New Glycosylated Dihydrochalcones Obtained by Biotransformation of 2′-Hydroxy-2-methylchalcone in Cultures of Entomopathogenic Filamentous Fungi

Flavonoids, including chalcones, are more stable and bioavailable in the form of glycosylated and methylated derivatives. The combined chemical and biotechnological methods can be applied to obtain such compounds. In the present study, 2′-hydroxy-2-methylchalcone was synthesized and biotransformed in the cultures of entomopathogenic filamentous fungi Beauveria bassiana KCH J1.5, Isaria fumosorosea KCH J2 and Isaria farinosa KCH J2.6, which have been known for their extensive enzymatic system and ability to perform glycosylation of flavonoids. As a result, five new glycosylated dihydrochalcones were obtained. Biotransformation of 2′-hydroxy-2-methylchalcone by B. bassiana KCH J1.5 resulted in four glycosylated dihydrochalcones: 2′-hydroxy-2-methyldihydrochalcone 3′-O-β-d-(4″-O-methyl)-glucopyranoside, 2′,3-dihydroxy-2-methyldihydrochalcone 3′-O-β-d-(4″-O-methyl)-glucopyranoside, 2′-hydroxy-2-hydroxymethyldihydrochalcone 3′-O-β-d-(4″-O-methyl)-glucopyranoside, and 2′,4-dihydroxy-2-methyldihydrochalcone 3′-O-β-d-(4″-O-methyl)-glucopyranoside. In the culture of I. fumosorosea KCH J2 only one product was formed—3-hydroxy-2-methyldihydrochalcone 2′-O-β-d-(4″-O-methyl)-glucopyranoside. Biotransformation performed by I. farinosa KCH J2.6 resulted in the formation of two products: 2′-hydroxy-2-methyldihydrochalcone 3′-O-β-d-(4″-O-methyl)-glucopyranoside and 2′,3-dihydroxy-2-methyldihydrochalcone 3′-O-β-d-(4″-O-methyl)-glucopyranoside. The structures of all obtained products were established based on the NMR spectroscopy. All products mentioned above may be used in further studies as potentially bioactive compounds with improved stability and bioavailability. These compounds can be considered as flavor enhancers and potential sweeteners.


Introduction
The widespread and well-documented evidence of health-promoting properties of dietary flavonoids results in a significant increase of interest in their application as dietary supplements and medicaments [1][2][3].
Chalcones are one of the subclasses of dietary flavonoids, which consist of two aromatic rings joined by a three-carbon α, β-unsaturated carbonyl system. Their mostly described biological activities can be highlighted as antifungal, antivirus, anti-diabetic, anti-inflammatory, and antitumor [1,[4][5][6].
The poor aqueous solubility of flavonoid aglycones limits their oral bioavailability and, as a consequence, pharmacological application. To overcome this obstacle, many attempts have been made, such as shifting the site of the absorption from the large intestine to the small intestine, increasing metabolic stability and aqueous solubility, inventing novel formulations for topical delivery [7,8].
As a result of biotransformation of 2′-hydroxy-2-methylchalcone in B. bassiana KCH J1.5 culture we obtained four glycosylated dihydrochalcones. In the case of I. fumosorosea KCH J2 as a biocatalyst only one glycosylation product was obtained. I. farinosa KCH J2.6 biotransformed 2′-hydroxy-2-methylchalcone into two products formed also by B. bassiana KCH J1. 5. According to our best knowledge, all obtained biotransformation products have not been previously known and can be used in biological activity and bioavailability assessment. Moreover, obtained dihydrochalcone glycosides may be concerned as potential flavor enhancers and health-promoting sweeteners.
Experiments were performed on a semi-preparative scale to determine the chemical structures of biotransformation products and their isolated yields.
