Beauveria bassiana , two of the species Beauveria caledonica , one of Isaria fumosorosea (KCh J2) and one of Isaria farinosa (KCh KW 1.1) were investigated. Both the number and the place of attachment of the methoxy groups in the flavonoid structure influenced the biotransformation rate and the amount of nascent products. Based on the structures of products and semi-products, it can be concluded that their formation is the result of a cascading process. As a result of enzymes produced in the cells of the tested strains, the test compounds undergo progressive demethylation and/or hydroxylation and 4-O-methylglucosylation. Thirteen novel flavonoid 4-O-methylglucosides and five hydroxy flavones were isolated and identified. The synthesis and biotransformation of five flavones containing methoxy substituents in the B ring: 2′-, 3′-, 4′-methoxyflavones, 2′,5′-dimethoxyflavone and 3′,4′,5′-trimethoxyflavone are described. Strains of entomopathogenic filamentous fungi were used as biocatalysts. Five strains of the species  Flavonoids are common in the world of plants as their secondary metabolites, mainly in citrus fruits, olive oil, seeds  and vegePrimula veris L.), which contains these compounds in the rhizomes, roots, flowers and leaves, and from which mainly expectorants are prepared [Methoxyflavones have well-described anti-cancer properties and are often more active than flavones without any functional groups . Due to prepared . The theAglycons are characterized by very low solubility in water ; howeverFlavonoids are not the only group of compounds in which the positive effect of the presence of a sugar moiety on the activity of the compound has been proven. The best examples are antibiotics, such as erythromycin or vancomycin, in which the presence of a sugar group is crucial for their high activity . The prede novo chemical synthesis [Carrying out the biotransformation of flavonoids to their glycosylated derivatives is less complicated and more effective than their ynthesis , which m1), 3′-(2), 4′-(3)-methoxyflavones, 2′,5′-dimethoxyflavone(4) and 3′,4′,5′-trimethoxyflavone(5). Listed compounds were biotransformed by the strains of entomopathogenic filamentous fungi belonging to the species Beauveria bassiana KCh J1.5, J2.1, J3.2, J1, BBT, Beauveria caledonica KCh J3.3, J3.4 [Isaria fumosorosea KCh J2 [Isaria farinosa KCh KW 1.1 [Aspergillus niger.The biotransformation of five methoxyflavones with substituents on the B ring is described: 2′- in cultures of Beauveria bassiana AM 446 and AM 278 [Beauveria bassiana ATCC 13144 is capable of simultaneous 4-O-methylglycosylation of the hydroxyl group located at the C-3 and hydroxylation of the C-4′ carbon [O-methylglycosylation of 3-hydroxyflavone, 6-hydroxyflavone, 7-hydroxyflavone, baicalein, quercetin, naringenin, luteolin, diosmetin, and daidzein in entomopathogenic filamentous fungal culture of the genus Isaria has been reported [Isaria fumosorosea KCh J2, I. farinosa KCh J1.4, I. farinosa KCh J1.6 and I. farinosa KCh KW1.2 cultures was preceded by hydroxylation of the C-4′ carbon. Subsequent C-4′ hydroxylation and 4-O-methylglycosylation was observed during the incubation of flavone, 3-methoxyflavone, 5-hydroxyflavone, 6-methoxyflavone, 6-methoxyflavanone and 6-methylflavone in the cultures of the genus Isaria [I. fumosorosea KCh J2 strain for simultaneous O-demethylation and glycosylation of 2′-, 3′- and 4′-methoxyflavanone was also described [Previous studies have shown the capacity for unique 4″- culture ,23. The ompounds ,25,26. Ad AM 278 ,29,30. Breported ,14,22. Ms Isaria . The capescribed . Based oBeauveria bassiana strains , two Beauveria caledonica strains (KCh J3.3 and J3.4), and two Isaria strains (I. fumosorosea KCh J2 and I. farinosa KCh KW 1.1) as biocatalysts. B. bassiana species are the most common among the entomopathogenic filamentous fungi used in biotransformations, including in the case of flavonoid compounds [B. bassiana. For the first time in flavonoid biotransformations, we also tested two strains of Beauveria caledonica species. The Isaria fumosorosea KCh J2 strain has already been described in our previous study as an effective biocatalyst of flavanones and flavones as substrates [I. fumosorosea KCh J2 to the results obtained from the transformations by the B. bassiana strains, we noted the similarity in the biotransformations of flavonoid compounds. In both cases, the main products are 4′-O-β-D-(4″-O-methyl)-glucopyranosides. We have also observed this ability for strains of I. farinosa [I. farinosa KCh KW 1.1 strain as a biocatalyst as well.Based on the previously described results concerning the biotransformation of flavonoids in the cultures of entomopathogenic strains, we decided to use five ompounds ,26,27. Iompounds . Therefobstrates ,22,32,33farinosa ,33. In t2 in DMSO. As a result of these reactions 2′-, 3′-, 4′-methoxyflavone, 2′,5′-dimethoxyflavone and 3′,4′,5′-trimethoxyflavone were obtained.All used substrates, containing in their structure from one to three methoxyl groups in the B ring, were obtained by a two-step chemical synthesis. In the first stage, five chalcones were synthesized from 2′-hydroxyacetophenone and the appropriate methoxybenzaldehyde in a basic medium. Then, they were transformed into the appropriate methoxyflavones, by reaction with J1) in the cultures of most of the tested strains resulted in one major product (6), with a retention time of 11.4 min according to HPLC (1) to the same product. This product was identified as 2′-O-β-d-(4″-O-methylglucopyranosyl)-flavone (6), obtained in all tested strains except Beauveria bassiana KCh J1. This compound was formed as a result of subsequent demethylation and 4-O-methylglycosylation. However, only in the culture of the B. bassiana KCh J1 strain, 2′-hydroxyflavone, was an intermediate product in the biosynthesis of 2′-O-β-D-(4″-O-methylglucopyranosyl)-flavone observed. This may be evidence that the substrate demethylation process is crucial for the rate of 2′-O-β-D-(4″-O-methylglucopyranosyl)-flavone formation. Among the strains used, only the B. bassiana KCh J1 strain does not have the ability of 4-O-methylglycosylation; thus only the 2′-hydroxy flavone (10) was formed during the biotransformation. The highest concentrations of 2′-O-β-D-(4″-O-methylglucopyranosyl)-flavone were identified in the cultures of Beauveria bassiana strains: KCh J3.2 (70%); KCh J2.1 (76%); KCh BBT (87%) after 10 days of biotransformation.Biotransformation of 2′-methoxyflavone ( to HPLC . Further1) turned out to be the I. fumosorosea KCh J2 strain, where two additional products were also observed   with a retention time of 14.8 min was observed. For this reason, a scale-up biotransformation of these two strains was performed to isolate and characterize the structure of the resulting products. As a result of the scale-up biotransformation of the 2′-methoxyflavone in the culture of the I. fumosorosea KCh J2, a total of four products were isolated (O-β-D-(4″-O-methylglucopyranosyl)-flavone is the major product (6). The formation of this compound is possible by successive demethylation and 4-O-methylglycosylation. The 1H NMR spectrum shows signals confirming that the structure of the flavone skeleton has not been changed. The presence, chemical shifts and multiplicities of the signals indicate that the only substituent is on the C-2′ carbon (as in a substrate). The structure of the flavone is confirmed by the COSY, HMQC and HMBC correlation spectra. However, instead of the signal from the protons of the CH3 group (visible in the 1H NMR substrate spectrum), signals from the sugar unit are visible. The multiplicities and positions of these signals in both the 1H and 13C NMR spectra indicate that a glucose molecule was introduced in place of the CH3 group. The HMBC spectrum shows the coupling of the signal from the H-1″ sugar unit proton with the signal from the C-2′ carbon of the flavone skeleton. This coupling indicates the exact place of attachment of the sugar substituent. Additionally, the 1H NMR spectrum shows a singlet derived from three protons (3.46 ppm). In the HMBC spectrum, this signal is coupled to the C-4″ carbon (sugar substituent), which indicates that the substituent is 4″-O-methylglucopyranoside.The exceptions during the biotransformation of 2′-methoxyflavone (rformed)  and the isolated . Based o1H NMR spectrum of the next product (8) (isolated with a yield of 20%), obtained by scale-up biotransformation of 2′-methoxyflavone in the culture of I. fumosorosea KCh J2, there are signals indicating that the product is 4-O-methylglycoside. However, the presence of a singlet derived from three protons at 3.90 ppm and its coupling to the C-2′ carbon of the flavone skeleton, visible in the HMBC spectrum, prove that the methoxy group has been preserved. Based on the position and multiplicities of the signals visible in the 1H NMR spectrum and the couplings visible in the correlation spectra, it can be concluded that an additional substituent was attached at the C-5′ carbon. Additionally, in the HMBC spectrum, the signal from this carbon is coupled with a doublet from the H-1″ proton of the sugar unit. Based on these data, this compound was identified as 5′-O-β-D-(4″-O-methylglucopyranosyl)-2′-methoxyflavone (8).In the 1H NMR spectrum of this compound and the couplings visible in the correlation spectra, it can be concluded that the substituent is 4″-O-methylglycoside, bound to carbon C-8. This compound was isolated in a yield of 4% and identified as 8-O-β-D-(4″-O-methylglucopyranosyl)-2′-methoxyflavone (7). Another product, identified as 3-O-β-D-(4″-O-methylglucopyranosyl)-2′-methoxyflavone (9), was isolated, but with a very low yield (<1%). From the 1H NMR spectrum it was found that the structure of 2′-methoxyflavone was retained. At the same time, as in the case of previously described products, signals from 4″-O-methylglycoside are visible. The lack of a signal of the H-3 proton indicates that the sugar substituent was introduced at the C-3 carbon by O-glycosylation reaction. Due to the low concentration of this compound, it was not possible to perform 13C NMR analysis and correlation spectra confirming its structure. Compounds 6, 7, 8 and 9 are the result of successive hydroxylation and 4-O-methylglycosylation.Based on NMR data, another product was also identified as a glycoside derivative of 2′-methoxyflavone. Based on the chemical shifts of the signals visible in the Beauveria bassiana KCh J1 strain, one product was isolated with 43.5% yield. On the basis of NMR analyses it was identified as 2′-hydroxyflavone (10)  at 10.03 ppm, the signal from the proton of the hydroxyl group located at C-2′ is visible.As a result of up-scale biotransformation of the 2′-methoxyflavone in the culture of the one (10) . This co2), was converted much faster by all tested biocatalysts. It is most likely related to easier access of the enzymes responsible for the demethylation process to the methoxy group situated meta in relation to the chromene substituent (1-benzopyran or chromene). Conversion of 3′-methoxyflavone in the cultures of four out of nine biocatalysts  was close to 100% after three days of incubation , during the transformation of 3′-methoxyflavone (2) in the culture of the B. bassiana KCh J1 strain, one of the main products was an effect of demethylation and was identified as 3′-hydroxyflavone (11) (B. bassiana KCh BBT strain.Similar to the biotransformation of 2′-methoxyflavone (one (11) . All NMRone (11)  confirm O-methylglycosylation, that is 3′-O-β-D-(4″-O-methylglucopyranosyl)-flavone (12) . The structure of the flavone is also confirmed by the 13C NMR spectra and the correlation with COSY, HMQC and HMBC spectra. The HMBC spectrum shows the coupling of the signal from the H-1″ proton of the sugar unit with the signal from the C-3′ carbon of the flavone skeleton. The 1H NMR spectrum shows a singlet from the