molecules

Article Microbial Transformation of by Isaria fumosorosea ACCC 37814

Fangmin Dou, Zhi Wang, Guiying Li and Baoqing Dun *

The National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, 12 Zhongguancun South Street, Beijing 100081, China; [email protected] (F.D.); [email protected] (Z.W.); [email protected] (G.L.) * Correspondence: [email protected]; Tel.: +86-10-8210-8746

 Received: 11 February 2019; Accepted: 11 March 2019; Published: 15 March 2019 

Abstract: Glycosylation is an efficient strategy to modulate the solubility, stability, bioavailability and bioactivity of drug-like natural products. Biological methods, such as whole-cell biocatalyst, promise a simple but highly effective approach to glycosylate biologically active small molecules with remarkable regio- and stereo-selectivity. Herein, we use the entomopathogenic filamentous fungus Isaria fumosorosea ACCC 37814 to biotransform a panel of phenolic natural products, including flavonoids and anthraquinone, into their . Six new flavonoid (4-O-methyl)glucopyranosides are obtained and structurally characterized using high resolution mass and nuclear magnetic resonance spectroscopic techniques. These compounds further expand the structural diversity of flavonoid glycosides and may be used in biological study.

Keywords: Isaria fumosorosea; flavonoids; microbial transformation; methylglycosylation

1. Introduction Flavonoids constitute the largest group of plant-derived secondary metabolites which could be found in various vegetables, fruits, nuts, grains, spices, wine and tea. The daily intake of flavonoids can range from 50 to 800 mg, depending on the consumption of flavonoid-containing foods and beverages [1]. Many studies have shown that flavonoids not only have beneficial effects to human health, such as antioxidative, cardioprotective and neuroprotective properties but also display a wide range of pharmacological activities including anticancer, anti-inflammatory, antibiotic and so forth [2–4]. The basic flavonoid structure consists of 15 carbon atoms that arranged in a C6-C3-C6 triple-ring system in which two aromatic rings (ring A and B) are connected by a C3 moiety (ring C). Based on the oxidation level and substitution pattern of ring C, flavonoids can be categorized into several subclasses such as flavones, flavanones, isoflavones, flavonols, flavanonols and flavan-3-ols (Figure1). The structural diversity of individual flavonoids within these subclasses, however, arises from the variations of substitution pattern on ring A and B [1]. Most naturally occurring flavonoids exist in the form of glycosides in which single or multiple sugar moieties are attached to the flavonoid scaffold via O- or C-glycosidic bonds [2]. Many clinically important natural products, such as the antibiotic vancomycin and erythromycin and the antineoplastic doxorubicin, require the sugar moiety to maintain their bioactivities [5–7]. However, glycosylation of flavonoids has a much more complicated impact on their biological activities which is not only influenced by the structural variations of the flavonoid core and the sugar unit but also by the number of sugar moieties and the position that the sugars are attached to [2,8,9]. Despite the fact that flavonoid aglycones usually exhibit stronger activities than their sugar-conjugates in cell-based bioassays, the potential application of aglycones are often limited by their insufficient solubility in organic and aqueous solvents, low bioavailability after oral administration and rapid metabolism in vivo [10,11]. glycosides,

Molecules 2019, 24, 1028; doi:10.3390/molecules24061028 www.mdpi.com/journal/molecules Molecules 2019, 24, 1028 2 of 13 on the other hand, have satisfying solubility and in many cases display improved bioavailability Molecules 2019, 24, x 0 2 of 13 when comparing to the aglycones. For instance, 3- and 4 -O-β-D-glucosides are much more efficiently absorbed than quercetin and quercetin 3-O-β-D-rutinoside in the small intestine [10,12], improved bioavailability when comparing to the aglycones. For instance, quercetin 3- and 4′-O--D- whichglucosides probably are duemuch to more the activeefficiently transport absorbed of glucosidesthan quercetin by Na-dependentand quercetin 3- glucoseO--D-rutinoside transporter in 1 (SGLT1)the small presented intestine in [10,12], the brush-border which probably membrane due to the of active the small transport intestine of glucosides [13]. As by glycosylation Na-dependent plays suchglucose a substantial transporter role 1 in(SGLT1) modulating presented the in physicochemical, the brush-border biologicalmembrane andof the metabolic small intestine properties [13]. of flavonoids,As glycosylation it is important plays such to a further substantial expand role thein modulating structural diversitythe physicochemical, of flavonoid biological glycosides and via chemicalmetabolic and/or properties biological of flavonoids, approaches. it is important to further expand the structural diversity of flavonoid glycosides via chemical and/or biological approaches.

FigureFigure 1. 1.Basic Basic structuresstructures of major flavonoid flavonoid subclasses. subclasses.

