Synthetic Routes toward Acidic Pentasaccharide Related to the O-Antigen of E. coli 120 Using One-Pot Sequential Glycosylation Reactions

Article abstract of DOI:10.1021/acs.joc.7b00561

Concise syntheses of the acidic pentasaccharide, related to the O-antigenic polysaccharide of Escherichia coli 120, as its p-methoxyphenyl glycoside, have been achieved using a one-pot sequential glycosylation technique. The glycosylations have been accom

Full text of DOI:10.1021/acs.joc.7b00561

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Article
Synthetic routes towards acidic pentasaccharide related to the O-
antigen of E. coli 120 using one-pot sequential glycosylation reactions
Mana Mohan Mukherjee, and Rina Ghosh
J. Org. Chem., Just Accepted Manuscript • Publication Date (Web): 10 May 2017
Downloaded from http://pubs.acs.org on May 11, 2017
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Synthetic routes towards acidic pentasaccharide related to the Oꢀ
antigen of E. coli 120 using oneꢀpot sequential glycosylation
reactions
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Mana Mohan Mukherjee, Rina Ghosh*
Department of Chemistry, Jadavpur University, Kolkata 700 032, India.
Abstract
Concise syntheses of the acidic pentasaccharide, related to the Oꢀantigenic
polysaccharide of Escherichia coli 120, as its pꢀmethoxyphenyl glycoside, have been achieved
utilizing oneꢀpot sequential glycosylation technique. The glycosylations have been accomplished
either by the activation of the thioglycosides using NIS in the presence of FeCl3 or by a
preactivation by Ph2SO, TTBP, Tf2O and the activation of the trichloroacetimidates using FeCl3
alone or TMSOTf. Most of the intermediate steps are high yielding, and the stereo outcomes of
the glycosylation steps were excellent. The syntheses of the targeted pentasaccharide have been
performed with both threeꢀ and fourꢀcomponent oneꢀpot sequential glycosylation reactions, and
in both cases the orthogonal glycosylations are carried out utilizing catalytic activity of FeCl3. A
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late stage TEMPOꢀmediated regioselective oxidation has been performed to achieve the required
uronic acid motif.
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Introduction
Being a major component of the cell wall of Gramꢀnegative bacteria, the Oꢀantigens often
play important roles during host infections, subsequent immune responses in the host and in
controlling its virulence property. The Oꢀantigen is one of the most variable cell constituent that
consists of a polysaccharide chain with a number of repeats of an oligosaccharide. Escherichia
coli (E. coli) are a facultative gramꢀnegative bacteria present predominantly in the human and
animal kingdoms. Identification of E. coli clones including the commensal and pathogenic
strains are normally done by the combination of their somatic (O), flagellar (H) and occasionally
by capsular (K) antigens.1 More than 180 Oꢀantigen forms of E. coli have been recognized so
far.2 The pathogenic E. coli strains in general cause three common infections like (i)
enteric/diarrhoeal (ii) septicaemia/meningitis and (iii) urinary track infections.3 The virulent E.
coli strains causing diarrhea are classified in six classes as (i) enteropathogenic E. coli (EPEC),
(ii) enterotoxigenic E. coli (ETEC), (iii) enteroinvasive E. coli (EIEC), (iv) diffusely adherent E.
coli (DAEC), (v) enteroaggregative E. coli (EAEC) and (vi) enterohaemorrhagic E. coli
(EHEC).4
The enterohaemorrhagic E. coli (EHEC) causes foodꢀborne diseases with life threatening
complications like haemorrhagic colitis (HC) and haemolyticꢀuraemic syndrome (HUS) in
human and animal kingdoms worldwide.4 Because of their toxic effect on the cultured Vero cells,
these EHEC strains are also called ‘verotoxigenic E. coli (VTEC)’. They are also termed as
Shiga toxin producing E. coli (STEC) as they produce a bacteriophageꢀmediated Shigaꢀlike
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toxin.5 The strains belonging to E. coli O120 isolated from their reservoirs like swine feces,
cattle and beef products are identified as STEC.6,7 Recently, Knirel et al. elucidated the structure
of the repeating unit of the Oꢀantigen from E. coli 120 and found to contain an acidic
hexasaccharide repeating unit (Figure 1).8
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Figure 1 Hexasaccharide repeating unit of the Oꢀantigen of E. coli type 120.
It has been well established that bacterial Oꢀantigens regulate immunoꢀchemical activity
of glycoꢀvaccines which make them attractive targets to the synthetic organic chemists for the
development of glycoconjugate vaccine candidates.9 Recently a number of reports have appeared
for the synthesis of glycoconjugate vaccines and their evaluation against bacterial interactions.9
Synthesis of an oligosaccharide with a temporary protecting group at the reducing end would be
useful for its easy removal whenever necessary as it is often required to attached the
oligosaccharide with a carrier protein through a spacer linker toward synthesis of
glycoconjigates.10 In this direction the synthesis of the oligosaccharide related to the repeating
unit of the Oꢀantigen from E. coli 120 was only reported by Mukhopadhyay et al,11 using a
conventional stepꢀwise approach. However introduction of a step economic multistep oneꢀpot
total synthesis is still necessary and needs refinement of the entire synthetic protocol. The
stepwise oligosaccharide syntheses12 demand extensive protecting group manipulation and
purification after each step making them expensive, time consuming, and tedious procedures.
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Figure 2 Structure of the target pentasaccharide.
In contrast, the oneꢀpot sequential oligosaccharide syntheses are step economic,
comparatively environment friendly, cost effective, and expeditious. We report herein concise
synthesis of the acidic pentasaccharide part of the hexasaccharide repeating unit in the form of its
4ꢀmethoxyphenyl glycoside via both three and four component oneꢀpot sequential glycosylation
reactions (1, Figure 2). The convergent strategy will give the scope for the preparation of this
important oligosaccharide structure in the pure form and in quantity that will pave the way for
understanding its role in the pathogenic cycle. Moreover, a selective oxidative removal of the pꢀ
methoxyphenyl aglycon group of the pentasaccharide derivative using ceric ammonium nitrate
(CAN) followed by formation of the corresponding trichloroacetimidate derivative will allow the
formation of glycoconjugate with suitable aglycons targeting potential vaccine candidates against
this deadly pathogen.
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Results and discussion
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The sequential oneꢀpot glycosylation technique was applied for these total syntheses.
Application of this in both threeꢀ and fourꢀcomponent oneꢀpot reactions requires different sets of
chemically distinct glycosyl donors requiring different activation conditions. For the convergent
synthesis of the targeted acidic pentasaccharide 1 two different pathways were contemplated, one
of which was a threeꢀcomponent sequential glycosylation i.e via [1+2+2] approach and the other
one was a fourꢀcomponent sequential glycosylation reactions i.e via [1+2+1+1] approach.
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Figure 3 A retrosynthetic analysis of the pentasaccharide derivative 2.
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For each pathway, a retroꢀsynthetic analysis of the fully protected pentasaccharide
derivative 2 led to two common building blocks, one is 2ꢀOꢀbenzoylꢀ3,4ꢀdiꢀOꢀbenzylꢀLꢀ
rhamnopyranosyl trichloroacetimidate donor 3 and other is the disaccharide acceptor 5, which
can be prepared from the monomeric units 8 and 9. Whereas the retrosynthetic analysis for the
second approach [1+2+1+1] of the total synthesis guided us for use of the monomeric units 10
and 11, the first synthetic approach [1+2+2] indicated us to deal with another disaccharide
segment 7 to be prepared from 10 and 11 (Figure 3). The disaccharide building blocks 5 and 7
can be obtained from their corresponding parent disaccharides 4 and 6 via selective removal of
acetate protection.
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Scheme 1 Synthesis of Lꢀrhamnose based building blocks.