three protons (3.47 ppm). In the HMBC spectrum, this signal is coupled to the C-4″ carbon (sugar substituent), which proves that the substituent is 4″-O-methylglucopyranoside.The major product of the transformation of 3′-methoxyflavone in most of the studied strains was the product of progressive demethylation and 4-one (12) . The che3) in the cultures of the tested strains were comparable to those obtained for 3′-methoxyflavone (2) at the seven days of biotransformation. Nearly 100% conversion was observed in the culture of Isaria farinosa KCh KW 1.1 and four strains of Beauveria bassiana  (1 and 2), the exception is the Beauveria bassiana KCh J1 strain, from the culture of which only the demethylation product 4′-hydroxyflavone was isolated (13)  .. Similated (13) . Spectra3) in the culture of the Beauveria bassiana KCh J1.5 strain, 36.5 mg of 4′-O-β-D- (4″-O-methylglucopyranosyl)-flavone (14) was isolated  was the 4-O-methylglucosylated derivative (the same trend as was observed in case monomethoxy flavone biotransformations). As a result of up-scaled biotransformation of this substrate in the culture of B. bassiana KCh J1.5, three products were isolated: 8, 15 and 16. These compounds were also observed during biotransformation in other cultures . Compound 8, with a retention time of 10.7 min (HPLC), was isolated in the highest yield -2′-methoxyflavone (8). The next product is 2′-O-β-D-(4″-O-methylglucopyranosyl)-5′-methoxyflavone (15), the signal of which is observed on the HPLC chromatogram with a retention time of 11.6 min. NMR spectra indicate that it is also a product of demethylation and 4-O-methylglucosylation. The HMBC spectrum shows the coupling of the signal from the H-1″ proton of the sugar unit with the signal from the C-2′ carbon of the flavone skeleton, which indicates that this product is 2′-O-β-D-(4″-O-methylglucopyranosyl)-5′-methoxyflavone (15). The third product with the lowest yield in the culture of B. bassiana KCh J1.5 has also been identified as 4-O-methylglucopyranoside. The 1H NMR spectrum shows three singlets from the protons of the -OCH3 groups. One methoxy group is bonded to the C-4″ carbon of the sugar unit, the other two to the C-2′ and C-5′ carbons of the flavone skeleton. The B-ring protons of this product give only two singlets at 7.00 and 7.56 ppm. Based on the chemical shifts, multiplicities and couplings observed in the correlation spectra, they were assigned to the protons H-3′ and H-6′, respectively. The NMR spectra (O-β-D-(4″-O-methylglucopyranosyl)-2′,5′-dimethoxyflavone (16) (The  spectra  made forone (16) .O-methylglucosylation of 2′,5′-dimethoxyflavone (4) take place in the cultures of the strains tested. The highest concentration (over 70%) of 5′-O-β-D-(4″-O-methylglucopyranosyl)-2′-methoxyflavone (8) in the reaction mixture was observed in the culture of I. fumosorosea KCh J2. In the cultures of B. bassiana KCh J1.5, KCh J2.1, KCh J3.2 and I. farinosa KCh KW1.1 the concentration of 5′-O-β-D-(4″-O-methylglucopyranosyl)-2′-methoxyflavone (8) is almost twice as high as 2′-O-β-D-(4″-O-methylglucopyranosyl)-5′-methoxyflavone (15). However, in the cultures of B. bassiana KCh BBT and B. caledonica KCh J3.3 strains, the concentration of these products was comparable. In the culture of the B. caledonica KCh J3.4 strain, several products were observed, but they were formed at low concentrations that prevented their isolation.Based on the NMR analyses of the obtained products, it can be seen that demethylation or hydroxylation and 4-4) products were observed in the culture of the B. bassiana KCh J1 strain. The main product was identified as 5′-hydroxy-2′-methoxyflavone (17). Compared to the spectrum of the substrate, instead of two, only one singlet from the three protons (3.84 ppm) is visible in the 1H NMR spectrum of this product. At the same time, at 9.41 ppm, a singlet derived from the hydroxyl group is observed. The HMBC spectrum shows connections of this signal with signals from carbons C-4′, C-5′ and C-6′, which proves that the methoxyl group situated at the C-5′ carbon was demethylated.Two 2′,5′-dimethoxyflavone (18) is produced in a much smaller amount in the culture of the B. bassiana KCh J1 strain. The 1H NMR spectrum shows two signals from protons of -CH3 groups in positions 3.84 and 3.85 ppm, respectively, and a singlet derived from one proton, in the field characteristic for signals from Ar-OH protons. These data prove the preservation of both methoxy groups and the introduction of an additional hydroxyl group to the substrate molecule. The chemical shifts and multiplicities of the signals from the protons in the A ring are almost identical to those observed in the 1H NMR of the substrate spectrum. However, the B-ring protons of this product only give two singlets at 6.70 and 7.53 ppm. Based on the shapes and couplings observed in the correlation spectra, H-3′ and H-6′ were assigned, respectively. Additionally, in the HMBC spectrum, the signal from the proton of the hydroxyl group is coupled with the signals from carbons C-3′, C-4′ and C-5′. This information indicates that this compound is 4′-hydroxy-2′,5′-dimethoxyflavone (18) (The second product (one (18) . In the B. bassiana KCh J1.5 and two strains of B. caledonica (KCh J3.3 and KCh J3.4). As a result of the scaled-up biotransformation in the culture of B. bassiana KCh J1.5, selected based on the results from HPLC (19–22) ). However, instead of two singlets, one for six protons, the other for three protons from the protons of the methoxy groups, three singlets from the protons of the -OCH3 groups are visible . In addition, the signals from the H-2′and H-6′ protons in the substrate spectrum generated a singlet at 7.15 ppm due to the same chemical environment. The spectrum of this product shows two doublets at 7.41 ppm from H-6′ and 7.49 ppm from H-2′. Such differentiation of these signals proves that the substitution took place within the B ring. Based on the correlation spectra, it can be stated that the 4-O-methyl glucose substituent is bound to the C-3′ carbon . The most common product of biotransformation of 3′,4′,5′-trimethoxyflavone (5) by B. bassiana KCh J1 was identified as 3′-O-β-D-(4″-O-methylglucopyranosyl)-4′,5′-dimethoxyflavone (19).The highest conversion (over 90% after the seventh day of the biotransformation process) of 3′,4′,5′-trimethoxyflavone was observed in the culture of rom HPLC  and TLC, (19–22) . The maiO-methylglucoside (20). However, the 1H NMR spectrum of 19 shows all signals from the flavonoid skeleton protons with multiplicity and positions similar to the 3′,4′,5′-trimethoxyflavone (5) spectrum. A singlet at 3.90 ppm derived from the six protons of methoxy groups (C-3′-OCH3 and C-5′-OCH3) is visible. The HMBC spectrum shows the coupling of the signal from the H-1″ proton with the signal from the C-4′ carbon, which proves that this product is 4′-O-β-D-(4″-O-methylglucopyranosyl)-3′,5′-dimethoxyflavone (20), which was identified in the cultures of most biocatalysts used. The main compound (19) was yielded in 80% of the reaction mixture in the culture of the B. bassiana KCh J1.5 strain and two B. caledonica strains (KCh J3.3 and KCh J3.4). In the cultures of these strains and the B. bassiana KCh J3.2 strain, the concentration of 19 was about five times higher than 4′-O-β-D-(4″-O-methylglucopyranosyl)-3′,5′-dimethoxyflavone (20). In the culture of B. bassiana KCh BBT, compounds 19 and 20 were formed in the ratio of 3:1, in the culture of I. farinosa KCh KW 1.1 in the ratio 10:1, and in the culture of I. fumosorosea KCh J2 in the ratio of 48:1.Furthermore, the second product is a 4-B. bassiana KCh J1 strain, even after ten days of 3′,4′,5′-trimethoxyflavone (5) incubation, nearly 60% unreacted substrate was observed. The resulting products were formed in very low concentrations, which made them impossible to isolate. Instead, scaled-up biotransformation in the B. bassiana KCh J1.5 culture gave two additional products which are formed at very low concentrations in the cultures of most of the tested biocatalysts. The compound with a retention time of 10.3 min was identified as 6-O-β-D-(4″-O-methylglucopyranosyl)-3′,4′,5′-trimethoxyflavone (21).In the culture of the 21, it was found that the sugar unit is bound to carbon C-6. This compound is an effect of hydroxylation and glycosylation. 13C NMR spectrum of 6-O-β-D-(4″-O-methylglucopyranosyl)-flavone (21) and the flavone previously described, which were used as a calculation standard [O-β-D-(4-O-methyl)-glucopyranosyl substituent on the C-6 carbon with respect to the position of analogous signals visible on the flavan spectrum are given. 13C NMR spectrum made for 3′,4′,5′-trimethoxyflavone (5) and the calculated signal positions for 6-O-β-D-(4″-O-methylglucopyranosyl)-3′,4′,5′-trimethoxyflavone (21). The 13C NMR data obtained for compound 21 are compatible with the calculated data -4′,5′-dimethoxyflavone. Very high compatibility of the calculated and measured values of the signal positions in the 13C NMR spectrum for 3′-O-β-D-(4″-O-methylglucopyranosyl)-6-hydroxy-4′,5′-dimethoxyflavone (22) was obtained. The NMR and MS spectra confirm the correct determination of the product structure  on ring B. Often the process of adding a sugar moiety to flavonoids is described as a one-step reaction, in which the end product is formed without the presence of intermediates [O-methylglucosylation, in which several enzymes and coenzymes of the biocatalyst are involved. In addition, the position of the substituent within the B ring had a significant impact on the speed and number of products formed. The demethylation and 4-O-methylglucosylation processes are fastest when the methoxy group is placed on the C-3′ carbon and slowest when on the C-2′ carbon. However, the largest number of products were formed when the methoxy substituent was in the 2′- position, where the 4-O-methylglycose molecule was attached not only within the ring where it was present, but also outside it. The same was true in the case of the flavonoid containing 3 methoxy groups, where attachment of 4-O-methylglycose within another ring was also observed.Flavonoid glycosylation in recent times has been increasingly described, which does not necessarily mean that we fully understood all the processes and enzymes responsible for these reactions. Nine strains of entomopathogenic filamentous fungi – mediates ,28,29,30The substrates 2-hydroxyacetophenone, 2-methoxybenzaldehyde, 3-methoxybenzaldehyde, 4-methoxybenzaldehyde, 2,5-dimethoxybenzaldehyde and 3,4,5-trimethoxybenzaldehyde were purchased from Sigma-Aldrich . Flavones used in biotransformations were synthesized from those substrates (the reactions are described below). The resulting chalcones were used as substrates for the flavone synthesis and their NMR spectral data are identical to those previously published ,35,36.1–5) were used as substrates for the biotransformation and their NMR spectral data are identical to those previously published [All used substrates were synthesized in the laboratory by carrying out two kinds of reactions. Firstly five different methoxychalcones were synthesized in the Claisen-Schmidt reaction of 2-hydroxyacetophenone with suitable methoxybenzaldehyde in reaction, described earlier and shown in ublished ,39.After 2 h of reflux, the product of the Claisen-Schmidt reaction was transferred into an acid environment and filtered using a Buchner funnel. The obtained product  was confirmed by NMR analysis. All other methoxychalcones were synthesized analogously. Methoxyflavones were synthesized from methoxychalcones by reaction with iodine in DMSO with 2–3 h incubation (until the substrate has reacted completely) at 130 °C , as pres1H NMR, 13C NMR, COSY, HMBC and HSQC) analysis.All substrates for biotransformations were synthesized in the same way. The obtained compounds were confirmed by NMR . Isolation and identification procedures were