Methyl and Benzyl (Ethyl 3,4-di-O-benzyl-2-O-benzoyl-1-thio-β-d-glucopyranosyl)uronate

Abstract

Methyl and benzyl (ethyl 3,4-di-O-benzyl-2-O-benzoyl-1-thio-β-D-glucopyranosyl)uronate were synthesised from a protected thioglycoside in three steps. A regioselective ring opening of the benzylidene acetal with BH₃^.THF generated C6-OH material, which was subsequently oxidised using biphasic TEMPO/BAIB conditions. The resultant uronic acid was esterified with either a methyl or benzyl moiety. The products were obtained on a multigram scale and fully characterised by ¹H, ¹³C and 2D NMR, alongside MS and IR analysis.

1. Introduction

Polysaccharides containing uronic acid moieties are prevalent in nature and display a multitude of physical properties and biological functions. Glycosaminoglycans (GAGs) are exemplars, in which glucuronic acid (GlcA) is a core building block in the repeating disaccharide units of heparin/heparan sulphate, chondroitin sulphate and hyaluronic acid [1]. Glycosaminoglycans are a family of linear heteropolysaccharides that are expressed ubiquitously on the cell surface and extracellular matrix [2]. They interact with growth factors, cytokines, chemokines and adhesion molecules, which play critical roles in cell division, gene expression, angiogenesis, inflammation and wound healing [3]. As part of a research programme targeting the synthesis of structurally defined glycosaminoglycans and related mimetic oligosaccharides [4,5,6,7,8], we required access to suitably protected GlcA building blocks. The oxidation state of C6 is at the carboxylic acid level in GlcA, as opposed to the primary alcohol in a pyranose. A variety of oxidation methods have been utilised to access this, with 2,2,6,6-tetramethyl-1-piperidinyloxyl/(diacetoxyiodo)benzene (TEMPO/BAIB) being frequently employed in carbohydrate syntheses due to its scalability, mild conditions and selectivity for primary alcohols [9,10]. Esterification of the uronic acid to form a uronate is desirable to protect the acid and enable facile purification [11]. Herein, we report multigram access to the title compounds, methyl- and benzyl-uronates 4 and 5, from known intermediate 2 [12], providing our record of their synthesis and full characterisation.

2. Results

Compound 2 was prepared in 42% yield over six steps from commercially available 1,2,3,4,6-penta-O-acetyl-β-D-glucose 1 according to literature procedures [12,13]. The 4,6-O-benzylidene acetal protection enabled a regioselective reductive ring opening using BH₃^.THF and TMSOTf to provide access to compound 3 in 91% yield, containing a free C6 hydroxyl group for oxidation (Scheme 1). The ¹H and ¹³C NMR data agreed with previously published results [13], however, additional 2D NMR (COSY, HSQC, HMBC) and HRMS data were collected to fully characterise compound 3. The COSY NMR displayed a correlation between a broad singlet at δ 1.97 ppm and H6 environments at δ 3.93 and 3.72 ppm, indicative of C6-OH. The resultant primary alcohol 3 was next oxidised using TEMPO/BAIB. The resultant uronic acid was identified by a doublet for H5 at δ 4.08 ppm with a large J_H4H5 coupling of 9.1 Hz. Subsequent esterification with either MeI or BnBr afforded the target GlcA building blocks 4 and 5 in 65% and 58% yields, respectively. Full characterisation was obtained for both compounds. HRMS data confirmed sulphur oxidation did not occur under these conditions. The analytical data collected for 4 and 5 supported the structural assignment and gave an indicative level of purity. Copies of NMR and MS data are included in the Supplementary Materials.