ChemicalChemical glycosylation glycosylation of of structurally structurally complex natural natural products products often often requires requires laborious laborious protection-deprotectionprotection-deprotection procedures procedures toto achieveachieve regio- and stereo-selectivity. stereo-selectivity. By By contrast, contrast, biological biological methods,methods, such such as as whole-cell whole-cell biocatalysis, biocatalysis, in vivo total biosynthesis biosynthesis and and inin vitro vitro enzymaticenzymatic reaction reaction usingusing isolated isolated glycosyltransferases, glycosyltransferases, offer offer alternative alternative approaches approaches to realizeto realize one-pot one-pot and and one-step one-step regio- andregio- stereospecific and stereospecific glycosylations glycosylations that proceedthat proceed under under mild mild conditions conditions and and avoid the the use use of oftoxic toxic reagentsreagents [9 ,14 [9,14].]. Filamentous Filamentous fungi, fungi, especially especially species of of genera genera AspergillusAspergillus andand BeauveriaBeauveria, are, are rich rich sources of whole-cell biocatalyst for the glycosylation of natural products [15–17]. Entomopathogenic sources of whole-cell biocatalyst for the glycosylation of natural products [15–17]. Entomopathogenic fungi are an ecologically highly specialized group of microorganisms that infect, kill or seriously fungi are an ecologically highly specialized group of microorganisms that infect, kill or seriously disable insects and many other arthropods. Isaria fumosorosea is an important member of this group disable insects and many other arthropods. Isaria fumosorosea is an important member of this group of fungi which is isolated from over 40 species of arthropods and also presents in soil, water and of fungi which is isolated from over 40 species of arthropods and also presents in soil, water and plants of every continent except for Antarctica [18]. I. fumosorosea alone or in combination with other plantsentomopathogenic of every continent fungal except species for Antarctica have been [18 successfully]. I. fumosorosea commercializedalone or in ascombination mycopesticides with other in entomopathogenicAmerica, Europe fungal and Asia species [11]. have Species been of successfullygenus Isaria commercializedproduce a wide asrange mycopesticides of biologically in America,active Europenatural and products, Asia [11 such]. Species as the ofimmunosuppressant genus Isaria produce sphingoid a wide ISP-I range [19], of the biologically antioxidative active pseudo- natural products,dipeptide such hanasanagin as the immunosuppressant [20], the insecticidal sphingoid cyclodepsipeptides, ISP-I [19], the antioxidative isariins, pseudo-dipeptide isarolides and hanasanaginsafelins/isaridins [20], the [21–24], insecticidal the carotane-type cyclodepsipeptides, sesquiterpenes, isariins, isarolides trichocaranes and E safelins/isaridins and F, CAF-603 [ and21–24 ], thetrichocarane carotane-type C sesquiterpenes, [25] and the novel trichocaranes skeleton polyketide E and F, CAF-603 tenuipyrone and trichocarane [26]. More importantly C [25] and the to us, novel skeletonseveral polyketide I. fumosorosea tenuipyrone species have [26]. shown More importantlyprominent ability to us, in several biotransformingI. fumosorosea a varietyspecies of have natural shown prominentproducts ability into their in biotransformingglycosides [11,27–29]. a variety of natural products into their glycosides [11,27–29]. ToTo further further exploit exploit the the biocatalytic biocatalytic potential potential of I. fumosorosea of I. fumosorosea and to expandand to the expand chemical the diversity chemical diversityof flavonoid of flavonoid glycosides, glycosides, we used Isaria we used fumosoroseaIsaria ACCC fumosorosea 37814 asACCC whole-cell 37814 biocatalyst as whole-cell to biocatalystglycosylate to eight glycosylate flavonoids eight which flavonoids cover the which major subclasses cover the of major flavonoid, subclasses such as of flavanone flavonoid, such( as flavanone 1 and (naringenin 6),1 flavoneand hesperetin (luteolin 26, ), diosmetin flavone 3 (luteolin and apigenin2, diosmetin 7), isoflavone3 and (formononetin 4 and 8) and flavonol ( 5), as well as another two phenolic 7), isoflavone (formononetin 4 and genistein 8) and flavonol (kaempferol 5), as well compounds (emodin 9 and curcumin 10). As a result, six new flavonoid methylglucopyranosides, as another two phenolic compounds (emodin 9 and curcumin 10). As a result, six new flavonoid namely 5,7-dihydroxyflavanone 4′-O--D-(4-O-methyl)glucopyranoside 1b, 5-hydroxyflavanone 4′,7- 0 β methylglucopyranosides, namely 5,7-dihydroxyflavanone 4 -O- -D-(4-O-methyl)glucopyranoside di-O- -D-(4-O-methyl)glucopyranoside0 1d, 4′,5,7-trihydroxyflavone0 3′-O- -D-(4-O- 1b, 5-hydroxyflavanone 4 ,7-di-O-β-D-(4-O-methyl)glucopyranoside 1d, 4 ,5,7-trihydroxyflavone methyl)glucopyranoside 2a, 3′,5,7-trihydroxyflavone 4′-O--D-(4-O-methyl)glucopyranoside 2b, 5,7- 0 β 0 0 β 3 -Odihydroxy-4′-methoxyflavone- -D-(4-O-methyl)glucopyranoside 3′-O--D-(4-2aO-methyl)glucopyranoside, 3 ,5,7-trihydroxyflavone 3a and 4'-methoxyisoflavone 4 -O- -D-(4-O-methyl) 0 0 glucopyranoside7-O--D-(4-O-methyl)glucopyranoside2b, 5,7-dihydroxy-4 -methoxyflavone 4a, along 3 with-O-β -D-(4- twoO -methyl)glucopyranoside known compounds 4′,5-3a and 0 4 -methoxyisoflavonedihydroxyflavanone 7-7-OO--β--D-(4-O-methyl)glucopyranoside-methyl)glucopyranoside 1a4a and, along 5,7-dihydroxyflavanone with two known compounds 4′-O-- 0 4 ,5-dihydroxyflavanoneD-glucopyranoside 1c were 7-O obtained-β-D-(4- andO-methyl)glucopyranoside structurally characterized. 1a and 5,7-dihydroxyflavanone 0 4 -O-β-D-glucopyranoside 1c were obtained and structurally characterized.

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2. Results and Discussion

2.1. Biotransformation Assay Small-scale fermentations of I. fumosorosea ACCC 37814 in the presence of each substrate were carried out to screen for all possible glycosylated products. The ethyl acetate extract of each fermentation culture was analyzed by HPLC-HRMS/MS and the potential glycosylated products were identified after examining their LC and HRMS/MS spectra. Satisfactorily, all tested compounds except for curcumin 10 were converted by I. fumosorosea ACCC 37814 into at least one glycosylated products. The LC peak areas (recorded at 300 nm) of all products and the unreacted substrate were used to calculate the substrate-to-product conversion rate for each compound (Figure2 and Supplementary Materials, Table S1). In all cases, mono-methylglucoside was produced as the major product, while mono-glucoside was also detected for some flavonoids such as naringenin 1, kaempferol 5 and hesperetin 6. Mono-methylglucoside regioisomers were presented in extracts of naringenin 1, luteolin 2, kaempferol 5, genistein 8 and emodin 9 but only naringenin had di-methylglucosylated product (Figure2 and Supplementary Materials, Table S1). The precise position to which the glycosyl and methyl substituents were attached, however, remained unclear at this point where NMR measurements wereMolecules required 2019, 24 to, x unambiguously characterize the structure. 3 of 13

Figure 2. The biotransformation of phenolic substrates by I. fumosorosea ACCC 37814. (A) Total Figure 2. The biotransformation of phenolic substrates by I. fumosorosea ACCC 37814. (A) Total conversion rate [%] of each tested substrate. All data represent the means ± SDs in three independent conversion rate [%] of each tested substrate. All data represent the means  SDs in three independent experiments. Mono-Glc: mono-glucoside; Mono-MeGlcA and –B: mono-(4-O-methyl)glucoside experiments. Mono-Glc: mono-glucoside; Mono-MeGlcA and –B: mono-(4-O-methyl)glucoside regioisomers; Di-MeGlc: di-(4-O-methyl)glucoside. NA: not applicable (B) Chemical structures of regioisomers; Di-MeGlc: di-(4-O-methyl)glucoside. NA: not applicable (B) Chemical structures of substrates 1–10. See Supplementary Materials, Table S1 for tabulated data. substrates 1–10. See SI Appendix, Table S1 for tabulated data. 2.2. Structure Characterization 2. Results and Discussion Scale-up biotransformations of flavonoids 1, 2, 3 and 4 by I. fumosorosea ACCC 37814 were carried2.1. Biotransformation out to obtain enough Assay amount of their glycosides for NMR measurements. The fermentation culture was extracted by ethyl acetate and the resulting crude extract was subjected to silica gel Small-scale fermentations of I. fumosorosea ACCC 37814 in the presence of each substrate were column chromatography and semi-preparative HPLC to give the single compounds 1a–1d, 2a, carried out to screen for all possible glycosylated products. The ethyl acetate extract of each fermentation culture was analyzed by HPLC-HRMS/MS and the potential glycosylated products were identified after examining their LC and HRMS/MS spectra. Satisfactorily, all tested compounds except for curcumin 10 were converted by I. fumosorosea ACCC 37814 into at least one glycosylated products. The LC peak areas (recorded at 300 nm) of all products and the unreacted substrate were used to calculate the substrate-to-product conversion rate for each compound (Figure 2 and SI Appendix, Table S1). In all cases, mono-methylglucoside was produced as the major product, while mono-glucoside was also detected for some flavonoids such as naringenin 1, kaempferol 5 and hesperetin 6. Mono-methylglucoside regioisomers were presented in extracts of naringenin 1, luteolin 2, kaempferol 5, genistein 8 and emodin 9 but only naringenin had di-methylglucosylated product (Figure 2 and SI Appendix, Table S1). The precise position to which the glycosyl and methyl substituents were attached, however, remained unclear at this point where NMR measurements were required to unambiguously characterize the structure.

