Stereoselective Glycal Fluorophosphorylation: Synthesis of ADP‐2‐fluoroheptose, an Inhibitor of the LPS Biosynthesis

Abstract

Heptosides are found in important bacterial glycolipids such as lipopolysaccharide (LPS), the biosynthesis of which is targeted for the development of novel antibacterial agents. This work describes the synthesis of a fluorinated analogue of ADP‐L‐glycero‐β‐D‐manno‐heptopyranose, the donor substrate of the heptosyl transferase WaaC, which catalyzes the incorporation of this carbohydrate into LPS. Synthetically, the key step for the preparation of ADP‐2F‐heptose is the simultaneous and stereoselective installation of both the fluorine atom at C‐2 and the phosphoryl group at C‐1 through a selectfluor‐mediated (selectfluor=1‐chloromethyl‐4‐fluorodiazoniabicyclo[2.2.2]octane bis(triflate)) electrophilic addition/nucleophilic substitution involving a heptosylglycal. Therefore, we detail in this article 1) the stereoselective preparation of the key intermediates heptosylglycals, 2) the development of a new fluorophosphorylation procedure allowing an excellent β‐gluco stereoselectivity with “all‐equatorial” glycals, 3) the synthesis of the target ADP‐2F‐heptose, and 4) some comments on the contacts observed between the fluorine atom of the final molecule and the protein in the crystallographic structure of heptosyltransferase WaaC.

The Inhibition of Liposaccharide Heptosyltransferase WaaC with Multivalent Glycosylated Fullerenes: A New Mode of Glycosyltransferase Inhibition

“…Based on the knowledge of the contacts between the heptose residue and the enzyme deduced from the crystallographic structure of heptosyltransferase WaaC in a complex with ADP-2F-heptose (Figure 1), [22] we have also decided to explore the binding properties of an octose bearing an additional ionic functionality (Figure 2, molecule C 60 (C) 12 ). Indeed, the amine of a lysine residue is located at the vicinity of the 6-position of the heptose (Figure 1) [23] and a properly positioned carboxylate group in the inhibitor structure might provide additional strong coulombic interactions.…”

“…Enzymatic assays: For determination of specific activity, WaaC activity was monitored by a coupled assay involving pyruvate kinase (PK) and luciferase as coupling enzymes. [22,23] Basically, the reaction product ADP was converted by PK to ATP, which was detected by luciferase activity. The reaction took place in a white 96-well plate (Costar) in a final volume of 60 mL.…”

“…[22,23] In brief, the specific activity of WaaC was followed by a coupled enzymatic assay involving pyruvate kinase to transform the ADP reaction product into ATP. The concentration of the latter was then monitored either by luminescence using luciferase or by fluorescence using lactate dehydrogenase.…”

Systematic Synthesis of Inhibitors of the Two First Enzymes of the Bacterial Heptose Biosynthetic Pathway: Towards Antivirulence Molecules Targeting Lipopolysaccharide Biosynthesis

“…[33] For the synthesis of fluoro-heptosides, we also explored the stereoselective epoxide formation followed by a cesium acetate opening. [10] The two strategies that are the most commonly used to date are a d-selective dihydroxylation of alkene 22 (Scheme 2, pathway D) [34] followed, if l-heptosides are required, by a Mitsunobu inversion, [35][36][37] and the l-selective addition of Grignard reagents (pathway L). [38][39][40][41][42][43][44][45][46] General synthesis of d-glycero-d-mannoheptosides: For the synthesis of all analogues of 2 with the d-glycero-d-mannoheptose scaffold, we followed a general synthetic pathway depicted in Scheme 3.…”

“…In 2000, Kosma et al described the first synthesis of the two anomers of ADP-l-heptose 6, [14] thus demonstrating the anomeric configuration of 6. [10] Fortunately, we were able to obtain a 3D structure of WaaC in complex with this inhibitor. In 2008, our group has described the synthesis and the inhibition properties of the 2fluoro analogue of 6.…”