
=
B
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Letter
mixture, (R),(S)-diastereomers at phosphorus) was prepared in
situ by treatment of GalN lactol 4 with N,N-diisopropyl
cyanoethylchlorophosphite in the presence of DIPEA.32 1H-
Tetrazole-mediated coupling of the latter to lactol 12 (α/β =
10:1) afforded a mixture of the intermediate anomeric
phosphite−triesters (31P NMR, major products δ, ppm: 138.9,
138.7). After in situ oxidation with tert-butyl hydroperoxide
(80% in di-tert-butyl peroxide) and treatment with Et3N to
remove the cyanoethyl protecting group from the phospho-
triester by β-elimination, extensive purification on silica gel
furnished the target phosphodiester 17 in 24% yield. This poor
outcome could be explained by the necessity to perform four
sequential transformations as a “one-pot” procedure owing to the
inherent instability and the impossibility of isolation of the
glycosyl phosphite/phosphate triester intermediates, such that
the residual reagents could become involved in side-reactions
during each following step.
Scheme 3. Assembly of the “Double Anomeric”
Phosphodiester Entailing Two 2-Amino-2-deoxy-sugars
Stepwise deprotection of 17 involved treatment with HF·Py to
remove the silyl protecting groups, reduction of the azido group
in 19 and final deacetylation to afford 21 (Scheme 4). The
presence of the terminal thiol in 19 precluded application of the
Pd-catalyzed hydrogenation for the reduction of the azido group,
so that the alternative procedures were investigated. Classical
Staudinger conditions did not result in any transformation,
apparently due to the steric inaccessibility of the 2″-N3-group
adjacent to the α,α-(1↔1)-phosphodiester linkage. Application
of the less sterically demanding trimethylphosphine (instead of
PPh3) in THF/aq NaOH33 was unsuccessful as well. Treatment
Scheme 4. Synthesis of βGlcN(1→6)-αGlcN(1→P←1)-
αGalN Epitope and BSA Conjugate
640 Hz) and the formation of the intermediate H-phosphonate
diesters (δ: 4.21 ppm, JP−H = 737 Hz and δ: 4.32 ppm, JP−H = 728
Hz for the 1-α- and 1-β-configured compounds, respectively,
both diastereomers at phosphorus). Oxidation by aqueous I2 in
pyridine at −15 °C and successive isolation by silica gel
chromatography afforded anomerically pure 13 as ammonium
salt in 53% yield. The α,α-(1→P←1) anomeric configuration
was confirmed by the downfield shifts of the H-1 (GlcN→P) and
H-1″ (GalN→P) signals and by J1,2 and J1,P coupling constants
(J1,2 = 3.5 Hz, J1,P = 7.3 Hz and J1″,2″ = 3.2 Hz, J1″,P = 7.9 Hz).
Desilylation of the GalN moiety by treatment with HF·Py
provided triol 14 which was subsequently deacetylated at pH
11.5 (MeOH-H2O-Et3N) to afford a “double anomeric”
phosphodiester 15 possessing an azido group at the GalN unit.
Hydrogenation over Pd/C in methanol followed by purification
by chromatography on Sephadex LH-20 resulted in a
quantitative formation of the zwitterionic trisaccharide 16. The
NMR spectra were fully assigned by 1D and 2D-spectroscopy
and were in excellent agreement with the data reported for the
carbohydrate backbone of Francisella lipid A isolated by alkaline
hydrolysis,15 thus confirming the structure of the α-GalN(1→P)-
modified diglucosamine backbone.
The same methodology was successfully employed for the
synthesis of the α,α-(1→P←1) trisaccharide 17 equipped with
an acetyl-protected sulfhydryl-containing spacer (Scheme 4).
Application of the nearly pure α-anomeric form (α/β = 10:1) of
the (1→6) diglucosamine lactol 12 allowed an enhancement of
the yield of the H-phosphonate coupling up to 85%.
Alternatively, to exploit the merits of the phosphoramidite
procedure, the N,N-diisopropyl-2-cyanoethyl phosphoramidite
18 (31P NMR, δ, ppm: 151.2, 150.5, 150.4, 149.0, α/β anomeric
C
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of 19 with a reducing tin(II) reagent, [Et3NH][Sn(SPh)3]
complex,34,35 resulted in a clean reduction of the 2″-N3-group
into the amino group. Prolonged reaction times in the presence
of an excess of tin(II) reagent caused a loss of GalN moiety,
presumably by a nucleophilic attack of the newly formed 2″-
NH2-group on the adjacent 1″-phosphate. To suppress this side
reaction, an excess of the tin(II) complex was trapped by the
treatment with the chelating agent, ethylenediaminetetraacetic
acid (EDTA), immediately after the N3-reduction was
completed. Final deacetylation was performed under basic
conditions (50% aqueous NH2OH) to afford, after isolation on
Superdex Peptide column, a zwitterionic phosphodiester 21 (as a
mixture with disulfide 20) in 44% yield. Reduction of the
disulfide bond in 20 was performed by tris(2-carboxyethyl)-
phosphine (TCEP)36 and monitored by H and HSQC NMR.
1
Coupling of 21 to a maleimide-activated BSA provided the
βGlcN(1→6)-αGlcN(1→P←1)-αGalN-containing neoglyco-
conjugate comprising up to an average of 13 pseudotrisaccharide
units per BSA molecule (according to MALDI-TOF mass
spectrometry data).
The unique structure of GalN-modified Francisella lipid A
renders a “double anomeric” phosphodiester αGlcN(1→P←
1)αGalN an attractive synthetic target. The lipid A-based
neoglycoconjugate containing an epitope βGlcN(1→6)αGlcN-
(1→P←1)αGalN, which is conserved in all Francisella strains,
might be of considerable use for the generation of specific
diagnostic antibodies which could be employed in immunoaf-
finity diagnostic assays for the prompt detection of Francisella
infection by the direct antigen determination in medical
samples.37 The epitope can potentially be attached to different
surfaces via its thiol-terminated spacer and utilized in diagnostic
immunoassays as a capture antigen.
(25) Paulsen, H.; Rauwald, W.; Weichert, U. Liebigs Ann. Chem. 1988,
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ASSOCIATED CONTENT
■
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* Supporting Information
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1
Experimental procedures and H, 13C, and 31P NMR spectra
(partially HSQC/COSY spectra) of all new compounds. This
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acs.org.
(30) Albert, R.; Dax, K.; Stutz, A. E.; Weidmann, H. J. Carbohydr. Chem.
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AUTHOR INFORMATION
■
(31) Froehler, B. C.; Matteucci, M. D. Tetrahedron Lett. 1986, 4, 469.
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Corresponding Author
*E-mail: alla.zamyatina@boku.ac.at.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Financial support by the Austrian Science Fund (FWF) (Grant
No. P-21276) is gratefully acknowledged.
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