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2b, 3a and 4a (Figure3). Their structures were unambiguously characterized by mass and NMR spectroscopicMolecules 2019, techniques.24, x 4 of 13

Figure 3. Product profiles of flavonoids 1–4 biotransformed in the I. fumosorosea ACCC 37814 cultures Figure 3. Product profiles of flavonoids 1–4 biotransformed in the I. fumosorosea ACCC 37814 cultures (reverse(reverse phase phase High High Performance Performance LiquidLiquid ChromatographyChromatography (HPLC) (HPLC) traces traces recorded recorded at at300 300 nm). nm). HPLC HPLC tracestraces of of each each standard standard substrate substrate (colored (colored inin red) were used used as as references. references.

2.2.2.3 Structure mg of Characterization1a, 3.2 mg of 1b, 6.5 mg of 1c and 1.5 mg of 1d were isolated from the scale-up fermentation culture of I. fumosorosea ACCC 37814 (Scheme1). The negative mode HRESIMS spectra of 1a andScale-up1b displayed biotransformations [M − H]− ion of flavonoids peaks at m/z 1, 2447.1289, 3 and 4 and by I. 447.1294, fumosorosea respectively, ACCC 37814 which were were carried out to obtain enough amount of their glycosides for NMR measurements. The fermentation 176 amu larger than that of naringenin 1 and thus indicated the presence of a methylated hexose unit culture was extracted by ethyl acetate and the resulting crude extract was subjected to silica gel in their structures. This was further supported by 1H- and 13C-NMR spectra of 1a and 1b, which column chromatography and semi-preparative HPLC to give the single compounds 1a–1d, 2a, 2b, 3a were quite similar, and both showed characteristic resonances of a naringenin core and a methylated and 4a (Figure 3). Their structures were unambiguously characterized by mass and NMR D-glucosylspectroscopic substituent techniques. (Table 1). Next, comprehensive analysis of HSQC and HMBC spectra revealed that 1a and 1b were regioisomers in which the sugar substituent was attached to different positions. The HMBC spectrum of 1a displayed correlations of the anomeric proton (δH 5.01, d, J = 7.8 Hz) and two aromatic protons (H-6, δH 6.15, brs; H-8, δH 6.13, brs) with the oxygen-bearing aromatic carbon at δC 165.1 (C-7), revealing that the D-glucosyl substituent was connected to OH-7 of naringenin (Scheme1). Moreover, the large coupling constant of the anomeric proton confirmed the β-configuration of the 0 glucosidic bond. Similarly, the D-glucose fragment in 1b was confirmed to be connected to C-4 of naringenin via an O-β-D-glycosidic bond because of the large coupling constant of the anomeric proton (δH 4.91, d, J = 7.7 Hz) and HMBC interaction between the anomeric proton and the oxygen-bearing 0 aromatic carbon at δC 157.4 (C-4 ). In both cases, HMBC correlations between the methoxy protons (1a: δH 3.44, s; 1b: δH 3.45, s) and C-4 of the glucose moiety (1a: δC 78.8; 1a: δC 79.0) confirmed that the methylation has been taken place on the OH-4 of the glucose fragment (Scheme1). Hence, the structures 0 of 1a andScheme1b were 1. The defined microbial as transformation 4 ,5-dihydroxyflavanone of naringenin 1 7-inO the-β I.-D fumosorosea-(4-O-methyl)glucopyranoside ACCC 37814 culture. and 0 5,7-dihydroxyflavanoneKey Heteronuclear Multiple 4 -O-β -BondD-(4- CorrelationsO-methyl)glucopyranoside, (HMBC) correlations respectively.of each product are indicated by Thered HRESIMS-determined arrows. molecular weight of 1c was 14 amu lower than those of 1a and 1b. Moreover, 1H- and 13C-NMR spectra of 1c were almost superimposable to those of 1b, except for 2.3 mg of 1a, 3.2 mg of 1b, 6.5 mg of 1c and 1.5 mg of 1d were isolated from the scale-up the absence of the methoxy signals and some chemical shift variations on C-3, C-4 and C-5 of the fermentation culture of I. fumosorosea ACCC 37814 (Scheme 1). The negative mode HRESIMS spectra glucosylof 1a and substituent 1b displayed (Table [M1 −). H] These− ion peaks evidence at m/z indicated 447.1289 that and 1c447.1294,has aglucosyl respectively, substituent, which were instead 176 of 0 a 4-amuO-methylated larger than that glucosyl of naringenin group, attached 1 and thus to indicated the 4 -OH the of presence naringenin. of a methylated Comprehensive hexose analysis unit in of HSQCtheir andstructures. HMBC This spectra was offurther1c further supported supported by 1H- this and speculation 13C-NMR spectra (Scheme of 1a1) andand thus1b, which the structure were 0 of 1cquitewas similar, elucidated and both as 5,7-dihydroxyflavanone showed characteristic resonances 4 -O-β-D -glucopyranoside.of a naringenin core and a methylated D- glucosyl substituent (Table 1). Next, comprehensive analysis of HSQC and HMBC spectra revealed that 1a and 1b were regioisomers in which the sugar substituent was attached to different positions.

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Figure 3. Product profiles of flavonoids 1–4 biotransformed in the I. fumosorosea ACCC 37814 cultures (reverse phase High Performance Liquid Chromatography (HPLC) traces recorded at 300 nm). HPLC traces of each standard substrate (colored in red) were used as references.

