A Versatile Synthesis of Pentacosafuranoside Subunit Reminiscent of Mycobacterial Arabinogalactan Employing One Strategic Glycosidation Protocol

Article abstract of DOI:10.1002/chem.201704009

Oligosaccharides are involved in a myriad of biological phenomena. Many glycobiological experiments can be undertaken if homogenous and well-defined oligosaccharides are accessible. Mycobacterial cell walls contain arabinogalactan as one of the major constituents that is challenging for chemical synthesis. Therefore, the major aim of this investigation is to synthesise a major oligosaccharide portion of the arabinogalactan. The pentacosafuranoside (25mer) synthesis involved installation of several arabinofuranosidic linkages through neighbouring group participation for 1,2-trans linkages and oxidation-reduction strategy for the 1,2-cis Araf. A strategically placed n-pentenyl moiety at the reducing end enables ligation of biomolecular probes through celebrated cross metathesis or thiol-ene click reactions. Several linear and branched oligosaccharides were synthesised ranging from trisaccharide to pentadecasaccharide during this endeavour. Synthesis of pentacosasaccharide was accomplished in 77 steps with 0.0012 % overall yield. These oligosaccharides are envisioned to be excellent probes for understanding disease biology thereby facilitating discovery of novel antitubercular agents, vaccines and/or diagnostics.

Full text of DOI:10.1002/chem.201704009

A Journal of
Accepted Article
Title: A Versatile Synthesis of Pentacosafuranoside Subunit
Reminiscent of Mycobacterial Arabinogalactan Employing One
Strategic Glycosidation Protocol
Authors: Sandip Pasari, Sujit Manmode, Gulab Walke, and Srinivas
Hotha
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To be cited as: Chem. Eur. J. 10.1002/chem.201704009
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10.1002/chem.201704009
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A Versatile Synthesis of Pentacosafuranoside Subunit
Reminiscent of Mycobacterial Arabinogalactan Employing One
Strategic Glycosidation Protocol
Sandip Pasari, Sujit Manmode, Gulab Walke and Srinivas Hotha*
Abstract: Oligosaccharides are involved in myriad of biological
phenomena. Many glycobiological experiments can be undertaken if
homogenous and well defined oligosaccharides are accessible.
Mycobacterial cell wall contains arabinogalactan as one of the major
constituents that is challenging for the chemical synthesis. Therefore,
the major aim of this investigation is to synthesize a major
oligosaccharide
portion
of
the
arabinogalactan.
The
pentacosafuranoside (25mer) synthesis involved installation of
several arabinofuranosidic linkages through the neighbouring group
participation for 1, 2-trans linkages and oxidation-reduction strategy
for the 1, 2-cis Araf respectively. Strategically placed n-pentenyl
moiety at the reducing end shall enable ligation of biomolecular
probes through celebrated cross metathesis or thiol-ene click
reactions. Several linear and branched oligosaccharides were
synthesized ranging from trisaccharide to pentadecasaccharide
during this endeavor. Synthesis of pentacosasaccharide was
accomplished in 77 steps with 0.0012% overall yield. These
oligosaccharides are envisioned to be excellent probes for
understanding disease biology thereby facilitating discovery of novel
anti-tubercular agents, vaccines and/or diagnostics.
Introduction
Figure 1. Cartoon representation of the mycobacterial cell wall and the
arabinogalactan
Tuberculosis (TB) still remains as a global health hazard in spite
of it being known for more than 132 years, 122 years since the
first vaccine trial and 96 years after the discovery of Bacille
Calmette-Guérin (BCG) vaccine.[1-4] Mycobacterial infections
have been receiving special attention recently due to the
increased incidence and emergence of multidrug-resistant
(MDR-TB) and extensively drug-resistant (XDR-TB) strains of
Mycobacterium tuberculosis (MTb), the organism that causes
TB.[5],[6] Usually, treatment of drug-susceptible tuberculosis
requires chemotherapy with multiple antibiotics administered
and galactose are in the furanosyl form.[13] Most of the
glycosidic linkages are in 1,2-trans or α-fashion between the
Araf-Araf or Galf-Galf or Araf-Galf except the terminal four which
are 1,2-cis or β-Araf residues which are in turn linked to the C2-
position of the penultimate 1,2-trans or α-Araf residue. All β-Araf
residues are esterified at the C-5 positions with mycolic acid.[13-
14]
The galactan portion was composed of β-D-Galf-(1→5)-β-D-
Galf-(1→6)-β-D-Galf- repeating unit containing pendant arabinan
polysaccharide at three sites through the C-5-D-Galf.[13] Access
to homogenous synthetic oligosaccharides could significantly
augment the discovery of carbohydrate-based vaccines and
diagnostics.
Unlike peptides and nucleotides, the synthesis of large
oligosaccharides still remains as a significant challenge although
many elegant methodologies for chemical glycosidation are
developed. Synthesis of oligosaccharides containing more than
20 saccharides in a stereoselective fashion is a herculean
task;[17,18] and as a result, fine tuning of the compatible glycosyl
donor chemistry is decisive for the success of any multi-step
over prolonged periods.
