First synthesis of bacillus cereus Ch HF-PS cell wall trisaccharide repeating unit

Article abstract of DOI:10.1021/ol5021527

The first total synthesis of Ch HF-PS, a cell wall trisaccharide repeating unit of B. cereus, is reported. The synthetic trisaccharide is appended with an aminopropyl linker at the reducing end to allow for conjugation to proteins and microarrays. The con

Full text of DOI:10.1021/ol5021527

Letter
pubs.acs.org/OrgLett
First Synthesis of Bacillus cereus Ch HF-PS Cell Wall Trisaccharide
Repeating Unit
Ananda Rao Podilapu and Suvarn S. Kulkarni*
Department of Chemistry, Indian Institute of Technology Bombay, Mumbai 400076, India
S
* Supporting Information
ABSTRACT: The first total synthesis of Ch HF-PS, a cell wall trisaccharide
repeating unit of B. cereus, is reported. The synthetic trisaccharide is appended with
an aminopropyl linker at the reducing end to allow for conjugation to proteins and
microarrays. The convergent synthesis involves transformation of D-mannose into
an orthogonally protected rare AAT sugar building block, two consecutive α-
stereoselective glycosylations, β-selective attachment of the linker by solvent
participation, and amide bond formation, as key steps.
Scheme 1. Retrosynthetic Analysis
Bacillus cereus is an aerobic, Gram-positive, spore-forming
bacterium which is a causative agent of self-limiting food
poisoning syndrome.1 The symptoms of the acute gastro-
intestinal illness include nausea, abdominal cramps, diarrhea,
and occasional vomiting and fever. The ubiquitous bacterium
which was once thought to be benign is increasingly being
acknowledged as a human pathogen. B. cereus can cause serious
opportunistic infections such as wound infections, bacteremia,
septicaemia, meningitis, pneumonia, infections of the central
nervous system, endocarditis, pericarditis, respiratory infections,
and peripheral infections.1−4 It is also associated with keratitis,
panopthalmitis, and other ocular ophthalmic infections which
usually result in the loss of the eye.5 B. cereus infections in
immunocompromised individuals may be lethal.6 Moreover,
inhalative exposure to B. cereus, containing B. anthracis toxin
genes, causes severe fatal pneumonia in healthy individuals.7
Furthermore, B. cereus infections are difficult to treat, as the
bacterium is resistant to several antibiotics including penicillin,
ampicillin, cephalosporins, and trimethoprim.8 Currently there
are no vaccines against B. cereus mediated infectious diseases.
Gram-positive bacteria synthesize various secondary cell wall
polysaccharides (SCWPs) which are covalently attached to the
peptidoglycan of the bacterial cell wall.9 These SCWPs mediate
various important functions such as bacterial adhesion, host
immunomodulation, virulence, and biofilm formation and are
thus regarded as potential carbohydrate antigens for the
development of vaccines as well as serodiagnostic markers.
to the glycan. Due to its unprecedented structure and
immunogenic potential, the trisaccharide repeating unit is an
attractive synthetic target. Over the past few years there have
been remarkable advances in the identification11,12 and
synthesis of rare sugar-containing bacterial glycans.13 The
total synthesis of a B. cereus glycan has yet to be reported. In
continuation of our research program on the synthesis of the
rare sugar containing bacterial glycoconjugates,14 we report
herein a first total synthesis of the unusual trisaccharide motif 1
́
Recently, Guerardel and co-workers analyzed B. cereus ATCC
14579 and characterized two structurally unrelated SCWPs
named as Ne HF-PS and Ch HF-PS.10 Of these, the charged
glycan Ch HF-PS (Scheme 1) is made up of a completely
original sequence, →6)-Gal(α1−2)(2-R-hydroxyglutar-5-
ylamido)Fuc2NAc4N(α1−6)GlcNAc(β1→, identified in the
Bacillus genus for the first time. A unique structural feature of
Ch HF-PS is the presence of the rare sugar and the
hydroxyglutaric acid spacer which imparts charge and flexibility
Received: July 22, 2014
Published: August 7, 2014
© 2014 American Chemical Society
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Letter
(Scheme 1). The synthetic trisaccharide is appended with a
terminal aminopropyl linker for subsequent conjugation to a
carrier protein or immobilization onto a microarray.