As a result of 2′-hydroxy-2-methylchalcone biotransformation in the B. bassiana KCH J1.5 culture four glycosylated dihydrochalcones were obtained. In the case of I. fumosorosea KCH J2 as a biocatalyst, only one product was obtained. The biotransformation in the culture of I. farinosa KCH J2.6 resulted in the formation of two products formed also in B. bassiana KCH J1.5 culture. In the experiment, 2′-hydroxy-2-methylchalcone (3) was synthesized in the Claisen-Schmidt condensation reaction (Scheme 1.) (4.1. Substrate). The structure of product (3) was confirmed based on NMR (Nuclear Magnetic Resonance)spectroscopy (Tables 1 and 2). In the 1 H NMR (Proton Nuclear Magnetic Resonance) spectrum were observed two characteristic signals: the first one from the hydroxyl moiety at δ = 12.88 ppm with the corresponding signal from C-2′ in the 13 C NMR (Carbon-13 Nuclear Magnetic Resonance) spectrum at δ = 164.5 ppm, and the second one from the three protons of the methyl moiety at δ = 2.51 ppm with the corresponding signal from C-2-CH3 in the 13 C NMR spectrum at δ = 19.8 ppm (Supplementary Materials: Figures S2 and S4). In the 1 H NMR spectrum were also observed two doublets from protons α and β (δ = 8.25 ppm and 7.94 ppm, respectively) and the signals from protons of the A and B rings (Supplementary Materials: Figure S3). In the 13 C NMR spectrum was also observed the signal from the carbonyl group at δ = 195.0 ppm (Supplementary Materials: Figure S4). The couplings between the three protons of the methyl moiety (δ = 2.51 ppm) and the signals from carbons C-1 (δ = 134.4 ppm), C-2 (δ = 139.5 ppm), and C-3 (δ = 131.8 ppm) confirm substitution with the methyl moiety at position C-2 (Supplementary Materials: Figure S180). Moreover, the protons at C-6′ (δ = 8.28 ppm) and at C-4′ (δ = 7.58 ppm) correlate with the signal from carbon at C-2′ Scheme 1. Synthesis of 2 -hydroxy-2-methylchalcone (3) in the Claisen-Schmidt condensation reaction.
Key COSY (on the left) and HMBC (on the right) correlations for the structure elucidation of product (3a). In in the 1 H NMR spectrum, the singlet from the proton of the hydroxyl group also appeared (δ = 8.16 ppm) and shifted signals from B ring were observed: one triplet from the proton at C-5 (δ = 6.92 ppm) and one doublet of doublets from the protons at C-6 and C-4 (δ = 6.72 ppm) indicating that the attachment of an electronegative hydroxyl group occurred at C-3 (Supplementary Materials: Figure  S39). Moreover, in the HMBC experiment hydroxyl group at C-3 (δ = 8.16 ppm) was correlated with C-2 signal (δ = 123.2 ppm) and C-3 signal (δ = 156.3 ppm shifted from δ = 131.8 ppm in substrate (3)) (Supplementary Materials: Figure S52). The signal from the methyl group at C-2 (δ = 2.21 ppm) was also correlated with shifted C-3 signal (Supplementary Materials: Figure S54)  In the case of product (3c) (2′-hydroxy-2-hydroxymethyldihydrochalcone 3′-O-β-D-(4″-O-methyl)-glucopyranoside), in 1 H NMR spectrum shifted signals from B ring were observed: one signal from the proton at C-3 (δ = 7.40 ppm), which merged with the signal from not shifted proton at C-4′, one multiplet from the proton at C-6 (δ = 7.28 ppm), and one merged signal from the protons at C-4 and C-5 (δ = 7.20 ppm) (Supplementary Materials: Figure S58). In the 1 H NMR spectrum, two additional signals In the product (3d), the substitution with the hydroxyl moiety at C-4 was confirmed by the appearance of singlet at δ = 8.02 ppm in the 1 H NMR spectrum and correlation between this signal and signals from carbon C-3 (δ = 117.9 ppm) and C-5 (δ = 113.7 ppm) Scheme 5. Key COSY (on the left) and HMBC (on the right) correlations for the structure elucidation of product (3c).

