Two new bibenzyl methylglucosides as SIRT3 activators obtained through microbial transformation

Abstract Microbial transformation of dihydroresveratrol (DHRSV) using Beauveria bassiana has produced two new methylglucosylated derivatives of DHRSV (1 and 2), whose structures were characterized as 4′-O-(4″-O-methyl-β-D-glucopyranosyl)-dihydroresveratrol (4′-O-MG DHRSV, 1) and 3-O-(4″-O-methyl-β-D-glucopyranosyl)-dihydroresveratrol (3-O-MG DHRSV, 2) on the basis of spectroscopic methods. They showed moderate SIRT3 agonistic activity, and compound 2 exhibited the best deacetylation of 406.63% at 10 μM. The activity of 2 increased by 3.12-fold compared with that of DHRSV, since 2 performed better in molecular docking assay (GScore −8.445).


Introduction
Sirtuin 3 (SIRT3), an important NAD þ -dependent deacetylase, is linked to a broad range of physiological and pathological processes, including aging and aging-related diseases [1].Besides, SIRT3 plays a significant role in regulating mitochondrial metabolism and energy production [2], protecting against many harmful oxidative stress-related phenotypes such as reactive oxygen species (ROS)-induced heart failure [3].
A large number of naturally-occurred bibenzyls are existing in nature, which have been reported to possess significant biological activities such as anti-oxidant [4] and anti-inflammatory properties [5].In recent years, it has been reported that bibenzyl derivatives could increase expression of SIRT3, preventing the accumulation of mitochondrial ROS [6].Therefore, considering the crucial role of SIRT3 in preventing cellular senescence, the production of potential SIRT3 activators based on bibenzyl skeleton is of great significance.
Microbial transformation is an effective approach to modify synthetic and natural products using microorganisms like fungi [7].In our previous work, Beauveria bassiana was used to transform the stilbenes, and five stilbene methylglucosides from the fermentation broth were identified [8].In the framework of our ongoing research for bibenzyl SIRT3 activators, we selected a representative bibenzyl dihydroresveratrol (DHRSV) as the substrate to be transformed by B. bassiana.Herein, we describe the progress of biotransformation, the isolation, structure elucidation and SIRT3 deacetylase activity evaluation of transformation products.

Biotransformation of dihydroresveratrol (DHRSV, 3) by Beauveria bassiana
After 3 days incubation of DHRSV with B. bassiana, the fermentation broth was extracted and analyzed by high performance liquid chromatography-mass spectrometry (HPLC-MS).The results revealed that the substrate DHRSV (3) was almost transformed to one major metabolite (2) and the other minor one (1) (Figure 1(A)).The molecular weight of the two metabolites was both deduced as 406 a.m.u.from the negative ion at m/z 405.20 [M-H] -, an increase of 176 a.m.u.over DHRSV (3) (m/z 229.10 [M-H] -), indicating the introduction of O-methyl-glucose (Figure 1(B)).In order to confirm the structure of the two metabolites, the crude extract was purified using preparative HPLC on a larger scale.Metabolites 1 and 2 were identified as 4 2), respectively, by high-resolution electrospray ionization mass spectra (HRESIMS) (Supplementary Figures S1, 8), one dimensional NMR (1D NMR), and two dimensional NMR (2D NMR) spectra (Supplementary Figures S3-S7, S10-S14).

