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Synthesis of the tetrasaccharide related to the repeating unit of the O-antigen from Azospirillum brasilense Jm125A2 in the form of its 2-aminoethyl glycoside
Accepted Manuscript Synthesis of the tetrasaccharide related to the repeating unit of the O-antigen from Azospirillum brasilense Jm125A2 in the form of its 2-aminoethyl glycoside Vikramjit Sarkar, Balaram Mukhopadhyay PII:
S0008-6215(18)30509-3
DOI:
10.1016/j.carres.2018.09.006
Reference:
CAR 7609
To appear in:
Carbohydrate Research
Received Date: 3 September 2018 Revised Date:
26 September 2018
Accepted Date: 26 September 2018
Please cite this article as: V. Sarkar, B. Mukhopadhyay, Synthesis of the tetrasaccharide related to the repeating unit of the O-antigen from Azospirillum brasilense Jm125A2 in the form of its 2-aminoethyl glycoside, Carbohydrate Research (2018), doi: https://doi.org/10.1016/j.carres.2018.09.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Synthesis of the tetrasaccharide related to the repeating unit of the O-antigen from Azospirillum brasilense Jm125A2 in the form of its 2-aminoethyl glycoside
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Vikramjit Sarkar, Balaram Mukhopadhyay
ACCEPTED MANUSCRIPT
Synthesis of the tetrasaccharide related to the repeating unit of the Oantigen from Azospirillum brasilense Jm125A2 in the form of its 2aminoethyl glycoside
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Vikramjit Sarkar, Balaram Mukhopadhyay
Sweet Lab, Department of Chemical Sciences, Indian Institute of Science Education and
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Research (IISER) Kolkata, Mohanpur, Nadia 741246
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Abstract: Total chemical synthesis of the linear tetrasaccharide repeating unit β-D-Glc-(1→2)-α-LRha-(1→3)-α-L-Rha-(1→2)-α-L-Rha-CH2CH2NH2 of the O-antigen from Azospirillum brasilense Jm125A2 is accomplished through rational protecting group manipulations of commercially available monosaccharides and stereoselective glycosylations. The target tetrasaccharide in the form of its 2aminoethyl glycoside is obtained in ~24% yield over 10 steps following a linear strategy. The structure is particularly suitable for further glycoconjugate formation through the terminal free amine without hampering the reducing end stereochemistry.
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Keywords: O-antigen; Oligosaccharide; Chloroacetate; 2-aminoethyl glycoside; H2SO4-silica 1. Introduction
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Bacterial O-polysaccharides (OPSs), constituents of the lipopolysaccharides are
located in the outer membrane of Gram-negative bacteria. They are responsible for determining and regulating various biological functions of the organism. The Opolysaccharide chains are composed of oligosaccharide repeating units containing Lrhamnose as a major constituent. Owing to their exposure in the outer surface, these Opolysaccharides are responsible for binding with the receptors of the host cells and play a pivotal role in the bacterial adhesion.1 Presence of different sugar residues in their structure provides these O-polysaccharides varied character in the host specific interactions. Various microorganisms use the roots of the crop plants as host for their living and in turn help the plants to grow better due to their adaptation to the soil and climate of the environment of the 1
ACCEPTED MANUSCRIPT area they inhabit. Increasing interest in aboriginal microorganisms as promising biofertilizers has prompted researchers to elucidate the specific pathway of recognition and attachment of various microorganisms to the host plants.
In the family of Rhodospirillaceae, Azospirillum is a genus initially comprised of two
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species namely Azospirillum brasilense and Azospirillum lipoferum.2 However, currently the genus Azospirillum includes 16 species, most of which have been found in the rhizosphere of important crops and forage cereals in various climatic zones.3 The lipopolysaccharides (LPSs) and the capsular polysaccharides (CPSs) of these organisms are responsible for the
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initial association with the host plants.4 Depending on the serological specificity and the structure of the O-polysaccharides (OPSs), strains of A. brasilense and A. lipoferum have been classified into three serogroups.5 The OPSs of serogroups I and III are linear
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homopolymers of D-rhamnose or branched polysaccharides having an L-rhamnan backbone and side chain glucose residues, respectively. Whereas, in the majority of the strains of serogroup II, two or more structurally different OPSs coexist, and the same OPS structures are shared by serologically heterogeneous strains.6
Recently, Sigida et al.7 reported the structure of the OPS related to the A. brasilense
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Jm125A2 isolated from the rhizosphere of pearl millet (Pennisetum americanum) in the USA.8 The same OPS structure was also previously reported for A. brasilense S17.9 Although discreet fragments have previously been synthesised,10 in this paper for the first time, we have demonstrated a concise strategy for the synthesis of the tetrasaccharide repeating unit of
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the OPS related to A. brasilense Jm125A2 in the form of its 2-aminoethyl glycoside.
Figure 1: Tetrasaccharide target structure related to the repeating unit of the O-antigen from Azospirillum brasilense Jm125A2 2
ACCEPTED MANUSCRIPT Retrosynthetic analysis indicated that a sequential glycosylation strategy with differentially protected monosaccharides will be the most suitable approach to furnish the target tetrasaccharide 1. The choice of aglycone at the reducing end is always very important to form suitable glyconjugates, for that we have chosen masked aminoethyl glycoside as the linker. Although azidoethyl moiety could have served the purpose, instead 2-
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(benzyloxycarbonyl)-aminoethyl11 moiety was used as it would be a more general aglycone for any glycoside. Additionally, conversion of 2-(benzyloxycarbonyl)-aminoethyl moiety to free amine does not require any harsh reaction condition.
The total synthesis of the tetrasaccharide began with furnishing the suitability
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protected rhamnosyl acceptor. Thus, the known rhamnose derivative 2 was subjected to Zemplén de-O-acetylation12 and the product obtained was directly treated with 2,2-dimethoxy
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propane in the presence of a catalytic amount of CSA13 to afford the derivative 3 in 91% yield with one free hydroxyl group. The free 4-OH was protected as benzoyl ester using BzCl in pyridine to generate the provisionally protected compound 4 in 94% yield. Further, the isopropylidene ketal was hydrolyzed by 80% AcOH at 80 °C14 to generate the rhamnosyl diol 5 in 85%. Finally, the equatorial 3-OH was selectively protected as benzyl ether by using stannylene chemistry15 to furnish the desired rhamnosyl acceptor 6 in 82% yield.
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The glycosylation between the acceptor 6 and the known donor 716 was accomplished through activation of the thioglycoside using combination of NIS and H2SO4-silica17 to form the desired 1,2-trans disaccharide 8 in 89% yield. The structure of the synthesized
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disaccharide was confirmed by the NMR spectroscopy which showed two anomeric protons at δ 4.85 (d, J1,2<1.0 Hz, H-1) and δ 5.14 (d, J1,2=1.5 Hz, H-1′). Moreover the anomeric
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carbon peaks at δ 98.9 (C-1) and δ 98.5 (C-1′) along with the 2D spectra unambiguously established the structure.
Removal of the orthogonal chloroacetate protection at the terminal rhamnosyl moiety
using thiourea in the presence of collidine18 furnished the disaccharide acceptor 9 in 92% yield. Glycosylation of the acceptor 9 with the known rhamnose donor 1019 through similar thioglycoside activation using NIS and H2SO4-silica afforded the desired trisaccharide 11 in 90% yield which was confirmed by NMR spectroscopy showing three anomeric protons at δ 5.22 (d, J1ʺ,2ʺ 1.0 Hz, H-1ʺ), 5.12 (d, J1ʺ,2ʺ 1.5 Hz, H-1′) and 4.82 (d, 1H, J1,2<1.0 Hz, H-1). Respective signals for the anomeric carbons appeared at δ 99.1 (C-1′), 99.0 (C-1) and 98.7 (C-1ʺ). Subsequent removal of the chloroacetate group from the C-2 position of the terminal 3
ACCEPTED MANUSCRIPT rhamnose moiety using thiourea in the presence of collidine produced the desired trisaccharide acceptor 12 in 87% yield. Attachment of the final glucosyl moiety in 1,2-trans fashion was found to be far more complicated than expected. A number of glucosyl donors were tried but failed to produce the desired tetrasaccharide (Table 1). The glycosylation reactions either resulted in the formation
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of the donor hemiacetal or produced complex inseparable mixture.
Table 1: Summary of the results obtained from the glycosylation of the trisaccharide acceptor
Donor
Reagents
Outcome
Complex mixture
H2SO4-silica
Dry CH2Cl2, -10 °C
Hemiacetal of the Donor
NIS and H2SO4-silica
Dry CH2Cl2, -10 °C
Complex mixture
H2SO4-silica
Dry CH2Cl2, -10 °C
Hemiacetal of the Donor
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Dry CH2Cl2, -5 °C
II21
IV23
Reaction condition
NIS and H2SO4-silica I20
III22
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with different glucosyl donors
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NIS and H2SO4-silica
Dry CH2Cl2, -10 °C
V24
4
Desired tetrasaccharide
ACCEPTED MANUSCRIPT Scheme 1: Synthesis of the target tetrasaccharide 1 O O
AcO AcO
O
i. NaOMe, MeOH
NHCBz ii. 2,2 DMP, CSA,
O
RO
91%
OAc
O
80% AcOH 80 oC
NHCBz
85%
O
NHCBz
O
BzO RO
O
OH
2 94%
Bu2SnO, CsF, BnBr
3R=H 4 R = Bz
82%
STol O
O
90%
OBz
Thiourea, collidine, 55 C
92%
OBz
O
BzO
BnO
8 R = CA 9R=H
o
O
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RO
O
O
BnO
NIS, H2SO4-silica, 0 oC
O
BnO
BnO
BnO OCA 10
NHCBz
O
BzO
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BnO
O
NHCBz BzO
O
BzO
NIS, H2SO4-silica, 0 oC
89%
O
STol
O BnO CAO OBz 7
OR
11 R = CA 12 R = H
Thiourea, collidine, 55 oC
87%
O
Ph
O O BnO
O
BnO
STol
AcO V
BnO
OBz
O
BzO O O BnO
BnO O AcO
HO
O
13
O
O
HO O
75%
OH
O
HO HO HO HO
NHCBz
HO O OH
O 1
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Ph
iii. NaOMe, MeOH
O
HO
i. 80% AcOH, 80 oC ii. H2, Pd-C
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O
82%
O
O
H2SO4-silica, 10 oC
O
NHCBz
O
BzO
5R=H 6 R = Bn
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BzCl, Py
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Finally, the donor V,25 in a NIS, H2SO4-silica promoted glycosylation furnished the
desired protected tetrasaccharide 13 in 82% yield. The proton NMR spectroscopy showed four distinct peaks at δ 4.81 (J1,2<1.0 Hz, H-1), δ 5.12 (J1′,2′<1.0 Hz, H-1′), δ 5.24 (H-1ʺ), and δ 4.70 (H-1‴) which corresponds to the four anomeric protons. The relevant anomeric carbon signals which appeared at δ 99.0 (C-1), δ 98.9 (C-1′), δ 100.4 (C-1ʺ) and δ 101.9 (C-1‴) along with the COSY and HSQC spectra undoubtedly confirmed the structure of the tetrasaccharide with proper configuration of the newly formed glycosidic linkage. Once the fully protected tetrasaccharide is in hand, global de-protection of the tetrasaccharide 15 was initiated with the hydrolysis of the benzylidene acetal using 80% aq. AcOH at 80 °C. Further, Zemplén de-O-acetylation using NaOMe in MeOH followed by hydrogenolysis using Pd-C 5
ACCEPTED MANUSCRIPT cartridge in a ThalesNano continuous flow hydrogenation assembly removed the benzyl groups along with the removal of CBz group to furnish the target tetrasaccharide 1 75% yield over three steps (Scheme 1).
