Chemical synthesis of a tetrasaccharide related to the exocellular polysaccharide from Rhodococcus sp. RHA1

Article abstract of DOI:10.1016/j.carres.2014.05.018

Chemical synthesis of the tetrasaccharide related to the exocellular polysaccharide from Rhodococcus sp. RHA1 is reported. The stereoselective glycosylations were achieved by activation of the thioglycoside donors using N-iodosuccinimide in the presence of La(OTf)₃ varying temperature per the need of 1,2-cis or 1,2-trans glycosylations. The target tetrasaccharide is reported in the form of its p-methoxyphenyl glycoside that can be cleaved selectively from the per-O-acetylated derivative allowing further glycoconjugate formation using trichloroacetimidate chemistry.

Full text of DOI:10.1016/j.carres.2014.05.018

Accepted Manuscript
Chemical synthesis of a tetrasaccharide related to the exocellular polysaccharide
from Rhodococcus sp. RHA1
Darshita Budhadev, Balaram Mukhopadhyay
PII:
S0008-6215(14)00217-1
DOI:
http://dx.doi.org/10.1016/j.carres.2014.05.018
CAR 6757
Reference:
Carbohydrate Research
To appear in:
Received Date:
Revised Date:
Accepted Date:
21 April 2014
23 May 2014
24 May 2014
Please cite this article as: Budhadev, D., Mukhopadhyay, B., Chemical synthesis of a tetrasaccharide related to the
exocellular polysaccharide from Rhodococcus sp. RHA1, Carbohydrate Research (2014), doi: http://dx.doi.org/
10.1016/j.carres.2014.05.018
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Chemical synthesis of a tetrasaccharide related to the exocellular polysaccharide
from Rhodococcus sp. RHA1
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Darshita Budhadev, Balaram Mukhopadhyay*
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Indian Institute of Science Education and Research Kolkata, Mohanpur, Nadia 741252, India
Abstract: Chemical synthesis of the tetrasaccharide related to the exocellular polysaccharide from
Rhodococcus sp. RHA1 is reported. The stereoselective glycosylations were achieved by activation of
the thioglycoside donors using N-iodosuccinimide in the presence of La(OTf)3 varying temperature per
the need of 1,2-cis or 1,2-trans glycosylations. The target tetrasaccharide is reported in the form of its p-
methoxyphenyl glycoside that can be cleaved selectively from the per-O-acetylated derivative allowing
further glycoconjugate formation using trichloroacetimidate chemistry.
Keywords: Exocellular Polysaccharide, bioremediation, La(OTf)3, TEMPO oxidation
1. Introduction
Exocellular polysaccharides (EPS) of Rhodococcus species are of great interest due to their role
in pathogenicity and their catabolic activities towards wide range of organic compounds. For example,
the EPS of pathogenic Rhodococcus equi is significantly responsible for the virulence to cause equine
necrotizing pneumonia. The detailed structural insights of the EPS from all R. equi species have
revealed their role in virulence, diagnostic serology and potential use as conjugate vaccines.i It is also
evident that the EPS of Rhodococcus species are responsible for the bacterial degradation of non-polar
aromatic and aliphatic compounds including aliphatic and aromatic hydrocarbons, polychlorinated
 Corresponding author’s E.mail: sugarnet73@hotmail.com; Tel: +91 9748 261742, Fax:
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biphenyls and even crude oil.ii,iii Therefore, these structures are attractive considering their potential as
agents for bioremediation of polluted environment. Recently, Perry et. al.iv reported the structure of the
EPS produced by Rhodococcus sp. RHA 1. The strain was isolated from hexachlorocyclohexane-
contaminated soil and found to be one of the most effective degrader of polychlorinated biphenyls.
Interestingly, the species can grow on a variety of aromatic compounds like ethylbenzene, benzoate,
phthalate, phenol, 4-hydroxybenzoate, toluene, o-xylene, benzene, terephthalate, dibenzothiophene, 2-
ethoxyphenol, guaiacol, 3-hydroxyphenylpropionic acid, isopropylbenzene, styrene, vanillate, veratrol
etc. The potential of the RHA 1 strain to adapt and survive in the stressed environmental condition has
made them attractive organism for bioremediation of contaminated soil and oil spills. For the better
understanding of the role played by the EPS towards the growth, survival and biosurfectant property, it
is essential to have reasonable quantity of the EPS structure in pure form. Herein, we report the
chemical synthesis of the repeating unit of the EPS isolated from Rhodococcus sp. RHA 1 (Figure 1) in
the form of its p-methoxyphenyl glycoside.
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Figure 1. Structure of the target tetrasaccharide related to the exocellular polysaccharide from
Rhodococcus sp. RHA1
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2. Results and discussion
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Retrosynthetic analysis on the target structure reveals that a sequential glycosylation strategy is
suitable as the (2+2) strategy will require the challenging -fucosyl linkage in the last step using a
disaccharide donor. With our previous experiences, we have chosen p-methoxyphenyl glycoside at the
reducing end to leave the scope of selective removal of the glycoside from the per-O-acetylated
derivative of the target structure. This will allow us to utilize trichloroacetimidate chemistry for further
glycoconjugate formation with suitable aglycon per need. Thus, known p-methoxyphenyl 3,4-O-
isopropylidene--D-galactopyranoside (2)v was benzylated using BnBr in the presence of NaH in
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DMF.vi The resulting dibenzyl derivative 3 was hydrolyzed using 80% AcOH at 80 Cvii to afford the
diol 4 in 92% yield. Formation of the orthoacetate of the cis-diol using trimethyl orthoacetate in the
presence of 10-camphorsulfonic acid (CSA) followed by rearrangement using 1N HClviii afforded
compound 5 in 85% yield. Further, glycosylation of acceptor 5 with known donor 6ix using N-
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iodosuccinimide (NIS) in the presence of La(OTf)3 at 0 C gave the disaccharide 7 in 83% yield. The
required 1,2-trans glycoside was the sole product as confirmed by 1H and 13C NMR experiments. Now,
the benzylidene acetal of the disaccharide 7 was selectively cleaved through reductive ring opening
using triethylsilane in the presence of BF3.Et2Oxi to afford the desired disaccharide acceptor 8 in 81%
yield. (Scheme 1).
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Scheme 1: Synthesis of the disaccharide acceptor 8
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Synthesis of the fucosyl donor started with known p-tolyl 3,4-O-isopropylidene-1-thio--L-
fucopyranoside (9).xii It was benzylated using BnBr in the presence of NaH in DMF to afford the
benzylated derivative 10 in 95% yield. Compound 10 was then hydrolyzed by 80% AcOH at 80 C
giving the diol 11 in 91% yield. Selective benzylation at the 3-position of the diol was accomplished by
the formation of the corresponding stannylene derivativexiii folowed by the reaction of BnBr in the
presence of Bu4NI resulting the dibenzyl derivative 12 in 82% yield. The sole free OH group was
protected by chloroacetate using chloroacetic anhydride in pyridinexiv to furnish the required fucosyl
donor 13 in 89% yield (Scheme 2).
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Scheme 2: Synthesis of the fucosyl donor 13
Glycosylation between the disaccharide acceptor 8 and the fucosyl donor 13 using NIS in the
presence of La(OTf)3 at -40 C afforded the trisaccharide 14 in 81% yield with the desired 1,2-cis
fucosyl linkage. It is worth noting that the 1,2-trans isomer was not found even after the
chromatographic purification. Further, the chloroacetate group was deprotected using thioureaxv to
furnish the trisaccharide acceptor 15 in 84% yield. Final glycosylation with the known donor 6 using
NIS in the presence of La(OTf)3 at 0 C gave the tetrasaccharide 16 in 87% yield. The benzylidene
acetal was hydrolyzed using 80% AcOH at 80 C to afford the corresponding diol 17 in 91% yield. The
primary OH group was selectively oxidized by TEMPOxvi,xvii mediated oxidation to give the desired
uronic acid moiety. The formation of the uronic acid was confirmed by mass spectroscopy. However,
we were unable to isolate the pure compound from the the salts generated during the oxidation reaction.
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Therefore, the crude compound was subjected to catalytic hydrogenation using H-cube (ThalesNano,
Hungary) flow hydrogenation assembly followed by de-O-acylation using NaOMe in MeOHxviii to
furnish the target tetrasaccharide 1 in 65% yield over last three steps (Scheme 3). The final compound
was purified by triturating in CH2Cl2 followed by HPLC using C18 column where all salts were
removed successfully.
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Scheme 3 . Synthesis of the target tetrasaccharide 1
3. Conclusion
In conclusion, we have achieved the chemical synthesis of the tetrasaccharide related to the O-
polysaccharide from Rhodococcus sp. RHA1 in the form of its p-methoxyphenyl glycoside.
Stereoselective glycosylations were accomplished by the activation of thioglycosides using NIS in the
presence of La(OTf)3. The p-methoxyphenyl group at the reducing end of the target tetrasaccharide has
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a particular advantage as it can be cleaved selectively from the per-O-acetylated derivative. The
anomeric OH group thus obtained may be converted to the corresponding trichloroacetimidate
derivative and further conjugated with suitable aglycon to form desired glycoconjugates.
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4. Experimental
4.1. General: All reagents and solvents were dried prior to use according to standard methods.xix
Commercial reagents were used without further purification unless otherwise stated. Analytical TLC
was performed on Silica Gel 60-F254 (Merck or Whatman) with detection by fluorescence and/or by
charring following immersion in a 10 % ethanolic solution of sulfuric acid. An orcinol dip carefully
prepared by the addition of concentrated sulfuric acid (20 mL) to an ice-cold solution of 3,5-
dihydroxytoluene (360 mg) in EtOH (150 mL) and H2O (10 mL) was used to detect deprotected
oligosaccharides by charring. Flash chromatography was performed with Silica Gel 230-400 mesh
(Merck, India). Optical rotations were measured at the sodium D-line at ambient temperature, with a