The structure elucidation of new metabolites 1 and 2
Metabolite 1, a brown amorphous powder, had the molecular formula C 21 H 26 O 8 , as determined by HRESIMS ion peak at m/z 407.1696 [M þ H] þ , which also indicated the introduction of O-methyl-glucose on DHRSV (Supplementary Figure S1).In the 13   S3), which were consistent with the chemical shift values of 4 00 -O-methyl-glucose moiety previously reported [8].Moreover, the location of methoxyl group was confirmed by the long-range correlation of OCH 3 (d 3.59) with C-4 00 (d 80.6) in the HMBC spectrum (Figure 2, Supplementary Figure S7).Detailed analysis of the HMBC spectra (Figure 2, Supplementary Figure S7) and the correlation of the anomeric proton H-1" (d 4.83, d, J ¼ 8.0 Hz) to C-4 0 (d 157.3) confirmed the connection of the sugar moiety to the aglycon at the C-4 0 position.Meanwhile, the same chemical shift signal of C-2 and C-6 in 13   optical rotation value, the absolute configuration of sugar was deduced as D-configuration.Finally, the structure of metabolite 1 was elucidated as 4 Metabolite 2 was obtained as faint yellow amorphous powder.It possessed a molecular formula of C 21 H 26 O 8 as established by the HRESIMS ion at m/z 407.1692S8), which was the isomer of 1.Similarly, the 13 C NMR spectrum of metabolite 2 (Table 1, Supplementary Figure S11) showed 21 signals, seven of which were attributed to the O-methyl-glucose moiety (d 102.0, 74.9, 77.9, 80.5, 76.9, 61.9, and 60.9).The corresponding signals in the 1 H NMR spectrum (Table 1, Supplementary Figure S10) were also consistent with the chemical shift values of -O-methyl-glucose moiety as mentioned above [8], confirmed by the longrange correlations of OCH 3 (d 3.58) with C-4 00 (d 80.5) in the HMBC spectrum (Figure 2).Furthermore, the HMBC correlation (Figure 2, Supplementary Figure S14) of the anomeric proton H-1" (d 4.76, d, J ¼ 8.0 Hz) to C-3 (d 159.9) was remarkable, showing the sugar unit of metabolite 2 should be connected to the C-3 position.Owing to the introduction of the O-methyl-glucose moiety, there was a slight difference between the chemical shift signals of C-3 (d 159.9) and C-5 (d 159.1) in 13 C-NMR spectrum (Table 1).In addition, the coupling constant of H-1" in metabolite 2 was large (J ¼ 8.0 Hz), indicating the b-configuration of the O-methyl-glucose (Table 1).The similar specific optical rotation values of 1 and 2 illustrated the absolute configurations of sugars were the same.On the basis of the above evidences, the structure of metabolite 2 was elucidated as 3
In order to find out the possible reason, structure-based molecular docking was performed.We selected 3-O-MG DHRSV (2) and DHRSV (3) to bind to SIRT3 protein at a suitable binding pocket (Figure 4).As shown in Figure 4(B,C), two bibenzyls (2 and 3) could form hydrogen bonding interactions with residues such as Asp 231 and Pro 176 , as well as pp interaction with Phe 157 .Additionally, the hydroxyl group on the glycosyl group of 2 was able to form hydrogen bonding interaction with Asp 156 (Supplementary Figure S15).The Glide GScore value of 2 was −8.445, which was lower than that of 3 (-7.607),indicating better affinity than the substrate 3.
In summary, we isolated and identified two new bibenzyl methylglucosides (1 and 2) from the fermentation broth of B. bassiana after feeding the substrate DHRSV.This indicated that B. bassiana has the capability to O-methylglucosylate bibenzyls and can be applied to produce a wide range of unnatural bibenzyl O-methylglucosides by biocatalysis.Specific methylglucosylated products were found to enhance bioactivity, solubility and stability [10].In vitro biological tests, methylglucoside of DHRSV (3-O-MG DHRSV, 2) displayed better SIRT3 agonistic activity than DHRSV, which might be due to the introduction of glycosyl group and the lower Glide GScore value.Considering the anti-aging and antioxidant functions of SIRT3, 3-O-MG DHRSV (2) could be a promising starting point for further functional optimization to treat a series of metabolic syndromes caused by excess ROS.

The substrate and culture medium
Dihydroresveratrol (DHRSV, purity � 98%) was purchased from Baoji Herbest Bio-Tech Co., Ltd.The biotransformation was performed in the liquid medium composed of 2% glucose, 0.5% yeast extract, 0.5% tryptone, 0.5% NaCl and 0.5% KH 2 PO 4 (pH 7.0).The culture medium was prepared by the following procedure: the corresponding five solids were weighed and added to the appropriate distilled water according to the volume of the culture medium required, followed by mixing thoroughly with a magnetic stirrer.The pH was adjusted to 7.0 with 30% sodium hydroxide.Then the medium was sterilized in an autoclave at 121 � C for 20 min before use.

Biotransformation procedure
First of all, a suitable amount of B. bassiana was scraped into 100 ml liquid medium and incubated at 28 � C with shaking speed at 160 rpm for 2-3 days to obtain the seed culture.Then, each of the six identical 500-ml flasks containing 90 ml of fresh liquid medium, 10 ml of the seed culture and 15 mg of dihydroresveratrol (dissolved in 250 ll DMSO) was incubated at 32 � C with shaking speed at 190 rpm for 3 days on a rotary shaker.The culture control was a fermentation blank in which B. bassiana was grown under the same conditions but without the substrate dihydroresveratrol.The substrate control was a sterile medium containing the same amount of substrate but without the B. bassiana strain.

