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.