Total Synthesis of the Complete Protective Antigen of Vibrio cholerae O139

Article abstract of DOI:10.1002/anie.201606116

The first chemical synthesis of the complete protective O-antigen of a human-disease-causing pathogenic bacterium is described. The synthesis involved a protecting-group strategy that facilitated the regioselectivity of the key transformations, stereosele

Full text of DOI:10.1002/anie.201606116

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Communications
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International Edition: DOI: 10.1002/anie.201606116
German Edition: DOI: 10.1002/ange.201606116
Oligosaccharides
Total Synthesis of the Complete Protective Antigen of Vibrio cholerae
O139
Sameh E. Soliman and Pavol Kovꢀ cˇ *
Abstract: The first chemical synthesis of the complete protec-
tive O-antigen of a human-disease-causing pathogenic bacte-
rium is described. The synthesis involved a protecting-group
strategy that facilitated the regioselectivity of the key trans-
formations, stereoselective glycosylation reactions, and enabled
the one-step global deprotection of the completely assembled,
fully protected, phosphorylated hexasaccharide by hydroge-
nation/hydrogenolysis. The final amino-group-functionalized,
linker-equipped antigen was obtained in a form ready for
conjugation to suitable carriers, for example, proteins, to yield
immunogens.
Scheme 1. Structure of the O-antigen of Vibrio cholerae O139.
C
holera in humans is caused by two strains of Vibrio
[1–4]
cholerae: Vibrio cholerae O1 and Vibrio cholerae O139.
The disease is endemic in over 50 countries; it affects 3–
million individuals each year and results in over 100000
towards a synthetic vaccine has been hampered because the
critical hexasaccharide has not been synthesized, although the
structure was elucidated two decades ago. The lack of such
a synthesis lies undoubtedly in the difficulties involved in the
synthesis, isolation, and purification of the charged substance.
Having first verified the methodology that would be
5
deaths annually. Programs aimed at reducing the global
burden of cholera by providing adequate sanitation and safe
water have been unsuccessful. The field has realized that the
development and deployment of an improved cholera vaccine
will be a critical component in cholera control programs, until
adequate sanitation and safe water are a reality for the most
impoverished individuals on the planet.
[12,13]
involved in the synthesis of the title antigen,
we now
report the first total synthesis of the complete protective O-
antigen of Vibrio cholerae O139 in a form ready for
conjugation. The synthesis started with the preparation of
the key disaccharide building blocks 3 and 8 (Scheme 2).
We have been involved in developing a glycoconjugate
vaccine for cholera from synthetic carbohydrates that mimic
the structure of the O-specific polysaccharides (O-SPs, O-
[
13]
Glycosidation of the a-glycosyl bromide 1 with the 4,6-
[5–7]
[14]
antigens) of bacterial pathogens for a number of years.
benzylidene acceptor 2 was successfully carried out in the
Generally, the prerequisite for developing such a vaccine is
the availability of a large fragment of the O-specific
polysaccharide characteristic of the bacterium, because O-
SPs are the protective antigens. Vibrio cholerae O139 is
unique among bacteria that cause disease in humans in that
the complete protective antigen is a single phosphorylated
hexasaccharide consisting of five different monosaccharides
presence of the promoter AgOTf by the use of our improved
protocol, which avoids the use of molecular sieves, to afford
[15]
exclusively the b-(1!3)-linked disaccharide 3 in excellent
[8]
yield (90%). The configuration of the b-interglycosidic
1
linkage was confirmed by both H NMR (d = 4.75 ppm, d,
H
II
13
J
= 8.1 Hz, H-1 ) and C NMR spectra (d = 99.4 ppm,
1
,2
C
1
II
JC1,H-1 = 165.2 Hz, C1 ).
