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Molecules 2016, 21(5), 614; doi:10.3390/molecules21050614

Article
Efficient Synthesis of the Lewis A Tandem Repeat
1
Department of Applied Bioorganic Chemistry, Gifu University, 1-1 Yanagido, Gifu-shi, Gifu 501-1193, Japan
2
Institute for Integrated Cell-Material Sciences (WPI Program), Kyoto University, Yoshida-ushinomiya-cho, Sakyo-ku, Kyoto-shi, Kyoto 606-8302, Japan
3
Faculty of Pharmaceutical Sciences, Aomori University, 2-3-1 Kobata, Aomori-shi, Aomori 030-0943, Japan
*
Authors to whom correspondence should be addressed.
Academic Editors: Vito Ferro and Trinidad Velasco-Torrijos
Received: 11 April 2016 / Accepted: 6 May 2016 / Published: 11 May 2016

Abstract

:
The convergent synthesis of the Lewis A (Lea) tandem repeat is described. The Lea tandem repeat is a carbohydrate ligand for a mannose binding protein that shows potent inhibitory activity against carcinoma growth. The Lea unit, {β-d-Gal-(1→3)-[α-l-Fuc-(1→4)]-β-d-GlcNAc}, was synthesized by stereoselective nitrile-assisted β-galactosylation with the phenyl 3-O-allyl-2,4,6-tri-O-benzyl-1-thio-β-galactoside, and ether-assisted α-fucosylation with fucosyl (N-phenyl)trifluoroacetimidate. This common Lea unit was easily converted to an acceptor and donor in high yields, and the stereoselective assembly of the hexasaccharide and dodecasaccharide as the Lea tandem repeat framework was achieved by 2-trichloroacetamido-assisted β-glycosylation and the (N-phenyl)trifluoroacetimidate method.
Keywords:
Lewis A tandem repeat; convergent synthesis; sugar-binding protein

1. Introduction

The Lewis A (Lea) trisaccharide, {β-d-Gal-(1→3)-[α-l-Fuc-(1→4)]-β-d-GlcNAc}, is a component of glycolipids that have been identified as antigens of the Lewis blood group. Recently, N-linked glycoproteins with Lea tandem repeats (Figure 1) consisting of four or more repeated sequences were isolated from the SW1116 human colorectal carcinoma cell line. This carbohydrate ligand forms a mannose binding protein–carbohydrate complex, which shows potent inhibitory activity against growth of human colorectal carcinoma cells [1,2,3]. To elucidate the inhibitory mechanism, it is necessary to determine and synthesize the minimum structure of the carbohydrate ligand; however, synthesis of the Lea tandem repeat has not been reported. We have recently developed and reported a new synthetic strategy for core 2 decasaccharide with four repeated type-II N-acetyllactsamines using a benzyl-protected N-trichloroacetyllactsaminyl imidate with high β-selectivity and high yield [4]. In this paper, we describe a synthetic method for the tetrameric Lea tandem repeat motif and the hexasaccharide and dodecasaccharide, via the synthesis of type-I lactosamine by β-selective galactosylation [5] and convergent synthesis with N-trichloroacetyllactosaminyl imidate.

2. Results and Discussion

In the retrosynthetic analysis of the Lea tetramer, we planned the convergent synthesis with a Lea trisaccharide common intermediate, which would be constructed with β-d-Gal-(1→3)-β-d-GlcNTCA synthesized by using β-selective galactosylation and 2-p-methoxybenzyl fucosyl imidate. The trisaccharide was designed as the suitably protected form equipped with TBDPS group on the 1-position of the glucosamine and an allyl group on the 3-position of the galactose for divergent synthesis of the acceptor and the donor. In addition, the trichloroacetyl (TCA) group on the 2-position of the glucosamine and the benzyl groups were expected to ensure high stereoselectivity and high yield during the later glycosylation [4,6,7,8,9,10,11,12,13]. We envisioned that the tetramer could be obtained by glycosylation promoted by a catalytic Lewis acid with haxasaccharyl acceptor and donor, which could be prepared from the hexasaccharide Lea dimer, and the hexasaccharide could be synthesized by coupling the acceptor and N-phenyl trichloroacetimidyl donor provided from the Lea trisaccharide common intermediate (Scheme 1).
Lea trisaccharide 11 was synthesized from galactosyl donor 2 [5], 2-azidoglucosyl acceptor 3 [14], and fucosyl donor 8 [15,16] (Scheme 2). First, disaccharide 4 was constructed from 2 and 3 by propionitrile-mediated β-selective galactosylation [5] under BSP-Tf2O-TTBP [17] conditions in 89% yield. Then, 4 was transformed into disaccharide acceptor 7 by reducing the azide group to give 5, TCA group protection to give 6, and reductive ring opening of the benzylidene to give 7 in 79% yield (three steps). The glycosylation of 7 with 8 was conducted in cyclopentyl methyl ether-dichloromethane (1:1) [16,18] at −40 °C to obtain trisaccharide 9 in 95% yield. Lea common intermediate 11 was provided by deprotection of a 4-methoxybenzyl group and subsequent acetylation.
Lea trisaccharide 11 was readily converted to donor 13 and acceptor 14 (Scheme 3). After desilylation of 11, the resulting hemiacetal 12 was treated with (N-phenyl)trifluoroacetimidoyl chloride [19] and K2CO3 to obtain (N-phenyl)trifluoroacetimidate 13. Acceptor 14 was prepared by selective deallylation with iridium-catalyzed olefin migration, followed by treatment with HgCl2 and HgO in aqueous acetone solution [20]. The glycosylation of 13 and 14 promoted by catalytic TMSOTf proceeded at −78 °C to give hexasaccharide 15 in 93% yield [4,21,22]. By using the same procedure, hexasaccharide donor 17 and acceptor 18 were prepared in high yields, respectively. In the next coupling, the oligosaccharides showed lower reactivity, and the reaction occurred at 0 °C to afford dodecasaccharide 19 (88%). Next, the linker for sugar probes was introduced (Scheme 4). Dodecasaccharide 19 was converted to (N-phenyl)trifluoroacetimidate 21 (85% over two steps) in an analogous manner, and coupled with N-Boc aminopropanol to give 22 in 79% yield. After deallylation, microwave-assisted reductive dehalogenation of the TCA group was attempted with excess Zn and AcOH in several solvents (ethyl acetate, 1,4-dioxane, AcOH, and THF) [10]. However, Boc group was cleaved, giving the mixture of aminopropyl and acetamidopropyl derivatives as the main products. It was found that THF was most suitable to obtain aminopropyl derivative 24 almost predominantly. Then, the terminal amino group of 24 was re-protected with Boc group to afford fine 25 in 76% over two steps.

3. Experimental Section

3.1. General Methods

1H- and 13C-NMR spectra were recorded with a spectrometer (Avance III 500, Bruker, Billerica, MA, USA). Chemical shifts are expressed in ppm (δ) relative to the Me4Si signal as an internal standard. Electrospray ionization time-of-flight high-resolution mass spectrometry was performed (micrOTOF, Bruker Daltonics, Billerica, MA, USA). Specific rotations were determined with a high-sensitivity polarimeter (SEPA-300, Horiba, Kyoto, Japan). Microwave irradiation was carried out in a microwave reactor (μReactor Ex, Shikoku Instrumentation Co., Ltd., Kagawa, Japan). TLC analysis was performed on glass TLC plates (silica gel 60F254, Merck, Darmstadt, Germany). Compounds were visualized either by exposure to UV light (254 nm) or by dipping in a solution of 10% H2SO4 in ethanol, in a solution of phosphomolybdic acid, H3PO4, and H2SO4, in H2O, or in ninhydrin reagent, followed by heating. Column chromatography was performed with the solvent system (v/v) specified on silica gel BW-80S, BW-300, PSQ-60B (Fuji Silysia Chemical Ltd. Kasugai, Japan), or Wakosil HC-N (Wako Pure Chemical Industries, Ltd. Osaka, Japan). Gel permeation chromatography was performed with the solvent system (v/v) specified on Sephadex LH-20 (GE Healthcare UK Ltd. Little Chalfont, UK), or Bio-beads S-X1, S-X3 (Bio-Rad Laboratories, Inc. Hercules, CA, USA). Evaporation and concentration were carried out in vacuo.

