Total synthesis of repeating unit of O-polysaccharide of Providencia alcalifaciens O22 via one-pot glycosylation

Article abstract of DOI:10.1021/acs.orglett.7b02791

The first total synthesis of the phosphorylated trisaccharide repeating unit of Providencia alcalifaciens O22 is reported. The trisaccharide contains rare deoxyamino sugar AAT at the reducing end and D-glyceramide 2-phosphate at the other end. The efficie

Full text of DOI:10.1021/acs.orglett.7b02791

Letter
pubs.acs.org/OrgLett
Total Synthesis of Repeating Unit of O‑Polysaccharide of Providencia
alcalifaciens O22 via One-Pot Glycosylation
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 the phosphorylated
trisaccharide repeating unit of Providencia alcalifaciens O22 is
reported. The trisaccharide contains rare deoxyamino sugar
AAT at the reducing end and D-glyceramide 2-phosphate at the
other end. The efficient synthesis involves one-pot assembly of
trisaccharide and late-stage phosphorylation as key steps.
Providencia is a genus of Gram-negative, rod-shaped bacteria
belonging to the family of Enterobacteriaceae. It is subdivided
into eight species including P. alcalifaciens, P. stuartii, P. rettgeri,
P. rustigianii, P. heimbachae, P. vermicola, P. sneebia, and P.
burhodogranariea.1 These species have been identified and
isolated from sputum, urine, perineum, axilla, stool, blood, and
wound specimens of humans as well as from other animals and
from soil and water sources.2 Medically important species,
including P. alcalifaciens, P. rustigianii, P. stuartii, and P. rettgeri,
are mostly associated with urinary tract infections and enteric
diseases.3 These opportunistic pathogens have also been
implicated in pericarditis,4 endocarditis,5 meningitis,6 and
ocular7 infections. P. alcalifaciens is particularly known to
cause diarrhea in children and travelers.3,8 Studies have shown
that some strains of P. alcalifaciens are invasive to intestinal
mucosa and other cell types in eukaryotes.9 Thus, accurate and
speedy diagnosis and effective vaccines are urgently required to
combat this pathogen. In this regard, the O-polysaccharides (O-
antigens) present on the cell surface of Gram-negative bacteria
which define serospecificity of strains and are used for the
serotyping of bacteria are important.
having to be conjugated to a carrier protein and elicit a stronger
immune response.11 Thus, the phosphorylated trisaccharide
repeating unit of P. alcalifaciens O22 is an important synthetic
target. Our laboratory has been involved in the synthesis of rare
sugar-containing bacterial glycoconjugates.12 Herein, we report
the first total synthesis of the repeating unit of P. alcalifaciens
O22 1 via one-pot glycosylation.
The challenges encountered in the synthesis of phosphory-
lated trisaccharide 1 are the procurement of the orthogonally
protected rare sugar AAT and phosphorylation of the
secondary alcohol adjacent to the amide functionality in the
D-glyceramide unit. Our retrosynthetic scheme is shown in
Figure 1. The target molecule 1 could be synthesized by global
deprotection of fully functionalized phosphorylated trisacchar-
ide 2. At the very outset, it was envisioned that the assembly of
the trisaccharide 3 could be carried out in a one-pot manner.
This would demand a careful selection of the protecting groups
not only to maintain the orthogonality but also to fine-tune the
electronic properties of the glycosyl donors and acceptors.13
Accordingly, a 9-fluorenylmethyloxycarbonyl (Fmoc) group
was employed as a temporary protecting group which could
potentially be selectively removed post glycosylation in the
same pot to obtain the 3-OH′′ trisaccharide 3, which could be
coupled with H-phosphonate 4 to obtain 2. The H-
phosphonate 4 could in turn be obtained from D-mannitol,
whereas the rare AAT building block 5 could be accessed
starting from cheaply available D-mannose via a one-pot double
serial SN2 displacement of the corresponding D-rhamnosyl 2,4-
bistriflate.12a Lastly, building blocks 6 and 7 could be obtained
by regioselective protection of D-galactose and D-glucosamine.