Scheme 5.
Key COSY (on the left) and HMBC (on the right) correlations for the structure elucidation of product (3c).
In the product (3d), the substitution with the hydroxyl moiety at C-4 was confirmed by the appearance of singlet at δ = 8.02 ppm in the 1 H NMR spectrum and correlation between this signal and signals from carbon C-3 (δ = 117.9 ppm) and C-5 (δ = 113.7 ppm) in the HMBC experiment (Supplementary Materials: Figures S76, S88) Key COSY (on the left) and HMBC (on the right) correlations for the structure elucidation of product (3d). (3)  In the product (3b) (2 ,3-dihydroxy-2-methyldihydrochalcone 3 -O-β-D-(4 -O-methyl)glucopyranoside), in the 1 H NMR spectrum, the singlet from the proton of the hydroxyl group also appeared (δ = 8.16 ppm) and shifted signals from B ring were observed: one triplet from the proton at C-5 (δ = 6.92 ppm) and one doublet of doublets from the protons at C-6 and C-4 (δ = 6.72 ppm) indicating that the attachment of an electronegative hydroxyl group occurred at C-3 (Supplementary Materials: Figure S39). Moreover, in the HMBC experiment hydroxyl group at C-3 (δ = 8.16 ppm) was correlated with C-2 signal (δ = 123.2 ppm) and C-3 signal (δ = 156.3 ppm shifted from δ = 131.8 ppm in substrate (3)) (Supplementary Materials: Figure S52). The signal from the methyl group at C-2 (δ = 2.21 ppm) was also correlated with shifted C-3 signal (Supplementary Materials: Figure S54) (Scheme 4).

Biotransformation of 2′-Hydroxy-2-methylchalcone
In the case of product (3c) (2 -hydroxy-2-hydroxymethyldihydrochalcone 3 -O-β-D-(4 -O-methyl)-glucopyranoside), in 1 H NMR spectrum shifted signals from B ring were observed: one signal from the proton at C-3 (δ = 7.40 ppm), which merged with the signal from not shifted proton at C-4 , one multiplet from the proton at C-6 (δ = 7.28 ppm), and one merged signal from the protons at C-4 and C-5 (δ = 7.20 ppm) (Supplementary Materials: Figure S58). In the 1 H NMR spectrum, two additional signals were observed: one doublet from two protons at the methylene group at C-2 (δ = 4.73 ppm) and one triplet from the proton at hydroxyl group attached to this methylene group (δ = 4.15 ppm). In the COSY experiment can be observed coupling between both signals, confirming hydroxylation of the methyl moiety at C-2 (Supplementary Materials: Figure S65). The two protons from the methylene group (δ = 4.73 ppm) was also correlated with C-1 (δ = 140.0 ppm) and C-3 (δ = 129.1 ppm) in the HMBC experiment, also indicating hydroxylation at C-2-CH 3 (Supplementary Materials: Figure S72) (Scheme 5). Such a reaction was observed previously in the case of the attachment of the hydroxyl moiety to the methyl moiety in the biotransformation of 6-methylflavanone performed also by B. bassiana KCH J1.5 [34].