Dry Lꢀrhamnose was acetylated by acetic anhydride and a catalytic amount of
magnesium (II) trifluromethanesulfonate [Mg(OTf)2]13 in neat condition; after full consumption
of the starting material (checked by TLC) into the same reaction vessel thiophenol followed by
BF3.Et2O were added to furnish phenyl 2,3,4ꢀtriꢀOꢀacetylꢀ1ꢀthioꢀαꢀLꢀrhamanopyranoside 13 in
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93% yield over two steps after purification by column chromatography. This phenyl 2,3,4ꢀtriꢀOꢀ
acetylꢀ1ꢀthioꢀαꢀLꢀrhamanopyranoside 13 was converted to phenyl 3,4ꢀdiꢀOꢀbenzylꢀ1ꢀthioꢀαꢀLꢀ
rhamanopyranoside 9 according to a reported procedure.14 Compound 9 was acetylated
quantitatively using Mg(OTf)2 and acetic anhydride to achieve phenyl 2ꢀOꢀacetylꢀ3,4ꢀdiꢀOꢀ
benzylꢀ1ꢀthioꢀαꢀLꢀrhamanopyranoside 10.15 On the other hand benzoylation of 9 produced
phenyl 2ꢀOꢀbenzoylꢀ3,4ꢀdiꢀOꢀbenzylꢀ1ꢀthioꢀαꢀLꢀrhamanopyranoside 14.14 Thioglycoside
hydrolysis of 14 was carried out following the method developed by us using
trichloroisocyanuric acid (TCCA)16 in aqueous acetone to give compound 15. Reaction of 15
with trichloroacetonitrile and DBU in dry DCM afforded 2ꢀOꢀbenzoylꢀ3,4ꢀdiꢀOꢀbenzylꢀLꢀ
rhamnopyranosyl trichloroacetimidate donor 3 in 90% yield. Thus Lꢀrhamnose based
monosaccharide units 3, 9 and 10 were gathered (Scheme 1).
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Scheme 2 Synthesis of Dꢀglucose based donor 8.
Dꢀglucose was used as the preliminary starting material to synthesize the glucose based
monomeric building block 8 (Scheme 2). Dry Dꢀglucose was acetylated using acetic anhydride
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and catalytic Mg(OTf)2 in neat condition, after completion of the reaction (checked by TLC)
into the same reaction vessel thiophenol followed by BF3.Et2O were added to produce phenyl
2,3,4,6ꢀtetraꢀOꢀacetylꢀ1ꢀthioꢀβꢀDꢀglucopyranoside 17 in 94% over all yield. Compound 17 was
transformed to phenyl 2,3ꢀdiꢀOꢀbenzylꢀ1ꢀthioꢀβꢀDꢀglucopyranoside 1817 according to the
reported procedure. Then compound 18 was chemoꢀselectively benzoylated using 1:1:0.5
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o
DCM:MeCN:NEt3 and benzoyl cyanide at ꢀ78 C for 2 hours to afford phenyl 6ꢀOꢀbenzoylꢀ2,3ꢀ
diꢀOꢀbenzylꢀ1ꢀthioꢀβꢀDꢀglucopyranoside 19 in 95% yield. Quantitative acetylation of 19 with
Mg(OTf)2 and acetic anhydride produced phenyl 4ꢀOꢀacetylꢀ6ꢀOꢀbenzoylꢀ2,3ꢀdiꢀOꢀbenzylꢀ1ꢀ
thioꢀβꢀDꢀglucopyranoside 20. Thioglycoside hydrolysis of 20 with TCCA in wet acetone
followed by base catalyzed formation of trichloroacetimidate furnished the desired glycosyl
donor 8 (Scheme 2).
Scheme 3 Synthesis of Dꢀgalactose based acceptor 11.
Dry Dꢀgalactose was converted to 3,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀgalactopyanosylꢀ1,2ꢀ(methyl
orthoacetate) 2318 according to the literature reported process. Then the reaction of this
benzylated orthoester 23 with pꢀmethoxyphenol using BF3.Et2O in dry MeCN and this followed
by Zemplén deacetylation19 gave the acceptor pꢀmethoxyphenyl 3,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀ
galactopyanoside 11 in 92% overall yield after two steps (Scheme 3). The αꢀconfiguration of the
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glycoside 11 was established from both H NMR spectrum (δ 5.50, H1, J1,2 = 3.5 Hz) and C
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spectrum (δ 98.9, JCꢀH = 169.8 Hz).
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Table 1 Optimization of αꢀDꢀglucosylation on acceptor 9.
Glycosylation of acceptor 9 was first attempted with phenyl 2,3ꢀdiꢀOꢀbenzylꢀ4,6ꢀOꢀ
benzylideneꢀ1ꢀthioꢀβꢀDꢀglucopyranoside donor 2420 based on a preꢀactivation technique using
1ꢀbenzenesulfinylpiperidine (BSP),20 2,4,6ꢀtriꢀtertꢀbutylpyrimidine (TTBP) and Tf2O in dry
DCM under standard reaction condition (entry 1, Table 1). Instead of formation of glycosylated
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product decomposition of the glycosyl donor was observed under this condition. Similar fate was
observed when the preactivation based glycosylation was attempted using diphenyl sulfoxide
(Ph2SO), TTBP and Tf2O in dry DCM (entry 2, Table 1).21 Changing the donor 24 with the more
reactive 2,3ꢀdiꢀOꢀbenzylꢀ4,6ꢀOꢀbenzylideneꢀDꢀglucopyranosyl trichloroacetimidate 25 was next
ventured. Coupling of 25 with rhamnopyranosyl acceptor 9 was explored using TMSOTf22 as
activator at different temperatures like 0 oC, ꢀ30 oC and ꢀ10 oC (entries 3, 4 and 5, Table 1).
Unfortunately in all the cases either decomposition of the reaction mixture was observed or
unreacted starting materials were recovered. Again another preꢀactivation based glycosylation
technique using BSP, TTBP, Tf2O was attempted on phenyl 4ꢀOꢀacetylꢀ6ꢀOꢀbenzoylꢀ2,3ꢀdiꢀOꢀ
benzylꢀ1ꢀthioꢀβꢀDꢀglucopyranoside 20, but the result was similar as before (entry 6, Table 1).
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Gratifyingly,
when
4ꢀOꢀacetylꢀ6ꢀOꢀbenzoylꢀ2,3ꢀdiꢀOꢀbenzylꢀDꢀglucopyranosyl
trichloroacetimidate donor 8 was allowed to couple with 9 using TMSOTf at ꢀ10 ℃ the desired
coupling product was obtained in 58% yield and α:β = 3:2 ratio (entry 7, Table 1). Pleasingly,
proper tuning of the reaction condition (entry 8, Table 1) allowed the isolation of the coupling
product in good yield but with poor steroselectivity. Then the glycosylations were run in a
mixture of DCM and Et2O, the ratio (2:1 to 3:2) of which was optimized (entries 9 and 10, Table
1) to comply with solubility requirements on the one hand and glycosylation stereoselectivity at
the other hand.
Scheme 4 Synthesis of disaccharide acceptor 5.
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Finally under the optimized condition 4ꢀOꢀacetylꢀ6ꢀOꢀbenzoylꢀ2,3ꢀdiꢀOꢀbenzylꢀDꢀ
glucopyranosyl trichloroacetimidate 8 and the glycosyl acceptor phenyl 3,4ꢀdiꢀOꢀbenzylꢀ1ꢀthioꢀ
αꢀLꢀrhamanopyranoside 9 were allowed to couple using TMSOTf22 at 0 oC in 3:2 DCM: Et2O to
produce the desired disaccharide in 90% yield with 9:1 α:β ratio. The NMR spectra of
disaccharide 4 showed signals characteristic of the presence of both donor and acceptor moieties,
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while an αꢀconfiguration of the interꢀglycosidic linkage was confirmed from the H and 13C
NMR spectra. A chemoꢀselective deacetylation in the presence of 6ꢀOBz of 4 using acetyl
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chloride in dry MeOH at 0 C produced the disaccharide acceptor 5 (Scheme 4) in 88% yield.
Compared to the NMR spectra of 4, the disappearance of one signal of carbonyl carbon from
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169.6 ppm in the corresponding C spectrum and the sharp singlet signal from 1.99 ppm in the
1H spectrum in 5 clearly indicated the selective removal of the acetate protecting group in the
presence of 6ꢀOBz.
Scheme 5 Synthesis of disaccharide acceptor 7.
A mixture of thioglycoside donor 10 and glycosyl acceptor pꢀmethoxyphenyl 3,4,6ꢀtriꢀOꢀ
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benzylꢀαꢀDꢀgalactopyanoside 11 in dry DCM at 0 °C was treated with NIS and FeCl3 to
produce disaccharide 6 in 92% yield. The NMR spectra of disaccharide 6 showed signals
characteristic of the presence of both donor and acceptor moieties, while an αꢀconfiguration of
the interglycosidic linkage was confirmed from the corresponding 1H (δ 5.01, H1', J1,2 = 0.8 Hz)
and 13C NMR (δ 97.8) spectra. Deacetylation of 6 with NaOMe ꢀ MeOH and dry DCM furnished
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quantitatively the disaccharide acceptor 7, necessary for the oneꢀpot sequential glycosylation via
an [1+2+2] approach (Scheme 5).