A number of researchers have
attributed such long drawn chemotherapy to the unusually dense,
waxy, and unique glycocalyx that acts as a robust permeability
barrier to the antibiotics.[7-11] The uniquely dense glycocalyx
architecture comprises trehalose, mycolic acid, D-arabinan, D-
galactan, D-mannan, linker disaccharide and a peptidoglycan.[12]
One of the major structural components of the glycocalyx is the
mycolyl arabinogalactan (AG) polysaccharide wherein the
mycolic acid (a cyclopropanated fatty acid) is esterified at the
non-reducing end of the arabinan; whereas, the reducing end is
attached as a pendant to the galactan (Figure 1).[13-16] Finer
structure of the mycolyl glycocalyx illustrated that the arabinose
assembly of oligosaccharides.
Nevertheless, synthesis of
polylactosamine with 25-saccharide residues by Ogawa[19] and
O-specific polysaccharide containing 24-residues by Pozsgay[20a]
were the conspicuous early efforts towards the synthesis of
large oligomers. Of mycobacterial origin, mannose-capped LAM
fragment by Fraser-Reid,[20b] independent syntheses of
Dr. Sandip Pasari, Mr. Sujit Manmode, Mr. Gulab Walke, Prof.
Srinivas Hotha, Department of Chemistry, Indian Institute of Science
Education and Research, Pune
–
411 008, MH, India,
s.hotha@iiserpune.ac.in, www.iiserpune.ac.in/~s.hotha
docosaarabinofuranoside
by
Lowary[21]
and
Ito,[18]
Supporting information for this article is given via a link at the end of
the document.
heneicosasaccharide (21-residues) of LAM[22] and AG[23]
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syntheses by our group are some of the noticeable efforts in the
literature that describe synthesis of large oligosaccharides.
Lowary invoked salient features of trichloroacetamidates and
thiotolyl donor chemistry whereas Ito utilized thioglycosides for
Both syntheses
accomplished the α-arabinofuranosylation by the neighbouring
group participation through the C-2 esters. β-
from Motifs A-E. In the retrosynthetic analysis, we envisioned
that the assembly of pentacosasaccharide (1) can be
comprehended by a stereoselective glycosidation between a
pentadecasaccharide (2) and two molar equivalents of a
pentasaccharide-carbonate (3) in a {2x5+15} fashion. The larger
component 2 was envisaged from two disaccharides 4, 5 and a
trisaccharide 6 (Scheme 1).
assembling the docosasaccharide.[18,21]
Arabinofuranosylation was accomplished by conformational bias
due to the silyl tethering in the Lowary’s synthesis whereas an
intramolecular aglycon delivery through NAP ether was utilized
in the Ito’s synthesis of docosasaccharide.[18],[21] Although
extensive research has been carried out on mycobacterial
arabinogalactan, very few studies exist which have conjugated
the arabinan moiety with the β-arabinofuranosides and the
galactan moiety until recently. While our studies are in progress,
a 92-mer arabinogalactan by Ye[24a] and a 50mer mannan using
automated synthesizer by Seeberger[24b] were reported.
The carbonate 3 was imagined from the disaccharide-
carbonate 7 and a diol 8. It is interesting to note that the
identified advanced building blocks are very similar to motifs A-E
as shown in Figure 2A. The highly convergent strategy would
enable (i) the impeccable quality control in every stage of the
synthesis and (ii) synthesis of enough quantity of the various
building blocks en route to the final assembly of the target
polysaccharide. Installation of the α-(1→x)-Araf linkage by the
[Au]/[Ag]-catalysed activation of alkynyl carbonate Araf donor
through neighbouring group participation was earlier reported by
our group.27 For the β-(1→2)-Araf linkage, we intended to use
the protocol that runs parallel to how mycobacterium
biosynthesizes the β-(1→2)-Araf linkage.[28] First, we install the
neighbouring group assisted 1,2-trans linkage on a Ribf-
derivative and subsequent saponification and oxidation-
reduction enables us to achieve desired 1,2-cis or β(1→2)Araf
linkage (Scheme 2).
Drawing upon these strands of research on the synthesis of
mycobacterial arabinogalactan, this study was therefore
conducted to synthesize a pentacosafuranoside containing 23-
Arafs and 2-Galf residues. A careful look at the pioneering work
by Brennan group identified five major structural fragments of
AG complex (Figure 2A).[13]
Later investigations on the
biosynthesis of AG showed that 1, 2-trans or α-linkages were
synthesized by arabinofuranosyl transferases (AraTs) and 1,2-
cis or β-arabinofuranosides are installed indirectly starting from
decaprenylphosphoryl D-ribofuranose as the substrate.[25],[26]
Fixing of the 1, 2-trans ribofuranoside with DprE1 followed by
oxidation-reduction with DprE2 transformed 1,2-trans Ribf into
1,2-cis Araf (Figure 2B).[25,26]
Scheme 2. Synthesis protocols for 1, 2-trans and 1, 2-cis Arafs
One furanosylation method, minimal number of complex
reactions and protecting groups are the key features of this
endeavour. With the identification of major motifs and generally
stereoselective arabinofuranosylation protocols, our exploration
towards the synthesis of pentacosasaccharide 1 started with the
synthesis of major structural motifs with appropriate protecting
groups in sufficient quantities.