Perkin Elmer 141 polarimeter. H NMR and 13C NMR spectra were recorded on Bruker Avance
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spectrometer at 500 and 125 MHz respectively, using Me4Si or CH3OH as internal standards, as
appropriate. The assignments of H and 13C peaks were done with the help of H-1H COSY and 1H-13C
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HSQC spectra.
4.2. p-Methoxyphenyl 3,4-O-isopropylidene-2,6-di-O-benzyl-β-D-galactopyranoside (3). The known
compound p-methoxyphenyl 3,4-O-isopropylidene-β-D-galactopyranoside (2) (4.0 g, 10.7 mmol) was
dissolved in dry DMF (25 mL). NaH (1.0 g, 42.8 mmol, 60% in mineral oil) was added to the solution
followed by BnBr (3.8 mL, 32.1 mmol). The reaction was completed after 1 hour as evident by TLC (n-
hexane-EtOAc; 5:1). MeOH (1.0 mL) was added to neutralize the excess NaH and the solvents were
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evaporated in vacuo. The residue was dissolved in CH2Cl2 (30 mL) and washed with brine solution
(250 mL). The organic layer was separated, dried over Na2SO4 and evaporated in vacuo. Flash
chromatography of the crude product using n-hexane-EtOAc (5:1) as eluent, afforded the pure product 3
(5.6 g, 90%) as white solid. [α]D25 +141 (c 1.1, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 7.45-7.27 (m, 10
H, ArH), 7.06, 6.81 (2d, 4H, C6H4OCH3), 4.95, 4.90, 4.64, 4.56 (4d, 4H, J 11.5 Hz, 2 × CH2Ph), 4.83 (d,
1H, J1,2 8.0 Hz, H-1), 4.26 (dd, 1H, J2,3 6.5 Hz, J3,4 5.5 Hz, H-3), 4.21 (dd, 1H, J3,4 5.5 Hz, J4,5 2.0 Hz,
H-4), 4.04 (m, 1H, H-5), 3.85 (m, 2H, H-6), 3.78 (s, 3H, C6H4OCH3), 3.68 (dd, 1H, J1,2 8.0 Hz, J2,3 6.5
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Hz, H-2), 1.43, 1.37 (2s, 6H, 2 × isopropylidene CH3). C NMR (CDCl3, 125 MHz) δ: 155.2, 151.4,
138.2, 138.1, 128.3 (2), 128.2(2), 128.1(2), 127.6(2), 127.5(2), 118.5(2), 114.5(2) (ArC), 110.2
[C(CH3)2], 102.0 (C-1), 79.3, 79.1, 73.6 (2), 73.5, 72.6, 69.5, 55.6 (C6H4OCH3), 27.7, 26.3 (2 ×
isopropylidene CH3). HRMS calcd. for C30H34O7Na (M+Na)+: 529.2202, found: 529.2205.
4.3. p-Methoxyphenyl 2,6-di-O-benzyl-β-D-galactopyranoside (4). A suspension of compound 3 (3.0
g, 5.7 mmol) in 80% aqueous AcOH (20 mL) was stirred at 80 °C for 2 hours when TLC (n-hexane-
EtOAc; 2:1) showed complete conversion of the starting material to a slower moving spot. Solvents
were evaporated in vacuo, coevaporated with toluene to remove traces of AcOH. The residue was
purified by flash chromatography using n-hexane-EtOAc (1.5:1) as eluent to give the pure product 4
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(2.5 g, 92%) as white solid. [α]D +103 (c 1.0, CHCl3). H NMR (500 MHz, CDCl3) δ: 7.38-7.29 (m,
10H, ArH), 7.06, 6.82 (2d, 4H, J 9.0 Hz, C6H4OCH3), 5.07, 4.79 (2d, 2H, J 11.5 Hz, CH2Ph), 4.86 (d,
1H, J1,2 7.5 Hz, H-1), 4.58 (s, 2H, CH2Ph), 4.02 (dd, 1H, J3,4 3.0 Hz, J4,5 <1.0 Hz, H-4), 3.81 (m, 2H, H-
6), 3.78 (m, 4H, H-2, C6H4OCH3), 3.71 (t, 1H, J5,6 5.5 Hz, H-5), 3.67 (dd, 1H, J2,3 9.0 Hz, J3,4 3.0 Hz,
H-3), 2.6 (bs, 2H, 2×OH). 13C NMR (CDCl3, 125 MHz) δ: 155.3, 151.3, 138.2, 137.8, 128.6(2),
128.4(2), 128.2(2), 128, 127.7, 127.7(2), 118.4(2), 114.5(2) (ArC), 102.8 (C-1), 78.9, 77.3, 74.8, 73.7,
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73.6, 73.1, 69.4, 68.9, 55.6 (C6H4OCH3). HRMS calcd. for C27H30O7Na (M+Na)+: 489.1889, found:
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489.1886.
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4.4. p-Methoxyphenyl 4-O-acetyl-2,6-di-O-benzyl-β-D-galactopyranoside (5). To a suspension of
compound 4 (2.2g, 4.5 mmol) in dry MeCN (20 mL), trimethyl orthoacetate (0.7 mL, 5.8 mmol) was
added followed by CSA (50 mg) and the mixture was stirred at room temperature for 30 minutes when
TLC (n-hexane-EtOAc; 1:1) showed complete conversion of starting material to a faster moving spot.
Et3N was added to neutralize the reaction and the solvents were evaporated. The residue was dissolved
in CH2Cl2 (30 mL) and shaken with 1M HCl (2×30 mL), and then washed with satd NaHCO3 solution
(30 mL) and brine (30 mL). The organic layer was separated, dried over Na2SO4, filtered and evaporated
in vacuo. The crude product thus obtained was purified by flash chromatography using n-hexane-EtOAc
(2.5:1) to furnish the monosaccharide acceptor 5 (2.0g, 85%) as white solid. [α]D25 +124 (c 1.2, CHCl3).
1H NMR (500 MHz, CDCl3) δ: 7.39-7.29 (m, 10H, ArH), 7.05, 6.81 (2d, 4H, C6H4OCH3), 5.42 (dd, 1H,
J3,4 3.0 Hz, J4,5 <1.0 Hz, H-4), 5.07, 4.78 (2d, 2H, J 11.5 Hz, CH2Ph), 4.90 (d, 1H, J1,2 8.0 Hz, H-1),
4.55, 4.47 (2d, 2H, J 12.0 Hz, CH2Ph), 3.85 (m, 2H, H-3, H-5), 3.78 (s, 3H, C6H4OCH3), 3.76 (t, 1H,
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J1,2, J2,3 8.0 Hz, H-2), 3.6 (m, 2H, J 6.5 Hz, H-6), 2.53 (bs, 1H, OH), 2.11 (s, 3H, COCH3). C NMR
(CDCl3, 125 MHz) δ: 170.9 (COCH3), 155.3, 151.2, 138, 137.7, 128.5 (2), 128.4 (2), 128.1 (2), 128.0,
127.8 (2), 127.7, 118.2 (2), 114.5 (2) (ArC), 102.7 (C-1), 79.0, 75.0, 73.6, 72.8, 71.9, 69.4, 68.3, 55.6
(C6H4OCH3), 20.8 (COCH3). HRMS calcd. for C29H32O8Na (M+Na)+: 531.1995, found: 531.1998.
4.5. p-Methoxyphenyl 2,3-di-O-benzoyl-4,6-O-benzylidene-β-D-glucopyranosyl-(1→3)-4-O-acetyl-
2,6-di-O-benzyl-β-D-galactopyranoside (7): A suspension of acceptor 5 (1.25 g, 2.15 mmol), the
known donor 6 (850 mg, 1.7 mmol) and MS 4Å (1.5g) in dry CH2Cl2 (15 mL) was stirred under
nitrogen for 30 minutes. After adding NIS (629 mg, 2.8 mmol), the mixture was cooled to 0 °C. After 10
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minutes, La(OTf)3 (50 mg) was added and the mixture was stirred at 0 °C for 20 minutes when TLC (n-
hexane-EtOAc; 1.5:1) revealed complete consumption of the donor. The reaction mixture was then
immediately filtered through a pad of celite and washed with CH2Cl2. The combined filtrate obtained
was washed successively with Na2S2O3 (2×50 mL), NaHCO3 (2×50 mL) and finally with brine (50 mL).
The organic layer was separated, dried (Na2SO4), filtered and evaporated in vacuo. The residue was then
purified by flash chromatography using n-hexane-EtOAc (3:1) as eluent to give the disaccharide 7 (2.0
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g, 83 %) as a white foam. [α]D +101 (c 1.1, CHCl3). H NMR (500 MHz, CDCl3) δ: 8.06-7.17 (m,
25H, ArH), 6.97, 6.77 (2d, 4H, J 9.0 Hz, C6H4OCH3), 5.77 (t, 1H, J2ʹ,3ʹ, J3ʹ,4ʹ 9.0 Hz, H-3ʹ ), 5.56 (s,
1H, CHPh), 5.52 (dd, 1H, J3,4 3.5 Hz, J4,5 <1.0 Hz, H-4), 5.48 (dd, 1H, J1ʹ ,2ʹ 7.5 Hz, J2ʹ ,3ʹ 9.0 Hz, H-
2′), 5.28 (d, 1H, J1ʹ ,2ʹ 7.5 Hz, H-1′), 4.86 (d, 1H, J1,2 8.0 Hz, H-1), 4.78 (d, 1H, J 11.0 Hz, CH2Ph),
4.54, 4.49 (2d, 2H, J 11.5 Hz, CH2Ph), 4.39 (m, 2H, H-6a′, CH2Ph), 4.02 (dd, 1H, J2,3 9.5 Hz, J3,4 3.5
Hz, H-3), 3.98 (d, 1H, J3ʹ ,4ʹ, J4ʹ,5ʹ 9.0 Hz, H-4′), 3.88 (m, 2H, H-6b, H-6b′), 3.76 (m, 4H, H-2,
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C6H4OCH3), 3.61 (m, 3H, H-5, H-5′, H-6a), 2.21 (s, 3H, COCH3). C NMR (CDCl3, 125 MHz) δ:
170.4 (COCH3), 165.6, 165.0 (2×COPh), 155.3, 151.2, 137.7 (2), 136.7, 133.2, 133.1, 129.7 (2), 129.6
(2), 129.3, 129.2, 129.0, 128.5 (2), 128.4 (2), 128.3 (3), 128.2 (2), 128.1 (2), 127.9, 127.8 (3), 127.7,
126.1 (2), 118.2 (2), 114.5 (2) (ArC), 102.6 (C-1), 101.4 (CHPh), 101.1 (C-1′), 79.3, 78.6, 76.1, 75,
73.7, 73.2, 72.9, 72.8, 69.6, 68.6, 68.5, 66.5, 55.6 (C6H4OCH3), 20.8 (COCH3). HRMS calcd. for
C56H54O15Na (M+Na)+: 989.3360, found: 989.3358.
4.6. p-Methoxyphenyl 2,3-di-O-benzoyl-6-O-benzyl-β-D-glucopyranosyl-(1→3)-4-O-acetyl-2,6-di-
O-benzyl-β-D-galactopyranoside (8): To a solution of compound 7 (1.4 g, 1.45 mmol) in dry CH2Cl2
(20 mL) was added triethylsillane (2.8 mL, 17.4 mmol) at 0°C and stirred for 10 minutes. BF3.Et2O
(0.37 mL, 2.9 mmol) was added and the reaction was continued for 5 min when TLC (n-hexane-EtOAc;
1.5:1) confirmed complete conversion of the starting material to a slower moving spot. The solution was
diluted with CH2Cl2 (10 mL) and washed successively with water (50 mL), NaHCO3 (50 mL) and brine
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(50 mL). The organic layer was collected, dried over Na2SO4, filtered and concentrated in vacuo. The
crude product thus obtained was purified by flash chromatography using n-hexane-EtOAc (2:1) as
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eluent to furnish the disaccharide acceptor 8 (1.1 g, 81%) as colourless foam. [α]D +112 (c 1.0,
6
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8
1
CHCl3). H NMR (500 MHz, CDCl3) δ: 7.98-7.20 (m, 25H, ArH), 6.96, 6.76 (2d, 4H, J 9 Hz,
9
C6H4OCH3), 5.51 (dd, 1H, J3,4 2.0 Hz, J4,5 <1.0 Hz, H-4), 5.46 (t, 1H, J2ʹ,3ʹ, J3ʹ,4ʹ 9.0 Hz, H-3′), 5.42
(dd, 1H, J1ʹ,2ʹ 7.5 Hz, J2ʹ,3ʹ 9.0 Hz, H-2′), 5.15 (d, 1H, J1ʹ,2ʹ 7.5 Hz, H-1′), 4.83 (d, 1H, J1,2 8.0 Hz, H-
1), 4.76, 4.4 (2d, 2H, J 10.5 Hz, CH2Ph), 4.65, 4.59, 4.53, 4.48 (4d, 4H, J 11.5 Hz, CH2Ph), 3.98 (m,
2H, H-3, H-4′), 3.83 (m, 3H, H-6a, H-6a′, H-6b′), 3.75 (m, 4H, H-2, C6H4OCH3), 3.59 (m, 3H, H-5, H-
5′, H-6b), 2.17 (s, 3H, COCH3). 13C NMR (CDCl3, 125 MHz) δ: 170.3 (COCH3), 166.9, 165.1
(2×COPh), 155.2, 151.2, 137.9, 137.8, 137.6, 133.3, 133.1, 129.8 (2), 129.5 (2), 129.3, 129.1, 128.5 (2),
128.4 (2), 128.3 (2), 128.3 (3), 127.8 (3), 127.7 (3), 127.6 (2), 127.5 (2), 118.2 (2), 114.4 (2) (ArC),
102.5 (C-1), 100.6 (C-1′), 79.3, 76.2, 76.1, 75, 74.3, 73.7, 73.6, 73, 71.8, 71.1, 70.0, 69.8, 68.8, 55.5
(C6H4OCH3), 20.7 (COCH3). HRMS calcd. for C56H56O15Na (M+Na)+: 991.3517, found: 991.3519.
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4.7. p-Tolyl 2-O-benzyl-3,4-O-isopropylidene-1-thio-β-L-fucopyranoside (10): To a solution of p-
tolyl 3,4-O-isopropylidene-1-thio-β-L-fucopyranoside (9) (1.6g, 5.2 mmol) DMF (20 mL), NaH (495 mg,
20.6 mmol) was added followed by BnBr (1.8 mL, 15.5 mmol) and the mixture was stirred for 1 hour at
room temperature when TLC (n-hexane-EtOAc; 2:1) revealed complete conversion of the starting
material to a faster moving spot. MeOH was added to neutralize excess NaH and the solvents were
evaporated in vacuo. The residue was dissolved in CH2Cl2 (20 mL) and washed with brine (20 mL). The
organic layer was separated, dried (Na2SO4), filtered and evaporated in vacuo. The crude product, thus
obtained was purified by flash chromatography using n-hexane-EtOAc (6:1) to give the pure product 10
25
1
(2.0 g, 95%) as colourless gel. [α]D -34 (c 1.0, CHCl3). H NMR (500 MHz, CDCl3) δ: 7.50, 7.12 (2d,
4H, J 8.0 Hz, C6H4SCH3), 7.49-7.30 (m, 5H, ArH), 4.87, 4.72 (2d, 2H, J 11.5 Hz, CH2Ph), 4.56 (d, 1H,
J1,2 9.5 Hz, H-1), 4.24 (t, 1H, J2,3, J3,4 6.0 Hz, H-3), 4.02 (dd, 1H, J3,4 6.0 Hz, J4,5 2.0 Hz, H-4), 3.77 (m,
10