3. Materials and Methods

3.1. General

Unless otherwise stated, all reagents used in the following experiments were bought commercially from Acros Organics (Antwerp, Belgium), Apollo Scientific (Manchester, UK), Fisher Scientific (Loughborough, UK), Fluorochem (Manchester, UK) or Sigma Aldrich (Gillingham, UK) and were used without further purification. Dry solvents were dried and stored under N₂ in Young’s flasks over 4 Å molecular sieves. Anhydrous DMF was purchased from Acros Organics (Antwerp, Belgium), fitted with AcroSeal™ packaging. Reactions were monitored by thin layer chromatography (TLC) using pre-coated 0.25 mm 60 F254 silica gel plates (Merck, London, UK) and eluent systems outlined in the respective experiments. Visualisation was achieved using UV light (λ = 254 nm) and 10% H₂SO₄:EtOH, followed by heating. Flash column chromatography was performed using silica gel [high purity grade, 60 Å pore size, 40–63 μm particle size]. ¹H NMR spectra were recorded at 400 MHz and ¹³C NMR spectra at 101 MHz on a Bruker AVIII400 spectrometer using deuterated solvent. Chemical shifts are reported in parts per million (ppm), coupling constants (J) are reported in hertz (Hz) and multiplicities are abbreviated as s (singlet), d (doublet), t (triplet) or m (multiplet) or combinations thereof. Chemical shifts were referenced to tetramethylsilane (TMS, where δ = 0.00 ppm). HRMS were recorded on a ThermoScientific LTQ Orbitrap XL at the ESPRC National Mass Spectrometry Facility at Swansea University. Infra-red spectra were recorded on a ThermoScientific Nicolet iS10 spectrometer; selected absorption frequencies (vmax) are reported in cm⁻¹. Relative intensities are described as follows: w (weak), m (medium) and s (strong). Melting points were recorded using open glass capillaries on a Gallenkamp melting point apparatus and are uncorrected. Optical rotations were recorded on a Bellingham + Stanley ADP430 (specific rotation, tube length: 50 mm, concentrations in g per 100 mL).

3.2. Ethyl 3,4-O-benzyl-2-O-benzoyl-1-thio-β-D-glucopyranoside (3)

Under an atmosphere of N₂, compound 2 (8.0 g, 15.6 mmol, 1.0 equiv.) was dissolved in DCM (60 mL) and cooled to 0 °C. A solution of 1 M BH₃^.THF (31 mL, 31.2 mmol, 2.0 equiv.) was added, followed by TMSOTf (0.4 mL, 2.34 mmol, 0.15 equiv.). The reaction was warmed to RT and stirred for 4 h, then cooled to 0 °C and quenched with sequential additions of Et₃N (2.4 mL, 17.2 mmol, 1.1 equiv.) and MeOH (2.5 mL, 546 mmol, 35 equiv.) until effervescence stopped. The resultant mixture was concentrated in vacuo, followed by co-evaporation with MeOH (100 mL). Purification via flash column chromatography (10:1 → 7:1 → 4:1 hexane:EtOAc) afforded 3 (7.2 g, 14.2 mmol, 91%) as a white solid. R_f 0.48 (2:1, hexane:EtOAc). m.p. 84–85 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.05–8.01 (m, 2H, Ar-H), 7.61–7.55 (m, 1H, Ar-H), 7.45 (dd, J = 10.7, 4.7 Hz, 2H, Ar-H), 7.38–7.29 (m, 5H, Ar-H), 7.17–7.11 (m, 5H, Ar-H), 5.28 (dd, J = 9.9, 9.2 Hz, 1H, H2), 4.87 (d, J = 11.0 Hz, 1H, CH₂OBn), 4.76 (d, J = 11.0 Hz, 1H, CH₂OBn), 4.68 (d, J = 11.0 Hz, 2H, CH₂OBn), 4.58 (d, J = 10.0 Hz, 1H, H1), 3.92 (d, J = 12.1 Hz, 1H, H6a), 3.87 (t, J = 9.0 Hz, 1H, H3), 3.76–3.73 (m, 1H, H6b), 3.71 (t, J = 9.4 Hz, H4), 3.49 (ddd, J = 9.7, 4.7, 2.6 Hz, 1H, H5), 2.75–2.67 (m, 2H, SCH₂CH₃), 1.97 (br s, 1H, C6-OH), 1.25 (m, 3H, SCH₂CH₃). ¹³C NMR (101 MHz, CDCl₃) δ 165.3 (C=O), 137.8 (Ar-C), 137.7 (Ar-C), 133.2 (Ar-C), 129.9 (Ar-CH), 129.7 (Ar-CH), 128.6 (Ar-CH), 128.4 (Ar-CH), 128.3 (Ar-CH), 128.1 (Ar-CH), 128.0 (Ar-CH), 127.7 (Ar-CH), 127.6 (Ar-CH), 84.2 (C1), 83.8 (C3), 79.8 (C5), 77.7 (C4), 75.3 (PhCH₂), 75.2 (PhCH₂), 72.5 (C2), 62.1 (C6), 24.2 (SCH₂CH₃), 14.9 (SCH₂CH₃). HRMS m/z (ESI⁺) found (M + NH₄)⁺ 526.2258, C₂₉H₃₆NO₆S required (M + NH₄)⁺, 526.2258. IR(FTIR) 3302 (s), 1693 (s), 1537 (s), 1242 (s), 1151 (m), 974 (m), 742 (m), 658 (s) cm⁻¹. These data are consistent with the literature data [13].