Results and Discussion
Figure 2. Major glycan motifs (A) of M. tuberculosis cell wall and biosynthesis
of 1, 2-trans and 1, 2-cis arabinofuranosides (B)
Synthesis of pentaarabinofuranosyl donor 3: One of the most
complex portions of the pentacosasaccharide (1) is the terminal
pentasaccharide which requires installation of two β-Araf-(1→2)-
Araf linkages that are notorious for their difficulty. In the
retrosynthetic plan of this motif, a {2x2+1} furanosylation
between the glycosyl donor 7 and the diol acceptor 8 was
recognized to be the most convenient. The glycosyl donor was
set out easily from two starting substrates 9 and 10 (Scheme 3).
The synthetic effort started with a known arabinofuranoside 11[29]
whose isopropylidene group was opened with allyl alcohol
Therefore, major challenges in the synthesis of
pentacosasaccharide are identified as: (i) stereoselective
installation of Araf-(1→2)-Araf linkage; (ii) synthesis of Galf-
(1→6)-Galf with free C5-OH for the attachment of arabinan; (iii)
a robust furanosylation method that enables preparation of both
1, 2-trans and 1, 2-cis linkages; (iv) minimum number of different
reactions; and (v) final assembly of the pentacosasaccharide
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Scheme 1. Retrosynthetic analysis of pentacosasaccharide motif of arabinogalactan of Mycobacterium tuberculosis
arabinofuranosyl donor 10 by reacting the hemiacetal 14 with
the reagent 15 in very high yield (Scheme 4).
Earlier studies from our group showed that the anomeric ratio in
the donor does not significantly affect the overall outcome of the
glycosidation reaction.[27] Hence, the carbonate 10 was not
separated into individual isomers. In continuation, the [Au]/[Ag]-
catalyzed glycosidation between the donor 10 and 12α
underwent smoothly giving the desired diarabinofuranoside 16 in
67% yield as pale yellow syrup. At this stage, chemoselective
saponification of acetates in the presence of benzoates became
a herculean task. Both chemical and lipase mediated hydrolysis
of acetate ester failed to give required alcohol in sufficient
quantities that has prompted us take a detour and continue our
synthetic effort. Without much difficulty, orthoacetate 13 was
transformed to di-O-silyl ether 17 that was treated with allyl
alcohol and AuBr3 conditions to obtain allyl arabinoside 18 with
acetate at the C-2 position. In continuation, acetate was
saponified easily under Zemplén conditions[34] and protected
again with the levulinic acid under DIC/DMAP conditions to
obtain levulinoate 19. The next step in the sequence is to
convert the allyl glycoside into the hemiacetal 23; however,
under normal conditions, the Pd-catalyzed allyl deprotection
resulted in the formation of compounds 20-22. The formation of
the compound 20 can be envisaged from the migration of
levulinoyl group from C-2 position to the more reactive
hemiacetal position where the formation of bicyclic compound 21
was more intriguing.[36] Departure of the allyl glycoside can
result in the formation of an oxocarbenium ion intermediate that
Scheme 3. Retrosynthetic analysis of pentasaccharide
under acidic conditions to afford a separable mixture of allyl
arabinofuranosides 12α and 12β in 1:1 ratio which are utilized
as glycosyl acceptors later.[30] 1,2-cis Arabinofuranosylation was
envisioned through oxidation-reduction strategy, hence, the 1, 2-
trans furanosyl donor of Ribf was required; in addition, the
protecting group at the C-2 position of the Ribf-donor needs to
be orthogonal to the remaining appendages present in the
molecule. Unmasking of acetates in the presence of benzoate
esters was reported and the installation of the acetate at the C-2
position is relatively easier if we start with an orthoacetate 13.[31-
33]
Accordingly, the orthoacetate 13 was synthesized by
invoking a protocol developed in our group and transformed to
hemiacetal 14 by
a
gold-catalyzed glycosidation using
AuCl3/H2O/CH3CN at 25 ⁰C and subsequently converted to the
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could be trapped by the ketone intramolecularly to give bicyclic
21 or be trapped by the methanol to afford the compound 22.
Nevertheless, the problem was circumvented by inviting a
method that was reported through a ternary solvent system.
Accordingly, compound 19 was hydrolyzed successfully to
hemiacetal 23 by employing 1,2-DME-CH2Cl2-MeOH (3:1:1)/25
⁰C in 83% yield.[40] Subsequently, the hemiacetal 23 was
conveniently converted into the desired carbonate donor 24 by
aforementioned conditions (Scheme 4).
alcohol 26 that was oxidized smoothly to its 2-ulose derivative
27;28 however, the reduction of the ketone by various reducing
agents gave back the original starting alcohol 26. Hence, the
glycosidation between glycosyl donor 24 and the acceptor 12α
was carried out to obtain the disaccharide 28 under gold/silver-
catalyzed glycosidation conditions. The levulinoate deprotection
of 28 to obtain alcohol 29 followed by oxidation afforded 2-ulose
derivative 30 that underwent smooth reduction with NaBH4 in
EtOAc-EtOH (2:1) converted β-Ribf-(1→2)-Araf into a much
desired β-Araf-(1→2)-Araf 31.