The β-selectivity in glycosylation would be controlled by
placement of participating groups (OBz, NHTroc, NHTCA) at
the C2 position in all of the building blocks 5−7. The NH2
Recently, Ovchinnikova and co-workers10 isolated a new
phosphorylated O-polysaccharide of the S-form lipopolysac-
charide (LPS) from P. alcalifaciens O22. The trisaccharide
repeating unit consists of two unusual components, 2-
acetamido-4-amino-2,4,6-trideoxy-D-galactose (D-FucNAc4N,
AAT) and D-glyceramide 2-phosphate (D-GroAN-2-P), the
latter being identified for the first time in bacterial
polysaccharides. The structure of the trisaccharide repeating
unit of the O-polysaccharide was established to be -4)-(D-
GroAN-2-P-3-)-β-D-GalNAc-(1−4)-β-D-Gal-(1−3)-β-D-Fuc-
NAc4N-(1-. Notably, the phosphate group and the free amino
group of the AAT residue imparts a zwitterionic character to
the O-polysaccharide. Over past few years, there has been great
interest in harnessing the immunological potential of bacterial
zwitterionic polysaccharides (ZPS) for vaccine development.
Such ZPS have been shown to directly activate T-cells without
Received: September 6, 2017
© XXXX American Chemical Society
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DOI: 10.1021/acs.orglett.7b02791
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Scheme 1. Synthesis of Rare Sugar AAT Building Block 5
17
group of 11 using NaBrO3 and Na2S2O4 furnished the key
AAT building block 5 (76%).
Our synthesis of the orthogonally protected β-thiogalactoside
donor 6 is outlined in Scheme 2. The easily accessible 2,3-diol
Scheme 2. Synthesis of 4-OH Thiogalactoside Donor 6
Figure 1. Retrosynthetic analysis.
group in 7 would be protected as NHTroc to enhance the
reactivity14 of O3 position, whereas TCA would be used to
mask the NH2 group in 5, as Troc would not be stable under
oxidative debenzylation conditions. Similarly the NH2 group of
D-glyceramide would be protected as N(Bn)2 so as to oblate the
hydrogen bonding and enhance the reactivity of the secondary
OH for phosphorylation. With these considerations we began
the total synthesis of 1.
Our synthesis started with procurement of the orthogonally
protected rare deoxy amino sugar building block 5 from D-
mannose along the lines of our earlier established protocol
(Scheme 1).12a Accordingly, the known D-rhamnosyl 2,4-diol
812g was first converted to the corresponding 2,4-bis-triflate by
treatment with Tf2O in pyridine. The crude triflate obtained
after a brief aqueous workup was subjected to a regioselective
C2-OTf displacement with a stoichiometric amount of
tetrabutylammonium azide (TBAN3)12,15 at −30 °C. After
complete conversion of the starting material as observed from
TLC analysis, benzyl carbamate was added in the same pot at rt
to displace the remaining C4-OTf to afford the desired
orthogonally protected AAT building block 9 in 61% yield over
three steps, after a single chromatographic purification. The
regioselectivity observed in the one-pot reaction can be nicely
explained by invoking Richardson−Hough rules for SN2
displacements of pyranosidic aryl and alkyl sulfonates, which
were recently updated for pyranoside and furanoside O-triflates
by Hale and co-workers.16 Reduction of azide in 9 to amine
using PPh3 followed by trichloroacetyl protection furnished
thioglycoside donor 10 in 78% yield over two steps.
Glycosylation of donor 10, with 2-propanol using NIS,
AgOTf as a promoter at −40 °C gave β-linked O-isopropyl
glycoside 11 in 86% yield. Oxidative removal of the benzyl
12, was first converted to the corresponding tin ketal by
treatment with dibutyltin oxide in toluene at 110 °C and
further reacted with benzyl bromide and tetrabutylammonium
bromide (TBAB) at 60 °C to afford the 3-OBn derivative 13, in
high regioselectivity, as a single isomer in 81% yield over two
steps. Benzoylation of the remaining 2-OH group in 13 using
benzoyl chloride and pyridine afforded the corresponding fully
protected β-thiophenyl galactoside which upon reductive
benzylidene ring opening using TFA and Et3SiH furnished 4-
OH thiogalactoside donor 6 in 81% yield.
