Synthesis of Rare Deoxy Amino Sugar Building Blocks Enabled the Total Synthesis of a Polysaccharide Repeating Unit Analogue from the LPS of Psychrobacter cryohalolentis K5T

Article abstract of DOI:10.1021/acs.joc.8b02037

Lipopolysaccharides (LPSs) play key roles in humoral immunity. Recently, the LPS structure of the Psychrobacter cryohalolentis K5^T strain was reported. Due to the presence of unnatural amino sugars and branched linkages, its structure is unique. Herein we report the total synthesis of an LPS analogue of P. cryohalolentis K5^T. After overcoming the issues like ring conformation changes and elimination of triflate, we were able to develop a strategy for the synthesis of the newly reported 2,3,4-triacetamido-2,3,4-trideoxy-l-arabinose derivative. Coupling of different donors with suitable acceptors from the nonreducing end to the reducing end and further functional group modifications delivered the protected LPS hexasaccharide repeating unit. After functional group modifications, we were unable to oxidize the hindered primary hydroxyl group to synthesize the target molecule. Alternatively, removal of the permanent protecting groups afforded the LPS hexasaccharide repeating unit analogue of Psychrobacter cryohalolentis K5^T.

Full text of DOI:10.1021/acs.joc.8b02037

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Article
Synthesis of Rare Deoxy Amino Sugar Building Blocks
Enabled the Total Synthesis of Polysaccharide Repeating Unit
Analogue from the LPS of Psychrobacter cryohalolentis K5T
Madhu Emmadi, and Suvarn S. Kulkarni
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b02037 • Publication Date (Web): 02 Nov 2018
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Synthesis of Rare Deoxy Amino Sugar Building Blocks Enabled the Total
Synthesis of Polysaccharide Repeating Unit Analogue from the LPS of
Psychrobacter cryohalolentis K5T
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Madhu Emmadi and Suvarn S. Kulkarni*
Department of Chemistry, Indian Institute of Technology Bombay, Mumbai 400076, India
suvarn@chem.iitb.ac.in
TOC Graphic
Abstract
Lipopolysaccharides (LPSs) play key roles in humoral immunity. Recently, the LPS
structure of Psychrobacter cryohalolentis K5T strain was reported. Due to the presence of
unnatural amino sugars and branched linkages, its structure is unique. Herein we report the
total synthesis of an LPS analogue of P. cryohalolentis K5T. After overcoming the issues like
ring conformation changes and elimination of triflate, we were able to develop a a strategy
for the synthesis of newly reported 2,3,4-triacetamido-2,3,4-trideoxy-L-arabinose derivative.
Coupling of different donors with suitable acceptors from non-reducing end to reducing end
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and further functional group modifications delivered the protected LPS hexasaccharide
repeating unit. After functional group modifications, we were unable to oxidize the hindered
primary hydroxyl group to synthesize the target molecule. Alternatively, removal of the
permanent protecting groups afforded the LPS hexasaccharide repeating unit analogue of
Psychrobacter cryohalolentis K5T.
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Introduction
Psychrobacter cryohalolentis K5T is a non-motile, non-pigmented and Gram-negative
bacterium isolated from Siberian permafrost.1 P. cryohalolentis K5T species are capable of
reproducing at temperatures ranging from -10 °C to 30 °C. The pathology of K5T strain is
not known, but related P. cryohalolentis strains are isolated from infected lungs of sheep2 and
human blood and tissues.3 Since P. cryohalolentis K5T is a Gram-negative bacterium, its cell
wall is heavily decorated with lipopolysaccharides (LPSs) in which O-antigen or O-specific
polysaccharides are attached to core oligosaccharides and further these core oligosaccharides
are attached to lipid A. Recently, Knirel and co-workers isolated O-specific polysaccharide
of Psychrobacter cryohalolentis K5T.4 The repeating unit of this LPS is hexasaccharide 1
(Figure 1), and it was characterized by sugar analysis, triflic acid solvolysis, Smith
degradation, and 2D NMR spectroscopy. Hexasaccharide 1 contains several rare sugar
building blocks such as a) 2,4-diacetamido-2,4,6-trideoxy-D-glucopyranose (D-Qui2,4NAc or
Bac), b) 2,3,4-triacetamido-2,3,4-trideoxy-L-arabinose (L-Arap2,3,4NAc), and c) 2,3-
diacetamido-2,3-dideoxy-D-glucuronamide (D-Glc2,3NAcA). Out of these three rare sugars,
L-Arap2,3,4NAc is reported for the first time. O-Antigens of LPS play vital roles in
immunogenicity: host immune system recognizes the O-antigen and generates antibodies
which can fight against pathogen.5 Since the O-antigen of P. cryohalolentis K5T has atypical
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Figure 1. LPS repeating unit of Psychrobacter cryohalolentis K5T.
rare amino sugars, immune response generated against them could be specific.6
Lipopolysaccharide structures of Psychrobacter have not been studied well and the biological
function remains unknown. Considering the problems associated with the isolation of LPS,
we embarked upon the total synthesis of 1. Herein we report the synthesis of P.
cryohalolentis K5T LPS repeating unit analogue of 1.
Retrosynthetic strategy for the synthesis of hexasaccharide 1 is outlined in Scheme 1.
For a convergent synthesis of 1, we envisaged that a [2+4] glycosylation would speed up the
synthesis. Target molecule 1 can be obtained from its fully protected form 2, upon global
deprotection. Hexasaccharide 2 can be accessed from the coupling of the rare sugars
containing disaccharide donor 3 with tetrasaccharide acceptor 4. Tetrasaccharide acceptor 4
can be obtained from monosaccharide building blocks 7, 8, 9 and 10 by conducting
glycosylation reactions from the reducing end to the non-reducing end. Disaccharide donor 3
can be synthesized from the rare monosaccharide building blocks 5 and 6 via orthogonal
glycosylation. Synthesis of the rare sugar building blocks (5, and 6) and their stereoselective
coupling are difficult tasks, especially building block 5, which has never been synthesized.
It was necessary to develop a synthesis of this rare sugar.
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Scheme 1. Retrosynthetic strategy.
Results and Discussion
Synthesis of rare sugar building block 5
To the best of our knowledge L-Arap2,3,4NAc was not reported in any bacterial
capsular polysaccharides or lipopolysaccharides or lipoteichoic acids or glycoproteins. For
the first time, Knirel and co-workers reported and confirmed its structure by NMR and triflic
acid solvolysis methods.4 L-Arabinose is a C5-sugar (pentose) trapped in pyranoside form.
Due to the lack of C6 carbon in L-arabinopyranoside, its synthesis and handling may not be
easy. Keeping all the challenges in our mind we decided to make the key intermediate (L-
Arap2,3,4N3 derivative 5) first. Since, D-arabinose is the enantiomer of L-arabinose, we
envisaged that the inversion of configuration at C2, C3 and C4 positions of D-arabinose with
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azide can afford the desired L-Arap2,3,4N3 derivative 5a in an expedient manner. Thus, per
O-acetylation7 of D-arabinose,
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Scheme 2. Attempted synthesis of L-arabinose derivative 5a.
and anomeric bromination followed by its nucleophilic displacement with thiophenol
afforded 11 in good yields (Scheme 2). Then, the thioglycoside 11 was subjected to Zemplén
deacetylation conditions to afford triol 12 in 96% yield. The next step was the conversion of
the triol into corresponding tri-triflate and its subsequent displacement with azide, but
unfortunately triflation of 12 did not work well. We rationalized that perhaps the
incorporation of three triflate groups in the strained ring could be difficult. To verify this, we
decided to work on the open chain form of D-arabinose, where no ring strain is present.
Accordingly, we synthesized the desired tetraol 13 as shown in Scheme 3. D-Arabinose was
treated with EtSH in acidic medium to provide the open chain dithio acetal derivative 13.8
The primary hydroxyl was protected as silyl ether by treatment with TBDPSCl and imidazole
to afford triol 14 in 90% yield (Scheme 3). With the open chain triol 14 in hand, we
conducted the triflation reaction by treating 14 with excess of Tf2O and pyridine, but to our
dismay, the triflation did not work on the open chain form as well. With these results, we
concluded that triflation of three adjacent hydroxyl groups may be difficult in our system,
though tosylation of C5 sugars has been reported, however with many difficulties.9 In this
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case, tosylation and mesylation approach may not serve to afford the desired product because
of their lower
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Scheme 3. Attempted synthesis of L-arabinose derivative 15.
reactivity. With these unsuccessful results, next we planned to carry out sequential
inversions at C4 followed by C2 and C3. For this purpose the 2,3-hydroxy groups in 13 were
protected as isopropylidene acetal (less stable 4,5 acetal formed during the course of reaction
was selectively cleaved)10 and the primary hydroxyl was selectively silylated using TBDPSCl
and imidazole to afford the desired 4-hydroxy derivative 16 in 71% yield (Scheme 4).