2.2. Structure Characterization

MoleculesScale-up 2019, 24 biotransformations, x of flavonoids 1, 2, 3 and 4 by I. fumosorosea ACCC 37814 5 were of 13 carried out to obtain enough amount of their glycosides for NMR measurements. The fermentation cultureThe HMBC was spectrum extracted of by 1a ethyl displayed acetate correlations and the resulting of the anomeric crude extract proton was (H 5.01, subjected d, J = to7.8 silica Hz) and gel columntwo aromatic chromatography protons (H-6, and H semi-preparative 6.15, brs; H-8, H 6.13,HPLC brs) to givewith thethe singleoxygen-bearing compounds aromatic 1a–1d, 2acarbon, 2b, 3aat Molecules 2019, 24, 1028 5 of 13 andC 165.1 4a (C-7), (Figure revealing 3). Their that the structures D-glucosyl were substituent unambiguously was connected characterized to OH-7 of by naringenin mass and (Scheme NMR spectroscopic1). Moreover, techniques.the large coupling constant of the anomeric proton confirmed the -configuration of the glucosidic bond. Similarly, the D-glucose fragment in 1b was confirmed to be connected to C-4′ of naringenin via an O--D-glycosidic bond because of the large coupling constant of the anomeric proton (H 4.91, d, J = 7.7 Hz) and HMBC interaction between the anomeric proton and the oxygen- bearing aromatic carbon at C 157.4 (C-4′). In both cases, HMBC correlations between the methoxy protons (1a: H 3.44, s; 1b: H 3.45, s) and C-4 of the glucose moiety (1a: C 78.8; 1a: C 79.0) confirmed that the methylation has been taken place on the OH-4 of the glucose fragment (Scheme 1). Hence, the structures of 1a and 1b were defined as 4′,5-dihydroxyflavanone 7-O--D-(4-O- methyl)glucopyranoside and 5,7-dihydroxyflavanone 4′-O--D-(4-O-methyl)glucopyranoside, respectively. The HRESIMS-determined molecular weight of 1c was 14 amu lower than those of 1a and 1b. Moreover, 1H- and 13C-NMR spectra of 1c were almost superimposable to those of 1b, except for the absenceSchemeScheme of 1.the 1.The methoxyThe microbial microbial signals transformation transformation and some chemical of naringenin shift variations11 inin the the I.I. fumosorosea on fumosorosea C-3, C-4 ACCC andACCC C-5 37814 37814 of theculture.culture. glucosyl substituentKeyKey Heteronuclear Heteronuclear (Table 1). Multiple TheseMultiple evidence Bond Bond Correlations Correlations indicated (HMBC) that 1c correlations correlationshas a glucosyl of of eacheach substituent, product product are areinstead indicated indicated of by a 4- byO- methylatedredred arrows. arrows. glucosyl group, attached to the 4′-OH of naringenin. Comprehensive analysis of HSQC and HMBC spectra of 1c further supported this speculation (Scheme 1) and thus the structure of 1c wasThe elucidated2.3 HRESIMS mg of 1aas , determined5,7-dihydroxyflavanone 3.2 mg of 1b molecular, 6.5 mg of weight4′- 1cO-  and-D of-glucopyranoside. 1.51d mgwas of 176 1d amu were larger isolated than from those the of scale-up1a and 1b 1 13 andfermentation its TheH- andHRESIMS cultureC-NMR determined of I. spectra fumosorosea showedmolecular ACCC resonance weight 37814 of(Scheme signals 1d was corresponding1). 176 The amu negative larger to modethan two those methylated-glucosylHRESIMS of 1a spectraand 1b substituentsofand 1a itsand 1 H-1b (Table displayed and1 ),13C-NMR suggesting [M − spectraH]− that ion peaks1d showedwas atthe m/z resonance di-(4- 447.1289O-methyl)glucopyranosyl signals and 447.1294, corresponding respectively, to substituted two which methylated- were derivative 176 of naringenin.amuglucosyl larger substituents than After that careful of naringenin (Table examination 1), 1 suggesting and of thus the indicated 2D-NMR that 1d the spectra was presence the (Scheme di-(4- of a methylatedO1-methyl)glucopyranosyl), the structure hexose of unit1d inwas 0 definedtheirsubstituted structures. as 5-hydroxyflavanone derivative This was of naringenin.further 4 ,7-di- supported OAfter-β-D careful -(4-by O1H--methyl)glucopyranoside. examination and 13C-NMR of spectrathe 2D-NMR of 1a and spectra 1b, which(Scheme were 1), quitetheThe structure similar, scale-up ofand biotransformation1d bothwas definedshowed ascharacteristic 5-hydroxyflavanone of luteolin resonances2 in the 4′,7-di- culture of a Onaringenin of--I.D-(4- fumosoroseaO-methyl)glucopyranoside. core andACCC a methylated 37814 resulted D- in 2.8glucosyl mgThe of substituentscale-up product biotransformation2a (Tableand 2.3 1). mg Next, of of productcomprehensive luteolin2b 2 (Schemein the analysis culture2). Theof of HSQC I. ( fumosorosea−)-HRESIMS and HMBC ACCC spectra spectra 37814 of revealed resulted2a and 2b displayedthatin 2.8 1a mg and [M of −1b productH] were− ions regioisomers 2a atandm/z 2.3461.1100 mg in of which product and the 461.1097, 2bsugar (Scheme substituent respectively, 2). The was (−)-HRESIMS which attached were to spectra consistent different of positions.2a with and that 2b of − (4-O displayed-methyl)glucosyl [M − H] substitutedions at m/z 461.1100 luteolin. and1H- 461.1097, and 13C-NMR respectively, spectra which of 2a wereand consistent2b showed with resonance that of (4-O-methyl)glucosyl substituted luteolin. 1H- and 13C-NMR spectra of 2a and 2b showed signals corresponding to a luteolin core structure, a D-glucose unit and a methoxy functionality. resonance signals corresponding to a luteolin core structure, a D-glucose unit and a methoxy Apparently, 2a and 2b were regio-isomers which have the sugar fragment attached to different positions functionality. Apparently, 2a and 2b were regio-isomers which have the sugar fragment attached to of luteolin. In the case of 2a, the coupling constant of the anomeric proton (δH 4.93, d, J = 7.8 Hz) different positions of luteolin. In the case of 2a, the coupling constant of the anomeric proton (H 4.93, along with the HMBC correlation between the anomeric proton and the oxygen-bearing aromatic d, J = 7.8 Hz) along with the HMBC correlation between the anomeric proton and the oxygen-bearing carbon at δ 145.7 (C-30) confirmed that the glucosyl substituent was connected to C-30 of luteolin aromaticC carbon at C 145.7 (C-3′) confirmed that the glucosyl substituent was connected to C-3′ of vialuteolin a O-β-glucosidic via a O--glucosidic linkage (Scheme linkage2 ).(Scheme By contrast, 2). By the contrast, HMBC the spectrum HMBC spectrum of 2b exhibited of 2b exhibited cross peak betweencross peak the anomericbetween the proton anomeric (δH 4.92,proton d, (JH= 4.92, 7.8 Hz) d, J and= 7.8 the Hz) oxygen-bearing and the oxygen-bearing aromatic aromatic carbon at 0 0 δC 148.4carbon (C-4 at ),C 148.4 confirming (C-4′), confirming an O-β-glucosidic an O--glucosidic linkage between linkage between the C-4 ofthe luteolin C-4′ of luteolin and the and anomeric the carbonanomeric of the carbon sugar of unit the (Scheme sugar unit2). Based(Scheme on 2). HMBC Based correlationon HMBC correlation between the between methoxy the protonsmethoxy ( 2a andprotons2b: δH (3.47,2a and s) and2b:  C-4H 3.47, of the s) and glucose C-4 of moiety the glucose (2a: δC moiety79.3; 2b (2a: δ: CC79.0), 79.3; the2b: methoxyC 79.0), the groups methoxy in both 2a andgroups2b inwere both confirmed 2a and 2b were to be confirmed attachedto to C-4be attached of the glucosyl to C-4 of substituent.the glucosyl substituent. Hence, the Hence, structures the of 0 0 2a andstructures2b were of established 2a and as2b 4 ,5,7-trihydroxyflavone were established as 3 -O 4′,5,7-trihydroxyflavone-β-D-(4-O-methyl)glucopyranoside 3′-O--D-(4-O and- 0 0 3 ,5,7-trihydroxyflavonemethyl)glucopyranoside 4 -O and-β-D -(4- 3′,5,7-trihydroxyflavoneO-methyl)glucopyranoside, 4′-O- respectively.-D-(4-O-methyl)glucopyranoside, respectively.

SchemeScheme 2. The2. The microbial microbial transformation transformation ofof luteolinluteolin 2 in the I.I. fumosorosea fumosorosea ACCCACCC 37814 37814 culture. culture. Key Key HMBCHMBC correlations correlations of of each each product product are are indicated indicated byby redred arrows.