Since D-galactosamine is expensive, we opted for its C4
epimer D-glucosamine as a cheap and abundant starting
material (Scheme 3). First, commercially available D-glucos-
amine was rapidly converted to the known NHTroc derivative
14, following the procedure reported by Galan.18 Regioselective
ring opening of 14 using Et3SiH and TFA furnished 4-OH
derivative 1518 in 82% yield. Triflation of 15 using Tf2O in
B
DOI: 10.1021/acs.orglett.7b02791
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donor 7 in the same pot in the presence of NIS/TMSOTf
promoter led to the formation of the corresponding fully
protected trisaccharide. Et3N was added to the reaction mixture
to quench the TfOH and simultaneously remove the Fmoc
group to afford 3-OH′′ trisaccharide 3 in 72% yield over three
steps, in a one-pot manner. The structural assignment of 3 was
Scheme 3. Synthesis of D-Galactosamine Donor 7
1
1
performed using H NMR, 13C NMR, H−13C HSQC, and
1H−1H COSY analysis (see the SI). Prior to conducting the
one-pot glycosylation, the corresponding disaccharide and
trisaccharide intermediates were isolated and characterized by
spectral means and then used as TLC standards during the one-
pot glycosylation.
For the synthesis of D-glyceramide building block 20, we
started with readily accessible dicyclohexylidene D-mannitol
(Scheme 5).19 Oxidation of diol using TEMPO and PhI(OAc)2
Scheme 5. Synthesis of D-Glyceramide Derivative 20
pyridine, followed by C4 inversion via intramolecular displace-
ment of 4-OTf by 3-OAc furnished the 3-OH D-galactosamine
derivative 16 in 91% yield over two steps. In this water-
mediated transformation, the C3-OAc migrates to C4-position
via orthoester type transition state. Fmoc protection of the
remaining 3-OH group in 16 using FmocCl and pyridine
afforded the corresponding fully protected β-thio galactosamine
donor 7 in 90% yield. All of the monosaccharide building
blocks were thoroughly characterized by in-depth 2D NMR
spectral analysis (see the Supporting Information).
With all the desired building blocks 5, 6 and 7 in hand, the
stage was set for one-pot glycosylation (Scheme 4).
Accordingly, a highly regioselective glycosylation of 4-OH
thiophenyl galactoside donor 6 and 3-OH AAT acceptor 5
using NIS/TMSOTf as the promoter cleanly afforded the
corresponding 4-OH′ disaccharide in 1 h. Addition of glycosyl
Scheme 4. One-Pot Assembly of Trisaccharide 3
afforded cyclohexylidene-protected D-glyceric acid, which upon
brief workup was as such coupled with benzylamine using DCC
and HOBt to obtain amide 18 in 62% yield over two steps. N-
Benzylation of 18 using NaH and BnBr followed by removal of
the cyclohexylidene group using 80% AcOH gave diol 19
(80%). Selective monobenzylation of the diol was achieved by
first converting it to the corresponding tin ketal by treatment
with dibutyltinoxide in toluene at 110 °C and further reacting
with benzyl bromide and TBAB at 60 °C to afford the requisite
2-OH D-glyceramide derivative 20, with high regioselectively, as
a single isomer in 90% yield over two steps.
As anticipated, phosphorylation of the D-glyceramide turned
out to be a difficult task. As shown in Scheme 6, the D-
glyceramide derivative 20 could be successfully phosphorylated
using imidazole, PCl3, and Et3N to afford H-phosphonate 4 in
89% yield, which was further coupled with trisaccharide 3 in the
presence of pivaloyl chloride and pyridine followed by
oxidation with I2 to furnish the phosphorylated trisaccharide
2 in 64% yield over two steps. Global deprotection of 2 was
done in three steps. First, NHTCA and NHTroc were
converted to NHAc by using Zn, AcOH, and Ac2O, followed
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Scheme 6. Phosphorylation and Global Deprotection
ACKNOWLEDGMENTS
■
We thank the Department of Science and Technology (Grant
No. EMR/2014/000235). A.R.P. thanks CSIR-New Delhi for a
fellowship.
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ASSOCIATED CONTENT
* Supporting Information
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1
terization data, and H and 13C spectra for all new
compounds (PDF)
AUTHOR INFORMATION
■
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Corresponding Author
*E-mail: suvarn@chem.iitb.ac.in.
ORCID
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Suvarn S. Kulkarni: 0000-0003-2884-876X
Notes
The authors declare no competing financial interest.
D
DOI: 10.1021/acs.orglett.7b02791
Org. Lett. XXXX, XXX, XXX−XXX