In the 1H NMR spectrum of
compound 31, the two anomeric protons were noticed at δ 5.22
and 5.33 ppm as two singlets. Additionally, the 13C NMR
spectrum confirmed the presence of 1,2-cis and 1,2-trans
linkages by displaying resonances at δ 101.5 and 104.6 ppm
respectively. Successfully synthesized disaccharide 31 was
transformed into the carbonate donor 32 in three steps (Scheme
5).[36]
Scheme 4. Synthesis of building blocks for the terminal
pentasaccharide motif: Reagents: (a) PTSA (0.2 eq), allylOH,
CH2Cl2, 55 ⁰C, 3 h, 43% for 12β and 46% for 12α; (b) 8mol%
AuCl3, CH3CN, H2O, 25 °C, 2 h, 88%; (c) 15, CH2Cl2, DMAP,
25 °C, 4 h, 83% for 10 and 94% for 24 (α:β = 1:3); (d) 8mol%
each of chloro[tris(2,4-di-tert-butylphenyl)phosphite]gold and
AgOTf, CH2Cl2, 4Å MS powder, 25 °C, 30 min, 67%; (e) NaOMe
(0.3 eq), CH2Cl2-MeOH (1:1), 25 °C, 2 h; (f) TBDPSCl, Im., DMF,
0-25 °C, 4 h, 68% over two steps; (g) 10mol% AuBr3, allylOH,
CH2Cl2, 4Å MS powder, 25 °C, 3 h, 89%; (h) Levulinic acid, DIC,
DMAP, CH2Cl2, 0-25 °C, 2 h, 82% over two steps; (i) PdCl2 (0.2
eq), CH2Cl2-MeOH (1:4), 25 °C, 4 h, 31% for 21, 18% for 22,
36% for 23; (j) PdCl2 (0.2 eq), 1,2-DME/CH2Cl2/MeOH (3:1:1),
25 °C, 4 h, 83%.
Scheme 5. Synthesis of the key β-Araf-(1→2)-Araf donor: Reagents: (a)
8mol% each of chloro[tris(2,4-di-tert-butylphenyl) phosphite]gold and AgOTf,
CH2Cl2, 4Å MS powder, 25 °C, 40 min, 83% for 25, 81% for 28; (b) 70%
N2H4.H2O (4.5 eq), CH2Cl2, AcOH (22 eq), Py (27 eq), 25 °C, 2 h, 89% for 26,
92% for 29; (c) PDC (0.6 eq), Ac2O (3.3 eq), CH2Cl2, 25 °C, 5 h; (d) NaBH4
(1.2 eq), EtOH/EtOAc (2:1), 0 °C, 2 h, 78% for 31 over two steps; (e) Py,
PhCOCl, CH2Cl2, 0-25 °C, 2 h, 93%; (f) PdCl2 (0.2 eq), CH2Cl2-MeOH (1:4),
25 °C, 4 h; (g) 15, DMAP, CH2Cl2, 25 °C, 5 h, 79% over two steps.
Synthesis of desired building blocks set the stage for the
preparation of β-Araf-(1→2)-Araf disaccharide.
Earlier
investigations from our group showed that the stereoelectronic
factors around the glycosyl acceptor govern the stereoselectivity
of β-arabinofuranosylation.[37] So, initially, donor 24 was treated
with excess amount of sterically hindered glycosyl acceptor 12β
under standard [Au]/[Ag]-catalyzed glycosidation conditions.
Gratifyingly, the disaccharide 25 was isolated in very high yield
whose structural homogeneity was confirmed by the
spectroscopic studies. For example, the two anomeric protons
of compound 25 were noticed at δ 5.51 (d, J = 4.6 Hz, 1H), 5.33
(s, 1H) ppm and δ 106.1, 100.9 ppm in the 1H and 13C NMR
spectra respectively (Scheme 5).[36]
Having achieved the key milestone encouraged us to move
forward to synthesize the pentasaccharide donor 3. The
synthesis started with the conversion of easily accessible
propargyl orthoester 33 to the di-O-TBDPS compound 34 that
was subjected to the gold-catalyzed glycosidation to obtain
desired allyl arabinofuranoside whose silyl ethers were cleaved
off under HF·py condition to afford the acceptor-diol 8 in 54%
yield over four steps (Scheme 6).