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With the present set of donors and acceptors in hand we set out for the oneꢀpot
glycosylation reactions towards targeted pentasaccharide via a [1+2+2] approach first.
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Disaccharide acceptor 5 was glycosylated with glycosyl donor 3 at ꢀ60 C to room temperature
using 10 mole% of FeCl3.23 After full consumption of the starting materials (checked by TLC),
into the same pot second disaccharide acceptor 7 followed by NIS were added. The reaction
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mixture was cooled down to 0 C and another 10 mole% of FeCl3 was added, TLC after 10
minutes showed complete consumption of the starting materials. Thus the targeted
pentasaccharide was prepared via a threeꢀcomponent one pot sequential glycosylation technique
in 78% yield (Scheme 6). The formation of the pentasaccharide derivative 2 was confirmed by
NMR spectroscopic techniques (1Hꢀ, 13Cꢀ, COSY, HSQC, HMBC, NOSEY) and also by HRMS.
Scheme 6 Synthesis of pentasaccharide derivative 2 via a threeꢀcomponent oneꢀpot sequential
glycosylation technique.
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′′′
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The anomeric protons of compound 2 appeared at δ 4.68 (bs, H1 ), 5.03 (2 bs, H1' and H1''), 5.21
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(bs, H1 ) and 5.23 (d, J 3.5 Hz, H1) ppm and the corresponding carbons at 94.1(C1'''), 98.2 (C1'),
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101.6 (C1''), 97.9 (C1'''') and 98.0 (C1) ppm, respectively, and the signal of the carbonyl carbons
appeared at 165.8 and 166.1 ppm.
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Scheme 7 Synthesis of pentasaccharide derivative 2 via a fourꢀcomponent oneꢀpot sequential
glycosylation technique.
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After successfully achieving a synthetic route towards pentasaccharide derivative 2 via a
threeꢀcomponent oneꢀpot sequential glycosylation technique we sought to synthesize the same in
a more step economic way. A fourꢀcomponent oneꢀpot sequential glycosylation reactions for
synthesis of the target 2 were then explored. For this, disaccharide acceptor 5 was first
glycosylated with 2ꢀOꢀ benzoylꢀ3,4ꢀdiꢀOꢀbenzylꢀLꢀrhamnopyranosyl trichloroacetimidate donor
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o
3 under previously standardized procedure using 10 mole% of FeCl3 at ꢀ60 C to room
temperature.23 After the reaction showed a clear conversion towards our desired trisaccharide
o
derivative (indicated by TLC) the reaction mixture was again cooled to ꢀ60 C. To this cold
mixture diphenyl sulfoxide (Ph2SO), TTBP and triflic anhydride (Tf2O) were added, and the
mixture was kept at that temperature for 10 minutes, and then the temperature was raised to ꢀ40
oC. After 1 hour at that temperature the second glycosyl acceptor 9 in dry DCM was injected into
the cold reaction vessel, and the reaction mixture was allowed to attain room temperature. After
consumption of both the starting materials (checked by TLC) the last acceptor 11 and NIS were
o
added into the same reaction vessel. Lowering the temperature to 0 C into this mixture FeCl3
was added again, and the resulting mixture was kept for 10 minutes at that temperature, and after
complete conversion towards desired pentasaccharide derivative 2 (indicated by TLC) the
reaction was quenched with NEt3. Thus the targeted pentasaccharide was prepared via a fourꢀ
component oneꢀpot sequential glycosylation technique in 72% yield (Scheme 7).
Finally debenzoylation under Zemplén condition, then regeoselective oxidation of
primary hydroxyl group in the presence of a secondary one using TEMPO, bisꢀacetoxy
iodobenzene (BAIB) in DCM water followed by benzylation using K2CO3, benzyl bromide in
dry DMF afforded the fully benzylated derivative of the acidic pentasaccharide derivative 26 in
79% yield over three steps (Scheme 8). Finally a global debenzylation using hydrogen and
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palladiumꢀcharcoal in a mixture of ethyl acetate, water and methanol ultimately afforded the
desired pentasaccharide 1 in 92% yield (Scheme 8). Compound 1 was characterized by NMR
spectroscopic techniques (1Hꢀ, 13Cꢀ, COSY) and also by HRMS.
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Scheme 8 Conversion of pentasaccharide derivative 2 to the target pentasaccharide 1.
Conclusion
In conclusion, we have developed an expeditious strategy for the synthesis of the acidic
pentasaccharide, related to the Oꢀantigen of E. coli 120, in the form of its pꢀmethoxyphenyl
glycoside (1) via oneꢀpot sequential glycosylation techniques. The synthesis of the target
compound is achieved through suitable protecting group manipulations on commercially
available monosaccharides and stereoꢀselective glycosylations. Protecting group manipulation
like perꢀOꢀacetylationꢀthioglycosidation was performed in oneꢀpot. The glycosylations were
achieved either by the activation of the thioglycosides using NIS in the presence of FeCl3 or by a
preactivation using Ph2SO, TTBP, Tf2O and the activation of the trichloroacetimidates using
FeCl3 alone or TMSOTf. The targeted pentasaccharide syntheses were performed with both
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threeꢀ and fourꢀ component oneꢀpot sequential glycosylation reactions, and in both cases the
orthogonal glycosylations were carried out utilizing the catalytic activity of FeCl3.
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Experimental
General procedure
All reactions were performed in flamedꢀdried flasks fitted with rubber septa under
a positive pressure of argon, unless otherwise stated. DCM was refluxed with P2O5 and
distilled before use and stored over 4Å molecular sieves. Traces of water in the starting
materials were removed by coꢀevaporation with toluene. Flash column chromatography
was performed employing Silica Gel 60 Sorbent (40ꢀ63 ꢁm, 230ꢀ400 mesh). Thinꢀlayer
chromatography (analytical and preparative) was performed using Merck silica gel plates
(60ꢀF254) to monitor the reactions and visualised under UV (254 nm) and/or by charring
1
13
with 5% ethanolic solution of sulfuric acid. H and C NMR spectra were recorded on a
Bruker DPXꢀ300 (300 MHz), a Bruker DPXꢀ400 (400 MHz), a Bruker DPXꢀ500 (500
MHz), or a Bruker DPXꢀ600 (600 MHz) spectrometer at ambient temperature in CDCl3
or D2O and assigned using 2Dꢀmethods (COSY, HSQC). Optical rotations were measured
using Jasco Pꢀ1020 digital polarimeter. High Resolution Mass Spectra (HRMS) were
measured in a QTOF I (quadrupoleꢀhexapoleꢀTOF) mass spectrometer with an orthogonal
Zꢀsprayꢀelectrospray interface on Micro (YAꢀ263) mass spectrometer (Manchester, UK).
2ꢀOꢀBenzoylꢀ3,4ꢀdiꢀOꢀbenzylꢀαꢀLꢀrhamnopyranosyl trichloroacetimidate (3)
To a solution of compound 14 (2 g, 3.7 mmol) in aqueous acetone (4:1), TCCA (1.2 g, 3.7
mmol) was added at 0 °C, and the reaction mixture was kept on stirring for 40 minutes. Then the
white precipitate was filtered, and the bed was washed with DCM (3×5 mL). The combined
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filtrate and washings was evaporated, and the resulting mixture was again dissolved in DCM.
This organic part was washed subsequently with saturated NaHCO3 solution (200 mL) and water
(200 mL). The organic layer was dried over anhydrous Na2SO4 and evaporated under vacuum to
furnish compound 19. Column filtration of the crude product furnished pure compound as white
foam (15, 1.6 g, 97 %). Then to this solution of compound 15 (1 g, 2.23 mmol) and CCl3CN
(0.34 mL, 3.34 mmol) in dry DCM (15 mL) DBU (0.1 mL, 0.67 mmol) was added at ꢀ5 °C, and
the reaction mixture was kept on stirring at that temperature. After 5 hours, excess solvent was
removed, and the resulting mixture was purified through flash column chromatography (PE/EA,
10
11
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5:1) to furnish pure compound 3 as colorless syrup (1.18 g, 90 %).; [α]25 4.2 (c 1.15, CHCl3).