3.2. Physical Data for All New Compounds

Molecules 21 00614 i001
tert-Butyldiphenylsilyl 3-O-allyl-2,4,6-tri-O-benzyl-β-d-galactopyranosyl-(1→3)-2-azido-4,6-O-benzylidene-2-deoxy-β-d-glucopyranoside (4). To a mixture of phenyl 3-O-allyl-2,4,6-tri-O-benzyl-1-thio-β-d-galactopyranoside 2 (438 mg, 0.75 mmol), tert-butyldiphenylsilyl 2-azido-4,6-O-benzylidene-2-deoxy-β-d-glucopyranoside 3 (200 mg, 0.38 mmol), benzenesulfinyl piperidine (237 mg, 1.13 mmol), tri-tert-butylpyrimidine (373 mg, 1.50 mmol), and molecular sieves 4A (1.41 g) in propionitrile (12.5 mL) was added dropwise trifluoromethanesulfonic anhydride (140 μL, 0.83 mmol) at −80 °C under Ar, and stirred for 1 h at −80 °C. The reaction mixture was quenched with sat. NaHCO3 aq., filtered through Celite, and diluted with EtOAc. The organic layer was separated, and the aqueous layer was extracted with EtOAc. The combined organic layer was successively washed with brine, dried over Na2SO4, and concentrated. The crude product was chromatographed on silica gel (PSQ-60B) with toluene–acetone (98:2) to give the title product 4 (338 mg, 89%). [α]D −31.3° (c 1.1, CHCl3); 1H-NMR (500 MHz, CDCl3) δ 7.70–7.15 (m, 30H, Ar), 5.93–5.85 (m, 1H, H2C=CHCH2), 5.39 (s, 1H, >CHPh), 5.31–5.27 (m, 1H, H2C=CHCH2), 5.16–5.13 (m, 1H, H2C=CHCH2), 4.96–4.89 (m, 2H, PhCH2 × 2), 4.80 (d, 1H, Jgem = 10.7 Hz, PhCH2), 4.63 (d, 1H, J1,2 = 7.9 Hz, H-1Gal), 4.57 (d, 1H, Jgem = 11.7 Hz, PhCH2), 4.48 (d, 1H, J1,2 = 7.8 Hz, H-1GlcN), 4.26–4.19 (m, 2H, PhCH2 × 2), 4.15–4.09 (m, 2H, H2C=CHCH2), 3.89 (dd, 1H, J5,6b = 4.9 Hz, Jgem = 10.1 Hz, H-6aGlcN), 3.78–3.69 (m, 4H, H-2Gal, H-4Gal, H-3GlcN, H-4GlcN), 3.59–3.51 (m, 3H, H-2GlcN, H-6bGlcN, H-6aGal), 3.36–3.31 (m, 2H, H-3Gal, H-6bGal), 3.25 (dd, 1H, J5,6a = J5,6b = 6.5 Hz, H-5Gal), 2.96–2.91 (m, 1H, H-5GlcN), 1.16 (s, 9H, tBu); 13C-NMR (125 MHz, CDCl3) δ 138.9, 138.8, 137.9, 137.3, 135.8, 134.9, 133.1, 132.5, 130.0, 129.8, 128.9, 128.4, 128.2, 128.2, 128.2, 128.1, 128.0, 127.9, 127.7, 127.6, 127.5, 127.4, 127.4, 126.0, 116.5, 102.7, 101.1, 97.2, 82.3, 79.9, 79.8, 78.7, 75.2, 74.4, 73.5, 73.1, 72.9, 71.7, 68.9, 68.8, 68.3, 66.2, 26.8, 19.1. HRMS (ESI) m/z: found [M + Na]+ 1026.4337, C59H65N3O10Si calcd. for [M + Na]+ 1026.4337.
Molecules 21 00614 i002
tert-Butyldiphenylsilyl 3-O-allyl-2,4,6-tri-O-benzyl-β-d-galactopyranosyl-(1→3)-2-amino-4,6-O-benzylidene-2-deoxy-β-d-glucopyranoside (5). A mixture of 4 (576 mg, 0.57 mmol), powdered Zn (1.50 g, 23.0 mmol), and AcOH (0.66 ml, 11.5 mmol) in CH2Cl2 (14 mL) was stirred for 30 min at room temperature under Ar. The mixture was diluted with CHCl3 and filtered through Celite. The filtrate was evaporated, and the residue was diluted with CHCl3. The organic layer was successively washed with sat. NaHCO3, water, and brine, dried over Na2SO4, and concentrated. The residue was chromatographed on silica gel with toluene–MeOH (95:5) to give the title product 5 (540 mg, 96%). [α]D −21.8° (c 1.3, CHCl3); 1H-NMR (500 MHz, CDCl3) δ 7.68–7.18 (m, 30H, Ar), 5.94–5.87 (m, 1H, H2C=CHCH2), 5.45 (s, 1H, >CHPh), 5.33–5.28 (m, 1H, H2C=CHCH2), 5.18–5.15 (m, 1H, H2C=CHCH2), 4.93–4.88 (m, 2H, PhCH2 × 2), 4.78 (d, 1H, Jgem = 10.9 Hz, PhCH2), 4.58 (d, 1H, Jgem = 11.6 Hz, PhCH2), 4.49 (d, 1H, J1,2 = 7.9 Hz, H-1Gal), 4.43 (d, 1H, J1,2 = 7.8 Hz, H-1GlcN), 4.31–4.25 (m, 2H, PhCH2 × 2), 4.17–4.08 (m, 2H, H2C=CHCH2), 3.96 (dd, 1H, J5,6a = 4.9 Hz, Jgem = 10.4 Hz, H-6aGlcN), 3.87–3.81 (m, 2H, H-2Gal, H-4Gal), 3.66–3.58 (m, 3H, H-4GlcN, H-6bGlcN, H-6aGlcN), 3.54 (t, 1H, J2,3 = J3.4 = 9.2 Hz, H-3GlcN), 3.41–3.37 (m, 3H, H-3Gal, H-5Gal, H-6bGal), 3.03–2.99 (m, 2H, H-2GlcN, H-5GlcN), 1.20 (s, 9H, tBu); 13C-NMR (125 MHz, CDCl3) δ 138.9, 138.5, 137.9, 137.6, 135.8, 135.8, 134.8, 133.4, 132.9, 129.8, 129.7, 128.6, 128.4, 128.3, 128.3, 128.1, 128.1, 127.8, 127.7, 127.7, 127.5, 127.4, 127.3, 126.1, 116.7, 104.3, 100.8, 99.1, 83.5, 82.7, 79.9, 79.4, 75.7, 74.5, 73.5, 73.1, 73.0, 71.4, 68.4, 68.2, 66.8, 60.1, 27.0, 19.2. HRMS (ESI) m/z: found [M + Na]+ 1000.4432, C59H67NO10Si calcd. for [M + Na]+ 1000.4432.
Molecules 21 00614 i003
tert-Butyldiphenylsilyl 3-O-allyl-2,4,6-tri-O-benzyl-β-d-galactopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-β-d-glucopyranoside (6); To a solution of 5 (12.9 g, 13.2 mmol) in pyridine (132 mL) was added dropwise trichloroacetyl chloride (1.76 mL, 15.8 mmol) at 0 °C under Ar, and stirred at 0 °C for 1 h. The mixture was evaporated, and the residue was diluted with CHCl3, successively washed with 2 M HCl, sat. NaHCO3, and brine, dried over Na2SO4, and concentrated. The residue was chromatographed on silica gel with toluene–EtOAc (97:3) to give the title product 6 (14.8 g, quant.). [α]D −9.8° (c 1.3, CHCl3); 1H-NMR (500 MHz, CDCl3) δ 7.67–7.18 (m, 30H, Ar), 7.02 (d, 1H, J2,NH = 7.0 Hz, NH), 5.95–5.87 (m, 1H, H2C=CHCH2), 5.45 (s, 1H, >CHPh), 5.34–5.30 (m, 1H, H2C=CHCH2), 5.24 (d, 1H, J1,2 = 7.9 Hz, H-1GlcN), 5.20–5.17 (m, 1H, H2C=CHCH2), 4.90 (d, 1H, Jgem = 11.6 Hz, PhCH2), 4.82 (d, 1H, Jgem = 10.7 Hz, PhCH2), 4.69 (d, 1H, PhCH2), 4.57 (d, 1H, PhCH2), 4.42 (d, 1H, J1,2 = 7.9 Hz, H-1Gal), 4.39–4.30 (m, 3H, PhCH2 × 2, H-3GlcN), 4.18–4.10 (m, 2H, H2C=CHCH2), 3.95 (dd, 1H, J5,6a = 4.9 Hz, Jgem = 10.4 Hz, H-6aGlcN), 3.82–3.79 (m, 2H, H-2Gal, H-4Gal), 3.65 (t, 1H, J3,4 = J4,5 = 9.2 Hz, H-4GlcN), 3.61–3.54 (m, 2H, H-6bGlcN, H-6aGal), 3.49 (dd, 1H, J5,6b = 5.4 Hz, Jgem = 9.0 Hz, H-6bGal), 3.44–3.39 (m, 2H, H-2GlcN, H-5Gal), 3.31 (dd, 1H, J2,3 = 9.8 Hz, J3,4 = 2.9 Hz, H-3Gal), 3.12–3.07 (m, 1H, H-5GlcN), 1.06 (s, 9H, tBu); 13C-NMR (125 MHz, CDCl3) δ 161.4, 138.9, 138.7, 137.8, 137.3, 135.9, 135.7, 134.8, 133.0, 132.5, 129.8, 129.8, 128.7, 128.5, 128.4, 128.1, 128.1, 128.0, 127.9, 127.9, 127.8, 127.5, 127.4, 127.4, 126.1, 116.8, 103.1, 100.8, 94.1, 92.2, 82.1, 79.9, 79.2, 77.6, 76.5, 75.8, 74.5, 73.6, 73.3, 73.2, 71.4, 68.3, 68.3, 65.9, 62.7, 26.9, 19.1. HRMS (ESI) m/z: found [M + Na]+ 1144.3368, C61H66Cl3NO11Si calcd. for [M + Na]+ 1144.3368.
Molecules 21 00614 i004
tert-Butyldiphenylsilyl 3-O-allyl-2,4,6-tri-O-benzyl-β-d-galactopyranosyl-(1→3)-6-O-benzyl-2-deoxy-2-tri-chloroacetamido-β-d-glucopyranoside (7). To a mixture of 6 (814 mg, 0.73 mmol) and molecular sieves 4A (4.20 g) in CH2Cl2 (7.3 mL) was added triethylsilane (463 μL, 2.90 mmol) and trifluoromethane sulfonic acid (127 μL, 1.45 mmol) at −78 °C under Ar, and stirred for 1 h at −78 °C, and 1.5 h at −40 °C. The reaction mixture was quenched with triethylamine, filtered through Celite, and diluted with CHCl3. The organic layer was successively washed with sat. NaHCO3, water, and brine, dried over Na2SO4, and concentrated. The crude product was chromatographed on silica gel with toluene–EtOAc (89:11) to give the title product 7 (665 mg, 82%). [α]D +1.5° (c 1.3, CHCl3); 1H-NMR (500 MHz, CDCl3) δ 7.72–7.16 (m, 30H, Ar), 6.70 (d, 1H, J2,NH = 7.1 Hz, NH), 5.93–5.86 (m, 1H, H2C=CHCH2), 5.32–5.29 (m, 1H, H2C=CHCH2), 5.19–5.17 (m, 1H, H2C=CHCH2), 5.11 (d, 1H, J1,2 = 8.0 Hz, H-1GlcN), 4.88 (d, 1H, Jgem = 11.6 Hz, PhCH2), 4.82 (d, 1H, Jgem = 11.3 Hz, PhCH2), 4.72 (d, 1H, PhCH2), 4.53 (d, 1H, PhCH2), 4.42–4.34 (m, 4H, PhCH2 × 4), 4.23 (d, 1H, J1,2 = 7.8 Hz, H-1Gal), 4.19–4.12 (m, 2H, H2C=CHCH2), 4.03 (dd, 1H, J2,3 = 10.1 Hz, J3,4 = 8.4 Hz, H-3GlcN), 3.80 (d, 1H, J3,4 = 2.9 Hz, H-4Gal), 3.75 (dd, 1H, J2,3 = 9.8 Hz, H-2Gal), 3.69 (s, 1H, OH), 3.58–3.47 (m, 6H, H-4GlcN, H-5Gal, H-6aGlcN, H-6aGal, H-6bGlcN, H-6bGal), 3.38–3.33 (m, 1H, H-2GlcN), 3.29 (dd, 1 H, H-3Gal), 3.17–3.13 (m, 1 H, H-5GlcN), 1.06 (s, 9 H, tBu); 13C-NMR (125 MHz, CDCl3) δ 161.5, 139.1, 138.6, 138.4, 137.6, 136.0, 135.8, 134.7, 133.1, 132.6, 129.7, 129.7, 128.4, 128.4, 128.2, 128.2, 127.9, 127.8, 127.8, 127.6, 127.5, 127.4, 127.3, 127.3, 116.8, 103.5, 93.7, 92.1, 81.7, 81.1, 79.5, 75.7, 75.1, 74.6, 73.6, 73.5, 73.4, 73.4, 71.7, 69.2, 69.1, 68.3, 61.3, 26.9, 19.1. HRMS (ESI) m/z: found [M + Na]+ 1146.3525, C61H68Cl3NO11Si calcd. for [M + Na]+ 1146.3525.