Molecules 2019, 24, 1028 6 of 13

1 13 Table 1. H- (600 MHz) and C-NMR (150 MHz) Data of 1a, 1b, 1c and 1d in DMSO-d6.

1a 1b 1c 1d No. δC, Type δH, Mult. (J in Hz) δC, Type δH, Mult. (J in Hz) δC, Type δH, Mult. (J in Hz) δC, Type δH, Mult. (J in Hz) 2 78.7, CH 5.50, brd (12.7) 78.0, CH 5.52, brd (12.2) 78.1, CH 5.52, brd (12.2) 3.35, m 3.27, m 3.27, m 3.36, m 3 42.0, CH 42.0, CH 42.1, CH 2 2.73, dt (17.4, 3.6) 2 2.73, dt (17.1, 3.2) 2.73, dt (17.1, 3.2) 2 2.80, dd (17.1, 2.8) 4 197.2, C 196.1, C 195.9, C 197.0, C 5 162.9, C 163.5, C 163.5, C 162.9, C 6 95.4, CH 6.15, brs 95.9, CH 5.89, brs 96.0, CH 5.87, brs 95.4, CH 6.17, brs 7 165.1, C 166.8, C 167.3, C 165.1, C 8 96.5, CH 6.13, brs 95.0, CH 5.88, brs 95.2, CH 5.86, brs 96.5, CH 6.14, brs 9 162.8, C 162.8, C 162.7, C 162.6, C 10 103.2, C 101.7, C 101.6, C 103.3, C 10 128.6, C 131.9, C 132.0, C 131.7, C 20 128.4, CH 7.33, d (8.2) 128.0, CH 7.43, d (8.2) 128.0, CH 7.43, d (8.2) 128.1, CH 7.45, d (8.2) 30 115.2, CH 6.80, d (8.2) 116.1, CH 7.06, d (8.2) 116.2, CH 7.06, d (8.2) 116.2, CH 7.06, d (8.2) 40 157.8, C 157.4, C 157.5, C 157.5, C 50 115.2, CH 6.80, d (8.2) 116.1, CH 7.06, d (8.2) 116.2, CH 7.06, d (8.2) 116.2, CH 7.06, d (8.2) 60 128.4, CH 7.33, d (8.2) 128.0, CH 7.43, d (8.2) 128.0, CH 7.43, d (8.2) 128.1, CH 7.45, d (8.2) 100 99.1, CH 5.01, d (7.8) 99.9, CH 4.91, d (7.7) 100.3, CH 4.88, d (7.4) 99.1, CH 5.01, d (7.8) 200 73.2, CH 3.24, t (8.1) 73.4, CH 3.24, m 73.2, CH 3.24, m 73.2, CH 3.24, t (8.1) 300 76.0, CH 3.40, m 76.3, CH 3.41, m 77.1, CH 3.33, m 76.0, CH 3.40, m 400 78.8, CH 3.01, t (9.3) 79.0, CH 3.03, t (9.4) 69.7, CH 3.16, t (9.0) 78.8, CH 3.01, t (9.3) 500 75.6, CH 3.43, m 75.6, CH 3.38, m 76.6, CH 3.26, m 75.6, CH 3.43, m 3.60, brd (11.8) 3.63, dd (11.8, 4.4) 3.69, btd (11.8) 3.60, brd (11.8) 600 60.1, CH 60.2, CH 60.7, CH 60.1, CH 2 3.47, m 2 3.49, m 2 3.45, m 2 3.47, m 00 4 -OCH3 59.6, CH3 3.44, s 59.7, CH3 3.45, s 59.6, CH3 3.44, s 1000 99.9, CH 4.91, d (7.7) 2000 73.4, CH 3.24, m 3000 76.3, CH 3.41, m 4000 79.0, CH 3.03, t (9.4) 5000 75.6, CH 3.38, m 3.63, dd (11.8, 4.4) 6000 60.2, CH 2 3.49, m 000 4 -OCH3 59.7, CH3 3.45, s 5-OH 12.01, s 12.13, s 12.15, s 12.02, s 40-OH 9.62, s Molecules 2019, 24, 1028 7 of 13

Molecules2.0 mg 2019 of, 24 compound, x 3a was isolated from the scale-up fermentation culture of I. fumosorosea 8 of 13 ACCCMolecules 37814. 2019, 24 The, x HRESIMS determined molecular weight of 3a was consistent with 8 that of 13 of 2.0 mg of compound 3a was isolated from the scale-up fermentation culture of I. fumosorosea (4-O-methyl)glucosylated derivative of diosmetin 3. Detailed analysis of 1D- and 2D-NMR spectra ACCC2.0 37814. mg of The compound HRESIMS 3a determinedwas isolated molecular from the weightscale-up of fermentation 3a was consistent culture with of I.that fumosorosea of (4-O- further confirmed that 3a is the methylglucosylated derivative of diosmetin. HMBC interaction methyl)glucosylatedACCC 37814. The HRESIMS derivative determined of diosmetin molecular 3. Detailed weight analysis of 3a of was1D- andconsistent 2D-NMR with spectra that of further (4-O- between the anomeric proton (δ 5.17, d, J = 7.8 Hz) and the oxygen-bearing aromatic carton confirmedmethyl)glucosylated that 3a is the derivative methylglucosylated ofH diosmetin derivative3. Detailed of analysis diosmetin. of 1D- HMBC and 2D-NMR interaction spectra between further the at δ 146.5 (C-30) indicated that the glucose unit was attached to OH-30 of diosmetin (Scheme3). anomericconfirmedC proton that 3a ( isH the 5.17, methylglucosylated d, J = 7.8 Hz) and derivative the oxygen-bearing of diosmetin. aromatic HMBC carton interaction at C between146.5 (C-3′) the The β-configuration of the glucosidic bond was proved by the coupling constant of the anomeric indicatedanomeric thatproton the (glucoseH 5.17, unitd, J was= 7.8 attached Hz) and to the OH-3′ oxygen-bearing of diosmetin aromatic (Scheme carton 3). The at β-configuration C 146.5 (C-3′) proton.ofindicated the HMBC glucosidic that the correlation glucose bond was unit between proved was attached the by the methoxy to coupling OH-3′ protons of constantdiosmetin (δH of 3.47,(Scheme the s) anomeric and3). The the proton.β-configuration oxygen-bearing HMBC 3 00 sp correlationof-carbon the glucosidic at betweenδC 79.1 bond the (C-4 methoxy was) confirmed proved protons by that ( theH 3.47, the coupling s) methylation and the constant oxygen-bearing has of been the anomeric taken sp3-carbon place proton. at on C 79.1 the HMBC (C- 4-OH 0 of the4′′)correlation confirmed glucose between moiety. that the the Thus,methylation methoxy the structureprotons has been (H oftaken 3.47,3a wasplaces) and defined on the the oxygen-bearing 4-OH as 5,7-dihydroxy-4 of the glucose sp3-carbon moiety.-methoxyflavone at  Thus,C 79.1 the(C- 0 3 -Ostructure4′′)-β confirmed-D-(4- O-methyl)glucopyranoside. of that 3athe methylation was defined has been as taken 5,7-dihydroxy-4′-methoxyflavone place on the 4-OH of the glucose moiety. 3′-O- Thus,-D-(4- theO- methyl)glucopyranoside.structure of 3a was defined as 5,7-dihydroxy-4′-methoxyflavone 3′-O--D-(4-O- methyl)glucopyranoside.

