Synthesis of the Campylobacter jejuni 81-176 Strain Capsular Polysaccharide Repeating Unit Reveals the Absolute Configuration of its O-Methyl Phosphoramidate Motif

Article abstract of DOI:10.1002/anie.201810222

The O-methyl phosphoramidate (MeOPN) motif is a non-stoichiometric modification of capsular polysaccharides (CPS) in ≈70 % of all Campylobacter jejuni strains. Infections by C. jejuni lead to food-borne illnesses and the CPS they produce are key virulence

Full text of DOI:10.1002/anie.201810222

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Angewandte
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Accepted Article
Title: Synthesis of the Campylobacter jejuni 81-176 strain capsular
polysaccharide repeating unit reveals the absolute configuration
of its O-methyl phosphoramidate motif
Authors: V. Narasimharao Thota, Michael J Ferguson, Ryan P
Sweeney, and Todd Lowary
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201810222
Angew. Chem. 10.1002/ange.201810222
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10.1002/anie.201810222
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Synthesis of the Campylobacter jejuni 81-176 strain capsular
polysaccharide repeating unit reveals the absolute configuration
of its O-methyl phosphoramidate motif
V. Narasimharao Thota, Michael J. Ferguson, Ryan P. Sweeney and Todd L. Lowary*[a]
Abstract: The O-methyl phosphoramidate (MeOPN) motif is a non-
stoichiometric modification of capsular polysaccharides (CPS) in
~70% of all Campylobacter jejuni strains. Infections by C. jejuni lead
to food-borne illnesses and the CPS they produce are key virulence
factors. The MeOPN phosphorus atom in these CPS is stereogenic
and is found as a single stereoisomer. However, to date, the
absolute stereochemistry at this atom has been undefined. We
report the synthesis of the three repeating units found in C. jejuni 81-
176 CPS; one of these possesses a MeOPN group. In the course of
these studies we established that the stereochemistry of the
phosphorus atom in this MeOPN group is R. These studies
represent the first unequivocal proof of stereochemistry of this group
in any C. jejuni CPS. The compounds produced are anticipated to be
useful tools in investigations targeting the function and biosynthesis
of this structurally-interesting modification, which so far has only
been identified in campylobacter.
The structures of the synthetic targets are shown in Figure 1B
(1–3). Each consists of N-acetylglucosamine, galactose and an
unusual 6-deoxyheptose[7] with the D-altro-stereochemistry. An
aminooctyl aglycone was incorporated to allow facile conjugation
to either proteins or surfaces. The heptose is present either in its
unmethylated (1) or O-3 methylated form (2) and the MeOPN
group is found on O-2 of the galactose residue (3).[8] The methyl
and MeOPN groups are phase-variable modifications.[9]
Trisaccharide 3, lacking both of these functionalities, has been
synthesized previously by Nam Shin and coworkers.[10] Our plan
was to synthesize all three trisaccharides via a single strategy
using building blocks 4–8 (Scheme 1).
Campylobacter jejuni is a food-borne pathogen that causes
significant gastrointestinal illness worldwide.[1] Although these
infections usually resolve in a few days, they sometimes lead to
the neurological disorder Guillain–Barré syndrome.[2] An
important C. jejuni virulence factor is its capsular polysaccharide
(CPS), which differentiates the 47 different serotypes identified
to date.[3] These CPS possess a number of unusual structural
motifs and prominent among these is the O-methyl
phosphoramidate (MeOPN) group (1, Figure 1A). This motif is a
non-stoichiometric substituent found in more than 70% of all C.
jejuni strains. The MeOPN group is believed to contribute
importantly to the virulence of the organism[4] and a vaccine
currently in development for the prevention of C. jejuni infections
incorporates CPS bearing this functionality.[5]
Figure 1 A) MeOPN motif present in Campylobacter CPS; B) structures of the
CPS repeating units present in C. jejuni strain 81-176 CPS (1–3).
The phosphorus atom in the MeOPN substituent is stereogenic
and it occurs as a single stereoisomer in the CPSs in which it is
found. Recent work has established the biosynthesis of the
MeOPN group: the immediate precursor is an activated CDP
derivative and the amino group is derived from glutamine.[6]
Despite these advances, the stereochemistry on phosphorus
remains unknown. We report here the synthesis of the three
repeating unit structures found in C. jejuni strain 81-176,
including one that contains a MeOPN group. In executing these
synthetic investigations, we established, for the first time, that
the stereochemistry on phosphorus in the MeOPN motif is R.[16]
[a]
V. N. Thota, Dr. M. J. Ferguson, Dr. R. P. Sweeney and Prof. Dr. T.
L. Lowary
Department of Chemistry
University of Alberta
Scheme 1. Retrosynthetic Analysis of 1–3
The major challenges in the synthesis of 1–3 are: 1) the
stereoselective installation of the α-galactoside residue; 2) the
preparation of the 6-deoxy heptose with the D-altro-
stereochemistry; and 3) introduction of the MeOPN moiety. We
Edmonton, Alberta
Canada T6G 2G2
E-mail: tlowary@ualberta.ca
Supporting information for this article is given via a link at the end of
the document.