Molecules 21 00614 i005
tert-Butyldiphenylsilyl 3-O-allyl-2,4,6-tri-O-benzyl-β-d-galactopyranosyl-(1→3)-[3,4-di-O-acetyl-6-2-O-p-methoxybenzyl-α-l-fucopyranosyl-(1→4)]-6-O-benzyl-2-deoxy-2-trichloroacetamido-β-d-glucopyranoside (9). To a mixture of 7 (1.96 g, 1.74 mmol), 3,4-di-O-acetyl-2-O-p-methoxybenzyl-l-fucipyranosyl (N-phenyl)-2,2,2-trifluoroacetimidate 8 (1.87 g, 3.47 mmol), and molecular sieves AW-300 (5.22 g) in CPME/CH2Cl2 (1:1, 58.0 mL) was added TMSOTf (15.7 μL, 0.087 mmol) dropwise at −40 °C under Ar, and stirred for 1 h at −40 °C. The reaction mixture was quenched with sat. NaHCO3, filtered through Celite, and diluted with CHCl3. The organic layer was separated, and the aqueous layer was extracted with CHCl3. The combined organic layer was successively washed with water and brine, dried over Na2SO4, and concentrated. The crude product was chromatographed on silica gel with hexane–acetone (80:20) to give the title product 9 (2.44 g, 95%). [α]D −19.6° (c 1.3, CHCl3); 1H-NMR (500 MHz, CDCl3) δ 7.72–7.11 (m, 32H, Ar), 6.93 (d, 1H, J2,NH = 6.8 Hz, NH), 6.81–6.78 (m, 2H, Ar), 5.95–5.87 (m, 1H, H2C=CHCH2), 5.35–5.32 (m, 1H, H2C=CHCH2), 5.21–5.11 (m, 5H, H2C=CHCH2, H-1GlcN, H-1Fuc, H-3Fuc, H-4Fuc), 5.09–5.05 (m, 1H, H-5Fuc), 4.88 (d, 1H, Jgem = 10.5 Hz, ArCH2), 4.74–4.69 (m, 2H, ArCH2 × 2), 4.55–4.49 (m, 3H, ArCH2 × 3), 4.46–4.39 (m, 3H, ArCH2 × 2, H-1Gal), 4.34 (d, 1H, Jgem = 12.6 Hz, ArCH2), 4.24–4.08 (m, 4H, H2C=CHCH2 × 2, ArCH2, H-3GlcN), 3.87–3.77 (m, 7H, OMe, H-4GlcN, H-4Gal, H-6aGal, H-2Fuc), 3.73–3.68 (m, 2H, H-6aGlcN, H-6bGal), 3.59 (dd, 1H, J1,2 = 8.0 Hz, J2,3 = 9.7 Hz, H-2Gal), 3.34 (dd, 1H, J5,6a = 4.9 Hz, J5,6b = 8.9 Hz, H-5Gal), 3.30–3.27 (m, 2H, H-2GlcN, H-3Gal), 3.09 (dd, 1H, J5,6b = 1.5 Hz, Jgem = 11.7 Hz, H-6bGlcN), 2.98–2.96 (m, 1H, H-5GlcN), 2.11 (s, 3H, Ac), 1.97 (s, 3H, Ac), 1.06 (s, 9H, tBu), 0.78 (d, 3H, J5,6 = 6.5 Hz, H-6Fuc); 13C-NMR (125 MHz, CDCl3) δ 170.3, 169.1, 160.9, 159.3, 138.9, 138.5, 138.5, 138.4, 135.8, 135.7, 134.9, 133.4, 132.6, 130.1, 129.7, 129.6, 128.8, 128.5, 128.5, 128.3, 128.3, 128.2, 128.1, 128.0, 127.6, 127.5, 127.4, 127.4, 127.3, 116.8, 113.7, 103.0, 97.1, 93.4, 92.2, 81.7, 79.9, 77.6, 76.0, 75.7, 75.0, 74.6, 73.5, 73.4, 73.3, 73.2, 73.1, 72.6, 72.3, 71.2, 70.8, 67.9, 67.0, 64.2, 55.2, 26.9, 20.9, 20.8, 19.2, 15.4. HRMS (ESI) m/z: found [M + Na]+ 1496.4890, C79H90Cl3NO18Si calcd. for [M + Na]+ 1496.4890.
Molecules 21 00614 i006
tert-Butyldiphenylsilyl 3-O-allyl-2,4,6-tri-O-benzyl-β-d-galactopyranosyl-(1→3)-[3,4-di-O-acetyl-α-l-fucopyranosyl-(1→4)]-6-O-benzyl-2-deoxy-2-trichloroacetamido-β-d-glucopyranoside (10). A solution of 9 (29.3 mg, 19.9 μmol) in trifluoroacetic acid/CH2Cl2 (1:9, 0.80 mL) was stirred for 20 min at room temperature. The reaction mixture was diluted with toluene, and evaporated. The residue was dissolved with CHCl3, successively washed with sat. NaHCO3, water, and brine, dried over Na2SO4, and concentrated. The crude product was chromatographed on silica gel with hexane–EtOAC (80:20) to give the title product 10 (27.0 g, quant.). [α]D −33.2° (c 1.1, CHCl3); 1H-NMR (500 MHz, CDCl3) δ 7.69–7.15 (m, 30H, Ar), 6.98 (d, 1H, J2,NH = 6.7 Hz, NH), 5.97–5.89 (m, 1H, H2C=CHCH2), 5.36 (dd, 1H, Jtrans = 17.3 Hz, Jgem = 1.6 Hz, H2C=CHCH2), 5.23–5.19 (m, 2H, H2C=CHCH2, H-1GlcN), 5.12–5.10 (m, 2H, H-1Fuc, H-4Fuc), 5.02–4.95 (m, 2H, H-5Fuc, H-3Fuc), 4.91 (d, 1H, Jgem = 10.4 Hz, PhCH2), 4.75 (d, 1H, Jgem = 11.1 Hz, PhCH2), 4.68 (d, 1H, PhCH2), 4.53 (d, 1H, PhCH2), 4.47–4.35 (m, 5H, PhCH2 × 4, H-1Gal), 4.22–4.11 (m, 3H, H-3GlcN, H2C=CHCH2 × 2), 3.92 (t, 1H, J3,4 = J4,5 = 9.4 Hz, H-4GlcN), 3.85–3.80 (m, 2H, H-2Fuc, H-4Gal), 3.70–3.56 (m, 4H, H-6aGal, H-6bGal, H-6aGlcN, H-2Gal), 3.34 (dd, 1H, J5,6a = 4.9 Hz, J5,6b = 8.7 Hz, H-5Gal), 3.30 (dd, 1H, J2,3 = 9.8 Hz, J3,4 = 2.8 Hz, H-3Gal), 3.22–3.19 (m, 2H, H-6bGlcN, H-2GlcN), 2.95–2.93 (m, 1H, H-5GlcN), 2.13 (s, 3H, Ac), 2.05 (s, 3H, Ac), 1.75 (d, 1H, J2,OH = 10.8 Hz, OH), 1.03 (s, 9H, tBu), 0.81 (d, 3H, J5,6 = 6.5 Hz, H-6Fuc); 13C-NMR (125 MHz, CDCl3) δ 170.4, 170.2, 161.0, 138.8, 138.4, 138.2, 138.0, 135.9, 135.7, 134.8, 133.2, 132.5, 129.7, 129.7, 128.7, 128.6, 128.5, 128.3, 128.3, 128.2, 128.2, 127.7, 127.6, 127.5, 127.3, 116.9, 103.0, 97.5, 93.2, 92.1, 81.8, 80.1, 77.6, 76.2, 75.9, 74.6, 73.4, 73.3, 73.0, 72.9, 72.4, 71.8, 71.3, 67.9, 67.3, 67.2, 64.6, 63.4, 29.7, 26.9, 21.0, 20.7, 19.2, 15.4. HRMS (ESI) m/z: found [M + Na]+ 1376.4315, C71H82Cl3NO17Si calcd. for [M + Na]+ 1376.4315.
Molecules 21 00614 i007
tert-Butyldiphenylsilyl 3-O-allyl-2,4,6-tri-O-benzyl-β-d-galactopyranosyl-(1→3)-[2,3,4-tri-O-acetyl-α-l-fucopyranosyl-(1→4)]-6-O-benzyl-2-deoxy-2-trichloroacetamido-β-d-glucopyranoside (11). To a solution of 10 (9.32 g, 6.87 mmol) in pyridine (458 mL) was added acetic anhydride (458 mL) at 0 °C under Ar, and stirred for 7 h at room temperature. The reaction mixture was concentrated. The crude product was chromatographed on silica gel with hexane–EtOAc (67:33) to give the title product 11 (9.45 g, 98%). [α]D −37.1° (c 1.2, CHCl3); 1H-NMR (500 MHz, CDCl3) δ 7.69–7.14 (m, 30H, Ar), 6.99 (d, 1H, J2,NH = 6.7 Hz, NH), 5.97–5.90 (m, 1H, H2C=CHCH2), 5.35 (dd, 1H, Jtrans = 17.3 Hz, Jgem = 1.7 Hz, H2C=CHCH2), 5.22–5.11 (m, 7H, H2C=CHCH2, H-1GlcN, H-1Fuv, H-2Fuc, H-3Fuc, H-4Fuc, H-5Fuc), 4.91 (d, 1H, Jgem = 10.3 Hz, PhCH2), 4.75 (d, 1H, Jgem = 11.0 Hz, PhCH2), 4.67 (d, 1H, PhCH2), 4.53–4.50 (m, 2H, PhCH2 × 2), 4.46–4.43 (m, 2H, PhCH2, H-1Gal), 4.38 (d, 1H, Jgem = 12.5 Hz, PhCH2), 4.31 (d, 1H, PhCH2), 4.25 (t, 1H, J2,3 = J3,4 = 9.4 Hz, H-3GlcN), 4.19–4.11 (m, 2H, H2C=CHCH2), 3.90 (t, 1H, J4,5 = 9.4 Hz, H-4GlcN), 3.84 (d, 1H, J3,4 =2.5 Hz, H-4Gal), 3.79–3.71 (m, 2H, H-6aGal, H-6bGal), 3.60 (dd, 1H, J1,2 = 8.0 Hz, J2,3 = 9.7 Hz, H-2Gal), 3.37–3.34 (m, 1H, H-5Gal), 3.31 (dd, 1H, H-3Gal), 3.24–3.15 (m, 3H, H-2GlcN, H-6aGlcN, H-6bGlcN), 2.99–2.97 (m, 1H, H-5GlcN), 2.15 (s, 3H, Ac), 1.97 (s, 3H, Ac), 1.96 (s, 3H, Ac), 1.03 (s, 9 H, tBu), 0.82 (d, 3H, J5,6 = 6.5 Hz, H-6Fuc); 13C-NMR (125 MHz, CDCl3) δ 170.5, 170.3, 169.3, 160.9, 138.7, 138.5, 138.4, 138.0, 135.9, 135.8, 134.9, 133.3, 132.7, 129.6, 128.7, 128.6, 128.5, 128.3, 128.3, 128.2, 127.6, 127.5, 127.4, 127.4, 127.3, 116.9, 103.1, 95.3, 93.1, 92.1, 81.8, 80.1, 76.2, 75.9, 74.7, 74.5, 73.3, 73.1, 73.1, 72.6, 72.5, 71.7, 71.2, 68.2, 68.1, 68.0, 66.8, 64.3, 63.6, 26.9, 20.8, 20.8, 20.7, 19.2, 15.3. HRMS (ESI) m/z: found [M + Na]+ 1418.4421, C73H84Cl3NO18Si calcd. for [M + Na]+ 1418.4421.
Molecules 21 00614 i008
3-O-Allyl-2,4,6-tri-O-benzyl-β-d-galactopyranosyl-(1→3)-[2,3,4-tri-O-acetyl-α-l-fucopyranosyl-(1→4)]-6-O-benzyl-2-deoxy-2-trichloroacetamido-d-glucopyranose (12). To a solution of 11 (1.67 g, 1.19 mmol) in THF (11.9 mL) were added acetic acid (0.68 mL, 11.9 mmol) and 1 M tetra-n-butylammonium fluoride in THF (4.76 mL, 4.76 mmol) at 0 °C under Ar, and stirred for 1 d at room temperature. The reaction mixture was concentrated. The residue was diluted with EtOAc and water, and extracted with EtOAc. The combined organic layer was successively washed with sat. NaHCO3, water, and brine, dried over Na2SO4, and concentrated. The crude product was purified by silica gel column chromatography with hexane–EtOAc (60:40) and gel permeation chromatography (LH-20, CHCl3-MeOH (50:50)) to give the title product 12 (1.38 g, quant.). 1H-NMR (500 MHz, CDCl3) δ 7.41–7.20 (m, 20H, Ar), 6.76 (d, 1H, J2,NH = 9.7 Hz, NH), 5.87–5.80 (m, 1H, H2C=CHCH2), 5.30–5.12 (m, 7H, H2C=CHCH2 × 2, H-1GlcN, H-1Fuc, H-2Fuc, H-3Fuc, H-4Fuc), 4.97 (dd, 1H, J4,5 = 12.8 Hz, J5,6 = 6.5 Hz, H-5Fuc), 4.87 (d, 1H, Jgem = 12.1 Hz, PhCH2), 4.72 (d, 1H, Jgem = 11.4 Hz, PhCH2), 4.63–4.47 (m, 6H, PhCH2 × 5, H-1Gal), 4.44 (d, 1H, Jgem = 11.6 Hz, PhCH2), 4.34–4.28 (m, 1H, H-2GlcN), 4.19–4.00 (m, 4H, H2C=CHCH2 × 2, H-3GlcN, H-6aGlcN), 3.96–3.91 (m, 1H, H-4GlcN), 3.81–3.76 (m, 2H, H-4Gal, H-6aGal), 3.70 (dd, 1H, J5,6b = 7.4 Hz, Jgem = 9.3 Hz, H-6bGal), 3.64–3.46 (m, 3H, H-2Gal, H-5GlcN, H-6bGlcN), 3.41–3.39 (m, 1H, H-5Gal), 3.22 (dd, 1H, J2,3 = 9.7 Hz, J3,4 = 2.7 Hz, H-3Gal), 3.09–3.08 (m, 1H, OH), 2.13 (s, 3H, Ac), 2.00 (s, 3H, Ac), 1.99 (s, 3H, Ac), 0.75 (d, 3H, H-6Fuc); 13C-NMR (125 MHz, CDCl3) δ 170.5, 170.3, 169.5, 161.3, 139.0, 138.6, 138.3, 137.5, 135.0, 134.7, 129.2, 128.8, 128.5, 128.5, 128.4, 128.3, 128.2, 128.2, 128.1, 127.9, 127.8, 127.8, 127.6, 127.5, 127.1, 116.9, 116.6, 103.8, 95.8, 92.7, 91.2, 81.9, 78.6, 77.6, 74.8, 74.3, 74.2, 73.7, 73.3, 73.2, 72.8, 72.3, 71.7, 71.7, 71.1, 68.7, 68.2, 68.1, 68.1, 67.4, 64.6, 64.5, 55.8, 20.8, 20.8, 20.7, 15.4, 15.4. HRMS (ESI) m/z: found [M + Na]+ 1180.3243, C57H66Cl3NO18 calcd. for [M + Na]+ 1180.3243.
