Enantioseparation and Absolute Configuration Determination of Angular-Type Pyranocoumarins from Peucedani Radix Using Enzymatic Hydrolysis and Chiral HPLC-MS/MS Analysis

Angular-type pyranocoumarins from Peucedani Radix (Chinese name: Qian-hu) have exhibited potential for use on treatment of cancer and pulmonary hypertension. Due to the existence of C-3′ and C-4′ chiral centers, compounds belonging to this chemical type commonly exist in enantiomers and/or diastereoisomers, which may elicit distinct activities during their interactions with the human body. In the present study, a new method, which combines enzymatic hydrolysis with chiral LC-MS/MS analysis, has been developed to determine the absolute configurations of these angular-type pyranocoumarins. Pyranocoumarins isolated from Qian-hu, their enantiomers, or metabolites were individually incubated with rat liver microsomes. As the common end product from enzymatic hydrolysis of all tested pyranocoumarins, cis-khellactone was collected and its absolute configuration was determined by comparison with (+)-cis-khellactone and (−)-cis-khellactone using chiral LC-MS/MS. The absolute configurations of all tested parent pyranocoumarins were determined by combination of LC-MS/MS, NMR and polarimetric analysis. The results revealed that the metabolite cis-khellactone retained the same absolute configurations of the stereogenic carbons as the respective parent compound. This method was proven to be rapid and sensitive and also has advantages in discriminating single enantiomers and mixtures of optical isomers with different ratios.

formed from enzyme-mediated reactions. On the other hand, chiral LC-MS/MS analysis affords high selectivity and sensitivity to allow the determination of drugs and metabolites at low concentrations, thus, has been widely adopted in bioanalysis. Therefore, in the present study, a method that combines enzymatic hydrolysis followed by chiral LC-MS/MS analysis of the metabolites formed was adopted for the first time to determine the absolute configurations of angular-type pyranocoumarin compounds from Qian-hu. The results obtained were consolidated with data from polarimetric analysis and NMR data or by comparison with the data of the reference compounds.

Identification of Pyranocoumarins from Qian-hu
By comparing the NMR, MS data and optical rotations with the literature values (Tables 1 and 2

Determination of Absolute Configuration of Angular-Type Pyranocoumarins
Our previous studies [14,21] and Ruan's report [22] on the metabolisms of (±)-praeruptorin A (2), (+)-praeruptorin A (2a), (−)-praeruptorin A (2b), (+)-praeruptorin B (3a) and (+)-praeruptorin E (4) revealed that all these angular-type pyranocoumarins underwent stepwise hydrolysis and generated cis-khellactone without alteration of the absolute configuration when they were incubated with liver microsomal proteins from rats or humans in the presence of a NADPH-regenerating system. This finding suggests that the absolute configurations of this type of coumarins can be determined from the configurations of C-3′ and C-4′ of khellactone produced from hydrolysis of the parent coumarins by rat or human hepatic phase I isozymes.   , were incubated with rat liver microsomes. The chemical structures and absolute configurations of 2a, 2b, 9 and 10 have been unambiguously identified using NMR analysis and optical rotation data in our laboratory [14], while 4 was commercially available and its chemical structure was double checked by comparing its mass spectral profile and optical rotation data [[α] 20 D +35° (c, 1.0)] with the literature report [8].
These results together with the optical rotation data of these parent pyranocoumarins (Table 1) support that their respective hydrolytic metabolite cis-khellactone keeps the absolute configurations during hydrolysis of the parent compounds in rat liver microsomal proteins.   (Tables 1 and 2). Peucedanocoumarins I, II and III, which are also trans-khellactone type coumarins, yielded cis-khellactone after alkaline hydrolysis [23]. Thus, epimerization might occur during alkaline treatment of pyranocoumarins and finally may cause misidentification of the absolute configurations of parent compounds.
It's interesting to note that compounds 5 and 6, which were obtained from Qian-hu to prepare 5a and 6a through crystallization, exhibited similar positive optical rotation and identical NMR data as compounds 5a and 6a, respectively. However, when subjected to enzymatic hydrolysis, both compounds formed the two optical isomers of cis-khellactone ( Figure 4C,D), indicating that compounds 5 and 6 were isolated from Qian-hu as enantiomerically enriched compounds. This was further confirmed by two peaks observed in chromatograms obtained from enantioseperation of compounds 5 and 6 (data not shown) and the enantiometric ratios (±) of cis-khellactone formed from 5 (7/1) and 6 (1/8) by RLMs (Table 1). Thus, the NMR data plus net optical rotation could not distinguish the enantiomerically enriched angular-type pyranocoumarin from its pure enantiomer.
The stereoselectivity in pharmacological actions [13], metabolism [14] and absorption [28] of praeruptorin A have been revealed by previous studies, indicating that it is of great importance to determine the absolute configurations of the angular-type pyranocoumarins when correlate their structures with the resultant biological interactions during pharmacological/toxicological evaluation and/or ADME screening. Furthermore, because most pyranocoumarins from Qian-hu naturally exist as enantiomers and/or diastereoisomers, misidentification of the identity of the compound studied has occurred [29][30][31]. Thus, it is crucial to establish a method which enables rapid determination of the absolute configurations of the pyranocoumarin compounds to be worked on. These findings obtained in the present study demonstrated that it is feasible to determine the absolute configurations of pyranocoumarins from that of the hydrolytic product khellactone using enzymatic hydrolysis coupled with chiral LC-MS/MS analysis. Because of the high sensitivity and selectivity afforded by LC-MS/MS techniques, this method can be used to determine the configuration of compounds of this chemical type with advantages in lower quantity of the tested compounds required for in vitro incubation.