Scheme 4. Attempted synthesis of L-arabinose derivative 15 via serial inversions.
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Treatment of 16 with Tf2O and pyridine, followed by displacement of the so formed O-
triflate with sodium azide provided the L-xylose derivative 17 in 81% yield. After inverting
the C4 center, isopropylidine group was removed to give diol 18 in 60% yield. The diol 18
was then treated with Tf2O and pyridine. However, in this case also the triflation was not
successful. At this stage we realized that instead of parallel inversions, serial inversions at
C2 and C3 chiral centres followed by ring closing may afford the desired product, but
selective protection of 2 or 3 hydroxy is difficult and synthetic route would then become
lengthy.
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With these considerations, we decided to exploit the protocol developed earlier in our
group, in which 2,4-bistriflates of -D-thiorhamnoside,11-14 2,4-bistriflates of -D-
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thiomannoside15 and 2,4-bistriflates of -L-rhamnosides as well as -L-fucosides
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sequentially displaced with various nucleophiles in a one-pot manner to afford various
bacterial rare sugar and glycosamine building blocks. To expand the scope of this protocol,
D-lyxose (due to its structural resemblance with D-mannopyranoside and rhamnopyranoside)
was selected as the precursor. Accordingly, D-lyxose was transformed into its corresponding
thioglycoside derivative 21 in good yields (Scheme 5). It is worthwhile to mention that for
the nucleophilic displacement of glycosyl bromide 19 with thiophenol, we tried several
reaction conditions and the best results were achieved by using phase transfer conditions.17
Tin mediated benzoylation18 of -D-thiolyxoside 21 with BzCl cleanly afforded the
desired 3-OBz derivative 22 in 80% yield. Next, the 2,4-diol 22 was treated with an excess
of Tf2O, pyridine and the so formed 2,4-bistriflate was displaced with sodium azide to obtain
diazido 23 in 82% yield over 2 steps. Then the 3-OBz group in 23 was deprotected and the
obtained 3-hydroxy derivative 24 was converted to the corresponding triflate which upon
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concomitant displacement with nitrite ion furnished 25 (70%) as a sole product in C4
conformation {1H NMR δ 5.34 (d, J = 4.0 Hz, 1H, H-1)} contrary to our anticipated 4C1
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Scheme 5. Attempted synthesis of L-arabinose derivative 5.
conformation. This could be due to the unfavourable arrangement of two axial adjacent
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groups at C3 and C4 in C1 conformer of 25. We expected that after the inversion of
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configuration at C-3 in 25, the triazido derivative 5 could be obtained in a C1 conformation
(Scheme 5). However, triflation of 25 afforded the elimination product 26. Structure of 26
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was confirmed by the H NMR {δ 5.45 (d, J = 1.8 Hz, 1H, H-3)}; C NMR {δ 139.5 (C3),
110.4 (C2)} and HRMS. This could be attributed to a strong β-trans axial effect (axial SPh at
C1) which retards SN2 at C3 via 1,3-diaxial repulsion (dipole-dipole repulsion) as well as
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vicinal triflate effect (axial C2 azide) in the C4 conformation.19 In the alternate C1
conformation, the presence of electron withdrawing axial C4 azido group hinders the azide
nucleophile to approach the C3 O-triflate from the rear side to achieve the SN2 transition
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state. Although, triflates have a better chance of overcoming the vicinal axial effect in SN2
displacements, as invoked by Hale and co-workers,19 in this particular case perhaps due to the
ease of ring flipping, owing to the presence of two axial group in each conformer, the
elimination took place in the first step itself. At this point, we realized that we need to invert
the stereocenters in such a way that no two axial groups be present at any stage. To avoid
two concomitant axial groups, first we decided to carry out inversion at C2 position, followed
by a double inversion at C3 position and finally at C4. This strategy avoids the presence of
two axial groups at any stage as depicted in Scheme 6.
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Accordingly, as shown in Scheme 6, -D-thiolyxoside 21 was treated with 2,3-
butanedione20 in the presence of CSA to form 3,4-butanediacetal and the so obtained 2-
hydroxy derivative was subjected to triflation and concomitant displacement with sodium
azide. Subsequent deprotection of 3,4-butanediacetal using TFA afforded the D-xylosamine
derivative 27 in excellent yields. Then, regioselective benzoylation of 27 was carried out
using dimethyltin dichloride as a catalyst to give 4-OBz derivative of D-xylosamine 28 in
90% yield. The D-xylosamine 28 was transformed into D-ribosamine derivative 29 via C3
epimerization using Lattrell-Dax reaction conditions.21-23 Triflation of the axial hydroxyl
group in 29 converted it to the corresponding triflate, which was subsequently displaced with
sodium azide in SN2 manner and the subsequent removal of 4-OBz group afforded the 4-
hydroxy derivative 30 in 78% yield over 3 steps. Gratifyingly, triflation and its concomitant
azide displacement of 4-hydroxyl group in 30 cleanly afforded the triazide derivative 5 in
84% yield. Since compound 5 has high N/C ratio, the reaction was conducted in a flask
which was free of any scratches. Rare sugar building block 5 and all synthetic intermediates
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were thoroughly characterized by NMR spectroscopic methods (1H, C, H-1H COSY and
HSQC; see SI). It should be noted that en route to the target L-arabino derivative 5, several
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important amino sugar derivatives (D-xylosamine derivative 27, 28 and 30, and D-ribosamine
derivatve 29) were synthesized (Scheme 6).
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Scheme 6. Synthesis of L-arabinose derivative 5.
Synthesis of 3-azido-3-deoxy-D-glucosaminic acid derivative
After the successful synthesis of L-arabinose derivative 5, synthesis of 3-azido-3-
deoxy-D-glucosaminic acid derivative was attempted. We envisaged that it can be obtained
from D-glucosamine by installation of amine functionality at C3 with retention of
configuration. In addition, this rare sugar is attached to the L-rhamnose derivative through a
-linkage, so installation of N-Troc functionality at C2 position of D-glucosamine will help in
getting -selectivity through neighboring group participation. Thus, D-glucosamine was
treated with TrocCl and aq. NaHCO3 to afford the corresponding N-trocylated derivative and
the free hydroxyl groups were acetylated by treating with Ac2O and pyridine (Scheme 7).
Nucleophilic displacement of the anomeric acetate with PhSH under acidic conditions
(BF3·Et2O) provided the desired thioglycoside 31 in 62% yield over 3 steps. Deacetylation of
31 was carried out by treating with 10% AcCl/MeOH and the so formed triol was treated with
benzaldehyde dimethyl acetal and catalytic amount of CSA to give 3-hydroxy derivative 3224
in 86% yield. Compound 32 was converted to 3-hydroxy D-allosamine derivative 33 via a C3
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epimerization using Lattrell-Dax reaction conditions.21-23
Owing to the ambident
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nucleophilicity of nitrite (ONO-), we also obtained the corresponding C3 nitro derivative 34
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Scheme 7. Attempted synthesis of 3-azido-3-deoxy-D-glucosaminic acid derivative 37.
as a minor side product (30%). When we changed the solvent from HMPA to DMF, the
desired product 33 was obtained in 80% yield, exclusively. 3-Hydroxy D-allosamine
derivative 33 upon treatment with Tf2O and pyridine followed by SN2 inversion with sodium
Scheme 8. Synthesis of rare glucosaminic acid derivative 42.