1H, H-5), 3.52 (dd, 1H, J1,2 9.5 Hz, J2,3 6.0 Hz, H-2), 2.35 (s, 3H, SC6H4CH3), 1.45, 1.39 (2s, 6H,
1
2
3
13
2×isopropylidene CH3), 1.42 (d, 3H, J5,6 6.5 Hz, C-CH3). C NMR (CDCl3, 125 MHz) δ: 137.9, 137.2,
4
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6
7
8
9
132.4 (2), 129.7, 129.2 (2), 128.0 (2), 127.9 (2), 127.4 (2) (ArC), 109.3 [C(CH3)2], 86.1 (C-1), 79.6, 78.0,
76.2, 73.1, 72.1, 27.7, 26.2 (2×isopropylidene CH3), 20.9 (SC6H4CH3), 16.7 (C-CH3). HRMS calcd. for
C23H28O4SNa (M+Na)+: 423.1606, found: 423.1603.
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4.8. p-Tolyl 2,3-di-O-benzyl-1-thio-β-L-fucopyranoside (12): A slurry of compound 10 (1.9g, 4.5
mmol) in 80% AcOH (20 mL) was heated at 80 °C for 2 hours when it was evident from TLC (n-
hexane-EtOAc; 2:1) that the starting material was completely converted to a slower moving spot. The
solvents were evaporated and coevaporated with toluene. The residue was purified by columm
chromatography using n-hexane-EtOAc (1:1) as eluent to furnish the pure product 11 which was used
directly for the next reaction. A mixture of compound 11 (1.5 g, 4.2 mmol) and Bu2SnO (1.4g, 5.46
mmol) in dry MeOH (20 mL) was refluxed for 3 hours. The resulting clear solution was concentrated in
vacuo, co-evaporated with toluene and the residue obtained was dried under vacuum for 30 min. It was
then dissolved in dry DMF (15 mL) and BnBr (0.65 mL, 5.5 mmol) was added followed by Bu4NI (1.7
g, 4.6 mmol). The solution was stirred at 60 ºC for 12 hours. After evaporating the solvents in vacuo, the
residue was dissolved in CH2Cl2 (40 mL) and washed successively with H2O (50 mL) and brine (50
mL). The organic layer was separated, dried over Na2SO4, filtered and evaporated in vacuo. The crude
product was purified by flash chromatography using n-hexane-EtOAc (5:1) as eluent to afford
compound 12 (1.8g, 82%) as pale yellow gel. [α]D25 -22 (c 1.1, CHCl3). 1H NMR (500 MHz, CDCl3) δ:
7.49-7.11 (m, 14H, ArH), 4.84, 4.75 (2d, 2H, J 10.5 Hz, CH2Ph), 4.72, 4.69 (2d, 2H, J 12.0 Hz, CH2Ph),
4.54 (d, 1H, J1,2 9.5 Hz, H-1), 3.82 (dd, 1H, J3,4 2.5 Hz, J4,5 <1.0 Hz, H-4), 3.67 (t, 1H, J1,2, J2,3 9.5 Hz,
H-2), 3.57 (dd, 1H, J2,3 9.5 Hz, J3,4 2.5 Hz, H-3), 3.54 (m, 1H, H-5), 2.34 (s, 3H, SC6H4CH3), 2.30 (bs,
1H, OH), 1.37 (d, 3H, J5,6 6 Hz, C-CH3). 13C NMR (CDCl3, 125 MHz) δ: 138.2, 137.7, 137.6, 132.6 (2),
129.6 (2), 128.5 (2), 128.3 (2), 128.2 (2), 128.0, 127.9 (2), 127.7, 126.9 (ArC), 87.7 (C-1), 82.9, 75.6,
11