3.3. Methyl (Ethyl 3,4-O-benzyl-2-O-benzoyl-1-thio-β-D-glucopyranoside)uronate (4)

To a vigorously stirred solution of 3 (6.00 g, 11.8 mmol, 1.0 equiv.) in DCM:H₂O (2:1 v/v, 30 mL), TEMPO (368 mg, 2.36 mmol, 0.2 equiv.) and BAIB (9.60 g, 29.6 mmol, 2.5 equiv.) were added at 0 °C. After 10 min., the solution was warmed to RT and stirred for a further 2 h. TLC analysis showed the formation of the desired product (7:1:1, EtOAc:MeOH:H₂O). The reaction was quenched with sat. aq. Na₂S₂O₃ (20 mL), and the layers were separated. The pH of the aqueous phase was reduced to 2 with 1 M HCl, then extracted with EtOAc (3 × 30 mL). The combined organic extracts were dried over MgSO₄, filtered and concentrated in vacuo. The crude material was then suspended in anhydrous DMF (30 mL) under an atmosphere of N₂. At 0 °C, MeI (2.20 mL, 35.4 mmol, 3.0 equiv.) and K₂CO₃ (4.90 g, 35.4 mmol, 3.0 equiv.) were added. The reaction was stirred at RT in the dark for 2 h before dilution with EtOAc (40 mL) and washed with H₂O (2 × 30 mL) and brine (1 × 30 mL). The organic phase was dried (MgSO₄), filtered and concentrated in vacuo. Purification via flash column chromatography (10:1 → 7:1 hexane:EtOAc) afforded 4 as a white solid (4.07 g, 7.55 mmol, 64% over two steps). R_f 0.60 (2:1 hexane:EtOAc). ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{24}$ = +25.4 (c 1, CHCl₃). m.p. 108–109 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.01 (dd, J = 8.4, 1.4 Hz, 2H, Ar-H), 7.60–7.56 (m, 1H, Ar-H), 7.45 (dd, J = 8.3, 7.2 Hz, 5H, Ar-H), 7.34–7.29 (m, 5H, Ar-H), 7.13 (d, J = 1.4 Hz, 3H, Ar-H), 5.34 (dd, J = 10.0, 9.0 Hz, 1H, H2), 4.78–4.64 (m, 5H, 2 × CH₂OBn, H1), 4.03–3.95 (m, 2H, H4, H5), 3.88–3.83 (t, J = 8.9 Hz, 1H, H3), 3.74 (s, 3H, OCH₃), 2.80–2.66 (m, 2H, SCH₂CH₃), 1.22 (t, J = 7.5 Hz, 3H, SCH₂CH₃). ¹³C NMR (101 MHz, CDCl₃) δ 168.4 (C=O), 165.2 (C=O), 137.7 (Ar-C), 137.5 (Ar-C), 133.2 (Ar-C), 129.8 (Ar-CH), 129.7 (Ar-CH), 128.4 (Ar-CH), 128.4 (Ar-CH), 128.3 (Ar-CH), 128.1 (Ar-CH), 128.0 (Ar-CH), 128.0 (Ar-CH), 127.8 (Ar-CH), 84.2 (C1), 83.4 (C3), 79.3 (C4), 78.4 (C5), 75.3 (PhCH₂), 75.2 (PhCH₂), 71.9 (C2), 52.6 (OCH₃), 23.9 (SCH₂CH₃), 14.7 (SCH₂CH₃). HRMS m/z (ESI+) found (M + NH₄)⁺ 554.2206, C₃₀H₃₆NO₇S required (M + NH₄)⁺ 554.2207. IR(FTIR) 1720 (m), 1714 (s), 1263 (m), 1066 (m), 1024 (w), 734 (m), 694 (m) cm⁻¹.

