Carbohydrate Chemistry

Proven Synthetic Methods, Volume 4

Christian Vogel, Paul Murphy, Christian Vogel, Paul Murphy

• Publisher: CRC Press
• Year: 2017
• Print ISBN:
  9781498726917
• eBook ISBN:
  9781351646611
• Topic: Medicine
• Subtopic: Biochemistry in Medicine

Volumes in the Proven Synthetic Methods Series address the concerns many chemists have regarding irreproducibility of synthetic protocols, lack of identification and characterization data for new compounds, and inflated yields reported in chemical communications—trends that have recently become a serious problem.

Featuring contributions from world-renowned experts and overseen by a highly respected series editor, Carbohydrate Chemistry: Proven Synthetic Methods, Volume 4 compiles reliable synthetic methods and protocols for the preparation of intermediates for carbohydrate synthesis or other uses in the glycosciences.

Exploring carbohydrate chemistry from both the academic and industrial points of view, this unique resource brings together useful information into one convenient reference. The series is unique among other synthetic literature in the carbohydrate field in that, to ensure reproducibility, an independent checker has verified the experimental parts involved by repeating the protocols or using the methods.

The book includes new or more detailed versions of previously published protocols as well as those published in not readily available journals. The essential characteristics of the protocols presented are reliability, updated characterization data for newly synthesized substances and the expectation of wide utility in the carbohydrate field. The protocols presented will be of wide use to a broad range of readers in the carbohydrate field and the life sciences, including undergraduates taking carbohydrate workshops.

Section I

Synthetic Intermediates

11 Synthesis of 4-Nitrophenyl β-D-galactofuranoside

A Useful Substrate for β-D-Galactofuranosidases Studies

Carla Marino^*, Santiago Poklepovich Caridea and Rosa M. de Lederkremer

Universidad de Buenos Aires, Pabellón II, Ciudad Universitaria

Sydney Villaume^†

University of Namur (UNamur)

CONTENTS

Experimental

General Methods

4-Nitrophenyl 2,3,5,6-tetra-O-benzoyl-β-D-galactofuranoside

4-Nitrophenyl β-D-galactofuranoside

Acknowledgments

References

β-D-Galactofuranosyl units (β-D-Galf) are constituents of microorganisms, some of them pathogenic, such as Mycobacteria, the trypanosomatids Trypanosoma cruzi and Leishmania,¹ and fungi such as Aspergillus fumigatus.² Since Galf has never been found in mammals, its biosynthesis and metabolism are good targets for chemotherapeutic strategies. In some species, the degradation of Galf-containing glycoconjugates is promoted by extracellular β-D-galactofuranosidases. For example, Penicillium and Apergillius species,³ Helminthosporium sacchari,⁴ and Trichoderma harzianum⁵ produce exo β-D-galactofuranosidases (EC 3.2.1.146).

First studies of β-D-galactofuranosidases involved the use of methyl β-D-galactofuranoside as substrate and the tedious measurement of the reducing sugar released by the enzyme.^4,6 The availability of the chromogenic substrate 4-nitrophenyl β-D-galactofuranoside (3), first described in our laboratory⁷ and later reported by Cousin et al.,⁸ significantly simplified this task. Since then, compound 3 has been extensively used for galactofuranosidase studies in P. fellutanum,⁹ and Aspergillius spp.⁸ Compound 3 has also been used as a substrate for chemoenzymatic syntheses.^10,11 It used to be commercially available (Merck) but it was later discontinued. In our case, the starting compound was per-O-benzoyl-α,β-D-Galf (1),¹² obtained in a single step from D-Gal, and the glycosylation was promoted by p-toluenesulfonic acid.⁷ The other laboratory started from per-O-acetyl-β-D-Galf, which required three steps for its synthesis, and the glycosylating agent was SnCl₄,⁸ which proved to be very efficient for the synthesis of different galactofuranosides.¹³ In this chapter, we describe the synthesis of 3 by SnCl₄-promoted glycosylation of easily available 1,¹² followed by de-O-acylation.