MeOH to afford 6 (62%) (Scheme 2). The absolute
stereochemistry of alcohol 6 was confirmed by converting it
to a known derivative 12 by selective hydrolysis of the tert-butyl
ester under acidic conditions followed by esterification with
methanol. The optical rotation of 12 matched perfectly well
with the reported value,17 confirming R stereochemistry in 6
(see Supporting Information).
Synthesis of the left-hand unit 3 using known thioglycoside
donor 5 and acceptor 6 was attempted next. Obtaining α-
selectivity in galactosylation is a challenging task. For this
reason, we screened various sets of reaction conditions, varying
the leaving groups, additives, and reaction temperatures (Table
1). The solvent participation effect was explored first, using
Structurally, target molecule 1 is composed of a terminal D-
galactopyranose α-linked to a 2-(R)-hydroxy-D-glutaric acid
moiety, which in turn is attached via an amide bond to the rare
sugar 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose (AAT/
Fuc2NAc4N) at C4′, which is further connected to the
linker-attached D-glucosamine unit at O6. The key challenges
encountered in the synthesis of trisaccharide 1 are the synthesis
of the orthogonally protected rare sugar AAT building block, β-
selective attachment of the linker, and installation of two
consecutive α-glycosidic linkages. Retrosynthetically, the most
obvious disconnection is the amide bond in the fully protected
trisaccharide 2. This dissects the molecule into two halves 3 and
4, which can in principle be separately synthesized and coupled
together, in a convergent manner, by selectively exposing the
acid and amine functionalities, respectively, to arrive at 2. The
left-hand unit 3 could be synthesized by stereoselective
glycosylation of easily accessible D-galactosyl donor 5 with
orthogonally protected 2-(R)-hydroxyl D-glutaric acid acceptor
6, under appropriate conditions. The key disaccharide 4 could
potentially be assembled by α-selective coupling of the
orthogonally protected AAT thioglycoside donor 7 with the
linker attached 6-OH glucosamine acceptor 8. The selection of
the protecting group in disaccharide 4 would demand careful
attention. Since the terminal C4′-NH2 group needs to be
revealed selectively postglycosylation, a phthalimido protection
was deemed suitable. The remaining C2 and C2′ NH2 groups
present on D-glucosamine and AAT units, respectively, were
masked as azido groups. While the C2′ azido group in 7 was
expected to favor α-selectivity, the C2 azido group in 8
restricted the choice to solvent participation for obtaining β-
selectivity with the linker acceptor. Finally, to maintain
orthogonality, we opted for a (benzyl)benzyloxycarbonyl-
amino linker that had been previously employed in
oligosaccharide synthesis.15 The hydroxyl groups in 7 and 8
were masked as benzyl ethers for enhancing the reactivity of the
donors in glycosylation. Moreover, benzyl groups are stable
under all the glycosylation and phthalimide deprotection
conditions and can be conveniently removed by hydrogenolysis
at the end.