3.4. Benzyl (Ethyl 3,4-O-benzyl-2-O-benzoyl-1-thio-β-D-glucopyranoside)uronate (5)

To a vigorously stirred solution of 3 (2.00 g, 3.94 mmol, 1.0 equiv.) in DCM:H₂O (2:1 v/v, 20 mL), TEMPO (62 mg, 0.79 mmol, 0.2 equiv.) and BAIB (3.18 g, 9.86 mmol, 2.5 equiv.) were added at 0 °C. After 10 min., the solution was warmed to RT and stirred for a further 2 h. TLC analysis showed the formation of the desired product (7:1:1, EtOAc:MeOH:H₂O). The reaction was quenched with sat. aq. Na₂S₂O₃ (20 mL), and the layers were separated. The pH of the aqueous phase was tuned to pH 2 with 1 M HCl, then extracted with EtOAc (3 × 30 mL). The combined organic extracts were dried over MgSO₄, filtered and concentrated in vacuo. The crude material was then suspended in anhydrous DMF (13 mL) under an atmosphere of N₂. At 0 °C, BnBr (0.70 mL, 5.92 mmol, 1.5 equiv.) and K₂CO₃ (820 mg, 5.92 mmol, 1.5 equiv.) were added. The reaction was stirred at RT for 18 h before dilution with EtOAc (25 mL) and washing with H₂O (3 × 30 mL). The organic phase was dried (MgSO₄), filtered and concentrated in vacuo. Purification via flash column chromatography (10/1 → 7/1 EtOAc/hexane) afforded 5 as a white solid (1.4 g, 2.23 mmol, 58%). R_f 0.63 (2/1, EtOAc/hexane). ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ = +13.4 (c 1, CHCl₃). m.p. 106–107 °C. ¹H NMR (400 MHz, CDCl₃) δ 8.04–7.97 (m, 2H, Ar-H), 7.57 (ddt, J = 8.7, 7.0, 1.4 Hz, 1H, Ar-H), 7.43 (dd, J = 8.3, 7.1 Hz, 2H, Ar-H), 7.38–7.22 (m, 8H, Ar-H), 7.11 (ddt, J = 7.4, 5.2, 3.8 Hz, 7H, Ar-H), 5.34 (dd, J = 10.0, 9.0 Hz, 1H, H2), 5.19 (s, 2H, 2 × CH₂-OBn) 4.72 (dd, J = 11.0, 1.5 Hz, 2H, 2 × CH₂-OBn), 4.64 (d, J = 11.1 Hz, 1H, CH₂-OBn), 4.58 (d, J = 10.0 Hz, 1H, H1), 4.49 (d, J = 10.7 Hz, 1H, CH2), 4.06–3.95 (m, 2H, H4, H5), 3.84 (t, J = 8.8 Hz, 1H, H3), 2.80–2.63 (m, 2H, SCH₂CH₃), 1.21 (t, J = 7.5 Hz, 3H, SCH₂CH₃). ¹³C NMR (101 MHz, CDCl₃) δ 167.8 (C=O), 165.1 (C=O), 137.6 (Ar-C), 137.5 (Ar-C), 135.0 (Ar-C), 133.2 (Ar-C), 129.8 (Ar-CH), 129.7 (Ar-CH), 128.6 (Ar-CH), 128.6 (Ar-CH), 128.5 (Ar-CH), 128.5 (Ar-CH), 128.4 (Ar-CH), 128.4 (Ar-CH), 128.3 (Ar-CH), 127.9 (Ar-CH), 127.8 (Ar-CH), 127.8 (Ar-CH), 84.2 (C1), 83.3 (C3), 79.4 (C4), 78.5 (C5), 75.3 (CH₂-OBn), 75.1 (CH₂-OBn), 71.8 (C2), 67.4 (CH₂-OBn), 23.9 (SCH₂CH₃), 14.8 (SCH₂CH₃). HRMS m/z (ES+) found (M + NH₄)⁺ 630.2517, C₃₆H₄₀NO₇S requires (M + NH₄)⁺, 630.2520. IR(FTIR) 1724 (m), 1718 (s), 1257 (m), 1170 (s), 1072 (s), 750 (m), 694 (s) cm⁻¹.