A simple synthesis of 2-keto-3-deoxy-D-erythro-hexonic acid isopropyl ester, a key sugar for the bacterial population living under metallic stress Claire Grison, Brice-Loïc Renard, Claude Grison To cite this version: Claire Grison, Brice-Loïc Renard, Claude Grison. A simple synthesis of 2-keto-3-deoxy-D-erythro- hexonic acid isopropyl ester, a key sugar for the bacterial population living under metallic stress. Bioorganic Chemistry, Elsevier, 2014, 52, pp.50-55. 10.1016/j.bioorg.2013.11.006. hal-03149058 HAL Id: hal-03149058 https://hal.archives-ouvertes.fr/hal-03149058 Submitted on 15 Mar 2021 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. A simple synthesis of 2-keto-3-deoxy-D-erythro-hexonic acid isopropyl ester, a key sugar for the bacterial population living under metallic stress ⇑ Claire M. Grison a, , Brice-Loïc Renard b, Claude Grison b a ICMMO UMR 8182, Equipe Synthèse Organique & Méthodologie, Université Paris Sud, Bât. 420, 15 rue Georges Clémenceau, 91405 Orsay cedex, France b CEFE UMR 5175, Campus CNRS, 1919 route de Mende, 34293 Montpellier cedex 5, France abstract 2-Keto-3-deoxy-D-erythro-hexonic acid (KDG) is the key intermediate metabolite of the Entner Doudoroff (ED) pathway. A simple, efficient and stereoselective synthesis of KDG isopropyl ester is described in five steps from 2,3-O-isopropylidene-D-threitol with an overall yield of 47%. KDG isopropyl ester is studied as an attractive marker of a functional Entner Doudoroff pathway. KDG isopropyl ester is used to promote Keywords: growth of ammonium producing bacterial strains, showing interesting features in the remediation of 2-Keto-3-deoxy-D-erythro-hexonic acid heavy-metal polluted soils. isopropyl ester Swern oxidation Modified Darzens reaction One-pot isomerization-reduction reaction Entner Doudoroff pathway Heavy-metal resistant rhizobia 1. Introduction vegetal and microbial diversity is the poorest. The ability to grow on a heavy-metal enriched soil devoid of carbon nutriment is a High ulosonic acids are well known to play an important struc- challenge. In such conditions, we assumed that their carbon tural role in the bacterial population. They are essential a-ketoac- metabolism is based on the ED pathway as the only glucose degra- ids such as 2-keto-3-deoxy-D-manno-octonic acid (KDO), which is dation way instead of the glycolytic pathway. This adaptation mostly found in the lipopolysaccharide (LPS) outer membrane of could be explained by the high selective pressure of a stressful Gram negative bacteria and such as 2-keto-3-deoxy-D-arabino- environment [3,7]. KDG uptake would prove the presence and heptonic acid (DAH), which plays a key role in the shikimic path- functionality of the ED pathway in bacterial strains. way of bacteria and plants. Therefore their chemical syntheses Therefore a rapid and efficient synthesis of KDG was needed. have often been studied to design new antibacterial agents for However, few stereoselective chemical syntheses of KDG had been inhibiting their own biosynthetic pathway [1]. described in the literature. In 1991, Plantier-Royon et al. [8] re- However, a little known ulosonic acid, 2-keto-3-deoxy-D-ery- viewed the first chemical routes of KDG and chose to improve thro-hexonic acid (or 2-keto-3-deoxy-D-gluconic acid, KDG), is the most promising way. However KDG was obtained with an the key intermediate metabolite in the Entner Doudoroff (ED) overall yield of 35% in 6 steps from D-glucose, using a Wittig– glycolytic pathway of certain microbial species, especially Gram Horner phosphonate reagent. During this period of years, Ramage negative bacterial species living in a stressful environment [2,3]. et al. [9] also reported a complicated chemical route to KDG with a Two novel heavy-metal resistant bacterial species were found to poor yield (<7% in 4 steps) from dioxalan-4-one, with a Wittig re- be of particular interest, Rhizobium metallidurans and Mesorhizobi- agent. Therefore a new chemical synthesis to KDG is needed. We um metallidurans [4,5]. They had been discovered growing on a have previously reported a general synthesis of a-ketoesters from highly polluted mining site of Les Avinières (Saint-Laurent-Le- carbonyl compounds, by using potassium alkyl dichloroacetates. Minier, Gard, South of France) in symbiosis with the Zn hyperaccu- Applied to a protected D-mannose, the method is convenient for