74.1, 72.1, 69.3, 65.3, 21.1 (SC6H4CH3), 16.7 (C-CH3). HRMS calcd. for C27H30O4SNa (M+Na)+:
1
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3
473.1762, found: 473.1765.
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8
9
4.9. p-Tolyl 2,3-di-O-benzyl-4-O-chloroacetyl-1-thio-β-L-fucopyranoside (13): Compound 12 (1.7 g,
3.8 mmol) was dissolved in dry CH2Cl2 (20 mL) and pyridine (0.50 mL, 5.7 mmol) and stirred at -5°C
for 10 minutes. Chloroacetic anhydride (975 mg, 5.7 mmol) was added and the solution was stirred at -
5°C for 1 hour when TLC (n-hexane-EtOAc; 4:1) revealed complete conversion of the starting material.
The solvents were evaporated and coevaporated with toluene for complete removal of pyridine. The
crude residue thus obtained was purified by flash chromatography using n-hexane-EtOAc (4:1) as eluent
to afford the desired donor 13 (1.8 g, 89%) as light yellow gel. [α]D25 -27 (c 1.0, CHCl3). 1H NMR (500
MHz, CDCl3) δ: 7.50- 7.11 (m, 14H, ArH), 5.43 (dd, 1H, J3,4 3.0 Hz, J4,5 <1.0 Hz, H-4), 4.78, 4.72 (2d,
2H, J 10.0 Hz, CH2Ph), 4.74, 4.52 (2d, 2H, J 11.0 Hz, CH2Ph), 4.58 (d, 1H, J1,2 9.0 Hz, H-1), 4.18 (m,
2H, CH2Cl), 3.71 (m, 1H, H-5), 3.68 (dd, 1H, J2,3 9.0 Hz, J3,4 3.0 Hz, H-3), 3.61 (t, 1H, J1,2, J2,3 9.0 Hz,
H-2), 2.35 (s, 3H, SC6H4CH3), 1.28 (d, 3H, J5,6 6.5 Hz, C-CH3). 13C NMR (CDCl3, 125 MHz) δ: 167.3
(COCH2Cl), 138.1, 137.8, 137.3, 132.8(2), 129.6 (2), 129.5, 128.4 (2), 128.3 (2), 128.2 (2), 128.1 (2),
127.9, 127.8 (ArC), 87.7 (C-1), 81.0, 76.3, 75.6, 72.6, 72.1, 71.9, 40.8 (CH2Cl), 21.1 (SC6H4CH3), 16.8
(C-CH3). HRMS calcd. for C29H31ClO5SNa (M+Na)+: 549.1478, found: 549.1481.
10
11
12
13
14
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4.10. p-Methoxyphenyl 2,3-di-O-benzyl-4-O-chloroacetyl-1-β-L-fucopyranosyl-(1→4)-2,3-di-O-
benzoyl-6-O-benzyl-β-D-glucopyranosyl-(1→3)-4-O-acetyl-2,6-di-O-benzyl-β-D-galactopyranoside
(14): A mixture of disaccharide acceptor 8 (900 mg, 0.93 mmol), donor 13 (525 mg, 1.21 mmol) and
MS 4Å (1.5 g) in dry CH2Cl2 (15 mL) was stirred under nitrogen for 30 minutes. The mixture was
cooled to -40 °C. NIS (353 mg, 1.6 mmol) was added followed by La(OTf)3 (30 mg) and the reaction
was continued for 30 minutes when TLC (n-hexane-EtOAc; 2:1) showed that the donor has been
12

