Chiral Recognition for the Two Enantiomers of Phenylalanine and Four Amino Acid Derivatives with (S)-Phenylethylamine Derived Nickel(II) Macrocyclic Complex

Experimental

Instruments and Materials. High performance liquid chromatography (HPLC) was performed on a JASCO HPLC system, consisting a JASCO PU-2080 Plus Intelligent Pump, a Rheodyne Model 7125 injector with a 20 μM sample loop, and a JASCO UV-2075 Plus Intelligent UV/VIS detector from JASCO (Tokyo, Japan). A Waters HPLC system (WATERS, Milford, USA), which consists of a Waters 2690 Separation Module and a Waters 996 Photodiode Array Detector, was also used to confirm the optical purities of the chiral samples studied. Methanol (MeOH), n-hexane and 2- propanol (IPA) of HPLC grade were obtained from J. T. Baker (USA). All racemic analytes tested in this study were purchased from Sigma-Aldrich (USA).

All HPLC solvents were supplied by Merck Korea (Seoul, Korea). The chiral columns and mobile phase conditions for each chiral sample were as follows: (1) phenylalanine; column, Chirosil (RStech, Korea); eluent, 10 mM acetic acid in an 80％ methyl alcohol aq. solution; flow rate, 0.5 mL/min; and detection at 254 nm UV. (2) N-Benzoyl-alanine, N-benzoyl-phenylalanine, N-benzoyl-methionine; column, Chiralhyun-LE(S)-1 (K-mac, Korea); eluent, 0.1％ TFA in a 10％ IPA in hexane; flow rate, 1.0 mL/min; and detection at 254 nm UV. (3) N-CBZ-alanine; column, Chiralcel OD-H (Daicel, Japan); eluent, 0.1％ TFA in a 10％ IPA in hexane; flow rate, 1.0 mL/min; and detection at 254 nm UV. The HPLC injection volume was 5 mL. The shaking incubator used to mix the chiral selector and racemic mixture was purchased from DAIHAN Scientific KMC-8480SF Model (Seoul, Korea). The chiral macrocyclic complex (1) used in this study was prepared using the same procedure with previous work.1112

Chiral Discrimination. Put 200 mg of the racemic samples into 200 mL of methyl alcohol (1000 mg/L sample solution). The racemic sample and macromolecule 1 were mixed at various molar ratios of 1:1, 1:2 and 1:5. [18 mg (1:1), 36 mg (1:2), 90 mg (1:5) of the macromolecule 1 was placed separately into three vials containing 1.62 mL of the 1000 mg/L racemic phenylalanine solution.] 1.90 mL of the 1000 mg/L N-benzoylalanine solution, 2.65 mL of the 1000 mg/L N-benzoylphenylalanine solution, 2.49 mL of the 1000 mg/L N-benzoylmethionine solution, and 2.19 mL of the 1000 mg/L N-CBZ-alanine solution were used instead of 1.62 mL of the 1000 mg/L of racemic phenylalanine solution. The mixed solution was shaken for two hours using a shaking incubator. After removing the solid particles by passing a silica micro column or filter paper, chiral HPLC was performed three times with the filtrate for each sample. The enatiomeric purity was measured by calculating the peak area for each peak of the (R)- and (S)-isomer from the chromatograms of racemic samples on each CSP.