An uneventful deprotection of levulinoate using 70% aqueous
solution of hydrazine hydrate in the presence of 1:1 mixture of
pyridine-acetic acid buffer in CH2Cl2 at 25 ⁰C afforded the
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Gold-catalyzed glycosidation with allyl alcohol followed by the
cleavage of the silyl ether using HF·py afforded the glycosyl
acceptor 37 for further exploration. Much to our satisfaction, the
gold-phosphite and AgOTf catalyzed glycosidation between
donor 32 and acceptor 37 resulted in a 4:1 diastereomeric
mixture of trisaccharides 38.[36]
Furthermore, the two
trisaccharides could be easily separated by flash silica gel
chromatography and enabled thorough characterization. The
three anomeric carbons of trisaccharide 38α were noticed at
δ105.5, 104.8, 100.0 ppm for two 1,2-trans and lone 1,2-cis
linkage respectively (Scheme 6).[36]
The major isomer of the trisaccharide 38α was treated with
hydrazine hydrate to afford alcohol 39 and further treated with
the glycosyl donor 32 under 8mol% each of Au-phosphite
catalyst and AgOTf to obtain structurally homogenous
pentasaccharide 40 in very high yield. In the 13C NMR spectrum
of pentasaccharide 40, resonances of the five anomeric carbons
were noticed at δ 105.7, 105.1, 104.4, 99.6 (2C) ppm along with
very good matching of molecular ion in the mass spectrum
(found: 2692.0864; calcd: 2692.0874 for C159H174NaO26Si6).[36]
The successfully synthesized pentasaccharide 40 was
converted easily into the corresponding carbonate donor 41 in
two easy steps (Scheme 6).
Synthesis of disaccharide donor
4 and trisaccharide
acceptor 6: Easily accessible propargyl orthoester 33 was
saponified under Zemplén conditions to obtain a diol which was
quickly split into two halves; one half was treated with 2.2
equivalents of TBDPS-Cl and imidazole in DMF at 25 ⁰C to
obtain a di-O-TBDPS orthoester and subsequently subjected to
Scheme 6. Synthesis of pentaarabinofuranosyl carbonate donor 40:
Reagents: (a) NaOMe (0.3 eq), CH2Cl2-MeOH (1:1), 25 °C, 2 h; (b) TBDPSCl,
Im., DMF, 0-25 °C, 4 h, 68% over two steps for 34; (c) 10mol% AuBr3, allylOH,
CH2Cl2, 4Å MS powder, 25 °C, 3 h, 86% towards 8 and 85% towards 37; (d)
Pyridine, HF.py, 0-25 °C, 5 h; 92% for 8 and 87% for 37; (e) 8mol% each of
chloro[tris(2,4-di-tert-butylphenyl)phosphite]gold and AgOTf, CH2Cl2, 4Å MS
powder, 25 °C, 40 min, 84% for 35, 76% for 38α, 19% for 38β and 84% for 40;
(f) Levulinic acid, DIC, DMAP, CH2Cl2, 0-25 °C, 2 h, 61% over three steps for
36; (g) 70% N2H4.H2O (4.5 eq), CH2Cl2, AcOH (22 eq), Py (27 eq), 25 °C, 2 h,
93%; (h) PdCl2 (0.2 eq), CH2Cl2-MeOH (1:4), 25 °C, 4 h; (i) 15, DMAP, CH2Cl2,
25 °C, 5 h, 77% over two steps.
gold-catalyzed
glycosidation
conditions
to
afford
monosaccharide 42 in 55% over three steps (Scheme 7). The
second half was treated with 1.1 equivalent of TBDPS-Cl, the
remaining C-3 hydroxyl group was protected as its benzoate
under BzCl/py./25 ⁰C and treated with pent-4-en-1-
ol/AuBr3/CH2Cl2/4Å MS powder/25 ⁰C to afford the required n-
pentenyl furanoside 44 in 46% overall yield. Hydrolysis of the n-
pentenyl glycoside 44 to the hemiacetal and subsequent
reaction with freshly prepared reagent 15 afforded the
arabinofuranosyl donor 45 as a fluffy solid.[38] Cleavage of the
silyl ether of compound 42 gave the diol 43 which will serve as
Now, the stage was set for the gold/silver-catalyzed
glycosidation in a {2x2+1} fashion. The glycosidation between
diol 8 and the acceptor 32 was performed affording an
inseparable mixture of pentasaccharides 35. Earlier reports on
the synthesis of pentasaccharide motifs indicated that the
diastereomeric ratio would likely be resulting from the C-5
position of the aglycon due to its inherently more reactive nature
the glycosyl acceptor.
Next, [Au]/[Ag]-catalyzed reaction
between donor 45 and acceptor 43 underwent glycosidation
resulting in a trisaccharide 46 which upon treatment with HF·py
afforded the desired motif B analogue 6 that is ready for further
explorations (Scheme 7).
The 1H NMR spectrum of compound 6 confirmed the
presence of the three anomeric protons by displaying
resonances at δ 5.24, 5.21 and 5.10 ppm and the presence of
olefin was evident from the resonances δ 4.96-4.86 (m, 2H) and
5.78-5.66 (m, 1H) ppm along with all other protons in the
aromatic and aliphatic regions. In the 13C NMR spectrum of
trisaccharide 6, resonances due to the three anomeric carbons
were noticed at δ106.0, 105.4, 105.3 ppm and the terminal olefin
carbon was observed at δ 115.1 ppm and resonances from five
carbonyls were identified at δ 166.4-165.1 ppm. In addition, MS
studies further confirmed the compound (found: 1025.3214;
compared to the C-3-OH.[18],[21-23]
Temperature controlled
glycosidations and reversing the addition sequence of glycosyl
donor and acceptor did not improve the diastereoselectivity and
more frustratingly, the isomers could not be separated. Hence,
sequential addition of the disaccharide was investigated and
installation at the C-5 position prior to the C-3 position would be
beneficial because the mixture of compounds, if any, shall be
easy to purify. With this idea, orthoester 33 was saponified,
monosilylated using 1.1 equivalent of TBDPS-Cl at 0 ⁰C and
installation of the levulinoate protection was carried-out at the C-
3 position afforded the compound 36 in 61% over three steps.
calcd: 1025.3207 for C55H54NaO18).[36]
In continuation, F-
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assisted cleavage of the silyl ether of monosaccharide 44
resulted in alcohol 47 that was glycosylated with the donor 45
prepared vide supra to obtain the disaccharide 48 in high yield.