consumed completely. The mixture was filtered through a pad of celite and washed with CH2Cl2. The
combined filtrate was washed successively with Na2S2O3 (2×50 mL), NaHCO3 (2×50 mL) and finally
with brine (50 mL). The organic layer was separated, dried (Na2SO4), filtered and evaporated in vacuo.
The crude residue was purified by flash chromatography using n-hexane-EtOAc (3.5:1) as eluent to
1
2
3
4
5
6
7
8
9
25
1
afford pure trisaccharide 14 (1.0 g, 81%) as colourless glass. [α]D +88 (c 1.0, CHCl3). H NMR (500
MHz, CDCl3) δ: 7.94-7.14 (m, 35H, ArH), 6.93, 6.75 (2d, 4H, J 9.0 Hz, C6H4OCH3), 5.66 (t, 1H, J2ʹ,3ʹ
10
11
12
13
14
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65
,
J3ʹ,4ʹ 9.5 Hz, H-3′), 5.52 (dd, 1H, J3,4 3.5 Hz, J4,5 <1.0 Hz, H-4), 5.33 (dd, 1H, J1ʹ,2ʹ 7.5 Hz, J2ʹ,3ʹ 9.5
Hz, H-2′), 5.22 (dd, 1H, J3″,4″ 3.0 Hz, J4″,5″ <1.0 Hz, H-4″), 5.06 (d, 1H, J1ʹ,2ʹ 7.5 Hz, H-1′), 5.03 (d,
1H, J1″,2″ 4.0 Hz, H-1″), 4.80 (d, 1H, J1,2 7.5 Hz, H-1), 4.76 (d, 1H, J 11.5 Hz, CH2Ph), 4.7, 4.34 (2d,
2H, J 10.5 Hz, CH2Ph), 4.66 (d, 1H, J 11.0 Hz, CH2Ph), 4.55-4.45 (m, 6H, 3×CH2Ph), 4.13 (t, 1H,
J3ʹ,4ʹ, J4ʹ,5ʹ 9.5 Hz, H-4′), 4.04 (s, 2H, CH2Cl), 3.94 (dd, 1H, J2,3 9.5 Hz, J3,4 3.5 Hz, H-3), 3.89 (m,
3H, H-3″, H-5″, H-6a′), 3.8 (m, 2H, H-5, H-6b′), 3.75 (s, 3H, C6H4OCH3), 3.72 (dd, 1H, J1,2 8.0 Hz, J2,3
9.5 Hz, H-2), 3.65 (dd, 1H, J1″,2″ 3.5 Hz, J2″,3″ 10.5 Hz, H-2″), 3.58 (m, 3H, H-5′, H-6), 2.16 (s, 3H,
13
COCH3), 0.6 (d, 3H, J5″,6″ 7.0 Hz, C-CH3). C NMR (CDCl3, 125 MHz) δ: 176.4 (COCH2Cl), 170.3
(COCH3), 167.2, 165.0 (2×COPh), 155.2, 151.3, 140.9, 138.5 (2), 138.1 (2), 137.9, 137.8 (2), 137.7,
133.8 (2), 133.1, 133.0, 130.1 (2), 129.7 (2), 129.5 (2), 129.4, 129.3, 128.4 (3), 128.3 (3), 128.1 (3),
128.0 (3), 127.8 (3), 127.7 (3), 127.5 (3), 118.2 (2), 114.4 (2) (ArC), 102.5 (C-1), 100.6 (C-1′), 99.4 (C-
1″), 79.2, 76, 75.9, 75.5, 75.1, 74.3, 74.2, 73.6 (2), 73.2, 73.1, 72.9, 72.8, 71.9, 69.9, 68.8, 68.0, 65.2,
55.6 (C6H4OCH3), 40.7 (CH2Cl), 20.8 (COCH3), 15.4 (C-CH3). HRMS calcd. for C78H79O20ClNa
(M+Na)+:1393.4751, found: 1393.4754.
4.11. p-Methoxyphenyl 2,3-di-O-benzyl-β-L-fucopyranosyl-(1→4)-2,3-di-O-benzoyl-6-O-benzyl-β-
D-glucopyranosyl-(1→3)-4-O-acetyl-2,6-di-O-benzyl-β-D-galactopyranoside (15): To a solution of
the trisaccharide 9 (1.2 g, 0.9 mmol) in CH2Cl2-CH3OH (2:3, 20 mL), thiourea (332 mg, 4.4 mmol) and
collidene (0.6 mL, 4.4 mmol) were added and it was refluxed at 40 C for 10 hours when TLC (n-
13