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2. Experimental section 2.1. General: All reagents and solvents were dried prior to use according to standardized methods.26 The commercially purchased reagents were used without any further purification unless mentioned otherwise. Dichloromethane was dried and distilled over P2O5 to make it
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anhydrous and moisture-free. All reactions were monitored by Thin Layer Chromatography (TLC) on Silica-Gel 60-F254 with detection by fluorescence followed by charring after
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immersion in 10% ethanolic solution of H2SO4. Flash chromatography was performed with Silica Gel 230-400 mesh. Optical rotations were measured on sodium-line at ambient temperature. 1H and
13
C NMR were recorded on Bruker spectrometer at 500 MHz and 125
MHz respectively. The assignments of 1H and
13
C peaks were done with the help of 1H-1H
COSY and 1H-13C HSQC spectra. In case of the tetrasaccharide, 1H NMR values were denoted as H for the reducing end rhamnose unit A, H′ for the rhamnose unit B, Hʺ for the
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rhamnose unit C and H‴ for the terminal glucose unit D (as numbered A-D in Figure 1).
2.2. 2-(benzyloxycarbonyl)-aminoethyl 2,3-O-isopropylidene-α-L-rhamnopyranoside (3):
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To a suspension of compound 2 (2.2g, 4.7 mmol) in dry MeOH (20 mL), NaOMe in MeOH (0.5M, 2 mL) was added and the mixture was stirred at room temperature for 8 hours when
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TLC (n-hexane-EtOAc; 1:1) suggested complete conversion of the starting material to a much slower moving spot. The reaction mixture was neutralised with DOWEX 50W H+ resin, filtered and the solvents were evaporated in vacuo. The crude product thus obtained was dried and dissolved in dry CH3CN (20 mL) followed by addition of 2,2-dimethoxy propane (820 µL, 6.7 mmol). After addition of CSA (50 mg), the mixture was allowed to stir at room temperature for 4 hours when TLC (n-hexane-EtOAc; 3:2) showed complete conversion of the starting material to a faster moving spot. The reaction mixture was neutralised with Et3N and evaporated in vacuo. The crude product thus obtained was purified by flash chromatography using n-hexane-EtOAc (1:1) as eluent to get the pure compound 3 (1.63g, 91%). [α]D25 +78 (c 1.1, CHCl3).1H NMR (500 MHz, CDCl3) δ: 7.35-7.30 (m, 5H, 6
ACCEPTED MANUSCRIPT ArH), 5.09 (s, 2H, CH2Ph), 4.94 (s, 1H, J1,2<1.0 Hz, H-1), 4.10 (d, 1H, J1,2<1.0 Hz, J2,3 5.5 Hz, H-2), 4.05 (dd, 1H, J2,3 5.5 Hz, J3,4 7.0 Hz, H-3), 3.75 (m, 1H, OCH2CH2), 3.59 (m, 1H, H-5), 3.51 (m, 1H, OCH2CH2), 3.42 (m, 1H, CH2CH2NH), 3.52 (m, 2H, H-4, CH2CH2NH), 3.06 (bs, 1H, OH), 1.50, 1.33 (2s, 6H, 2×isopropylidene-CH3), 1.25 (d, 3H, J5,6 6.5 Hz, CCH3). 13C NMR (CDCl3, 125 MHz) δ: 156.3 (COCH2Ph), 128.5 (3), 128.1 (3) (ArC), 109.4
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(isopropylidene-C), 97.2 (C-1), 78.4, 75.7, 74.3, 66.8, 66.6, 65.9, 40.7 (CH2CH2NH), 27.9, 26.1 (2×isopropylidene-CH3), 17.2 (CH3). HRMS calcd for C19H27NO7Na (M+Na)+:
2.3.
2-(benzyloxycarbonyl)-aminoethyl
4-O-benzoyl-2,3-O-isopropylidene-α-L-
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rhamnopyranoside (4):
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404.1685, found: 404.1682.
To a solution of compound 3 (1.6 g, 4.19 mmol) in pyridine (15 mL), benzoyl chloride (810 µL, 7.0 mmol) was added and the mixture was stirred for 2 hours at room temperature. When the TLC (n-hexane-EtOAc; 2:1) showed that whole of the starting material has been converted into a faster moving spot, solvents were evaporated in vacuo and co-evaporated with toluene until the last trace of pyridine was removed. The crude product thus obtained
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was purified by flash chromatography using n-hexane-EtOAc (3:1) as eluent to obtain the pure compound 4 (1.91 g, 94%). [α]D25 +68 (c 1.0, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 8.04-7.29 (m, 10H, ArH), 5.12 (m, 3H, H-4, CH2Ph), 5.05 (d, 1H, J1,2<1.0 Hz, H-1), 4.32 (dd, 1H, J2,3 5.5 Hz, J3,4 7.5 Hz, H-3), 4.19 (d, 1H, J1,2<1.0 Hz, J2,3 5.5 Hz, H-2), 3.85 (m, 1H, H-
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5), 3.79 (m, 1H, OCH2CH2), 3.54 (m, 1H, OCH2CH2), 3.45 (m, 1H, CH2CH2NH), 3.41 (m, 1H, CH2CH2NH),1.61, 1.34 (2s, 6H, 2×isopropylidene-CH3), 1.20 (d, 3H, J5,6 6.0 Hz, C-
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CH3). 13C NMR (CDCl3, 125 MHz) δ: 165.6 (COPh), 156.3 (COCH2Ph), 133.1 (2), 129.6 (3), 128.4 (2), 128.2 (2), 128.1 (2), 128.0 (ArC), 109.7 (isopropylidene-C), 97.1 (C-1), 75.8, 75.6, 74.7, 66.7, 64.3, 40.6 (CH2CH2NH), 27.6, 26.2 (2×isopropylidene-CH3), 16.9 (CH3). HRMS calcd for C26H31NO8Na (M+Na)+: 508.1947, found: 508.1944.
2.4. 2-(benzyloxycarbonyl)-aminoethyl 4-O-benzoyl-α-L-rhamnopyranoside (5): A suspension of compound 4 (1.7 g, 3.5 mmol) in 80% aq. AcOH (30 mL) was allowed to stir for 2 hours at 80 °C when the TLC (n-hexane-EtOAc; 1:1) suggested complete
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ACCEPTED MANUSCRIPT conversion of the starting material into a slower moving spot. Solvents were evaporated in vacuo and co-evaporated with toluene to remove trace of AcOH. Flash chromatography using n-hexane-EtOAc (3:2) as eluent afforded pure compound 5 (1.3g, 85%). [α]D25 +103 (c 1.0, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 8.02-7.26 (m, 10H, ArH), 5.48 (m, 1H, NH), 5.13 (t, 1H, J3,4, J4,5 9.5 Hz, H-4), 5.07 (s, 2H, CH2Ph), 4.84 (s, 1H, J1,2<1.0 Hz, H-1), 4.00 (m, 2H,
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H-2, H-3), 3.89 (m, 1H, H-5), 3.74 (m, 1H, OCH2CH2), 3.50 (m, 1H, OCH2CH2), 3.41 (m, 1H, CH2CH2NH), 3.34 (m, 1H, CH2CH2NH),1.21 (d, 3H, J5,6 6.0 Hz, C-CH3).13C NMR (CDCl3, 125 MHz) δ: 166.8 (COPh), 156.5 (COCH2Ph), 136.3, 133.1, 129.7 (2), 129.5, 128.4 (2), 128.3 (2), 128.0 (2), 127.9 (ArC), 99.6 (C-1), 75.2, 70.7, 69.8, 66.6 (2), 66.2, 40.6
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(CH2CH2NH), 17.3 (CH3). HRMS calcd for C23H27NO8Na (M+Na)+: 468.1634, found:
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468.1631.
2.5. 2-(benzyloxycarbonyl)-aminoethyl 4-O-benzoyl-3-O-benzyl-α-L-rhamnopyranoside (6):
A mixture of compound 5 (1.2 g, 2.7 mmol) and Bu2SnO (870 mg, 3.5 mmol) in dry toluene (25 mL) was refluxed at 110 °C for 2 hours until the reaction mixture became clear. Toluene
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was evaporated in vacuo and the crude product thus obtained was well dried before it was dissolved in dry DMF. CsF (450 mg, 3.0 mmol) was added to it and the reaction mixture was kept under N2 atmosphere for 30 min. Finally BnBr (420 µL, 3.5 mmol) was added to the reaction mixture and it was allowed to stir at room temperature for 12 hours till TLC (n-
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hexane-EtOAc; 3:2) showed that all of the starting material was converted to a faster moving spot. Solvents were evaporated in vacuo and the crude product was dissolved in EtOAc (20
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mL). It was washed with water (2×30 mL). The organic layer was separately collected over Na2SO4, filtered and evaporated in vaccuo. The crude product thus obtained was subjected to flash chromatography using n-hexane-EtOAc (2:1) as eluent to furnish the pure acceptor 6 (1.2g, 82%). [α]D25 +84 (c 0.9, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 8.02-7.13 (m, 15H, ArH), 5.34 (t, 1H, J3,4, J4,5 9.5 Hz, H-4), 5.13 (s, 2H, CH2Ph), 4.89 (s, 1H, J1,2<1.0 Hz, H-1), 4.64, 4.51 (ABq, 2H, JA,B12.0 Hz, CH2Ph), 4.07 (bs, 1H, H-3), 3.87 (m, 2H, H-2, H-5), 3.79 (m, 1H, OCH2CH2), 3.59 (m, 1H, OCH2CH2), 3.45 (m, 1H, CH2CH2NH), 3.42 (m, 1H, CH2CH2NH), 2.82 (bd, 1H, J 13.0 Hz, OH), 1.23 (d, 3H, J5,6 6.5 Hz, C-CH3).13C NMR (CDCl3, 125 MHz) δ: 165.7 (COPh), 156.3 (COCH2Ph), 137.3, 136.4, 133.1 (2), 129.8, 129.7 (2), 128.5 (2), 128.4 (2), 128.3 (3), 128.2, 127.8 (2), 127.7 (2) (ArC), 99.3 (C-1), 76.6, 72.8, 8
ACCEPTED MANUSCRIPT 71.7, 68.3, 67.0, 66.8, 66.5, 40.8 (CH2CH2NH), 17.4 (CH3). HRMS calcd for C30H33NO8Na (M+Na)+: 558.2104, found: 558.2101.