Conversion of the n-pentenyl glycoside 48 to the carbonate 4
was carried out by aforementioned two-step sequence (Scheme
7). Presence of the carbonate of disaccharide 4 was confirmed
by its transmittance at 1726 cm-1 in the IR spectrum.
led to the synthesis of compound 52. Unblocking of the O-trityl
ether under acidic conditions afforded the galactofuranosyl
acceptor 53 in 33% yield over eight steps (Scheme 8).
Scheme 8. Synthesis of Motif E: Reagents: (a) CH2Cl2, CH3COBr (6.5 eq),
MeOH (4.5 eq), 0 °C, 3 h; (b) CH2Cl2, propargyl alcohol, 2,6-lutidine (2.2 eq),
TBAI (0.5 eq), 25 °C, 10 h, 76% over two steps for 50; (c) 10mol% AuBr3,
CH2Cl2, pent-4-ene-1-ol, 4Å MS powder, 25 °C, 3 h, 82%; (d) NaOMe (0.3 eq),
CH2Cl2-MeOH (1:1), 25 °C, 2 h; (e) PTSA (0.2 eq.), 2,2-DMP, Acetone, 8 h; (f)
Py, PhCOCl, CH2Cl2, 0-25 °C, 2 h, 64% over three steps for 51; (g) 80% AcOH,
aq. THF, 60 °C, 3 h, 85%; (h) TrCl, py, CH2Cl2, 60 °C, 24 h; (i) Levulinic acid,
DIC, DMAP, CH2Cl2, 25 °C, 3 h, 74% over two steps for 52; (j) Et3SiH, TFA,
CH2Cl2, 0 °C, 5 min, 96%; (k) CH3CN, H2O, 25 °C, 6 h; (l) 15, DMAP, CH2Cl2,
25 °C,
4 h, 58% over three steps (a,k,l) for 55; (m) 8mol% each of
chloro[tris(2,4-di-tert-butylphenyl)phosphite]gold and AgOTf, CH2Cl2, 4Å MS
powder, 25 °C, 40 min, 89%; (n) 70% N2H4.H2O (4.5 eq), CH2Cl2, AcOH (22
eq), Py (27 eq), 25 °C, 2 h, 80%.
In parallel, methyl furanoside 49 was converted into the
hemiacetal 54 under strongly acidic conditions (excess
AcBr/MeOH) and subsequently treated with reagent
15/DMAP/CH2Cl2 to obtain the galactofuranosyl donor 55 in 58%
yield over two steps. Galactofuranosyl donor 55 and the
Scheme 7. Synthesis of disaccharide 4 and trisaccharide 6: Reagents: (a)
NaOMe (0.3 eq), CH2Cl2-MeOH (1:1), 25 °C, 2 h; (b) TBDPSCl, Im., DMF, 0-
25 °C, 4 h; (c) 10mol% AuBr3, 4-pentene-1-ol, CH2Cl2, 4Å MS powder, 25 °C,
3 h, 55% for 42 and 75% for 44 over three steps; (d) Pyridine, HF.py, 0-25 °C,
5 h; 86% for 43, 82% for 47 and 87% for 6; (e) BzCl, py., 25 ⁰C; (f) NIS (2.2
eq), TfOH (0.2 eq) CH2Cl2/CH3CN/H2O (5:3:0.5), -10 °C to 0 °C, 1 h, 81%
towards 45, and 78% towards 4; (g) 15, DMAP, CH2Cl2, 25 °C, 4 h, 85% for 45
acceptor
53
were
conveniently
glycosylated
under
aforementioned [Au]/[Ag]-catalyzed glycosidation conditions to
afford the digalactofuranoside 56 which was transformed to
compound 5 by unmasking the levulinoate employing 70%
and 82% for 4; (h)
8
mol% each of chloro[tris(2,4-di-tert-
hydrazine hydrate solution (Scheme 8).
In the 1H NMR
butylphenyl)phosphite]gold and AgOTf, CH2Cl2, 4Å MS powder, 25 °C, 40 min,
95% for 46 and 92% for 48.
spectrum of disaccharide 5, characteristic resonances of the two
anomeric protons are identified as singlets at δ 5.12, 5.29 ppm
and the 13C NMR spectral studies further confirmed presence of
the two 1,2-trans linkages by displaying C-1 carbons at δ 105.9
and 106.4 ppm. In addition, disaccharide 5 gave good
matching of molecular weight to that of calculated molecular
weight (found: 1057.3266; calcd: 1057.3259 for C59H54NaO17).[36]
Synthesis of octasaccharide. Accomplishing sufficient
quantities of all major structural motifs encouraged us to move
ahead towards the total synthesis of pentacosasaccharide 1.