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10.1002/anie.201810222
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chose to address the first challenge through the use of an α-
selective donor with a di-tert-butyl silyl acetal on O-4 and O-6.[11]
Triflation and inversion of an advanced intermediate (See
Supporting Information) from the known 6-deoxy-D-manno
heptopyranoside[12] was our approach in addressing the second.
Finally, we envisioned that the MeOPN group could be
introduced from 8, or one of two other methods previously
developed for introducing this structure into carbohydrates.[13]
Monosaccharide 4[14] is known and the synthesis of 5–7 is
provided in the Supporting Information.
Scheme 2. A) Synthesis of 1. B) Synthesis of 2.
Accessing the parent trisaccharide
1
and the derivative
with hydrogen fluoride in pyridine, giving 13, which was then
subjected to hydrogenolysis and azide reduction, providing
target 1 in 67% yield over the two steps. With this route
established, the preparation of 2 was achieved (Scheme 2B) by
glycosylation of 6 with 10 and then application of the same
functional group transformations and deprotection reactions
used for synthesizing 1.
containing the phase-variable methyl group (2) began with the
synthesis of disaccharide 10 (Scheme 2A). Thus, thioglycoside 4
was converted into trichloroacetimidate 9, which was then used
immediately to glycosylate alcohol 5 employing trimethylsilyl
trifluoromethanesulfonate as the activator. This reaction
furnished the β-linked disaccharide 10 in 85% yield. Subsequent
glycosylation of the 6-deoxy altro-heptoside acceptor 6 with 10,
promoted by N-iodosuccimide and triflic acid, afforded an 88%
yield of trisaccharide 11. Treatment of 11 with ethylenediamine
at 90 °C cleaved both the phthalimide and the benzoate ester;
selective N-acetylation of the intermediate product was achieved
by reaction with acetic anhydride, triethylamine and methanol.
This sequence yielded trisaccharide 12 in 70% yield over the
two steps. Deprotection of 12 was achieved by first treatment
Next, we turned our attention to the synthesis of 3, which
contains both of the phase-variable modifications: the methyl
and MeOPN groups. We initially explored two previously
reported methods (Scheme 3) to carry out this transformation.
We first attempted to introduce the MeOPN onto 15 using methyl
benzylphosphoramidochloridate,[13b] but under these conditions,
no product was formed; only the starting material was isolated.
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The use of a two-step approach[13a] involving an activated O-
methyl H-phosphonate pivalate ester (obtained by treatment of
the parent O-methyl H-phosphonate with pivaloyl chloride
immediately before addition of the alcohol) and subsequent
oxidation via the Atherton–Todd reaction,[15] again resulted only
in the re-isolation of 15. We also explored two derivatives of 15
in which the silyl acetal was replaced with other groups (See
Supporting Information, Scheme S4), but these substrates too
gave, at best, only very low yields of the desired methyl
phosphoramidate product.
Scheme 3. Attempted synthesis of phosphoramidate 17 from 15 using two reported methods[13]
O
P
H
Si
O
Si
O
O
NBu
O
H3CO
4
O
BnO
BnO
BnO
BnO
O
O
O
O
O
18
O
O
BnO
BnO
O
O
O
P
1. 15, CH2Cl2, pyridine, 55 °C
AcHN
AcHN
+
P
Ac2O, CH2Cl2,
pyridine, 55 °C
BnHN
MeO
2. CBrCl3, Et3N,BnNH2,
CH2Cl2, rt
CH3O
BnHN
O
O
BnO
BnO
BnO
BnO
65% (1:1.2, 19:20)
O
O
O
P
19
H3CO
OAc
20
H3CO
O(CH2)8N3
H3CO
O(CH2)8N3
H
1. HF·pyridine, THF, pyridine, rt
2. H2, Pd(OH)2–C, CH2Cl2,
CH3OH, HOAc, rt
1. HF·pyridine, THF, pyridine, rt
2. H2, Pd(OH)2–C, CH2Cl2,
CH3OH, HOAc, rt
65%
77%
(S)-3
(R)-3
Scheme 4. Successful introduction of the methyl phosphoramidate into 15 and elaboration into target trisaccharide 3.