2.6. 2-(benzyloxycarbonyl)-aminoethyl 3-O-chloroacetyl-2-O-benzoyl-4-O-benzyl-α-L-
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rhamnopyranosyl-(1→ →2)-4-O-benzoyl-3-O-benzyl-α-L-rhamnopyranoside (8): A mixture of the acceptor 6 (1.1g, 2.1 mmol), donor 7 (1.4g, 2.6 mmol) and 4Ǻ MS (1.5 g) in dry CH2Cl2(15 mL) was kept stirring under N2 atmosphere for 45 min. NIS (780 mg, 3.5 mmol) was added to the reaction mixture and it was cooled to 0 °C under N2 atmosphere.
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After 30 min, H2SO4-silica (50 mg) was added to initiate the reaction. The mixture was allowed to stir at 0 °C for 15 min when the TLC (n-hexane-EtOAc; 2:1) showed complete
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consumption of the acceptor. The reaction mixture was then filtered through a pad of Celite. The filtrate was washed successively with aq. Na2S2O3 (2 × 30 mL), aq. NaHCO3 (2 × 30 mL) and brine (30 mL). Organic layer was separated, dried over Na2SO4 and evaporated in vacuo. The syrupy crude product thus obtained was purified by flash chromatography using n-hexane-EtOAc (5:2) as eluent to afford pure disaccharide 8 (1.8g, 89%) as white foam. [α]D25 +78 (c 1.0, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 8.05-7.06 (m, 25H, ArH), 5.67 (dd,
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1H, J1′,2′ 1.5 Hz, J2′,3′ 3.5 Hz, H-2′), 5.56 (dd, 1H, J2′,3′ 3.5 Hz, J3′,4′ 10.0 Hz, H-3′), 5.35 (t, 1H, J3,4, J4,5 9.5 Hz, H-4), 5.14 (m, 3H, H-1′, CH2Ph), 4.85 (d, 1H, J1,2<1.0 Hz, H-1), 4.74-4.55 (ABq, 4H, 2×CH2Ph), 4.05 (bs, 1H, H-3), 4.02 (m, 1H, H-5′), 3.97, 3.89 (ABq, 2H, JA,B15.0 Hz, CH2Cl), 3.93 (m, 1H, H-2), 3.86 (m,1H, H-5), 3.78 (m, 1H, OCH2CH2), 3.66 (t, 1H, J3′,4′,
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J4′,5′ 9.5 Hz, H-4′), 3.56 (m, 1H, OCH2CH2), 3.48 (m, 1H, CH2CH2NH), 3.43 (m, 1H, CH2CH2NH),1.41 (d, 3H, J5′,6′ 6.0 Hz, C-CH3), 1.27 (d, 3H, J5,6 6.0 Hz, C-CH3).
13
C NMR
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(CDCl3, 125 MHz) δ: 166.0 (COCH2Cl), 165.3, 165.2 (2×COPh), 156.3 (COCH2Ph), 137.7 (2), 133.3, 133.0, 129.9, 129.8 (2), 129.7 (2), 129.6, 128.5 (2), 128.4 (4), 128.3 (2), 128.2 (3), 127.9 (5), 127.6 (2), 127.5 (2) (ArC), 98.9 (C-1), 98.5 (C-1′), 78.5, 76.1, 75.2, 73.9, 73.6, 73.4, 71.9, 70.3, 68.3, 67.2, 66.9, 66.8, 40.8 (CH2CH2NH), 40.6 (COCH2Cl), 18.0, 17.6 (2×CH3). HRMS calcd for C52H54ClNO14Na (M+Na)+: 974.3131, found: 974.3128.
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ACCEPTED MANUSCRIPT 2.7. 2-(benzyloxycarbonyl)-aminoethyl 2-O-benzoyl-4-O-benzyl-α-L-rhamnopyranosyl(1→ →2)-4-O-benzoyl-3-O-benzyl-α-L-rhamnopyranoside (9): A mixture of disaccharide 8 (1.6g, 1.7 mmol), thiourea (1.2g, 16.8 mmol) and 2,4,6-collidine (2.2 mL, 16.8 mmol) in CH2Cl2-MeOH (2:3, 20 mL) was refluxed for 10 hours when TLC (nhexane-EtOAc; 3:2) confirmed the complete conversion of the starting material to a slower
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moving spot. The solvents were evaporated in vacuo; the resulting solid residue was dissolved in CH2Cl2 (20 mL) and washed with 1(N) HCl (2×30 mL) and H2O (30 mL). The organic layer was separated, filtered, dried over Na2SO4 and evaporated in vacuo. The crude residue was then subjected to flash chromatography using n-hexane-EtOAc (2:1) as eluent to
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give the pure disaccharide acceptor 9 (1.4g, 92%).[α]D25 +96 (c 0.8, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 8.08-7.09 (m, 25H, ArH), 5.67 (dd, 1H, J1′,2′ 1.5 Hz, J2′,3′ 3.5 Hz, H-2′), 5.35
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(t, 1H, J3,4, J4,5 9.5 Hz, H-4), 5.15 (m, 2H, CH2Ph), 5.05 (d, 1H, J1′,2′<1.0 Hz, H-1′), 4.92-4.54 (ABq, 4H, 2×CH2Ph), 4.85 (d, 1H, J1,2<1.0 Hz, H-1), 5.56 (m, 1H, H-3′), 3.95 (m, 3H, H-2, H-3, H-5′), 3.84 (m, 1H, H-5), 3.78 (m, 1H, OCH2CH2), 3.53 (m, 1H, OCH2CH2), 3.49 (t, 1H, J3′,4′, J4′,5′ 9.5 Hz, H-4′), 3.47 (m, 1H, CH2CH2NH), 3.42 (m, 1H, CH2CH2NH),1.38 (d, 3H, J5′,6′ 6.5 Hz, C-CH3), 1.23 (d, 3H, J5,6 6.0 Hz, C-CH3).13C NMR (CDCl3, 125 MHz) δ: 166.1, 165.5 (2×COPh), 156.3 (COCH2Ph), 138.2, 137.7, 133.2, 133.0, 129.9, 129.8 (5),
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128.5 (2), 128.4 (2), 128.3 (5), 128.2 (4), 128.1 (3), 127.8, 127.7 (2), 127.6 (ArC), 99.3 (C1′), 99.0 (C-1), 81.3, 75.8, 75.3, 75.2, 73.3 (2), 71.9, 70.4, 68.1, 67.1, 67.0, 66.8, 40.8 (CH2CH2NH), 18.2, 17.6 (2×CH3). HRMS calcd for C50H53NO13Na (M+Na)+: 898.3415,
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found: 898.3412.
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2.8. 2-(benzyloxycarbonyl)-aminoethyl 2-O-chloroacetyl-4-O-benzoyl-3-O-benzyl-α-Lrhamnopyranosyl-(1→ →3)-2-O-benzoyl-4-O-benzyl-α-L-rhamnopyranosyl-(1→ →2)-4-Obenzoyl-3-O-benzyl-α-L-rhamnopyranoside (11): A mixture of the disaccharide acceptor 9 (1.2g, 1.4 mmol), known donor 10 (960 mg, 1.8 mmol) and 4Ǻ MS (1.5g) in dry CH2Cl2 (15 mL) was kept stirring under N2 atmosphere for 45 min. NIS (520 mg, 2.3 mmol) was added and the mixture was cooled to 0 oC keeping under continuous N2 flow. H2SO4-silica (30 mg) was added to the mixture and allowed to stir at the same temperature for 30 min when TLC (n-hexane-EtOAc; 4:1) showed complete consumption of the acceptor. The mixture was then filtered through a Celite padandthe
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ACCEPTED MANUSCRIPT filtrate was successively washed with aq. Na2S2O3 (2 × 30 mL), aq. NaHCO3 (2 × 30 mL) and brine (30 mL). The organic layer was collected, dried over Na2SO4 and evaporated in vacuo. Crude product thus obtained was purified by flash chromatography using n-hexaneEtOAc (3:1) as the eluent to give the pure trisaccharide 11 (1.6 g, 90%) as white foam. [α]D25 +118 (c 0.9, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 8.07-6.96 (m, 35H, ArH), 5.55 (dd, 1H,
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J1′,2′ 1.5 Hz, J2′,3′ 3.0 Hz, H-2′), 5.44 (dd, 1H, J1ʺ,2ʺ 1.5 Hz, J2ʺ,3ʺ 3.0 Hz, H-2ʺ), 5.30 (t, 1H, J3ʺ,4ʺ, J4ʺ,5ʺ 9.5 Hz, H-4ʺ), 5.22 (d, 1H, J1ʺ,2ʺ 1.0 Hz, H-1ʺ), 5.19 (t, 1H, J3,4, J4,5 9.5 Hz, H-4), 5.12 (m, 3H, H-1′, CH2Ph), 4.82 (d, 1H, J1,2<1.0 Hz, H-1), 4.80-4.25 (m, ABq, 6H, 3×CH2Ph), 4.40 (dd, 1H, J2′,3′ 3.0 Hz, J3′,4′ 9.5 Hz, H-3′), 4.20, 4.12 (ABq, 2H, JA,B15.5 Hz,
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CH2Cl), 4.09 (m, 1H, H-5), 3.97 (m, 2H, H-2, H-5′), 3.88 (m, 2H, H-3, H-3ʺ), 3.81 (m, 1H, H-5ʺ), 3.77 (m, 1H, OCH2CH2), 3.61 (t, 1H, J3′,4′, J4′,5′ 9.5 Hz, H-4′), 3.53 (m, 1H, OCH2CH2), 3.45 (m, 1H, CH2CH2NH), 3.41 (m, 1H, CH2CH2NH),1.37 (d, 3H, J5′,6′ 6.5 Hz,
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C-CH3), 1.21 (d, 3H, J5ʺ,6ʺ 6.5 Hz, C-CH3), 1.17 (d, 3H, J5,6 6.0 Hz, C-CH3).13C NMR (CDCl3, 125 MHz) δ: 166.7 (COCH2Cl), 165.5, 165.2 (2) (3×COPh), 156.3 (COCH2Ph), 137.8, 137.5, 137.3, 133.2, 133.0 (2), 130.0 (2), 129.9 (2), 129.7 (3), 128.6 (4), 128.5 (4), 128.3 (6), 128.2 (4), 128.1 (4), 128.0, 127.7 (4), 127.5 (2) (ArC), 99.1 (C-1′), 99.0 (C-1), 98.7 (C-1ʺ), 80.8, 75.9, 75.6, 75.5, 74.2, 73.3, 72.7, 72.5, 71.9, 71.5, 70.5, 68.5, 67.6, 67.2, 66.9
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(2), 40.9 (COCH2Cl), 40.8 (CH2CH2NH), 18.1, 17.7, 17.2 (3×CH3). HRMS calcd for C72H74ClNO19Na (M+Na)+: 1314.4441, found: 1314.4438.
