Stereoselective synthesis of α- and β-L-Ara4N glycosyl H-phosphonates and a neoglycoconjugate comprising glycosyl phosphodiester linked β-L-Ara4N

Article abstract of DOI:10.1021/acs.orglett.6b03358

Stereoselective synthesis of variably protected α- and β-L-Ara4N glycosyl H-phosphonates as key intermediates in the syntheses of β-L-Ara4N-modified LPS structures and α-L-Ara4N-containing biosynthetic precursors is reported. A facile one-pot approach toward β-L-Ara4N glycosyl H-phosphonates includes anomeric deallylation of protected 4-azido β-L-Ara4N via terminal olefin isomerization followed by ozonolysis and methanolysis of formyl groups to furnish exclusively β-configured lactols that are phosphitylated with retention of configuration. The carbohydrate epitope of β-L-Ara4N-modified Lipid A, βGlcN(1→6)αGlcN(1→P←1)β-L-Ara4N, was stereoselectively synthesized and linked to maleimide-activated bovine serum albumin.

Full text of DOI:10.1021/acs.orglett.6b03358

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Letter
pubs.acs.org/OrgLett
Stereoselective Synthesis of α- and β‑L‑Ara4N Glycosyl
H‑Phosphonates and a Neoglycoconjugate Comprising Glycosyl
Phosphodiester Linked β‑L‑Ara4N
Ralph Hollaus, 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: Stereoselective synthesis of variably protected
α- and β-L-Ara4N glycosyl H-phosphonates as key inter-
mediates in the syntheses of β-L-Ara4N-modified LPS
structures and α-L-Ara4N-containing biosynthetic precursors
is reported. A facile one-pot approach toward β-L-Ara4N
glycosyl H-phosphonates includes anomeric deallylation of
protected 4-azido β-L-Ara4N via terminal olefin isomerization
followed by ozonolysis and methanolysis of formyl groups to
furnish exclusively β-configured lactols that are phosphitylated
with retention of configuration. The carbohydrate epitope of β-L-Ara4N-modified Lipid A, βGlcN(1→6)αGlcN(1→P←1)β-L-
Ara4N, was stereoselectively synthesized and linked to maleimide-activated bovine serum albumin.
odification of the terminal portion of lipopolysaccharide
(LPS), a glycophospholipid Lipid A, with 4-amino-4-
deoxy-β-L-arabinose (L-Ara4N) is an adaptive mechanism that
attributed to incorporation of L-Ara4N into the Lipid A moiety
3,4,10
M
of LPS.
L-Ara4N biosynthesis occurs in the cytoplasm,
whereas in the final step, L-Ara4N is delivered by the long-chain
enables Gram-negative bacteria to resist recognition by the
isoprene lipid carrier undecaprenyl phosphate (UndP) to the
1
,2
11
components of the host immune system. Inducible addition of
L-Ara4N to at least one of the phosphate groups of Lipid A (at the
periplasmic face of the inner bacterial membrane. There, a
membrane lipid-to-lipid glycosyltransferase ArnT catalyzes
1
and/or 4′ positions) in Escherichia coli, Salmonella enterica
transfer of L-Ara4N from undecaprenylphosphate-α-L-Ara4N to
11,12
serovar Typhimurium, Yersinia pestis, Pseudomonas aeruginosa,
and Burkholderia cepacia complex is required for bacterial
viability and largely contributes to antibiotic resistance and
the phosphate groups of Lipid A.
ArnT family is the last
enzyme in the Ara4N biosynthesis pathway in Gram-negative
bacteria and is thus an attractive target for development of
antibacterial agents affecting LPS biosynthesis, which neces-
sitates a synthetic access to α-L-Ara4N-containing UndP
3
−9
bacterial virulence (Figure 1).
Inducible addition of cationic
13
derivatives.
Ara4N-modified LPS structures can hardly be obtained in pure
form by isolation from bacterial sources, due to the inherent
lability of the anomeric phosphodiester functionality. To clarify
immuno-modulating and immunogenic potential of the Ara4N
modification, a reliable synthetic approach toward β-L-Ara4N
glycosyl phosphate-containing LPS partial structures is highly
desirable. The Lipid A-based neoglycoconjugate, containing
conserved epitope βGlcN(1→6)αGlcN(1→P←1)β-L-Ara4N of
highly virulent Gram-negative human pathogens, is an important
antigen that could be applied to help generate specific
monoclonal antibodies. Such mAbs could be used in diagnostic
immunoaffinity assays for rapid antigen determination in clinical
samples and applied to screen not-yet-identified Ara4N-
producing mutants in, for example, Y. pestis flea infection
Figure 1. Structure of β-L-Ara4N-modified Lipid A and a biosynthetic
precursor undecaprenyl phosphate-α-L-Ara4N.
sugar L-Ara4N reduces the net negative charge of the bacterial
membrane, which protects it from recognition by the cationic
antimicrobial peptides (CAMPs) that comprise the foremost
component of the innate immune response at epithelial surfaces.