We postulated that the lack of success in introducing the
phosphoramidate onto 15 was due to steric hindrance near the
C-2 hydroxyl group of the galactose residue. We further
surmised that using a reagent smaller than those shown in
Scheme 3, and more forcing conditions, would provide the
desired target. With this approach in mind, 15 was subjected to
a modified version of the H-phosphonate formation (Scheme 4).
approach,[13a] products corresponding to both diastereomers on
phosphorus were formed (19 and 20). While often these two
diastereomers are inseparable, in this case they could be
separated. Fortuitously, one of the diastereomers (19) was a
solid and we obtained an X-ray structure of the compound
(Figure 3).[16] Analysis of the structure allowed us to assign the
stereochemistry at phosphorous in 19 as R.[17] By inference then,
the phosphorus atom in 20 has the S stereochemistry.
Activation of the precursor H-phosphonate (18) was done using
acetic anhydride instead of pivaloyl chloride, and the coupling
was done at 55 °C instead of room temperature. Under these
conditions, a new product was formed that, when subjected to
the Atherton–Todd reaction,[15] furnished the desired O-methyl
phosphoramidate in 65% yield. As in other examples using this
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With both (R)-3 and (S)-3 in hand, comparison of the NMR data
with that previously reported for the CPS[5b,8c] was carried out
(Table 1). It should be noted that for these deprotected
compounds the restricted rotation seen in 19 and 20 was not
observed; thus in all NMR spectra only single resonances were
present.
Table 1. Comparison of the NMR data for (R)-3 and (S)-3 with natural CPS
31P NMR[a]
14.291
1H NMR[a],[b]
4.54
(R)-3
(S)-3
CPS
Figure 3. ORTEP of 19, showing the R-stereochemistry on phoshorus
In further characterizing 19 we were surprised to find that the 31
13.849
4.48
14.257[8c]
4.52[5b]
P
NMR spectrum of the compound showed two signals instead of
the anticipated single peak (ratio 2:1); a similar thing was seen
in the 31P spectrum of 20 (ratio 4:1). Given the difficulty we had
in installing the MeOPN group, presumably due to steric
congestion, we hypothesized that these two resonances arise
from restricted rotation around one of the phosphoramidate
bonds. To test this, we measured the 31P spectrum on 19 and 20
at 60 °C. Under these conditions, the two 31P signals for both
compounds changed in relative intensity, leading to spectra that
contained one major signal; in the case of 20 a single resonance
was observed (See Supporting Information, Page S92). This
experiment provides support for restricted rotation leading to the
doubling of the signals. Unfortunately, the solvent used (CDCl3)
limited the temperature at which we could heat the sample and
thus it was never possible to obtain spectra of 19 that had a
single resonance, indicative of free rotation in this molecule.
Nevertheless, this observation, and the difficulty with which it is
install the MeOPN group onto the molecule, provides insight into
the sterically hindered nature of this region of the molecule.
[a] Chemical shifts in ppm
[b] Chemical shift of H-2 in galactose residue
The 31P NMR spectrum of (R)-3 showed a signal at 14.291 ppm
and the resonance for this atom in the CPS is in close
agreement (14.257 ppm). On the other hand, the 31P NMR
spectrum of (S)-3 showed a resonance at 13.849 ppm. Both
spectra contained a single resonance, which suggests that there
was no loss of stereochemical integrity on phosphorus during
the deprotection. Had scrambling of the phosphorus
stereochemistry occurred, a mixture of the two diastereomers
would be expected, not complete conversion to the opposite
stereoisomer. When considering the H-2 signal of galactose
1
residue in the H NMR spectra, the data for (R)-3 (4.54 ppm) is
in close agreement with that reported for the CPS (4.52 ppm),
whereas the data for (S)-3 differs. Based on these comparisons,
we conclude that MeOPN group in the C. jejuni 81-176 CPS is R.
In conclusion, we have successfully synthesized the three
repeating units present in CPS produced by C. jejuni 81-176,
including one that contains a MeOPN motif. While carrying out
these syntheses we obtained a crystal structure of a key
intermediate that, following its deprotection and comparison with
data for the polysaccharide, established the phosphorous
stereochemistry of the MeOPN in the native CPS as R. This
represents the first unambiguous determination of the
stereochemistry of a campylobacter CPS MeOPN group, which
exists naturally as a single stereoisomer. It remains to be
determined if MeOPN-functionalized CPS produced by other
campylobacter have the same stereochemistry. However, we
note that the biosynthesis of the group is done enzymatically,[6]
which, depending on the similarities of enzymes across species,
could suggest it is the same in all campylobacters. The use of
(R)-3 and its enantiomer (S)-3 in probing the specificity of sera
raised against vaccination with the native CPS is ongoing and
these compounds will be useful probes in studies of MeOPN
function and assembly. We anticipate this study will also
motivate the development of methods for the diastereoselective
introduction of MeOPN groups into carbohydrates.