Synthesis
of
2-(benzyloxycarbonyl)-aminoethyl
4-O-benzoyl-3-O-benzyl-α-L-
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2.9.
rhamnopyranosyl-(1→ →3)-2-O-benzoyl-4-O-benzyl-α-L-rhamnopyranosyl-(1→ →2)-4-O-
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benzoyl-3-O-benzyl-α-L-rhamnopyranoside (12): A mixture of the trisaccharide 11 (1.5g, 1.2 mmol), thiourea (880 mg, 11.5 mmol) and collidine (1.5 mL, 11.5 mmol) in CH2Cl2-MeOH (2:3, 20 mL) was refluxed for 12 hours when TLC (n-hexane-EtOAc; 3:1) suggested complete conversion of the starting material to a slower moving spot. The mixture was evaporated in vacuo and the solid residue thus obtained was dissolved in CH2Cl2 and washed with 1(N) HCl (2×30 mL) and brine (30 mL). The organic layer was separated, dried over Na2SO4, filtered and evaporated in vacuo to obtain a syrupy residue which was further subjected to flash chromatography using n-hexane-EtOAc (3:2) as eluent to give the pure trisaccharide acceptor 12 (1.3g, 87%) as white foam.[α]D25 +112 (c 0.8, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 8.07-6.96 (m, 35H, ArH), 5.59 (m, 1H, 11
ACCEPTED MANUSCRIPT H-2′), 5.30 (m, 3H, H-1ʺ, H-4, H-4ʺ), 5.13 (m, 3H, H-1′, CH2Ph), 4.84 (d, 1H, J1,2<1.0 Hz, H1), 4.77-4.38 (m, ABq, 6H, 3×CH2Ph), 4.40 (m, 1H, H-3′), 4.06 (m,1H, H-5), 4.00 (m, 1H, H-2), 3.96 (m, 1H, H-5′), 3.90 (m, 2H, H-2ʺ, H-3ʺ), 3.83 (m, 1H, H-5ʺ), 3.77 (dd, 1H, J2,3 3.0 Hz, J3,4 9.5 Hz, H-3), 3.75 (m, 1H, OCH2CH2), 3.60 (t, 1H, J3′,4′, J4′,5′ 9.5 Hz, H-4′), 3.54 (m, 1H, OCH2CH2), 3.46 (m, 1H, CH2CH2NH), 3.41 (m, 1H, CH2CH2NH),1.36 (d, 3H, J5′,6′ 6.0
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Hz, C-CH3), 1.22 (d, 3H, J5ʺ,6ʺ 6.5 Hz, C-CH3), 1.17 (d, 3H, J5,6 6.5 Hz, C-CH3).13C NMR (CDCl3, 125 MHz) δ: 165.6, 165.2 (2) (3×COPh), 156.3 (COCH2Ph), 137.8, 137.7, 137.5, 133.1, 132.9 (2), 130.2, 130.0 (2), 129.8 (2), 129.7 (3), 128.5 (5), 128.5 (2), 128.2 (3), 128.2 (6), 128.1 (3), 128.0, 127.7 (4), 127.6 (2), 127.5 (2) (ArC), 100.5 (C-1ʺ), 99.0 (C-1′), 98.9 (C-
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1), 80.6, 76.6, 75.9, 75.8, 75.4, 75.0, 73.3, 73.0, 72.7, 71.8 (2), 68.8, 68.4, 67.2, 67.1, 66.9, 66.8, 40.8 (CH2CH2NH), 18.1, 17.7, 17.2 (3×CH3). HRMS calcd for C70H73NO18Na
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(M+Na)+: 1238.4725, found: 1238.4721.
2.10. 2-(benzyloxycarbonyl)-aminoethyl 2-O-acetyl-3-O-benzyl-4,6-O-benzylidene -β-D→3)-2-Ogalactopyranosyl-(1→ →2)-4-O-benzoyl-3-O-benzyl-α-L-rhamnopyranosyl-(1→ benzoyl-4-O-benzyl-α-L-rhamnopyranosyl-(1→ →2)-4-O-benzoyl-3-O-benzyl-α-L-
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rhamnopyranoside (13):
A mixture of trisaccharide acceptor 12 (1.4g, 1.2 mmol), donor V (940 mg, 1.7 mmol) and 4Ǻ MS (1.5 g) in dry CH2Cl2 (15 mL) was stirred at 10 °C for 30 min under nitrogen atmosphere. NIS was then added and the reaction mixture was cooled to -10 °C. After 30 min
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H2SO4-silica (30 mg) was added to it and the mixture was allowed to stir at the same temperature for 15 min when TLC (n-hexane-EtOAc; 3:2) showed complete consumption of
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the acceptor. The mixture was then neutralized with Et3N and filtered through a Celite pad. The filtrate was successively washed with aq. Na2S2O3 (2 × 30 mL), aq. NaHCO3 (2×30 mL) and brine (30 mL). The organic layer was collected, dried (Na2SO4) and evaporated in vacuo. Crude product thus obtained was purified by flash chromatography using n-hexane–EtOAc (2:1) as the eluent to afford pure tetrasaccharide 13 (1.5g, 82%) as white foam. [α]D25 +148 (c 0.9, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 8.12-7.00 (m, 45H, ArH), 5.58 (m, 1H, H-2′), 5.48 (s, 1H, CHPh), 5.29 (m, 1H, H-4), 5.24 (s, 1H, H-1ʺ), 5.17 (t, 1H, J3ʺ,4ʺ, J4ʺ,5ʺ 9.5 Hz, H4ʺ), 5.13 (m, 2H, CH2Ph), 5.12 (d, 1H, J1′,2′<1.0 Hz, H-1′), 5.06 (t, 1H, J1‴,2‴, J2‴,3‴ 8.5 Hz, H2‴), 4.93-4.27 (m, 9H, H-4‴, 4×CH2Ph), 4.81 (d, 1H, J1,2<1.0 Hz, H-1), 4.70 (m, 1H, H-1‴), 4.37 (m, 1H, H-3′), 4.03-3.97 (m, 3H, H-2, H-2ʺ, H-5′), 3.89 (m, 3H, H-5, OCH2CH2), 3.84 12
ACCEPTED MANUSCRIPT (m, 2H, H-3, H-5ʺ), 3.70 (m, 3H, H-3ʺ, H-3‴, H-6a), 3.51 (m, 1H, H-6b), 3.55 (t, 1H, J3′,4′, J4′,5′ 9.5 Hz, H-4′), 3.44 (m, 1H, CH2CH2NH), 3.40 (m, 1H, CH2CH2NH), 3.22 (m, 1H, H-5‴), 2.09 (s, 3H, COCH3), 1.32 (d, 3H, J5,6 6.0 Hz, C-CH3), 1.21 (d, 3H, J5ʺ,6ʺ 6.0 Hz, C-CH3), 1.14 (d, 3H, J5′,6′ 6.0 Hz, C-CH3).13C NMR (CDCl3, 125 MHz) δ: 169.8 (COCH3), 165.2 (3×COPh), 156.3 (COCH2Ph), 138.4, 138.0, 137.9, 137.8, 133.2, 132.9, 132.8, 130.3 (2),
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130.0, 129.8 (5), 128.9, 128.5 (8), 128.2 (10), 128.1 (3), 127.8 (3), 127.7 (2), 127.6 (3), 127.5 (4), 126.0 (4) (ArC), 101.9 (C-1‴), 101.1 (CHPh), 100.4 (C-1ʺ), 99.0 (C-1), 98.9 (C-1′),81.6, 81.4, 80.7, 78.3, 75.7, 75.4, 75.1, 74.9, 74.0, 73.5, 73.3, 73.1, 73.0, 72.7, 72.6, 71.8, 68.5, 68.3, 67.9, 67.2, 66.9 (2), 66.1, 40.8 (CH2CH2NH), 20.8 (COCH3), 18.0, 17.7, 17.4 (3×CH3).
2-(benzyloxycarbonyl)-aminoethyl
β-D-galactopyranosyl-(1→ →2)-
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2.11.
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HRMS calcd for C87H91NO25Na (M+Na)+: 1572.5778, found: 1572.5773.
α-L-
rhamnopyranosyl-(1→ →3)- α-L-rhamnopyranosyl-(1→ →2)- α-L-rhamnopyranoside (1): A suspension of compound 16 (1.3g, 0.8 mmol) in AcOH-H2O (8:1, 18 mL) was stirred at 80 °C for 2 hours when the starting material was completely converted to a slower moving spot as suggested by TLC (n-hexane–EtOAc; 2:1). The solvents were evaporated and co-
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evaporated twice with toluene. The residue was dissolved in MeOH (50 mL) and passed through a 10% Pd–C cartridge in a ThalesNano flow hydrogenation assembly under continuous flow of H2 at atmospheric pressure. As evident from mass spectroscopy the hydrogenolysis of the benzyl groups were complete after 2 such cycles. In the next step
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NaOMe in MeOH (0.5 M, 1 mL) was added to the solution and it was stirred at room temperature for 12 hours. The solution was neutralized by DOWEX 50W H+ resin, filtered
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and evaporated in vacuo to afford the final tetrasaccharide 1 (447 mg, 75%) as white amorphous mass. [α]D25 +62 (c 0.6, MeOH).1H NMR (MeOD, 500 MHz) δ: 5.45 (d, 1H, J1,2< 1.0 Hz, H-1′), 5.11 (d, 1H, J1,2< 1.0 Hz, H-1ʺ), 5.06 ppm (d, 1H, J1,2< 1.0 Hz, H-1), 4.90 (m, 1H, H-1ʺʺ)., 3.30 (m, 2H, OCH2CH2NH2).
C NMR (MeOD, 125 MHz) δ: 104.1 (C-1‴),
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103.9 (C-1ʺ), 103.4 (C-1′), 101.4 (C-1), 83.0, 82.8, 81.4, 80.0, 77.4, 77.2, 76.9, 76.4, 75.2, 74.4, 74.2, 71.4, 71.2, 70.8, 70.5, 68.5, 68.3, 42.7 (CH2CH2NH2), 19.3, 19.2, 19.0 (3×CH3). HRMS calcd for C26H47NO18Na (M+Na)+: 684.2691, found: 684.2687.
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ACCEPTED MANUSCRIPT Acknowledgement VS is thankful to Indian Institute of Science Education and Research (IISER) Kolkata for fellowship. The work is supported by Science and Engineering Research Board (SERB), New
Reference
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Delhi, India through the grant SB/S1/OC-48/2013 to BM.
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(a) Boiko, A. S.; Smol’kina, O. N.; Fedonenko, Y. P.; Zdorovenko, E. L.; Kachala, V. V.; Konnova, S. A. et al. Microbiology 2010, 79, 197–205. (b) Fedonenko, Y. P.; Boiko, A. S.; Zdorovenko, E. L.; Konnova, S. A.; Shashkov, A. S.; Ignatov, V. V. et
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al. Biochemistry (Mosc) 2011, 76, 797–802. (c) Sigida, E. N.; Fedonenko, Y. P.; Zdorovenko, E. L.; Burygin, G. L.; Konnova, S. A.; Ignatov, V. V. Microbiology 2014, 83, 326–34. 6.
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Ignatov, V. V.; Knirel, Y. A. Carbohydr. Res. 2015, 416, 37–40.
