3.2 Purification and identification of compound A
Succeeding to the above finding, we used the Agilent semi-preparative HPLC equipped with a preparative column (Kromasil) for the initial purification of the target compound (compound A ). An eluent tube was collected every 0.2~0.3 min, and the preparative liquid chromatogram was shown in Fig. 2a. The eluent collected at 22.4, 23.1, 24.1 and 26.3 min (Fig. 2a inset) was analyzed by analytical HPLC, and it was found that the fractions around Rt 22.4~24.1 mainly contained compound A , while the fraction at Rt 26.3 mainly had interference compounds (Fig. 2b). The crude product was obtained by combining those fractions between 22.4~24.1 min of Rt. Then, the collection was further subjected to Agilent semi-preparative HPLC equipped with a preparative Hypesil ODS-BP column. The eluent fractions between 20.2~22.5 min were again collected every 0.2 min (Fig. 2c), and detected by analytical HPLC. It was seen that the fractions at Rt 20.2 min or 22.5 min are impurities (Fig. 2d). The eluent around Rt 21.2 min was collected. After the removal of methanol and water by rotary evaporation and freeze-drying, finally 2.4 mg purified compoundA was obtained.
To determine the chemical structure of compound A , we did various NMR analyses besides LC-MS/MS. The 1H-NMR and13C-NMR spectra of compound A (Table 1, Fig. S2 and S3) indicated six methyl groups and one hydroxymethyl group, while the substrate HLDOA has seven methyl groups and no hydroxymethyl group. The fact showed that compound A was a hydroxylated product of a methyl group of HLDOA. Comparing these spectroscopic data with those of HLDOA (Wang et al., 2018), the shift values of C-28 and C-29, both of which are connected to C-4 carbon atom, are significantly different (Table 1), suggesting that one of the two methyl groups was hydroxylated. The NOESY (nuclear overhauser effect spectroscopy) spectrum data of compound A revealed that 3-β-OH interacted with 29-β-CH3, but did not with 28-CH2OH (Fig. 3a), indicating that the orientation of hydroxylated methyl group and 3-β-OH was different, that is, the hydroxylation reaction occurred at the 28-α-CH3 of HLDOA. Integrated with the data of distortionless enhancement by polarization transfer (DEPT) spectra, heteronuclear multiple-bond correlation (HMBC), heteronuclear single quantum coherence (HSQC) and homonuclear correlation spectroscopy (COSY) (Fig. S4, S5, S6 and S7), the structure of compound A was finally determined to be 3,28-dihydroxy-lanosta-8, 24-dien-26-oic acid (DHLDOA) (Fig. 3b), whose structure has not yet been reported.