Synthesis of the Aeromonas veronii Strain Bs8 Disaccharide Repeating Unit

Abstract

Presented herein is the synthesis of the Aeromonas veronii disaccharide repeating unit which has been achieved in 11 steps starting from D-fucose and D-galactosamine.

Graphical Abstract

Graphical Abstract

1. Introduction

The genus Aeromonas consists of gram-negative rods widely distributed in freshwater environments and soils where they are symbionts of the leech, mussel, and zebrafish.[1, 2] Aeromonas research is common due to their relevance to both human and veterinarian health. For example, A. salmonicida is important to fishery biologists and hatchery operators due to its ability to cause furunculosis - boils on fish skin.[3] Additionally, A. hydrophila and A. veronii cause epizootic ulcerative syndrome in farmed fish and play an etiological role in fish kills.[4, 5] Regarding human health, Aeromonas species are connected to gastroenteritis, wound infection, and sepsis – bacterial blood poisoning.[6–9] Indeed, Aeromonas species were the leading cause of skin and soft tissue infections found in survivors of the 2004 tsunami in Thailand.[10–12]

The pathogenesis of Aeromonas species involves a series of virulence factors including flagella, extracellular enzymes, and toxins.[13–15] Additionally, cell surface lipopolysaccharides (LPS) contribute to virulence by modulating the interface with eukaryotic cells. For example, an O-specific polysaccharide determines strain immunospecificity and is the basis for their serological classification. Several O-antigens have been described for Aeromonas species (Figure 1).[16–21] Described herein, we report the synthesis of an O-specific disaccharide from A. veronii strain Bs8.[22]

Figure 1.

a. Aermonas polysaccharides; b. Synthetic analysis.

From an analysis standpoint the disaccharide features a fucose residue united to C3 of N-Acetylgalactosamine (GalNAc) by a problematic a-glycosidic bond. Regardless of the conditions used, installing glycosidic bonds to C3 of GalNAc with stereocontrol is usually problematic.[23–25] While not accompanied by full diastereocontrol, we have had success installing this glycosidic bond in a previous total synthesis by leveraging the equatorial participating powers of thiophene as an additive.²³ To reach the glycosylation step, we would need to synthesize two known building blocks: perbenzylated fucosyl thioglycoside 7[26] and the galactosamine benzylidene acetal 8.[27]

2. Results and discussion

In the forward direction, building block preparation started from fucose and involved peracetylation which occurred in near quantitative yield. Exchange of the anomeric acetate for its thioglycoside was achieved after exposure of the substrate to boron trifluoride diethyl etherate (BF₃•OEt₂). Lastly, to arm the donor, and enable a more facile deprotection sequence, the acetate protecting groups were exchanged for electron releasing benzyl ethers. For the GalNAc residue, we first masked the acetamide as its trichloro-derivative. After this step, peracetylation provide 13 in 80% yield over two steps. From here, the anomeric acetate was exchanged for a permanent paramethoxyphenyl group under Lewis acidic conditions. Following saponification, treatment with benzylaldehydedimethylacetal provided acceptor 8 in 75% yield.

With the key building blocks in hand, we evaluated several glycosylation reactions to discover conditions that would favor the α-anomer. Ultimately, the best conditions that we discovered to favor the desired diastereomer was to run the reaction in ether at low temperature. These conditions provided the glycosidic bond in favor of the α-anomer (3.5 to 1). With the protected disaccharide in hand, exposure to exhaustive hydrogenolysis provided the repeating unit (as its reducing end PMP-acetal) in 85% yield. This reaction removed a single benzylidene acetal, three benzyl ethers, and converted the trichloroacetamide to the acetimade. The spectral data showed homology with the published characterization data for the naturally occurring polymer.

3. Conclusion

Herein, we report an 11-step synthesis of the disaccharide repeating unit from A. veronii strain Bs8. Future work on this project will involve efforts to generate synthetic polymers, from this building block, to study structural and biological homoloy with naturally occurring polymers.