Occurrence of profound resistance to exogenous CAMP
polymyxin B, a “last line of defence” antibiotic for the treatment
of multi-drug-resistant Gram-negative infections, is also
6
models.
In contrast to the abundantly prevailing phosphodiester bonds
connecting one anomeric and one non-anomeric sugar hydroxyl
Received: November 9, 2016
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.6b03358
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Organic Letters
Letter
group, the L-Ara4N-modified Lipid A involves a glycosyl
phosphodiester linkage connecting anomeric centers of amino-
sugars Ara4N and GlcN (Figure 1). Assembly of such a binary
glycosyl phosphodiester requires both rigorous anomeric
stereocontrol and very mild reaction conditions that allow for
preservation of the labile glycosyl phosphoester intermediates.
We have recently shown that application of the H-phosphonate
approach is advantageous for such a purpose, compared to other
dine yielded anomeric H-phosphonates 17 and 18 (α/β = 1:3).
2,3-O-TIPDS-protected 17-β was smoothly isolated in pure
form, though in a moderate 35% yield (Scheme 2). In contrast,
separation of the 2,3-di-O-TBDMS-protected α/β mixture 18
was challenging and ineffective.
Scheme 2. Stereocontrolled Synthesis of α-L-Ara4N and β-L-
Ara4N Glycosyl H-Phosphonates
14,15
phosphitylation methodologies.
Preparation of anomerically
pure α- and β- H-phosphonate monoesters of orthogonally
protected L-Ara4N in a stereoselective manner comprises a major
synthetic challenge in the synthesis of Ara4N-containing
phosphodiesters (Figure 2).
A predominant formation of the β-configured H-phosphonate
was achieved by application of fairly reactive SalPCl, which
quickly trapped the excess of axial β-lactol in 12, such that the
initial α/β ratio was preserved. To guide in situ anomerization in
favor of the α-lactol, a less reactive phosphitylation agent that
would primarily react with the more nucleophilic equatorial 1-
OH group, to shift the α/β ratio in favor of α-anomer, could be of
use. Indeed, slow addition of diphenylphosphite to a solution of
Figure 2. Synthetic neoglycoconjugate based on the diglucosamine
backbone of Lipid A modified with β-L-Ara4N at the anomeric
phosphate group.
Our synthesis of L-Ara4N glycosyl H-phosphonates relied on
the initial protection of hydroxyl groups in positions 2 and 3 in
1
6
1
2 (α/β = 1:1) in pyridine resulted in preponderant formation of
the kinetic product, an equatorial glycosyl H-phosphonate (α/β
2:1) 17-α readily isolated in 62% yield (Scheme 2, Table 1).
Monitoring the progress of I -mediated hydrolysis of the prop-
azide 1 to furnish 2,3-O-tetraisopropyldisiloxane-1,3-diyl
TIPDS), 2, 2,3-di-O-tert-butyldimethylsilyl (TBDMS), 3, 2,3-
(
=
di-O-benzyl, 4, PMB, 5, and 2,3-di-O-acetyl (Ac), 6, protected β-
allyl glycosides (Scheme 1). Stereoselectivity of phosphitylation
2
1
1
-enyl group in 7 by H NMR indicated that exclusively the β-
lactol was formed when the reaction was performed at 0 °C (α/β
ratio varied from 0:10 to 1:10), while the proportion of the α-
configured lactol increased at a higher reaction temperature (25
Scheme 1. Synthesis of L-Ara4N Glycosyl H-Phosphonates via
Conventional Approach
°
C, α/β = 1:1). Insight into the mechanistic pathway of I2-
assisted prop-1-enyl glycoside cleavage in 7 suggested formation
of the intermediate halohydrin (at 0 °C), which is cleaved
without affecting the anomeric configuration, to form exclusively
20,21
the β-configured lactol (Figures S1 and S2).
At 25 °C, and
upon chromatography on silica gel, a rapid anomerization toward
α-lactol sets in. Attempts to avoid the purification step and to
conduct the phosphitylation of the crude reaction mixture 12
enriched with the β-lactol (α/β = 1:10) failed due to formation of
numerous byproducts.
To elaborate an efficient approach toward anomerically pure
β-L-Ara4N H-phosphonates, we scrutinized the options for
traceless hydrolysis of β-allyl glycosides without affecting the
axial anomeric configuration at C-1. To this end, after allyl group
isomerization, the anomeric prop-1-enyl ether could be oxidized
by ozonolysis to give a stable formyl intermediate under mild
22−24
conditions (Scheme 3).
The formate group could be
at the anomeric center generally relies on the ultimate anomeric
selectively hydrolyzed to give a β-lactol and volatile methyl
formate, so that the crude β-lactol could be directly subjected to
phosphitylation without needing chromatographic purification.
Accordingly, prop-1-enyl intermediates 7 and 9−11 were
treated with ozone at −78 °C followed by addition of thiourea.