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azide afforded the 3-azido-D-glucosamine derivative 35 in good yields. Benzylidine acetal
removal in 35 under acidic conditions gave diol 36 in 87% yield. Selective oxidation of diol
36 using TEMPO, BAIB25 did not work. In this reaction, the starting material was
completely consumed to give a polar spot on TLC. However, the product was decomposed
during work-up or direct column chromatography. We rationalized that the decomposition of
the product was perhaps due to the presence of NHTroc group. So we changed the N-
protecting group from NHTroc in 32 to its corresponding azide derivative 38 in a 2 step
sequence (Scheme 8). In the first step, NHTroc was converted to the corresponding amine
using Zn/AcOH26 and the so formed amine was transformed into the desired 2-azido
derivative 38 by using TfN3.27 The 3-hydroxyl D-glucosamine derivative 38 was then
transformed into D-allosamine derivative 39 via C3 epimerization using Lattrell-Dax reaction
conditions.21-23 Triflation of axial hydroxyl group in 39 and its concomitant displacement
with sodium azide and followed by benzylidine deprotection under acidic conditions afforded
the desired diol in good yields. Selective oxidation of primary hydroxyl group in 40 using
TEMPO and BAIB furnished the C6 carboxylic acid which was esterified using MeI and
K2CO3 to give 41 {1H NMR δ 3.87 (s, 3H, CO2CH3); 13C NMR δ 169.3} in 74% yield. For
obtaining the requisite β-selectivity in the ensuing [2+4] glycosylation, via neighboring group
participation, the azide groups were swapped with NHTroc at this stage. Reduction of azide
groups to corresponding amines and N-trocylation using TrocCl and NaHCO3 afforded the
desired acceptor 42 in good yield.
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Synthesis of tetrasaccharide acceptor 4
Our strategy for the assembly of tetrasaccharide acceptor 4 relied on the activation of
various thioglycoside donors and their coupling with requisite acceptors starting from the
reducing end to the non reducing end (vide supra Scheme 1). The synthesis of 4 started with
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the preparation of D-galactose building block 8 (Scheme 9a). Fischer glycosylation of D-
galactose using Amberlite resin gave  mixture of methyl D-galactoside,28 which was
treated with 2,2-DMP in acidic medium to afford the corresponding 2,6-diol. At this stage,
we were unable to separate the -isomers. Then 2,6-diol was treated with benzyl bromide
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and sodium hydride to afford the fully protected -D-galactose derivative 43 in 52% yield.
After benzylation we were able to separate the -isomers. The acetonide group in 43 was
cleaved with 80% AcOH and the so formed 3,4-diol was subjected to tin mediated18 acylation
using Me2SnCl2 and FmocCl to afford the 4-hydroxy galactose derivative 8 {1H NMR δ 5.11
(dd, J = 10.0, 3.0 Hz, 1H, H-3)} in 84% yield.
Scheme 9. Synthesis of D-galactose and bacillosamine derivatives.
For the synthesis of bacillosamine derivative 7, the azide functional groups in 2,4-diazido
bacillosamine 4512 were reduced with Pd/C and the so revealed amine groups were trocylated
to give the desired bacillosamine derivative 7 in good yield (Scheme 9b). To obtain the
desired β-selectivity through neighboring group participation in the subsequent [2+1]
glycosylation, it was necessary to covert the azides to the corresponding NHTroc derivative
7.
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Next, we moved on to synthesize L-rhamnose thioglycosides 9 and 10. Recently, an
elegant approach was developed for the protection of methyl -L-rhamnoside using
dimethyltin dichloride as the catalyst.18 We extended this study to thiophenyl L-rhamnoside
(Scheme 10). Accordingly, -L-thiorhamnoside 46 was treated with catalytic (0.05 equiv)
amount of Me2SnCl2 and 1.1 equiv of BzCl to afford the 3-O-benzoyl-L-rhamnose derivative
47 in 90% isolated yield. The diol 47 was again subjected to tin mediated acetylation using
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Scheme 10. Synthesis of L-rhamnose building blocks 9 & 10.
catalytic (0.05 equiv) amount of Me2SnCl2 and 1.1 equiv of AcCl to give the 4-acetylated
product 48 in 80% yield (Scheme 10a). Onomura and co-workers,18 in their preliminary
communication on Me2SnCl2 mediated acylations reported that, in the case of methyl -L-
rhamnoside the first electrophile (benzoyl group) goes to the C3 position and the second
electrophile (tosyl group) goes to C2 position. But in the case of thioglycoside, we observed
that, the second electrophile (acetyl group) went to C4 position. The structure of 48 was
1
unambiguously confirmed by the downfield movement of H-4 in H NMR and a detailed
COSY and HSQC analysis (See Supporting Information). The free C2 hydroxyl group in 48
was benzoylated to give the desired fully protected donor 10 (90%). On the other hand, 2,3-
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orthoester formation and its regioselective opening under acidic conditions furnished the 2-O-
acetate-3,4-diol which was benzoylated to give the thioglycoside donor 9 as shown in
Scheme 10b.
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Having all the desired building blocks for the synthesis of tetrasaccharide acceptor 4 in
hand, we carried out the coupling reactions of the glycosyl donors with the appropriate
acceptors as shown in Scheme 11. First thioglycoside 9 was activated with NIS and TMSOTf
promoter system and coupled with acceptor 8. After complete consumption of the acceptor
and donor, Fmoc group was smoothly removed by treating the reaction mixture with Et3N to
give the desired -linked disaccharide 3-OH acceptor 49 {1H NMR δ 5.28 (d, J = 1.4 Hz, 1H,
H-1)} in 81% yield. Activation of thioglycoside in bacillosamine derivative 7 followed by
coupling with acceptor 49 gave the desired trisaccharide 50 [13C NMR δ 100.6, 100.4, 99.0]
in 76% yields. Acetate group in trisaccharide derivative 50 was selectively removed with
Scheme 11. Synthesis of tetrasaccharide acceptor 4.
10% AcCl in MeOH to afford the trisaccharide acceptor 51 in good yield. Finally, coupling
of 51 and thioglycoside 10 using the same conditions as mentioned above and subsequent
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deprotection of the acetate group under mild acidic conditions gave the desired
5
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tetrasaccharide acceptor 4 in 66% yield.
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9
Synthesis of the rare disaccharide 56 and hexasaccharide 57
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After successful assembly of the tetrasaccharide acceptor 4, we focused our attention
towards the synthesis of the rare disaccharide 53. For this purpose, thioglycoside 5 was
converted to its corresponding bromide derivative and the in situ generated glycosyl bromide
was activated with AgOTf and coupled with the acceptor 42 to obtain the -linked
disaccharide 53 in 54% yield {1H NMR δ 5.07 (d, J = 2.9 Hz, 1H, H-1)} as shown in Scheme
12. In this reaction, disaccharide 53 was obtained as an inseparable mixture with minor
glycal impurities formed due to decomposition of the donor. Next we conducted the [2+4]
glycosylation to get the desired hexasaccharide 54. Activation of donor 53 with various
promoters (NIS/AgOTf,29 Ph2SO,Tf2O25) and its coupling with acceptor 4 did not deliver the
desired hexasaccharide 54. The corresponding trichloroacetimidate donor also did not work.
We believe that due to the presence of C-6 ester, the donor became disarmed and it was
unable to couple with the acceptor.
Scheme 12. Attempted synthesis of hexasaccharide 54.
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So we changed the C-6 ester group to an O-benzoate to achieve the [2+4] glycosylation.
Benzylidine deprotection in 35 and selective benzoylation30 of the so-formed diol furnished
the desired acceptor 55 in 82% yield. After having donor 5 and acceptor 55, we went ahead
for the synthesis of key disaccharide 56. Thus, thioglyoside 5 was converted to its
corresponding bromide by treating with bromine and the so formed glycosyl bromide was
activated with AgOTf and coupled with the acceptor 55 to furnish the left hand disaccharide
56 {exclusive -product 1H NMR δ 5.41 (d, J = 2.9 Hz, 1H, H-1); 13C NMR δ 98.8 (C1)} in
70% yield, as shown in Scheme 13. Having the disaccharide donor 56 and the
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Scheme 13. Synthesis of hexasaccharide 57.
tetrasaccharide acceptor 4 in hand, we conducted the [2+4] glycosylation. Thioglycoside 56
was activated with NIS, TMSOTf and coupled with the acceptor 4 to give the desired
hexasaccharide 57 in low yield of 30%. Under these conditions, the donor was decomposed
before it could couple with the acceptor 4. By employing a mild promoter system
NIS/AgOTf,29 we obtained the desired hexasaccharide 57 in 72% yield. The structure of
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hexasaccharide was adequately characterized by H, 13C and HRMS (See the supporting
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6
information).
