半制备SFC在ECD和VCD光谱仪手性物质结构分析中的应用

圆二色性（CD）光谱学是一种有用的技术，因为它不需要大量的样品量，而且与其他方法相比易于操作。通过比较计算得到的理论谱和实验谱，可以确定手性分子的绝对构型。在本研究中，我们利用半制备SFC分离和分离手性物质的对映体，并将获得的对映体样品应用于ECD和VCD光谱仪进行光谱测量。此外，我们通过计算理论的ECD和VCD光谱，确定了每个对映体的绝对构型。SFC 2018 Utilization of Semi-prep SFC for Structural Analysis of Chira l Substances b y ECD and VCD Spectrometer s Satoe lijima， Jiawei Xu， Kengo Yoshida ， Keisuke Watanabe， Yoshiro Kondo， Satoko Suzuki， Masao Bounoshita， Yasuyo Sato， Miki Kuwajima JASCO Corporation， 2967-5 /shikawa-machi， Hachioji， Tokyo 192-8537 E-mail： satoe.iijima@jasco.co.jp Introduction The methods of generat i ng enantiomers are classi f ied roughly i nto asymmetr i c syn t hesis，crystallization， enzyme reaction， and optical resolution by chroma t ography. I n par t icular， normal phase chromatography with chiral stationary phases has been used i n various fields such as pharmaceuticals， agr i chemicals， natural products， biomolecu l es， and functional materials because of the cost efficiency. Recently， preparative SFC， which provides faster， higher-separation analysis and easier post handling of fractions than HPLC， has become a main stream as an eff i cient method of producing enantiomers i n the drug discovery stage. Nuclear magnetic resonance (NMR)， X-ray diffraction (XRD)， electronic circular dichroism (ECD)， vibrat i onal circular dichroism (VCD)， and tandem mass spectrometry (MS/MS) are used for qualitative analysis and structural analysis of enantiomers of chiral substa n ces. In general ，these analyses require t he sample amount from 1 to 100 mg. Circular dichroism (CD)spectroscopy is a useful t echnique because it doesn’t require a lot of sample amount and is easy to operate compared to other methods. It i s possible to determine the absolute configuration of chiral molecules by comparing t he calculated t heoretical spectrum and exper i mental one. In th i s presentation， we separated and fract i onated enant i omers of chiral substances using semi-preparative SFC and applied the obtained enantiomer samples to spectra measurements by ECD and VCD spectrometers . Fur t hermore， we determined t he absolute conf i guration of each enantiomer by calcu l ating t heoretical ECD and VCD spectra. Experimental Appara t us Figure 1 shows the appearance and schematic diagram of JASCO semi-prep SFC system used i n t his experiment . Figure 2 and 3 show the appearance of JASCO ECD and VCD spectrometers. Fractionated enantiomers f rom semi -prep SFC system were applied t o spectral measurements by ECD and VCD systems. 12 11 13 14 1. COz cyl ind er 2. Modi fi er sol v ent 3. CO， delivery pump (PU-4387)4. Modif i er sol v ent p ump (PU-4087)65.. Stop valve 6..M i xing unit 7. Preheater (HE-02)+H eater c on t roller 15产品产品 169. Col u mn oven (CO-4060)10.Chiral column (CHIRALPAK I A)11.Detector (CD-4095 wi th Prep. High Pressure cell)12.Ba ck pressure regulat o r (BP-4340)13.Preheater (HE -4340-01-100)14.Fraction valve uni t (FV-4000-06)15.Fraction col l ec t or (Gi l son 223)+F u me hoo d ((HC -4068-01) (FH-4388) 8. Autosampler (AS-4358) 16.Fraction vials wi t h Cyclone separator 87 Fig. 1 The JASCO semi-prep SFC system (UFC-M). Analysis Conditio n s Semi-Prep SFC Chiral column： CHIRALPAK IA (20 mml.D.x250 mmL，5 pm) Flavanone Mobile phase ： Co，/ methanol Flow rate ： CO，；35 mL/min， Methanol； 10 mL/min Column temperature： 40°C CD detection： 310 nm (Flavanone)， 270 nm (Warfarin) Back pressure： 20 MPa Sample： 10 mg/mL racemic flavanone in methanol 10 mg/mL racemic warfarin in methanol Warfarin Injection volume： 1.0 mL (Flavanone)， 0.5 mL (Warfarin) Injection number： 40 (Flavanone)， 80 (Warfarin) Injection interval： 3.5 min Fraction mode： Timed fraction Pretreatme n t P rocedure o f f ra ctio n ated enantiomers Results and Discussion Results of Semi-Pre p S FC Figure 4 shows the collection r es u lts o f f la vanone and warfarin i n a single inject i on. Fi r st and second peaks were C collected into vial 1 and 3 by timed fraction. The range between t hose p eaks was collected into another vial (v i al 2) to enhance t he pu r ity of ea ch peak. In order t o obtain 100 to 200 mg of each enan ti omer ， r epeat i n ject i o n s were executed (40 times for f l avanone and 80 times for warfar i n). The total r un time and so l vent s consumption were reduced 1.6 t imes using stacked injection m ode. Figure 5 s h ows stacked inject i on r esults of f l avanone and warfar in， respective l y. Table 1shows t he recovered amount (dry weight ) of each ena n t i omer by sem i -prep SFC. T h ese e n ant i omer sample s were applied to later ECD and VCD meas u rements. Flav a no ne Warfarin Fig.5 Stacked injection results of f lavanone and warfarin from 1st to 20th injection. Peak 1 Peak 2 181 mg (91%) 159 mg (79%) Results of ECDand VCD Measu r eme n ts Figure 6 shows t he experimental and calculated ECD and absorbance spectra of flavanone and Figure 7shows the experimental ECD a n d absorbance spect r a of warfar i n. F i gure 8 shows the exper i mental and calculated i n f rared (I R) and VCD spectra of f lavanone and Figure 9 shows the exper im enta l I R and VCD spectra of warfar i n . As shown in these f i gure， the symmet r ical ECD and VCD spectra were obtained in exper i mental spectra ，respectively. The trace of calculated ECD and VCD spectra (DF T and TD-DFT， B3PW91/CC-pVDZ level ) of f l avanone signif i cantly corresponded to those of experimental spectra. Compared to calculated spect r a， the enantiomers from peak 1 a n d 2 can be i den t if ied to R for m and S form of flavanone. Fig.6 Experimental and calculated ECD and absorbance spectra of flavanone. Fig. 7 Exper i mental ECD and absorbance spectra of warfarin. Conclusion ●The total run time and s olvents consumpt i on by semi-prep SFC were r educed 1.6 times using stacked i n j ection mode (2.5 hours for 150 to 180 mg of each flavanone enantiomer). The symmet r ica l ECD and VCD spectra were obtained in experimental spectra. The trace of calculated ECD and VCD spectra of fl avanone were signi f icantly corresponded t o those of E experimental spectra and t h e enantiomers from peak 1 and 2 can be i dentified to R form and S form of Tlavanone . ●The enant i omer sample amount required for ECD and VCD measu r ements was 3 to 4 mg (twice i nj ection in semi-prep SFC system provide enough sample amount). Acknowled ge ments The authors are gratefu l to prof. M. Uchiyama f r om the University of Tokyo for th e support on t h e DFT and TD-DFT calc u lat i ons. References 1.N. M. Maier， P. Franco， W. Linder， J. of Chromatography A ， 906， 3-33(2001). 2.L. Miller， J. of Chromatography A ， 1250， 250-255(2012).