Biosynthesis and antioxidant activity of 4NRC β-glycoside

Tetrahedron Letters 54 (2013) 6656–6659

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Biosynthesis and antioxidant activity of 4NRC b-glycoside Kelly C. F. Araújo Cordeiro a, Kênnia R. Rezende a, Boniek G. Vaz b, Wanderson Romão c, Luciano M. Lião b, Eric de Souza Gil a, Valéria de Oliveira a,⇑ a

Faculdade de Farmácia, Universidade Federal de Goiás, Caixa Postal 131, 74.605-220 Goiânia, GO, Brazil Instituto de Química, Universidade Federal de Goiás, Caixa Postal 131, 74.605-220 Goiânia, GO, Brazil c Departamento de Química, Universidade Federal do Espírito Santo, 29075-910 Vitória, ES, Brazil b

a r t i c l e

i n f o

Article history: Received 29 August 2013 Revised 27 September 2013 Accepted 28 September 2013 Available online 3 October 2013 Keywords: Glycosylation 4-Nerolidylcatechol Cunninghamella 4NRC-b-glycoside

a b s t r a c t This Letter describes glycosylation of 4-Nerolidylcatechol (4-NRC) the major secondary metabolite from Pothomorphe peltata and Pothomorphe umbellata showing remarkable antioxidant, antimalarial, antiinflammatory, and anti-HIV activities. One step biosynthesis was catalyzed by Cunninghamella echinulata ATCC 9245 and the reaction was undertaken in PDSM medium at 27 °C, 200 rpm for 96 h. After purification by silica gel flash column chromatography the 4-NRC b-glycoside was identified by ultrahigh resolution mass spectrometry and 1H NMR. The antioxidant activity was evaluated by differential pulse voltammetry. Ó 2013 Elsevier Ltd. All rights reserved.

4-Nerolidylcatechol (4-NRC) is the major secondary metabolite from Pothomorphe peltata and Pothomorphe umbellata traditionally used for the treatment of hepatic disorders in Brazilian Southwest and Amazon area.1 Pharmacological studies have shown remarkable antioxidant,2 antimalarial,3 anti-inflammatory,4 and anti-HIV activities.5 Despite of its promising pharmaceutical profile, the instability often presents a shortcoming for its medical applications. Glycosylation is useful for preparing more stable glycosides from unstable compounds; improving their bioavailability and pharmacological properties. Usually, the glycosylation reactions by classical organic chemistry are conducted in four steps. First, the per-O-acetylation of glucose is carried out, followed by halogenation at anomeric per-Oacetylation of glucose position. Next a Lewis acid catalizes the reaction of the target compound with 2, 3, 4, 6-tetra-O-acetyl-aD-glucopyranosyl bromide and the glycosylated compound is unprotected in the presence of sodium methoxide.6,7 Examples of oxidation, reduction, and glycosylation reactions using filamentous fungi as catalysts have been reported in the literature.8,9 One of the benefits of using the microbial metabolism system, instead of the traditional or chemical synthesis, is the possibility of production of several metabolites or derivatives drugs that are difficult to be synthesized.10 Glycosylation is an example of a reaction that takes several steps in chemical synthesis and via microbial reactions can be done in a single step, also under mild reaction conditions.11,6 ⇑ Corresponding author. Tel.: +55 62 3209 6432; fax: +55 62 3209 6037. E-mail address: [email protected] (V. de Oliveira). 0040-4039/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tetlet.2013.09.133