SIRT3 activity assay in vitro
SIRT3 activity assay in vitro was carried out using the fluorometric SIRT3 Activity Assay Kit (#ab156067, Abcam, Cambridge, MA, USA) according to the manufacturer's instructions.The test compounds were dissolved with DMSO at 100 lM to obtain the mother solutions.Following the instruction manual, ddH 2 O, SIRT3 assay buffer, fluoro-substrate peptide and NAD were added in sequence to microtiter plate wells correspondingly.Then, 5 ll of test compounds or just 5 ll of solvent DMSO and developer were added to each well of the microtiter plate and mixed thoroughly.Finally, 5 ll of recombinant SIRT3 was added to initiate reaction.The fluorescence intensity reflecting SIRT3 deacetylation activity was measured by a microplate reader with excitation at 355 nm and emission at 460 nm for 30 min at 2 min intervals.The fluorescence value of the corresponding response time point was recorded while the reaction velocity remains constant.All experimental groups were performed in triplicate.Calculations and the construction of the bar graph were performed with GraphPad Prism software.

Molecular docking
The molecular docking studies were carried out using grid-based ligand docking program by Maestro 2021 molecular docking suite incorporated in the Schrodinger package (Schrodinger, Inc., NY, US) [11].The crystal structure of human SIRT3 protein obtained by crystal X-ray diffraction was retrieved from Protein Data Bank (PDB ID: 4C7B, resolution: 2.10 Å) [12].In addition, optimized potential for liquid simulations (OPLS_2005) force field was applied for the structure of SIRT3 protein closest to the active receptor in the organisms [13].After preparing ligands, proteins, and preparation of grid formation of the active site of protein, Glide docking suites output GScore (empirical scoring function) by predicting the best binding orientation to the protein target [14].Compounds 2 and 3 were based on the docking region, aligning the docking posture with the ligand bibenzyl derivative mentioned previously in the crystal structure 4C7B [6], and selecting the most similar conformation as the output result.
C-NMR spectrum at d 108.0 further supported the 4 00 -O-methyl-glucose moiety was introduced into B-ring of DHRSV.A large coupling constant (J ¼ 8.0 Hz) of the anomeric proton H-1" (d 4.83) was observed, confirming the b-configuration of the glucosidic bonds.Considering the same catalytic mechanism and the similar specific

Figure 1 .
Figure 1.HPLC-MS results for the biotransformation products of DHRSV (3) by B. bassiana.(A) The BB þ DHRSV shows HPLC chromatogram of the products (1 and 2) of DHRSV transformed by B. bassiana for 3 days at 190 rpm/32 � C. Substrate control refers to liquid medium containing the same amount of substrate under the same culture conditions but without the B. bassiana strain.Culture control is a fermentation blank in which B. bassiana was grown under the same conditions but without the substrate dihydroresveratrol.(B) MS data of 1-3.(C) Conversion route of DHRSV (3) by B. bassiana.

Figure 2 .
Figure 2. The key HMBC and 1 H-1 H COSY correlations of compounds 1 and 2.

Figure 3 .
Figure 3.The SIRT3 deacetylase activity of substrate dihydroresveratrol (DHRSV) and its methylglucosylated derivatives (compounds 1 and 2) at 10 lM in vitro.The no additive group refers to DMSO without test compounds.The left ordinate represents the deacetylation activity of SIRT3 in each group, in which the value of the no additive group is set to 100%.The right ordinate represents the increasing multiples of the two conversion products compared with dihydroresveratrol.All data were expressed as the mean ± SEM (n ¼ 3).

Figure 4 .
Figure 4. Interactions between bibenzyl derivatives (2 and 3) and SIRT3 protein predicted by molecular docking.(A) Molecular docking of compounds 2 and 3 with SIRT3 protein.(B) Compound 2 bound to SIRT3 protein, and the red represents compound 2. (C) Compound 3 bound to SIRT3 protein, and the yellow represents the substrate 3.
of Bioactive Substance and Function of Natural Medicines & NHC Key Laboratory of Biosynthesis of Natural Products, CAMS Key Laboratory of Enzyme and Biocatalysis of Natural Drugs, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China CONTACTTing Gong gongting@imm.ac.cn;Ping Zhu zhuping@imm.ac.cnState Key Laboratory Supplemental data for this article can be accessed online at https://doi.org/10.1080/10286020.2023.2283483.