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9
Global deprotection of hexasaccharide 57 involved conversion of azide and NHTroc
groups to the corresponding NHAc by treating with Zn/AcOH/Ac2O,25 and debenzoylation
using NaOMe/MeOH to afford the C6′′′′′ primary hydroxy compound. Unfortunately,
TEMPO oxidation of the so formed primary hydroxyl group did not work and we failed to
obtain the desired acid 58. At this stage we do not have any evidence to explain the failure
but possible reason might be the steric hindrance at the C6′′′′′ primary hydroxy group and the
presence of several NHAc groups which makes the compound very polar. On the other hand,
a [2+4] glycosylation did not work with disachharide 53 in which the acid group was already
incorporated as shown in Scheme 12. Nevertheless, de-benzylation using Pd/C in presence of
hydrogen afforded the hexasaccharide repeating unit analogue of P. cryohalolentis K5T 59 in
52% yield over 3 steps (Scheme 14).
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Scheme 14. Synthesis of lipopolysaccharide analogue 59 of P. cryohalolentis K5T.
Conclusions
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In conclusion, we report the synthesis of a polysaccharide repeating unit analogue
from the lipopolysaccharide of Psychrobacter cryohalolentis K5T. This structurally well
defined LPS analogue can be exploited for its immunogenicity and competitive ELISA
characterization. In this manuscript we also report the synthesis of variety of azido sugar
building blocks (D-ribosamine, D-xylosamine, L-arabinosamine and L-lyxosamine) and
development of an elegant strategy for the synthesis of L-Arap2,3,4N3 derivative. Given the
application of various azido sugars for probing glycans in cells, the building blocks made
herein could prove to be precious tools.31
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Experimental Section
General
All reactions were conducted under a dry nitrogen atmosphere. Solvents (CH2Cl2 >99%,
THF 99.5%, Acetonitrile 99.8%, DMF 99.5%) were purchased in capped bottles and dried
under sodium or CaH2. All other solvents and reagents were used without further
purification. All glasswares used were oven dried before use. TLC was performed on pre-
coated Aluminium plates of Silica Gel 60 F254 (0.25 mm, E. Merck). Developed TLC plates
were visualized under a short-wave UV lamp and by heating plates that were dipped in
ammonium molybdate/cerium (IV) sulfate solution. Silica gel column chromatography was
performed using Silica gel (100-200 mesh) and employed a solvent polarity correlated with
TLC mobility. NMR experiments were conducted on 400 and 500 MHz instrument using
CDCl3 (D, 99.8%), CD3OD (99.9%), D2O (99.9%) or (CD3)2CO (D, 99.9%) as solvents.
Chemical shifts are relative to the deuterated solvent peaks and are in parts per million (ppm).
1H-1H COSY was used to confirm proton assignments. Mass spectra were acquired in the
ESI-QTOF mode. Melting points were determined by capillary apparatus. Specific rotation
experiments were measured at 589 nm (Na) and 25 °C. IR spectra were recorded on an FT-IR
spectrometer using CsCl plates.
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Phenyl 2,3,4-O-Triacetyl-1-thio--D-arabinopyranoside (11)
Ac2O (7.8 mL, 83.0 mmol) and DMAP (0.2 g, 1.6 mmol) were added to a turbid solution of
D-arabinose (2.5 g, 16.6 mmol) in CH2Cl2 (35 mL) and pyridine (13.5 mL) and the reaction
mixture was kept for strirring at room temparature 3 h. Then solvents were removed under
reduced pressure. The residue which was obtained after solvents removal was dissolved in
EtOAc and washed with brine. Separated organic layer was dried over Na2SO4, and
concentrated to afford the peracetylated compound. The crude acetylated product was
dissolved in CH2Cl2 (10 mL), to this HBr/AcOH (30 mL) was added and kept for stirring at
room temparature for 1 h. After complete consumption of starting material, reaction mixture
diluted with CH2Cl2 and washed several times with water until the pH reached to 7. Separated
organic layer was dried over Na2SO4, and concentrated to give glycosyl bromide as foam.
NaH (1.1 g, 48.3 mmol) was added at room temperature to a stirred solution of glycosyl
bromide in DMF (40 mL) and PhSH (5.0 mL, 48.3 mmol). The reaction was stirred for 1 h at
the same temperature and then diluted with EtOAc. Then, this reaction mixture was poured
into ice cold water and the organic layer was washed with 0.1 M HCl and then with ice cold
water until the pH reached tpo neautrality. The organic extract was dried over Na2SO4,
filtered and concentrated in vacuo to obtain the desired product 11 as a pale yellowish
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coloured liquid (6.1 g, 61%). [α]20 -10.2 (c 9.41, CHCl3); IR (CHCl3) ῡ 3022, 2860, 1748,
D
1
1479, 1372, 1219, 1060, 758, 668 cm-1; H NMR (400 MHz, CDCl3) δ 7.50-7.48 (m, 2H,
ArH), 7.33-7.28 (m, 3H, ArH), 5.28-5.23 (m, 2H, H-2 & H-4), 5.10 (dd, J = 8.4, 3.4 Hz, 1H,
H-3), 4.81 (d, J = 7.8 Hz, 1H, H-1), 4.16 (d, J = 12.8, 4.2 Hz, 1H, H-5a), 3.68 (dd, J = 12.8,
2.0 Hz, 1H, H-5b), 2.11 (s, 6H, 2CH3), 2.07 (s, 3H, CH3); 13C{1H} NMR (100 MHz, CDCl3)
δ 170.4, 170.1, 169.6, 132.4, 129.1, 128.1, 87.0 (C1), 77.4, 70.6, 68.6, 67.6, 21.05, 21.01,
20.9; HR-ESI-MS (m/z): [M + Na]+ calcd. for C17H20O7NaS, 391.0822; found, 391.0823.
Phenyl 1-Thio-a-D-arabinopyranoside (12)
NaOMe (1.2 g) was added to a stirred solution of 11 (12.0 g, 27.3 mmol) in MeOH (110 mL).
After 1 h, reaction mixture was neutralized with Amberlite IR 120 (H+) (10 g) resin. The
reaction mixture was filtered and concentrated under reduced pressure to afford desired
tetraol 12 as a foam (6.35 g, 96%). [α]20 +12.5 (c 1.47, MeOH); IR (MeOH) ῡ 3350, 2934,
D
1451, 1116, 1026, 667 cm-1; 1H NMR (400 MHz, D2O) δ 7.52-7.47 (m, 2H, ArH), 7.31-7.21
(m, 3H, ArH), 4.64 (d, J = 8.2 Hz, 1H, H-1), 3.97 (dd, J = 12.2, 3.6 Hz, 1H, H-5a), 3.89-3.87
13
(m, 1H, H-4), 3.69 (t, J = 8.2 Hz, 1H, H-2), 3.60-3.56 (m, 2H, H-3, H-5b); C{1H} NMR
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(100 MHz, D2O) δ 136.2, 132.5, 129.9, 128.2, 90.4 (C1), 75.0, 71.7, 69.65, 69.60; HR-ESI-
MS (m/z): [M + Na]+ calcd. for C11H14O4NaS, 265.0505; found, 265.0509.
7
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6-tert-O-Butyl diphenylsilyl-D-arabinose diethyl dithio acetal (14)
To a clear solution of D-arabinose diethyl dithio acetal 1310 (3.14 g, 12.24 mmol) in CH3CN
(40 mL), were added imidazole (2.08 g, 30.6 mmol), and TBDPSCl (3.5 g, 13.4 mmol) and it
was stirred at room temperature for 1 h. After complete consumption of starting material, the
solvents were removed under reduced pressure and chromatographed on silica gel (20% ethyl
9
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acetate and petroleum ether) to afford 14 as a viscous liquid (4.08 g, 90%). [α]20 -29.0 (c
D
1
3.95, CHCl3); IR (CHCl3) ῡ 3449, 3017, 2931, 1428, 1216, 1113, 759 cm-1; H NMR (400
MHz, CDCl3) δ 7.69-7.66 (m, 4H, ArH), 7.46-7.38 (m, 6H, ArH), 4.10 (t, J = 8.3 Hz, 1H),
4.05 (d, J = 9.4 Hz, 1H), 3.89-3.77 (m, 4H), 3.38 (s, 1H, OH), 2.83 (d, J = 7.0 Hz, 1H, OH),
2.78-2.51 (m, 4H, 2CH2), 2.48 (d, J = 9.4 Hz, 1H, OH), 1.28 (t, J = 7.4 Hz, 1H, 2CH3), 1.07
(s, 9H, (CH3)3C); 13C{1H} NMR (100 MHz, CDCl3) δ 135.76, 135.71, 133.1, 133.06, 130.04,
128.0, 72.5, 70.6, 70.1, 65.2, 55.8, 27.0, 25.8, 23.6, 19.4, 14.8, 14.6; HR-ESI-MS (m/z): calcd
for C25H38NaO4S2Si [M+Na] + 517.1873, found 517.1872.