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London: Pergamon; 1996
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ACCEPTED MANUSCRIPT Highlights 1. Total synthesis of the tetrasaccharide repeating unit of the O-polysaccharide 2. Activation of thioglycoside using NIS in the presence of H2SO4-silica
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3. Use of chloroacetate group as the orthogonal protecting group
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4. Extensive study on the successful glycosylation of the terminal glucose unit
S0008-6215(18)30509-3
DOI:
10.1016/j.carres.2018.09.006
Reference:
CAR 7609
To appear in:
Carbohydrate Research
Received Date: 3 September 2018 Revised Date:
26 September 2018
Accepted Date: 26 September 2018
Please cite this article as: V. Sarkar, B. Mukhopadhyay, Synthesis of the tetrasaccharide related to the repeating unit of the O-antigen from Azospirillum brasilense Jm125A2 in the form of its 2-aminoethyl glycoside, Carbohydrate Research (2018), doi: https://doi.org/10.1016/j.carres.2018.09.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Synthesis of the tetrasaccharide related to the repeating unit of the O-antigen from Azospirillum brasilense Jm125A2 in the form of its 2-aminoethyl glycoside
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Vikramjit Sarkar, Balaram Mukhopadhyay
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Synthesis of the tetrasaccharide related to the repeating unit of the Oantigen from Azospirillum brasilense Jm125A2 in the form of its 2aminoethyl glycoside
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Vikramjit Sarkar, Balaram Mukhopadhyay
Sweet Lab, Department of Chemical Sciences, Indian Institute of Science Education and
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Research (IISER) Kolkata, Mohanpur, Nadia 741246
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Abstract: Total chemical synthesis of the linear tetrasaccharide repeating unit β-D-Glc-(1→2)-α-LRha-(1→3)-α-L-Rha-(1→2)-α-L-Rha-CH2CH2NH2 of the O-antigen from Azospirillum brasilense Jm125A2 is accomplished through rational protecting group manipulations of commercially available monosaccharides and stereoselective glycosylations. The target tetrasaccharide in the form of its 2aminoethyl glycoside is obtained in ~24% yield over 10 steps following a linear strategy. The structure is particularly suitable for further glycoconjugate formation through the terminal free amine without hampering the reducing end stereochemistry.
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Keywords: O-antigen; Oligosaccharide; Chloroacetate; 2-aminoethyl glycoside; H2SO4-silica 1. Introduction
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Bacterial O-polysaccharides (OPSs), constituents of the lipopolysaccharides are
located in the outer membrane of Gram-negative bacteria. They are responsible for determining and regulating various biological functions of the organism. The Opolysaccharide chains are composed of oligosaccharide repeating units containing Lrhamnose as a major constituent. Owing to their exposure in the outer surface, these Opolysaccharides are responsible for binding with the receptors of the host cells and play a pivotal role in the bacterial adhesion.1 Presence of different sugar residues in their structure provides these O-polysaccharides varied character in the host specific interactions. Various microorganisms use the roots of the crop plants as host for their living and in turn help the plants to grow better due to their adaptation to the soil and climate of the environment of the 1
ACCEPTED MANUSCRIPT area they inhabit. Increasing interest in aboriginal microorganisms as promising biofertilizers has prompted researchers to elucidate the specific pathway of recognition and attachment of various microorganisms to the host plants.
In the family of Rhodospirillaceae, Azospirillum is a genus initially comprised of two
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species namely Azospirillum brasilense and Azospirillum lipoferum.2 However, currently the genus Azospirillum includes 16 species, most of which have been found in the rhizosphere of important crops and forage cereals in various climatic zones.3 The lipopolysaccharides (LPSs) and the capsular polysaccharides (CPSs) of these organisms are responsible for the
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initial association with the host plants.4 Depending on the serological specificity and the structure of the O-polysaccharides (OPSs), strains of A. brasilense and A. lipoferum have been classified into three serogroups.5 The OPSs of serogroups I and III are linear
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homopolymers of D-rhamnose or branched polysaccharides having an L-rhamnan backbone and side chain glucose residues, respectively. Whereas, in the majority of the strains of serogroup II, two or more structurally different OPSs coexist, and the same OPS structures are shared by serologically heterogeneous strains.6
Recently, Sigida et al.7 reported the structure of the OPS related to the A. brasilense
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Jm125A2 isolated from the rhizosphere of pearl millet (Pennisetum americanum) in the USA.8 The same OPS structure was also previously reported for A. brasilense S17.9 Although discreet fragments have previously been synthesised,10 in this paper for the first time, we have demonstrated a concise strategy for the synthesis of the tetrasaccharide repeating unit of
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EP
the OPS related to A. brasilense Jm125A2 in the form of its 2-aminoethyl glycoside.
Figure 1: Tetrasaccharide target structure related to the repeating unit of the O-antigen from Azospirillum brasilense Jm125A2 2
ACCEPTED MANUSCRIPT Retrosynthetic analysis indicated that a sequential glycosylation strategy with differentially protected monosaccharides will be the most suitable approach to furnish the target tetrasaccharide 1. The choice of aglycone at the reducing end is always very important to form suitable glyconjugates, for that we have chosen masked aminoethyl glycoside as the linker. Although azidoethyl moiety could have served the purpose, instead 2-
RI PT
(benzyloxycarbonyl)-aminoethyl11 moiety was used as it would be a more general aglycone for any glycoside. Additionally, conversion of 2-(benzyloxycarbonyl)-aminoethyl moiety to free amine does not require any harsh reaction condition.
The total synthesis of the tetrasaccharide began with furnishing the suitability
SC
protected rhamnosyl acceptor. Thus, the known rhamnose derivative 2 was subjected to Zemplén de-O-acetylation12 and the product obtained was directly treated with 2,2-dimethoxy
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propane in the presence of a catalytic amount of CSA13 to afford the derivative 3 in 91% yield with one free hydroxyl group. The free 4-OH was protected as benzoyl ester using BzCl in pyridine to generate the provisionally protected compound 4 in 94% yield. Further, the isopropylidene ketal was hydrolyzed by 80% AcOH at 80 °C14 to generate the rhamnosyl diol 5 in 85%. Finally, the equatorial 3-OH was selectively protected as benzyl ether by using stannylene chemistry15 to furnish the desired rhamnosyl acceptor 6 in 82% yield.
TE D
The glycosylation between the acceptor 6 and the known donor 716 was accomplished through activation of the thioglycoside using combination of NIS and H2SO4-silica17 to form the desired 1,2-trans disaccharide 8 in 89% yield. The structure of the synthesized
EP
disaccharide was confirmed by the NMR spectroscopy which showed two anomeric protons at δ 4.85 (d, J1,2<1.0 Hz, H-1) and δ 5.14 (d, J1,2=1.5 Hz, H-1′). Moreover the anomeric
AC C
carbon peaks at δ 98.9 (C-1) and δ 98.5 (C-1′) along with the 2D spectra unambiguously established the structure.
Removal of the orthogonal chloroacetate protection at the terminal rhamnosyl moiety
using thiourea in the presence of collidine18 furnished the disaccharide acceptor 9 in 92% yield. Glycosylation of the acceptor 9 with the known rhamnose donor 1019 through similar thioglycoside activation using NIS and H2SO4-silica afforded the desired trisaccharide 11 in 90% yield which was confirmed by NMR spectroscopy showing three anomeric protons at δ 5.22 (d, J1ʺ,2ʺ 1.0 Hz, H-1ʺ), 5.12 (d, J1ʺ,2ʺ 1.5 Hz, H-1′) and 4.82 (d, 1H, J1,2<1.0 Hz, H-1). Respective signals for the anomeric carbons appeared at δ 99.1 (C-1′), 99.0 (C-1) and 98.7 (C-1ʺ). Subsequent removal of the chloroacetate group from the C-2 position of the terminal 3
ACCEPTED MANUSCRIPT rhamnose moiety using thiourea in the presence of collidine produced the desired trisaccharide acceptor 12 in 87% yield. Attachment of the final glucosyl moiety in 1,2-trans fashion was found to be far more complicated than expected. A number of glucosyl donors were tried but failed to produce the desired tetrasaccharide (Table 1). The glycosylation reactions either resulted in the formation
RI PT
of the donor hemiacetal or produced complex inseparable mixture.
Table 1: Summary of the results obtained from the glycosylation of the trisaccharide acceptor
Donor
Reagents
Outcome
Complex mixture
H2SO4-silica
Dry CH2Cl2, -10 °C
Hemiacetal of the Donor
NIS and H2SO4-silica
Dry CH2Cl2, -10 °C
Complex mixture
H2SO4-silica
Dry CH2Cl2, -10 °C
Hemiacetal of the Donor
EP
TE D
M AN U
Dry CH2Cl2, -5 °C
II21
IV23
Reaction condition
NIS and H2SO4-silica I20
III22
SC
with different glucosyl donors
AC C
NIS and H2SO4-silica
Dry CH2Cl2, -10 °C
V24
4
Desired tetrasaccharide
ACCEPTED MANUSCRIPT Scheme 1: Synthesis of the target tetrasaccharide 1 O O
AcO AcO
O
i. NaOMe, MeOH
NHCBz ii. 2,2 DMP, CSA,
O
RO
91%
OAc
O
80% AcOH 80 oC
NHCBz
85%
O
NHCBz
O
BzO RO
O
OH
2 94%
Bu2SnO, CsF, BnBr
3R=H 4 R = Bz
82%
STol O
O
90%
OBz
Thiourea, collidine, 55 C
92%
OBz
O
BzO
BnO
8 R = CA 9R=H
o
O
M AN U
RO
O
O
BnO
NIS, H2SO4-silica, 0 oC
O
BnO
BnO
BnO OCA 10
NHCBz
O
BzO
SC
BnO
O
NHCBz BzO
O
BzO
NIS, H2SO4-silica, 0 oC
89%
O
STol
O BnO CAO OBz 7
OR
11 R = CA 12 R = H
Thiourea, collidine, 55 oC
87%
O
Ph
O O BnO
O
BnO
STol
AcO V
BnO
OBz
O
BzO O O BnO
BnO O AcO
HO
O
13
O
O
HO O
75%
OH
O
HO HO HO HO
NHCBz
HO O OH
O 1
EP
Ph
iii. NaOMe, MeOH
O
HO
i. 80% AcOH, 80 oC ii. H2, Pd-C
TE D
O
82%
O
O
H2SO4-silica, 10 oC
O
NHCBz
O
BzO
5R=H 6 R = Bn
RI PT
BzCl, Py
AC C
Finally, the donor V,25 in a NIS, H2SO4-silica promoted glycosylation furnished the
desired protected tetrasaccharide 13 in 82% yield. The proton NMR spectroscopy showed four distinct peaks at δ 4.81 (J1,2<1.0 Hz, H-1), δ 5.12 (J1′,2′<1.0 Hz, H-1′), δ 5.24 (H-1ʺ), and δ 4.70 (H-1‴) which corresponds to the four anomeric protons. The relevant anomeric carbon signals which appeared at δ 99.0 (C-1), δ 98.9 (C-1′), δ 100.4 (C-1ʺ) and δ 101.9 (C-1‴) along with the COSY and HSQC spectra undoubtedly confirmed the structure of the tetrasaccharide with proper configuration of the newly formed glycosidic linkage. Once the fully protected tetrasaccharide is in hand, global de-protection of the tetrasaccharide 15 was initiated with the hydrolysis of the benzylidene acetal using 80% aq. AcOH at 80 °C. Further, Zemplén de-O-acetylation using NaOMe in MeOH followed by hydrogenolysis using Pd-C 5
ACCEPTED MANUSCRIPT cartridge in a ThalesNano continuous flow hydrogenation assembly removed the benzyl groups along with the removal of CBz group to furnish the target tetrasaccharide 1 75% yield over three steps (Scheme 1).