To allow the determination of the phosphorous stereochemistry
in the natural CPS, both 19 and 20 were deprotected (Scheme
4). Treatment of 19 with HF•pyridine furnished a diol that was
then subjected to hydrogenolysis using Pd(OH)2 to afford (R)-3
in 77% yield over the two steps. Similar treatment of 20 provided
an 65% yield of (S)-3. We found that the hydrogenolysis needed
significant optimization (See Supporting Information, Table S1)
and the best results were obtained when a small amount of
acetic acid was added to the reaction mixture. Without acetic
acid we found that the majority of the MeOPN group was
cleaved. The deprotected compounds were also found to have
limited stability in D2O; most of the MeOPN group was cleaved
within few hours during storage in D2O. The lability of this group
in the native CPS was not reported previously,[8c] although we
note that the NMR spectra of the polysaccharide were recorded
in the presence of 1% acetic acid. It thus appears that the
presence of a small amount of acid is required for the molecule
to be stable in solution. This is also consistent with our finding
(above) that in the absence of acetic acid in the solvent mixture,
the hydrogenolysis led to cleavage of the MeOPN group. The
extraction of these CPS was also done using hot phenol, an
acidic medium.[8c]
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[14] B. M. Swarts, Y.-C. Chang, H. Hu, Z. Guo, Carbohydr. Res. 2008, 343,
2894.
Acknowledgements
[15] a) F. R. Atherton, H. T. Openshaw, A. R. Todd, J. Chem. Soc. 1945,
660, b) S. S. Le Corre, M. Berchel, H. Couthon-Gourvès, J.-P. Haelters,
P.-A. Jaffrès, Beilstein J. Org. Chem. 2014, 10, 1166.
This work was funded by the Alberta Glycomics Centre, the
University of Alberta and the Natural Sciences and Engineering
Research Council of Canada. We thank Dr. Mark Miskolzie
(NMR Laboratory, University of Alberta) for help with high
temperature NMR studies of 19 and 20.
[16] Crystallographic data for 19: C81H110N5O18PSi, Mr = 1500.79, colourless
needle, 0.38 x 0.06 x 0.05 mm, orthorhombic, P212121 (No. 19), a =
15.1429(3) Å, b = 20.0680(4) Å, c = 26.8195(6) Å, V = 8150.1(3) Å3, Z =
4, rcalcd = 1.223 g cm-3, µ = 1.008 mm-1, Cu Ka (1.54178), T = –100 °C,
2q limit = 144.77°, no. of measured (48945) and independent (15989)
reflections, Rint = 0.0513, R1 = 0.0436, wR2 = 0.1137, largest difference
peak and hole 0.282 and –0.333 e Å-3. Data were collected on a Bruker
D8 diffractometer, equipped with a Cu microfocus source and an APEX
II CCD detector, using 1.0º w and f scans and 15 s exposures. Face-
indexed absorption corrections were performed using SADABS-2016.
The structure was solved using intrinsic phasing method of SHELXT-
2014 and refined with the program SHELXL-2018. The CIF has been
deposited at the Cambridge Crystallographic Data Centre (CCDC
Keywords: Capsular polysaccharide • 6-deoxyheptose •
glycosylation • NMR spectroscopy • phosphoramidate
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COMMUNICATION
COMMUNICATION
A single approach enabled the
synthesis of three trisaccharide
fragments of the capsular
polysaccharide produced by C. jejuni
81-176. The targets all include 6-
deoxy-D-altro-heptose and one is
functionalized with an O-methyl
phosphoramidate motif, which
V. Narasimharao Thota, Michael J.
Ferguson, Todd L. Lowary*
Si
O
O
Page No. – Page No.
Synthesis of the Campylobacter
BnO
BnO
BnO
O
O
O
O
jejuni
81-176
strain
capsular
O
polysaccharide repeating unit reveals
the absolute configuration of its O-
methyl phosphoramidate motif
AcHN
P
BnHN
CH3O
contains a stereogenic phosphorus
atom. X-ray analysis of an advanced
intermediate allowed, for the first time,
the phosphorus stereochemistry in the
natural glycan to be determined.
O
BnO
BnO
R
from X-ray
O
H3CO
O(CH2)8N3
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