Total Synthesis of the Lipid-Anchor-Attached Core Trisaccharides of Lipoteichoic Acids of Streptococcus pneumoniae and Streptococcus oralis Uo5

Article abstract of DOI:10.1021/acs.orglett.9b04264

Herein we report an efficient total synthesis of lipid-anchor-appended core trisaccharides of lipoteichoic acids of Streptococcus pneumoniae and Streptococcus oralis Uo5. The key features include the expedient synthesis of the rare sugar 2,4,6-trideoxy-2-acetamido-4-amino-d-Galp building block via one-pot sequential S_N2 reactions and the α-selective coupling of d-thioglucoside with the diacyl glycerol acceptor to construct a common disaccharide acceptor, which was utilized in the total synthesis of target molecules 1 and 2.

Full text of DOI:10.1021/acs.orglett.9b04264

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Total Synthesis of the Lipid-Anchor-Attached Core Trisaccharides of
Lipoteichoic Acids of Streptococcus pneumoniae and Streptococcus
oralis Uo5
Balasaheb K. Ghotekar, Ananda Rao Podilapu, and Suvarn S. Kulkarni*
Department of Chemistry, Indian Institute of Technology Bombay, Mumbai 400076, India
S
* Supporting Information
ABSTRACT: Herein we report an efficient total synthesis of
lipid-anchor-appended core trisaccharides of lipoteichoic acids
of Streptococcus pneumoniae and Streptococcus oralis Uo5. The
key features include the expedient synthesis of the rare sugar
2,4,6-trideoxy-2-acetamido-4-amino-D-Galp building block via
one-pot sequential SN2 reactions and the α-selective coupling
of D-thioglucoside with the diacyl glycerol acceptor to
construct a common disaccharide acceptor, which was utilized
in the total synthesis of target molecules 1 and 2.
acteria often contain unique deoxy amino glycans on their
surfaces that are absent in host cells.1 The bacterial rare-
core trisaccharide 1 itself displays interesting immunological
properties.9 S. oralis is rarely associated with diseases such as
endocarditis.10,11 The biological activity of 1 and structural
similarities between 1 and 2 make them attractive synthetic
targets.
B
sugar-containing carbohydrates play important roles in the
bacterial growth, survival and motility, entry of pathogen,
infection, as well as the immune response.2 For example, the
Gram-positive bacteria Streptococcus pneumoniae and Strepto-
coccus oralis Uo5 both contain lipoteichoic acids (LTAs) as
major components of the cell wall, with core trisaccharides 1
and 2 composed of a common disaccharide unit containing a
rare 2,4,6-trideoxy-2-acetamido-4-amino-D-Galp (AAT) sugar
unit α-linked to diacylglycerol (Figure 1).3 Streptococcus
The synthesis of trisaccharides 1 and 2 poses two main
challenges: the synthesis of a rare-sugar AAT building block
and the installation of a 1,2-cis linkage in the glucosylation with
the diacylglycerol acceptor at the reducing end. The first total
synthesis of the LTA of S. pneumoniae was reported by
Schmidt and coworkers in 2010.12 The heroic synthesis of
lipid-anchored LTA nonasaccharide relied on glycosylation
with trichloroacetimidate donors, and they synthesized 1 with
myristic acid lipid chains en route to the synthesis of LTA.9
The acyl chains on the glycerol were introduced in a late stage,
and the rare AAT sugar was synthesized from D-glucosamine.