described in our previous paper [The microorganisms us paper ,22.4, concentrated in vacuo and analyzed using TLC and HPLC methods.Erlenmeyer flasks (300 mL), each containing 100 mL of the sterile cultivation medium , were inoculated with a suspension of each entomopathogenic strain and then incubated for 3 days at 24 °C on a rotary shaker. After this time, 10 mg of a substrate was dissolved in 1 mL of dimethyl sulfoxide (DMSO) and added to the interior. Samples were collected on the 1st, 3rd, 7th and 10th day of the process. Then, all products were extracted using ethyl acetate, and extracts were dried using MgSOth day of the process. Products were extracted three times using ethyl acetate and then analyzed using TLC, HPLC and NMR spectroscopy  analysis.For the scale-up process we used Erlenmeyer flasks (2000 mL), each containing 500 mL of the same cultivation medium , which were inoculated in the same way as described above. Three days after inoculation, 100 mg of a substrate was dissolved in 2 mL of DMSO and added to the interior. Samples were collected on the 142, DC Alufolien Kieselgel 60 F254 (0.2 mm thick), Merck, Darmstadt, Germany). The mobile phase contained a mixture of chloroform and methanol in 9:1 (v/v) relation. The plates were observed using a UV lamp (254 and 365 nm).Initial tests were carried out using TLC plates (SiOv/v) ratio. Separation products were scraped out and extracted twice using ethyl acetate.The scale-up biotransformation products were separated using 1000 µm preparative TLC silica gel plates . The mobile phase contained a mixture of chloroform and methanol in a 9:1  and a Guard-Pak Inserts µBondapak C18 pre-column was used to perform HPLC analyses. The mobile phase consisted of eluent A (80% acetonitrile in 4.5% acetic acid solution) and eluent B (4.5% acetic acid) with gradient elution: 0–7 min, 10% A/90% B; 7–10 min, 50% A/50% B; 10–13 min. 60% A/40% B; 15–20 min 80% A/20% B; 20–30 min, 90% A/10% B; 30–40 min, 100% A. The flow rate was 1.0 mL/min, injection volume was 10 µL, detection wavelength 280 nm.A Thermo Scientific Dionex Ultimate 3000 UHPLC+ instrument  with a photodiode array detector (detection in wavelength: 210–450 nm) with a C-18 analytical column ZORBAX Eclipse XDB  was used to performed UHPLC analyses. Chromatographic separation was achieved using an isocratic elution of 50% A (0.05% formic acid water solution) and 50% B (methanol containing 0.05% of formic acid) for 2 min, then a linear gradient of B from 50% to 95% for 10 min and isocratic elution of 95% B for 5 min. The flow rate was 1.0 mL/min.6 or Acetone-d6.The NMR analysis was performed with a DRX 600 MHz Bruker spectrometer  with an UltraShield Plus magnet and measured in DMSO-d1H NMR (600 MHz) (DMSO) δ (ppm): 3.07 , 3.29-3.35 , 3.41-3.54 , 3.46 , 3.64 , 4.72 , 5.16 , 5.27 , 5.39 , 7.08 , 7.22 , 7.35 , 7.50 , 7.50 , 7.74 , 7.83 , 7.93 , 8.06 . 13C NMR  δ = 59.74 (C-4″-OCH3), 60.15 (C-6″), 73.50 (C-2″), 75.70 (C-5″), 76.55 (C-3″), 78.89 (C-4″), 99.73 (C-1″), 112.17 (C-3), 115.37 (C-3′), 118.58 (C-8), 120.72 (C-1′), 122.00 (C-5′), 123.20 (C-4a), 124.75 (C-5), 125.37 (C-6), 129.26 (C-6′), 132.70 (C-4′), 134.22 (C-7), 155.30 (C-2′), 156.01 (C-8a), 160.37 (C-2), 177.27 (C-4).1H NMR (600 MHz) (Acetone-d6) δ (ppm): 3.27 , 3.53 , 3.61 , 3.64-3.74 , 3.85 , 4.03 , 5.18 , 7.10 , 7.16 , 7.25 , 7.35 , 7.56 , 7.61 , 7.74 , 8.22 . 13C NMR  δ = 56.36 (C-4″-OCH3), 60.58 (C-4″-OCH3), 61.99 (C-6″), 75.02 (C-2″), 77.21 (C-5″), 78.13 (C-3″), 80.01 (C-4″), 102.24 (C-1″), 112.68 (C-3), 113.06 (C-3′), 118.59 (C-5), 120.84 (C-7), 121.26 (C-1′), 121.83 (C-5′), 125.43 (C-6), 125.76 (C-4a), 130.59 (C-6′), 133.51 (C-4′), 147.70 (C-8a), 148.02 (C-8), 159.23 (C-2′), 160.88 (C-2), 178.08 (C-4).1H NMR (600 MHz) (DMSO) δ (ppm): 3.02 , 3.26 , 3.40-3.45 , 3.46 , 3.53 , 3.67 , 3.90 , 4.77 , 4.87 , 5.29 , 5.43 , 6.99 , 7.20 , 7.27 , 7.50 , 7.63 , 7.76 , 7.83 , 8.05 . 13C NMR  δ = 56.39 (C-2′-OCH3), 59.71 (C-4″-OCH3), 60.39 (C-6″), 73.48 (C-2″), 75.73 (C-5″), 76.28 (C-3″), 79.20 (C-4″), 101.11 (C-1″), 111.71 (C-3), 113.68 (C-3′), 116.93 (C-6′), 118.61 (C-8), 120.14 (C-1′), 120.88 (C-4′), 123.10 (C-4a), 124.71 (C-5), 125.45 (C-6), 134.33 (C-7), 151.17 (C-5′), 152.88 (C-2′), 155.91 (C-8a), 160.05 (C-2), 177.18 (C-4).1H NMR (600 MHz) (DMSO) δ (ppm): 3.07 , 3.27-3.37 , 3.40 , 3.41-3.55 , 3.79 , 4.45 , 5.14 , 5.27 , 5.31 , 7.06 , 7.16 , 7.50-7.55 , 7.66 , 7.73 , 7.82 , 8.14 .1H NMR (600 MHz) (Acetone-d6) δ (ppm): 7.07 , 7.13 , 7.19 , 7.42 , 7.48 , 7.70 , 7.80 , 7.99 , 10.03 . 13C NMR  δ = 112.54 (C-3), 117.93 (C-3′), 119.18 (C-8), 119.58 (C-1′), 120.91 (C-5′), 124.71 (C-4a), 125.85 (C-6), 125.89 (C-5), 129.73 (C-6′), 133.25 (C-4′), 134.62 (C-7), 157.16 (C-2′), 157.32 (C-8a), 161.76 (C-2), 178.25 (C-4).1H NMR (600 MHz) (DMSO) δ (ppm): 6.94 , 7.01 , 7.38 , 7.44 , 7.51 , 7.53 , 7.77 , 7.86 , 8.06 , 9.90 . 13C NMR  δ = 106.93 (C-3), 112.85 (C-2′), 117.23 (C-6′), 118.53 (C-8), 118.88 (C-4′), 123.36 (C-4a), 124.82 (C-5), 125.57 (C-6), 130.29 (C-5′), 132.45 (C-1′), 134.38 (C-7), 155.68 (C-8a), 157.92 (C-3′) 162.73 (C-2), 177.10 (C-4).1H NMR (600 MHz) (DMSO) δ (ppm): 3.05 , 3.25-3.34 , 3.42-3.57 , 3.47 , 3.68 , 4.80 , 5.07 , 5.31 , 5.46 , 7.07 , 7.26 , 7.47-7.55 , 7.73 , 7.76 , 7.79-7.87 , 8.06 . 13C NMR  δ = 59.76 (C-4″-OCH3), 60.35 (C-6″), 73.48 (C-2″), 75.75 (C-5″), 76.34 (C-3″), 79.19 (C-4″), 100.02 (C-1″), 107.24 (C-3), 113.84 (C-2′), 118.69 (C-6′), 119.79 (C-4′), 120.00 (C-8), 123.36 (C-4a), 124.81 (C-5), 125.63 (C-6), 130.31 (C-5′), 132.52 (C-1′), 134.43 (C-7), 155.72 (C-8a), 157.81 (C-3′), 162.25 (C-2), 177.23 (C-4).1H NMR (600 MHz) (DMSO) δ (ppm): 6.88 , 6.92-6.96 , 7.48 , 7.75 , 7.81 , 7.95-7.99 , 8.03 , 10.32 . 13C NMR  δ = 104.83 (C-3), 115.97 (C-3′ and C-5′), 118.39 (C-8), 121.60 (C-1′), 123.34 (C-4a), 124.75 (C-5), 125.35 (C-6), 128.39 (C-2′ and C-6′), 134.06 (C-7), 155.61 (C-8a), 161.00 (C-4′), 163.09 (C-2), 176.91 (C-4).1H NMR (600 MHz) (DMSO) δ (ppm): 3.06 , 3.29 , 3.42-3.49 , 3.47 , 3.52 , 3.65 , 4.74 , 5.07 , 5.30 , 5.48 , 6.99 , 7.18-7.22 , 7.50 , 7.78 , 7.83 , 8.04 , 8.06-8.10 . 13C NMR  δ = 59.74 (C-4″-OCH3), 60.18 (C-6″), 73.37 (C-2″), 75.69 (C-5″), 76.25 (C-3″), 78.94 (C-4″), 99.50 (C-1″), 105.76 (C-3), 116.54 (C-3′ and C-5′), 118.50 (C-8), 123.33 (C-4a), 124.41 (C-5), 124.78 (C-6), 125.46 (C-1′), 128.11 (C-2′ and C-6′), 134.20 (C-7), 155.65 (C-8a), 160.05 (C-4′), 162.46 (C-2), 177.02 (C-4).1H NMR (600 MHz) (DMSO) δ (ppm): 3.05 , 3.25-3.29 , 3.40-3.45 , 3.45 , 3.51-3.55 , 3.63 , 3.81 , 4.69 , 5.03 , 5.24 , 5.34 , 7.13 , 7.30 , 7.44 , 7.50 , 7.77 , 7.83 , 8.05 . 13C NMR  δ = 55.73 (C-2′-OCH3), 59.72 (C-4″-OCH3), 60.21 (C-6″), 73.55 (C-2″), 75.70 (C-5″), 76.56 (C-3″), 78.96 (C-4″), 100.56 (C-1″), 112.36 (C-3), 113.87 (C-6′), 117.13 (C-3′), 117.99 (C-4′), 118.71 (C-8), 121.58 (C-1′), 123.20 (C-4a), 124.71 (C-5), 125.37 (C-6), 134.18 (C-7), 149.34 (C-5′), 153.94 (C-2′), 155.99 (C-8a), 160.14 (C-2), 177.27 (C-4).1H NMR (600 MHz) (DMSO) δ (ppm): 3.00 , 3.32-3.37 , 3.39-3.43 , 3.46 , 3.47-3.54 , 3.64-3.69 , 3.84 , 3.90 , 4.79 , 5.14 , 5.32 , 5.49 , 6.98 , 7.00 , 7.48 , 7.56 , 7.79 , 7.81 , 8.03 . 13C NMR  δ = 56.41 (C-2′-OCH3), 56.69 (C-6′-OCH3), 59.75 (C-4″-OCH3), 60.39 (C-6″), 73.27 (C-2″), 75.96 (C-5″), 76.67 (C-3″), 79.36 (C-4″), 99.48 (C-1″), 101.40 (C-3′), 110.56 (C-3), 111.82 (C-1′), 113.06 (C-6′), 119.60 (C-8), 123.11 (C-4a), 124.66 (C-5), 125.29 (C-6), 134.33 (C-7), 142.81 (C-4′), 150.23 (C-5′), 153.23 (C-2′), 155.78 (C-8a), 160.31 (C-2), 177.12 (C-4).1H NMR (600 MHz) (DMSO) δ (ppm): 3.84 , 6.94 , 6.97 , 7.09 , 7.32 , 7.50 , 7.71 , 7.83 , 8.05 , 9.41 . 13C NMR  δ = 56.33 (C-2′-OCH3), 111.57 (C-3), 114.07 (C-3′), 115.01 (C-6′), 118.43 (C-8), 119.36 (C-4′), 120.29 (C-1′), 123.13 (C-4a), 124.77 (C-5), 125.42 (C-6), 134.33 (C-7), 150.80 (C-2′), 151.12 (C-5′), 155.89 (C-8a), 160.53 (C-2), 177.16 (C-4).1H NMR (600 MHz) (DMSO) δ (ppm): 3.84 , 3.85 , 6.70 , 6.95 , 7.47 , 7.53 , 7.78 , 7.81 , 8.02 , 10.07 . 13C NMR  δ = 56.17 (C-5′-OCH3), 56.64 (C-2′-OCH3), 101.11 (C-3′), 109.36 (C-1′), 109.90 (C-3), 112.87 (C-6′), 118.51 (C-8), 123.11 (C-4a), 124.63 (C-5), 125.16 (C-6), 133.89 (C-7), 141.87 (C-4′), 151.41 (C-5′), 153.86 (C-2′), 155.74 (C-8a), 160.67 (C-2), 177.08 (C-4).J = 9.1 Hz, H-4″), 3.31-3.36 , 3.48 , 3.46-3.58 , 3.70 , 3.81 , 3.91 , 4.84 , 5.07 , 5.32 , 5.46 , 7.11 , 7.41 , 7.49 , 7.54 , 7.81-7.84 , 8.04 . 13C NMR  is presented in 3.04 , 3.31-3.36 , 3.44 , 3.46-3.58 , 3.69 , 3.90 , 4.52 , 5.14 , 5.19 , 5.23 , 7.14 , 7.39 , 7.49 , 7.81-7.84 , 8.04 . 13C NMR  δ = 56.76 (C-3′-OCH3 and C-5′-OCH3), 59.66 (C-4″-OCH3), 60.34 (C-6″), 74.40 (C-2″), 76.08 (C-5″), 76.42 (C-3″), 79.18 (C-4″), 101.90 (C-1″), 104.89 , 106.78 (C-3), 118.73 (C-8), 123.33 (C-4a), 124.77 (C-5), 125.53 (C-6), 126.27 (C-1′), 134.21 (C-7), 137.35 (C-4′), 152.93 (C-3′ and C-5′), 155.71 (C-8a), 162.44 (C-2), 177.22 (C-4).3.03  (DMSO) δ (ppm): 3.08 , 3.28 , 3.36-3.39 , 3.41-3.46 , 3.47 , 3.62-3.67 , 3.75 , 3.91 , 4.74 , 5.01 , 5.28 , 5.46 , 7.14 , 7.39 , 7.51 , 7.58 , 7.83 . 13C NMR  is presented in 1H NMR (600 MHz) (DMSO) δ (ppm): 3.03 , 3.30-3.35 , 3.47 , 3.43-3.57 , 3.68-372 , 3.80 , 3.90 , 4.81 , 5.05 , 5.29 , 5.43 , 7.02 , 7.25 , 7.31 , 7.38 , 7.50 , 7.68 , 10.02 . 13C NMR  is presented in Molecular formulas of products were confirmed by LC-MS 8045 SHIMADZU analysis. The mobile phase was a mixture of 0.1% aqueous formic acid v/v (A) and acetonitrile (B). The program was as follows: 80% B and 20% A in 5 min. The flow rate was 0.3 mL/min and the injection volume was 2 µl. The column  was operated at 30 °C. The major operating parameters were as follows: nebulizing gas flow: 3 L/min, heating gas flow: 10 L/min, interface temperature: 300°C, drying gas flow: 10 L/min, data acquisition range m/z 100–1000 Da; ionization mode—positive. Data were collected with LabSolutions  software.O-methylglucosides and 5 hydroxy flavones. To the best of our knowledge, these flavone glucosides have never been reported before.To sum up, obtaining 18 novel flavonoids via whole-cell fungal biotransformations was described, 13 of them being 4-O-methylglycosyl attachment to the hydroxyl group in the flavonoid molecule is unique for entomopathogenic filamentous fungi. In this publication, we have demonstrated the capacity of these strains to produce 4-O-methylglucoside flavonoids, the formation of which is preceded by the effective O-demethylation and/or hydroxylation of the methoxyflavones obtained by chemical synthesis.Our previous studies have shown that the ability of 4-The performed biotransformations resulted in products that may indicate that glycosylation is not a one-step process, but is likely the result of an enzyme cascade as intermediates also appear. Obtaining these products is, therefore, possible due to the use of whole cells cultures of biocatalysts containing many interacting enzymes.B. bassiana KCh J1.5 or I. fumosorosea KCh J2) were able to introduce a sugar group into the flavone ring by hydroxylation and then 4-O-methylglycosylation. This sequence resulted in four different glycosylated products in a one-pot reaction. The demethylation and 4-O-methylglucosylation processes are fastest when the methoxy group is on the C-3′ carbon and slowest when the methoxy substituent is on the C-2′ carbon. However, placing the substituent at the C-2′ position results in the highest number of end products. We have obtained 13 new glycosylated flavones (4-O-methylglucopyranosides) and five hydroxy derivatives. Each of the obtained products was determined by means of HPLC, LC-MS and NMR (Some of the entomopathogenic fungi used (e.g.,  and NMR .Additionally, research on the properties of flavonoids, including their glycoside derivatives, is still ongoing, and efficient methods for obtaining such compounds are sought continuously. The methods presented in this publication allow significant amounts of glycoside derivatives to be obtained in an efficient and relatively cheap way while following the principles of “green chemistry”.