SchemeScheme 3. The3. The microbial microbial transformation transformation ofof diosmetindiosmetin 3 inin the the I.I. fumosorosea fumosorosea ACCCACCC 37814 37814 culture. culture. Key Key HMBC correlations of each product are indicated by red arrows. HMBCScheme correlations 3. The microbial of each transformation product are indicated of diosmetin by red 3 in arrows. the I. fumosorosea ACCC 37814 culture. Key HMBC correlations of each product are indicated by red arrows. 2.02.0 mg mg of of compound compound4a 4awas was purified purified fromfrom thethe scale-up biotransformation biotransformation ofof formononetin formononetin 4 in4 in thethe culture culture2.0 mg of of I.of fumosoroseaI. compound fumosorosea ACCC4a ACCC was purified 37814. 37814. The Thefrom negativenegative the scale-up mode mode biotransformation HRESIMS HRESIMS spectrum spectrum of formononetin of of4a4a displayeddisplayed 4 in a a − [M[Mthe− H] −culture H]−ion ion of at at I.m mfumosorosea//zz 489.1422,489.1422, ACCC indicating 37814. 4a4a The waswas negative the the methylglucosylated methylglucosylated mode HRESIMS derivative spectrum derivative of 4aformononetin. of formononetin.displayed a ThisThis[M speculation − H] speculation− ion at was m /z wassupported 489.1422, supported indicating by the by presence the4a was presenceof the resonance methylglucosylated of resonance signals corresponding signals derivative corresponding of to formononetin. a formononetin to a 1 1 13 13 core,formononetinThis a D speculation-glucose core, unit wasa D and-glucose supported a methoxy unit and by functionala the methoxy presence functional group of resonance in group the H-in signalsthe and H- andC-NMR corresponding C-NMR spectra spectra to of a 4a. H 1 13 HMBCofformononetin 4a. interaction HMBC interaction core, between a D-glucose between the unit anomeric the and anomeric a methoxy proton proton ( δfunctionalH (5.14, 5.14, d, group d,J =J =7.8 7.8 in the Hz ) )H- and and and the the oxygen-bearingC-NMR oxygen-bearing spectra aromatic carton at C 161.3 (C-7) as well as the large coupling constant of this anomeric proton aromaticof 4a. HMBC carton interaction at δC 161.3 between (C-7) the as wellanomeric as the proton large (H coupling 5.14, d, J = constant 7.8 Hz ) and of this the oxygen-bearing anomeric proton  indicatedindicatedaromatic that carton that the the glucose at glucoseC 161.3 fragment fragment (C-7) was as well was attached attachedas the to large C-7 to of C-7 coupling formononetin of formononetin constant via of an via thisO -β an anomeric-glucosidic O- -glucosidic proton linkage. linkage. The HMBC spectrum of 4a also displayed cross peak between the methoxy protons (H 3.47, Theindicated HMBC spectrum that the glucose of 4a also fragment displayed was cross attached peak to between C-7 of formononetin the methoxy protonsvia an O (-δH-glucosidic3.47, s) and 3 C s)linkage. and the The oxygen-bearing HMBC 3spectrum sp -carbon of 4a also at displayed 78.900 (C-4′′), cross confirming peak between that the methoxyC-4 of the protons glucose ( moietyH 3.47, the oxygen-bearing sp -carbon at δC 78.9 (C-4 ), confirming that the C-4 of the glucose moiety has a has a methoxy substituent3 (Scheme 4). Hence, the structure of 4a was elucidated as 4′- methoxys) and substituentthe oxygen-bearing (Scheme sp4).-carbon Hence, at theC 78.9 structure (C-4′′),of confirming4a was elucidated that the C-4 as of 4 0the-methoxyisoflavone glucose moiety methoxyisoflavonehas a methoxy substituent 7-O--D-(4- O (Scheme-methyl)glucopyranoside. 4). Hence, the structure of 4a was elucidated as 4′- 7-O-β-D-(4-O-methyl)glucopyranoside. methoxyisoflavone 7-O--D-(4-O-methyl)glucopyranoside.

Scheme 4. The microbial transformation of formononetin 3 in the I. fumosorosea ACCC 37814 culture. Scheme 4. The microbial transformation of formononetin 3 in the I. fumosorosea ACCC 37814 culture. KeyScheme HMBC 4. The correlations microbial of transformation each product areof formononetin indicated by red 3 in arrows. the I. fumosorosea ACCC 37814 culture. Key HMBC correlations of each product are indicated by red arrows. Key HMBC correlations of each product are indicated by red arrows. Most of the naturally occurring flavonoids exist in the form of O-glycosides and flavone and Most of the naturally occurring flavonoids exist in the form of O-glycosides and flavone and flavonolMost O of-glycosides the naturally account occurring for the flavonoids largest two exist groups in the of formflavonoid of O -glycosidesglycosides [2].and Theoretically flavone and flavonol O-glycosides account for the largest two groups of flavonoid glycosides [2]. Theoretically speaking,flavonol O any-glycosides free phenolic account hydroxyl for the group largest on two the groupsflavonoid of scaffoldflavonoid can glycosides be glycosylated [2]. Theoretically except for speaking,OH-5speaking, which any any forms free free phenolic phenolichydrogen hydroxylhydroxyl bond with group group the C-4 on on carbonylthe the flavonoid flavonoid group scaffold and scaffold thus can has canbe relatively glycosylated be glycosylated low exceptreactivity except for for[8].OH-5 OH-5 However, which which forms nature forms hydrogen seems hydrogen tobond favor bond with some the with C-4positions the carbonyl C-4 rather carbonyl group than and groupothers. thus has and The relatively thusmajority has low of relatively reactivitynaturally low reactivityoccurring[8]. However, [8 ]., However, nature flavanones seems nature to and seemsfavor some to favor positions tend some to rather positionsbe glycosylated than rather others. at than OH-7,Theothers. majority while Theflavonols of naturally majority and of naturallyflavanolsoccurring occurring usually flavones, have flavones, flavanones sugar fragments flavanones and isoflavones attached and isoflavonestend to OH-3 to be and/or glycosylated tend OH-7 to be [2]. at glycosylated OH-7, Therefore, while it atis interesting OH-7, and while flavonolsthatflavanols I. fumosorosea and usually flavanols have ACCC usually sugar 37814 fragments have prefers sugar toattached fragmentsglycosylate to OH-3 attachedthe and/orOH-3′ to orOH-7 OH-3 OH-4′ [2]. and/or on Therefore, ring OH-7 B of it flavones [ 2is]. interesting Therefore, such it is interestingasthat luteolin I. fumosorosea thatand diosmetin.I. fumosoroseaACCC 37814 By contrast,ACCC prefers 37814 tothe glycosylate widely prefers studied tothe glycosylate OH-3′ biocatalyst, or OH-4′ the B. OH-3 onbassiana ring0 or B, OH-4whichof flavones0 onis also ring such an B of as luteolin and diosmetin. By contrast, the widely studied biocatalyst, B. bassiana, which is also an