In the product (3d), the substitution with the hydroxyl moiety at C-4 was confirmed by the appearance of singlet at δ = 8.02 ppm in the 1 H NMR spectrum and correlation between this signal and signals from carbon C-3 (δ = 117.9 ppm) and C-5 (δ = 113.7 ppm) in the HMBC experiment (Supplementary Materials: Figures S76 and S88) (Scheme 6). The isolated yield of (3e) was 5.3% (4.8 mg) (Scheme 7). The structure of product 3e was established based on the NMR spectroscopy (Tables 1 and 2, Scheme 8 (with key COSY and HMBC correlations). The resulting compound was 4 -O-methylglycosylated dihydrochalcone, like products (3a-3d). However, in this case, glycosylation occurred at C-2. In the 1 H NMR spectrum signal from the hydroxyl moiety at C-2 (δ = 12.88 ppm) disappeared, indicating substitution at this position. At δ = 8.08 ppm appeared singlet from one proton demonstrating substrate hydroxylation at another position (Supplementary Materials: Figure S95). The three-proton signal of the methyl moiety at C-2 was shifted from δ = 2.51 ppm to δ = 2.19 ppm (like product (3b)) indicating an electronegative atom's attachment in the adjacent position at C-3. Moreover, in the HMBC experiment, the signal from the hydroxyl group at C-3 (δ = 8.08 ppm) was coupled with the shifted signal from the carbon at C-3 (δ = 131.8 ppm in (3), δ = 156.1 ppm in (3e)) and triplet from one proton at C-5 (δ = 6.90 ppm) was also coupled with the signal from carbon at C-3 (Supplementary Materials: Figure S107). The substitution with the 4 -Omethylglucosyl moiety at C-2 was confirmed by coupling between the signal from proton at the anomeric carbon atom (δ = 5.04 ppm) and the signal from carbon at C-2 , which was shifted from δ = 164.5 ppm to δ = 157.1 ppm (Supplementary Materials: Figure S109). The coupling between proton at 6 (δ = 7.58 ppm), proton at C-4 (δ = 7.47 ppm), proton at C-3 (δ = 7.30 ppm) and carbon at C-2 also confirmed glycosylation at C-2 (Supplementary Materials: Figure S107). The structure of product 3e was established based on the NMR spectroscopy (Tables 1 and 2, Scheme 8 (with key COSY and HMBC correlations). The resulting compound was 4″-O-methylglycosylated dihydrochalcone, like products (3a-3d). However, in this case, glycosylation occurred at C-2. In the 1 H NMR spectrum signal from the hydroxyl moiety at C-2′ (δ = 12.88 ppm) disappeared, indicating substitution at this position. At δ = 8.08 ppm appeared singlet from one proton demonstrating substrate hydroxylation at another position (Supplementary Materials: Figure S95). The three-proton signal of the methyl moiety at C-2 was shifted from δ = 2.51 ppm to δ = 2.19 ppm (like product (3b)) indicating an electronegative atom's attachment in the adjacent position at C-3. Moreover, in the HMBC experiment, the signal from the hydroxyl group at C-3 (δ = 8.08 ppm) was coupled with the shifted signal from the carbon at C-3 (δ = 131.8 ppm in (3), δ = 156.1 ppm in (3e)) and triplet from one proton at C-5 (δ = 6.90 ppm) was also coupled with the signal from carbon at C-3 (Supplementary Materials: Figure S107). The substitution with the 4″-O-methylglucosyl moiety at C-2′ was confirmed by coupling between the signal from proton at the anomeric carbon atom (δ = 5.04 ppm) and the signal from carbon at C-2′, which was shifted from δ = 164.5 ppm to δ = 157.1 ppm (Supplementary Materials: Figure S109).The coupling between proton at 6′ (δ = 7.58 ppm), proton at C-4′ (δ = 7.47 ppm), proton at C-3′ (δ = 7.30 ppm) and carbon at C-2′ also confirmed glycosylation at C-2′ (Supplementary Materials: Figure S107).
However, to the best of our knowledge, we are the first team to describe biotransformations leading to the reduction of the double bond together with the glycosylation of the chalcone substrate. All three used strains of entomopathogenic filamentous fungi B. bassiana KCH J1.5, I. fumosorosea KCH J2, and I. farinosa KCH J2.6 were able to carry out such reactions.