Formyl glycosides 19−22 were isolated in pure form by
chromatography on deactivated silica gel. Finally, methanolysis
17,18
ratio in the lactol precursors
and demands the preparation of
anomerically enriched hemiacetals which should be straightfor-
wardly converted into the H-phosphonates. Anomeric deal-
lylation of 2 and 3 was carried out by sequential double bond
+
−
isomerization with [Ir(1,5-Cod)(PMePh ) ] PF followed by
2
2
6
I2-assisted prop-1-enyl cleavage, to furnish anomeric mixtures 12
and 13, respectively (α/β = 1:1). Lactols 12 and 13 could be
enriched with the β-anomer (α/β = 1:3) by treatment with
diluted AcOH. Subsequent phosphitylation by reaction with 2-
(NEt /MeOH) of the formate esters at −40 °C furnished solely
3
β-configured lactols 12−16, which were used without further
purification. Subsequent phosphitylation with SalPCl provided
anomerically pure β-glycosyl H-phosphonates 17-β, 23-β, 24-β,
19
chloro-1,3,2-benzodioxaphosphorin-4-one (SalPCl) in pyri-
B
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Letter
Table 1. Stereoselective Synthesis of α- and β-Glycosyl H-
Phosphonates of L-Ara4N
phosphonates in 84% yield, in which the α-configured product
prevailed (α/β = 2:1, Scheme S1).
Glycosyl H-phosphonate 17-β was coupled to the β(1→6)-
14
linked diglucosamine lactol 26 (α/β = 2:1) in pyridine using
pivaloyl chloride (PivCl) as activating agent to furnish H-
phosphonate glycosyl phosphodiester 27 as an anomeric mixture
1
31
at GlcN moiety (α/β = 2:1, according to H and P NMR data)
Scheme 4). Oxidation of 27 by treatment with aq I at −40 °C
(
2
Scheme 4. Synthesis of βGlcN(1→6)-αGlcN(1→P←1)-β-L-
Ara4N Epitope and BSA Conjugate
a
Method for removal of anomeric prop-1-enyl ether and phosphity-
lation: (A) (1) I2 (2 equiv), THF/H O (2:1, v/v), rt; (2) slow
2
addition (4 h) of (PhO) P(O)H (4 equiv), Py; (3) NEt /H O, rt, 30
2
3
2
min; (B) (1) I (2 equiv), THF/H O (2:1, v/v), 0 °C; (2) in situ
2
2
anomerization with AcOH; (3) SalPCl (2 equiv), Py; (4) NEt /H O,
3
2
rt, 30 min; (C) (1) O , −78 °C, 5 min, thiourea (1.3 equiv); (2)
3
MeOH, NEt , −40 °C; (3) SalPCl (2 equiv), Py; (4) NEt /H O, rt, 30
3
3
2
b
min. Yield of glycosyl β-L- or α-L-Ara4N H-phosphonate after
conventional column chromatography on silica gel (not HPLC).
Scheme 3. Stereoselective Synthesis of β-L-Ara4N Glycosyl H-
Phosphonates
followed by chromatographic purification afforded anomerically
pure binary glycosyl phosphodiester 29 in 58% yield, whereas an
undesired β-anomeric product was expectedly destroyed upon
aqueous I -mediated oxidation and isolation by chromatography
2
25
on silica gel. Despite the apparent simplicity, this procedure
requires strict control of reaction conditions since minor
alterations can result in hydrolysis of the binary glycosyl
phosphodiester or the appearance of byproducts. PivCl-
26
mediated coupling can instigate formation of P-acyl byproducts
resulting from an over-reaction of 17 or 27 with PivCl and the
27
formation of GlcNAc-derived oxazolines. Oxidation of 27 into
the P(V) counterpart 29 by treatment with aq I could result in
2
rapid hydrolysis, with the loss of Ara4N, if the optimized reaction
conditions were not rigorously followed. To circumvent these
possible drawbacks, we exchanged the activating agent to 3-nitro-
1,2,4-triazol-1-yl-tris(pyrrolidin-1-yl)-phosphonium hexafluoro-
phosphate (PyNTP), which selectively reacted with the
electrophilic phosphorus atom of 17 to form a P−N-activated
and 25-β in 62−78% yield (Table 1). Exchange of the 4-azido
group in 2 for the Fmoc-protected amino group, followed by
anomeric deallylation and phosphitylation with SalPCl, furnished
an inseparable mixture of α- and β-L-Ara4N glycosyl H-
15,28
intermediate.
Formation of the binary glycosyl H-phospho-
C
DOI: 10.1021/acs.orglett.6b03358
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
nate diester 27 was confirmed by appearance of the P−H-
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AUTHOR INFORMATION
■
(
Corresponding Author
*E-mail: alla.zamyatina@boku.ac.at.
ORCID
Alla Zamyatina: 0000-0002-4001-3522
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
2
Financial support by Austrian Science Fund (FWF) Grant P-
1276 and P-28915 is gratefully acknowledged.
D
DOI: 10.1021/acs.orglett.6b03358
Org. Lett. XXXX, XXX, XXX−XXX