Molecules 2019, 24, 1028 8 of 13

flavones such as luteolin and diosmetin. By contrast, the widely studied biocatalyst, B. bassiana, which is also an entomopathogenic filamentous fungus and genetically close to I. fumosorosea, favors OH-7 as methylglucosylation site [14]. The biosynthesis of natural flavonoid O-glycosides in plant cells are catalyzed by various uridine diphosphate-dependent glycosyltransferases (UGTs). Over the past two decades, highly efficient biosynthesis of flavonoid O-glycosides has been achieved by using genetically engineered Escherichia coli strains that harbor UGTs from Arabidopsis thaliana, Oryza sativa, Medicago truncatula and so forth [30–32]. Bacteria also utilize a wide range of UGTs to glycosylate their secondary metabolites. E. coli strains that expressing heterologous bacterial UGTs were used to biosynthesize not only natural flavonoid O-glucosides [33–35] but also “unnatural” flavonoid glycosides which incorporate amino- and deoxysugars in their structures [36,37]. Despite the fact that fungi have long been used as whole-cell biocatalyst for the glycosylation of a variety of natural products, only a few fungal UGTs have been identified and functional characterized so far [9,14,38]. Very recently, Xie et al. identified a glycotransferase-methyltransferase (GT-MT) biosynthetic module from B. bassiana which proficiently synthesized (4-O-methyl)glucopyranosides of various phenolic substances, including benzendiol lactones, and flavonoids, in Saccharomyces cerevisiae host [14]. Recent studies and our present work demonstrated that I. fumosorosea is capable of methylglucosylating a number of flavonoid scaffolds and prefers the free aromatic hydroxyl group on ring B as glycosylation site [11,27–29]. These results indicate that I. fumosorosea is a promising source to exploit novel fungal GT-MT module that exhibit different chemo- and regio-selectivity than those from B. bassiana. Many studies have proved that glycosylation significantly improves the aqueous solubility of natural products such as flavonoids, while methylation on one of the hydroxyl groups of the sugar moiety does not significantly alter the solubility [5,14]. However, the biological and pharmacological significance of the 4-O-methylation of the sugar moiety is less clear. Flavonoids and their glycosides undergo intense metabolism by both intestine epithelium and gut microbiota after they are taken into human body, which eventually diminishes the beneficial effects of flavonoids observed in vitro [12]. A recent study has shown that attaching methyl group to the OH-4 of glucose moiety can substantially avert the rapid de-glucosylation process of benzendiol lactone glucosides in both yeast and mammalian cell models [14] and thus may increase the in vivo stability and bioavailability of these drug-like compounds. Consequently, the effect of methylglucosylation of flavonoids would be an interesting topic to further explore.

3. Conclusions The current study describes the microbial transformation of natural phenolics using the entomopathogenic filamentous fungus I. fumosorosea ACCC 37814. Nine out of ten substrates were converted to their glycosides in the culture of I. fumosorosea ACCC 37814 after four days of biotransformation. Mono-methylglucosides are the major products for all substrates but di- methylglucosylated and mono-glucosylated products were also detected in several cases. Four substrates representing different sub-classes of flavonoid were selected for scale-up fermentation. As a result, six new flavonoid glycosides and two known compounds were isolated from the scale-up cultures and their structures were unambiguously characterized. The results indicated that I. fumosorosea ACCC 37814 prefers free aromatic hydroxyl groups on ring B of flavonoid scaffold as glycosylation site and could be developed as regio-selective biocatalyst for the methylglucosylation of phenolic natural products.

4. Materials and Methods

4.1. General Experimental Orocedures All substrates were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Samples were routinely analyzed on an Agilent 1260 Infinity II HPLC instrument (Santa Clara, Molecules 2019, 24, 1028 9 of 13

CA, USA). The HPLC was equipped with an Agilent Eclipse Plus C18 RRHD column (1.8 µm, 2.1 mm × 50 mm) and eluted with a linear gradient of 10–95% of acetonitrile-water for 20 min, 95% acetonitrile-water for 10 min and drop down to 10% in 5 min, then 10% acetonitrile-water for 5 min at a flow rate of 0.5 mL/min. 1H-, 13C-, HSQC- and HMBC-NMR spectra were recorded on an Agilent 600 DD2 spectrometer. Chemical shift values (δ) are given in parts per million (ppm) and the coupling constants (J values) are in Hz. Chemical shifts were referenced to the residual solvent peaks of DMSO-d6. HPLC-HRESIMS spectra were acquired on an Agilent 1290 Infinity II HPLC coupled with an Agilent QTOF 6530 instrument operated in negative ion mode using capillary and cone voltages of 3.6 kV and 40–150 V, respectively. For accurate mass measurements the instrument was calibrated each time using a standard calibration mix (Agilent) in the range of m/z 150–1900.

4.2. Culture and Biotransformation Procedures The fungus Isaria fumosorosea ACCC 37814, acquired from the Agricultural Culture Collection of China (ACCC), was maintained on potato-dextrose agar (PDA, Difco, Sparks, MD, USA) at 26 ◦C for 7 days. Spores were collected after 10 days of growth by flooding the plates with sterile water (0.1% Triton-X100). For small scale biotransformation experiment, the collected spores with 106/mL concentration were inoculated into a 100 mL Erlenmeyer flask containing 50 mL potato-dextrose broth (PDB, Difco, Sparks, MD, USA) medium. The flask was placed in a rotary shaker at 25 ◦C and shook at 220 rpm for three days. After that, 0.5 mg of substrates (10 uL DMSO solution of substrate at the concentration of 50 mg/mL) was added and the flask was maintained under the same conditions for an additional four days. Two controls were incubated under the same conditions: (1) I. fumosorosea ACCC 37814 fermentation broth with the addition of substrate-free DMSO solution and (2) microorganism-free PDB medium with the addition of DMSO solution containing the substrate. For large scale fermentation, a total of 3 L fermentation culture and 75 mg of substrates were used (2.5 mg/flask) in the 250 mL flasks, each containing 100 mL of PDB and incubated under the same conditions as described for the small scale experiment.

4.3. Extraction, Isolation and Purification of the Products The scale-up fermentation cultures were combined and extracted with an equal volume of ethyl acetate for three times. The extracts were pooled and dried in vacuo to give the crude extracts I (481.6 mg/L), II (844.0 mg/L), III (868.1 mg/L) and IV (868.1 mg/L) for substrates 1–4, respectively. Each crude extract was first subjected to silica gel column chromatography and eluted with a gradient of dichloromethane/methanol (100:0, 98:2, 95:5, 93:7 and 90:10, v/v) to give five fractions (Fr. A–E). These fractions were analyzed by LC-MS instrument (Agilent Technologies, Santa Clara, CA, USA) to detect potential products. Fractions containing the target compounds were subsequently purified by semi-preparative HPLC on an Agilent Eclipse XDB-C18 reversed-phase column (5 µm, 9.4 mm × 250 mm) using an Agilent 1260 Infinity II instrument. Specifically, Fr. D of extract I was isolated using acetonitrile-water (25:75, v/v) as eluent at the flow rate of 2 mL/min to give compound 1a (2.3 mg, tR = 23.2 min), compound 1b (3.2 mg, tR = 27.3 min), compound 1c (6.5 mg, tR = 18.5 min) and compound 1d (1.5 mg, tR = 13.4 min); Fr. D of extract II was isolated using acetonitrile-water (30:70, v/v) as eluent at the flow rate of 2 mL/min to give compound 2a (2.8 mg, tR = 16.2 min) and compound 2a (2.3 mg, tR = 14.7 min). Fr. D of extract III was isolated using acetonitrile-water (30:70, v/v) as eluent at the flow rate of 2 mL/min to give compound 3a (2.0 mg, tR = 17.5 min). Fr. E of extract IV was isolated using acetonitrile-water (50:50, v/v) as eluent at the flow rate of 2 mL/min to give compound 4a (4.0 mg, tR = 8.5 min). Molecules 2019, 24, 1028 10 of 13

1 13 Table 2. H- (600 MHz) and C-NMR (150 MHz) Data of 2a, 2b, 3a and 4a in DMSO-d6.