D
1HꢀNMR (300 MHz, CDCl3): δ 8.67 (s, 1H, NH), 8.11ꢀ8.09 (d, J = 8.1 Hz, 1H, ArH), 7.64ꢀ7.26
(m, 13H, ArH), 6.31 (s, 1H, H1), 5.72 (s, 1H, H2), 4.93 (d, J = 10.8 Hz, 1H, BnH), 4.80 (d, J =
11.4 Hz, 1H, BnH), 4.66 (d, J = 11.1 Hz, 1H, BnH), 4.61 (d, J = 11.5 Hz, 1H, BnH), 4.11 (dd, J
= 3.0, 9.4 Hz, 1H, H3), 4.00 (m, 1H, H5), 3.65 (app t, J =9.5 Hz, 1H, H4), 1.39 (d, J = 6.2 Hz, 3H,
13
CH3).; CꢀNMR (75 MHz, CDCl3): δ 165.5 (C=O), 160.2 (C=NH), 138.1, 137.6, 133.4, 130.0,
129.7, 128.5, 128.4, 128.3, 128.1, 127.9, 127.8, 95.4 (C1), 90.9, 79.4, 77.4, 75.6, 71.9, 70.8, 68.1,
18.2 (CH3).; HRMS (ESIꢀTOF): Calculated for C29H28Cl3NO6Na (M+Na) 614.088 found
614.0882.
Phenyl 6ꢀOꢀbenzoylꢀ2,3ꢀdiꢀOꢀbenzylꢀ1ꢀthioꢀβꢀDꢀglucopyranoside (19)
To a solution of phenyl 2,3ꢀdiꢀOꢀbenzylꢀ1ꢀthioꢀβꢀDꢀglucopyranoside 18 (3 g, 6.64 mmol) in
1:1:0.5 mixture of DCM:MeCN:NEt3 (10 mL) benzoyl cyanide ( 0.9 mL, 7.3 mmol) diluted in 2
mL of dry DCM was added at ꢀ78 oC, and the reaction mixture was stirred at that temperature for
2 hours. The reaction mixture was diluted in DCM and washed subsequently with saturated
NaHCO3 solution (200 mL) and water (200 mL). The organic layer was dried over anhydrous
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Na2SO4 and evaporated under vacuum to furnish crude product 19. Purification of 19 by silica
gel column chromatography (PE/EA, 3:1) yielded phenyl 6ꢀOꢀbenzoylꢀ2,3ꢀdiꢀOꢀbenzylꢀ1ꢀthioꢀβꢀ
Dꢀglucopyranoside as white solid (19, 3.5 g, 95%).; mp (EA/PE) 90ꢀ92 °C.; [α]25D ꢀ21.8 (c 1.65,
CHCl3).; 1HꢀNMR (300 MHz, CDCl3): δ 8.09ꢀ8.06 (d, J = 7.3 Hz, 2H, ArH), 7.61ꢀ7.14 (m, 18H,
ArH), 4.95ꢀ4.92 (m, 2H), 4.81ꢀ4.70 (m, 3H), 4.64 (bs, 1H), 3.52ꢀ3.59 (m, 4H), 3.51 (m, 1H).;
13CꢀNMR (75 MHz, CDCl3): δ 166.9 (C=O), 138.3, 137.9, 133.5, 133.3, 132.2, 129.9, 129.8,
128.9, 128.7, 128.5, 128.4, 128.3, 128.1, 128.0, 127.7, 87.5 (C1), 85.9, 80.4, 77.8, 75.7, 75.4,
70.1, 63.9.; HRMS (ESIꢀTOF): Calculated for C33H32O6SNa (M+Na) 579.1818 found
579.1819.
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Phenyl 4ꢀOꢀacetylꢀ6ꢀOꢀbenzoylꢀ2,3ꢀdiꢀOꢀbenzylꢀ1ꢀthioꢀβꢀDꢀglucopyranoside (20)
Acetic anhydride (0.56 mL, 5.94 mmol) and Mg(OTf)2 (9.4 mg, 0.027 mmol) were added to a
solution of compound 19 (3 g, 5.39 mmol) in dry DCM (15 mL) at 0 °C, and the resulting
mixture was kept on stirring for 30 minutes. After completion of the reaction (indicated by TLC),
excess acetic anhydride was removed using rotatory evaporator. The resulting syrup was
dissolved in DCM and washed subsequently with saturated NaHCO3 solution (200 mL) and
water (200 mL). The organic layer was dried over anhydrous Na2SO4 and concentrated to afford
the crude product. It was purified by column chromatography on silica gel. Column elution by
PE/EA, 4:1 furnished pure compound 20 as white solid (3.13 g, 97%).; mp (EA/PE) 84ꢀ86 °C.;
[α]27D ꢀ27.8 (c 2.8, CHCl3).; 1HꢀNMR (300 MHz, CDCl3): δ 8.08ꢀ8.05 (d, J = 7.5 Hz, 2H, ArH),
7.60ꢀ7.10 (m, 18H, ArH), 4.74ꢀ4.64 (m, 3H, H1, H6, BnH), 4.54 (d, J = 12.1 Hz, 1H, BnH), 5.14
(t, J = 9.7 Hz, 1H, H4), 4.93ꢀ4.82 (m, 2H, BnH), 4.32 (m, 1H, H6), 3.77ꢀ3.68 (m, 2H, H3, H5),
3.58 (t, J = 9.3 Hz, 1H, H2), 1.93 (s, 3H, COCH3).; 13CꢀNMR (75 MHz, CDCl3): δ 169.7 (C=O),
166.2 (C=O), 138.0, 137.8, 133.2, 133.1, 132.4, 129.9, 129.8, 128.9, 128.5, 128.4, 128.3, 128.1,
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127.9, 127.8, 87.5 (C1), 83.9, 80.6, 76.0, 75.6, 69.9, 63.2, 20.8 (CH3CO).; HRMS (ESIꢀTOF):
Calculated for C35H34O7SNa (M+Na) 621.1923 found 621.1940.
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4ꢀOꢀAcetylꢀ6ꢀOꢀbenzoylꢀ2,3ꢀdiꢀOꢀbenzylꢀDꢀglucopyranose (21)
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To a solution of compound 20 (2 g, 3.3 mmol) in aqueous acetone (4:1), TCCA (1.2 g, 3.3
mmol) was added at ambient temperature, and the mixture was kept on stirring for 20 minutes.
Then the white precipitate was filtered, and the bed was washed with DCM (3×15 mL). The
combined filtrate and washings was evaporated, and the resulting mixture was again dissolved in
DCM. This organic part was washed subsequently with saturated NaHCO3 solution (200 mL)
and water (200 mL). The organic layer was dried over anhydrous Na2SO4 and evaporated under
vacuum to furnish compound 21. Column filtration of the crude product furnished white foam of
pure compound as mixture of anomers (21, 1.62 g, 96 %).; HRMS (ESIꢀTOF): Calculated for
C29H30O8Na (M+Na) 529.1839 found 529.1840.
4ꢀOꢀAcetylꢀ6ꢀOꢀbenzoylꢀ2,3ꢀdiꢀOꢀbenzylꢀDꢀglucopyranosyl trichloroacetimidate (8)
To a solution of compound 21 (1.5 g, 2.96 mmol) and CCl3CN (0.46 mL, 4.44 mmol) in dry
DCM (15 mL) DBU (0.14 mL, 0.9 mmol) was added at ꢀ5 °C, and the reaction mixture was kept
on stirring at that temperature. After 5 hours, excess solvent was removed, and the resulting
crude product was purified through flash column chromatography (PE/EA, 5:1) to furnish pure
compound 8 as colorless syrup (1.75 g, 91 %).; 1HꢀNMR (300 MHz, CDCl3): δ 8.63 (s, 1H, NH),
8.02ꢀ8.00 (d, J = 8.0 Hz, 2H, ArH), 7.55ꢀ7.25 (m, 13H, ArH), 6.84 (d, J = 3.0 Hz, 1H, H1), 5.21
(t, J = 10.0 Hz, 1H, H4), 4.89 (d, J = 11.5 Hz, 1H, BnH), 4.76ꢀ4.67 (m, 3H, H5, BnH), 4.47 (d, J
= 12.0 Hz, 1H, BnH), 4.27 (dd, J = 12.5, 5.0 Hz, 1H, H6), 4.21 (dd, J = 10.5, 4.5 Hz, 1H, H6),
4.02 (t, J = 9.5 Hz, 1H, H3), 3.83 (dd, J = 9.5, 3.5 Hz, 1H, H2), 1.97 (s, 3H, COCH3).; 13CꢀNMR
(75 MHz, CDCl3): δ 169.6 (C=O), 166.2 (C=O), 162.3, 160.9, 138.2, 137.7, 133.1, 129.8, 128.5,
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128.4, 128.3, 128.0, 127.9, 127.8, 127.7, 93.9 (C1), 91.1, 79.2, 78.4, 75.4, 73.1, 70.5, 69.2, 62.6,
20.8 (COCH3).; Calculated for C31H30NCl3O8Na (M+Na) 672.0935 found 672.0930.
pꢀMethoxylphenyl 3,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀgalactopyranoside (11).