hexane-EtOAc; 2:1) showed complete conversion of the starting material to a slower moving spot. After
evaporating the solvents in vacuo, the residue was dissolved in CH2Cl2 (50 mL) and washed with 1M
HCl (50 mL) and H2O (50 mL). The organic layer was collected, dried (Na2SO4), filtered and
evaporated. The crude product thus obtained was purified by flash chromatography using n-hexane-
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4
5
6
7
8
9
25
EtOAc (2:1) as eluent to afford the pure trisaccharide acceptor (950 mg, 84%) as colourless foam. [α]D
10
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12
13
14
15
16
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65
+101 (c 1.1, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 7.92-7.09 (m, 35H, ArH), 6.97, 6.77 (2d, 4H, J 9.0
Hz, C6H4OCH3), 5.69 (t, 1H, J2′,3′ , J3′,4′ 9.5 Hz, H-3′), 5.55 (dd, 1H, J3,4 3.5 Hz, J4,5 <1.0 Hz, H-4), 5.35
(dd, 1H, J1′,2′ 8.0 Hz, J2′,3′ 9.5 Hz, H-2′), 5.09 (d, 1H, J1′,2′ 8.0 Hz, H-1′), 5.03 (d, 1H, J1″,2″ 4.0 Hz, H-1″),
4.84 (d, 1H, J1,2 8.0 Hz, H-1), 4.78, 4.59 (2d, 2H, J 11.5 Hz, CH2Ph), 4.72, 4.37 (2d, 2H, J 11.0 Hz,
CH2Ph), 4.71, 4.65 (2d, 2H, J 11.5 Hz, CH2Ph), 4.53, 4.48 (2d, 2H, J 12.5 Hz, CH2Ph), 4.54, 4.5 (2d,
2H, J 11.5 Hz, CH2Ph), 4.12 (t, 1H, J3′,4′, J4′,5′ 9.5 Hz, H-4′), 3.98 (dd, 1H, J2,3 9.5 Hz, J3,4 3.5 Hz, H-3),
3.89 (m, 2H, H-6a′, H-6b′), 3.81 (m, 3H, H-5, H-3″, H-5″), 3.75 (m, 5H, H-2, H-2″, C6H4OCH3), 3.60
13
(m, 4H, H-4″, H-5′, H-6), 2.19 (s, 3H, COCH3), 0.79 (d, 3H, J5″,6″ 7.0 Hz, C-CH3). C NMR (CDCl3,
125 MHz) δ: 170.3 (COCH3), 166.0, 165.0 (2×COPh), 155.1, 151.2, 138.5, 138.2, 137.9 (2), 137.8,
133.0, 132.9, 129.6 (3), 129.5 (3), 129.3, 128.5 (3), 128.4 (3), 128.3 (3), 128.2 (3), 128.0 (3), 127.8 (2),
127.7 (3), 127.6 (2), 127.6, 127.4 (3), 127.3 (2), 118.1 (2), 114.4 (2) (ArC), 102.4 (C-1), 100.6 (C-1′),
99.2 (C-1″), 79.1, 77.9, 76.5, 75.7, 75.4, 75, 74.2, 74.1, 73.6, 73.1, 73.0, 72.8, 72.1 (2), 69.9, 69.7, 68.8,
68.2, 66.2, 55.5 (C6H4OCH3), 20.7 (COCH3), 15.4 (C-CH3). HRMS calcd. for C76H78O19Na (M+Na)+:
1317.5035, found: 1317.5038.
4.12. p-Methoxyphenyl 4,6-O-benzylidene-2,3-di-O-benzoyl-β-D-glucopyranosyl-(1→4)-2,3-di-O-
benzyl-β-L-fucopyranosyl-(1→4)-2,3-di-O-benzoyl-6-O-benzyl-β-D-glucopyranosyl-(1→3)-4-O-
acetyl-2,6-di-O-benzyl-β-D-galactopyranoside (16): A mixture of trisacchaide acceptor 15 (345 mg,
0.27 mmol), known donor 6 (202 mg, 0.35 mmol) and MS 4Å (1 g) in dry CH2Cl2 (10 mL) was stirred
under nitrogen for 30 minutes. Then the mixture was cooled to 0 C and NIS (100 mg, 0.45 mmol) was
14

