ANISODAMINE

Racanisodamine is one of the racemic isomers of anisodamine, resembles anisodamine in pharmacological effect. Racanisodamine is a non-selective muscarinic antagonist, used as a component of eye drops for myopic control[1].

General procedure: The hydroxylase activity and epoxidase activity were assayed by measuring the formation of 6beta-hydroxyhyoscyamine and scopolamine respectively. The reaction mixture contained: 50 mM Tris/HCl buffer (pH 7.6), 0.4 mM FeSO4, 4 mM sodium ascorbate, 1 mM 2-oxoglutaric acid, 0.2 mM (-)-hyoscyamine hydrobromide or 6beta-hydroxyhyoscyamine, 2 mg/mL of catalase (Sigma-Aldrich, cat. no. C9322) and AbH6H. [5] and [7] In a final volume of 20 mL, the enzyme assay was incubated at 34 °C for 2 h, and then quenched with 300 muL of saturated Na2CO3 solution to raise the pH to .similar.9.5, followed by extraction with 50 mL of EtOAc and drying over Na2SO4. After removing EtOAc under reduced pressure, the residue was dissolved in 0.5 mL of diluted HCl (20 mM). The quantification of the alkaloids was performed by HPLC analysis.32Using the previously described AbH6H assay, the Km and Vmax of the hydroxylation reaction catalyzed by AbH6H were determined by varying the concentration of (-)-hyoscyamine (0.01, 0.02, 0.04, 0.08, 0.15, 0.25 mM) while keeping the amount of AbH6H constant (0.61 muM) with 1 h incubation time. To test the conversion of analogues, each analogue was added to the assay at a final concentration of 0.2 mM. For analogues that were not recognized as substrates by AbH6H, their inhibitory properties were tested by the same AbH6H assay (containing 0.2 mM (-)-hyoscyamine) with 0.1 mM of each analogue, and HPLC quantification was performed to observe a potential reduction of the production of 6beta-hydroxyhyoscyamine.General procedure: The hydroxylase activity and epoxidase activity were assayed by measuring the formation of 6beta-hydroxyhyoscyamine and scopolamine respectively. The reaction mixture contained: 50 mM Tris/HCl buffer (pH 7.6), 0.4 mM FeSO4, 4 mM sodium ascorbate, 1 mM 2-oxoglutaric acid, 0.2 mM (-)-hyoscyamine hydrobromide or 6beta-hydroxyhyoscyamine, 2 mg/mL of catalase (Sigma-Aldrich, cat. no. C9322) and AbH6H. [5] and [7] In a final volume of 20 mL, the enzyme assay was incubated at 34 °C for 2 h, and then quenched with 300 muL of saturated Na2CO3 solution to raise the pH to .similar.9.5, followed by extraction with 50 mL of EtOAc and drying over Na2SO4. After removing EtOAc under reduced pressure, the residue was dissolved in 0.5 mL of diluted HCl (20 mM). The quantification of the alkaloids was performed by HPLC analysis.32Using the previously described AbH6H assay, the Km and Vmax of the hydroxylation reaction catalyzed by AbH6H were determined by varying the concentration of (-)-hyoscyamine (0.01, 0.02, 0.04, 0.08, 0.15, 0.25 mM) while keeping the amount of AbH6H constant (0.61 muM) with 1 h incubation time. To test the conversion of analogues, each analogue was added to the assay at a final concentration of 0.2 mM. For analogues that were not recognized as substrates by AbH6H, their inhibitory properties were tested by the same AbH6H assay (containing 0.2 mM (-)-hyoscyamine) with 0.1 mM of each analogue, and HPLC quantification was performed to observe a potential reduction of the production of 6beta-hydroxyhyoscyamine.

anticholinergic, antispasmodic