Molecules 21 00614 i009
3-O-Allyl-2,4,6-tri-O-benzyl-β-d-galactopyranosyl-(1→3)-[2,3,4-tri-O-acetyl-α-l-fucopyranosyl-(1→4)]-6-O-benzyl-2-deoxy-2-trichloroacetamido-d-glucopyranosyl (N-phenyl)-2,2,2-trifuoroacetimidate (13). A mixture of 12 (1.35 g, 1.16 mmol), (N-phenyl)-2,2,2-trifluoroacetoimidoyl chloride (482 mg, 2.32 mmol), and K2CO3 (802 mg, 5.80 mmol) in acetone (23.2 mL) was stirred for 1 h at room temperature. The reaction mixture was filtered through Celite, and concentrated. The crude product was purified by gel permeation chromatography [S-X3, toluene–EtOAc (75:25)] and silica gel column chromatography with hexane–EtOAc (71:29) to give the title product 13 (1.31 g, 85%). [α]D −0.7° (c 1.4, CHCl3); 1H-NMR (500 MHz, CDCl3) δ 7.38–7.08 (m, 23H, Ar), 6.75–6.71 (m, 3H, Ar, NH), 6.32 (br, 1H, H-1GlcN), 5.88–5.80 (m, 1H, H2C=CHCH2), 5.31–5.21 (m, 5H, H2C=CHCH2, H-1Fuc, H-2Fuc, H-3Fuc, H-4Fuc), 5.14 (dd, 1H, Jtrans = 10.5 Hz, Jgem = 1.4 Hz, H2C=CHCH2), 4.95 (dd, 1H, J4,5 = 10.5 Hz, J5,6 = 6.4 Hz, H-5Fuc), 4.84 (d, 1H, Jgem = 11.9 Hz, PhCH2), 4.73 (d, 1H, Jgem = 11.4 Hz, PhCH2), 4.64–4.49 (m, 7H, PhCH2 × 5, H-2GlcN, H-1Gal), 4.44 (d, 1H, Jgem = 11.6 Hz, PhCH2), 4.16 (t, 1H, J2,3 = J3,4 = 9.2 Hz, H-3GlcN), 4.10–4.05 (m, 3H, H2C=CHCH2 × 2, H-4GlcN), 3.87–3.81 (m, 3H, H-4Gal, H-6aGal, H-5GlcN), 3.70 (dd, 1H, J5,6b = 7.5 Hz, Jgem = 9.2 Hz, H-6bGal), 3.62–3.56 (m, 3H, H-2Gal, H-6aGlcN, H-6bGlcN), 3.46–3.44 (m, 1H, H-5Gal), 3.24 (dd, 1H, J2,3 = 9.7 Hz, J3,4 = 2.7 Hz, H-3Gal), 2.14 (s, 3H, Ac), 2.02 (s, 3H, Ac), 2.00 (s, 3H, Ac), 0.78 (d, 3 H, H-6Fuc); 13C-NMR (125 MHz, CDCl3) δ 170.4, 120.2, 169.5, 161.2, 142.9, 138.7, 138.4, 138.2, 137.5, 134.9, 129.2, 129.0, 128.8, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.8, 127.8, 127.7, 127.6, 127.3, 124.6, 119.3, 116.6, 103.8, 95.9, 92.4, 81.9, 78.5, 77.6, 74.9, 74.8, 74.2, 73.7, 73.7, 73.3, 73.2, 72.2, 71.9, 71.7, 71.6, 68.7, 68.1, 68.0, 66.7, 64.8, 54.7, 20.8, 20.8, 20.7, 15.4. HRMS (ESI) m/z: found [M + Na]+ 1351.3539, C65H70Cl3F3N2O18 calcd. for [M + Na]+ 1351.3539.
Molecules 21 00614 i010
tert-Butyldiphenylsilyl 2,4,6-tri-O-benzyl-β-d-galactopyranosyl-(1→3)-[2,3,4-tri-O-acetyl-α-l-fucopyranosyl-(1→4)]-6-O-benzyl-2-deoxy-2-trichloroacetamido-β-d-glucopyranoside (14). A mixture of Ir(COD)(PMe2Ph)2PF6 (14.2 mg, 16.8 μmol) in THF (14.0 mL) was stirrede at room temperature for 15 min under H2, and the atmosphere was replaced by Ar. To the mixture of activated Ir complex in THF was added a solution of 11 (781 mg, 0.56 mmol) in THF (14.0 mL) under Ar, and stirred for 30 min at room temperature. The reaction mixture was concentrated. The residue was dissolved with 90% acetone aq. (28.0 mL). To the solution were added HgCl2 (380 mg, 1.40 mmol) and HgO (48.5 mg, 0.22 mmol), and stirred for 1 h at room temperature. The reaction mixture was diluted with CHCl3 and water, and extracted with CHCl3. The combined organic layer was successively washed with 10% KI aq., water, and brine, dried over Na2SO4, and concentrated. The crude product was purified by silica gel column chromatography with hexane–EtOAc (75:25) and gel permeation chromatography (LH-20, CHCl3–MeOH (50:50)) to give the title product 14 (727 mg, 96%). [α]D −41.9° (c 1.1, CHCl3); 1H-NMR (500 MHz, CDCl3) δ 7.68–7.14 (m, 30H, Ar), 6.85 (d, 1H, J2,NH = 7.2 Hz, NH), 5.24–5.18 (m, 4H, H-1Fuc, H-2Fuc, H-3Fuc, H-4Fuc), 5.10 (dd, 1H, J4,5 = 12.9 Hz, J5,6 = 6.4 Hz, H-5Fuc), 5.05 (d, 1H, J1,2 = 6.7 Hz, H-1GlcN), 4.80 (d, 1H, Jgem = 11.1 Hz, PhCH2), 4.76 (d, 1H, PhCH2), 4.64 (d, 1H, Jgem = 11.2 Hz, PhCH2), 4.58–4.52 (m, 3H, PhCH2 × 3), 4.47 (d, 1H, J1,2 = 7.8 Hz, H-1Gal), 4.38 (d, 1H, Jgem = 12.5 Hz, PhCH2), 4.32 (d, 1H, PhCH2), 4.22 (t, 1H, J2,3 = J3,4 = 9.3 Hz, H-3GlcN), 3.91 (t, 1H, J4,5 = 9.3 Hz, H-4GlcN), 3.83–3.79 (m, 3H, H-4Gal, H-6aGal, H-6bGal), 3.51–3.43 (m, 2H, H-3Gal, H-5Gal), 3.36–3.32 (m, 2H, H-2GlcN, H-2Gal), 3.25 (dd, 1H, J5,6a =2.5 Hz, Jgem = 11.0 Hz, H-6aGlcN), 3.17 (dd, 1H, J5,6b = 1.4 Hz, H-6bGlcN), 2.98–2.96 (m, 1H, H-5GlcN), 2.17 (s, 3H, Ac), 2.10 (d, 1H, J3,OH = 6.8 Hz, OH), 1.99 (s, 3H, Ac), 1.96 (s, 3H, Ac), 1.16 (s, 9 H, tBu), 0.89 (d, 3H, H-6Fuc); 13C-NMR (125 MHz, CDCl3) δ 170.5, 170.2, 169.4, 161.1, 138.8, 138.4, 138.1, 137.9, 135.9, 135.8, 133.2, 132.6, 129.7, 129.7, 128.9, 128.6, 128.5, 128.3, 128.3, 128.3, 128.1, 128.1, 127.9, 127.6, 127.5, 127.5, 127.3, 102.8, 95.4, 93.6, 92.2, 81.2, 77.6, 75.8, 75.4, 75.3, 75.3, 74.6, 74.1, 73.4, 73.2, 73.2, 72.7, 71.7, 68.1, 68.1, 66.8, 64.3, 62.8, 26.9, 20.8, 20.8, 20.8, 19.2, 15.6. HRMS (ESI) m/z: found [M + Na]+ 1378.4108, C70H80Cl3NO18Si calcd. for [M + Na]+ 1378.4108.
Molecules 21 00614 i011
tert-Butyldiphenylsilyl 3-O-allyl-2,4,6-tri-O-benzyl-β-d-galactopyranosyl-(1→3)-[2,3,4-tri-O-acetyl-α-l-fucopyranosyl-(1→4)]-6-O-benzyl-2-deoxy-2-trichloroacetamido-β-d-glucopyranosyl-(1→3)-2,4,6-tri-O-benzyl-β-d-galactopyranosyl-(1→3)-[2,3,4-tri-O-acetyl-α-l-fucopyranosyl-(1→4)]-6-O-benzyl-2-deoxy-2-trichloroacetamido-β-d-glucopyranoside (15). To a mixture of the glycosyl donor 13 (343 mg, 0.26 mmol), the glycosyl acceptor 14 (234 mg, 0.17 mmol), and molecular sieves AW-300 (516 mg) in CH2Cl2 (5.7 mL) was added dropwise TMSOTf (3.1 μL, 17.2 μmol) at −40 °C under Ar, and stirred for 20 h at −40 °C. The reaction mixture was quenched with sat. NaHCO3, filtered through Celite, and diluted with CHCl3. The organic layer was separated, and the aqueous layer was extracted with CHCl3. The combined organic layer was successively washed with water and brine, dried over Na2SO4, and concentrated. The crude product was purified by gel permeation chromatography [S-X1, toluene–EtOAc (75:25)] and silica gel column chromatography with toluene–EtOAc (89:11) to give the title product 15 (399 mg, 93%). [α]D −58.2° (c 1.4, CHCl3); 1H-NMR (500 MHz, CDCl3) δ 7.65–6.98 (m, 50H, Ar), 6.62–6.60 (m, 2H, NH × 2), 5.92–5.84 (m, 1H, H2C=CHCH2), 5.47 (d, 1H, J1,2 = 7.2 Hz, H-1GlcN), 5.32–4.99 (m, 13H, H-1GlcN, H-1Fuc × 2, H-2Fuc × 2, H-3Fuc × 2, H-4Fuc × 2, H-5Fuc × 2, H2C=CHCH2 × 2), 4.78–4.70 (m, 5H, PhCH2 × 5), 4.64 (d, 1H, Jgem = 10.7 Hz, PhCH2), 4.60–4.52 (m, 3H, PhCH2 × 3), 4.50–4.40 (m, 6H, PhCH2 × 5, H-1Gal), 4.36–4.32 (m, 2H, PhCH2, H-1Gal), 4.29–4.17 (m, 3H, PhCH2, H-3GlcN × 2), 4.14–4.06 (m, 2H, H2C=CHCH2 × 2), 4.00 (t, 1H, J3,4 = J4,5 = 9.4 Hz, H-4GlcN), 3.90 (d, 1H, J3,4 = 2.3 Hz, H-4Gal), 3.85–3.77 (m, 5H, H-4Gal, H-4GlcN, H-3Gal, H-6aGal × 2), 3.70–3.69 (m, 2H, H-6bGal × 2), 3.61–3.57 (m, 3H, H-2Gal × 2, H-6aGlcN), 3.50–3.38 (m, 5H, H-6bGlcN, H-2GlcN, H-5GlcN, H-5Gal × 2), 3.26 (dd, 1H, J2,3 = 9.7 Hz, J3,4 = 2.6 Hz, H-3Gal), 3.22 (dd, 1H, J5,6a = 2.4 Hz, Jgem = 11.3 Hz, H-6aGlcN), 3.12–3.10 (m, 1H, H-6bGlcN), 2.98–2.90 (m, 2H, H-2GlcN, H-5GlcN), 2.16 (s, 3H, Ac), 2.13 (s, 3H, Ac), 2.08 (s, 3H, Ac), 2.02 (s, 3H, Ac), 1.97 (s, 3H, Ac), 1.93 (s, 3H, Ac), 1.00 (s, 9H, tBu), 0.79 (d, 3H, J5,6 = 6.5 Hz, H-6Fuc), 0.74 (d, 3H, J5,6 = 6.5 Hz, H-6Fuc); 13C-NMR (125 MHz, CDCl3) δ 170.5, 170.3, 170.2, 169.4, 169.3, 161.0, 160.8, 139.2, 138.7, 138.5, 138.4, 138.3, 138.0, 137.5, 135.8, 135.7, 134.8, 133.2, 132.6, 129.7, 129.1, 129.0, 128.9, 128.7, 128.4, 128.3, 128.3, 128.2, 128.2, 128.1, 127.9, 127.8, 127.7, 127.6, 127.5, 127.4, 127.3, 116.7, 103.2, 103.2, 95.6, 95.5, 92.2, 92.1, 81.8, 81.2, 79.3, 77.6, 76.1, 75.6, 75.4, 75.2, 75.1, 74.9, 74.6, 74.5, 73.5, 73.3, 73.1, 73.0, 72.9, 72.8, 72.4, 71.7, 71.7, 71.4, 68.2, 68.2, 68.1, 68.0, 66.8, 66.7, 64.5, 64.3, 26.9, 20.9, 20.8, 20.8, 20.7, 19.1, 15.3. HRMS (ESI) m/z: found [M + Na]+ 2517.7349, C127H144Cl6N2O35Si calcd. for [M + Na]+ 2517.7348.