added followed by La(OTf)3 (20 mg). After stirring for 20 minutes TLC (toluene-EtOAC; 5:1) revealed
complete consumption of the donor. The reaction mixture was immediately filtered through a pad of
celite and washed with CH2Cl2. The combined filtrate was successively washed with Na2S2O3 (2×50
mL), NaHCO3 (2×50 mL) and brine (50 mL). The organic layer was separated, dried (Na2SO4), filtered
and evaporated in vacuo. The residue was purified by flash chromatography using toluene-EtOAc (11:1)
1
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3
4
5
6
7
8
9
10
11
12
13
14
15
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25
as eluent to furnish pure tetrasaccharide (410 mg, 87%) as colourless foam. [α]D +98 (c 0.9, CHCl3).
1H NMR (500 MHz, CDCl3) δ: 7.94-7.13 (m, 50H, ArH), 6.95, 6.76 (2d, 4H, J 9.0 Hz, C6H4OCH3),
5.64 (t, 1H, J2′,3′, J3′,4′ 9.0 Hz, H-3′), 5.61 (d, 1H, J2″′,3″′, J3″′,4″′ 9.0 Hz, H-3′′′), 5.52 (m, 2H, H-2′′′, H-4),
5.45 (s, 1H, CHPh), 5.32 (dd, 1H, J1′,2′ 7.5 Hz, J2′,3′ 9.5 Hz, H-2′), 5.04 (d, 1H, J1′,2′ 7.5 Hz, H-1′), 4.95
(d, 1H, J1″,2″ 4.0 Hz, H-1″), 4.88 (d, 1H, J1′′′,2′′′ 7.5 Hz, H-1′′′), 4.83 (d, 1H, J1,2 8.0 Hz, H-1), 4.82 (d, 1H,
J 10.5 Hz, CH2Ph), 4.72 (s, 2H, CH2Ph), 4.7 (d, 1H, J 10.5 Hz, CH2Ph), 4.54 (d, 1H, J 11.5 Hz, CH2Ph),
4.53 (d, 1H, J 12.5 Hz, CH2Ph), 4.51 (d, 1H, J 11.0 Hz, CH2Ph), 4.49 (d, 1H, J 11.5 Hz, CH2Ph), 4.41
(d, 1H, J 12.5 Hz, CH2Ph), 4.35 (d, 1H, J 10.5 Hz, CH2Ph), 4.21 (dd, 1H, J3′′′,4′′′ 9.5 Hz, J4′′′,5′′′ 5.0 Hz, H-
4′′′), 4.09 (t, 1H, J3′,4′, J4′,5′ 9.5 Hz, H-4′), 3.92 (m, 3H, H-2″, H-3, H-6a′′′), 3.81 (m, 4H, H-3″, H-4″, H-
6′), 3.75 (s, 3H, C6H4OCH3), 3.71 (m, 3H, H-2, H-6), 3.59 (m, 4H, H-5, H-5′, H-5″, H-6b′′′), 3.49 (dd,
1H, J4′′′,5′′′ 5 Hz, J5′′′,6′′′ 10 Hz, H-5′′′), 2.16 (s, 3H, COCH3), 0.49 (d, 3H, J5″,6″ 6.5 Hz, C-CH3). 13C NMR
(CDCl3, 125 MHz) δ: 170.3 (COCH3), 166, 165.6, 165, 164.9 (4×COPh), 155.2, 151.3, 138.5, 138.4,
138.3, 137.9, 137.8, 136.8, 133.1, 133.0, 132.9, 129.7 (2), 129.65 (2), 129.62, 129.5, 129.4, 129.3,
129.2, 129.0, 128.9, 128.4 (3), 128.35 (3), 128.34 (3), 128.33 (3), 128.26 (2), 128.24, 128.16, 128.13
(3), 128 (3), 127.75 (3), 127.73 (3), 127.65 (2), 127.62, 127.5 (3), 127.4, 127.3 (2), 126.1 (3), 118.1 (2),
114.4 (2) (ArC), 102.4 (C-1), 101.7 (C-1′′′), 101.3 (ChPh), 100.6 (C-1′), 98.9 (C-1″), 79.1, 78.4, 78.2,
77.6, 76.6, 75.5, 75.4, 75, 74.8, 74.2, 74.1, 73.6, 73.1 (2), 73, 72.8, 72.4 (2), 69.9, 68.9, 68.4, 67.9, 66.6,
66.5, 55.5 (C6H4OCH3), 20.7 (COCH3), 15.5 (C-CH3). HRMS calcd. for C103H100O26Na (M+Na)+:
1775.6401, found: 1775.6405.
15

4.13.
p-Methoxyphenyl
2,3-di-O-benzoyl-β-D-glucopyranosyl-(1→4)-2,3-di-O-benzyl-β-L-
1
2
fucopyranosyl-(1→4)-2,3-di-O-benzoyl-6-O-benzyl-β-D-glucopyranosyl-(1→3)-4-O-acetyl-2,6-di-O-
benzyl-β-D-galactopyranoside (17): A mixture of compound 16 (400 mg, 0.23 mmol) in 80% aqueous
AcOH (10 mL) was stirred at 80 °C for 2 hours when TLC (n-hexane-EtOAc; 1:1) showed complete
conversion of the starting material to a slower moving spot. The solvents were evaporated in vacuo and
coevaporated with toluene to remove traces of AcOH. The residue obtained was purified by flash
chromatography using n-hexane-EtOAc (1.5:1) as eluent to give pure tetrasaccharide diol 17 (345 mg,
91%) as colourless foam. [α]D25 +138 (c 0.9, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 7.94-7.11 (m, 45H,
ArH), 6.93, 6.74 (2d, 4H, J 9.0 Hz, C6H4OCH3), 5.60 (t, 1H, J2′,3′, J3′,4′ 9.5 Hz, H-3′), 5.50 (dd, 1H, J3,4
4.0 Hz, J4,5 <1.0 Hz, H-4), 5.38 (dd, 1H, J1′′′,2′′′ 7.0 Hz, J2′′′,3′′′ 8.5 Hz, H-2′′′), 5.3 (dd, 1H, J1′,2′ 8.0 Hz,
J2′,3′ 9.5 Hz, H-2′), 5.22 (t, 1H, J2′′′,3′′′, J3′′′,4′′′ 8.5 Hz, H-3′′′), 5.03 (d, 1H, J1′,2′ 7.5 Hz, H-1′), 4.92 (d, 1H,
J1″,2″ 3.5 Hz, H-1″), 4.79 (d, 1H, J1,2 8.0 Hz, H-1), 4.77 (d, 1H, J1′′′,2′′′ 7 Hz, H-1′′′), 4.76 (d, 1H, J 11.5
Hz, CH2Ph), 4.74 (d, 1H, J 11.5 Hz, CH2Ph), 4.69 (d, 1H, J 11.5 Hz, CH2Ph), 4.68 (d, 1H, J 11.0 Hz,
CH2Ph), 4.52 (d, 1H, J 12.0 Hz, CH2Ph), 4.5 (d, 1H, J 11.5 Hz, CH2Ph), 4.49 (d, 1H, J 11.0 Hz,
CH2Ph), 4.47 (d, 1H, J 11.5 Hz, CH2Ph), 4.42 (d, 1H, J 12.5 Hz, CH2Ph), 4.33 (d, 1H, J 11.0 Hz,
CH2Ph), 4.07 (t, 1H, J3′,4′, J4′,5′ 9.5 Hz, H-4′), 3.97 (t, 1H, J3′′′,4′′′, J4′′′,5′′′ 9.5 Hz, H-4′′′), 3.92 (dd, 1H, J2,3
9.5 Hz, J3,4 3.5 Hz H-3), 3.85 (dd, 1H, J1″,2″ 3.5 Hz, J2″,3″ 10.5 Hz, H-2″), 3.79 (m, 4H, H-3″, H-4″, H-
6′), 3.74 (m, 4H, H-6a′′′, C6H4OCH3), 3.7 (m, 2H, H-2, H-6b′′′), 3.58 (m, 5H, H-5, H-5′, H-5″, H-6),
3
4
5
6
7
8
9
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
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
13
3.37 (m, 1H, H-5′′′), 2.15 (s, 3H, COCH3), 0.47 (d, 3H, J5″,6″ 6.5 Hz, C-CH3). C NMR (CDCl3, 125
MHz) δ: 170.3 (COCH3), 167.6, 166, 165.1, 165.0 (4×COPh), 155.2, 151.3, 138.4 (2), 138.3 (2), 138.2
(2), 138 (2), 137.9 (2), 133.5 (2), 133.2 (2), 133 (2), 130 (2), 129.6 (2), 129.5 (2), 129.4, 129.34, 129.31,
128.8, 128.5 (2), 128.45 (2), 128.43 (3), 128.34 (3), 128.32 (2), 128.2, 128.1 (2), 127.9, 127.8 (3),
127.76 (3), 127.74 (2), 127.64, 127.61 (2), 127.5, 118.2 (2), 114.4 (2) (ArC), 102.5 (C-1), 101.2 (C-1′′′),
100.6 (C-1′), 99 (C-1″), 79.7, 79.1, 77.7, 77.6, 76.6, 75.7, 75.6, 75.5, 75, 74.3, 74.2, 73.6 (2), 73.2, 73.1,
16

