Novel Regioselective Synthesis of Urolithin Glucuronides─Human Gut Microbiota Cometabolites of Ellagitannins and Ellagic Acid

Abstract

Urolithins (dibenzo-pyran-[b,d]-6 one derivatives) are human gut microbiota metabolites produced from the natural food antioxidant ellagic acid. Urolithins are better absorbed than ellagic acid and demonstrate biological activities that suggest that they are responsible for the health effects observed after consuming ellagitannin- and ellagic acid-containing foods. Urolithins occur in the systemic circulation as glucuronide conjugates following phase II metabolism. These phase II conjugates are essential for testing the urolithin mechanisms of action in human cell line bioassays. Urolithin glucuronides are not commercially available, and their biosynthesis leads to mixtures of regional isomers. This study describes a novel and regioselective synthesis of urolithin A (3,8-dihydroxy urolithin) 3- and 8-glucuronides and isourolithin A (3,9-dihydroxy urolithin) 3- and 9-glucuronides. The metabolites were characterized using ¹H and ¹³C NMR spectroscopy and UV spectrophotometry. The presence of these metabolites in human subjects belonging to different urolithin metabotypes was also investigated.

Keywords: ellagic acid; glucuronides; gut microbiota metabolites; synthesis; urolithins.

Figure 1

Structures of the urolithin glucuronide metabolites. *(Nomenclature following Kay et al., 2020).

Scheme 1. Synthesis of Isourolithin A 9-Glucuronide (8)

Scheme 2. Synthesis of Isourolithin A 3-Glucuronide (12)

Scheme 3. Synthesis of Urolithin A Intermediate 16

Scheme 4. Synthesis of Urolithin A 8-Glucuronide (18) and 3-Glucuronide (22)

Reagents and conditions: (i) 6, BF₃.Et₂O (0.15 equiv), CH₂Cl₂, rt, 1 h., 76% yield; (ii) KF (2 equiv), K₂CO₃ (2 equiv), MeOH/H₂O, rt, 16 h., 46% yield; (iii) pivaloyl chloride (20 equiv), Py, rt, 16 h; 71% yield; (iv) KF (1.1 equiv), MeOH, rt, 1 h., 75% yield; (v) 6, BF₃.Et₂O (0.15 equiv), CH₂Cl₂, rt, 1 h, 99% yield; and (vi) KF (2 equiv), K₂CO₃ (2 equiv), MeOH/H₂O (5:1), rt, 16 h, 46%.

Figure 2

HPLC–DAD chromatogram (305 nm) (A) and UV spectra (B) of the synthesized urolithin glucuronides. Urolithin A 8-glucuronide (18); urolithin A 3-glucuronide (22); isourolithin A 9-glucuronide (8); and isourolithin A 3-glucuronide (12).

Figure 3

HPLC–DAD chromatograms at 305 nm of urine samples from volunteers belonging to urolithin metabotype A (A) and urolithin metabotype B (B). Urolithin A 8-glucuronide (18); urolithin A 3-glucuronide (22); isourolithin A 3-glucuronide (12); and urolithin B 3-glucuronide (23).