The physical data, including color and form, melting point ( • C), molecular ion mass, molecular formula, retention time t R (min), retardation factor Rf, andNMR spectral data of the resulting compound (3)are presented below, in Tables 1 and 2 Table 1, 13 C-NMR, see Table 2, Supplementary Materials: Figures S1-S18.

Microorganisms
The biotransformations were performed using three strains of entomopathogenic filamentous fungi B. bassiana KCH J1.5, I. fumosorosea KCH J2, and I. farinosa KCH J2.6. Microorganisms were assembled from the Department of Chemistry of Wrocław University of Environmental and Life Sciences, Poland.
The description of material collection, fungi propagation, and genetic identification have already been described in our previous papers [33,40]. The filamentous fungi were maintained on potato slants at 4 • C and were subcultured to a liquid medium before use in the experiments [33,40].

Analysis
The course of the biotransformation was tracked by chromatographic methods (TLC, HPLC). TLC analysis was carried out using TLC Silica gel 60/Kieselguhr F254 (0.2 mm thick) plates (Merck, Darmstadt, Germany). As the developing system was used a mixture of chloroform (Chempur, PiekaryŚląskie, Poland) and methanol (Chempur, PiekaryŚląskie, Poland) (9:1 v/v). The products were observed under the ultraviolet lamp at λ = 254 nm and λ = 365 nm without additional visualization.
HPLC analyses were performed on a Dionex Ultimate 3000 instrument (Thermo Fisher Scientific, Waltham, MA, USA) with a DAD-3000 diode array detector using analytical octadecyl silica (ODS) 2 column (4.6 mm × 250 mm, Waters, Milford, MA, USA) and pre-column. The gradient program was as follows: initial conditions-32.5% B in A, 4 min-40% B in A, 8 min-40% B in A, 10 min-45% B in A, 15 min-95% B in A, 18 min-95% B in A, 19 min-32.5% B in A, 23 min-32.5% B in A. The flow rate was 1 mL min −1 , the injection volume was 5 µL, and the detection wavelength was 280 nm [34].
Separation of the products obtained by the scale-up biotransformation was achieved using 500 and 1000 µM preparative TLC silica gel plates (Analtech, Gehrden, Germany). The elution of the post-reaction products from the adsorbent on TLC plate was performed with the use of mixture of chloroform and methanol (9:1 v/v). Afterward, separate gel fractions were extracted with 20 mL of ethyl acetate (Chempur, PiekaryŚląskie, Poland) three times. The extracts from a single fraction were combined. The solvent was evaporated from all fractions under reduced pressure and 0.9 cm 3 of deuterated acetone was added to each sample prior to the NMR analysis [34].
Molecular formulas of all products were confirmed by analysis performed on the LC-MS 8045 SHIMADZU Triple Quadrupole Liquid Chromatograph Mass Spectrometer with electrospray ionization (ESI) source (Shimadzu, Kyoto, Japan). Identification of compounds was performed as described previously, with minor modifications [41]. Analyses were conducted using method "MRM event from precursor ion search". It means that in each analysis, in a sample with a pure compound, only a specific ion with a known molecular mass (determined by previous NMR analysis) was searched. The separation was achieved on the Kinetex column (2.6 µM C18 100 Å, 100 mm × 3 mm, Phenomenex, Torrance, CA, USA) operated at 30 • C. The mobile phase was a mixture of 0.1% aqueous formic acid v/v (A) and acetonitrile (B). The gradient program was as follows: initial conditions-80% B in A, 6.5 min-100% B, 7 min-80% B in A. The flow rate was 0.4 mL min −1 and the injection volume was 5 µL. The principal operating parameters for the LC-MS were set as follows: nebulizing gas flow: 3 L min −1 , heating gas flow: 10 L min −1 , interface temperature: 300 • C, drying gas flow: 10 L min −1 , data acquisition range, m/z 100-1000 Da; ionization mode, negative and positive. Data were collected with LabSolutions version 5.97 (Shimadzu, Kyoto, Japan) software.