One of the key steps in their synthesis of the core trisaccharide
unit 1 was the incorporation of the reducing-end α-glucosidic
linkage with cyclohexylidene-protected glycerol, with moderate
stereoselectivity (α/β 3.2:1). The structure of the LTA of S.
oralis Uo5 has recently been elucidated,3a and the total
synthesis of the lipid anchor trisaccharide 2 has not been
reported so far. Both 1 and 2 have been shown to contain
palmitic acid as a major lipid component. We have developed
an efficient protocol to synthesize rare bacterial amino deoxy
building blocks via highly regioselective one-pot SN2 displace-
ments of 2,4-bis-triflates derived from D-mannose.13 The
protocol has been successfully applied to the synthesis of a
variety of bacterial glycoconjugates.14,15
Figure 1. Target molecules.
pneumoniae causes a variety of life-endangering diseases such
as severe infection in the upper respiratory track, pneumonia,
bacteremia, and meningitis, thereby resulting in high mortality
rates.4−6 The teichoic acid of Streptococcus pneumoniae
performs a crucial role in cell division, cation homeostasis,
and the regulation of cell-wall elongation.7,8 Moreover, the
Received: November 29, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.9b04264
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Herein we report an extension of our protocol to synthesize
the appropriately protected AAT thioglycoside building block
and its coupling to a D-glucoside to obtain the common
disaccharide. Chain elongation of the common disaccharide
unit with D-glucoside and D-galactoside donors paved the way
to an efficient synthesis of 1 and the first total synthesis of 2.
As shown in the retrosynthetic strategy (Scheme 1), the
target molecules 1 and 2 can be synthesized from fully
flate.13 The α-linked D-glucose acceptor 9 can be assembled
from the stereoselective glycosylation of thioglycoside donor
10 and linker derivative 11 as an acceptor using solvent
participation, followed by Nap deprotection. Glucose donor 10
and linker acceptor 11 could be prepared from commercially
available D-glucose and D-mannitol, respectively.
Our synthesis began with the preparation of D-glycerol
acceptor 11 (Scheme 2). The known D-mannitol derived diol
Scheme 1. Retrosynthetic Strategy for Target Molecules 1
and 2
Scheme 2. Preparation of Glycerol Acceptor 11
1216 on esterification using palmitic acid, DCC, and cat.
DMAP delivered compound 13 in 82% yield. The cleavage of
benzyl ether by using H2 (1 atm) in the presence of Pd(OH)2
afforded linker acceptor 11 in 88% yield.
The rare sugar AAT thioglycoside donor 8 was synthesized
along the lines of our established one-pot protocol (Scheme
3).13 A highly regioseletive naphthylmethylation of triol 14
Scheme 3. Synthesis of Orthogonally Protected AAT
Building Block 8
using Bu2SnO furnished the desired O3-Nap derivative 15.15
Previously, we have used compound 15 for the synthesis of the
D-fucosamine derivative through a one-pot sequential
nucleophilic displacement reaction using TBAN3 and
TBANO2.15 The same diol 15 was employed in the synthesis
of AAT building block 16 by using TBAN3 and CbzNH2.
Accordingly, diol 15 was first converted to the corresponding
2,4-bis-triflate by the treatment with Tf2O in pyridine, and the
crude bis-triflate, upon a brief workup, was as such 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 indicated by TLC, CbzNH2 was
added in the same pot at room temperature to displace the
remaining C4-OTf to afford the desired orthogonally protected
compound 16 in 60% yield over three steps. The
regioselectivity observed in the displacement of triflates is in
accordance with the recently proposed Hale−Hough−
Richardson rules for the nucleophilic displacement of
pyranosidic O-triflates.17 The reaction works well on 0.2 g
protected trisaccharides 3 and 4 by the selective removal of the
chloroacetyl group, followed by the hydrogenolytic removal of
Bn, Cbz, as well as benzylidine groups and the conversion of
NHTCA to NHAc in a single step. Trisaccharide 3 and 4 can
be assembled by [1 + 2] glycosylation in a convergent manner
by the coupling of easily accessible donors 5 and 6 individually
with the common disaccharide 7. The β-selectivity in
glycosylation could be ascertained by the installation of a
participating chloroacetyl group at the C2 position, which is
orthogonal to the palmitic esters. The disaccharide acceptor 7,
in turn, could be synthesized by the glycosylation of a suitably
protected AAT donor 8 with glucose acceptor 9, followed by
the removal of the temporary 2-naphthylmethyl (Nap)
protection. The key AAT building block 8 could, in turn, be
synthesized starting from commercially available D-mannose
via a multistep sequence involving a one-pot double serial SN2
displacement of the corresponding D-rhamnosyl 2,4-bistri-
B
DOI: 10.1021/acs.orglett.9b04264
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Letter
scale, but the yield dropped in scale-up operations. This
limitation could be circumvented by setting up multiple small-
scale reactions at a time in parallel to get the desired quantity
of compound 16. The reduction of azide 16 to amine using Zn,
AcOH condition, followed by trichloroacetyl chloride
protection afforded AAT 8 in 71% yield over two steps.