EXPERIMENTAL

General Methods

Thin-layer chromatography (TLC) was performed on 0.2 mm silica gel 60 F₂₅₄ (Merck) aluminum-supported plates. Detection was effected by UV light and by charring with 10% (v/v) H₂SO₄ in EtOH. Column chromatography was performed on silica gel 60 (230–400 mesh, Merck). The proton (¹H) and carbon (¹³C) nuclear magnetic resonance (NMR) spectra were recorded with a Bruker AM 500 spectrometer at 500 MHz (¹H) and 125.8 MHz (¹³C). Assignments were supported by homonuclear correlation spectroscopy (COSY) and heteronuclear single quantum coherence (HCQC) experiments. Optical rotations were measured with a Perkin-Elmer 343 polarimeter, with a path length of 1 dm. Melting points were determined with a Fisher-Johns apparatus and are uncorrected. CH₂Cl₂ was distilled from P₂O₅ and stored over 4 Å MS. MeOH was dried by refluxing over magnesium turnings and a little iodine, distilled, and stored over 4 Å MS.

Sodium methoxide was prepared by carefully reacting a sub-stoichiometric amount of sodium (∼0.020–0.025 g) with dried methanol (5 mL).

4-Nitrophenyl 2,3,5,6-tetra-O-benzoyl-β-D-galactofuranoside (2)

A 50 mL, single-neck round-bottom flask equipped with a rubber septum was charged with 1,2,3,5,6-penta-O-benzoyl-α,β-D-galactofuranose (1, 1.05 g, 1.50 mmol),^* dry CH₂Cl₂ (10.0 mL), and 4 Å MS (0.1 g). The reaction vessel was flushed with argon and, with magnetic stirring and external cooling in an ice-water bath, SnCl₄ (0.25 mL, 2.1 mmol, 99% purity)^* was added, followed after 10 min by addition of 4-nitrophenol (0.25 g, 1.8 mmol)^†. The cooling was removed, and the reaction mixture was allowed to reach room temperature.

After 3 h of stirring, TLC showed consumption of the starting material (R_f ∼0.6 both anomers, 9:1 toluene–EtOAc) and presence of main product (R_f 0.7). Excess 4-nitrophenol is also observed by TLC. The mixture was filtered, diluted with CH₂Cl₂ (50 mL), and successively washed with NaHCO₃ (2.5%, 2 × 20 mL)^‡ and sat aq NaCl (20 mL). The organic layer was dried (Na₂SO₄), filtered, and concentrated. The syrup obtained (1.95 g) was chromatographed (49:1 toluene–EtOAc) to afford syrupy 2 (654 mg, 61%), [α]_D −11 (c 1, CHCl₃).^{7 1}H NMR (500 MHz, CDCl₃) δ 8.16–7.15 (H-aromatic), 6.10 (m, 1 H, H-5), 6.07 (s, J < 0.5 Hz, 1 H, H-1), 5.78 (s, J < 0.5 Hz, 1 H, H-2), 5.77 (d, J 4.8 Hz, 1 H, H-3), 4.80 (apparent t, J 4.3 Hz, 1 H, H-4), 4.75 (m, 2 H, H-6, 6′); ¹³C NMR (50.3 MHz, CDCl₃) δ 165.9, 165.7, 165.6, 165.4 (PhCO), 142.7–116.5 (C-aromatic), 103.8 (C-1), 83.1 (C-4), 81.9 (C-2), 77.3 (C-3), 69.9 (C-5), 63.0 (C-6). Anal. Calc. for C₄₀H₃₁NO₁₂: C, 66.94; H, 4.35; N, 1.95. Found: C, 67.17; H, 4.36; N, 1.77.⁷

4-Nitrophenyl β-D-galactofuranoside (3)

A dry 100 mL, single-neck round-bottom flask with a magnetic stirring bar and a rubber septum was charged with 2 (0.5 g, 0.69 mmol) and dried in vacuo for 1 h at room temperature. Anhydrous CH₂Cl₂ (8 mL) was added and with stirring and external cooling in an ice-water bath 0.2 M methanolic NaOMe in anhydrous MeOH (2.5 mL) was added in one portion. After 30 min, TLC analysis (7:1:2 PrOH-28% aq NH₃) showed complete conversion of the starting material into a more polar product (R_f 0.5), faster-moving than a galactose standard (R_f 0.2). The solution was concentrated to a small volu...

Table of contents

• Cover Page
• Title Page
• Copyright Page
• Dedication Page
• Contents
• Foreword
• Introduction
• Editors
• Series Editor
• Contributors
• SECTION I Synthetic Methods
• SECTION II Synthetic Intermediates
• Index