Molecules 21 00614 i012
3-O-Allyl-2,4,6-tri-O-benzyl-β-d-galactopyranosyl-(1→3)-[2,3,4-tri-O-acetyl-α-l-fucopyranosyl-(1→4)]-6-O-benzyl-2-deoxy-2-trichloroacetamido-β-d-glucopyranosyl-(1→3)-2,4,6-tri-O-benzyl-β-d-galactopyranosyl-(1→3)-[2,3,4-tri-O-acetyl-α-l-fucopyranosyl-(1→4)]-6-O-benzyl-2-deoxy-2-trichloroacetamido-d-glucopyranose (16). Compound 15 (453 mg, 0.18 mmol) was desilylated with 1 M TBAF/THF (0.72 mL, 0.72 mmol) and AcOH (0.10 mL, 1.81 mmol) in THF (3.6 mL) as described for 12. The crude product was purified by gel permeation chromatography [S-X1, toluene–EtOAc (75:25)] and silica gel column chromatography with toluene-EtOAc (67:33) to give the title product 16 (395 mg, 97%). Analysis of compound 16 was too difficult for anomeric isomer, so the product was analyzed in next step. HRMS (ESI) m/z: found [M + Na]+ 2279.6171, C111H126Cl6N2O35 calcd for [M + Na]+ 2279.6170.
Molecules 21 00614 i013
3-O-Allyl-2,4,6-tri-O-benzyl-β-d-galactopyranosyl-(1→3)-[2,3,4-tri-O-acetyl-α-l-fucopyranosyl-(1→4)]-6-O-benzyl-2-deoxy-2-trichloroacetamido-β-d-glucopyranosyl-(1→3)-2,4,6-tri-O-benzyl-β-d-galactopyranosyl-(1→3)-[2,3,4-tri-O-acetyl-α-l-fucopyranosyl-(1→4)]-6-O-benzyl-2-deoxy-2-trichloroacetamido-d-gluco-pyranosyl (N-phenyl)-2,2,2-trifuoroacetimidate (17). Compound 16 (97 mg, 42.9 μmol) was reacted with (N-phenyl)-2,2,2-trifluoroacetoimidoyl chloride (17.8 mg, 85.8 μmol) and K2CO3 (29.7 mg, 215 μmol) in acetone (1.7 mL) as described for 13. The crude product was purified by silica gel column chromatography with hexane–EtOAc (83:17) and gel permeation chromatography [S-X1, toluene–EtOAc (75:25)] to give the title product 17 (94.7 mg, 91%). [α]D −35.2° (c 1.3, CHCl3); 1H-NMR (500 MHz, CDCl3) δ 7.38–7.07 (m, 43H, Ar), 6.82 (d, 1H, J2,NH = 9.4 Hz, NH), 6.71–6.69 (m, 2H, Ar), 6.26 (br, 1H, H-1GlcN), 6.00 (d, 1H, J2,NH = 4.7 Hz, NH), 5.88–5.81 (m, 1H, H2C=CHCH2), 5.30–5.14 (m, 10H, H2C=CHCH2 × 2, H-1Fuc × 2, H-2Fuc × 2, H-3Fuc × 2, H-4Fuc × 2), 5.07 (d, 1H, J1,2 = 8.1 Hz, H-1GlcN), 5.03–5.00 (m, 2H, H-5Fuc, PhCH2), 4.94 (dd, 1H, J4,5 = 12.7 Hz, J5,6 = 6.4 Hz, H-5Fuc), 4.78 (d, 1H, Jgem = 11.6 Hz, PhCH2), 4.71 (d, 1H, Jgem = 11.2 Hz, PhCH2), 4.63 (d, 1H, Jgem = 11.9 Hz, PhCH2), 4.60–4.37 (m, 13H, PhCH2 ×11, H-1Gal, H-2GlcN), 4.32 (d, 1H, J1,2 = 7.8 Hz, H-1Gal), 4.20 (t, 1H, J2,3 = J3,4 = 9.2 Hz, H-3GlcN), 4.07–4.01 (m, 3H, H2C=CHCH2 × 2, H-4GlcN), 3.93–3.77 (m, 8H, H-4GlcN, H-2GlcN, H-6aGal, H-6aGal, H-4Gal, H-5GlcN, H-4Gal, H-6bGal), 3.76–3.65 (m, 3H, H-3Gal, H-3GlcN, H-2Gal), 3.62–3.52 (m, 5H, H-6aGlcN, H-6bGlcN, H-6aGlcN, H-6bGal, H-2Gal), 3.49–3.44 (m, 2H, H-5Gal, H-6bGlcN), 3.38 (dd, 1H, J5,6a = 5.5 Hz, J5,6b = 7.5 Hz, H-5Gal), 3.21–3.18 (m, 2H, H-3Gal, H-5GlcN), 2.16 (s, 3H, Ac), 2.15 (s, 3H, Ac), 2.14 (s, 3H, Ac), 2.05 (s, 3H, Ac), 2.01 (s, 6H, Ac × 2), 0.85 (d, 3H, H-6Fuc), 0.69 (d, 3H, J5,6 = 6.4 Hz, H-6Fuc); 13C-NMR (125 MHz, CDCl3) δ 170.4, 170.3, 170.2, 170.1, 169.4, 160.9, 160.7, 142.7, 139.0, 138.8, 138.4, 138.3, 138.2, 138.1, 137.8, 137.6, 137.3, 134.9, 129.3, 129.0, 129.0, 128.8, 128.5, 128.4, 128.4, 128.3, 128.2, 128.2, 128.2, 128.1, 128.1, 127.9, 127.8, 127.7, 127.7, 127.6, 127.5, 127.3, 127.3, 125.2, 124.6, 119.2, 117.0, 116.6, 114.8, 103.4, 103.2, 99.4, 96.0, 95.6, 93.3, 92.6, 92.4, 81.8, 80.8, 78.5, 78.1, 77.6, 76.5, 75.1, 74.9, 74.7, 74.6, 74.4, 74.3, 74.1, 73.4, 73.3, 73.2, 72.9, 72.8, 72.4, 72.0, 71.6, 71.5, 71.4, 68.8, 68.5, 68.2, 68.1, 68.0, 67.9, 67.1, 66.7, 64.8, 64.5, 58.7, 54.6, 30.9, 29.6, 21.4, 20.9, 20.8, 20.8, 20.7, 20.7, 15.7, 15.2, 14.1. HRMS (ESI) m/z: found [M + Na]+ 2450.6466, C119H130Cl6F3N3O35 calcd. for [M + Na]+ 2450.6466.
Molecules 21 00614 i014
tert-Butyldiphenylsilyl 2,4,6-tri-O-benzyl-β-d-galactopyranosyl-(1→3)-[2,3,4-tri-O-acetyl-α-l-fucopyranosyl-(1→4)]-6-O-benzyl-2-deoxy-2-trichloroacetamido-β-d-glucopyranosyl-(1→3)-2,4,6-tri-O-benzyl-β-d-galactopyranosyl-(1→3)-[2,3,4-tri-O-acetyl-α-l-fucopyranosyl-(1→4)]-6-O-benzyl-2-deoxy-2-trichloroacetamido-β-d-glucopyranoside (18). Compound 15 (104 mg, 41.6 μmol) was deallylated by Ir(COD)(PMe2Ph)2PF6 (1.1 mg, 1.25 μmol) in THF (1.0 mL × 2) and deprotected by HgCl2 (28.2 mg, 104 μmol) and HgO (3.6 mg, 16.6 μmol) with 90% acetone aq. (2.1 mL) as described for 14. The crude product was purified by silica gel column chromatography with hexane–EtOAc (83:17) and gel permeation chromatography [S-X1, toluene–EtOAc (75:25)] to give the title product 18 (92.2 mg, 90%). [α]D −46.2° (c 1.0, CHCl3); 1H-NMR (500 MHz, CDCl3) δ 7.65–7.12 (m, 50H, Ar), 6.63 (d, 1H, J2,NH = 7.3 Hz, NH), 6.27 (br, 1H, NH), 5.33–5.08 (m, 10H, H-1GlcN, H-1Fuc × 2, H-2Fuc × 2, H-3Fuc × 2, H-4Fuc × 2, H-5Fuc), 5.00 (dd, 1H, J4,5 = 12.3 Hz, J5,6 = 6.4 Hz, H-5Fuc), 4.91 (br, 1H, H-1GlcN), 4.84–4.82 (m, 2H, PhCH2 × 2), 4.74 (d, 1H, Jgem = 10.9 Hz, PhCH2), 4.67 (d, 1H, Jgem = 11.3 Hz, PhCH2), 4.62–4.53 (m, 6H, PhCH2 × 6), 4.51–4.46 (m, 3H, PhCH2 × 3), 4.43–4.28 (m, 5H, H-1Gal × 2, PhCH2 × 3), 4.15–4.12 (m, 1H, H-3GlcN), 4.04–4.00 (m, 2H, H-3GlcN, H-4GlcN), 3.90–3.87 (m, 2H, H-4Gal, H-6aGal), 3.85–3.81 (m, 3H, H-6bGal, H-4Gal, H-4GlcN), 3.79–3.68 (m, 4H, H-3Gal, H-6aGal, H-6bGal, H-2GlcN), 3.64–3.60 (m, 2H, H-2Gal, H-6aGlcN), 3.52 (dd, 1H, J5,6b = 2.0 Hz, Jgem = 10.7 Hz, H-6bGlcN), 3.49–3.40 (m, 3H, H-5Gal × 2, H-3Gal), 3.36–3.32 (m, 2H, H-5GlcN, H-2Gal), 3.24–3.12 (m, 3H, H-6GlcN × 2, H-2GlcN), 2.92 (d, J4,5 = 9.6 Hz, H-5GlcN), 2.18 (s, 3H, Ac), 2.14 (s, 3H, Ac), 2.09 (s, 3H, Ac), 2.03 (s, 3H, Ac), 2.00 (d, 1H, J3,OH = 5.2 Hz, OH), 1.98 (s, 3H, Ac), 1.93 (s, 3H, Ac), 1.00 (s, 9H, tBu), 0.84 (d, 3H, J5,6 = 6.4 Hz, H-6Fuc), 0.76 (d, 3H, H-6Fuc); 13C-NMR (125 MHz, CDCl3) δ 170.5, 170.3, 170.2, 170.1, 169.5, 169.3, 161.0, 160.7, 139.2, 138.6, 138.5, 138.4, 138.2, 138.1, 137.9, 137.8, 137.4, 135.8, 135.7, 133.1, 132.5, 129.7, 129.6, 129.1, 129.0, 129.0, 128.7, 128.4, 128.4, 128.3, 128.3, 128.2, 128.2, 128.1, 128.0, 128.0, 127.8, 127.8, 127.8, 127.7, 127.6, 127.5, 127.4, 127.4, 127.3, 125.3, 103.1, 99.0, 95.7, 95.5, 93.6, 92.4, 92.3, 81.5, 80.1, 77.6, 77.5, 76.2, 75.3, 75.2, 75.1, 75.1, 75.0, 74.9, 74.9, 74.7, 74.1, 73.9, 73.7, 73.5, 73.1, 73.1, 72.9, 72.9, 72.8, 71.6, 71.6, 68.2, 68.1, 68.1, 68.0, 67.9, 66.8, 66.6, 64.4, 64.3, 62.2, 59.3, 31.9, 29.6, 29.3, 26.8, 26.7, 22.6, 21.4, 20.9, 20.8, 20.8, 20.7, 20.7, 19.1, 18.8, 15.6, 15.4, 14.1. HRMS (ESI) m/z: found [M + Na]+ 2477.7035, C124H140Cl6N2O35Si calcd. for [M + Na]+ 2477.7035.
Molecules 21 00614 i015