For the synthesis of the right-hand sugar unit 9, easily
accessible 2,3-diol 1718 was first converted to the correspond-
ing tin ketal and further reacted with 2-naphthylmethyl
bromide and TBAB at 80 °C to afford the O3-Nap compound
18, in high regioselectivity, as a single isomer in 70% yield over
two steps (Scheme 4). Benzylation of the remaining 2-OH
Scheme 5. Synthesis of Disaccharide Acceptor
Scheme 4. Synthesis of α-Glucopyranoside Acceptor
Scheme 6. Synthesis of D-Glucosyl and D-Galactosyl Donors
group in 18 using benzyl bromide and NaH conditions
furnished donor 10 in 84% yield. The solvent participation
effect was explored here to obtain α-selectivity using Et2O as
the cosolvent.19 The glycosylation of thioglycoside donor 10
under NIS/TMSOTf activation conditions in a CH2Cl2/Et2O
solvent mixture (1:1) with acceptor 11 at 0 °C afforded the
desired α-linked product 19 in 78% yield as a single isomer. In
the absence of ether, the glycosylation furnished 19 in 75%
yield in 9:1 α/β selectivity. The characteristic NMR signals
(1H NMR δ 4.76 (d, 1H, J1,2 = 3.6 Hz, H-1), 13C NMR δ 98.8)
confirmed the α-linkage of the newly formed glycosidic bond
in 19. The selective deprotection of the Nap group using DDQ
under Bennett’s conditions20 gave compound 9 in 81% yield. It
should be noted that use of β-pinene in this reaction increased
the yield from 64 to 81%.
After successfully synthesizing acceptor 9, we went ahead
with further glycosylation. Initial attempts to directly
glycosylate AAT thioglycoside 8 with acceptor 9 under NIS
and TMSOTf promotion at 0 and −30 °C met little success, so
we resorted to the imidate donor. For that, thioglycoside donor
8 was first converted to its corresponding hemiacetal by NBS
and THF/H2O and was further treated with Cl3CCN and
DBU in CH2Cl2 to afford imidate donor 20 in 82% yield over
two steps (Scheme 5). The glycosylation of imidate donor 20
with acceptor 9 using TMSOTf at −30 °C resulted in the
formation of disaccharide 21 in 74% yield. Compound 21 on
Nap deprotection using DDQ furnished disaccharide acceptor
7 in 69% yield
benzylidene ring opening using BH3·THF21 furnished 6-OH
compound 22 in 88% yield. Compound 22 upon treatment
with FmocCl and pyridine afforded glucose donor 23.
Alternatively, the benzylation of 22 using benzyl imidate and
TfOH furnished glucose donor 5 in 71% yield. On similar lines
(Scheme 6b), the chloroacetylation of the 2-OH group of
compound 2514g and its subsequent regioselective, borane-
reductive benzylidene ring opening at O6 afforded 6-OH
derivative 26 in 91% yield. The 6-OH group of 26 was
subjected to levulinate protection using LevOH, EDC·HCl,
and DMAP conditions to get glucose donor 27 in 93% yield.
On the contrary, the benzylation of 26 using benzyl imidate
and TfOH gave O6-benzylated galactosyl donor 6 in 76%
yield. It should be noted that the Fmoc protection of 26 also
proceeded well to give the corresponding O6-Fmoc-protected
donor. However, the donor was not able to couple to the
acceptor 7, presumably due to the steric hindrance imposed by
the axially oriented 4-OBn group in the galactose series that
prohibits free rotation of the bulky Fmoc group.