(
a-Colp-(1!2)-4,6-P-b-d-Galp-(1!3)-[a-Colp-(1!4)]-b-d-
GlcpNAc-(1!4)-a-d-GalpA-(1!3)-b-d-QuipNAc-(1!;
Scheme 1) and not a chain of oligosaccharide repeating
units, which is the usual scenario within the Gram-negative
Zemplꢀn de-O-acylation of the a-(1!3)-linked disac-
[12]
charide 4 gave tetraol 5 in virtually quantitative yield, and
subsequent p-methoxybenzylidenation of 5 gave selectively
[
9,10]
II II
the 4 ,6 -acetal derivative 6 (92%). Controlled benzylation
[
11]
bacteria. In the case of Vibrio cholerae O139, the work
of 6 with benzyl bromide and sodium hydride in DMF–DME
at low temperature (6!7, 86%), to minimize elimination at
I
C6 in the presence of a strong base, followed by regioselec-
[
*] Dr. S. E. Soliman, Dr. P. Kovꢀcˇ
NIDDK, LBC, Section on Carbohydrates
National Institutes of Health (NIH)
Bethesda, MD 20892-0815 (USA)
E-mail: kpn@helix.nih.gov
tive reductive opening of the alkylidene ring in 7, afforded the
linker-equipped disaccharide acceptor 8 in 89% yield. As
13
II
compared to the C NMR spectrum of 7, the signal for C4 in
(d = 68.2 ppm) was shifted upfield (by ca. 6 ppm), which
8
C
confirmed that the reductive opening of the p-methoxyben-
Dr. S. E. Soliman
Department of Chemistry, Faculty of Science, Alexandria University
Alexandria 21321 (Egypt)
II
II
zylidene acetal led to the HO-4 -free, 6 -p-methoxybenzyl
ether.
With the two building blocks 3 and 8 in hand, we focused
on the 2+2 coupling. Accordingly, NIS/AgOTf-promoted
Supporting information for this article can be found under:
http://dx.doi.org/10.1002/anie.201606116.
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activation of ethyl 2,4-di-O-
benzyl-3,6-dideoxy-1-thio-b-l-
[
18]
xylo-hexopyranoside (14) with
[19,20]
CuBr /Bu NBr.
However,
2
4
the reaction was largely incom-
plete. Activation of the freshly
prepared a-colitosyl bromide 15
under halide-assisted 1,2-cis gly-
[21]
cosylation conditions was suc-
cessful (Scheme 4) and afforded
the desired hexasaccharide 16 as
the major product (66%) along
with two isomeric pentasacchar-
ides (ca. 23% combined yield),
which were readily separable by
chromatography. The two penta-
saccharides can be either colito-
sylated to give more of the
desired hexasaccharide 16, or
they can be deprotected to give
fragments of the O-antigen and
used in antigenicity studies
related to Vibrio cholerae O139.
1
3
The C NMR spectra for 16
showed the expected downfield
III
shift of the signals for C4 and
IV
C2 as a result of colitosylation at
these positions. Furthermore, sig-
nals for the two anomeric hydro-
gen atoms of the colitose moiet-
Scheme 2. Synthesis of the key disaccharide building blocks 3 and 8 (TCA=trichloroacetyl, Tf=trifluoro-
methanesulfonyl, TMU=1,1,3,3-tetramethylurea, PMP=p-methoxyphenyl, PMB=p-methoxybenzyl,
CSA=10-camphorsulfonic acid, Bn=benzyl, DMF=N,N-dimethylformamide, DME=1,2-dimethoxy-
ethane, MS=molecular sieves).
ies appeared as doublets in the
1
H NMR spectrum at d = 5.24
H
and 5.03 ppm (J = 3.2 and 3.7 Hz,
glycosylation of the spacer-equipped disaccharide acceptor 8
with the b-thioglycoside disaccharide donor 3 at ꢀ258C
proceeded stereoselectively to afford the desired linear
tetrasaccharide 9 (84%; Scheme 3). The acidity of the
reaction medium was optimized by using excess AgOTf to
minimize the conversion of the donor into the corresponding
respectively), which confirmed the formation of the desired a-
glycosidic linkages.
Subsequent de-O-acetylation (Zemplꢀn) of 16, followed
by selective phosphorylation with 2,2,2-trichloroethyl phos-
[
22]
phorodichloridate at ꢀ208C, gave predominantly the (S )-
P
IV IV
4 ,6 -cyclic 2,2,2-trichloroethyl phosphate 18 (S/R 9:1,
P NMR, ca. 91% combined yield). Global deprotection of
[
16]
31
stable oxazoline.
The structure of the tetrasaccharide
1
13
product 9 was confirmed by its H and C NMR spectra,
which showed signals characteristic of both the acceptor and
the donor moieties.
18 (by the transformation of 13 functional groups in a one-pot
reaction) was successfully carried out by catalytic hydro-
genation/hydrogenolysis (Pd/C, H , 1 atm) at pH 7 (0.1m
2
Selective removal of the bromoacetyl ester in 9 by
potassium phosphate buffer, to neutralize HCl formed).