6-tert-O-Butyl diphenylsilyl-2,3-O-isopropylidine-D-arabinose diethyl dithio acetal (16)
2,2-Dimethoxypropane (1.0 mL, 8.2 mmol) and p-toluenesulfonic acid (0.05 g) were added to
a suspension of D-arabinose diethyl dithioacetal 13 (0.94 g, 3.6 mmol) in acetone (14 mL).
After complete consumption of starting material, reaction mixture was quenched with Et3N
and solvents were removed under reduced pressure. The obtained residue was dissolved in
MeOH (14 mL) and water (0.15 mL) and the reaction mixture was stirred at 50 °C for 3 h.
Then, solvents were removed under vacuum and the residue was used for the next step with
any purification. The residue which was obtained in above step was dissolved in acetonitrile
(10 mL) and to this clear solution imidazole (0.6 g, 9.12 mmol), TBDPSCl (1.12 mL, 4.38
mmol) were added. After stirring at room temperature for 1 h, solvents were removed under
reduced pressure and the crude product was purified by silica gel chromatography to afford
16 as a viscous liquid (1.06 g, 71%). [α]20D -21.1 (c 2.28, CHCl3); IR (CHCl3) ῡ 3440, 3016,
1
2930, 1428, 1217, 1113, 762 cm-1; H NMR (400 MHz, CDCl3) δ 7.72-7.65 (m, 4H, ArH),
7.45-7.29 (m, 6H, ArH), 4.36 (dd, J = 6.8, 2.8 Hz, 1H), 4.17 (t, J = 7.2 Hz, 1H), 4.06 (d, J =
3.2 Hz, 1H), 3.87 (dd, J = 10.0, 3.4 Hz, 1H), 3.70-3.68 (m, 1H), 2.78-2.64 (m, 4H, 2CH2),
1.42 (s, 3H, CH3), 1.30-1.10 (m, 9H, 3CH3), 1.02 (s, 9H, (CH3)3C); 13C{1H} NMR (100
MHz, CDCl3) δ 135.8, 135.7, 135.0, 133.2, 130.0, 128.1, 127.9, 110.3, 84.0, 73.7, 65.4, 53.4,
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29.9, 27.5, 27.3, 27.0, 26.7, 14.7; HR-ESI-MS (m/z): [M + Na]+ calcd. for C28H42O4S2SiNa,
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557.2186; found, 557.2186.
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9
4-Azido-6-tert-O-butyl diphenylsilyl-2,3-O-isopropylidine-D-arabinose diethyl dithio
acetal (17)
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Trifluoromethanesulfonic anhydride (0.15 mL, 0.92 mmol ) was added dropwise at 0 oC to a
stirred solution of 16 (0.31 g, 0.77 mmol ) in pyridine (3.9 mL, 47.6 mmol) and CH2Cl2 (5
mL) and this solution was stirred at the same temperature for 1 h and then concentrated in
vacuo and the crude product was used for the next step without purification.
The crude product which was obtained in above step was dissolved in DMF (6 mL) and
to this, NaN3 (0.5 g, 7.7 mmol) was added. This reaction mixture was stirred at room
temperature for 8 h and then reaction mixture was diluted with EtOAc and washed with brine.
Separated aqueous layer was washed twice with EtOAc. The combined organic layers were
dried over Na2SO4 and concentrated in vacuo. The desired product was purified by column
chromatography (10% ethyl acetate: pet ether) to obtain 17 as a pale yellowish liquid (0.29 g,
81%). [α]20 -18.9 (c 53, CHCl3); IR (CHCl3) ῡ 3020, 2933, 2112, 1519, 1216, 1113, 761
D
cm-1; 1H NMR (400 MHz, CDCl3) δ 7.71-7.68 (m, 4H, ArH), 7.46-7.37 (m, 6H, ArH), 4.31(t,
J = 7.5 Hz, 1H), 4.15 (dd, J = 7.5, 2.0 Hz, 1H), 3.99-3.89 (m, 2H), 3.85 (d, J = 6.2 Hz, 1H),
3.69-3.66 (m, 1H), 2.77-2.64 (m, 4H, 2CH2), 1.43 (s, 3H, CH3), 1.39 (s, 3H, CH3), 1.28-1.23
(m, 6H, 2CH3), 1.07 (s, 9H, (CH3)3C); 13C{1H} NMR (100 MHz, CDCl3) δ 135.8, 133.1,
130.0, 127.9, 110.3, 79.8, 78.7, 64.4, 63.0, 53.0, 27.2, 27.0, 26.9, 25.6, 25.2, 19.3, 14.6, 14.4;
HR-ESI-MS (m/z): [M + Na]+ calcd. for C28H41O3N3S2SiNa, 582.2251; found, 582.2253.
4-Azido-6-tert-O-butyl diphenylsilyl-D-arabinose diethyl dithio acetal (18)
A solution of 17 (0.15 g, 0.26 mmol) in 80% AcOH (6 mL) was stirred at 80 °C for 8 h. After
complete consumption of starting material, the reaction mixture was concentrated on rotary
evaporator and the residue was chromatographed on silica gel to afford 18 as a pale yellowish
liquid (88 mg, 60%). [α]20D -6.8 (c 1.0, CHCl3); IR (CHCl3) ῡ 3440, 3020, 2930, 2111, 1428,
1216, 1113, 760, 669 cm-1; 1H NMR (400 MHz, CDCl3) δ 7.70-7.67 (m, 4H, ArH), 7.46-7.37
(m, 6H, ArH), 4.19 (t, J = 7.4 Hz, 1H), 4.00 (d, J = 8.4 Hz, 1H), 3.89-3.82 (m, 2H), 3.63 (q, J
= 5.6 Hz, 1H), 3.56 (td, J = 4.6, 2.4 Hz, 1H), 3.26 (s, 1H, OH), 2.75-2.59 (m, 4H), 2.55 (s,
1H, OH), 1.29-1.24 (m, 6H, 2CH3), 1.09 (s, 9H, C(CH3)3);; 13C{1H} NMR (100 MHz,
CDCl3) δ 135.8, 135.7, 133.0, 132.9, 130.0, 128.0, 72.0, 70.0, 66.2, 64.1, 55.6, 26.9, 26.0,
23.9, 19.3, 14.7, 14.6; HR-ESI-MS (m/z): [M + Na]+ calcd. for C25H37O3N3S2SiNa, 542.1938;
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Phenyl 2,3,4-O-Triacetyl-1-thio--D-lyxopyranoside (20)
Ac2O (15.7 mL, 166.5 mmol) and DMAP (0.4 g, 3.33 mmol) were added to a turbid solution
of D-lyxose (5.0 g, 33.3 mmol) in CH2Cl2 (70 mL) and pyridine (27 mL) and the reaction
mixture was kept for strirring at room temparature 3 h. Then solvents were removed under
reduced pressure. The residue which was obtained after solvents removal was dissolved in
CH2Cl2 (10 mL) and to this HBr in AcOH (60 mL) was added. After stirring at room
temparature for 1 h, reaction mixture was diluted with CH2Cl2 and washed sevral times with
water untill the pH reaches to 7. Separated organic layer dried over Na2SO4, concentrated to
give 19 (10.1 g, 88%) as a yellowish liquid. To this clear solution of glycosyl bromide 19 in
EtOAc (80 mL) were added TBAHS (10.2 g, 31.2 mmol) and PhSH (9.7 mL, 93.7 mmol).
After 5 min. 1M Na2CO3 (80 mL) was added dropwise and kept for stirring at room
temparature 1 h. Then, this reaction mixture was poured in cold water and the organic layer
was washed with brine. The organic extract was dried over Na2SO4, filtered and concentrated
in vacuo to obtain the desired product 20 as a pale yellowish coloured liquid (8.63 g, 75%).
[α]20D -115.5 (c 2.89, CHCl3); IR (CHCl3) ῡ 3020, 2912, 1750, 1217, 1053, 768, 669 cm-1; 1H
NMR (400 MHz, CDCl3) δ 7.48-7.45 (m, 2H, ArH), 7.31-7.28 (m, 3H, ArH), 5.72 (t, J = 3.1
Hz, 1H, H-2), 5.19 (d, J = 2.8 Hz, 1H, H-1), 5.15-5.10 (m, 2H, H-3 & H-4), 4.37 (d, J = 12.3,
3.8 Hz, 1H, H-5a), 3.49 (dd, J = 12.3, 2.8 Hz, 1H, H-5b), 2.14 (s, 3H, CH3), 2.08 (s, 3H,
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CH3), 2.06 (s, 3H, CH3); C{1H} NMR (100 MHz, CDCl3) δ 170.1, 170.0, 169.9, 131.8,
129.2, 127.9, 86.2 (C1), 69.6, 69.2, 67.5, 21.0, 20.9, 20.8; HR-ESI-MS (m/z): [M + Na]+
calcd. for C17H20O7NaS, 391.0822; found, 391.0824.