tert-Butyldiphenylsilyl 3-O-allyl-2,4,6-tri-O-benzyl-β-d-galactopyranosyl-(1→3)-[2,3,4-tri-O-acetyl-α-l-fucopyranosyl-(1→4)]-6-O-benzyl-2-deoxy-2-trichloroacetamido-β-d-glucopyranosyl-(1→3)-2,4,6-tri-O-benzyl-β-d-galactopyranosyl-(1→3)-[2,3,4-tri-O-acetyl-α-l-fucopyranosyl-(1→4)]-6-O-benzyl-2-deoxy-2-trichloroacetamido-β-d-glucopyranosyl-(1→3)-2,4,6-tri-O-benzyl-β-d-galactopyranosyl-(1→3)-[2,3,4-tri-O-acetyl-α-l-fucopyranosyl-(1→4)]-6-O-benzyl-2-deoxy-2-trichloroacetamido-β-d-glucopyranosyl-(1→3)-2,4,6-tri-O-benzyl-β-d-galactopyranosyl-(1→3)-[2,3,4-tri-O-acetyl-α-l-fucopyranosyl-(1→4)]-6-O-benzyl-2-deoxy-2-trichloroacetamido-β-d-glucopyranoside (19). To a mixture of the glycosyl donor 17 (276 mg, 113 μmol), the glycosyl acceptor 18 (210 mg, 85.0 μmol), and molecular sieves AW-300 (255 mg) in CH2Cl2 (2.8 mL) was added dropwise TMSOTf (3.0 μL, 17.0 μmol) at 0 °C under Ar, and stirred for 1 h at 0 °C. The reaction mixture was quenched with sat. NaHCO3, filtered through Celite, and diluted with CHCl3. The organic layer was separated, and the aqueous layer was extracted with CHCl3. The combined organic layer was successively washed with brine, dried over Na2SO4, and concentrated. The crude product was purified by gel permeation chromatography [S-X1, toluene–EtOAc (75:25)] and silica gel column chromatography with toluene–EtOAc (83:17) to give the title product 19 (352 mg, 88%). [α]D −50.5° (c 1.0, CHCl3); 1H-NMR (500 MHz, CDCl3) δ 7.65–7.02 (m, 90H, Ar), 6.67 (d, 1H, J2,NH = 7.1 Hz, NH), 6.27 (brs, 1H, NH), 5.97 (m, 2H, NH × 2), 5.86-5.78 (m, 1H, H2C=CHCH2), 5.43–4.87 (m, 26H, H-1Fuc × 4, H-2Fuc × 4, H-3Fuc × 4, H-4Fuc × 4, H-5Fuc × 4, H-1GlcN × 4, H2C=CHCH2 × 2), 4.84–4.27 (m, 37H, H-1Gal × 4, H-2GlcN, PhCH2 × 32), 4.22–3.12 (m, 48H, H-2Gal × 4, H-3Gal × 4, H-4Gal × 4, H-5Gal × 4, H-6aGal × 4, H-6bGal × 4, H-2GlcN × 3, H-3GlcN × 4, H-4GlcN × 4, H-5GlcN × 3, H-6aGlcN × 4, H-6bGlcN × 4, H2C=CHCH2 × 2), 2.93 (d, 1H, J4,5 = 9.6 Hz, H-5GlcN), 2.21–1.88 (m, 36H, Ac × 12), 1.01 (s, 9H, tBu), 0.89–0.67 (m, 12H, H-6Fuc × 4); 13C-NMR (125 MHz, CDCl3) δ 171.1, 170.5, 170.4, 170.3, 170.2, 170.2, 169.6, 169.5, 169.4, 160.9, 160.7, 139.4, 139.2, 138.9, 138.6, 138.6, 138.5, 138.5, 138.4, 138.0, 137.9, 137.7, 137.6, 137.5, 136.0, 135.8, 135.0, 133.2, 132.6, 130.0, 129.8, 129.3, 129.2, 129.2, 129.1, 128.8, 128.7, 128.6, 128.5, 128.5, 128.4, 128.4, 128.3, 128.3, 128.2, 128.1, 128.1, 128.0, 127.9, 127.8, 127.8, 127.8, 127.7, 127.6, 127.6, 127.4, 125.4, 116.7, 103.5, 103.3, 103.1, 99.2, 99.1, 98.9, 96.0, 95.9, 95.7, 95.6, 93.7, 92.8, 92.6, 92.4, 81.9, 81.5, 81.4, 78.7, 77.6, 77.4, 76.6, 76.2, 75.6, 75.6, 75.4, 75.3, 75.3, 75.1, 75.0, 75.0, 74.8, 74.5, 74.1, 74.0, 73.9, 73.6, 73.3, 73.3, 73.3, 73.1, 73.0, 73.0, 72.9, 72.6, 72.8, 71.8, 71.7, 68.6, 68.5, 68.3, 68.2, 68.1, 67.0, 66.8, 64.6, 64.5, 64.4, 62.4, 60.4, 59.4, 58.9, 32.0, 29.7, 29.4, 27.0, 22.8, 21.5, 21.1, 21.0, 20.9, 20.9, 20.8, 20.8, 19.2, 15.8, 15.6, 15.4, 14.3, 14.2. HRMS (ESI) m/z: found [1/2M + Na]+ 2369.6540, C235H264Cl12N4O69Si calcd for [1/2M + Na]+ 2369.6544.
Molecules 21 00614 i016
3-O-Allyl-2,4,6-tri-O-benzyl-β-d-galactopyranosyl-(1→3)-[2,3,4-tri-O-acetyl-α-l-fucopyranosyl-(1→4)]-6-O-benzyl-2-deoxy-2-trichloroacetamido-β-d-glucopyranosyl-(1→3)-2,4,6-tri-O-benzyl-β-d-galactopyranosyl-(1→3)-[2,3,4-tri-O-acetyl-α-l-fucopyranosyl-(1→4)]-6-O-benzyl-2-deoxy-2-trichloroacetamido-β-d-glucopyranosyl-(1→3)-2,4,6-tri-O-benzyl-β-d-galactopyranosyl-(1→3)-[2,3,4-tri-O-acetyl-α-l-fucopyranosyl-(1→4)]-6-O-benzyl-2-deoxy-2-trichloroacetamido-β-d-glucopyranosyl-(1→3)-2,4,6-tri-O-benzyl-β-d-galactopyranosyl-(1→3)-[2,3,4-tri-O-acetyl-α-l-fucopyranosyl-(1→4)]-6-O-benzyl-2-deoxy-2-trichloroacetamido-d-glucopyranose (20). Compound 19 (324 mg, 68.9 μmol) was desilylated with 1 M TBAF/THF (0.28 mL, 0.28 mmol) and AcOH (39.0 μL, 0.69 mmol) in THF (1.4 mL) as described for 12. The crude product was purified by gel permeation chromatography [S-X1, toluene–EtOAc (75:25)] and silica gel column chromatography with toluene–EtOAc (71:29) to give the title product 20 (298 mg, 97%). [α]D −42.5° (c 1.0, CHCl3); 1H-NMR (500 MHz, CDCl3) δ 7.65–7.02 (m, 83H, Ar, NH × 3), 6.70 (d, 1H, J2,NH = 9.9 Hz, NH), 5.86–5.79 (m, 1H, H2C=CHCH2), 5.63–3.11 (m, 112H, H-1Gal × 4, H-2Gal × 4, H-3Gal × 4, H-4Gal × 4, H-5Gal × 4, H-6aGal × 4, H-6bGal × 4, H-1GlcN × 4, H-2GlcN × 4, H-3GlcN × 4, H-4GlcN × 4, H-5GlcN × 4, H-6aGlcN × 4, H-6bGlcN × 4, H-1Fuc × 4, H-2Fuc × 4, H-3Fuc × 4, H-4Fuc × 4, H-5Fuc × 4, H2C=CHCH2 × 2, H2C=CHCH2 × 2, PhCH2 × 32), 2.17–1.99 (m, 36H, Ac × 12), 0.89–0.67 (m, 12H, H-6Fuc × 4); 13C-NMR (125 MHz, CDCl3) δ 170.4, 170.3, 170.3, 170.3, 170.2, 170.2, 169.5, 169.5, 169.4, 160.7, 160.3, 139.5, 139.5, 139.4, 138.9, 138.5, 138.4, 138.4, 138.4, 138.3, 138.2, 138.1, 137.8, 137.6, 137.5, 137.5, 137.4, 134.9, 129.1, 129.1, 129.0, 128.7, 128.6, 128.5, 128.4, 128.4, 128.4, 128.3, 128.3, 128.2, 128.2, 128.2, 128.1, 128.1, 128.1, 128.0, 127.8, 127.8, 127.7, 127.7, 127.7, 127.7, 127.6, 127.6, 127.6, 127.4, 127.3, 127.3, 127.3, 127.2, 125.3, 116.5, 103.5, 103.4, 103.1, 99.4, 99.3, 99.0, 96.0, 95.8, 95.6, 93.0, 92.9, 92.8, 92.8, 92.6, 91.2, 82.0, 81.8, 81.6, 78.3, 77.6, 76.6, 76.5, 75.7, 75.5, 75.4, 75.3, 75.1, 75.0, 74.8, 74.8, 74.7, 74.7, 74.6, 74.3, 74.2, 74.1, 74.0, 73.4, 73.3, 73.2, 73.2, 73.0, 72.8, 72.8, 72.4, 71.7, 71.6, 71.6, 70.8, 68.6, 68.5, 68.2, 68.1, 68.0, 67.2, 66.9, 66.8, 64.6, 64.4, 57.8, 57.6, 55.6, 29.7, 29.3, 29.3, 21.4, 20.9, 20.9, 20.8, 20.8, 15.8, 15.7, 15.6, 15.5, 15.2, 14.1. HRMS (ESI) m/z: found [1/2M + Na]+ 2250.5955, C219H246Cl12N4O69 calcd for [1/2M + Na]+ 2250.5955.
Molecules 21 00614 i017
3-O-Allyl-2,4,6-tri-O-benzyl-β-d-galactopyranosyl-(1→3)-[2,3,4-tri-O-acetyl-α-l-fucopyranosyl-(1→4)]-6-O-benzyl-2-deoxy-2-trichloroacetamido-β-d-glucopyranosyl-(1→3)-2,4,6-tri-O-benzyl-β-d-galactopyranosyl-(1→3)-[2,3,4-tri-O-acetyl-α-l-fucopyranosyl-(1→4)]-6-O-benzyl-2-deoxy-2-trichloroacetamido-β-d-glucopyranosyl-(1→3)-2,4,6-tri-O-benzyl-β-d-galactopyranosyl-(1→3)-[2,3,4-tri-O-acetyl-α-l-fucopyranosyl-(1→4)]-6-O-benzyl-2-deoxy-2-trichloroacetamido-β-d-glucopyranosyl-(1→3)-2,4,6-tri-O-benzyl-β-d-galactopyranosyl-(1→3)-[2,3,4-tri-O-acetyl-α-l-fucopyranosyl-(1→4)]-6-O-benzyl-2-deoxy-2-trichloroacetamido-d-glucopyranosyl (N-phenyl)-2,2,2-trifuoroacetimidate (21). Compound 20 (761 mg, 0.17 mmol) was reacted with (N-phenyl)-2,2,2-trifluoroacetoimidoyl chloride (55.0 μL, 0.34 mmol) and K2CO3 (117 mg, 0.85 mmol) in acetone (6.8 mL) as described for 13. The crude product was purified by silica gel column chromatography with hexane–EtOAc (83:17) to give the title product 21 (693 mg, 88%). Analysis of compound 21 was too difficult for a few isomers, so the product was analyzed in the next step. 1H-NMR of product 21 could not be assigned because all peaks were shown as broad peaks in all range. 13C-NMR (125 MHz, CDCl3) δ 170.4, 170.3, 170.2, 170.2, 170.2, 170.2, 169.5, 169.5, 160.8, 160.7, 160.4, 142.7, 139.4, 139.3, 139.1, 138.8, 138.5, 138.4, 138.4, 138.3, 138.3, 138.2, 138.1, 137.8, 137.6, 137.5, 137.5, 137.3, 134.9, 129.2, 129.1, 129.0, 129.0, 128.8, 128.6, 128.5, 128.4, 128.4, 128.3, 128.3, 128.2, 128.2, 128.1, 127.9, 127.8, 127.7, 127.6, 127.6, 127.6, 127.6, 127.4, 127.4, 127.3, 127.3, 127.2, 125.3, 124.7, 119.2, 117.0, 116.5, 114.8, 103.5, 103.4, 103.1, 99.3, 99.2, 99.0, 96.1, 95.8, 95.8, 95.6, 93.4, 92.9, 92.7, 92.4, 81.8, 81.6, 78.4, 77.6, 76.6, 76.5, 76.4, 75.6, 75.5, 75.4, 75.3, 75.2, 75.0, 74.8, 74.8, 74.7, 74.3, 74.1, 74.1, 74.0, 73.4, 73.3, 73.2, 73.0, 72.9, 72.9, 72.8, 72.8, 72.4, 72.0, 71.7, 71.6, 71.5, 68.5, 68.1, 68.1, 68.0, 67.9, 67.0, 66.9, 66.8, 66.6, 64.7, 64.5, 64.4, 58.3, 57.9, 54.7, 31.9, 30.9, 29.7, 29.3, 22.7, 21.4, 20.9, 20.9, 20.8, 20.8, 20.7, 15.8, 15.7, 15.6, 15.2, 14.1. HRMS (ESI) m/z: found [1/2M + Na]+ 2336.1103, C227H250Cl12F3N5O69 calcd for [1/2M + Na]+ 2329.1103.
Molecules 21 00614 i018