Materials
Qian-hu was obtained from Ningguo, Anhui Province, China and the crude drug was authenticated as the dried roots of P. praeruptorum DUNN by Professor Pengfei Tu from Department of Natural Medicines, Peking University (Beijing, China). The specimens were stored at the State Key Laboratory of Quality Research in Chinese Medicine, University of Macau.

Polarimetric and NMR Analysis
The polarimetric analysis was performed on a Perkin-Elmer 243B digital polarimeter (PerkinElmer, Netherlands) in chloroform at 589.3 nm, 20 °C. Compounds 1-11 were dissolved in CDCl 3 containing 0.3% trimethylsilane (TMS) and their 1 H NMR spectra were acquired on a Bruker Avance 600 spectrometer (600 MHz, Bruker GmbH, Bremen, Germany). Chemical shifts were reported in δ scale in units of part per million (ppm) with TMS as the reference, and coupling constants (J) were expressed in units of Hertz (Hz).

Hydrolysis of Angular-type Pyranocoumarins from Qian-hu by Rat Liver Microsomes
Enzymatic hydrolysis of each of the angular-type pyranocoumarins (final concentration 25 µM) was carried out in a 200 µL reaction system containing 1 mg/mL of rat liver microsomal proteins in 0.1 M potassium phosphate buffer (pH 7.4) at 37 °C. The reactions were initiated by adding the NADPH-regenerating system (4 mM MgCl 2 , 1 mM β-NADP + , 1 mM G-6-P and 1 U/mL of G-6-PD). Reactions were stopped at 60 min by adding an equal volume of ice-cold methanol and the samples centrifuged to remove the proteins before subjected to achiral HPLC-UV analysis.

Achiral HPLC-UV Analysis
The achiral HPLC-UV analysis was performed on an Agilent 1200 series liquid chromatographic system (Agilent Technologies, Palo Alto, CA, USA) that equipped with a vacuum degasser, a quaternary pump, an autosampler and a diode array detector (DAD) system. Data acquisition was controlled by an Agilent ChemStation B 3.0 software. Sample separation was performed on an ODS reversed-phase C 18 column (250 mm × 4.6 mm I.D., particle size 5 µm, Agilent) at 35 °C. The mobile phase consisted of water containing 0.1% formic acid (A) and methanol (B). A gradient elution was carried out at 1.

Chiral HPLC-MS/MS Analysis
Chiral LC-MS/MS analysis was performed on an Agilent 1200SL series (Agilent) liquid chromatography coupled online with an API 4000 Q-trap @ mass spectrometer (Applied Biosystems, Foster City, CA, USA). The LC system consisted of a vacuum degasser, a binary pump, and an autosampler. Chromatographic separation was performed on an analytical CHIRALPAK AD-RH column (5 × 150 mm I.D., particle size 5 µm, Daicel, Tokyo, Japan), which was eluted with acetonitrile-H 2 O (30:70, v/v) at a flow rate of 0.65 mL/min. The mass spectrometer was equipped with a Turbo V TM source and a Turbo Ion Spray probe (500 °C) and operated in positive ion multiple reactions monitoring (MRM) mode. Ion optics was tuned using polypropylene glycol (PPG) standard dilution solvents. Nitrogen was used as the nebulizer, heater, curtain and collision gases. Optimum ion source parameters for cis-khellactone were as follows: nebulizer (GS1), heater (GS2) and curtain gas flow rates 50, 50, 10 instrument units, respectively; ionspray voltage 4500 V; heater gas temperature 550 °C; declustering potential (DP) 100 V. The ion pairs used for monitoring cis-khellactone were 263.1 > 245.1 and 263.1 > 203.1. Collision energies (CE) were set as 25 eV and 30 eV for the two ion pairs, respectively. The Applied Biosystems Analyst Software package (Version 1.5) was used for instrument control, data acquisition and processing.

Conclusions
In the present study, a new method using enzymatic hydrolysis coupled with chiral LC-MS/MS analysis was developed to assist the determination of the absolute configuration of angular-type pyranocoumarins. It has been proved to be rapid and sensitive and demonstrated success in identification of this chemical type from Qian-hu.