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To a solution of 3,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀgalactose 1,2ꢀ(methyl orthoacetate) 23 (2.50 g, 4.94
mmol), in dry CH3CN (30 mL) containing 4 Ǻ molecular sieves, pꢀmethoxyphenol (1.23 g, 9.96
mmol) were added at room temperature. BF3.Et2O (1.9 mL, 14.94 mmol) was added to this
mixture on iceꢀbath and kept for 30 minutes. The reaction mixture was filtered through a celite
bed, and the residue was washed with DCM (3×15 mL). The combined filtrate and washings was
washed subsequently with cold 5% aqueous NaOH (250 mL) followed by water (100 mL). The
organic layer was dried over anhydrous Na2SO4 and concentrated. The crude residue was used
for further reaction. To a solution of this crude material in a mixture of dry DCM (10 ml) and dry
MeOH (8 mL) 1 M methanolic NaOMe (2 mL) solution was added, and the reaction mixture was
stirred for 2 hours at room temperature. The reaction mixture was then neutralized with Dowexꢀ
50W cation exchange resin (H+) and filtered. The resin was washed with MeOH, and the
combined filtrate and washings was concentrated under reduced pressure. The residue was
purified by column chromatography on silica gel (PE/EA, 9:1) to afford 11 as white solid (2.56
1
g, 92% over 2 steps).; mp (EA/PE) 96ꢀ98 °C.; [α]29 +100.6 (c 2.1, CHCl3).; HꢀNMR (300
D
MHz, CDCl3): δ 7.43ꢀ7.26 (m, 15H, ArH), 7.07ꢀ7.04 (d, J = 8.9 Hz, 2H, ArH), 6.83ꢀ6.79 (d, J =
8.9 Hz, 2H, ArH), 5.51 (d, J = 3.5 Hz, 1H, H1), 4.76 (d, J = 11.3 Hz, 1H, BnH), 4.84ꢀ4.75 (ABq,
J = 11.5 Hz, 2H, BnH), 4.62 (d, J = 11.3 Hz, 1H, BnH), 4.51ꢀ4.35 (m, 3H, OH, BnH), 4.17 (app
t, J = 6.3, 6.1 Hz, 1H, H6), 4.10 (bs, 1H, H4), 3.92 (dd, J = 9.9, 2.4 Hz, 1H, H3), 3.77 (s, 3H,
OCH3), 3.69ꢀ3.56 (m, 3H, H2, H5, H6).; 13CꢀNMR (75 MHz, CDCl3): δ 155.3, 150.8, 138.5,
138.2, 137.9, 128.6, 128.4, 128.3, 128.2, 127.8, 127.76, 118.7, 114.6, 98.9 (C1, JCꢀH = 169.8),
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79.6, 74.8, 74.0, 73.5, 72.6, 70.4, 69.0, 68.8, 55.6 (OCH3).; HRMS (ESIꢀTOF): Calculated for
5
6
C34H36O7Na (M+Na) 579.2359 found 579.2379.
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Phenyl 2ꢀOꢀacetylꢀ6ꢀOꢀbenzoylꢀ2,3ꢀdiꢀOꢀbenzylꢀ
benzylꢀ1ꢀthioꢀ ꢀLꢀrhamnopyranoside (4)
To solution of 2ꢀOꢀacetylꢀ6ꢀOꢀbenzoylꢀ2,3ꢀdiꢀOꢀbenzylꢀ
trichloroacitimidate 8 (200.0 mg, 0.31 mmol) and phenyl 3,4ꢀdiꢀOꢀbenzylꢀ1ꢀthioꢀ
αꢀDꢀglucopyranosylꢀ(1→2)ꢀ3,4ꢀdiꢀOꢀ
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α
a
αꢀDꢀglucopyranosyl
αꢀLꢀ
rhamnopyranoside 9 (120.9 mg, 0.28 mmol) in 3:2 (v/v) mixture of dry DCM:Et2O (30 mL)
activated molecular sieves (4Å) were added, and the reaction mixture was kept on stirring under
argon atmosphere for 45 minutes. Then the reaction vessel was placed in a 0 °C cold bath, and
TMSOTf (10 µL, 0.06 mmol) was added to it via a micro syringe. After 10 minutes complete
consumption of both the starting materials was observed. The reaction mixture was quenched by
Et3N, and then filtered through celite bed, and the bed was washed with DCM (3×15 mL). The
combined filtrate and washings was washed subsequently with saturated NaHCO3 (2x50 mL)
and water (2x50 mL). The organic layer was dried over anhydrous Na2SO4 and concentrated to
furnish a syrupy compound. The crude product was purified by flash column chromatography
(eluent: PE/EA, 3:1) to afford the desired disaccharide derivative 4 as white foam (230.6 mg,
1
90%).; [α]28 +11.8 (c 3.6, CHCl3).; HꢀNMR (400 MHz, CDCl3): δ 8.08ꢀ8.06 (d, J = 8.0 Hz,
D
2H, ArH), 7.99ꢀ7.18 (m, 28H, ArH), 5.48 (s, 1H, H1), 5.18 (app t, J = 9.6, 10.0 Hz, 1H, H4'),
5.04ꢀ4.89 (m, 3H, H1', BnH), 4.82ꢀ4.61 (m, 5H, BnH), 4.54 (d, J = 11.6 Hz, 1H, BnH), 4.38ꢀ4.23
(m, 4H, H2, H5, H5', H6'), 4.06 (t, J = 9.6 Hz, 1H, H3'), 3.97ꢀ3.89 (m, 2H, H3, H6′), 3.77 (t, J =
9.2 Hz, 1H, H4), 3.76 (dd, 1H, J = 9.6, 2.8 Hz, H2'), 1.99 (s, 3H, COCH3), 1.45 (d, 3H, J = 6.0
Hz, CH3).; 13CꢀNMR (75 MHz, CDCl3): δ 169.6 (C=O), 166.2 (C=O), 138.4, 138.6, 138.2,
138.0, 134.6, 133.1, 131.7, 129.9, 129.8, 129.1, 128.5, 128.4, 128.1, 127.9, 127.8, 127.7, 127.5,
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127.3, 94.6 (C1), 84.9 (C1'), 80.4, 79.9, 79.2, 78.7, 75.3, 75.2, 74.5, 73.0, 72.3, 69.7, 69.4, 67.8,
62.5, 20.9 (COCH3), 17.9 (CH3).; Calculated for C55H56O11SNa (M+Na) 947.3441 found
947.3457.
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56
57
58
59
60
Phenyl 6ꢀOꢀbenzoylꢀ2,3ꢀdiꢀOꢀbenzylꢀ
thioꢀ ꢀLꢀrhamnopyranoside (5)
αꢀDꢀglucopyranosylꢀ(1→2)ꢀ3,4ꢀdiꢀOꢀbenzylꢀ1ꢀ
α
Compound 4 (200 mg, 0.22 mmol) was dissolved in dry MeOH (15 mL), and the solution is
o
cooled to 0 C on an iceꢀbath. Acetyl chloride (0.16 mL, 2.2 mmol) was added drop wise, with
continuous stirring, and the solution was kept on stirring under anhydrous condition for 2 hours.