[
17]
treatment with thiourea and sym-collidine (9!10, 95%),
Compound 20 was obtained (87%) in a pure state (TLC,
II
[23]
and subsequent oxidative removal of the 6 -O-p-methoxy-
NMR ) by HPLC.
benzyl group with DDQ in CH Cl –H O, gave diol 11 (88%).
An important feature of the design of this synthetic
sequence is that the phosphorylation is performed at a very
late stage of the overall synthesis. Although the two isomeric
(S,R) cyclic phosphates can be separated by chromatography,
a mixture of the isomeric phosphates formed can be used
directly for the reductive deprotection step. Because the
phosphorus atom is no longer asymmetric after removal of the
2
2
2
Regioselective oxidation of the primary hydroxy group in 11
with a combination of a catalytic amount of TEMPO free
radical and a slight excess of BAIB in a diphasic CH Cl –H O
2
2
2
solvent system, followed by benzylation (BnBr/K CO3 in
2
DMF) of the formed carboxylic acid, furnished uronate 12
II II
(
89% over two steps). Reductive ring opening of the 4 -6 -O-
[
24,25]
benzylidene acetal in 12 with sodium cyanoborohydride and
m HCl–Et O in THF at room temperature gave the
trichloroethyl group,
the same product is formed from S
2
and R isomers, and the amount of the product can be
increased. Accordingly, similar treatment of a mixture of the
two isomeric cyclic phosphates 18 and 19 gave, as expected,
the desired compound 20 in comparable yield.
2
tetrasaccharide diol acceptor 13 (85%) with complete regio-
selectivity.
The stereoselective installation of the two colitose resi-
IV
III
dues at positions 2 and 4 in diol 13 was first attempted by
2
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Scheme 4. Synthesis of the spacer-equipped, phosphorylated hexasac-
charide 21.
Scheme 3. Synthesis of the linear tetrasaccharide diol acceptor 13
NIS=N-iodosuccinimide, DDQ=2,3-dichloro-5,6-dicyano-1,4-benzo-
(
quinone, TEMPO=2,2,6,6-tetramethyl-1-piperidinyloxy free radical,
BAIB=[bis(acetoxy)iodo]benzene).
Acknowledgements
This research was supported by the Intramural Research
Program of the National Institutes of Health (NIH) and
National Institute of Diabetes and Digestive and Kidney
Diseases (NIDDK).
Finally, saponification (0.1m KOH in H O at pH 11) of the
methyl ester 20 was followed by HPLC purification to give the
pure spacer-equipped, phosphorylated hexasaccharide 21
2
(
83% yield), and its structure was confirmed by NMR and
[
26]
HRMS data.
Keywords: glycosylation · oligosaccharides ·
In conclusion, we have completed synthesis of the full O-
antigen of the human-disease-causing pathogen Vibrio chol-
erae O139, which is a complex, branched hexasaccharide
consisting of five different monosaccharides and a cyclic
phosphate (Scheme 1). The highlights of the synthesis include
regio- and stereoselective transformations and a protecting-
group strategy that allows global deprotection. The final
hexasaccharide is equipped with a linker, which is function-
alized for conjugation to yield a vaccine. Both conjugation
and related antigenicity studies with the hexasaccharide
antigen and a wide spectrum of fragments thereof are in
progress.
O-specific antigens · pathogenic bacteria · stereoselectivity
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[
[
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13
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[
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to prevent any acyl-group migration during the selective O-
debromoacetylation reaction.
Received: June 23, 2016
Published online: && &&, &&&&
4
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Oligosaccharides
The whole shebang: The protective O-
antigen (see structure) of a human-dis-
ease-causing pathogenic bacterium was
synthesized by a route involving a pro-
tecting-group strategy that enabled one-
step global deprotection of the com-
pletely assembled phosphorylated hexa-
saccharide by hydrogenation/hydroge-
nolysis. The final antigen was obtained
with a linker functionalized for conjuga-
tion to suitable carriers, such as proteins,
to yield immunogens.
S. E. Soliman, P. Kovꢁ cˇ *
&&&& — &&&&
Total Synthesis of the Complete Protective
Antigen of Vibrio cholerae O139
Angew. Chem. Int. Ed. 2016, 55, 1 – 5
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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5
These are not the final page numbers!