The next major milestone will be the synthesis of an
Synthesis of α-Galf-(1→6)-Galf 5: Synthesis of
disaccharide 5 started with the easily accessible methyl 2,3,5,6-
tetra-O-benzoyl galactofuranoside 49[39] which was converted to
the orthoester 50 in 76% yield over two steps. Characteristic
resonances due to the orthoester moiety of 50 are noticed at δ
123.4 ppm for the quaternary carbon of the orthoester. Global
saponification under Zemplén conditions (NaOMe/MeOH),
isopropylidenation and esterification of the remaining two
hydroxyl moieties under BzCl/py resulted in the formation
pentenyl galactoside 51. Hydrolysis of the isopropylidene of
compound 51, regioselective protection of C-6 position as its
trityl ether and further treatment with levulinic acid/DIC/DMAP
octasaccharide and a heptasaccharide.
The glycosidation
between arabinofuranosyl donor 4 and digalactoside 5 enabled
us to conveniently synthesize the arabinogalactan 57 in 92%
yield.
Unmasking of the silyl ether using HF·py at 25 ⁰C
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10.1002/chem.201704009
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followed by the repetition of the gold-phosphite and AgOTf
assisted glycosidation afforded the hexasaccharide 58 and one
more repetition of this two-step sequence gave us an
octasaccharide with two Galf- and six Araf- residues.[36] Fluoride
ion mediated cleavage of silyl ether afforded the required
acceptor 59 that will be utilized in futuro for the synthesis of
pentadecasaccharide (Scheme 9).
carbonate 61 in 77% yield reinvoking a protocol that was
delineated above (Scheme 10).
Scheme 10. Synthesis of heptasaccharide: Reagents: (a) 8mol% each of
chloro[tris(2,4-di-tert-butylphenyl)phosphite]gold and AgOTf, CH2Cl2, 4Å MS
powder, 25 °C, 40 min, 91%; (b) NIS (2.2 eq), TfOH (0.2 eq)
CH2Cl2/CH3CN/H2O (5:3:0.5), -10 °C to 0 °C, 1 h; (c) 15, DMAP, CH2Cl2,
25 °C, 4 h, 77% over two steps.
Structural authenticity of the heptasaccharide 60 was evident
1
from the H NMR spectrum wherein the presence of the seven
anomeric protons were identified at δ 5.62, 5.50, 5.46, 5.45, 5.41,
5.38 and 5.26 ppm and the presence of olefin was confirmed by
the characteristic resonances at δ 5.11-5.01 (m, 2H) and 5.87
(m, 1H) ppm. The 13C NMR spectrum of heptasaccharide 60
confirmed the formation of α-linkages by displaying resonances
due to the anomeric carbons at δ 106.1(2C), 106.0 (4C), 105.3
ppm; the terminal olefinic CH2- was identified at δ 115.0 ppm.[36]
Assembly of the pentadecasaccharide. Stapling of the
heptasaccharyl donor 61 and the octasaccharyl acceptor 59 was
accomplished successfully by re-inviting the gold-catalyzed,
silver assisted glycosidation reaction in the presence of 8mol%
each of Au-phosphite and AgOTf catalysts in CH2Cl2 at 25 ⁰C for
40 min. The two silyl ethers at the non-reducing end of the
pentasaccharide 62 were unmasked by the treatment of HF·py
to afford the aglycon 63 in 79% yield (Scheme 11).
Scheme 9. Synthesis of octasaccharide: Reagents: (a) 8mol% each of
chloro[tris(2,4-di-tert-butylphenyl)phosphite]gold and AgOTf, CH2Cl2, 4Å MS
powder, 25 °C, 40 min, 92% for 57, 87% for 58, 83% for 59 ; (b) Pyridine,
HF.py, 0-25 °C, 5 h, 85% towards 58, 81% towards 59.
Structural homogeneity of octasaccharide 59 was confirmed
The 1H NMR spectrum of
1
by spectroscopic techniques.
The H NMR spectrum of pentadecasaccharide 63 showed
octasaccharide 59 showed the presence of the eight anomeric
protons from δ 5.42 to 5.15 ppm and the presence of olefin was
evident from the resonances δ 4.87-4.78 (m, 2H) and 5.95 (m,
1H) ppm along with all other protons in the aromatic and
aliphatic regions. Further, 13C NMR spectrum confirmed the
formation of α-linkages of octasaccharide 59 by showing
resonances due to the anomeric carbons of Galf- at δ 107.0 and
105.7 ppm whereas the remaining six anomeric carbons of Araf-
residues were noticed between δ 105.9 and 105.7 ppm; the
terminal olefinic carbon was identified at δ 114.9 ppm and
resonances from eighteen carbonyls were identified from δ166.1
to 165.1 ppm. In addition, MS studies further confirmed
formation of the octasaccharide 59.[36]
the presence of n-pentenyl moiety by displaying characteristic
resonances at δ 6.02 (m, 1H), 4.91 (m, 2H), 3.48 (m, 2H), 2.06
(m, 2H) and 1.59 (m, 2H). No resonances were observed from
the TBDPS- moiety and many overlapping signals were noticed
in the anomeric and aromatic regions.