After completion of the reaction (indicated by TLC), excess MeOH and acetyl chloride were
removed in vacuo. The resulting syrup was dissolved in DCM (15 mL), and the solution was
washed subsequently with saturated aqueous NaHCO3 (200 mL) and brine (100 mL). The
organic layer was dried over Na2SO4 and concentrated. This was purified by column
chromatography on silica gel (PE/EA, 4:1) to afford 5 as colorless syrup (168 mg, 88%).; [α]25
D
+48.5 (c 1.0, CHCl3).; 1HꢀNMR (400 MHz, CDCl3): δ 8.07ꢀ8.05 (d, J = 7.6 Hz, 2H, ArH), 7.24ꢀ
7.61 (m, 28H, ArH), 5.51 (s, 1H, H1), 5.04ꢀ4.98 (m, 2H, BnH), 4.96 (d, J = 3.4 Hz, 1H, H1'), 4.87
(d, J = 11.2 Hz, 1H, BnH), 4.78ꢀ4.63 (m, 5H, BnH), 4.46 (dd, J = 12.0, 4.0 Hz, 1H, H6'), 4.37
(bs, 1H, H2), 4.26ꢀ4.20 (m, 3H, H5, H5', H6'), 3.97 (t, J = 9.2 Hz, 1H, H4'), 3.90 (dd, J = 2.6, 9.2
Hz, 1H, H3), 3.75 (t, J = 9.6 Hz, 1H, H3'), 3.62ꢀ3.56 (m, 2H, H2', H4), 1.43 (d, J = 6.0 Hz, 3H,
CH3).; 13CꢀNMR (75 MHz, CDCl3): δ 166.9 (C=O), 138.7, 138.4, 138.3, 137.9, 133.2, 131.5,
129.8, 129.7, 129.1, 128.54, 128.45, 128.4, 128.3, 128.1, 128.0, 127.9, 127.8, 127.7, 127.6,
127.4, 95.2 (C1), 85.1 (C1'), 80.6, 80.4, 79.7, 79.2, 75.4, 75.1, 72.7, 72.4, 70.0, 69.4, 63.4, 17.9
(CH3).; Calculated for C53H54O10SNa (M+Na) 905.3336 found 905.3337.
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pꢀMethoxylphenyl
2ꢀOꢀacetylꢀ3,4ꢀdiꢀOꢀbenzylꢀαꢀLꢀrhamnopyranosylꢀ(1→2)ꢀ3,4,6ꢀtriꢀOꢀ
5
6
benzylꢀαꢀDꢀgalactopyranoside (6)
7
8
9
To a mixture of phenyl 2ꢀOꢀacetylꢀ3,4ꢀdiꢀOꢀbenzylꢀ1ꢀthioꢀ
αꢀLꢀrhamnopyranoside 10
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(189.3 mg, 0.39 mmol) and pꢀmethoxyphenyl 3,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀgalactopyranoside
11 (200 mg, 0.36 mmol) in dry DCM (15 mL), flame activated molecular sieves (4Å)
were added. It was stirred at room temperature under argon atmosphere. After 40 minutes
the mixture was cooled to 0 °C, and NIS (90 mg, 0.39 mmol) was added to it. Then FeCl3
(13.0 mg, 0.08 mmol) was added. After 15 minutes when the acceptor was consumed
completely (checked by TLC) reaction mixture was filtered off through celite bed. The
filtrate was diluted with DCM and washed subsequently with saturated sodium
thiosulphate (100 mL), NaHCO3 solution (100 mL), and water (100 mL). The organic
layer was dried over anhydrous Na2SO4 and concentrated to afford the glycosylated
product. The residue was purified by flash column chromatography (PE/EA, 5:1) to
afford the title compound
CHCl3).; 1HꢀNMR (CDCl3, 400 MHz): δ 7.42ꢀ7.21 (m, 25H, Ar
6
as colorless syrup (305.8 mg, 92%).; [α]25 +19.9 (c 6.5,
D
H), 7.01ꢀ6.99 (m, 2H,
Ar
3.6 Hz, 1H,
10.8 Hz, 1H, Bn
Hz, 2H, Bn ), 4.31 (dd,
= 9.6, 3.2 Hz, 1H,
H
), 6.78ꢀ6.75 (d,
1), 5.10 (d,
), 4.79ꢀ4.68 (m, 3H), 4.59ꢀ4.48 (m, 3H, Bn
= 10.0, 3.6 Hz, 1H, 2), 4.13ꢀ4.04 (m, 3H, H3
3'), 3.78 (m, 1H, 5'), 3.75 (s, 3H, OC 3), 3.53ꢀ3.60 (m, 2H,
4'), 2.13 (s, 3H, COC 3), 1.00 (d, = 6.4 Hz,
=O), 155.0, 151.0, 138.6, 138.4, 138.3,
J
= 9.2 Hz, 2H, Ar
H
), 5.58 (dd,
J
= 2.8, 2.0 Hz, 1H,
= 11.2 Hz, 1H, Bn
), 4.43ꢀ4.35 (ABq,
H4
H
2'), 5.50 (d,
J
=
H
J
= 0.8 Hz, 1H,
H1'), 4.96 (d,
J
H), 4.86 (d,
J
=
H
H
J
= 11.6
H
J
H
,
, H6), 3.95
(dd,
H5
J
H
H
H
,
H6), 3.37 (app t,
J
= 9.6, 9.2 Hz, 1H,
H
H
J
3H, C
H
3).; 13CꢀNMR (CDCl3, 75 MHz): δ 170.1 (
C
138.1, 137.9, 128.5, 128.4, 128.3, 128.1, 127.8, 127.7, 127.6, 118.1, 114.6, 99.9 (C1'),
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97.9 (C1), 79.9, 78.3, 77.9, 76.3, 75.1, 75.0, 74.8, 73.4, 73.2, 71.7, 69.9, 68.6, 68.4, 55.6
5
6
(OCH3), 21.1 (COCH3), 17.7 (CH3).; Calculated for C56H60O12Na (M+Na) 947.3983
7
8
9
found 947.4022.
10
11
12
13
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15
16
17
18
19
20
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23
24
25
26
27
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60
pꢀMethoxylphenyl
3,4ꢀdiꢀOꢀbenzylꢀαꢀLꢀrhamnopyranosylꢀ(1→2)ꢀ3,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀ
galactopyranoside (7)
To a solution of 6 (250 mg, 0.27 mmol) in dry DCM (5 mL) and dry MeOH (5 mL) 1 M
methanolic NaOMe (0.2 mL) solution was added, and the reaction mixture was stirred for 3
hours at room temperature. The solvent was removed under pressure and the crude mixture was
diluted with DCM (10 mL), and subsequently washed with brine solution (100 mL), and
washings were concentrated under reduced pressure. The residue was purified by column
chromatography on silica gel (PE/EA, 5:1) to give glassy syrupy product 7 (236 mg, 99%).;
[α]27 +21.9 (c 3.12, CHCl3).; HꢀNMR (400 MHz, CDCl3): δ 7.31ꢀ7.22 (m, 25H, ArH), 7.05ꢀ
1
D
7.02 (d, J = 8.8 Hz, 2H, ArH), 6.79ꢀ6.77 (d, J = 9.2 Hz, 2H, ArH), 5.57 (d, J = 3.2 Hz, 1H, H1),
5.13 (s, 1H, H1'), 4.96 (d, J = 11.6 Hz, 1H, BnH), 4.83 (d, J = 11.2 Hz, 1H, BnH), 4.85 ꢀ4.70
(ABq, J = 11.6 Hz, 2H, BnH), 4.69ꢀ4.62 (ABq, J = 11.6 Hz, 2H, BnH), 4.61ꢀ4.58 (d, J = 11.2
Hz, 2H, BnH), 4.44ꢀ4.36 (q, J = 11.6 Hz, 2H, BnH), 4.31 (dd, J = 10.0, 3.6 Hz, 1H, H2), 4.13ꢀ
'
4.04 (m, 4H, H3/ H3 H4, H2', H5'), 3.87 (dd, J = 3.0, 9.0 Hz, 1H, H3/H3'), 3.80 (dd, J = 9.6, 6.4
Hz, 1H, H5), 3.75 (s, 3H, OCH3), 3.62 (m, 1H, H6), 3.54 (dd, J = 9.2, 6.0 Hz, 1H, H6), 3.41 (t, J
= 9.2 Hz, 1H, H4'), 0.95 (d, J = 6.0 Hz, 3H, CH3).; 13CꢀNMR (75 MHz, CDCl3): δ 154.9, 151.0,
138.6, 138.5, 138.4, 138.0, 137.9, 128.6, 128.5, 128.4, 128.32, 128.3, 127.9, 127.82, 127.8,
127.7, 127.4, 118.0, 114.6, 101.8 (C1'), 97.8 (C1), 79.9, 79.8, 78.1, 77.4, 75.1, 74.9, 73.4, 73.1,
72.0, 69.9, 68.8, 68.7, 68.0, 55.6 (OCH3), 17.6 (CH3).; Calculated for C54H58O11Na (M+Na)
905.3877 found 905.3943.