72.8, 72.7, 72.1, 69.9, 69.6, 68.9, 68, 66.8, 62, 55.6 (C6H4OCH3), 20.8 (COCH3), 15.6 (C-CH3). HRMS
1
2
3
calcd. for C96H96O26Na (M+Na)+: 1687.6088, found: 1687.6085.
4
5
6
7
8
9
4.14. p-Methoxyphenyl β-D-glucopyranosyluronic acid-(1→4)-β-L-fucopyranosyl-(1→4)-O-β-D-
glucopyranosyl-(1→3)-β-D-galactopyranoside (1): To the CH2Cl2 solution (1.5 mL) of the diol 17
(310 mg, 0.19 mmol), TEMPO (1.0 mg) followed by a solution of saturated aqueous NaHCO3 (1.2 mL),
containing potassium bromide (6.0 mg) and Bu4NBr (8.0 mg) were added. The reaction mixture was
cooled to 0 °C. To it, a solution of sodium hypochlorite (1.3 M, 1.5 mL), sodium bicarbonate (0.6 mL)
and brine (1.2 mL) were added dropwise over 45 min. The reaction was stirred at room temperature for
overnight. The solution was acidified by adding 4M HCl. The reaction mixture was washed with EtOAc
(3 × 25 mL). The organic layer was collected, dried (Na2SO4), evaporated and dried in vacuum. The
dried compound was dissolved in MeOH (100 mL) and it was passed through an H-cube flow
hydrogenation assembly using a 10% Pd-C cartridge with a flow rate of 1 mL min-1. Complete removal
of the five benzyl groups was achieved after four cycles as confirmed by the mass spectra. MeOH was
evaporated and the compound was dried well. The dried residue was redissolved in MeOH (10 mL) and
NaOMe (1 mL, 0.5M in MeOH) was added to it. The reaction mixture was stirred at room temperature
for 10 hours. DOWEX 50W H+ resin was added to neutralize excess NaOMe. The solution was filtered,
evaporated in vacuo and the residue was triturated with CH2Cl2 to remove the organic soluble side
products/salts generated during TEMPO oxidation. The residue obtained thereafter was further purified
by HPLC using C18 column and CH3CN-H2O mixture as eluent to afford the desired tetrasaccharide 1
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
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
25
1
(90 mg, 65%) as white amorphous mass. [α]D +61 (c 1.0, MeOH). H NMR (500 MHz, CDCl3) δ:
7.05, 6.82 (2d, 4H, J 9.0 Hz, C6H4OCH3), 4.99 (d, 1H, J1″,2″ 3.0 Hz, H-1″), 4.79 (d, 1H, J1′,2′ 7.5 Hz, H-
13
1′), 4.57 (d, 1H, J1,2 7.5 Hz, H-1), 4.35 (d, 1H, J1′′′,2′′′ 7.0 Hz, H-1′′′). C NMR (CDCl3, 125 MHz) δ:
170.0 (COOH), 156.7, 153.2, 119.4 (2), 115.5 (2) (ArC), 105.6 (C-1), 105.3 (C-1′′′), 103.6 (C-1′), 101.4
(C-1′′), 84.7, 83.4, 79.6, 79.0, 78.0, 77.3, 76.7, 76.5 (2), 76.2, 75.6, 74.8, 74.4, 71.3, 70.9, 69.8, 69.5,
17

68.6, 62.4, 56.1 (C6H4OCH3), 16.4 (C-CH3). HRMS calcd. for C31H46O22Na (M+Na)+: 793.2378, found:
1
2
3
793.2380.
4
5
6
7
8
9
Acknowledgement
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DB is thankful to UGC, New Delhi for Junior Research Fellowship. The work is supported by the
research grant 01(2370)/10/EMR-II from CSIR, New Delhi to BM.
Reference:
i. Woolcock, J. B.; Mutimer, M. J. Gen. Microbiol. 1978, 109, 127-130.
ii. Warhurst, A. M.; Fewson, C. A. Crit. Rev Biotechnol. 1994, 14, 29-73.
iii. Iwabuchi, N.; Sunairi, M.; Anzai, H.; Nakajima, M.; Harayama, S. Appl. Environ. Microbiol., 2006,
66, 5073-5077.
iv. Perry, M. B.; MacLean, L. L.; Patrauchanb, M. A.; Vinogradova, E. Carbohydr. Res. 2007, 342,
2223-2229.
v. Lv, X.; Cheng, S.; Wei, G.; Du, Y. Carbohydr. Res. 2010, 345, 2272-2276.
vi. Thollas, B.; Biosset, C. Synlett 2007, 11, 1736–1738.
vii. Gent, P. A.; Gigg, R. J. Chem. Soc., Perkin Trans. 1 1974, 1446–1455.
viii. Mukhopadhyay, B.; Field, R. A. Carbohydr. Res. 2003, 338, 2149-2152.
ix. Lin, C. –C.; Hsu, T. –S.; Lu, K. –C.; Huang, I. –T. J. Chin. Chem. Soc. 2000, 47, 921-928.
x. Mukherjee, S.; Mukhopadhyay, B. Synlett 2010, 19, 2853-2856.
xi. Debenham, S. D.; Toone, E. J. Tetrahedron: Asymm. 2000, 11, 385–387.
xii. Verma, P. R.; Mukhopadhyay, B. RSC Adv. 2013, 3, 201–207.
xiii. Rajput, V. K.; Mukhopadhyay, B. J. Org. Chem. 2008, 73, 6924-6927.
xiv. Bertolini, M.; Glaudemans, C. P. J. Carbohydr. Res. 1970, 15, 263–270.
18

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2
3
xv. Takeo, K.; Maki, K.; Wada, Y.; Kitamura, S. Carbohydr. Res. 1993, 245, 81–96.
xvi. Davis, J. N.; Flitsch, L. S. Tetrahedron Lett. 1993, 34, 1181-1184.
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5
6
7
8
9
xvii.Huang, L.; Teumelsan, N.; Huang, X. Chem–Eur J. 2006, 12, 5246–5252.
xviii. Zemplén, G. Ber. Deutch. Chem. Ges. 1926, 59, 1254–1266.
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58
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60
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xix. Perrin, D. D.; Amarego, W. L.; Perrin, D. R. Purification of Laboratory Chemicals; Pergamon:
London, 1996.
19

Chemical synthesis of a tetrasaccharide related
to the exocellular polysaccharide from
Rhodococcus sp. RHA1
Darshita Budhadev, Balaram Mukhopadhyay

Highlights
1.
2.
3.
Synthesis of the repeating unit of the EPS from Rhodococcus sp. RHA1
Activation of thioglycoside using NIS and La(OTf)3
Late stage TEMPO mediated oxidation