Screening Procedure
The screening procedure was performed to assess time needed for the complete microbial transformation of substrate (3). As a growth medium for entomopathogenic filamentous fungi was used a modified Sabouraud medium (10g aminobac (purchased from BTL, Warsaw, Poland), 30 g saccharose (purchased from Chempur, PiekaryŚląskie, Poland), 1 L distilled water). Firstly, the culture of filamentous fungi strain was transferred from potato slants to a 300 mL Erlenmeyer flask with 100 mL of liquid medium. This preincubation culture was bred on a rotary shaker (DHN, Warsaw, Poland) (140 rpm) at 25 • C for 72 h. Secondly, the pre-grown culture (0.5 mL) was transferred to another 300 mL Erlenmeyer flask with 100 mL of liquid medium and also incubated for 72 h at 25 • C on a rotary shaker (140 rpm). Afterward, 10 mg of the substrate, 2 -hydroxy-2-methylchalcone (3), dissolved in 0.5 mL of dimethyl sulfoxide (Chempur, PiekaryŚląskie, Poland), was added to the flask. The molar concentration of substrate (3) was 0.42 mM. The samples were collected and extracted (with 30 mL of ethyl acetate) after 3, 6 and 9 days of incubating the substrate. All biotransformations were ended after confirming complete substrate conversion (or lack of further substrate conversion) after 9 days. The extracts were dried for five minutes with anhydrous magnesium sulfate (Chempur, PiekaryŚląskie, Poland), concentrated using a rotary evaporator (Heidolph, Schwabach, Germany) at temperature 55 • C, and analyzed by TLC and HPLC methods. Two controls were performed: stability of the substrates under biotransformation conditions and microorganisms cultivation with no substrate added [34].

The Semi-Preparative Biotransformations
The semi-preparative biotransformations were performed in 2 L flasks with 500 mL of the modified Sabouraud medium each. For each biotransformation was used one flask. Firstly, 1 mL of the preincubation culture was transferred to the flask and incubated for 72 h under the same conditions as during the screening procedure. Afterward, 50 mg of the substrate, 2 -hydroxy-2-methylchalcone (3), dissolved in 0.5 mL of dimethyl sulfoxide, was added and biotransformation was performed for 9 days. The molar concentration of substrates (3) was also 0.42 mM. The biotransformation was ended after confirming the complete substrate conversion (or lack of further substrate conversion). The metabolites were extracted two times (with 300 mL of ethyl acetate each time). The joined extracts were dried for five minutes with anhydrous magnesium sulfate and concentrated using a rotary evaporator. The products of biotransformation were separated using preparative TLC plates, and then analyzed by NMR, HPLC, and LC-MS [34].

Conclusions
In this study,2 -hydroxy-2-methylchalcone was synthesized and biotransformed in the cultures of three entomopathogenic filamentous fungi strains B. bassiana KCH J1.5, I. fumosorosea KCH J2, and I. farinosa KCH J2.6. The strain B. bassiana KCH J1.5 was capable of the reduction of the double bond of 2 -hydroxy-2-methylchacone, its glycosylation at position C-3 , and hydroxylation at C-3, C-4 and C2-CH 3 . It can be assumed that the presence of the hydroxyl moiety at C-2 is responsible for the directing effect on glycosylation at position C-3 . The strain I. farinosa KCH J2.6 also formed dihydrochalcones with 4 -Omethyl-glycosyl group at C-3 and hydroxylated at C-3. The last strain I. fumosorosea KCH J2 was capable of the reduction of the double bond of 2 -hydroxy-2-methylchacone and its glycosylation at C-2 . In all formed products, the methyl moiety at C-2 remained intact. All the above-mentioned biotransformation products have not been previously described in the literature. Their biological activity and bioavailability can be assessed in further in vitro and in vivo studies. Above and beyond, dihydrochalcones may be considered as flavor enhancers and potential sweeteners.