According to our research, the proportion of (R)-bavachinin in plants is considerably lower than that of (S)-bavachinin. To obtain the
two bavachinin enantiomers in high optical purity and quantity for additional bioassays and further chiral separation, racemic
bavachinin 1 was prepared via the synthetic route (Scheme 1), which is a previously reported classical synthetic route of racemic
bavachinin.17 4-Hydroxybenzaldehyde 2 was treated with chloromethyl methyl ether in DMF in the presence of N,N-
diisopropylethylamine to obtain 4-(methoxymethoxy)benzaldehyde 3 in an 82.8% yield.18 The 2′-hydroxyacetophenone 4 was treated
with prenyl bromide in acetone with potassium carbonate by using Williamson ether synthesis to obtain 5, which then yielded 56.1%
(two steps) of 6 in N,N-diethylaniline through Claisen rearrangement.19 The intermediate 6 was treated with 3 and potassium
trimethylsilanolate in ethanol to obtain chalcone 7 by using base-catalyzed Claisen-Schmidt condensation; the obtained yield was
60.8%. Compound 7 was refluxed with potassium fluoride in methanol to obtain the corresponding methoxymethylated flavanone 8,
which was formed through cyclization of 7 in a 64.4% yield. The methoxymethylated flavanone was demethoxymethylated by using
hydrogen chloride in methanol to obtain the desired racemic bavachinin 1 in a 79.1% yield.20
Before the enantiomeric separation, an analytical SFC method was established. The racemic bavachinin solutions were separated on
a Chiralpak OZ-H column by using isocratic elution. The racemic bavachinin was then purified using a customized preparative SFC
system. The sample was separated on a preparative Chiralpak OZ-H column. Isocratic elution, similar to that used in the analytical
method, was performed to obtain (S)- and (R)-bavachinin. The ee and optical rotation values of the two enantiomers are shown in
Figure 3 and Table 1. According to a previous report, (-)-flavanones have S-chirality at the C-2 position, and the S configuration
accounts for most natural bavachinin forms.5 The optical rotation value of natural bavachinin was -10.4° ([α]D30 , CHCl3),5 which was
consistent with the ee value of natural bavachinin that we investigated.
To evaluate the PPAR-γ agonist activity of bavachinin enantiomers, we used a time-resolved fluorescence resonance energy
transfer21 (TR-FRET)-based competitive binding assay to further validate PPAR-γ ligands according to manufacturer protocol.
Competitive ligand binding to a nuclear receptor is detected on the basis of the ability of a test compound to displace labeled ligands
from the receptor, which results in the loss of FRET signals (520 nm/495 nm).
The test results before the assays were performed revealed that the PerkinElmer EnVision Multilabel reader can detect changes in
TR-FRET signals. IC50 values were calculated using GraphPad Prism 5.0. The inhibition constant (Ki) of the competing ligand was
obtained from IC50 values by applying the Cheng-Prusoff equation. A commercially available PPAR-γ agonist, rosiglitazone, was used
as a positive control for PPAR-γ activity. Natural bavachinin, (S)-bavachinin, and (R)-bavachinin were tested separately. The results
suggested that (S)- and (R)-bavachinin demonstrated similar PPAR-γ agonist activities (Figure 4, Table 2).
To elucidate the interaction between bavachinin and PPAR-γ, we performed molecular docking of the two enantiomers onto the
PPAR-γ receptor-binding domain. Before docking, the crystal structure of PPAR-γ (PDB ID: 1FM9)22 was prepared using “Protein
Preparation Wizard”.23-26 Protons were added, and bond orders and atomic charges were assigned. Protonation states for basic and acidic
residues were assigned according to the optimization of hydrogen bonding patterns. Default parameters were adapted for the final
minimization of the protein. Ligands were prepared using LigPrep27 with Epik generating different protonation states at target pH values
ranging from 5.0 to 9.0.
According to the docking result (Figure 5, Table 2), (S)- and (R)-bavachinin are located in the binding site of PPAR-γ, with similar
orientations. Both enantiomers interact with the protein mainly through hydrophobic interactions with residues such as Ile281, Cys285,
Leu330, Ile341, Met348, and Leu353 and a hydrogen-bond with the backbone of Ser342 (Figure 5). Because of the chirality, the
phenolic hydroxyl group of (R)-bavachinin can form a hydrogen-bond with Glu259, whereas (S)-bavachinin cannot, which may explain
the slightly higher affinity of (R)-bavachinin. The docking score achieved from molecular docking was consistent with the IC50 values
obtained in the competitive binding assay experiment.
In conclusion, we established a concise and efficient method for rapid small-scale separation of bavachinin enantiomers in high
optical purity by taking advantage of SFC. We also completed the initial evaluation of the PPAR-γ agonist activity of the two
enantiomers. The results suggest that (S)- and (R)-bavachinin demonstrate similar PPAR-γ agonist activities. Further investigation on
the structure-activity relationships of bavachinin and its synthetic analogs is ongoing in our laboratories to determine their PPAR-γ
agonist activities.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (Grant No. 81173518), the Eastern Scholar Tracking
Program of Shanghai Municipal Education Commission (2012-90), International Collaboration Project (2014DFA31130) and the
program of Shanghai E-Institute of bioactive constituents in Traditional Chinese Medicine.
References and notes:
1. Bharathi, E.; Jagadeesan, G. Biomedicine & Preventive Nutrition. 2014, 4, 131.
2. Li, F.; Chow, S.; Cheung, W.; Chan, F.L.; Chen, S.; Leung, L.K. Journal of Nutritional Biochemistry. 2013, 24, 1112.
3. Erlund, I.; Meririnne, E.; Alfthan, G.; Aro, A. Journal of Nutrition. 2001, 131, 235.
4. Chtourou, Y.; Fetoui, H.; Jemai, R.; Slima, A.B.; Makni, M.; Gdoura, R. European Journal of Pharmacology. 2015, 746, 96.
Du, Guoxin