The reactions were generally rapid; the yields were comparable to yields reported in the literature for the conventional batch reactionDibromocyclopropanations are conventionally done by addition of dibromocarbene to alkenes under phase-transfer conditions in batch reactions using a strong base (50% NaOH (aq)), vigorous stirring and long reaction times. We have shown that cyclopropanation of unsaturated alcohols can be done under ambient conditions using continuous flow chemistry with 40% ( One of the disadvantages of the Doering-Hoffman reaction is its high sensitivity to water, often reducing the yields significantly.Traditionally, reaction , in whic3, X = Cl, Br), with a concentrated (50% (w/w)) aqueous solution of sodium hydroxide, and a phase-transfer catalyst, partially solved this problem when published a decade later [gem-dihalocyclopropanes [A two-phase dihalocyclopropanation reaction, achieved by vigorous stirring of a solution of the starting alkene and haloform  solution of NaOH is used [w/w) [w/w) NaOH solution in an aqueous-to-organic flow ratio (AO ratio) of 4, good to excellent yields of dibromocyclopropanes could be obtained [In the traditional two-phase system, a 50%  . Other bobtained .Employing the same conditions to 3-methyl-2-buten-1-ol gave a yield of 70% of the corresponding dibromocyclopropane 1 , entry 12 was obtained in 89% yield.  in 57% yield when 2.5 equivalents of bromoform  were used  after chromatography (6 (3%) was isolated. The presence of the formate ester can be explained by insertion of dibromocarbene at the O-H bond and subsequent hydrolysis   , entry 8When the tertiary alcohol, 2-methyl-3-buten-2-ol, was used as a substrate only small amounts of the starting material could be seen , entry 99 as a mixture of diastereomers (1:1) when (E)-2-methyl-2-buten-1-ol was used as the starting material. Under our flow chemistry conditions, only one diastereomer was detected and the adduct 9 was obtained in 62% yield  according to 1H NMR. Purification of the mixture through a plug of silica yielded the dibromocarbene adduct 10 (49%) as a mixture of diastereomers (1:3).The secondary alcohol, 3-methyl-3-buten-2-ol ((Z)-and (E)-dec-4-en-1-ol were tested -(3-(tert-butoxy)prop-1-en-1-yl)benzene (w/w) NaOH (aq) solution, which, as previously mentioned, does not work well with the narrow tubes in the microreactor. Even when doubling the reaction times, our attempts were unsuccessful.Next, we turned to styrene derived alcohols -3-phenylbut-2-en-1-ol was tested in the reaction -3-p(E)-3-prop-2-en-1-ol (E)-3-acrylate  NaOH (aq) as the base. Moderate to excellent yields were achieved in less time than for batch chemistry, depending on the structure of the alcohol. The trisubstituted alkenes  using 40% -2-methyl-2-buten-1-ol [H-pyran [(E)-(3-(tert-butoxy)prop-1-en-1-yl)benzene [(E)-3-phenylbut-2-en-1-ol [(E)-3--prop-2-en-1-ol [All chemicals were purchased from commercial suppliers and used without further purification unless otherwise stated. Preparation of 3-methyl-3-buten-2-ol , (E)-2-mten-1-ol , 2--(3-)benzene ,43, (E)--en-1-ol , and (E)-en-1-ol  was done4 stain. Flash column chromatography was performed on silica gel  or VWR Chemicals/BDH Prolabo Normasil 60 (40–63 µm). In order to degas the dichloromethane, it was sonicated for 15 min prior to use in the flow system. Mass Spectrometry was performed on an Autospec Ultima  using electron ionization .Analytical thin layer chromatography (TLC) was performed on Merck DC-Alufolien Kieselgel 60 F254. Compounds were visualized by KMnO3 as a solvent and TMS as a reference. 1H NMR spectra were recorded at 400 MHz, and 13C NMR spectra were recorded at 100 MHz. IR spectra were recorded on a Perkin-Elmer Spectrum BX series FT-IR spectrophotometer using a ZnSe HATR cell . The flow instrumentation apparatus used was the flow chemistry toolkit FRX200 from Syrris Ltd. fitted with 2 Frx200 pumps, a reagent module containing one Syrris sample loop  and one 1.0 mL sample loop , a Y-mixer (Tube Reactor 3 input Adaptor (PCTFE) from Syrris Ltd.), and a 25 mL tube reactor .NMR spectra were recorded on a Bruker AscendTM 400 using CDCl3 (2.86 mmol), 4.3 mol% TEBA  and 0.6 vol.% ethanol (absolute) in CH2Cl2, and a 5 mL sample loop  containing 40% (w/w) NaOH (aq) solution, was used. The filling of the 40% (w/w) NaOH (aq) solution into the sample loop was done very slowly and with great care, due to the high viscosity of the strongly basic NaOH solution and danger of spillage due to pressure build-up. The two solutions were simultaneously introduced into the flow system at a total flow rate of 0.50 mL/min (flow rate NaOH (aq): 0.40 mL/min, flow rate organic solution: 0.10 mL/min) at room temperature, i.e., a residence time of 50 min and an AO flow ratio of 4. The mixture was fed into brine (50 mL), and the flow was collected for 77 min at this flow rate, and then for 4 min at 2 × 1.5 mL/min (to flush the system). The pressure in the system was 1–4 bar. The reaction mixture was extracted with ethyl acetate (100 mL + 3 × 50 mL), and the combined organic phases were washed with brine (2 × 50 mL), dried (MgSO4), filtered and concentrated in vacuo. The residue was purified by filtering it through a plug made of 0.5 cm silica and 0.5 cm Celite 545 coarse  using ethyl acetate as the eluent. Concentration in vacuo yielded a mixture (0.31 g) containing -methanol (1): 3-methyl-2-buten-1-ol: bromoform; 88:2:10 according to 1H NMR. Estimated yield of 1: 0.27 g, 74%, corresponding to a space time yield of ~0.25 moL L−1 h−1. The spectral data were in accordance with the literature [A 1.0 mL sample loop (PTFE) containing a solution of 3-methyl-2-buten-1-ol (1.43 mmol), CHBrterature .2: linalool: bromoform; 87:9:4, according to 1H and 13C NMR. Estimated yield of the dibromide 2, 0.42 g, 89%. The spectral data were in accordance with the literature [Yield: 0.48 g of a mixture containing the dibromide terature .3 per equivalent of 3,7-dimethyl-6-octen-1-ol were used. The crude mixture was purified by column chromatography  yielding the dibromide 4  as a mixture of diastereomers (approximately 1:1), according to 1H and 13C NMR. IR (HATR) νmax: 3338 , 2954 (s), 2926 (s), 2870 (s), 1456 (s), 1375 (s), 1147 (m), 1109 (m), 1060 , 1006 (m), 963 (m), 756 (s), 740 (s) cm−1; 1H NMR  δ 0.89 , 1.07–1.52 , 1.14 , 1.34 , 1.52–1.65 , 1.75 , 3.57–3.72 ; 13C NMR  δ 19.2 and 19.3 (CH3), 19.5 and 19.6 (CH3), 25.4 and 25.5 (CH2), 27.4 (CH3), 27.9 and 28.0 (C), 29.3 and 29.4 (CH), 35.5 and 35.6 (CH2), 39.6 (CH2), 40.01 and 40.05 (CH), 48.3 and 48.4 (C), 60.9 (CH2). MS, m/z (%) = 246 /248 , 228 (10)/230 (10), 167 (40), 163 (34), 162 (33), 149 (28), 107 (13)/109 (14), 93 (24)/95 (23), 83 (100), 81 (56), 69 (74) and 67 (49). HRMS (EI+) m/z 325.9878. .2.5. equivalents of CHBr5  and 2-ethyl formate (6) , both as oils. The spectral data for the dibromoalcohol 5 were in accordance with literature [6).2-ethyl formate ( IR (HATR) νmax: 2963 (s), 2928 (s), 2873 (m), 1725 (s), 1454 (m), 1430 (m), 1383 (m), 1260 (m), 1167 (s), 733 (s), 694 (s) cm-1; 1H NMR  δ 1.40 , 1.44 , 1.51 , 1.92–2.13 , 4.36 , 8.05 ; 13C NMR  δ 22.6 (CH3), 27.4 (C), 34.5 (CH2), 36.9 (CH2), 37.7 (C), 61.3 (CH2), 160.9 (CH). MS, m/z (%) = 238 /240 /242 , 211 (10)/213 (18)/215 (9), 159 (72)/161 (70), 131 (11)/133 (12), 119 (4)/121 (3), 80 (100) and 79 (87). HRMS (EI+) m/z 237.8994 .The crude mixture was purified by column chromatography  yielding the dibromide terature . 