Table 1. α-Stereoselective Glycosylation of 5 and 6
yield
entry
1
conditions
T (°C)
−20
time
2 h
α/β
(%)
NIS, TMSOTf
8:1
48
CH2Cl2/Et2O 1:1
NIS, TMSOTf
2
3
4
−40
−70
−70
2 h
2 h
1 h
9:1
12:1
−
50
62
−
CH2Cl2/Et2O 1:1
NIS, TMSOTf
CH2Cl2/Et2O 1:1
1. NBS, THF, H2O
2. K2CO3, CCl3CN
3. TMSOTf
CH2Cl2/Et2O 1:1
1. Br2, CH2Cl2
5
6
7
−50/−30
40
1 h
3 d
1 h
−
−
2. AgOTf, CH2Cl2
1. Br2, CH2Cl2
1:0
1:0
25
75
2. TBAI
Ph2SO, Tf2O, CH2Cl2
−60
diethyl ether as a cosolvent (entries 1−3).18 Glycosylation of
thioglycoside 5 under NIS/TMSOTf activation conditions in a
dichloromethane/diethyl ether solvent mixture (1:1) with
acceptor 6 at −20 °C afforded the desired product 3 with
good selectivity (8:1). Lowering of the temperature to −40 °C
(entry 2) and −70 °C (entry 3), under identical conditions,
progressively improved the α/β selectivity to 9:1 and 12:1,
respectively. Since the two isomers could not be separated by
silica gel flash column chromatography even after repeated
attempts, it became imperative for us to tune the conditions so
as to obtain a single α-isomer. Thioglycoside 5 being a stable
and flexible donor could be readily converted into the
corresponding trichloroacetimidate (−O-C(NH)CCl3)19 and
bromide (entries 4 and 5). However, the trichloroacetimidate
donor was too reactive and decomposed spontaneously under
the reaction conditions even at −70 °C (entry 4). Similarly,
AgOTf promoted the activation of the corresponding galactosyl
bromide at −30 and −50 °C in the presence of acceptor 6 led
to complex mixtures (entry 5). We did observe clean α-
selectivity under in situ anomerization conditions20 using TBAI
to obtain pure 3, albeit in a low yield and after prolonged
stirring at 40 °C (entry 6). This outcome was still far from
Our synthesis started with the preparation of orthogonally
protected 2-(R)-hydroxy D-glutaric acid unit 6 (Scheme 2).
Readily available D-glutamic acid 9 was converted to (R)-
butyrolactone 10 (78%) following the procedure reported by
Austin and co-workers.16 Esterification of 10 was carried out by
DCC mediated coupling with t-BuOH to obtain 11 (76%),
which was subjected to lactone ring opening by NaOMe in
Scheme 2. Preparation of D-Glutaric Acid Derivative
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Letter
satisfactory from a practical point of view. Gratifyingly,
diphenylsulfoxide and Tf2O mediated21 thioglycoside activation
of 5 at −60 °C with acceptor 6 rapidly furnished only α-linked
product 3 (1H NMR H-1 δ 5.12, J = 3.9 Hz, 13C NMR C-1 δ
95.8) in 75% yield (entry 7). Selective deprotection of the
methyl ester in 3 under mild basic conditions furnished acid 13
(85%).
Scheme 4. Synthesis of the Linker Attached Reducing End 6-
OH D-Glucosamine Unit
The synthesis of the key AAT building block 7 from known
D-rhamnosyl triol 1814b is outlined in Scheme 3. Earlier, we had
Scheme 3. Synthesis of the Orthogonally Protected AAT
Building Block
Scheme 5. Assembly of the Trisaccharide and Global
Deprotection
successfully synthesized a 3-OBz derivative corresponding to 7
from 18.14b However, since the 3-OBz group was not
compatible with phthalimide deprotection conditions, we
decided to employ a 3-OBn derivative instead. Accordingly,
triol 3 was first converted to the corresponding tin ketal by
treatment with dibutyltin oxide in toluene at 100 °C and
further reacted with benzyl bromide and TBAB at 60 °C to
afford the 3-OBn derivative 19, in high regioselectivity, as a
single isomer in 85% yield over two steps. Following our
recently established protocol of regioselective nucleophilic
displacements of triflates,14b the 2,4-diol 19 was first converted
to the corresponding 2,4-bis-triflate by treatment with Tf2O in
pyridine. The crude triflate obtained after aqueous workup was
subjected to a regioselective C2-OTf displacement with a
stoichiometric amount of TBAN3 at −30 °C. After the
complete conversion of the starting material as observed from
TLC analysis, potassium phthalimide was added in the same
pot at rt to displace the remaining C4-OTf to afford the desired
orthogonally protected AAT building block 7 in 52% yield over
three steps after a single chromatographic purification.