Phenyl 1-Thio-β-D-lyxopyranoside (21)
NaOMe (0.6 g) was added to a stirred solution of 20 (6.0 g, 13.5 mmol) in MeOH (60 mL).
After 1 h, reaction mixture was neutralized with Amberlite IR 120 (H+) (6.0 g) resin. The
reaction mixture was filtered and concentrated under reduced pressure to afford desired
tetraol 21 as a white solid (3.14 g, 92%). [α]20 -68.4 (c 1.11, MeOH); mp 185 °C; IR
D
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(MeOH) ῡ 3350, 2934, 1667, 1451, 1116, 1026, 667 cm-1; H NMR (500 MHz, MeOD) δ
7.44-7.31 (m, 2H, ArH), 7.29-7.28 (m, 3H, ArH), 5.06 (s, 1H, H-1), 4.13 (s, 1H, H-2), 4.00-
3.97 (m, 1H, H-5a), 3.83-3.78 (m, 1H, H-4), 3.59-3.56 (m, 1H, H-3), 3.29-3.19 (m, 1H, H-
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5b); C{1H} NMR (125 MHz, MeOD) δ 134.8, 131.6, 130.4, 128.7, 89.0 (C1), 74.5, 72.3,
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69.1, 67.5; HR-ESI-MS (m/z): [M + Na]+ calcd. for C11H14O4NaS, 265.0505; found,
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265.0499.
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Phenyl 3-O-Benzoyl-1-thio-β-D-lyxopyranoside (22)
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Me2SnCl2 (25 mg, 0.11 mmol) and DIPEA (0.78 mL, 4.53 mmol) were added to a stirred
solution of 21 (0.55 g, 2.27 mmol) in THF (16 mL). To this, BzCl (0.29 mL, 2.49 mmol) was
added, and after 1 h, the reaction mixture was quenched with 3% HCl (30 mL) and extracted
with EtOAc (50 mL x 2). The combined organic layers were dried over Na2SO4, concentrated
in vacuo, and purified by column chromatography (40% ethyl acetate: petroleum ether) to
afford 22 as a white solid (0.63 g, 80%). [α]20D -20.4 (c 3.29, CHCl3); mp 215 °C; IR (CHCl3)
ῡ 3474, 3019, 1719, 1436, 1216, 1052, 758, 669 cm-1;1H NMR (400 MHz, CDCl3) δ 8.20 (d,
J = 7.2 Hz, 2H, ArH), 7.61-7.46 (m, 5H, ArH), 7.36-7.30 (m, 3H, ArH), 5.40 (d, J = 3.3 Hz,
1H, H-1), 5.26 (dd, J = 8.0, 3.6 Hz, 1H, H-3), 4.52 (t, J = 3.7 Hz, 1H, H-2), 4.41 (d, J = 12.6,
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2.8 Hz, 1H, H-5a), 4.15-12 (m, 1H, H-4), 3.62 (dd, J = 12.6, 4.4 Hz, 1H, H-5b); C{1H}
NMR (125 MHz, CDCl3) δ 166.4, 133.9, 132.4, 132.2, 130.3, 130.0, 129.3, 128.8, 128.0,
90.8 (C1), 74.3, 66.9, 66.7; HR-ESI-MS (m/z): [M + Na]+ calcd. for C18H18O5NaS, 369.0767;
found, 369.0765.
Phenyl 2,4-Diazido-2,4-dideoxy-3-O-benzoyl-1-thio-β-L-arabinopyranoside (23)
o
Trifluoromethanesulfonic anhydride (1.3 mL, 7.8 mmol) was added dropwise at -10 C to a
stirred solution of 22 (0.45 g, 1.3 mmol) and pyridine (1.36 mL, 17.0 mmol) in CH2Cl2 (20
o
mL) and this solution was gradually brought to 10 C over 2 h. After complete consumption
of starting material, as indicated by TLC, the reaction mixture was concentrated in vacuo and
the crude product was used for the next step without any purification.
The crude product which was obtained in the above step was dissolved in DMF (10
mL) and to this, NaN3 (1.7 g, 26.0 mmol) was added. The reaction mixture was stirred at rt
for 8 h and then it was diluted with EtOAc (50 mL) and washed with water. Separated
aqueous layer was washed with EtOAc (50 mL x 2). The combined organic layers were dried
over Na2SO4 and concentrated in vacuo. The desired product was purified by column
chromatography (10% ethyl acetate: pet ether) to obtain 23 as a pale yellowish liquid (0.42 g,
82%). [α]20D -20.0 (c 0.53, CHCl3); IR (CHCl3) ῡ 3019, 2926, 2114, 1725, 1265, 1216, 1093,
759, 669 cm-1; 1H NMR (400 MHz, CDCl3) δ 8.12 (dd, J = 8.2, 1.2 Hz, 2H, ArH), 7.66-7.63
(m, 3H, ArH), 7.53-7.49 (m, 2H, ArH), 7.41-7.39 (m, 3H, ArH), 5.21 (dd, J = 9.2, 3.6 Hz,
1H, H- 3), 4.60 (d, J = 9.2 Hz, 1H, H-1), 4.23 (d, J = 12.6, 3.0 Hz, 1H, H-5a), 4.18-4.16 (m,
1H, H-4), 3.93 (t, J = 9.2 Hz, 1H, H-2), 3.73 (dd, J = 12.6, 1.7 Hz, 1H, H-5b); 13C{1H} NMR
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(100 MHz, CDCl3) δ 165.7, 134.1, 133.7, 130.2, 129.3, 128.8, 128.6, 87.3 (C1), 74.4, 67.0,
60.4, 58.2; HR-ESI-MS (m/z): [M + Na]+ calcd. for C18H16N6O3NaS, 419.0897; found,
419.0891.
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Phenyl 2,4-Diazido-2,4-dideoxy-1-thio-β-L-arabinopyranoside (24)
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NaOMe (50 mg) was added to a stirred solution of 23 (0.34 g, 0.86 mmol) in MeOH (5 mL)
and DCM (2 mL). After 1 h, reaction mixture was neutralized with Amberlite IR 120 (H+)
(0.1 g) resin. The reaction mixture was filtered and concentrated under reduced pressure to
afford desired product 24 as foam (0.23 g, 92%). [α]20D +21.5 (c 1.38, CHCl3); IR (CHCl3) ῡ
3019, 2924, 2113, 1446, 1217, 1085, 770, 669 cm-1; 1H NMR (400 MHz, CDCl3) δ 7.63-7.61
(m, 2H, ArH), 7.38-7.29 (m, 3H, ArH), 4.47 (d, J = 9.4 Hz, 1H, H-1), 4.20 (dd, J = 9.4, 3.6
Hz, 1H, H- 3), 3.93 (bs, 1H, H-4), 3.75-3.56 (m, 3H, H-2, H-5a & H-5b); 13C{1H} NMR (125
MHz, CDCl3) δ 133.5, 129.3, 128.7, 87.3 (C1), 73.2, 67.0, 63.2, 60.1; HR-ESI-MS (m/z): [M
+ Na]+ calcd. for C11H12N6O2NaS, 315.0635; found, 315.0638.
Phenyl 2,4-Diazido-2,4-dideoxy-1-thio-β-L-lyxopyranoside (25)
Trifluoromethanesulfonic anhydride (0.16 mL, 0.96 mmol ) was added dropwise at -10 oC to
a stirred solution of 24 (0.23 g, 0.8 mmol) and pyridine (0.39 mL, 4.8 mmol) in CH2Cl2 (4
o
mL) and this solution was gradually brought to 10 C over 2 h. After complete consumption
of starting material, as indicated by TLC, the reaction mixture was concentrated in vacuo and
the crude product was used for the next step without any purification.
The crude product which was obtained in the above step was dissolved in DMF (4
mL) and to this, NaNO2 (0.55 g, 8.0 mmol) was added. The reaction mixture was stirred at rt
for 8 h and then it was diluted with EtOAc (50 mL) and washed successively with aq.