2- as an oil . IR (HATR) νmax: 3343 , 2962 (s), 2926 (s), 2868 (s), 1456 (s), 1374 (s), 1335 (m), 1308 (m), 1128 , 1090 (s), 773 (s), 745 (s) cm-1; 1H NMR  δ 1.16 and 1.17 , 1.20 , 1.35 , 1.58 , 1.15–1.75 , 3.75–3.87 ; 13C NMR  δ 19.3 (CH3), 23.7 (CH3), 24.2 and 24.4 (CH2), 27.42 and 27.44 (CH3), 28.0 and 28.1 (C), 37.6 and 37.8 (CH2), 39.7 and 39.9 (CH), 48.0 and 48.3 (C), 67.6 and 67.7 (CH). MS, m/z (%) = 280 /282 /284 , 238 (3)/240 (5)/242 (2), 173 (37)/175 (35), 159 (4)/161 (4), 122 (40), 121 (83), 107 (46), 94 (100), 79 (53) and 77 (40). HRMS (EI+) m/z 279.9461 .The crude product was purified by column chromatography , yielding the dibromide 9): (E)-2-methyl-2-buten-1-ol: ethyl acetate: bromoform; 87:3:2:8 according to 1H NMR. Estimated yield of 9: 0.22 g, 62%. The spectral data were in accordance with the literature [1H NMR  δ 1.11 , 1.25 , 1.49 , 1.85–1.98 , 3.63 , 3.80 ; 13C NMR  δ 11.6 (CH3), 14.6 (CH3), 31.8 (CH), 33.2 (C), 45.4 (C), 71.3 (CH2).Yield: a mixture (0.25 g) containing methanol (terature . 1H NMR 1H NMR) was purified by filtration through a small plug of silica/Celite 545 coarse , using hexane, then hexane: ethyl acetate (9:1), as eluents. The dibromide 10 was obtained as a mixture of diastereomers  as an oil . The spectral data were in accordance with the literature [1H NMR  δ 1.32–1.37 , 1.35 , 1.46 , 1.64 , 1.60–1.75 , 3.69 ; 13C NMR  δ 16.7 (CH3), 19.1 (CH3), 33.3 (C), 34.6 (CH2), 35.0 (C), 73.9 (CH). Minor isomer: 1H NMR  δ 1.27 , 1.38 , 1.41 , 1.47 , 3.77 ; 13C NMR  δ 15.6 (CH3), 19.2 (CH3), 32.7 (CH2), 34.2 (C), 38.1 (C), 73.7 (CH).The crude product .1H NMR  δ 7.44–7.15 , 3.93 , 3.84 , 2.23 , 1.49 . 13C NMR  δ 155.48, 128.37, 128.34, 128.18, 127.16, 125.68, 59.81, 41.51, 38.22, 29.61, 24.56.The crude product was purified by filtration through a small plug of silica/celite 545 coarse , using hexane, then hexane: ethyl acetate (9:1), as eluents to yield compound


The strategy relies on a highly regio- and trans-stereoselective hydroboration–oxidation reaction of the 4,5-unsubstituted 2,3-dihydroisoxazoles with basic hydrogen peroxide. The consecutive oxidation/reduction route, sequentially employing Dess–Martin periodinane and ʟ-selectride, is used for the inversion of the C-3/4-trans relative configuration of the isoxazolidine ring. The significance of the method lies in its variability and applicability to a concise synthesis of various 4-hydroxyisoxazolidines, starting from the readily available C-alkyl/aryl-nitrones. The resemblance to 3-hydroxypyrrolidines certainly makes the 4-hydroxyisoxazolidines important and valuable structural fragments in drug discovery.Isoxazolidines represent a very important class of N/O-containing heterocycles used as the key intermediates in the synthesis of more complex cyclic and acyclic compounds, including various biologically active molecules. Here, we present a fast and highly stereoselective approach towards both C-3/4- Their1 and 4-hydroxyisoxazolidines 2 . The dihydroxylation reactions proceeded with an excellent trans selectivity with respect to the substituent at the C-3 carbon atom. The obtained products were benzoylated with benzoyl chloride/pyridine in the presence of DMAP to give the fully benzoylated isoxazolidine-4,5-diols 6a and 6b, which were subsequently treated with Et3SiH (3 equiv) and TMSOTf (2 equiv) in anhydrous CH2Cl2 at room temperature for 2 h + calcd for C16H18NO2, 256.1333; found 256.1329.(±)-2-Benzyl-3-phenylisoxazolidin-4-one (9a): A Schlenk flask was charged with the isoxazolidinol 8a , evacuated, and filled with argon. The starting material was dissolved in anhydrous CH2Cl2 (18 mL), the reaction mixture was cooled down to 0 °C, and solid Dess–Martin periodinane  was slowly added under a stream of argon. The reaction was stirred at 0 °C for 12 h, and after the complete conversion of the starting material , a saturated aq NaHCO3 (20 mL) and a saturated aq Na2S2O3∙5H2O (20 mL) solution were added. The mixture was stirred for 15 min at 0 °C and then, the solution was allowed to warm to rt. The organic layer was separated and washed with water (2 × 20 mL), dried over MgSO4, and evaporated in vacuo. The residue was purified by FCC  to give the isoxazolidin-4-one 9a  as yellowish waxy solid that gradually decomposed over time. mp 35–38 °C; Rf 0.61 ; IR (ATR) νmax: 3032, 2871, 2814, 1770, 1495, 1454, 1123, 1048, 737, 695, 615, 545, 470 cm−1; 1H NMR  δ 3.97 , 3.98 , 4.14 , 4.24 , 4.29 , 7.28–7.41 ; 13C NMR  δ 60.7 (PhCH2), 70.7 (C-5), 75.1 (C-3), 127.9 (CH-Ph), 128.5 (CH-Ph), 128.7 (CH-Ph), 128.8 (CH-Ph), 129.0 (CH-Ph), 129.3 (CH-Ph), 133.6 (C-Ph), 136.0 (C-Ph), 210.8 (C=O); HRMS (APCI) (m/z): [M + H]+ calcd for C16H16NO2, 254.1176; found, 254.1174.(±)-2-Benzyl-3-phenylisoxazolidin-4-ol (10a): ʟ-Selectride  was added dropwise to a solution of the isoxazolidin-4-one 9a  in anhydrous THF (11 mL) under an argon atmosphere at 0 °C, and the reaction mixture was stirred for 30 min. When TLC analysis showed that the starting isoxazolidinone had disappeared , a saturated aq NH4Cl solution was added slowly (20 mL), and the mixture was stirred for additional 10 min. Afterwards, the mixture was extracted with CH2Cl2 (3×20 mL). The combined organic layers were washed with water (50 mL), dried over MgSO4, and concentrated under reduced pressure. The product was isolated by FCC  to give the isoxazolidinol 10a  as colorless oil. Rf 0.41 ; IR (ATR) νmax: 3421, 3028, 2924, 2868, 1495, 1454, 1107, 1028, 749, 696, 599, 531 cm−1; 1H NMR  δ 1.65 , 3.70 , 3.79 , 3.85 , 4.07 , 4.38 , 4.59 , 7.24–7.47 ; 13C NMR  δ 60.4 (PhCH2), 74.5 (C-5), 75.3 (C-3), 76.8 (C-4), 127.4 (CH-Ph), 128.3 (CH-Ph), 128.4 (CH-Ph), 128.9 (CH-Ph), 129.0 (CH-Ph), 129.2 (CH-Ph), 134.4 (C-Ph), 137.3 (C-Ph); HRMS (ESI) (m/z): [M + H]+ calcd for C16H18NO2, 256.1333; found 256.1329.(±)-2,2,2-Trichloroethyl 4-(benzoyloxy)-3-isopropylisoxazolidine-2-carboxylate (11): 2,2,2-Trichloroethyl chloroformate  was added dropwise to a stirred solution of the N-benzylisoxazolidine 7b  and lithium iodide  in anhydrous acetonitrile (4 mL) under argon. The reaction mixture was stirred at 60 °C for 8 h. When the TLC analysis showed that the starting isoxazolidine had disappeared , a saturated aq NaHCO3 (10 mL) solution and CH2Cl2 (20 mL) were added. After vigorous stirring for additional 5 min, the organic layer was separated, and the aqueous phase was extracted with CH2Cl2 (10 mL). The combined organic layers were washed with water (20 mL), dried over MgSO4, and concentrated under reduced pressure. The product was purified by FCC  to give the N-Troc-isoxazolidine 11  as colorless sticky oil. Rf 0.20 ; IR (ATR) νmax: 3067, 2963, 2881, 1755, 1717, 1374, 1315, 1265, 1107, 1052, 805, 708, 572 cm−1; 1H NMR : δ 1.09 , 1.14 , 1.89–1.97 2), 4.12 , 4.22 , 4.51 , 4.72 , 4.86 , 5.64 , 7.43–7.46 , 7.57–7.60 , 7.95–7.98 ; 13C NMR  δ 19.1 (CH3), 19.2 (CH3), 29.7 (CH(CH3)2), 72.1 (C-3), 74.4, 75.3 , 78.9 (C-4), 94.8 (CCl3), 128.6 (CH-Ph), 128.9 (C-Ph), 129.7 (CH-Ph), 133.7 (CH-Ph), 157.1 (CO2CH2), 165.9 (COPh); HRMS (ESI) (m/z): [M + H]+ calcd for C16H19Cl3NO5, 410.0324; found 410.0329.(±)-3-Isopropylisoxazolidin-4-ol (12): The NaOH solution  was added to a solution of the N-Troc-isoxazolidine 11  in isopropyl alcohol (1.2 mL), and the mixture was stirred at rt for 1 h. After the disappearance of the starting material , the solution was neutralized with HCl (6 M). Afterwards, a saturated aq NaHCO3 solution (2 mL) and solid NaCl were added, and the resulting slurry was vigorously extracted with EtOAc (3×5 mL). The combined organic layers were dried over MgSO4 and concentrated under reduced pressure. The product was purified by FCC (EtOAc) to give the isoxazolidinol 12  as colorless sticky oil. Rf 0.25 (EtOAc); IR (ATR): νmax = 3160, 2971, 2899, 2874, 1473, 1093, 1036, 1013, 935, 886, 754, 718, 647 cm−1; 1H NMR  δ 1.02 , 1.04 , 1.58–1.66 2), 2.90 , 3.83 , 3.89 , 4.51 ; 13C NMR  δ 19.8 (CH3), 20.0 (CH3), 29.1 (CH(CH3)2), 75.7 (C-3), 78.0 (C-5), 78.4 (C-4); HRMS (ESI) (m/z): [M + H]+ calcd for C6H14NO2, 132.1020; found 132.1020.File 1Detailed experimental procedures, analytical data, and NMR spectra of all compounds.