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pꢀMethoxyphenyl 2ꢀOꢀbenzoylꢀ3,4ꢀdiꢀOꢀbenzylꢀαꢀLꢀrhamnopyranosylꢀ(1→4)ꢀ6ꢀOꢀbenzoylꢀ
2,3ꢀdiꢀOꢀbenzylꢀαꢀDꢀglucopyranosylꢀ(1→2)ꢀ3,4ꢀdiꢀOꢀbenzylꢀαꢀLꢀrhamnopyranosylꢀ(1→2)ꢀ
3,4ꢀdiꢀOꢀbenzylꢀαꢀLꢀrhamnopyranosylꢀ(1→2)ꢀ3,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀgalactopyranoside
(2).
10
11
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13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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47
48
49
50
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52
53
54
55
56
57
58
59
60
A mixture of 3 (73.6 mg, 0.13 mmol) and 5 (100.0 mg, 0.113 mmol) and flame activated 4Å
molecular sieves were stirred in dry DCM (10 mL) for 40 minutes at room temperature
under argon atmosphere. The mixture was cooled to ꢀ60 ºC, and FeCl3 (2.2 mg, 0.013
mmol) was added, and the reaction mixture was allowed to achieve room temperature. After 45
minutes complete consumption of both the starting materials was observed (Checked by TLC).
Acceptor 7 (89.7 mg, 0.10 mmol) and NIS (25.4 mg, 0.113 mmol) were then added to the same
vessel. After the addition reaction vessel was placed in a 0 °C cold bath and another 10 mol% of
FeCl3 (2.2 mg, 0.013 mmol) was added to it. The second step of the reaction was completed
within 10 minutes (indicated by TLC). The reaction mixture was quenched by Et3N and then
filtered through celite bed. The bed was washed with DCM (3×15 mL). The combined filtrate
and washings was washed subsequently with saturated sodium thiosulphate, aqueous NaHCO3
(2x50 mL) and water (2x50 mL). The organic layer was dried over anhydrous Na2SO4 and
concentrated to furnish a syrupy compound. The crude product was purified by flash column
chromatography (eluent: toluene/Et2O, 9.5:0.5) to afford the desired fully protected
1
pentasaccharide 2 as white foam (184.5 mg, 78%).; [α]27D +28.1 (c 1.1, CHCl3).; HꢀNMR (500
MHz, CDCl3): δ 8.06ꢀ8.03 (d, J = 7.0 Hz, 2H, ArH), 8.01ꢀ7.98 (d, J = 7.5 Hz, 2H, ArH), 7.57ꢀ
7.48 (m, 2H, ArH), 7.43ꢀ7.03 (m, 56H, ArH), 7.01ꢀ6.98 (d, J = 9.0 Hz, 2H, ArH), 6.77ꢀ6.74 (d, J
= 9.0 Hz, 2H, ArH), 5.53 (d, J = 3.5 Hz, 1H, H1), 5.46 (t, J = 2.5 Hz, 1H, H2''''), 5.21 (bs, 1H,
H1''''), 5.03 (bs, 2H, H1', H1''), 4.97ꢀ4.93 (m, 3H, BnH), 4.87ꢀ4.75 (m, 5H, BnH), 4.73ꢀ4.53 (m,
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10H, H1''', BnH), 4.43 (d, J = 12.0 Hz, 1H, BnH), 4.41ꢀ4.33 (ABq, J = 11.5 Hz, 2H, BnH), 4.32ꢀ
4.24 (m, 3H, H2, H6''', BnH), 4.19ꢀ4.12 (m, 4H, H2', H2'', H6''', H5''''), 4.06 (app t, J = 6.5, 6.0 Hz,
1H, H5'''), 4.04ꢀ4.01 (m, 2H, H4, H3''''), 3.97ꢀ3.86 (m, 5H, H3, H3'', H3''', H4''', BnH), 3.84ꢀ3.78 (m,
1H, H5'', 1dd, J = 10.0, 3.0 Hz, 1H, H3'), 3.75ꢀ3.68 (1s, OCH3, m, 1H, H5'), 3.64 (app t, J = 7.0
Hz, 1H, H4''), 3.61ꢀ3.49 (m, 4H, H5, H6, H6, H4'''), 3.40 ꢀ3.34 (m, 2H, H4', H2'''), 1.27 (d, J = 6.5
10
11
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19
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22
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26
27
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48
49
50
51
52
53
54
55
56
57
58
59
60
13
Hz, 3H, CH3''''), 1.19 (d, J = 6.0 Hz, 3H, CH3''), 0.94 (d, J = 6.0 Hz, 3H, CH3').; CꢀNMR (125
MHz, CDCl3): δ 166.1 (C=O), 165.8 (C=O), 155.1, 151.2, 138.9, 138.87, 138.8, 138.7, 138.6,
138.5, 138.4, 138.3, 138.2, 133.1, 133.0, 130.3, 130.26, 130.0, 129.9, 128.7, 128.69, 128.64,
128.5, 128.4, 128.31, 128.3, 128.2, 128.1, 127.9, 127.89, 127.86, 127.83, 127.77, 127.7, 127.65,
127.60, 127.5, 127.4, 127.1, 118.2, 114.8, 101.6 (C1''), 98.2 (C1'), 98.0 (C1), 97.9 (C1''''), 94.1
(C1'''), 80.62, 80.6, 80.5, 80.1, 79.8, 78.2, 78.1, 78.0, 75.6, 75.5, 75.34, 75.3, 75.2, 75.13, 75.10,
74.3, 73.5, 73.2, 73.1, 72.6, 72.4, 71.9, 71.6, 70.1, 69.8, 69.04, 69.0, 68.8, 68.7, 68.67, 62.9, 55.8
(OCH3), 18.3 (CH3), 18.1 (CH3), 17.9 (CH3).; Calculated for C128H132O26Na (M+Na)
2107.8905 found 2107.8908.
pꢀMethoxyphenyl 2ꢀOꢀbenzoylꢀ3,4ꢀdiꢀOꢀbenzylꢀαꢀLꢀrhamnopyranosylꢀ(1→4)ꢀ6ꢀOꢀbenzoylꢀ
2,3ꢀdiꢀOꢀbenzylꢀαꢀDꢀglucopyranosylꢀ(1→2)ꢀ3,4ꢀdiꢀOꢀbenzylꢀαꢀLꢀrhamnopyranosylꢀ(1→2)ꢀ
3,4ꢀdiꢀOꢀbenzylꢀαꢀLꢀrhamnopyranosylꢀ(1→2)ꢀ3,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀgalactopyranoside
(2).
A mixture of 3 (73.7 mg, 0.13 mmol) and 5 (100.0 mg, 0.0.113 mmol) and flame activated 4Å
molecular sieves were stirred in dry DCM (10 mL) for 40 minutes at room temperature
under argon atmosphere. The mixture was cooled to ꢀ60 ºC, and FeCl3 (2.2 mg, 0.013
mmol) was added, and the reaction mixture was allowed to achieve room temperature. After 45
minutes complete consumption of both the starting materials was observed (Checked by TLC).
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The reaction was cooled to ꢀ60o C, and to this Ph2SO (50.2 mg, 0.25 mmol), TTBP (42.1 mg,
0.17 mmol) and Tf2O (0.03 ml, 0.14 mmol) were added one by one. Then the reaction mixture
was slowly brought to ꢀ40o C, and kept at that temperature for another 1hour, in which phenyl
3,4ꢀdiꢀOꢀbenzylꢀ1ꢀthioꢀαꢀLꢀrhamnopyranoside 9 (44.4 mg, 0.10 mmol) in dry DCM (2 mL) was
added. The reaction mixture was slowly warmed to room temperature. After full consumption of
starting materials (checked by TLC) acceptor 11 (58.0 mg, 0.10 mmol) and NIS (22.5 mg, 0.10
mmol) were then added to the same vessel. After the addition reaction vessel was placed in a 0
°C cold bath and another 10 mol% of FeCl3 (2.2 mg, 0.0078 mmol) was added to it. The
reaction was completed within 10 minutes (indicated by TLC). The reaction mixture was
quenched by Et3N and then filtered through celite bed. The bed was washed with DCM (3×15
mL). The combined filtrate and washings was washed subsequently with saturated sodium
thiosulphate (2x50 mL), aqueous NaHCO3 (2x50 mL) and water (2x50 mL). The organic layer
was dried over anhydrous Na2SO4 and concentrated to furnish a syrupy compound. The crude
product was purified by flash column chromatography (eluent: toluene/diethyl ether, 9.5:0.5) to
afford the desired fully protected pentasaccharide 2 as white foam (188.7 mg, 72%); Spectral
data matches with the substrate synthesized previously.