mulating legume Anthyllis vulneraria (Hyperaccumulating the total synthesis of KDO [10]. This strategy is also very suitable rate = 154 mg kgÀ1 of zinc) [6]. The novel strains had been found to prepare the protected KDO under isopropyl carboxylic ester where the pollution is the highest, on the tailing basins (Zn: form, which is better for bacterial cell wall penetration [11].We 160 g kgÀ1, Pb: 84 g kgÀ1 Cd: 1.3 g kgÀ1 of soil) and where the propose here an original but simple and enantiomerically pure synthesis of KDG isopropyl ester and microbiological assays of ⇑ Corresponding author. KDG isopropyl ester uptake, as a selective bacterial growth E-mail address: [email protected] (C.M. Grison). stimulator. 2. Experimental protocols (5:1 hexane/EtOAc) = 0.5) was used immediately in the synthesis of compound 5. All starting materials were commercially available research- grade chemicals and used without further purification. Reactions 2.1.3. Synthesis of isopropyl dichloroacetate 4 were monitored by TLC (Merck – 5535 – Kieselgel 60-F254), To 150 mL of anhydrous isopropanol (117.90 g, 1.961 mol, detection being carried out by UV, by iodine vapor or by spraying 13 equiv) were added dichloroacetic acid (19.35 g, 150 mmol, solution of H2SO4 15% in ethanol followed by heating. NMR spectra 1 equiv) and 2 mL of sulfuric acid (36 N). The solution was stirred were recorded on a Bruker DRX-300. Chemical shifts are expressed for 26 h at 100 °C. Isopropanol was then evaporated under reduced as parts per million downfield from the internal standard pressure. The remaining yellow oil was dissolved in 60 mL of dieth- 1 13 tetramethylsilane for H and C. Multiplicities are indicated by s ylether, washed with an aqueous solution of NaHCO3 8% until (singlet), d (doublet), dd (dedoubled doublet), t (triplet), q (quadru- pH = 8 and extracted three times with diethylether. The organic plet), m (multiplet), bs (broadened singlet); coupling constants are layer was dried over MgSO4, filtered and concentrated under re- reported in Hertz (Hz). IR spectra were recorded on a PerkinElmer duced pressure to give 24 g of compound 4 as yellow oil (96% Spectrum 100 FT-IR spectrometer, in ATR mode, and are given in yield). The analytical data were consistent with literature [10]. cmÀ1. High-resolution mass spectrometry (HRMS) data were re- corded using the electrospray ionization technique in positive 2.1.4. Synthesis of 2,3-anhydro-2-C-chloro-6-O-[(1,1- mode (ESI+) with a tandem Q-TOF analyser (Bruker, 2009). Optical dimethylethyl)dimethylsilyl]-4,5-O-isopropylidene-D-erythro-hexonic rotations were measured using a 10 cm quartz cell (Jasco, P-1010). acid 1-methylethyl ester 5 T Values for ½/D were obtained with the D-line of sodium at the indi- A suspension of solid potassium (0.52 g, 13.2 mmol, 2 equiv) in cated temperature T, using solutions of concentration (c) in units of 41 mL of isopropanol under nitrogen was stirred until complete gÁ100 mLÀ1. Column chromatography was performed on silica gel dissolution of potassium. After addition of 8 mL of diethylether, (MN Kieselgel 60, 0.063–0.2 mm/70–230 mesh, Macherey–Nagel). the solution was cooled to 0 °C and a mixture of 3 (6.63 mmol, All primers were purchased from Invitrogen (Saint-Aubin, France). 1 equiv) and isopropyl dichloroacetate 4 (2.26 g, 13.2 mmol, 2 equiv) in 32 mL of diethylether, was added dropwise for 30 min. The mixture was stirred for 2 h, neutralized by addition 2.1. Chemical synthesis of 2-keto-3-deoxy-D-erythro-hexonic acid of saturated HCl solution in diethylether and centrifuged isopropyl ester 8 (1000 rpm, 15 min). The supernatant was taken off, concentrated and purified by silica chromatography (5:1 hexane/EtOAc) to give 2,21 g of compound 5 as brownish oil (71% yield). 2.1.1. Synthesis of 4-O-tert-butyldimethylsilyl-2,3-O-isopropylidene- 5a (60%): Rf (5:1 hexane/EtOAc) = 0.5; 1H RMN (300 MHz, D-threitol 2 CDCl3) d 0.09 (s, 6 H, Si–H3), 0.91 (s, 9 H, Si–tBu), 1.43–1.44 (s, 6 To a suspension of NaH (60% in oil, 0.18 g, 6.63 mmol, 1 equiv) H, C–CH3), 1.46–1.48 (m, 6 H, CH–CH3), 3.50–3.52 (d, 1 H, CH–O), in THF (10 mL) was added, over 15 min under a stream of nitrogen 3.80–3.90 (2dd, 2 H, CH2–OTBDMS), 4.05–4.30 (m, 2 H, 2 CH–O), at 5 °C, a solution of 2,3-O-isopropylidene-D-threitol (1.30 g, 13 5.09–5.18 (h, 1 H, O–CH–); C NMR (75 MHz, CDCl3) d À5.3 (Si– 6.63 mmol, 1 equiv) in THF (12 mL).