4. Experimental

4.1. General materials and methods

Commercial reagents were used as received. Anhydrous solvents were taken from an MBRAUN solvent purification system (MB SPS) and stored over 4 Å or 3 Å molecular sieves. All moisturesensitive reactions were performed in flame- or oven-dried round bottom flasks under an argon atmosphere. All air- or moisturesensitive liquids were transferred via oven-dried stainless steel syringes or cannula. Reaction temperatures were monitored and controlled via thermocouple thermometer and corresponding hot plate stirrer. Flash column chromatography was performed as described by Still et al. using silica gel 230–400 mesh. Analytical thin-layer chromatography (TLC) was performed on glass-backed Silica gel 60 F254 plates (EMD/Merck KGaA) and visualized using UV, cerium ammonium molybdate stain, and anisaldehyde stain. 1 H NMR spectra were obtained on a Bruker 400 or 600 MHz spectrometer with reporting relative to residual solvent signals (CDCl₃, 7.26 ppm; CH₃OD, 3.31 ppm; D₂O, 4.79 ppm). ¹H NMR spectral data are presented as follows: chemical shifts (d ppm), multiplicity (s = singlet, d = doublet, dd = doublet of doublets, t = triplet, q = quartet, m = multiplet, br = broad), coupling constants (Hz), integration, proton assignment. ¹³C NMR spectra were obtained on a Bruker 100 MHz spectrometer with reporting relative to residual solvent signals (CDCl₃, 77.16 ppm; CH₃OD, 49.0 ppm). ¹³C NMR spectral data are presented as follows: chemical shifts (d ppm), carbon assignment. Proton and carbon assignments were made with the aid of 2D NMR techniques (COSY, HSQC, and HMBC). High resolution mass spectra were recorded on a high resolution Thermo Electron Corporation MAT 95XP-Trap by use of electro-spray ionization (ESI) by the Indiana University Mass Spectrometry facility and a SYNAPT G2 or SYNAPT G2-S spectrometer (Waters, for TOFMS) by the McLean lab of Vanderbilt University. Optical rotations were obtained using a Perkin Elmer 341 polarimeter.

4.2. Synthetic procedures

4.2.1. 2,2,2-trichloro-N-((2R,4aR,6S,7R,8R,8aR)-6-(4-methoxyphenoxy)-2-phenyl-8-(((2S,3R,4S,5S,6R)-3,4,5-tris(benzyloxy)-6-methyltetrahydro-2H-pyran-2-yl)oxy)hexahydropyrano[3,2-d][1,3]dioxin-7-yl)acetamide (6)