3
4
5
6
7
8
9
10
11
12
13
14
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16
17
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pꢀMethoxyphenyl 3,4ꢀdiꢀOꢀbenzylꢀαꢀLꢀrhamnopyranosylꢀ(1→4)ꢀbenzylꢀ2,3ꢀdiꢀOꢀbenzylꢀαꢀ
Dꢀglucopyranosyluronateꢀ(1→2)ꢀ3,4ꢀdiꢀOꢀbenzylꢀαꢀLꢀrhamnopyranosylꢀ(1→2)ꢀ3,4ꢀdiꢀOꢀ
benzylꢀαꢀLꢀrhamnopyranosylꢀ(1→2)ꢀ3,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀgalactopyranoside (26).
To a solution of 2 (150 mg, 0.075 mmol) in DCM (5 mL) and dry MeOH (5 mL) 1 M methanolic
NaOMe (0.2 mL) solution was added, and the reaction mixture was stirred for 3 hours at room
temperature. The solvent was removed under pressure, coevaporated with toluene and the crude
mixture was diluted with DCM (10 mL), and subsequently washed with brine solution (100 mL).
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The combined organic layer was dried over anhydrous Na2SO4 concentrated under reduced
pressure. The crude material was used for the next step without any purification. To this crude
debenzoylated pentasaccharide derivative in DCMꢀH2O (2:1, 10 mL), TEMPO (5.9 mg, 0.038
mmol) was added followed by BAIB (72.4 mg, 0.23 mmol) and the twoꢀphase reaction mixture
was stirred vigorously at room temperature for 8 hours till TLC (4:1 toluene/acetone) indicated
complete conversion of the starting material to a lower running spot. The mixture was diluted
with DCM (10 mL) and subsequently washed with saturated sodium thiosulphate solution (100
mL) and saturated NaHCO3 solution (100 mL). The combined organic layer was dried over
anhydrous Na2SO4 concentrated under reduced pressure and used for further reaction without
any purification. Anhydrous K2CO3 (12.4 mg, 0.09 mmol) followed by BnBr (10 ꢁL, 0.09 mmol)
10
11
12
13
14
15
16
17
18
19
20
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22
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25
26
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44
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48
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52
53
54
55
56
57
58
59
60
o
was added at 0 C under argon to a solution of the foregoing material in dry DMF (10 mL), and
the mixture was allowed to warm to room temperature. The organic layer was dried,
concentrated, and coꢀevaporated with toluene. The crude mixture was diluted with DCM (10
mL), and subsequently washed with brine solution (100 mL). The combined organic layer was
dried over anhydrous Na2SO4 concentrated under reduced pressure. Purification of this crude
mass by silica gel Column chromatography (toluene/acetone 9:1) yielded the desired benzyl
urinate 26 (124.7 mg, 79% over three steps).; [α]25D +15.1 (c 1.0, CHCl3).; 1HꢀNMR (500 MHz,
CDCl3): δ 7.34ꢀ7.18 (m, 60H, ArH), 6.99ꢀ6.97 (d, J = 9.2 Hz, 2H, ArH), 6.77ꢀ6.74 (d, J = 8.8
Hz, 2H, ArH), 5.51 (d, J = 3.2 Hz, 1H), 5.09ꢀ4.99 (m, 4H), 4.94ꢀ4.74 (m, 9H), 4.69ꢀ4.53 (m,
10H), 4.50ꢀ4.45 (m, 2H), 4.41ꢀ4.23 (m, 5H), 4.11ꢀ3.81 (m, 11H), 3.78ꢀ3.74 (m, 2H), 3.75 (s, 3H,
OCH3), 3.68 (m, 1H), 3.59ꢀ3.49 (m, 4H), 3.37 (m, 1H), 3.26 (app t, J = 9.6, 9.2 Hz, 1H), 1.23 (d,
13
J = 6.0 Hz, 3H, CH3), 1.00 (d, J = 6.0 Hz, 3H, CH3), 0.86 (d, J = 6.0 Hz, 3H, CH3).; CꢀNMR
(100 MHz, CDCl3): δ 170.0 (C=O), 155.1, 151.2, 139.4, 138.9, 138.8, 138.7, 138.6, 138.5,
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138.4, 138.3, 138.2, 138.1, 135.0, 128.7, 128.68, 128.6, 128.5, 128.45, 128.4, 128.35, 128.3,
128.2, 128.1, 128.0, 127.7, 127.6, 127.56, 127.52, 127.5, 127.2, 118.1, 114.7, 114.2, 101.4, 99.5,
98.5, 97.9, 95.7, 80.2, 79.9, 78.0, 77.5, 77.4, 75.5, 75.3, 75.2, 75.1, 74.6, 73.5, 73.2, 72.23, 72.2,
72.0, 71.83, 71.8, 70.8, 70.0, 69.0, 68.9, 68.7, 68.3, 68.2, 67.6, 55.8 (OCH3), 18.1 (CH3), 18.0
(CH3), 17.9 (CH3).; Calculated for C121H128O25K (M+K) 2020.8416 found 2020.8438.
pꢀMethoxyphenyl αꢀLꢀrhamnopyranosyl (1→4)ꢀαꢀDꢀglucopyranosyluronic acidꢀ(1→2)ꢀαꢀLꢀ
rhamnopyranosylꢀ(1→2)ꢀαꢀLꢀrhamnopyranosylꢀ(1→2)ꢀαꢀDꢀgalactopyranoside (1)
A mixture of protected acidic pentasaccharide 35 (75 mg, 0.038 mmol) the resulting mixture and
10% PdꢀC (90 mg) was taken in ethyl acetate (1 mL), methanol (3 mL) and H2O (1 mL), and
kept on stirring under H2 atmosphere for 3 hours. The catalyst was filtered through celite bed,
and the bed was washed with methanol (3 x 5 mL). The combined filtrate and washings was
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25
26
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28
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31
32
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34
35
36
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45
46
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concentrated under reduced pressure. It was passed through a 0.45µm Millipore membrane, and
1
lyophilized to afford 1 as white foam (31.3 mg, 92 %).; [α]25 +45.5 (c 0.37, H2O).; HꢀNMR
D
(400 MHz, D2O): δ 7.08ꢀ7.01 (d, J = 8.8 Hz, 2H, ArH), 6.95ꢀ6.92 (d, J = 9.2 Hz, 2H, ArH), 5.60
(d, J = 3.6 Hz, 1H, H1), 5.09 (s, 1H, H1'), 5.03 (s, 1H, H1''), 4.99 (d, J = 3.6 Hz, 1H, H1'''), 4.59 (s,
1H, H1''''), 4.57 (s, 1H, H5'''), 4.00 (dd, J = 10.4, 3.2 Hz, 1H, H3), 4.05 (m, 1H, H2'), 4.02ꢀ3.99 (m,
3H, H4, H5', H2''), 3.97ꢀ3.91 (m, 2H, H2, H5), 3.88 (m, 1H, H3'), 3.83 and 3.81 (each t, J = 3.2 Hz,
1H, H3'', H3''''), 3.75 (s, 3H, OCH3), 3.78ꢀ3.68 (m, 3H, H3, H5''), 3.66 and 3.64 (bs, 2H, H3''',
H4'''), 3.62ꢀ3.53 (m, 4H, H4'', H2''', H2'''', H5''''), 3.49ꢀ3.43 (t, J = 10.0 Hz, 2H, H4', H4''''), 3.40ꢀ
3.35 (m, 2H, H6, H6), 1.25 (d, J = 6.4 Hz, 3H, CH3''), 1.18 (d, J = 6.4 Hz, 3H, CH3'), 0.91 (d, J =
6.0 Hz, 3H, CH3'''').; 13CꢀNMR (100 MHz, D2O): δ 173.0 (C=O), 154.6, 150.2, 118.1, 115.2,
101.4, 101.0, 99.9, 97.9, 97.1, 79.2, 78.6, 77.3, 77.0, 71.9, 71.5, 71.34, 71.3, 70.5, 70.3, 70.2,
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