N-(tert-Butoxycarbonyl)-3-aminopropyl 3-O-allyl-2,4,6-tri-O-benzyl-β-d-galactopyranosyl-(1→3)-[2,3,4-tri-O-acetyl-α-l-fucopyranosyl-(1→4)]-6-O-benzyl-2-deoxy-2-trichloroacetamido-β-d-glucopyranosyl-(1→3)-2,4,6-tri-O-benzyl-β-d-galactopyranosyl-(1→3)-[2,3,4-tri-O-acetyl-α-l-fucopyranosyl-(1→4)]-6-O-benzyl-2-deoxy-2-trichloroacetamido-β-d-glucopyranosyl-(1→3)-2,4,6-tri-O-benzyl-β-d-galactopyranosyl-(1→3)-[2,3,4-tri-O-acetyl-α-l-fucopyranosyl-(1→4)]-6-O-benzyl-2-deoxy-2-trichloroacetamido-β-d-glucopyranosyl-(1→3)-2,4,6-tri-O-benzyl-β-d-galactopyranosyl-(1→3)-[2,3,4-tri-O-acetyl-α-l-fucopyranosyl-(1→4)]-6-O-benzyl-2-deoxy-2-trichloroacetamido-β-d-glucopyranoside (22). To a mixture of the glycosyl donor 21 (144 mg, 31.0 μmol), N-Boc-3-amino-1-propanol (53.0 μL, 310 μmol), and molecular sieves AW-300 (93.0 mg) in CH2Cl2 (1.0 mL) was added dropwise 1% TMSOTf in CH2Cl2 solution (56.0 μL, 3.1 μmol) at 0 °C under Ar, and stirred for 1 h at 0 °C. The reaction mixture was quenched with sat. NaHCO3, filtered by Celite, and diluted with CHCl3. The organic layer was separated, and the aqueous layer was extracted with CHCl3. The combined organic layer was successively washed with brine, dried over Na2SO4, and concentrated. The crude product was purified by gel permeation chromatography [S-X1, toluene–EtOAc (75:25)] and silica gel column chromatography with toluene–EtOAc (71:29) to give the title product 22 (113 mg, 79%). [α]D −51.8° (c 1.0, CHCl3); 1H-NMR (500 MHz, CDCl3) δ 7.65–7.02 (m, 80H, Ar), 6.67 (d, 1H, J2,NH = 6.5 Hz, NH), 6.19 (brs, 1H, NH), 6.02–5.78 (m, 3H, NH × 2, H2C=CHCH2), 5.31–4.87 (m, 26H, H-1Fuc × 4, H-2Fuc × 4, H-3Fuc × 4, H-4Fuc × 4, H-5Fuc × 4, H-1GlcN × 4, H2C=CHCH2 × 2), 4.84–4.27 (m, 46H, H-1Gal × 4, H-2GlcN × 3, H-3GlcN × 3, H-4GlcN × 3, PhCH2 × 32, NHCH2CH2CH2O), 4.20–3.25 (m, 38H, H-2Gal × 4, H-3Gal × 3, H-4Gal × 4, H-5Gal × 4, H-6aGal × 4, H-6bGal × 4, H-3GlcN, H-4GlcN, H-5GlcN × 3, H-6aGlcN × 3, H-6bGlcN × 3, H2C=CHCH2 × 2, NHCH2CH2CH2O × 2), 3.27–3.01 (m, 7H, H-3Gal, H-2GlcN, H-5GlcN, H-6GlcN × 2, NHCH2CH2CH2O × 2), 2.21–1.95 (m, 36H, Ac × 12), 1.71–1.59 (m, 2H, NHCH2CH2CH2O × 2), 1.42 (s, 9H, tBu), 0.84–0.67 (m, 12H, H-6 Fuc × 4). 13C-NMR (125 MHz, CDCl3) δ 170.4, 170.3, 170.3, 170.2, 169.5, 169.5, 169.4, 161.2, 160.8, 160.7, 160.6, 156.0, 139.3, 139.1, 139.0, 138.9, 138.5, 138.5, 138.5, 138.4, 138.4, 138.4, 138.3, 137.7, 137.5, 137.5, 135.0, 129.2, 129.1, 129.0, 128.8, 128.6, 128.5, 128.5, 128.4, 128.3, 128.3, 128.2, 128.2, 128.2, 128.2, 128.1, 128.1, 128.0, 127.8, 127.8, 127.8, 127.7, 127.7, 127.7, 127.6, 127.5, 127.4, 116.6, 103.5, 103.1, 99.1, 98.5, 95.9, 95.9, 95.7, 92.7, 92.5, 92.3, 81.9, 81.3, 81.2, 79.2, 78.7, 77.9, 77.6, 76.5, 76.2, 75.5, 75.3, 75.2, 75.1, 75.1, 75.0, 74.9, 74.9, 74.4, 73.9, 73.8, 73.5, 73.3, 73.2, 73.2, 73.1, 73.0, 72.9, 72.5, 71.8, 71.7, 71.6, 68.5, 68.4, 68.3, 68.2, 68.1, 68.1, 67.3, 66.8, 64.6, 64.5, 37.3, 29.7, 28.5, 20.9, 20.8, 20.8, 20.8, 20.8, 15.7, 15.7, 15.5, 15.3. HRMS (ESI) m/z: found [1/2M + Na]+ 2329.1507, C227H261Cl12N5O71 calcd. for [1/2M + Na]+ 2329.1506.
Molecules 21 00614 i019
N-(tert-Butoxycarbonyl)-3-aminopropyl 2,4,6-tri-O-benzyl-β-d-galactopyranosyl-(1→3)-[2,3,4-tri-O-acetyl-α-l-fucopyranosyl-(1→4)]-6-O-benzyl-2-deoxy-2-trichloroacetamido-β-d-glucopyranosyl-(1→3)-2,4,6-tri-O-benzyl-β-d-galactopyranosyl-(1→3)-[2,3,4-tri-O-acetyl-α-l-fucopyranosyl-(1→4)]-6-O-benzyl-2-deoxy-2-trichloroacetamido-β-d-glucopyranosyl-(1→3)-2,4,6-tri-O-benzyl-β-d-galactopyranosyl-(1→3)-[2,3,4-tri-O-acetyl-α-l-fucopyranosyl-(1→4)]-6-O-benzyl-2-deoxy-2-trichloroacetamido-β-d-glucopyranosyl-(1→3)-2,4,6-tri-O-benzyl-β-d-galactopyranosyl-(1→3)-[2,3,4-tri-O-acetyl-α-l-fucopyranosyl-(1→4)]-6-O-benzyl-2-deoxy-2-trichloroacetamido-β-d-glucopyranoside (23). Compound 22 (231 mg, 50.0 μmol) was deallylated by Ir(COD)(PMe2Ph)2PF6 (1.3 mg, 1.50 μmol) in THF (2.5 mL × 2) and deprotected by HgCl2 (34.0 mg, 125 μmol) and HgO (4.3 mg, 20.0 μmol) with 90% acetone aq. (5.0 mL) as described for 14. The crude product was purified by silica gel column chromatography with hexane–EtOAc (75:25) to give the title product 23 (219 mg, 96%). [α]D −59.0° (c 1.0, CHCl3); 1H-NMR (500 MHz, CDCl3) δ 7.65–7.02 (m, 80H, Ar), 6.82 (d, 1H, J2,NH = 7.0 Hz, NH), 6.22 (brs, 1H, NH), 5.78–5.63 (m, 2H, NH × 2), 5.31–4.88 (m, 23H, H-1Fuc × 4, H-2Fuc × 4, H-3Fuc × 4, H-4Fuc × 4, H-5Fuc × 4, H-1GlcN × 4), 4.87–4.27 (m, 38 H, H-1Gal × 4, H-1GlcN, PhCH2 × 32, NHCH2CH2CH2O), 4.20 (m, 1H, H-3GlcN), 4.03–3.02 (m, 52H, H-2Gal × 4, H-3Gal × 4, H-4Gal × 4, H-5Gal × 4, H-6aGal × 4, H-6bGal × 4, H-2GlcN × 4, H-3GlcN × 3, H-4GlcN × 4, H-5GlcN × 4, H-6aGlcN × 4, H-6bGlcN × 4, NHCH2CH2CH2O × 2, NHCH2CH2CH2O × 2), 2.21–1.95 (m, 37H, Ac × 12, OH), 1.71–1.59 (m, 2H, NHCH2CH2CH2O × 2), 1.42 (s, 9H, tBu), 0.90–0.73 (m, 12H, H-6Fuc × 4); 13C-NMR (125 MHz, CDCl3) δ 170.5, 170.3, 170.3, 170.2, 170.2, 169.6, 169.6, 169.5, 169.4, 167.8 161.3, 161.0, 160.7, 160.5, 156.0, 139.5, 139., 139.0, 138.9, 138.6, 138.5, 138.5, 138.4, 138.3, 138.3, 138.3, 137.9, 137.7, 137.7, 137.6, 137.5, 132.5, 130.9, 129.2, 129.2, 129.1, 129.0, 128.8, 128.8, 128.6, 128.6, 128.5, 128.5, 128.4, 128.4, 128.3, 128.3, 128.3, 128.2, 128.2, 128.1, 128.1, 127.8, 127.8, 127.8, 127.8, 127.7, 127.7, 127.6, 127.5, 127.4, 125.3, 103.3, 103.0, 99.6, 99.2, 98.6, 95.9, 95.8, 95.7, 92.8, 92.7, 92.4, 81.9, 81.5, 81.3, 79.6, 79.2, 77.9, 76.3, 76.1, 75.5, 75.3, 75.2, 75.2, 75.0, 74.9, 74.7, 74.2, 74.1, 73.8, 73.8, 73.3, 73.3, 73.2, 73.2, 73.1, 73.0, 72.9, 71.7, 71.6, 68.7, 68.6, 68.6, 68.2, 68.1, 67.3, 66.8, 64.6, 64.5, 38.8, 37.2, 30.4, 29.7, 29.0, 28.5, 23.8, 23.0, 21.5, 21.0, 20.9, 20.9, 20.9, 20.8, 20.8, 15.8, 15.6, 14.1, 14.1. HRMS (ESI) m/z: found [1/2M + Na]+ 2309.1352, C224H257Cl12N5O71 calcd. for [1/2M + Na]+ 2309.1350.
Molecules 21 00614 i020
N-(tert-Butoxycarbonyl)-3-aminopropyl 2,4,6-tri-O-benzyl-β-d-galactopyranosyl-(1→3)-[2,3,4-tri-O-acetyl-α-l-fucopyranosyl-(1→4)]-2-acetamido-6-O-benzyl-2-deoxy-β-d-glucopyranosyl-(1→3)-2,4,6-tri-O-benzyl-β-d-galactopyranosyl-(1→3)-[2,3,4-tri-O-acetyl-α-l-fucopyranosyl-(1→4)]-2-acetamido-6-O-benzyl-2-deoxy-β-d-glucopyranosyl-(1→3)-2,4,6-tri-O-benzyl-β-d-galactopyranosyl-(1→3)-[2,3,4-tri-O-acetyl-α-l-fucopyranosyl-(1→4)]-2-acetamido-6-O-benzyl-2-deoxy-β-d-glucopyranosyl-(1→3)-2,4,6-tri-O-benzyl-β-d-galactopyranosyl-(1→3)-[2,3,4-tri-O-acetyl-α-l-fucopyranosyl-(1→4)]-2-acetamido-6-O-benzyl-2-deoxy-β-d-glucopyranoside (25). A mixture of 23 (60.0 mg, 13.1 μmol), powdered Zn (1.71 g, 26.2 mmol), and AcOH (1.89 mL, 32.8 mmol) in THF (1.3 mL) was stirred under microwave irradiation at 250 W for 1 h under Ar. The mixture was diluted with CHCl3 and filtered through Celite. The filtrate was successively washed with sat. NaHCO3, water, and brine, dried over Na2SO4, and concentrated. The residue was chromatographed on silica gel with CHCl3–MeOH (92:8). The crude product was dissolved in DMF (1.3 mL), and stirred with Boc2O (4.3 μL, 20.0 μmol) and Et3N (5.4 μL, 39.0 μmol) at room temperature for 1 d. The reaction mixture was concentrated, and purified by gel permeation chromatography [S-X1, toluene–EtOAc (75:25)] and silica gel column chromatography with CHCl3–acetone (80:20–67:33) to give the title product 25 (41.0 mg, 76% in 2steps). 1H-NMR of the product 25 could not be assigned because all peaks were shown as broad peaks in all range. [α]D −6.5° (c 1.0, CHCl3); 13C-NMR (125 MHz, CDCl3) δ 170.6, 170.4, 170.3, 170.2, 169.5, 169.4, 169.4, 156.0, 139.1, 139.0, 138.8, 138.7, 138.6, 138.6, 138.2, 137.9, 137.8, 137.7, 137.6, 135.9, 129.4, 129.2, 129.1, 129.0, 129.0, 129.0, 128.7, 128.6, 128.6, 128.5, 128.4, 128.3, 128.2, 128.2, 128.2, 128.1, 128.1, 128.0, 128.0, 127.9, 127.7, 127.7, 127.6, 127.6, 127.6, 127.4, 127.3, 127.1, 127.1, 125.3, 103.4, 103.3, 103.2, 100.9, 100.5, 99.0, 95.8, 95.5, 80.5, 79.8, 79.3, 77.6, 76.4, 75.4, 75.2, 75.0, 74.7, 74.6, 74.3, 73.3, 73.3, 73.2, 73.1, 71.8, 68.2, 67.6, 66.7, 64.4, 37.1, 33.7, 32.8, 31.9, 30.2, 30.1, 29.7, 29.5, 29.4, 28.5, 27.1, 23.4, 23.2, 23.2, 22.7, 21.5, 20.8, 20.8, 15.6, 15.5, 15.4, 15.3, 14.1. HRMS (ESI) m/z: found [1/2M + Na]+ 2105.3693, C224H269N5O71 calcd for [1/2M + Na]+ 2105.3688.