RI PT
2. Experimental section 2.1. General: All reagents and solvents were dried prior to use according to standardized methods.26 The commercially purchased reagents were used without any further purification unless mentioned otherwise. Dichloromethane was dried and distilled over P2O5 to make it
SC
anhydrous and moisture-free. All reactions were monitored by Thin Layer Chromatography (TLC) on Silica-Gel 60-F254 with detection by fluorescence followed by charring after
M AN U
immersion in 10% ethanolic solution of H2SO4. Flash chromatography was performed with Silica Gel 230-400 mesh. Optical rotations were measured on sodium-line at ambient temperature. 1H and
13
C NMR were recorded on Bruker spectrometer at 500 MHz and 125
MHz respectively. The assignments of 1H and
13
C peaks were done with the help of 1H-1H
COSY and 1H-13C HSQC spectra. In case of the tetrasaccharide, 1H NMR values were denoted as H for the reducing end rhamnose unit A, H′ for the rhamnose unit B, Hʺ for the
TE D
rhamnose unit C and H‴ for the terminal glucose unit D (as numbered A-D in Figure 1).
2.2. 2-(benzyloxycarbonyl)-aminoethyl 2,3-O-isopropylidene-α-L-rhamnopyranoside (3):
EP
To a suspension of compound 2 (2.2g, 4.7 mmol) in dry MeOH (20 mL), NaOMe in MeOH (0.5M, 2 mL) was added and the mixture was stirred at room temperature for 8 hours when
AC C
TLC (n-hexane-EtOAc; 1:1) suggested complete conversion of the starting material to a much slower moving spot. The reaction mixture was neutralised with DOWEX 50W H+ resin, filtered and the solvents were evaporated in vacuo. The crude product thus obtained was dried and dissolved in dry CH3CN (20 mL) followed by addition of 2,2-dimethoxy propane (820 µL, 6.7 mmol). After addition of CSA (50 mg), the mixture was allowed to stir at room temperature for 4 hours when TLC (n-hexane-EtOAc; 3:2) showed complete conversion of the starting material to a faster moving spot. The reaction mixture was neutralised with Et3N and evaporated in vacuo. The crude product thus obtained was purified by flash chromatography using n-hexane-EtOAc (1:1) as eluent to get the pure compound 3 (1.63g, 91%). [α]D25 +78 (c 1.1, CHCl3).1H NMR (500 MHz, CDCl3) δ: 7.35-7.30 (m, 5H, 6
ACCEPTED MANUSCRIPT ArH), 5.09 (s, 2H, CH2Ph), 4.94 (s, 1H, J1,2<1.0 Hz, H-1), 4.10 (d, 1H, J1,2<1.0 Hz, J2,3 5.5 Hz, H-2), 4.05 (dd, 1H, J2,3 5.5 Hz, J3,4 7.0 Hz, H-3), 3.75 (m, 1H, OCH2CH2), 3.59 (m, 1H, H-5), 3.51 (m, 1H, OCH2CH2), 3.42 (m, 1H, CH2CH2NH), 3.52 (m, 2H, H-4, CH2CH2NH), 3.06 (bs, 1H, OH), 1.50, 1.33 (2s, 6H, 2×isopropylidene-CH3), 1.25 (d, 3H, J5,6 6.5 Hz, CCH3). 13C NMR (CDCl3, 125 MHz) δ: 156.3 (COCH2Ph), 128.5 (3), 128.1 (3) (ArC), 109.4
RI PT
(isopropylidene-C), 97.2 (C-1), 78.4, 75.7, 74.3, 66.8, 66.6, 65.9, 40.7 (CH2CH2NH), 27.9, 26.1 (2×isopropylidene-CH3), 17.2 (CH3). HRMS calcd for C19H27NO7Na (M+Na)+:
2.3.
2-(benzyloxycarbonyl)-aminoethyl
4-O-benzoyl-2,3-O-isopropylidene-α-L-
M AN U
rhamnopyranoside (4):
SC
404.1685, found: 404.1682.
To a solution of compound 3 (1.6 g, 4.19 mmol) in pyridine (15 mL), benzoyl chloride (810 µL, 7.0 mmol) was added and the mixture was stirred for 2 hours at room temperature. When the TLC (n-hexane-EtOAc; 2:1) showed that whole of the starting material has been converted into a faster moving spot, solvents were evaporated in vacuo and co-evaporated with toluene until the last trace of pyridine was removed. The crude product thus obtained
TE D
was purified by flash chromatography using n-hexane-EtOAc (3:1) as eluent to obtain the pure compound 4 (1.91 g, 94%). [α]D25 +68 (c 1.0, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 8.04-7.29 (m, 10H, ArH), 5.12 (m, 3H, H-4, CH2Ph), 5.05 (d, 1H, J1,2<1.0 Hz, H-1), 4.32 (dd, 1H, J2,3 5.5 Hz, J3,4 7.5 Hz, H-3), 4.19 (d, 1H, J1,2<1.0 Hz, J2,3 5.5 Hz, H-2), 3.85 (m, 1H, H-
EP
5), 3.79 (m, 1H, OCH2CH2), 3.54 (m, 1H, OCH2CH2), 3.45 (m, 1H, CH2CH2NH), 3.41 (m, 1H, CH2CH2NH),1.61, 1.34 (2s, 6H, 2×isopropylidene-CH3), 1.20 (d, 3H, J5,6 6.0 Hz, C-
AC C
CH3). 13C NMR (CDCl3, 125 MHz) δ: 165.6 (COPh), 156.3 (COCH2Ph), 133.1 (2), 129.6 (3), 128.4 (2), 128.2 (2), 128.1 (2), 128.0 (ArC), 109.7 (isopropylidene-C), 97.1 (C-1), 75.8, 75.6, 74.7, 66.7, 64.3, 40.6 (CH2CH2NH), 27.6, 26.2 (2×isopropylidene-CH3), 16.9 (CH3). HRMS calcd for C26H31NO8Na (M+Na)+: 508.1947, found: 508.1944.
2.4. 2-(benzyloxycarbonyl)-aminoethyl 4-O-benzoyl-α-L-rhamnopyranoside (5): A suspension of compound 4 (1.7 g, 3.5 mmol) in 80% aq. AcOH (30 mL) was allowed to stir for 2 hours at 80 °C when the TLC (n-hexane-EtOAc; 1:1) suggested complete
7
ACCEPTED MANUSCRIPT conversion of the starting material into a slower moving spot. Solvents were evaporated in vacuo and co-evaporated with toluene to remove trace of AcOH. Flash chromatography using n-hexane-EtOAc (3:2) as eluent afforded pure compound 5 (1.3g, 85%). [α]D25 +103 (c 1.0, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 8.02-7.26 (m, 10H, ArH), 5.48 (m, 1H, NH), 5.13 (t, 1H, J3,4, J4,5 9.5 Hz, H-4), 5.07 (s, 2H, CH2Ph), 4.84 (s, 1H, J1,2<1.0 Hz, H-1), 4.00 (m, 2H,
RI PT
H-2, H-3), 3.89 (m, 1H, H-5), 3.74 (m, 1H, OCH2CH2), 3.50 (m, 1H, OCH2CH2), 3.41 (m, 1H, CH2CH2NH), 3.34 (m, 1H, CH2CH2NH),1.21 (d, 3H, J5,6 6.0 Hz, C-CH3).13C NMR (CDCl3, 125 MHz) δ: 166.8 (COPh), 156.5 (COCH2Ph), 136.3, 133.1, 129.7 (2), 129.5, 128.4 (2), 128.3 (2), 128.0 (2), 127.9 (ArC), 99.6 (C-1), 75.2, 70.7, 69.8, 66.6 (2), 66.2, 40.6
SC
(CH2CH2NH), 17.3 (CH3). HRMS calcd for C23H27NO8Na (M+Na)+: 468.1634, found:
M AN U
468.1631.
2.5. 2-(benzyloxycarbonyl)-aminoethyl 4-O-benzoyl-3-O-benzyl-α-L-rhamnopyranoside (6):
A mixture of compound 5 (1.2 g, 2.7 mmol) and Bu2SnO (870 mg, 3.5 mmol) in dry toluene (25 mL) was refluxed at 110 °C for 2 hours until the reaction mixture became clear. Toluene
TE D
was evaporated in vacuo and the crude product thus obtained was well dried before it was dissolved in dry DMF. CsF (450 mg, 3.0 mmol) was added to it and the reaction mixture was kept under N2 atmosphere for 30 min. Finally BnBr (420 µL, 3.5 mmol) was added to the reaction mixture and it was allowed to stir at room temperature for 12 hours till TLC (n-
EP
hexane-EtOAc; 3:2) showed that all of the starting material was converted to a faster moving spot. Solvents were evaporated in vacuo and the crude product was dissolved in EtOAc (20
AC C
mL). It was washed with water (2×30 mL). The organic layer was separately collected over Na2SO4, filtered and evaporated in vaccuo. The crude product thus obtained was subjected to flash chromatography using n-hexane-EtOAc (2:1) as eluent to furnish the pure acceptor 6 (1.2g, 82%). [α]D25 +84 (c 0.9, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 8.02-7.13 (m, 15H, ArH), 5.34 (t, 1H, J3,4, J4,5 9.5 Hz, H-4), 5.13 (s, 2H, CH2Ph), 4.89 (s, 1H, J1,2<1.0 Hz, H-1), 4.64, 4.51 (ABq, 2H, JA,B12.0 Hz, CH2Ph), 4.07 (bs, 1H, H-3), 3.87 (m, 2H, H-2, H-5), 3.79 (m, 1H, OCH2CH2), 3.59 (m, 1H, OCH2CH2), 3.45 (m, 1H, CH2CH2NH), 3.42 (m, 1H, CH2CH2NH), 2.82 (bd, 1H, J 13.0 Hz, OH), 1.23 (d, 3H, J5,6 6.5 Hz, C-CH3).13C NMR (CDCl3, 125 MHz) δ: 165.7 (COPh), 156.3 (COCH2Ph), 137.3, 136.4, 133.1 (2), 129.8, 129.7 (2), 128.5 (2), 128.4 (2), 128.3 (3), 128.2, 127.8 (2), 127.7 (2) (ArC), 99.3 (C-1), 76.6, 72.8, 8
ACCEPTED MANUSCRIPT 71.7, 68.3, 67.0, 66.8, 66.5, 40.8 (CH2CH2NH), 17.4 (CH3). HRMS calcd for C30H33NO8Na (M+Na)+: 558.2104, found: 558.2101.