The linker attached reducing end D-glucosamine building
block 8 was prepared from known trichloroacetimidate donor
1515c and linker 1415d as shown in Scheme 4. Glycosylation of
15 with 14 under TMSOTf activation using acetonitrile as a
participating solvent22 at −40 °C cleanly afforded the β-linked
product 16 (1H NMR H-1 δ4.33, 13C NMR C-1 δ 102.06) in
72% yield. Removal of the acetates under mild conditions
(AcCl, MeOH, 94%) followed by benzylidene formation in
acetonitrile23 as a solvent (90%) furnished 3-OH derivative 17
which was benzylated (92%) and subjected to a regioselective
BH3-reductive ring opening reaction24 (80%) to fashion the
requisite 6-OH acceptor 8.
acceptor 8 in the presence of AgOTf at −30 °C to cleanly
afford the desired α-linked disaccharide 4 as a single isomer in
64% yield. Selective removal of the phthalimide protecting
group using ethylene diamine in n-BuOH at an elevated
temperature (95 °C)25 and its subsequent EDC-HOBT26
mediated coupling with acid 13 afforded the fully protected
trisaccharide 2 (82% over two steps). Global deprotection of 2
via azide to NHAc conversion and hydrolysis of the tert-butyl
ester, followed by hydrogenolytic removal of benzyl groups and
purification on Sephadex G-25, furnished the target trisacchar-
ide 1 (84% over three steps). Target molecule 1 and all the
1
synthetic intermediates were thoroughly characterized by H,
13C, and 2D NMR as well as HRMS (see Supporting
Information).
In conclusion, we have reported the first total synthesis of a
Ch HF-PS trisaccharide repeating unit from B. cereus. The
glycan is equipped with a β-O-linked aminopropyl linker at the
reducing end to allow for further attachment to carrier proteins.
Our convergent approach also enables rapid synthesis of the
rare sugar containing disaccharide. Immunological studies of 1
and its derivatives may provide valuable insight regarding the
feasibility of developing a carbohydrate-based vaccine and
serodiagnostic markers for B. cereus.
Assembly of the target molecule, its functional group
transformation, and global deprotection are delineated in
Scheme 5. The key AAT thioglycoside 7 was converted to
the corresponding glycosyl bromide in situ and treated with
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ASSOCIATED CONTENT
* Supporting Information
Experimental details and procedures, compound character-
ization data, and copies of 1H and 13C spectra for new
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
■
S
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AUTHOR INFORMATION
Corresponding Author
■
(18) Demchenko, A. V. General aspects of the glycoside bond
formation. In Handbook of Chemical Glycosylation: Advances in
Stereoselectivity and Therapeutic Relevance; Demchenko, A. V., Ed.;
Wiley-VCH: 2008; pp 1−27.
*E-mail: suvarn@chem.iitb.ac.in.
Notes
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and Biochemistry; Horton, D., Ed.; Academic Press: San Diego, 1994;
Vol. 50, p 21.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(20) Kulkarni, S. S.; Gervay-Hague, J. Glycosyl Chlorides, Bromides
and Iodides. In Handbook of Chemical Glycosylation: Advances in
Stereoselectivity and Therapeutic Relevance; Demchenko, A. V., Ed.;
Wiley-VCH: 2008; pp 59−93.
We thank the Department of Science and Technology (Grant
No. SR/S1/OC-40/2009), Council of Scientific and Industrial
Research (Grant No. 01(2376)/10/EMR-II), and Board of
Research in Nuclear Sciences (Grant No. 2013/37C/51/
BRNS) for financial support. A.R.P. thanks CSIR-New Delhi
for a fellowship.
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