High-throughput determination of enantiopurity in atroposelective synthesis of aryl triazoles

Atropisomeric scaffolds are a common design element found in pharmaceuticals, many deriving from an N–C axis of chirality. The handedness associated with atropisomeric drugs is oftentimes crucial for their efficacy and/or safety. With the increased use of high-throughput screening (HTS) for drug discovery, the need for rapid enantiomeric excess (ee) analysis is needed to keep up with the fast workflow. Here, we describe a circular dichroism (CD) based assay that could be applied to the ee determination of N–C axially chiral triazole derivatives. Analytical samples for CD were prepared from crude mixtures by three sequential steps: liquid–liquid extraction (LLE), a wash-elute, and complexation with Cu(ii) triflate. The initial ee measurement of five samples of atropisomer 2 was conducted by the use of a CD spectropolarimeter with a 6-position cell changer, resulting in errors of less than 1% ee. High-throughput ee determination was performed on a CD plate reader using a 96-well plate. A total of 28 atropisomeric samples (14 for 2 and 14 for 3) were screened for ee. The CD readings were completed in 60 seconds with average absolute errors of ±7.2% and 5.7% ee for 2 and 3, respectively.


General procedure
All reagents were purchased from commercial sources.Acetonitrile was spectrophotometric grade.All other solvents were ACS grade and used without further purification.Compounds 1, 2, and 3 were synthesized according to literature procedure.s1 Compound characterization for these samples may be found in reference s1.Chiral HPLC data were acquired using an Agilent 1100 series analytical chiral HPLC equipped with a photodiode array detector (254 nm) with a Daicel Chiralpak IA column (5 μm particle size, 4.5 x 250 mm), flow rate of 1.0 mL/min, and either 18% EtOH/hexanes (for compound 2) or 20 % EtOH/hexanes (for compound 3).CD screening of the atropisomers 1, 2, and 3 were performed using either a Jasco J-815 spectropolarimeter in the Targeted Therapeutic Drug Discovery and Development Program facility at the University of Texas at Austin or an Ekko CD microplate reader manufactured by Hinds Instruments.Titration study was conducted by using a Cary 100 UV-Vis spectrophotometer from Agilent Technology.HyperSep™ silica cartridges (bed weight 50 mg, column capacity 1 mL) were used for a washelute step.All stock solutions for CD analysis were prepared using acetonitrile as the solvent: 1 (17.5 mM), 2 (17.5 mM), 3 (16 mM), and Cu(OTf) 2 (17.5 mM for complexation with 1 and 2, 16 mM for complexation with 3).

Titration study
The titration study was carried out using a Cary 100 UV-Vis spectrophotometer from Agilent Technology (10 mm cell, UV quartz).The spectra were obtained at 320 nm where only the complexes can absorb light.The concentration of copper(II) triflate in acetonitrile was set to 0.175 mM.The absorbance curve reached a plateau at approximately 3:1 ligand-metal molar ratio.
-A solution (1mL) in DCM layer was loaded onto the silica cartridge.
-Dry the sample with N 2 -blowing.
-Dissolve the dried sample in acetonitrile (2mL) and determine the concentration by measuring the UV absorbance (241 nm) of solution (200 μL per well) with an Ekko microplate reader.
-A solution of copper(II) triflate in acetonitrile (3 eq.) was added to the solution.
-The concentration was adjusted to 1.75 mM for 2 and 1.6 mM for 3 by adding acetonitrile.
-A solution of the complex (40 μL) was added to a 96-well plate and analyzed by a CD microplate reader.