2.6. 2-(benzyloxycarbonyl)-aminoethyl 3-O-chloroacetyl-2-O-benzoyl-4-O-benzyl-α-L-
RI PT
rhamnopyranosyl-(1→ →2)-4-O-benzoyl-3-O-benzyl-α-L-rhamnopyranoside (8): A mixture of the acceptor 6 (1.1g, 2.1 mmol), donor 7 (1.4g, 2.6 mmol) and 4Ǻ MS (1.5 g) in dry CH2Cl2(15 mL) was kept stirring under N2 atmosphere for 45 min. NIS (780 mg, 3.5 mmol) was added to the reaction mixture and it was cooled to 0 °C under N2 atmosphere.
SC
After 30 min, H2SO4-silica (50 mg) was added to initiate the reaction. The mixture was allowed to stir at 0 °C for 15 min when the TLC (n-hexane-EtOAc; 2:1) showed complete
M AN U
consumption of the acceptor. The reaction mixture was then filtered through a pad of Celite. The filtrate was washed successively with aq. Na2S2O3 (2 × 30 mL), aq. NaHCO3 (2 × 30 mL) and brine (30 mL). Organic layer was separated, dried over Na2SO4 and evaporated in vacuo. The syrupy crude product thus obtained was purified by flash chromatography using n-hexane-EtOAc (5:2) as eluent to afford pure disaccharide 8 (1.8g, 89%) as white foam. [α]D25 +78 (c 1.0, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 8.05-7.06 (m, 25H, ArH), 5.67 (dd,
TE D
1H, J1′,2′ 1.5 Hz, J2′,3′ 3.5 Hz, H-2′), 5.56 (dd, 1H, J2′,3′ 3.5 Hz, J3′,4′ 10.0 Hz, H-3′), 5.35 (t, 1H, J3,4, J4,5 9.5 Hz, H-4), 5.14 (m, 3H, H-1′, CH2Ph), 4.85 (d, 1H, J1,2<1.0 Hz, H-1), 4.74-4.55 (ABq, 4H, 2×CH2Ph), 4.05 (bs, 1H, H-3), 4.02 (m, 1H, H-5′), 3.97, 3.89 (ABq, 2H, JA,B15.0 Hz, CH2Cl), 3.93 (m, 1H, H-2), 3.86 (m,1H, H-5), 3.78 (m, 1H, OCH2CH2), 3.66 (t, 1H, J3′,4′,
EP
J4′,5′ 9.5 Hz, H-4′), 3.56 (m, 1H, OCH2CH2), 3.48 (m, 1H, CH2CH2NH), 3.43 (m, 1H, CH2CH2NH),1.41 (d, 3H, J5′,6′ 6.0 Hz, C-CH3), 1.27 (d, 3H, J5,6 6.0 Hz, C-CH3).
13
C NMR
AC C
(CDCl3, 125 MHz) δ: 166.0 (COCH2Cl), 165.3, 165.2 (2×COPh), 156.3 (COCH2Ph), 137.7 (2), 133.3, 133.0, 129.9, 129.8 (2), 129.7 (2), 129.6, 128.5 (2), 128.4 (4), 128.3 (2), 128.2 (3), 127.9 (5), 127.6 (2), 127.5 (2) (ArC), 98.9 (C-1), 98.5 (C-1′), 78.5, 76.1, 75.2, 73.9, 73.6, 73.4, 71.9, 70.3, 68.3, 67.2, 66.9, 66.8, 40.8 (CH2CH2NH), 40.6 (COCH2Cl), 18.0, 17.6 (2×CH3). HRMS calcd for C52H54ClNO14Na (M+Na)+: 974.3131, found: 974.3128.
9
ACCEPTED MANUSCRIPT 2.7. 2-(benzyloxycarbonyl)-aminoethyl 2-O-benzoyl-4-O-benzyl-α-L-rhamnopyranosyl(1→ →2)-4-O-benzoyl-3-O-benzyl-α-L-rhamnopyranoside (9): A mixture of disaccharide 8 (1.6g, 1.7 mmol), thiourea (1.2g, 16.8 mmol) and 2,4,6-collidine (2.2 mL, 16.8 mmol) in CH2Cl2-MeOH (2:3, 20 mL) was refluxed for 10 hours when TLC (nhexane-EtOAc; 3:2) confirmed the complete conversion of the starting material to a slower
RI PT
moving spot. The solvents were evaporated in vacuo; the resulting solid residue was dissolved in CH2Cl2 (20 mL) and washed with 1(N) HCl (2×30 mL) and H2O (30 mL). The organic layer was separated, filtered, dried over Na2SO4 and evaporated in vacuo. The crude residue was then subjected to flash chromatography using n-hexane-EtOAc (2:1) as eluent to
SC
give the pure disaccharide acceptor 9 (1.4g, 92%).[α]D25 +96 (c 0.8, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 8.08-7.09 (m, 25H, ArH), 5.67 (dd, 1H, J1′,2′ 1.5 Hz, J2′,3′ 3.5 Hz, H-2′), 5.35
M AN U
(t, 1H, J3,4, J4,5 9.5 Hz, H-4), 5.15 (m, 2H, CH2Ph), 5.05 (d, 1H, J1′,2′<1.0 Hz, H-1′), 4.92-4.54 (ABq, 4H, 2×CH2Ph), 4.85 (d, 1H, J1,2<1.0 Hz, H-1), 5.56 (m, 1H, H-3′), 3.95 (m, 3H, H-2, H-3, H-5′), 3.84 (m, 1H, H-5), 3.78 (m, 1H, OCH2CH2), 3.53 (m, 1H, OCH2CH2), 3.49 (t, 1H, J3′,4′, J4′,5′ 9.5 Hz, H-4′), 3.47 (m, 1H, CH2CH2NH), 3.42 (m, 1H, CH2CH2NH),1.38 (d, 3H, J5′,6′ 6.5 Hz, C-CH3), 1.23 (d, 3H, J5,6 6.0 Hz, C-CH3).13C NMR (CDCl3, 125 MHz) δ: 166.1, 165.5 (2×COPh), 156.3 (COCH2Ph), 138.2, 137.7, 133.2, 133.0, 129.9, 129.8 (5),
TE D
128.5 (2), 128.4 (2), 128.3 (5), 128.2 (4), 128.1 (3), 127.8, 127.7 (2), 127.6 (ArC), 99.3 (C1′), 99.0 (C-1), 81.3, 75.8, 75.3, 75.2, 73.3 (2), 71.9, 70.4, 68.1, 67.1, 67.0, 66.8, 40.8 (CH2CH2NH), 18.2, 17.6 (2×CH3). HRMS calcd for C50H53NO13Na (M+Na)+: 898.3415,
EP
found: 898.3412.
AC C
2.8. 2-(benzyloxycarbonyl)-aminoethyl 2-O-chloroacetyl-4-O-benzoyl-3-O-benzyl-α-Lrhamnopyranosyl-(1→ →3)-2-O-benzoyl-4-O-benzyl-α-L-rhamnopyranosyl-(1→ →2)-4-Obenzoyl-3-O-benzyl-α-L-rhamnopyranoside (11): A mixture of the disaccharide acceptor 9 (1.2g, 1.4 mmol), known donor 10 (960 mg, 1.8 mmol) and 4Ǻ MS (1.5g) in dry CH2Cl2 (15 mL) was kept stirring under N2 atmosphere for 45 min. NIS (520 mg, 2.3 mmol) was added and the mixture was cooled to 0 oC keeping under continuous N2 flow. H2SO4-silica (30 mg) was added to the mixture and allowed to stir at the same temperature for 30 min when TLC (n-hexane-EtOAc; 4:1) showed complete consumption of the acceptor. The mixture was then filtered through a Celite padandthe
10
ACCEPTED MANUSCRIPT filtrate was successively washed with aq. Na2S2O3 (2 × 30 mL), aq. NaHCO3 (2 × 30 mL) and brine (30 mL). The organic layer was collected, dried over Na2SO4 and evaporated in vacuo. Crude product thus obtained was purified by flash chromatography using n-hexaneEtOAc (3:1) as the eluent to give the pure trisaccharide 11 (1.6 g, 90%) as white foam. [α]D25 +118 (c 0.9, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 8.07-6.96 (m, 35H, ArH), 5.55 (dd, 1H,
RI PT
J1′,2′ 1.5 Hz, J2′,3′ 3.0 Hz, H-2′), 5.44 (dd, 1H, J1ʺ,2ʺ 1.5 Hz, J2ʺ,3ʺ 3.0 Hz, H-2ʺ), 5.30 (t, 1H, J3ʺ,4ʺ, J4ʺ,5ʺ 9.5 Hz, H-4ʺ), 5.22 (d, 1H, J1ʺ,2ʺ 1.0 Hz, H-1ʺ), 5.19 (t, 1H, J3,4, J4,5 9.5 Hz, H-4), 5.12 (m, 3H, H-1′, CH2Ph), 4.82 (d, 1H, J1,2<1.0 Hz, H-1), 4.80-4.25 (m, ABq, 6H, 3×CH2Ph), 4.40 (dd, 1H, J2′,3′ 3.0 Hz, J3′,4′ 9.5 Hz, H-3′), 4.20, 4.12 (ABq, 2H, JA,B15.5 Hz,
SC
CH2Cl), 4.09 (m, 1H, H-5), 3.97 (m, 2H, H-2, H-5′), 3.88 (m, 2H, H-3, H-3ʺ), 3.81 (m, 1H, H-5ʺ), 3.77 (m, 1H, OCH2CH2), 3.61 (t, 1H, J3′,4′, J4′,5′ 9.5 Hz, H-4′), 3.53 (m, 1H, OCH2CH2), 3.45 (m, 1H, CH2CH2NH), 3.41 (m, 1H, CH2CH2NH),1.37 (d, 3H, J5′,6′ 6.5 Hz,
M AN U
C-CH3), 1.21 (d, 3H, J5ʺ,6ʺ 6.5 Hz, C-CH3), 1.17 (d, 3H, J5,6 6.0 Hz, C-CH3).13C NMR (CDCl3, 125 MHz) δ: 166.7 (COCH2Cl), 165.5, 165.2 (2) (3×COPh), 156.3 (COCH2Ph), 137.8, 137.5, 137.3, 133.2, 133.0 (2), 130.0 (2), 129.9 (2), 129.7 (3), 128.6 (4), 128.5 (4), 128.3 (6), 128.2 (4), 128.1 (4), 128.0, 127.7 (4), 127.5 (2) (ArC), 99.1 (C-1′), 99.0 (C-1), 98.7 (C-1ʺ), 80.8, 75.9, 75.6, 75.5, 74.2, 73.3, 72.7, 72.5, 71.9, 71.5, 70.5, 68.5, 67.6, 67.2, 66.9
TE D
(2), 40.9 (COCH2Cl), 40.8 (CH2CH2NH), 18.1, 17.7, 17.2 (3×CH3). HRMS calcd for C72H74ClNO19Na (M+Na)+: 1314.4441, found: 1314.4438.