2a 2b 3a 4a No. δC, Type δH, Mult. (J in Hz) δC, Type δH, Mult. (J in Hz) δC, Type δH, Mult. (J in Hz) δC, Type δH, Mult. (J in Hz) 2 163.4, C 163.1, C 163.0, C 153.6, CH 8.44, s 3 103.0, CH 6.79, s 104.0, CH 6.82, s 103.7, CH 6.89, s 123.4, C 4 181.7, C 181.7, C 181.7, C 174.6, C 5 161.4, C 161.4, C 161.4, C 127.0, CH 8.06, d (8.8) 6 98.9, CH 6.18, brs 98.9, CH 6.20, brs 99.1, CH 6.17, brs 115.6, CH 7.15, dd (8.8, 1.8) 7 164.3, C 164.3, C no show 161.3, C 8 94.1, CH 6.49, brs 94.0, CH 6.50, brs 94.2, CH 6.49, brs 103.3, CH 7.24, d (1.8) 9 157.3, C 157.3, C 157.4, C 157.0, C 10 103.6, C 103.8, C 103.4, C 118.5, C 10 121.1, C 124.7, C 122.9, C 124.0, C 20 114.4, C 7.74, d (2.2) 113.6, C 7.50, brs 112.8, C 7.70, d (2.0) 130.1, CH 7.53, d (8.4) 30 145.7, C 146.9, C 146.5, C 115.6, CH 7.00, d (8.4) 40 151.4, C 148.4, C 152.1, C 159.0, C 50 116.6, CH 6.95, d (8.5) 115.8, CH 7.22, d (8.5) 112.4, C 7.16, d (8.6) 115.6, CH 7.00, d (8.4) 60 122.0, CH 7.64, dd (8.5, 2.2) 118.5, CH 7.51, brd (9.0) 121.0, CH 7.64, dd (8.6, 2.0) 130.1, CH 7.53, d (8.4) 100 101.5, CH 4.93, d (7.8) 100.7, CH 4.92, d (7.8) 99.4, CH 5.17, d (7.8) 99.6, CH 5.14, d (7.8) 200 73.5, CH 3.34, m 73.4, CH 3.34, m 73.4, CH 3.30, m 73.3, CH 3.30, m 300 75.8, CH 3.52, m 75.8, CH 3.52, m 76.6, CH 3.46, m 76.2, CH 3.46, m 400 79.3, CH 3.04, t (9.2) 79.0, CH 3.06, t (9.3) 79.1, CH 3.05, t (9.3) 78.9, CH 3.06, t (9.1) 500 75.7, CH 3.46, m 75.6, CH 3.46, m 75.6, CH 3.52, m 75.7, CH 3.52, m 3.70, brs (11.4) 3.66, brs (11.4) 3.64, brs (11.0) 3.66, brs (11.0) 600 60.4, CH 60.2, CH 60.2, CH 60.2, CH 2 3.56, m 2 3.52, m 2 3.52, m 2 3.52, m 0 4 -OCH3 55.8, CH3 3.86, s 55.2, CH3 3.79, s 00 4 -OCH3 59.7, CH3 3.47, s 59.7, CH3 3.47, s 59.7, CH3 3.47, s 59.7, CH3 3.47, s 5-OH 12.96, s 12.90, s 12.90, s Molecules 2019, 24, 1028 11 of 13

0 4 ,5-Dihydroxyflavanone 7-O-β-D-(4-O-methyl)glucopyranoside (1a): 1H- and 13C-NMR data see − Table1;( −)-HRESIMS m/z 447.1289 [M − H] (calcd. for C22H23O10, 447.1291). 0 5,7-Dihydroxyflavanone 4 -O-β-D-(4-O-methyl)glucopyranoside (1b): 1H- and 13C-NMR data see − Table1;( −)-HRESIMS m/z 447.1294 [M − H] (calcd. for C22H23O10, 447.1291). 0 5,7-Dihydroxyflavanone 4 -O-β-D-glucopyranoside (1c): 1H- and 13C-NMR data see Table1; − (−)-HRESIMS m/z 433.1122 [M − H] (calcd. for C21H21O10, 433.1135). 0 5-Hydroxyflavanone 4 ,7-di-O-β-D-(4-O-methyl)glucopyranoside (1d): 1H- and 13C-NMR data see − Table1;( −)-HRESIMS m/z 623.1950 [M − H] (calcd. for C29H35O15, 623.1976). 0 0 4 ,5,7-Trihydroxyflavone 3 -O-β-D-(4-O-methyl)glucopyranoside (2a): 1H- and 13C-NMR data see − Table2;( −)-HRESIMS m/z 461.1100 [M − H] (calcd. for C22H21O11, 461.1084). 0 0 3 ,5,7-Trihydroxyflavone 4 -O-β-D-(4-O-methyl)glucopyranoside (2b): 1H- and 13C-NMR data see − Table2;( −)-HRESIMS m/z 461.1097 [M − H] (calcd. for C22H21O11, 461.1084). 0 0 5,7-Dihydroxy-4 -methoxyflavone-3 -O-β-D-(4-O-methyl)glucopyranoside (3a): 1H- and 13C-NMR − data see Table2;( −)-HRESIMS m/z 475.1261 [M − H] (calcd. for C23H23O11, 475.1240). 0 4 -methoxyisoflavone 7-O-β-D-(4-O-methyl)glucopyranoside (4a): 1H- and 13C-NMR data see Table2; − (−)-HRESIMS m/z 489.1422 [M − H] (calcd. for C24H25O11, 489.1397).

Supplementary Materials: The supplementary materials are available online. Author Contributions: Resources, B.D.; conceptualization, Z.W. and B.D.; formal analysis, Z.W.; investigation, F.D. and Z.W.; methodology, F.D. and Z.W.; supervision, G.L. and B.D.; validation, F.D. and Z.W.; visualization, F.D. and G.L.; writing—original draft, F.D. and Z.W.; writing—review & editing, Z.W. and B.D.; funding acquisition, B.D. Funding: This research was funded by the Agricultural Science and Technology Innovation Program (CAAS-ASTIP-2017-ICS) and Special Fund for Agroscientific Research in the Public Interest of China (201503135). Acknowledgments: We thank Jingang Gu of Agricultural Culture Collection of China for generously providing the fungus Isaria fumosorosea ACCC 37814. We also thank the Research Facility Center of the Biotechnology Institute for providing analytical instruments and software. Conflicts of Interest: The authors declare no conflict of interest.

References

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Sample Availability: Samples of the compounds 1–10, 1a–1d, 2a, 2b, 3a and 4a are available from the authors.

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