Additionally, the 13C
NMR confirmed the presence of 15-saccharide residues by
displaying fifteen signals at δ 107.0, 106.0 (8C), 105.9 (3C),
105.8, 105.7 and 105.5 ppm. ESI-MS spectrum of compound 63
revealed m/z= 5377.5426 for the corresponding sodium adduct
(calcd. 5377.5407 for C299H258NaO94).[36]
Synthesis of pentacosasaccharide 1. The glycosidation
by {2x5+15} fashion was envisioned to accomplish the targeted
pentacosasaccharide 1. Accordingly, pentadecasaccharide 63
and the pentafuranosyl donor 41 were called upon to complete
the assembly of pentacosasaccharide 1. The glycosidation was
carried out by the catalytic protocol containing 8mol% each of
Au-phosphite and AgOTf at 25 ⁰C for 40 min.
Synthesis of heptasaccharide. In continuation, synthesis of
the pentacosasaccharide 1 required access to the middle
heptasaccharide. In view of this, the glycosidation between the
glycosyl donor 4 and the acceptor 6 was carried-out using
[Au]/[Ag]-catalyzed glycosidation procedure under Argon
atmosphere at 25 ⁰C (Scheme 10).
Furthermore,
heptasaccharide 60 was transformed into the ethynylcyclohexyl
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10.1002/chem.201704009
Chemistry - A European Journal
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Scheme 11. Synthesis of pentadecasaccharide: Reagents: (a) 8mol% each of
chloro[tris(2,4-di-tert-butylphenyl)phosphite]gold and AgOTf, CH2Cl2, 4Å MS
powder, 25 °C, 40 min, 88%; (b) Pyridine, HF.py, 0-25 °C, 5 h, 79%.
The pentacosasaccharide 64 was carefully isolated by the
flash silica gel column chromatography. The presence of large
number of benzoates and furanose ring protons have obscured
1
anomeric protons in the H NMR spectrum; however, evidence
of pentenyl moiety at δ 6.04 (m, 1H), 4.96-4.88 (m, 2H), 3.34 (m,
2H), 2.07 (m, 2H) and 1.60 (m, 2H) ppm and tert-butyl of
TBDPS-moieties at 0.99, 0.96, 0.91, 0.88, 0.80 and 0.79 ppm as
six pairs of singlets supported the successful formation of the
pentacosasaccharide 64 (Scheme 12). Full confirmation and the
structural integrity of polysaccharide 64 were obtained from the
high field 13C NMR spectrum (150.97 MHz). All the twenty five
anomeric carbons were identified between δ 107.0, 106.2 (3C),
106.0-105.9 (13C), 105.7 (2C), 105.3, 105.0, 99.9 (2C) and 99.8
(2C) ppm. The anomeric carbon at the reducing end of the
arabinogalactan 64 was confirmed at the δ 99.9 and 99.8 ppm
as anticipated. Besides, the pentacosasaccharide showed
satisfactory matching of sodium adduct of the molecular ion.[36]
Subsequently, cleavage of the silyl ether under standard HF·py
conditions followed by Zemplén saponification conditions
afforded the free pentacosasaccharide as a pentenyl glycoside 1
(Scheme 12). The free pentacosasaccharide 1 was purified by
gel filtration chromatography using Bio-Rad P4 gel followed by
lyophilization to afford polysaccharide 1 as a fluffy white powder
(13 mg).
Conclusions
In summary, the synthesis of pentacosasaccharide with twenty
four interglycosidic linkages achieved by invoking salient
features of alkynyl carbonate glycosyl donor strategy involving
[Au]/[Ag]-catalysis. Major features of this endeavor are: (i)
installation of all 1,2-trans linkages by the neighbouring group
assistance from the C-2 acyl group, (ii) conversion of β- or 1,2-
trans Ribf to β- or 1,2-cis Araf through an oxidation-reduction
Scheme 12. Synthesis of pentacosasaccharide motif of Arabinogalactan:
Reagents: (a) 8mol% each of chloro[tris(2,4-di-tert-butylphenyl)phosphite]gold
and AgOTf, CH2Cl2, 4Å MS powder, 25 °C, 40 min, 87%; (b) Pyridine, HF.py,
0-25 °C, 5 h; (c) 0.5M NaOMe (2 mL), CH2Cl2-MeOH (1:1), 25 °C, 2 h; 67%
over two steps.
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10.1002/chem.201704009
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Keywords: Tuberculosis • Arabinogalactan • Oligosaccharide •
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A 77 step synthesis of a
pentacosafuranoside of
Sandip Pasari, Sujit Manmode, Gulab
Walke and Srinivas Hotha*
Mycobacterium tuberculosis
glycocalyx employing single
glycosidation protocol is accomplished
in 0.0012% yield.
Page No. – Page No.
A Versatile Synthesis of
Pentacosafuranoside Subunit
Reminiscent of Mycobacterial
Arabinogalactan Employing One
Strategic Glycosidation Protocol
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