Donor 7 (1.0 eq, 0.500g, 1.04 mmol) and acceptor 8 (1.5 eq, 0.813g, 1.6 mmol) were coevaporated with benzene (2 × 8 mL) and placed in a vacuum desiccator containing P₂O₅ overnight. The donor/ acceptor mixture was dissolved in diethyl ether (14 mL) and the resulting solution was cannulated into a reaction flask containing 4 Å powdered molecular sieves. The mixture was stirred under argon 1 hour then cooled to −78C and TfOH (0.1 eq, 0.038 mL in 0.2 mL CH₂Cl₂) was added. The reaction was stirred 2 hour then quenched with Et₃N. The reaction was diluted with CH₂Cl₂, filtered through celite, dried (MgSO₄), filtered, and concentrated in vacuo. The crude residue was purified via flash column chromatography (2:1 hexanes/EtOAc) to yield diasaccharide 6 (0.890g, 0.95 mmol, 91%) as a yellow foam: Rf 0.35 (1:1 hexanes/EtOAc); [α]D²⁵ = +38.7° (c = 0.012, CHCl₃) (α major product),¹H NMR (600 MHz, CDCl₃) δ 7.39 – 7.25 (m, 20H), 7.02 (d, J = 9.1 Hz, 2H), 6.93 (d, J = 8.4 Hz, 1H,-NH), 6.85 (d, J = 9.0 Hz, 2H), 5.71 (d, J = 3.5 Hz, 1H), 5.26 (s, 1H), 5.16 (d, J = 3.5 Hz, 1H), 5.02 (d, J = 11.5 Hz, 1H), 4.85 (d, J = 11.9 Hz, 1H), 4.79 – 4.77 (m, 1H), 4.75 (d, J = 9.2 Hz, 1H), 4.72 (d, J = 7.2 Hz, 1H), 4.65 (d, J = 11.5 Hz, 1H), 4.62 (d, J = 11.4 Hz, 1H), 4.40 (d, J = 3.3 Hz, 1H), 4.24 (d, J = 13.3 Hz, 1H), 4.20 (dd, J = 11.0, 3.2 Hz, 1H), 4.13 (dd, J = 10.1, 3.5 Hz, 1H), 4.07 – 4.01 (m, 2H), 3.92 (d, J = 13.3 Hz, 1H), 3.80 (m, 1H), 3.78 (s, 3H), 3.64 (m, 1H), 1.20 (d, J = 6.6 Hz, 3H). ¹³C NMR (150 MHz, CDCl₃) δ 161.6, 155.5, 150.4, 138.8, 138.5, 137.5, 128.4, 128.2, 128.2, 128.1, 128.0, 127.4, 117.9, 114.8, 100.6, 97.5, 96.8, 92.6, 78.9, 78.0, 75.9, 75.0, 73.3, 73.1, 72.8, 72.0, 69.2, 67.7, 63.7, 55.6, 50.5, 16.8. HRMS (ESI) calcd for C₄₉H₅₀Cl₃NO₁₁[M+Na]⁺ 956.2347, found 956.2348.

4.2.2. N-((2R,3R,4R,5R,6R)-5-hydroxy-6-(hydroxymethyl)-2-(4-methoxyphenoxy)-4-(((2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)tetrahydro-2H-pyran-3-yl)acetamide (5)

To a solution of 6 (1.0 eq, 0.500 g, 0.535 mmol) in CH₃OH (10 mL) and AcOH (1 mL) and Pd(OH)₂ was added (2.0 eq, 0.75g g, 1.07 mmol). The reaction was stirred under H₂ for 3 days then was diluted with CH₃OH, filtered through celite, concentrated in vacuo. The crude material was purified by size exclusion chromatography (Bio-Gel P2 gel) using a 1:1 mixture of deionized H₂O:CH₃OH as an eluant. Fractions containing the desired product (determined from MS) were combined and lyophilized to yield 5 (0.214 g, 0.452 mmol, 85%) as a white solid; [α]D²⁵ = +60.7° (c = 0.012, CH₃OH), ¹H NMR (600 MHz, CD₃OD) δ 7.18 (d, J = 9.2 Hz, 2H), 7.01 (d, J = 9.3 Hz, 2H), 5.50 (s, 1H), 5.12 (s, 1H), 4.62 – 4.51 (m, 1H), 4.35 – 4.28 (m, 1H), 4.18–4.12 (m, 2H), 4.06–4.05 (m, 1H), 3.85 (s, 3H), 3.84 – 3.83–3.75(m, 5H), 2.11 (s, 3H), 1.31 (s, 3H). ¹³C NMR (150 MHz, CD₃OD) δ 175.4, 156.2, 151.8, 120.2, 120.0, 116.3, 98.8, 97.9, 75.8, 73.2, 72.6, 71.0, 69.3, 68.7, 66.7, 62.3, 57.0, 50.2, 23.3, 16.9. HRMS (ESI) calcd for C₂₁H₃₁NO₁₁ [M+Na]⁺ 496.1794, found 496.1101. [α]D²⁵ = +60.7° (c = 0.015, CDCl₃)

Figure 2.

a. Building block synthesis; b. Glycosylation and global deprotection.

Highlights:

Repeating unit, disaccharide, glycosylation, cell-surface glycan