NaHCO3, water and brine. Separated aqueous layer was dried over Na2SO4 and concentrated
in vacuo. The desired product was purified by column chromatography (10% ethyl acetate:
pet ether) to obtain 25 as a pale yellowish liquid (0.16 g, 70%). [α]20D -5.12 (c 0.50, CHCl3);
IR (CHCl3) ῡ 3019, 2926, 2855, 2113, 1523, 1424, 1216, 1025, 760, 669 cm-1; 1H NMR (400
MHz, CDCl3) δ 7.54-7.51 (m, 2H, ArH), 7.38-7.34 (m, 3H, ArH), 5.34 (d, J = 4.0 Hz, 1H, H-
1), 4.06-3.76 (m, 5H, H-2, H-3, H-4, H-5a & H-5b); 13C{1H} NMR (125 MHz, CDCl3) δ
132.4, 129.4, 128.4, 86.0 (C1), 70.3, 63.3, 62.4, 59.6; HR-ESI-MS (m/z): [M + Na]+ calcd.
for C11H12N6O2NaS, 315.0635; found, 315.0613.
Phenyl 2,4-Diazido-2,3,4-trideoxy-2-dehydro-1-thio-β-D-lyxopyranoside (26)
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Trifluoromethanesulfonic anhydride (0.05 mL, 0.33 mmol) was added dropwise at 0 C to a
stirred solution of 25 (82 mg, 0.28 mmol) and pyridine (0.14 mL, 1.7 mmol) in CH2Cl2 (2
mL) and this solution was stirred at the same temperature for 1 h. After complete
consumption of starting material, as indicated by TLC, the reaction mixture was concentrated
in vacuo and the crude product was purified by column chromatography (5% ethyl acetate:
pet ether) to obtain 26 as a pale brownish liquid (69 mg, 90%). [α]20D -35.2 (c 0.2, CHCl3); IR
(CHCl3) ῡ 3019, 2111, 1648, 1522, 1478, 1216, 1078, 928, 770, 669 cm-1;1H NMR (400
MHz, CDCl3) δ 7.47-7.41 (m, 2H, ArH), 7.28-7.27 (m, 3H, ArH), 5.45 (d, J = 1.8 Hz, 1H, H-
3), 5.34 (s, 1H, H-1), 4.16-4.12 (m, 1H, H-4), 4.01 (t, J = 11.3 Hz, 1H, H-5a), 3.91 (dd, J =
11.3, 6.3 Hz, 1H, H-5b); 13C{1H} NMR (100 MHz, CDCl3) δ 139.5, 133.7, 132.5, 129.3,
128.3, 110.4 (C3), 83.4 (C1), 60.9, 53.5; HR-ESI-MS (m/z): [M + Na]+ calcd. for
C11H10N6ONaS, 297.0529; found, 297.0520.
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Phenyl 2-Azido-2-deoxy--D-1-thio-xylopyranoside (27)
Camphorsulphonic acid (4 mg) was added to a clear solution of 21 (0.45 g, 1.86 mmol), 2,3-
butanedione (0.2 mL, 2.2 mmol) and CH(OMe)3 (0.6 mL, 5.6 mmol) in MeOH (6 mL) and
the reaction mixture was stirred at 70 C for 15 h. After complete consumption of starting
materials, solvents were removed under reduced presure. The obtained residue was dissolved
in CH2Cl2 (9 mL) and to this pyridine (0.92 mL, 11.3 mmol), Tf2O (0.92 mL, 11.3 mmol)
were added at 0 C. After 1 h, solvents were removed and to this crude triflate, NaN3 (1.2 g,
18.9 mmol) was added in DMF (10 mL). After 8 h, reaction mixture was diluted with EtOAC
(40 mL) and washed twice with brine. Separated organic layer was dried over Na2SO4 and
concentrated in vacuo.
The crude product which was obtained after solvents removal was dissolved in 90%
aq. TFA (13.5 mL) and stirred at rt for 5 min. Then, the reaction mixture was diluted with
EtOAc (60 mL) and washed successively with water and aq. NaHCO3. Separated organic
layer was dried over Na2SO4 and concentrated in vacuo and the obtained crude residue was
purified by column chromatography (45% ethyl acetate: pet ether) to obtain 27 (61% 0.3 g) as
a yellowish liquid. [α]20 -7.2 (c 0.34, CHCl3); IR (CHCl3) ῡ 3430, 3019, 2929, 2111, 1521,
D
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1424, 1216, 1069, 763, 669 cm-1; H NMR (500 MHz, CDCl3) δ 7.58-7.57 (m, 2H, ArH),
7.35-7.28 (m, 3H, ArH), 4.45 (d, J = 9.8 Hz, 1H, H-1), 4.09-4.04 (m, 1H, H-5a), 3.66-3.61
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(m, 1H, H-4), 3.43-3.40 (m, 1H, H-3), 3.39-3.22 (m, 2H, H-2 & H-5b); C{1H} NMR (125
MHz, CDCl3) δ 133.4, 131.5, 129.3, 128.6, 87.6 (C1), 69.6, 69.4, 65.2; HR-ESI-MS (m/z):
[M + Na]+ calcd. for C11H13N3O3NaS, 290.0570; found, 290.0566.
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Phenyl 2-Azido-2-deoxy-4-benzoyl--D-1-thio-xylopyranoside (28)
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Me2SnCl2 (11 mg, 0.05 mmol) and DIPEA (0.35 mL, 2.0 mmol) were added to a stirred
solution of 27 (0.27 g, 1.0 mmol) in THF (5 mL). To this, BzCl (0.13 mL, 1.12 mmol) was
added. After 1 h, the reaction mixture was quenched with 3% HCl (30 mL) and extracted
with EtOAc (50 mL x 2). The combined organic layers were dried over Na2SO4, concentrated
in vacuo, and purified by column chromatography (20% ethyl acetate: petroleum ether) to
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afford 28 as a foam (90%, 0.34 g). [α]20 -67.19 (c 1.58, CHCl3); IR (CHCl3) ῡ 3420, 3019,
D
2112, 1725, 1217, 1113, 706, 670 cm-1; 1H NMR (500 MHz, CDCl3) δ 8.02 (dd, J = 8.2, 1.2
Hz, 2H, ArH), 7.63-7.57 (m, 3H, ArH), 7.46-7.43 (m, 2H, ArH), 7.38-7.28 (m, 3H, ArH),
5.08-5.03 (m, 1H, H-4), 4.52 (d, J = 9.8 Hz, 1H, H-1), 4.31 (dd, J = 11.4, 5.3 Hz, 1H, H-5a),
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3.84 (t, J = 9.0 Hz, 1H, H-3), 3.44-3.38 (m, 2H, H-2 & H-5b); C{1H} NMR (125 MHz,
CDCl3) δ 166.4, 133.76, 133.71, 129.9, 129.2, 128.7, 128.6, 87.0 (C1), 75.0, 71.7, 66.6, 65.1;
HR-ESI-MS (m/z): [M + Na]+ calcd. for C18H17N3O4NaS, 394.0832; found, 394.0833.
Phenyl 2-Azido-2-deoxy-4-benzoyl--D-1-thio-ribopyranoside (29)
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Trifluoromethanesulfonic anhydride (0.16 mL, 0.95 mmol) was added dropwise at 0 C to a
stirred solution of 28 (0.29 g, 0.79 mmol) and pyridine (0.39 mL, 4.8 mmol) in CH2Cl2 (5
mL) and this solution was stirried at rt for 1 h. After complete consumption of starting
material, as indicated by TLC, the reaction mixture was concentrated in vacuo and the crude
product was used for the next step without any purification.
The crude product which was obtained in the above step was dissolved in DMF (5
mL) and to this, NaNO2 (0.55 g, 8.0 mmol) was added. The reaction mixture was stirred at rt
for 10 h and then it was diluted with EtOAc (40 mL) and washed successively with aq.