Pulicaria jaubertii is a medicinal herb that alleviates inflammations and fever. Chromatographic separation, phytochemical characterization, and in vitro biological activities of the plant n-hexane extract were conducted for the first time in this study. Six compounds were isolated for the first time from the n-hexane fraction of Pulicaria jaubertii aerial parts and were identified on the bases of NMR and MS analyses as pseudo-taraxaterol (1), pseudo-taraxasterol acetate (2), 3β-acetoxytaraxaster-20-en-30-aldehyde (3), calenduladiol-3-O-palmitate (4), stigmasterol (5), and α-tocospiro B (6). Compound (6) was a rare tocopherol-related compound and was isolated for the first time from family Asteraceae, while compound (3) was isolated for the first time from genus Pulicaria. The total alcoholic extract and n-hexane fraction were tested for their anti-inflammatory, antidiabetic, and cytotoxic activities. The n-hexane fraction has dose dependent red blood cells (RBCs) membrane stabilization and inhibition of histamine release activities with IC50: 60.8 and 72.9 µg/mL, respectively. As antidiabetic activity, the alcoholic extract exerted the most inhibition on the activity of yeast α-glucosidase, with an IC50: 76.8 µg/mL. The n-hexane fraction showed cytotoxic activity against hepatocarcinoma (HepG-2), breast carcinoma (MCF-7), and prostate carcinoma (PC-3) cell lines with IC50: 51.8, 90.8 and 62.2 µg/mL, respectively. In conclusion, the anti-inflammatory effect of Pulicaria jaubertii might be attributed to the triterpenoid constituents of the n-hexane extract of the plant. Pulicaria, belonging to the Asteraceae family, tribe Inuleae, consists of 100 species distributed in Europe, North Africa, and Asia, especially in Yemen and Saudi Arabia and is known for its medicinal properties  is one of the Pulicaria species indigenous in Yemen. It is a perennial aromatic plant used by local people as a flavoring agent for food preparation +. The IR spectrum exhibited absorption bands corresponding to a hydroxyl group (3400 cm−1) and a trisubstituted double bond (1650 cm−1). The 1H-NMR spectrum of compound 1 exhibited the signals of seven singlet tertiary methyls at δ 0.73 (Me-28), 0.76 (Me-24), 0.85 (Me-25), 0.95 (Me-27), 0.97 (Me-23), 1.04 (Me-26), and 1.63 (Me-30), one doublet secondary methyl at δ 0.99 , an oxymethine at δ 3.20 , and an olefinic proton at δ 5.26 . The 13C-NMR spectrum of 1 displayed 30 carbon resonances, including two olefinic carbons at δ 139.86 (C-20) and 118.87 (C-21) and one oxygen bearing carbon at δ 79.03 (C-3), indicating a pentacyclic triterpene alcohol with one trisubstituted double bond. According to these data and through comparison with the literature +. An ester carbonyl (1733 cm−1) and an α,β-unsaturated carbonyl (1692 and 1645 cm−1) absorptions were observed in its IR spectrum. It showed an absorption band at 235 nm in the UV spectrum indicated the presence of conjugation in the molecule. The 1H and 13C-NMR spectral data of 3 were almost identical to that of 2, suggested the presence of a monounsaturated pentacyclic 3β-acetoxytriterpene of the pseudotaraxasterane skeleton with a methyl group oxidized to aldehyde in 3 versus 2. The presence of an aldehyde group in the molecule was established from the observed downfield proton signal at δ 9.36  in the 1H-NMR spectrum and the downfield carbon signal at δ 194.11 (C-30) in the 13C-NMR spectrum of 3. Additionally, the downfield shifted proton at δ 6.71  and carbon at δ 149.28 (C-21) compared to those of 1 and 2 confirmed the presence of the aldehyde group at C-20 position. From the above findings, the structure of 3 was assigned to be 3β-acetoxytaraxaster-20-en-30-aldehyde + indicating a molecular formula C29H48O. Its IR spectrum showed a characteristic absorption band for a hydroxyl group at 3433 cm−1 and olefinic bonds at 1645 cm−1. The 1H-NMR spectrum of compound 5 exhibited the signals of six methyls between δ 0.69 and 1.02, an oxymethine at δ 3.60  and olefinic protons at δ 5.02 , 5.15  and 5.35  that are characteristic for Δ5,22-3β-hydroxy sterols. The 13C-NMR spectrum of compound 5 displayed 29 carbon signals of which 6 signals  were assigned to 6 methyls, one signal (δ 71.83) for an oxymethine carbon and four signals  for the four olefinic carbons. Comparison of all these data with those described in the literature confirmed the identity of compound 5 to be a stigmasterol [Compound (6) was obtained as a colorless oil and had an [M − H]- ion peak at m/z 461 in the negative ESIMS, which is compatible with the molecular formula C29H50O4. The IR spectrum of 6 showed absorptions for hydroxyl (3435 cm−1), five-membered ring conjugated carbonyl (1720 cm−1) and olefinic (1655 cm−1) groups. Its UV spectrum showed an absorption band at 236 nm. The 1H-NMR spectrum exhibited signals for a hydroxyl proton at δ 4.69 , a methylketone at δ 2.02 , a tertiary methyl at δ 1.31 , two olefinic methyls at δ 1.81  and 1.84  and four secondary methyls at δ 0.83 , 0.84 , 0.86 . The 13C-NMR spectrum of 6 showed 29 carbon signals, including one methyl ketone carbon [δ 24.87 (C-3a)], two vinyl methyl carbons [δ 11.78 (C-5a) and 8.75 (C-6a)], two olefinic carbons [δ 139.51 (C-6) and 163.21 (C-5)], three oxygenated carbons  and two carbonyl carbons [δ 205.18 (C-1) and 207.01 (C-3)]. The above data suggested that 6 was a spiro compound of two five-membered rings with a branched hydrocarbon chain and was considered to be a α-tocopherol-related compound. The relative stereochemistry of the spiro moiety in 6 was also assigned on the basis of comparison of the chemical shift of H3-9a (δ 1.31) of 6 with those of H3-9a (δ 1.02) of α-tocospiro A and H3-9a (δ 1.29) of α-tocospiro B previously isolated from Ficus microcarpa +, m\z 425 [M − H]-.Pseudo-taraxaterol (2): Colorless needles; IR υmax (KBr) cm-1: 1735 (CO), 1642 (C=C); 1H-NMR  δ 0.73 , 0.77 , 0.84 , 0.85 , 0.87 , 0.94 , 0.99 , 1.04 , 1.63 , 2.04 , 4.48 , 5.26 ; 13C-NMR  δ 14.71 (C-27), 16.05 (C-26), 16.38 (C-25), 16.53 (C-24), 17.72 (C-28), 18.20 (C-6), 21.35 (CO3CH), 21.64 , 22.55 (C-29), 23.70 (C-2), 27.03 (C-15), 27.61 (C-12), 27.96 (C-23), 34.17 (C-7), 34.40 (C-17), 36.33 (C-19), 36.70 (C-16), 37.07 (C-10), 38.74 (C-1), 38.85 (C-4), 39.20 (C-13), 41.09 (C-8), 42.18 (C-22), 42.34 (C-14), 48.69 (C-18), 50.34 (C-9), 55.39 (C-5), 81.01 (C-3), 118.89 (C-21), 139.88 (C-20), 171.06 (COCH3); EI-MS m\z 468 [M]+.Pseudo-taraxasterol acetate (3): Colorless needles; UV λmax (MeOH) nm: 235; IR υmax (KBr) cm−1: 1733 (CO), 1692, 1645 (C=C-C=O); 1H-NMR  δ 0.67 , 0.84 , 0.85 , 0.88 , 0.96 , 1.02 , 1.03 , 9.36 , 2.04 , 4.48 , 6.71 ; 13C-NMR  δ 14.69 (C-27), 15.98 (C-26), 16.35 (C-25), 16.52 (C-24), 17.53 (C-28), 18.17 (C-6), 21.35 (CO3CH), 21.45 (C-11), 23.18 (C-29), 23.69 (C-2), 26.88 (C-15), 27.25 (C-12), 27.99 (C-23), 34.13 (C-7), 34.80 (C-17), 29.38 (C-19), 36.47 (C-16), 37.03 (C-10), 38.44 (C-1), 37.80 (C-4), 39.01 (C-13), 41.05 (C-8), 43.03 (C-22), 42.27 (C-14), 48.20 (C-18), 50.30 (C-9), 55.37 (C-5), 80.96 (C-3), 148.44 (C-20), 149.28 (C-21), 171.07 (COCH3), 194.11 (C-30); EI-MS m\z 482 [M]+.β-acetoxytaraxaster-20-en-30-aldehyde : An amorphous white solid; IR υmax (KBr) cm−1: 3440 (OH), 1730 (C=O), 1640, 883 (C=C), 725 ; 1H-NMR ; calenduladiol δ 0.79 , 0.83 , 0.84 , 0.85 , 0.98 , 1.03 , 1.68 , 2.49 , 3.60 , 4.47 , 4.59 , 4.70 , fatty acid δ 0.87 , 1.19 , 1.21 , 1.25-1.30 (H-4`-H-13`), 1.61 , 2.28 ; 13C-NMR  δ 11.69 (C-28), 16.00 (C-26), 16.17 (C-27), 16.19 (C-25), 16.56 (C-24), 18.19 (C-6), 19.31 (C-30), 20.89 (C-11), 21.30 (C-2`), 23.73 (C-2), 24.74 (C-12), 27.97 (C-23), 29.90 (C-21), 34.18 (C-7), 36.87 (C-15), 37.06 (C-10), 37.25 (C-13), 37.72 (C-22), 37.85 (C-4), 38.41 (C-1), 40.95 (C-8), 44.09 (C-14), 48.61 (C-17), 47.63 (C-19), 47.70 (C-18), 49.92 (C-9), 55.41 (C-5), 77.09 (C-16), 80.55 (C-3), 109.85 (C-29), 149.98 (C-20), fatty acid δ 14.14 (C-16`), 22.71 (C-15`), 25.18 (C-3`), 29.71-29.18 (C-4`-C-13), 31.94 (C-14`), 34.87 (C-2`), 173.76 (C-1`), EI-MS m\z 680 [M]+.Calenduladiol-3-5): A colorless crystalline mass; IR υmax (KBr) cm−1: 3433 (OH), 1645 (C=C); 1H-NMR  δ 0.69 , 0.87 , 0.89 , 0.92 , 1.01 , 1.02 , 3.52 , 3.60 , 5.02 , 5.15 , 5.35 ; 13C-NMR  δ 12.06 (C-18), 12.27 (C-29), 18.99 (C-19), 19.41 (C-26), 21.08 (C-27), 21.10 (C-21), 21.23 (C-11), 24.38 (C-15), 25.42 (C-28), 29.38 (C-16), 31.67 (C-2), 31.94 , 36.52 (C-10), 37.26 (C-1), 39.69 (C-12), 40.51 (C-20), 42.23 (C-4), 42.31 (C-13), 45.84 (C-25), 50.16 (C-9), 51.25 (C-24), 55.96 (C-17), 56.87 (C-14), 71.83 (C-3), 121.73 (C-6), 129.28 (C-23), 138.34 (C-22), 140.76 (C-5); ESI-MS m\z 413 [M + H]+.Stigmasterol (6): Colorless oil; UV λmax (MeOH) nm: 236; IR υmax (KBr) cm−1: 3435 (OH), 1720 (CO), 1655 (C=C); 1H-NMR  δ 0.83 , 0.84 , 0.86 , 1.31 , 1.62 , 1.70 , 1.77 , 1.81 , 1.84 , 1.88 , 2.35 , 2.02 , 4.69 ; 13C-NMR  δ 8.75 (C-6a), 11.78 (C-5a), 19.62 (C-13a), 19.77 (C-17a), 22.41 (C-11), 22.64 (C-22), 22.73 (C-21a), 24.48 (C-19), 24.81 (C-15), 24.87 (C-3a), 25.42 (C-9a), 27.99 (C-21), 32.76 (C-17), 32.81 (C-13), 33.42 (C-7), 36.82 (C-8), 37.30 (C-18), 37.44 (C-14), 37.47 (C-16), 37.48 (C-12), 39.38 (C-20), 42.09 (C-10), 87.24 (C-9), 89.43 (C-4), 92.59 (C-2), 139.51 (C-6), 163.21 (C-5), 205.18 (C-1), 207.01 (C-3); ESI-MS m\z 461 [M − H]-, 497 [M − Cl]-.α-Tocospiro B (n-hexane extracts in concentrations of 7.81–1000 µg/mL or standard indomethacin were mixed with 0.5 mL of the stock red blood cells (RBCs) suspension and 5 mL of the hypotonic solution consists of sodium phosphate buffer (10 mM) and NaCl (50 mM) adjusted to pH 7.4. Control samples were prepared by similar manner without extracts or indomethacin. Afterward, previous mixtures were withstanding for 10 min in room condition and subjected to 10 min centrifugation at 3000 rpm. Then absorbance was measured for the supernatant at 540 nm and the hemolysis percentage inhibition was calculated from the following equation: The method was conducted according to the literature . Erythron-hexane extracts was measured on U937 human monocytes . In accordance with literature [Histamine release effect of total alcoholic and terature , extractn-hexane extracts or standard acarbose in a concentration range of 7.81–1000 μg/mL were incubated at 25 °C for 10 min. One percent of starch solution (1 mL) in sodium phosphate buffer  was added to the mixtures and incubated for 10 min at room temperature. 