The β-thioglucoside donors 23 and 5 were prepared as
shown in Scheme 6a. Chloroacetylation of the 2-OH group of
easily accessible 2018 using chloroacetyl chloride and pyridine
afforded the corresponding fully protected β-thiophenyl
glucoside (92%), which upon TMSOTf-catalyzed reductive
The completion of the synthesis of lipid anchor-attached
trisaccharides 1 and 2 employing the common disaccharide
C
DOI: 10.1021/acs.orglett.9b04264
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Scheme 7. Synthesis of Trisaccharides 1 and 2
acceptor 7 is delineated in Scheme 7. The glycosylation of
thioglycoside glucose donor 5 and galactose donor 6,
individually, under NIS/TMSOTf activation conditions in
CH2Cl2 as a solvent with common disaccharide acceptor 7 at 0
°C afforded the desired glucose trisaccharide product 3 (77%)
and galactose trisaccharide 4 (73%), respectively. Because the
O6′′-phosphitylated intermediate is a key intermediate in the
total synthesis of the LTA of Streptococcus pneumonia, we
required O6′′ orthogonally protected trisaccharide.9,12 For this
purpose, thioglucoside donor 23 and disaccharide acceptor 7
were glycosylated under NIS and TMSOTf conditions to
furnish the fully protected trisaccharide 28. Triethylamine was
added to the reaction mixture to quench the TMSOTf and
simultaneously remove the Fmoc group to afford 6-OH′′
trisaccharide 29 in 66% yield over two steps in a one-pot
manner. Similarly, the glycosylation of D-galactose donor 27
with disaccharide acceptor 7 using NIS and TMSOTf as a
promoter at 0 °C gave the β-linked trisaccharide 30 in 91%
yield. The levulinoyl group was selectively cleaved by using
NH2NH2·H2O and Py/AcOH (4:1) to afford 6-OH′′
trisaccharide 31 in 88% yield. The selective removal of the
chloroacetyl group in 3 and 4 by using thiourea in pyridine and
the concomitant hydrogenolysis using H2 and 20 wt %
Pd(OH)2/C in EtOH furnished target molecules 1 and 2 in 79
and 81% yield over two steps, respectively (Scheme 7).
steps starting from D-mannose, with an overall yield of 4.6% for
1 and 4.5% for 2. Schmidt’s route for the synthesis of 1
involved 19 steps from D-glucosamine with an overall yield of
0.76%. Efforts to assemble LTAs and study the immunological
properties of 1 and 2 are currently underway.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.orglett.9b04264.
Experimental procedures, characterization data of
synthetic compounds, and 1D and 2D NMR spectra
(PDF)
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: suvarn@chem.iitb.ac.in
ORCID
Suvarn S. Kulkarni: 0000-0003-2884-876X
Notes
The authors declare no competing financial interest.
The phosphitylation of trisaccharides 29 and 31 would allow
the synthesis of the LTA of S. pneumoniae and S. oralis Uo5.
In conclusion, we synthesized the O3-Nap-protected rare
sugar AAT thioglycoside in an expedient manner from D-
mannose and glycosylated it with a 1,2-cis-linked glucoside to
construct a common disaccharide that was, in turn, utilized to
complete an efficient synthesis of 1 and the first total synthesis
of 2. Our synthetic route entails a longest linear sequence of 20
ACKNOWLEDGMENTS
■
S.S.K. thanks the Science and Engineering Research Board
(grant no. CRG/2019/000025) and the Department of
Biotechnology (BT/INF/22/SP23026/2017) for financial
support. B.K.G. and A.R.P. thank the University Grants
Commission and the Council of Scientific and Industrial
Research, respectively, for research fellowships.
D
DOI: 10.1021/acs.orglett.9b04264
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Letter
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DOI: 10.1021/acs.orglett.9b04264
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