NaHCO3, water and brine. Separated aqueous layer was dried over Na2SO4 and concentrated
in vacuo. The desired product was purified by column chromatography (5% ethyl acetate: pet
ether) to obtain 29 as a foam (0.2 g, 70%). [α]20 -36.6 (c 0.3, CHCl3); IR (CHCl3) ῡ 3424,
D
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2928, 2111, 1749, 1219, 770 cm-1; H NMR (500 MHz, CDCl3) δ 8.09 (d, J = 8.2 Hz, 2H,
ArH), 7.63-7.58 (m, 3H, ArH), 7.49-7.46 (m, 2H, ArH), 7.38-7.28 (m, 3H, ArH), 5.30 (d, J =
6.7 Hz, 1H, H-1), 5.16 (q, J = 4.0 Hz, 1H, H-4), 4.44 (d, J = 2.6 Hz, 1H, H-3), 4.22 (dd, J =
11.3, 3.5 Hz, 1H, H-5a), 4.03 (dd, J = 7.6, 1.0 Hz, 1H, H-5b), 3.66 (dd, J = 6.7, 2.6 Hz, 1H,
H-2), 2.63 (d, J = 4.2 Hz, 1H, OH); 13C{1H} NMR (125 MHz, CDCl3) δ 166.1, 133.7, 133.0,
131.9, 130.0, 129.4, 129.3, 128.7, 128.5, 84.3 (C1), 69.5, 67.9, 62.9, 61.9; HR-ESI-MS (m/z):
[M + Na]+ calcd. for C18H17N3O4NaS, 394.0832; found, 394.0827.
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Trifluoromethanesulfonic anhydride (0.48 mL, 2.8 mmol) was added dropwise at 0 C to a
stirred solution of 29 (0.89 g, 2.4 mmol) and pyridine (1.15 mL, 14.4 mmol) in CH2Cl2 (12
mL) and the reaction mixture was kept for stirring at rt for 1 h. After complete consumption
of starting material, as indicated by TLC, the reaction mixture was concentrated in vacuo and
the crude product was used for the next step without any purification.
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The crude product which was obtained in the above step was dissolved in DMF (12
mL) and to this, NaN3 (1.6 g, 24.0 mmol) was added. The reaction mixture was stirred at rt
for 8 h and then it was diluted with EtOAc (50 mL) and washed with water. Separated
aqueous layer was washed with EtOAc (50 mL x 2). The combined organic layers were dried
over Na2SO4 and concentrated in vacuo. NaOMe (80 mg) in MeOH (8 mL) was added to the
crude residue which was obtained in above step and after stirring at rt for 1 h, reaction
mixture was neutralized with Amberlite IR 120 (H+) (0.8 g) resin. The reaction mixture was
filtered, concentrated and purified by column chromatography (10% ethyl acetate: pet ether)
to obtain 30 as foam (0.54 g, 78%). [α]20 -143.6 (c 0.5, CHCl3); IR (CHCl3) ῡ 3020, 2113,
1522, 1217, 1077, 765, 669 cm-1; H NMR (500 MHz, CDCl3) δ 7.59-7.57 (m, 2H, ArH),
D
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7.37-7.28 (m, 3H, ArH), 4.47 (d, J = 9.6 Hz, 1H, H-1), 4.12 (dd, J = 11.6, 5.2 Hz, 1H, H-5a),
3.64-3.58 (m, 1H, H-4), 3.32-3.23 (m, 3H, H-2, H-3 & H-5b); C{1H} NMR (125 MHz,
CDCl3) δ 133.7, 129.3, 128.8, 87.8 (C1), 69.9, 69.8, 68.7, 64.1; HR-ESI-MS (m/z): [M + Na]+
13
calcd. for C11H12N6O2NaS, 315.0635; found, 315.0622.
Phenyl 2,3,4-Triazido -2,3,4-trideoxy -1-thio --L-arabinopyanoside (5)
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Trifluoromethanesulfonic anhydride (0.3 mL, 1.8 mmol) was added dropwise at 0 C to a
stirred solution of 35 (0.43 g, 1.5 mmol) and pyridine (0.72 mL, 0.9 mmol) in CH2Cl2 (6 mL)
and the reaction mixture was kept for stirring at rt for 1 h. After complete consumption of
starting material, as indicated by TLC, the reaction mixture was concentrated in vacuo and
the crude product was used for the next step without any purification.
The crude product which was obtained in the above step was dissolved in DMF (6
mL) and to this, NaN3 (0.97 g, 15.0 mmol) was added. The reaction mixture was stirred at rt
for 6 h and then it was diluted with EtOAc (40 mL) and washed with water. Separated
aqueous layer was washed with EtOAc (40 mL x 2). The combined organic layers were dried
over Na2SO4, concentrated and purified by column chromatography (8% ethyl acetate: pet
ether) to obtain 5 as a pale yellowish liquid (0.39 g, 84%). [α]20 -23.9 (c 4.39, CHCl3); IR
(CHCl3) ῡ 3019, 2926, 2113, 1515, 1216, 1093, 759, 669 cm-1; H NMR (500 MHz, CDCl3)
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The Journal of Organic Chemistry
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δ 7.63-7.61 (m, 2H, ArH), 7.39-7.28 (m, 3H, ArH), 4.46 (d, J = 9.3 Hz, 1H, H-1), 4.17 (dd,
J = 12.8, 2.5 Hz, 1H, H-5a), 3.87 (bs, 1H, H-4), 3.67-3.60 (m, 2H, H-2 & H-5b), 3.54 (dd, J =
13
9.6, 3.5 Hz, 1H, H-3); C{1H} NMR (125 MHz, CDCl3) δ 133.7, 131.0, 129.3, 128.8, 87.8
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(C1), 67.7, 64.6, 61.5, 58.9; HR-ESI-MS (m/z): [M + Na]+ calcd. for C11H11N9ONaS,
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340.0699; found, 340.0692.
Phenyl 4,6-O-Benzylidene-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-1-thio-β-D-
allopyranoside (33)
o
Trifluoromethanesulfonic anhydride (0.1 mL, 0.6 mmol) was added dropwise at 0 C to a
stirred solution of 3224 (0.26 g, 0.5 mmol) and pyridine (0.24 mL, 3.0 mmol) in CH2Cl2 (4
mL) and this solution was stirred at rt for 1 h. After complete consumption of starting
material, as indicated by TLC, the reaction mixture was concentrated in vacuo and the crude
product was used for the next step without any purification.
The crude product which was obtained in the above step was dissolved in DMF (6
mL) and to this, NaNO2 (0.35 g, 4.9 mmol) was added. The reaction mixture was stirred at rt
for 10 h and then it was diluted with EtOAc (40 mL) and washed successively with aq.
NaHCO3, water and brine. Separated aqueous layer was dried over Na2SO4 and concentrated
in vacuo. The desired product was purified by column chromatography (20% ethyl acetate:
pet ether) to obtain 33 as foam (0.21 g, 80%). [α]20 -13.9 (c 18.24, CHCl3); IR (CHCl3) ῡ
3429, 3018, 2871, 1736, 1515, 1216, 1075,997, 758, 668 cm-1; H NMR (500 MHz, CDCl3)
D
1
δ 7.74-7.62 (m, 4H, ArH), 7.49-7.46 (m, 4H, ArH), 7.42-7.32 (m, 2H, ArH), 5.78 (d, J = 9.0
Hz, 1H, NH), 5.57 (s, 1H, benzylidene), 5.01 (d, J = 10.0 Hz, 1H, H-1), 4.91 (d, J = 12.0 Hz,
1H, H2CCCl3), 4.74 (d, J = 12.0 Hz, 1H, H2CCCl3), 4.43 (dd, J = 10.0, 5.0 Hz, 1H, H-6a),
4.32 (bs, 1H, H-3), 4.09-3.94 (m, 2H, H-2 & H-5), 3.83 (t, J = 10.0 Hz, 1H, H-6b), 3.69-3.66
(m, 1H, H-4), 2.61 (s, 1H, OH); 13C{1H} NMR (125 MHz, CDCl3) δ 154.2, 136.9, 133.3,
132.2, 129.6, 129.1, 128.6, 128.0, 126.3, 101.9 (benzylidine), 95.6 (-CCl3), 85.9 (C1), 78.5,
76.9, 69.1, 68.6, 66.3, 53.2; HR-ESI-MS (m/z): [M
+
Na]+ calcd. for
C22H22NCl3O6NaS,558.0102; found, 558.0101.
Phenyl
4,6-O-Benzylidene-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-3-nitro-1-
thio-β-D- allopyranoside (34)
[α]20 -15.5 (c 6.35, CHCl3); IR (CHCl3) ῡ 3324, 2870, 1741, 1516, 1219, 1116, 1076, 997,
D
1
755, 699 cm-1; H NMR (500 MHz, CDCl3) δ 7.56-7.49 (m, 4H, ArH), 7.38-7.31 (m, 6H,
ArH), 5.61 (s, 1H, benzylidene), 5.57 (d, J = 8.0 Hz, 1H, NH), 5.03 (d, J = 10.0 Hz, 1H, H-
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