Synthesis
of
2-(benzyloxycarbonyl)-aminoethyl
4-O-benzoyl-3-O-benzyl-α-L-
EP
2.9.
rhamnopyranosyl-(1→ →3)-2-O-benzoyl-4-O-benzyl-α-L-rhamnopyranosyl-(1→ →2)-4-O-
AC C
benzoyl-3-O-benzyl-α-L-rhamnopyranoside (12): A mixture of the trisaccharide 11 (1.5g, 1.2 mmol), thiourea (880 mg, 11.5 mmol) and collidine (1.5 mL, 11.5 mmol) in CH2Cl2-MeOH (2:3, 20 mL) was refluxed for 12 hours when TLC (n-hexane-EtOAc; 3:1) suggested complete conversion of the starting material to a slower moving spot. The mixture was evaporated in vacuo and the solid residue thus obtained was dissolved in CH2Cl2 and washed with 1(N) HCl (2×30 mL) and brine (30 mL). The organic layer was separated, dried over Na2SO4, filtered and evaporated in vacuo to obtain a syrupy residue which was further subjected to flash chromatography using n-hexane-EtOAc (3:2) as eluent to give the pure trisaccharide acceptor 12 (1.3g, 87%) as white foam.[α]D25 +112 (c 0.8, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 8.07-6.96 (m, 35H, ArH), 5.59 (m, 1H, 11
ACCEPTED MANUSCRIPT H-2′), 5.30 (m, 3H, H-1ʺ, H-4, H-4ʺ), 5.13 (m, 3H, H-1′, CH2Ph), 4.84 (d, 1H, J1,2<1.0 Hz, H1), 4.77-4.38 (m, ABq, 6H, 3×CH2Ph), 4.40 (m, 1H, H-3′), 4.06 (m,1H, H-5), 4.00 (m, 1H, H-2), 3.96 (m, 1H, H-5′), 3.90 (m, 2H, H-2ʺ, H-3ʺ), 3.83 (m, 1H, H-5ʺ), 3.77 (dd, 1H, J2,3 3.0 Hz, J3,4 9.5 Hz, H-3), 3.75 (m, 1H, OCH2CH2), 3.60 (t, 1H, J3′,4′, J4′,5′ 9.5 Hz, H-4′), 3.54 (m, 1H, OCH2CH2), 3.46 (m, 1H, CH2CH2NH), 3.41 (m, 1H, CH2CH2NH),1.36 (d, 3H, J5′,6′ 6.0
RI PT
Hz, C-CH3), 1.22 (d, 3H, J5ʺ,6ʺ 6.5 Hz, C-CH3), 1.17 (d, 3H, J5,6 6.5 Hz, C-CH3).13C NMR (CDCl3, 125 MHz) δ: 165.6, 165.2 (2) (3×COPh), 156.3 (COCH2Ph), 137.8, 137.7, 137.5, 133.1, 132.9 (2), 130.2, 130.0 (2), 129.8 (2), 129.7 (3), 128.5 (5), 128.5 (2), 128.2 (3), 128.2 (6), 128.1 (3), 128.0, 127.7 (4), 127.6 (2), 127.5 (2) (ArC), 100.5 (C-1ʺ), 99.0 (C-1′), 98.9 (C-
SC
1), 80.6, 76.6, 75.9, 75.8, 75.4, 75.0, 73.3, 73.0, 72.7, 71.8 (2), 68.8, 68.4, 67.2, 67.1, 66.9, 66.8, 40.8 (CH2CH2NH), 18.1, 17.7, 17.2 (3×CH3). HRMS calcd for C70H73NO18Na
M AN U
(M+Na)+: 1238.4725, found: 1238.4721.
2.10. 2-(benzyloxycarbonyl)-aminoethyl 2-O-acetyl-3-O-benzyl-4,6-O-benzylidene -β-D→3)-2-Ogalactopyranosyl-(1→ →2)-4-O-benzoyl-3-O-benzyl-α-L-rhamnopyranosyl-(1→ benzoyl-4-O-benzyl-α-L-rhamnopyranosyl-(1→ →2)-4-O-benzoyl-3-O-benzyl-α-L-
TE D
rhamnopyranoside (13):
A mixture of trisaccharide acceptor 12 (1.4g, 1.2 mmol), donor V (940 mg, 1.7 mmol) and 4Ǻ MS (1.5 g) in dry CH2Cl2 (15 mL) was stirred at 10 °C for 30 min under nitrogen atmosphere. NIS was then added and the reaction mixture was cooled to -10 °C. After 30 min
EP
H2SO4-silica (30 mg) was added to it and the mixture was allowed to stir at the same temperature for 15 min when TLC (n-hexane-EtOAc; 3:2) showed complete consumption of
AC C
the acceptor. The mixture was then neutralized with Et3N and filtered through a Celite pad. The filtrate was successively washed with aq. Na2S2O3 (2 × 30 mL), aq. NaHCO3 (2×30 mL) and brine (30 mL). The organic layer was collected, dried (Na2SO4) and evaporated in vacuo. Crude product thus obtained was purified by flash chromatography using n-hexane–EtOAc (2:1) as the eluent to afford pure tetrasaccharide 13 (1.5g, 82%) as white foam. [α]D25 +148 (c 0.9, CHCl3). 1H NMR (500 MHz, CDCl3) δ: 8.12-7.00 (m, 45H, ArH), 5.58 (m, 1H, H-2′), 5.48 (s, 1H, CHPh), 5.29 (m, 1H, H-4), 5.24 (s, 1H, H-1ʺ), 5.17 (t, 1H, J3ʺ,4ʺ, J4ʺ,5ʺ 9.5 Hz, H4ʺ), 5.13 (m, 2H, CH2Ph), 5.12 (d, 1H, J1′,2′<1.0 Hz, H-1′), 5.06 (t, 1H, J1‴,2‴, J2‴,3‴ 8.5 Hz, H2‴), 4.93-4.27 (m, 9H, H-4‴, 4×CH2Ph), 4.81 (d, 1H, J1,2<1.0 Hz, H-1), 4.70 (m, 1H, H-1‴), 4.37 (m, 1H, H-3′), 4.03-3.97 (m, 3H, H-2, H-2ʺ, H-5′), 3.89 (m, 3H, H-5, OCH2CH2), 3.84 12
ACCEPTED MANUSCRIPT (m, 2H, H-3, H-5ʺ), 3.70 (m, 3H, H-3ʺ, H-3‴, H-6a), 3.51 (m, 1H, H-6b), 3.55 (t, 1H, J3′,4′, J4′,5′ 9.5 Hz, H-4′), 3.44 (m, 1H, CH2CH2NH), 3.40 (m, 1H, CH2CH2NH), 3.22 (m, 1H, H-5‴), 2.09 (s, 3H, COCH3), 1.32 (d, 3H, J5,6 6.0 Hz, C-CH3), 1.21 (d, 3H, J5ʺ,6ʺ 6.0 Hz, C-CH3), 1.14 (d, 3H, J5′,6′ 6.0 Hz, C-CH3).13C NMR (CDCl3, 125 MHz) δ: 169.8 (COCH3), 165.2 (3×COPh), 156.3 (COCH2Ph), 138.4, 138.0, 137.9, 137.8, 133.2, 132.9, 132.8, 130.3 (2),
RI PT
130.0, 129.8 (5), 128.9, 128.5 (8), 128.2 (10), 128.1 (3), 127.8 (3), 127.7 (2), 127.6 (3), 127.5 (4), 126.0 (4) (ArC), 101.9 (C-1‴), 101.1 (CHPh), 100.4 (C-1ʺ), 99.0 (C-1), 98.9 (C-1′),81.6, 81.4, 80.7, 78.3, 75.7, 75.4, 75.1, 74.9, 74.0, 73.5, 73.3, 73.1, 73.0, 72.7, 72.6, 71.8, 68.5, 68.3, 67.9, 67.2, 66.9 (2), 66.1, 40.8 (CH2CH2NH), 20.8 (COCH3), 18.0, 17.7, 17.4 (3×CH3).
2-(benzyloxycarbonyl)-aminoethyl
β-D-galactopyranosyl-(1→ →2)-
M AN U
2.11.
SC
HRMS calcd for C87H91NO25Na (M+Na)+: 1572.5778, found: 1572.5773.
α-L-
rhamnopyranosyl-(1→ →3)- α-L-rhamnopyranosyl-(1→ →2)- α-L-rhamnopyranoside (1): A suspension of compound 16 (1.3g, 0.8 mmol) in AcOH-H2O (8:1, 18 mL) was stirred at 80 °C for 2 hours when the starting material was completely converted to a slower moving spot as suggested by TLC (n-hexane–EtOAc; 2:1). The solvents were evaporated and co-
TE D
evaporated twice with toluene. The residue was dissolved in MeOH (50 mL) and passed through a 10% Pd–C cartridge in a ThalesNano flow hydrogenation assembly under continuous flow of H2 at atmospheric pressure. As evident from mass spectroscopy the hydrogenolysis of the benzyl groups were complete after 2 such cycles. In the next step
EP
NaOMe in MeOH (0.5 M, 1 mL) was added to the solution and it was stirred at room temperature for 12 hours. The solution was neutralized by DOWEX 50W H+ resin, filtered
AC C
and evaporated in vacuo to afford the final tetrasaccharide 1 (447 mg, 75%) as white amorphous mass. [α]D25 +62 (c 0.6, MeOH).1H NMR (MeOD, 500 MHz) δ: 5.45 (d, 1H, J1,2< 1.0 Hz, H-1′), 5.11 (d, 1H, J1,2< 1.0 Hz, H-1ʺ), 5.06 ppm (d, 1H, J1,2< 1.0 Hz, H-1), 4.90 (m, 1H, H-1ʺʺ)., 3.30 (m, 2H, OCH2CH2NH2).
C NMR (MeOD, 125 MHz) δ: 104.1 (C-1‴),
13
103.9 (C-1ʺ), 103.4 (C-1′), 101.4 (C-1), 83.0, 82.8, 81.4, 80.0, 77.4, 77.2, 76.9, 76.4, 75.2, 74.4, 74.2, 71.4, 71.2, 70.8, 70.5, 68.5, 68.3, 42.7 (CH2CH2NH2), 19.3, 19.2, 19.0 (3×CH3). HRMS calcd for C26H47NO18Na (M+Na)+: 684.2691, found: 684.2687.
13
ACCEPTED MANUSCRIPT Acknowledgement VS is thankful to Indian Institute of Science Education and Research (IISER) Kolkata for fellowship. The work is supported by Science and Engineering Research Board (SERB), New
Reference
RI PT
Delhi, India through the grant SB/S1/OC-48/2013 to BM.
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AC C
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London: Pergamon; 1996
15
ACCEPTED MANUSCRIPT Highlights 1. Total synthesis of the tetrasaccharide repeating unit of the O-polysaccharide 2. Activation of thioglycoside using NIS in the presence of H2SO4-silica
RI PT
3. Use of chloroacetate group as the orthogonal protecting group
AC C
EP
TE D
M AN U
SC
4. Extensive study on the successful glycosylation of the terminal glucose unit