4. Conclusions

We have developed a convenient synthesis of the fourth repeated Lea tandem repeat framework. Lea trisaccharide was synthesized by β-selective galactosylation and α-selective fucosylation with high selectivity. Glycosyl acceptors and donors of Lea derivatives were obtained readily in a few steps, and the Lea tandem repeat derivatives, the hexasaccharide and dodecasaccharide, were constructed in high yields. This convergent synthetic strategy can efficiently produce oligosaccharides with repeating structures, which will make an important contribution to biological studies.

Acknowledgments

The authors acknowledge Kazuhiro Mori, Yuko Nakahara, Yoshiaki Nakahara (Tokai University), and Hironobu Hojo (Osaka University) for their contribution to the preliminary experiments on this work. The iCeMS is supported by World Premier International Research Center Initiative (WPI), MEXT, Japan. We thank Kiyoko Ito (Gifu University) for providing technical assistance.

Author Contributions

A.U., A.I., H.A., M.K., and H.I. conceived and designed the experiments; D.K., T.Y., K.N., and A.U. performed the experiments; D.K. and T.Y. analyzed the data; A.U. and H.I. wrote the paper, and all authors participated in the revisions of the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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  • Sample Availability: Not available.
Figure 1. Structure of the Lea tandem repeat.
Figure 1. Structure of the Lea tandem repeat.
Molecules 21 00614 g001
Scheme 1. Retrosynthetic scheme.
Scheme 1. Retrosynthetic scheme.
Molecules 21 00614 sch001
Scheme 2. Synthesis of the Lea trisaccharide common intermediate.
Scheme 2. Synthesis of the Lea trisaccharide common intermediate.
Molecules 21 00614 sch002
Scheme 3. Synthesis of Lea tetramer.
Scheme 3. Synthesis of Lea tetramer.
Molecules 21 00614 sch003
Scheme 4. Introduction of a linker to the dodecasaccharide.
Scheme 4. Introduction of a linker to the dodecasaccharide.
Molecules 21 00614 sch004
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