3,5-Dinitrosalicylic acid (2 mL) was added and the mixtures were then incubated in a boiling water bath for 5 min. After cooling, 10 mL of distilled water was added to the mixtures. A control test was conducted in a similar manner without test samples. The absorbance was then recorded at 565 nm and the percentage inhibition of amylase was calculated from equation: According to the reported method , mixture control .n-hexane extracts was determined by the method of Shai et al. with minor modification [Saccharomyces cerevisiae, Sigma Aldrich, Bangalore, India) and preincubated for 20 min at 37 °C. Then, p-nitrophenyl-α-D-glucopyranoside (20 μL of 5.0 mM) was added to the mixtures and incubated further for 20 min at 37 °C. The reaction was stopped by adding Na2CO3 (50 μL of 1 M). Absorbance was recorded at 405 nm to determine the p-nitrophenol amount released from the reaction mixture. The reaction mixture without test samples was used as a control where alpha glucosidase enzyme percentage inhibition was calculated from using following equation: α-Glucosidase inhibitory activity of the alcoholic and fication . In 96-w control .n-hexane extract was conducted against three human tumor cell lines: hepatocarcinoma (HepG-2), breast carcinoma (MCF7), and prostate carcinoma (PC-3) cell lines using MTT assay according to the reported method [2 and were sub cultured two to three times a week. Cell viability percentage was calculated by the equation: The antiproliferative activity of d method . All celt-test was applied for detecting the significance (p < 0.05) of difference between the values of the treated groups and the control. Biological experiments were conducted in independent triplicate, mean were calculated ± St. Error mean. Student’s O-palmitate, stigmasterol, and α-tocospiro B were isolated using different chromatographic techniques including VLC, silica gel and Sephadex LH-20 columns from Pulicaria jaubertii n-hexane extract. The compounds were characterized by the IR, NMR, and MS spectroscopy and by the comparison with literatures. The rare α-tocospiro B was isolated for the first time from family Asteraceae and genus Pulicaria, however, all other compounds were recorded for the first time from the plant. The in vitro biological investigation of the plant revealed the significant higher activity of the n-hexane extract as an anti-inflammatory agent compared to the total alcoholic extract which showed better antidiabetic and cytotoxic activities. The anti-inflammatory effect of the n-hexane extract could be attributed to the extract constituents including triterpenes, which have been prevalently known for this effect. Further animal-based in vivo anti-inflammatory testing of the n-hexane extract is an essential future plan to confirm the activity.Six compounds, pseudo-taraxaterol, pseudo-taraxasterol acetate, 3β-acetoxytaraxaster-20-en-30-aldehyde, calenduladiol-3-


Suberea clavata led to the isolation of eight new bromotyrosine metabolites named subereins 1–8 (2–9) along with twelve known co-isolated congeners. The detailed configuration determination of the first representative major compound of this family 11-epi-fistularin-3  (1) is described. Their chemical characterization was achieved by HRMS and integrated 1D and 2D NMR (nuclear magnetic resonance) spectroscopic studies and extensive comparison with literature data. For the first time, a complete assignment of the absolute configurations for stereogenic centers C-11/17 of the known members  11-epi-fistularin-3 (1) and 17-deoxyfistularin-3 (10) was determined by a combination of chemical modifications, Mosher’s technology, and ECD spectroscopy. Consequently, the absolute configurations of all our new isolated compounds 2–9 were determined by the combination of NMR, Mosher’s method, ECD comparison, and chemical modifications. Interestingly, compounds 2–7 were obtained by chemical transformation of the major compound 11-epi-fistularin-3 (1). Evaluation for acetylcholinesterase inhibition (AChE), DNA methyltransferase 1 (DNMT1) modulating activity and antifouling activities using marine bacterial strains are also presented.Chemical investigation of the South-Pacific marine sponge  Pseudovibrio denitrificans Ab134 isolated from the sponge Arenosclera brasiliensis methoxyphenyl]-2-oxazolidinone (18) + corresponding to the molecular formula C31H3079Br381Br3N4O11 . This isomer of fistularin-3 presented some differences on the 1H NMR spectrum. The singlet H-5 expected at δH 6.55 ppm was not observed, but another one appeared at δH 7.56 ppm, a chemical shift in accordance with an aromatic proton. An interesting change was observed for the CH2-7′ observed at δH 3.81 ppm as a singlet associated by HSQC with a carbon observed at the low chemical shift δC 25.5 ppm. The COSY spectrum  and 122.1(C6′). Furthermore, the proton H-5′ at δH 7.56 was also correlated to C-1′, C-6′, and to those at δC 106.3 (C-4′) and 108.9 (C-2′). The COSY spectrum (recorded in acetone-d6) revealed a spin system including a methylene group CH2-10 at δH 3.78/3.52, followed by an -NH proton at δH 7.62 and oxymethine CH-11 at δH 4.24, and terminated with two diastereotopic protons CH2-12 at δH 4.02/4.05. The HMBC spectrum displayed key correlations between H-10 and H-7 with the amidic carbonyl C-9 at δC 160.4  and 290 nm (Δє + 2.17). Compound 2 was named suberein-1.In a slightly less polar fraction, we identified a mixture of two compounds mpound 2  was isolspectrum  displayeδC 160.4 , which c3 ). The HRESIMS spectrum showed the predominant peak at m/z 1136.6864 [M+Na]+, corresponding to a molecular formula C31H30Br6N4O11 . The 1H and 13C NMR spectral data  and 286 nm (Δє + 2.19), in its ECD spectrum. Compound 3 was named suberein-2.Compound 3  was a reral data  were simδC 166.6 . The abs4 + 5 were isolated together as an inseparable 1/1 mixture. Their HRESIMS spectrum exhibited a predominant peak at m/z 1100.6935 [M+H]+ corresponding to a molecular formula C30H28Br6N4O11 , which indicated 14 a.m.u less than 11-epi-fistularin-3 (1). The 1H NMR spectrum (H 3.73 instead of two in 11-epi-fistularin-3 (1). Indeed, the 13C NMR spectrum disclosed two characteristic resonances at δC 183.6 (C-3′) assigned to a carbonyl group, and δC 75.3 attributed to C-1′, which were not recorded for 11-epi-fistularin-3 (1). The COSY spectrum showed key correlations between the proton at δH 5.08 (H-2′) and the oxymethine at δH 4.41 (H-1′), with a coupling constant J = 11.4 Hz. Significant HMBC correlations were observed between the proton H-5′ and the carbons at δC 75.3 (C-1′), 57.4 (C-2′), 183.6 (C-3′), 122.6 (C-4′), and 91.8 (C-6′). Additionally, the oxymethine H-1′ was correlated with C-2′ and meanwhile, H-2′ was correlated with C-3′. Thus, the structure of 4 was determined as described in 5 in the left side of the molecule (C-1 to C-6 instead of C-1′ to C-6′). The observed NOE correlation between H-2′ and one of the H-5′ together with the absence of NOE correlations between H-1′ and H2′ suggested a trans configuration for 4 + 5. Compounds 4 and 5 are named suberein-3 and suberein-4, respectively.Compounds spectrum  showed o4 + 5, the following compounds 6 + 7 were also isolated together as an inseparable in 1/1 mixture. Their molecular formulas were established as C30H28Br6N4O11 from their positive HRESIMS spectrum with a predominant peak at m/z 1100.6935 [M+H]+, . Their 1H NMR spectrum  suggesting a cis configuration, which was further confirmed by the NOE correlations. Therefore, the structure of 6 and 7 were assigned as described in As for spectrum  showed s4 + 5 and 6 + 7 epimerize within two days in solution into a mixture of 4/5/6/7: 1/1/1/1. Their purification just allowed their characterisation as mixtures of the structures of the cis stereoisomers on the one hand and of the trans stereoisomers on the other hand.It should be noted that due to the keto-enolic equilibrium, carbons C-2 and C-2′ in both mixtures 8 was isolated as an amorphous powder ). Its HRESIMS showed an isotopic cluster indicating six bromines with a principal peak at m/z 1110.6661 [M−H]− corresponding to a molecular formula C31H28Br6N4O11 . Its 1H NMR spectrum (H 4.87 (H-18) together with the absence of the H-17 signal observed for fistularin-3 at δH 4.90. The chemical shift of the aromatic protons (H-15/15′) was observed at δH 8.28. The 13C NMR spectrum , H-15/15′ (δH 8.28), and the non-protonated carbon at δC 192.2, thus assigning the carbonyl group to C-17. Further key HMBC correlations were consistent with the backbone of fistularin-3. Thus, the structure was depicted as 17-oxo-11-epi-fistularin-3 for compound 8 and named suberein-7 ) showed in its HRESIMS an isotopic cluster of peaks indicating a tetrabrominated compound with a principal peak at m/z 761.8695 [M+H]+ corresponding to the molecular formula C23H26Br4N3O6 . The 1H NMR spectrum (epi-fistularin-3 (1). The COSY spectrum allowed the identification of additional spin system including an oxymethylene group (CH2-16) at δH 4.07 based on its chemical shift and CH2 group at δH 2.11 (CH2-17), and terminated with an azomethylene group (CH2-18) at δH 2.87. A singlet at δH 2.47 integrated for six protons was assigned to the N,N-dimethyl terminus (CH3-19/20). The HMBC spectrum displayed key correlations between the oxymethylene group at δH 4.07 (CH2-16) and the quaternary carbon C-15 at δC 152.1, linking this partial structure to the tetrasubstituted phenyl ring. Furthermore, the signal corresponding to the N,N-dimethyl functionality  displayed HMBC correlation with the carbon C-18 at δC 56.0. The other HMBC correlations were consistent with the proposed structure for 9 and named suberein-8 ) showed in its negative HRESIMS a characteristic isotopic cluster of a hexabrominated compound with a predominant peak at m/z 1096.6923 [M−H]− corresponding to the molecular formula C31H30Br6N4O10 . Its 1H NMR spectral pattern was identical to the one of 17-deoxyfistularin-3 Suberea clavata. The sponge was deep frozen on board until work up. It was then grounded, freeze-dried, and extracted.The sponge was collected by hand using SCUBA in the Solomon Islands, Russell Group, during the sampling cruise BSMS-1  aboard tSuberea clavata (200 g) was extracted at room temperature with MeOH/DCM (1/1) to give 57.8 g of dried extract. The crude extract was submitted to n-butanol/H2O partition to obtain a desalted butanol extract (30 g). A portion (25 g) of the later extract was submitted to reversed-phase C18 SPE  and eluted with a gradient H2O/MeOH (100/0 to 0/100) and MeOH/DCM (100/0 to 0/100) to give six fractions f1 to f6. The fraction f4 (10 g) was submitted to reversed-phase HPLC  yielding 13 subfractions, F1 to F13. The sub-fraction F1 afforded pure aerophobin-2 (16) .The lyophilized sponge epi-fistularin-3 (1)  and F9 pure 11,17-dideoxyfistularin-3 (14) .Fraction F6 only contained 11-2/MeOH (75/25), isocratic condition) and yielded 17-deoxy-epi-fistularin-3 (10) , 11-deoxyfistularin-3 (14) , and 17-oxo-11-epi-fistularin-3 (8) .Fraction F7 was purified by SFC , suberein-1 (2) , and suberein-1 (3) .Fraction F8 was purified by SFC , isocratic condition) to yield 11-18, H2O + 0.1% HCOOH/CH3CN + 0.1% HCOOH, 85/15 to 25/75 in 25 min). Agelorin A (11)  and B (12) , 11-hydroxyaerothionin (15) , as well as a mixture of 4 + 5  and a mixture of 6 + 7 , were obtained.Fraction F5 was submitted to reversed-phase HPLC (Waters Sunfire C18 silica-gel cartridge (23 × 110 mm); H2O + 0.1% HCOOH/MeOH+0.1% HCOOH, 100/0 to 0/100, in 35 min). It yielded eight subfractions F’1 to F’8. Pseudoceralidinone A (20)  was obtained from F’1. Fraction F’7 was submitted to reversed-phase HPLC  to afford aplysinamisin-1 (17) , subereaphenol K (19) , and 7R, 11S methoxyphenyl]-2-oxazolidinone (18) . Fraction F’8 afforded suberein-8 (9)  by reversed-phase HPLC purification using an Xbridge C18 column / CH3CN + 0.15% NH4OH, 90/10 to 0/100 in 35 min).Fraction F1 (1.5 g) was purified by MPLC (Versapak CS-epi-fistularin-3 (1): yellowish amorphous solid (3 g); [α]D25 + 148.0° ; [α]D25 + 169.0 ; ECD  λmax (Δє) 253 (+7.5), 285 (+8.0); 1H NMR : δH ppm 7.67 , 7.66 , 7.62 , 6.53 , 5.41 , 5.00 , 4.90 , 4.44 , 4.25 , 4.18 , 4.04 , 3.85 , 3.82 , 3.80 , 3.73 , 3.63 , 3.54 , 3.49 , 3.19 , 3.16 ; 13C NMR : δC ppm 160.5 , 155.2 (C-8′), 155.1 (C-8), 152.7 (C-13), 148.8 , 143.3 (C-16), 132.4 (C-5′), 132.3 (C-5), 131.5 , 122.1 , 118.4 , 113.9 (C-2′), 113.8 (C-2), 91.8 , 75.9 (C-12), 75.2 , 71.0 (C-17), 69.9 (C-11), 60.2 , 47.7 (C-18), 43.6 (C-10), 40.0 ; HRESIMS m/z 1136.6934 [M+Na]+ .171  and anhydrous Pyridine  in anhydrous DCM ) to give the S-MTPA-epi-fistularin-3 ester  as a white solid: 1H NMR : δH 7.95 , 7.63 , 7.46 , 7.40 , 6.52 , 6.13 , 5.42 , 4.48 , 4.25 , 4.18 , 4.08 , 3.98 , 3.88–3.82 , 3.86–3.80 , 3.82 , 3.73 , 3.62 , 3.55 , 3.23–3.17 , 3.19–3.13 ; HRESIMS m/z 1352.7357 [M+Na]+, .To a stirred solution of compound 1  and anhydrous Pyridine  in anhydrous DCM  = 0.066 M) at room temperature, S-(+)-MTPA-Cl  and DMAP (0.5 mg) were added. The reaction progress was monitored by thin-layer chromatography (TLC) on silica gel (95:5 DCM/MeOH). After 4 h, compound 1 was totally consumed. The reaction mixture was quenched by the addition of water and EtOAc. The aqueous layer was extracted with two additional portions of EtOAc, and the combined organic layers were dried on MgSO4, filtered, and concentrated in vacuo. The crude product mixture was purified by reversed-phase HPLC ) to give the R-MTPA-epi-fistularin-3 ester  as a white solid: 1H NMR : δH 7.73 , 7.68 , 7.48 , 7.30 , 6.52 , 6.21 , 5.43 , 4.49 , 4.26 , 4.17 , 4.09 , 3.88–3.83 , 3.82–3.77 , 3.80 , 3.78 , 3.73 , 3.60 , 3.50 , 3.23–3.16 , 3.12–3.06 ; HRESIMS m/z 1352.7282 [M+Na]+ .To a stirred solution of compound 2): colourless amorphous solid (10 mg); [α]D25 + 61.0 ; [α]D25 + 32.5 ; UV (MeOH) λmax (є) 208 (38200), 286 (10100) nm; ECD  λmax (Δє) 214 (−2.79), 256 (2.55), 290 (2.17) nm; IR νmax 3380, 2990, 2900, 1655, 1630, 1545, 1460, 1400, 1255 cm-1; 1H and 13C NMR data, m/z 1136.6924 [M+Na]+ .Suberein-1 (3): colourless amorphous solid (10 mg); [α]D25 +70.7 ; [α]D25 + 32.0 ; UV (MeOH) λmax (є) 208 (38200), 286 (10100) nm; ECD  λmax (Δє) 215 (–1.73), 255 (2.33), 286 (2.19) nm; IR νmax 3360, 2985, 2900, 1655, 1545, 1460, 1415, 1260 cm-1; 1H and 13C NMR data, m/z 1136.6864 [M+Na]+ .Suberein-2 (4 and 5): amorphous off-white powder (5 mg); UV (MeOH) λmax (є) 210 (22500), 232 (7800), 260 (5100) nm; 1H and 13C NMR data, m/z 1100.6935 [M+H]+ Suberein-3 and suberein-4 (6 and 7): amorphous off-white powder (5 mg); UV (MeOH) λmax (є) 210 (22500), 232 (7800), 260 (5000) nm; 1H and 13C NMR data, m/z 1100.6935 [M+H]+ .Suberein-5 and Suberein-6 (8): yellow amorphous solid (9 mg); [α]D25 +149.5 ; [α]D25 + 14.5 ; UV (MeOH) λmax (є) 210 (29500), 235 (15800), 283 (8800) nm; ECD  λmax (Δє) 253 (7.5), 285 (8.0) nm; IR νmax 3380, 2930, 1670, 1580, 1540, 1270 cm-1; 1H and 13C NMR data, m/z 1110.6661 [M−H]− .Suberein-7 (9): yellow amorphous solid (3 mg); [α]D25 +84.0 ; UV (MeOH) λmax (є) 208 (27800), 283 (6700) nm; ECD  λmax (Δє) 245 (7.2), 285 (7.8); IR νmax 3350, 2930, 2855, 1665, 1600, 1540, 1460, 1400, 1380, 1250 cm−1; 1H and 13C NMR data, 1H NMR : δ 4.09 , 6.41 , 3.75 , 3.06 , 3.74 , 3.40 , 4.76 , 7.62 , 4.07 , 2.11 , 2.87 , 2.47 , 2.47 , 3.73 ; 13C NMR : δ 74.2 (C-1), 112.6 (C-2), 147.9 (C-3), 121.5 (C-4), 130.7 (C-5), 91.1 (C-6), 38.3 (C-7), 153.0 (C-8), 160.8 (C-9), 47.3 (C-10), 70.1 (C-11), 142.5 (C-12), 130.2 , 117.6 , 152.1 (C-15), 70.8 (C-16), 26.5 (C-17), 56.0 (C-18), 43.8 (C-19), 43.8 (C-20), 59.0 (OMe); HRESIMS m/z 761.8695 [M+H]+ .Suberein-8 (epi-fistularin-3 (10): yellow amorphous solid (8 mg); [α]D25 +71.0 ; [α]D25 +20.0 ; ECD ) λmax (Δє) 251 (7.7), 286 (7.6); 1H NMR : δ 7.74 , 7.63 , 7.52 , 6.52 , 5.45 , 4.47 , 4.24 , 4.18 , 4.03 , 3.86 , 3.82 , 3.78 , 3.73 , 3.57 , 3.52 , 3.20 , 3.15 , 2.88 ; HRESIMS m/z 1096.6923 [M−H]− .17-deoxy-11-1, 2, 3, 8, and 10 for the preparation of the 1-aminodiol for Marfey derivatization: the compounds (2 mg of each) were dissolved in a mixture of TFA/H2O  and heated in a closed tube at 120 °C for 36 h. Each solution was evaporated to dryness to obtain the corresponding crude hydrolysate.General acid hydrolysis of compounds 3 . The solution was heated at 60 °C for 5 min, then cooled. The mixture was filtered on 0.2 μm Millex-LG. Standards (R) and (S)-3-amino-1,2-propanediol were converted to their L-FDAA derivatives following the same procedure. (a) Marfey derivatization: the hydrolysate was dissolved in water (100 μL) and combined with 1-fluoro-2,4-dinitrophenyl-5-L-alaninamide (L-FDAA)  and aqueous NaHCO2O + 0.1% HCOOH/CH3CN+ 0.1% HCOOH (98/2 to 70/30 in 10 min). The (R)- and (S)-DAA derivatives of 3-amino-1,2-propanediol were eluted at RT = 6.59 min and RT = 6.68 min, respectively. Analysis of 1-L-DAA derivative gave a retention time of RT = 6.58 min, corresponding to an 11R configuration for epi-fistularin-3 (1).(b) LC analysis: the solutions of DAA derivatives were analyzed directly by UPLC/MS using HSS C18 column and eluted with H3 . The solution was heated at 60 °C for 5 min, then cooled. The mixture was filtered on 0.2 μm Millex-LG. Standards (R) and (S)-3-amino-1,2-propanediol were converted to their L-FDAA derivatives following the same procedure. (a) Marfey derivatization: the hydrolysate was dissolved in water (100 μL) and combined with 1-fluoro-2,4-dinitrophenyl-5-L-alaninamide (L-FDAA)  and aqueous NaHCO2O + 0.1% HCOOH/CH3CN + 0.1% HCOOH (98/2 to 70/30 in 10 min). The (R)- and (S)-DAA derivatives of 3-amino-1,2-propanediol were eluted at RT = 7.01 min and RT = 7.10 min, respectively. Analysis of the compound L-DAA derivative gave a retention time of RT = 7.01 min, corresponding to an 11R configuration for compounds 2 and 3.(b) LC analysis: the solutions of DAA derivatives were analyzed directly by UPLC/MS using HSS C18 column and eluted with H(a) Marfey derivatization: the hydrolysate was dissolved in water (100 μL) and combined with 1-fluoro-2,4-dinitrophenyl-5-L-alaninamide (L-FDAA)  and aqueous NaHCO3 . The solution was heated at 60 °C for 5 min, then cooled. The mixture was filtered on 0.2 μm Millex-LG. Standards (R) and (S)-3-amino-1,2-propanediol were converted to their L-FDAA derivatives following the same procedure. 2O + 0.1% HCOOH/CH3CN+ 0.1% HCOOH (98/2 to 70/30 in 10 min). The (R)- and (S)-DAA derivatives of 3-amino-1,2-propanediol were eluted at RT = 10.14 min and RT = 10.30 min, respectively. Analysis of 8-L-DAA derivative gave a retention time of RT = 10.13 min, corresponding to an 11R configuration for compound 8.(b) LC analysis: the solutions of DAA derivatives were analyzed directly by UPLC/MS using HSS C18 column and eluted with H(a) Marfey derivatization: the hydrolysate was dissolved in water (100 μL) and combined with 1-fluoro-2,4-dinitrophenyl-5-L-alaninamide (L-FDAA)  and aqueous NaHCO3 . The solution was heated at 60 °C for 5 min, then cooled. The mixture was filtered on 0.2 μm Millex-LG. Standards (R) and (S)-3-amino-1,2-propanediol were converted to their L-FDAA derivatives following the same procedure.2O + 0.1% HCOOH/CH3CN + 0.1% HCOOH (98/2 to 70/30 in 10 min). The (R)- and (S)-DAA derivatives of 3-amino-1,2-propanediol were eluted at RT = 6.78 min and RT = 6.88 min, respectively. Analysis of 10-L-DAA derivative gave a retention time of RT = 6.80 min, corresponding to an 11R configuration for 17-deoxy-epi-fistularin-3 (10).(b) LC analysis: the solutions of DAA derivatives were analyzed directly by UPLC/MS using HSS C18 column and eluted with Hepi-fistularin-3 (1) to compounds 2–7 and agelorins A/B: compound 1 was dissolved in a mixture 1/1 of acetonitrile (+0.1% HCOOH)/water (+0.1% HCOOH) and stored at room temperature for two weeks. The reaction mixture was then dried under vacuum and submitted for HPLC analysis. Compound 1 gave agelorin A, agelorin B, and the compounds 2–7 by comparison with the retention time of each product.Conversion of 11-Preparation of the compounds: antifouling efficiency of the compounds was assessed towards the inhibition of growth of key bacteria involved in marine and terrestrial surface colonisation. All bioassays were run in six replicates and using two batches of organisms. Compounds were tested at 0.01, 0.1, and 1 µg/mL. 96 well-plates were used and were coated with the compounds. Methanol was used as a carrier solvent and after its evaporation plates were sterilized under UV exposure for 30 min .Halomonas aquamarina (ATCC 14400), Polaribacter irgensii (ATCC 700398), Shewanella putrefaciens (ATCC 8071), Roseobacter littoralis (ATCC 495666), Pseudoalteromonas elyakovii (ATCC 700519), Vibrio aestuarianus (ATCC 35048), Vibrio natriegens (ATCC 14058), and three strains of terrestrial bacteria, i.e., Escherichia coli (ATCC 11775), Staphylococcus aureus (ATCC 126000), and Pseudomonas aeruginosa (ATCC 16145) were used to assess the antibacterial potency of the compounds.Seven strains of marine bacteria i.e., For maintenance, bacterial strains were kept on solid media: autoclaved seawater, 0.5% peptone (oxoid), and 1% agar for marine strains .8 cells/mL) in LB medium , (supplemented with NaCl (35 g/L) for marine strains), at 30 °C for 48 h. After incubation, the intensity of growth in the presence of the tested compounds and control (media only) was assessed by measurement of the OD 620 nm using a spectrophotometer . Results are expressed as minimum inhibitory concentrations .Inhibition of bacterial growth: experiments were run as previously described by Maréchal et al. . 96-well2.6H2O). Acetylcholinesterase from Electrophorus electricus  was dissolved in buffer A, 5,5′-dithiobis-2-nitrobenzoic acid (DTNB-Ellman’s reagent−3 mM) in buffer B, and acetylthiocholine iodide (ACTI−6 mM) in H2O. Tested compounds were dissolved in DMSO at 10 mg/mL, stored at −20 °C, and subsequently 100 times diluted in H2O before the assay. 10 μL AChE was preincubated for 10 min with 70 μL DTNB and 10 μL compounds solution in 96-well microplates. The enzymatic reaction was initiated with the addition of 10 μL ACTI, a synthetic substrate of AChE. After 30 min at 25 °C, absorbance of the plate was measured at 405 nm using a microplate reader (Tecan Infinite M200). Percentage of inhibition was calculated using the following equation: 100 × (Acontrol − Asample)/(Acontrol − A0), where Acontrol is the average absorbance of the wells in which tested compounds were replaced by water, Asample is the absorbance of the well with tested compound, and A0 the average absorbance of blank wells where AChE was omitted. Products leading to 50% inhibition of AChE were then submitted to dose-response experiments. Serial dilutions of the products were prepared . Enzymatic assay was performed according to the same procedure with 10 µL of each concentration added. IC50 values (concentration leading to 50% inhibition of enzymatic activity) were graphically determined by plotting the percentage of inhibition against the compound concentrations.This bioassay was set up after Ellman . This meEach assay was performed in triplicate and results are expressed as mean values ± SD.DNMT1 activity was measured using in vitro DNMT activity/inhibition assay  according to manufacturer’s instructions. All compounds were used at a final 50 µM concentration. The methylation reaction was performed by incubating 25 ng of purified DNMT1 with compounds for 2 h at 37 °C in the presence of 0.01% Triton X-100. The methylated DNA was then recognized by the His-tagged methyl-CpG binding domain protein 2b. The addition of a poly-histidine antibody conjugated to horseradish peroxidase provided a colorimetric readout quantified with a spectrophotometer  at the wavelength of 450 nm. Data of DNMT1 activity are reported as percentage of control.