Cunninghamella is a filamentous fungus typically found in soil and plant material, they are able to metabolize a wide range of xenobiotics in a regio- and stereo-selective manner. Generally, the fungi produce a metabolite by a mode similar to those in mammalian enzyme system.12 It is known that the Cunninghamella has the ability to perform a variety of selective reactions, including phase I (oxidative) and phase II (conjugative) biotransformation.13,14 There are a great number of studies describing the ability of filamentous fungi of the species C. echinulata to biotransform drugs, including aromatic xenobiotics, one of the Cunninghamella strains, Cunninghamella echinulata ATCC 9245, has been an important microorganism to perform b-glycosylation reactions.15,16 Then, the C. echinulata appears as a promising tool for the production of molecules with improved, different, or less toxic activities. Glycoside is probably obtained by the action of fungal glycosidase to a direct reaction using a primary or secondary alcohol as a nucleophile.17 The aim of this study was to perform the glycosylation of 4-NRC (1)18 using the filamentous fungi, C. echinulata ATCC 9245 for further evaluation of similar, new pharmacological activities, and toxicological studies of 4-NRC b-glycoside A spore suspension of Cunninghamella echinulata ATCC 9245 strain (American Type Culture Collections) incubated at 27 °C for 7 days on potato agar, was transferred to liquid culture medium. Incubation was performed in ten 250 mL Erlenmeyer flasks containing 100 mL of culture medium (5 g bacteriological peptone, 5 g soy lecithin, 5 g KH2PO4, 5 g NaCl, 20 g dextrose, and 3 g yeast extract) and grown at 27 ± 1 °C, 200 rpm for 65 h. After the growth of mycelium 1 mL of the ethanolic solution of 1 (25 mg ml1) was

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K. C. F. Araújo Cordeiro et al. / Tetrahedron Letters 54 (2013) 6656–6659 1

HO

2'

3'

HO

2

14

13

1'

3

5

4

6

7

concentrated in vacuum. Purification was performed by silica gel column chromatography (2 cm  20 cm) eluting with ethyl acetate:methanol (95:05). The product 2 (Scheme 1) was characterized by Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS). This technique plays an important role for high accuracy mass measurements at ultrahigh resolution in many scientific and industrial areas.19 Very high accurate mass measurements at the partper-billion (ppb) level on product ions enable high confident determination of the elemental composition as unambiguous assignments of fragment ions. Typically, exact mass measurements at high resolution are used to determine the elemental composition of a compound which is either unknown or the by-products or metabolites of a known compound.20 For characterization by FT-ICR MS, the compound 2 was dissolved in 1 mL of methanol. For ESI in the negative mode, 2 lL of an aqueous solution of 1% ammonium hydroxide was added to the solution and analyzed by 9.4 T Solarix mass spectrometer (Bruker Daltonics, Bremen, Germany). Fig. 1 displays the ESI() FT-ICR mass spectrum of 2. Note the detection of 2 the [MH] anion of m/z 475.27024 with the characteristic cluster of isotopologue ions. Compound 2 was measured with exceptional mass accuracy, 240 ppb of the exact mass. The molecular formula assigned to the ion of m/z 475.27024, C27H39O7, closely matches with the molecular formula of the deprotonated ion of compound 2. This match corroborates the proposed structure for the compound 2. The 1H NMR analyses were obtained on a Bruker Avance III 500 instrument (operating at 500.13 MHz for 1H). NMR spectra of 1 were recorded in CDCl3 and its product in CD3OD using tetramethyl silane (TMS) as an internal standard. Compound 2 (Scheme 1) presented one additional glucose unit characterized by typical 1H NMR signals at d 4.69 (d, J = 7.5 Hz, 1H), 3.87 (dd, J = 12.1, 5.1 Hz, 1H), 3.74 (dd, J = 12.1, 2.4 Hz), 3.50 (dd, J = 9.8, 7.5 Hz, 1H), 3.36 (ddd, J = 7.4, 5.1, 2.4 Hz, 1H) and multiplets in the d 3.433.52 region (Table 1). The glucose b-configuration was confirmed through its coupling constant (J = 7.5 Hz), and its position was determined by 2D NMR and NOE experiments. In the former experiment, a cross-peak between the anomeric hydrogen at d 4.69 and the carbon at d 146.6 (C-30 ) was observed.

15

8

9 10

11

12

6'

4'

5'

4-Nerolidylcatechol (1)

Cunninghamella echinulata 4"

HO

6"

OH HO

ATCC 9245

3" 5"

O

2"

OH

1

1"

O

HO

3'

4'

2'

2 1'

14

13 3

4

5

6

7

15 9

8

10

11

12

6' 5'

4-Nerolidylcatechol-3'-β-O-glycoside (2) Scheme 1. Glycosylation of 4NRC by Cunninghamella echinulata ATCC 9245.

added to each flask. Two controls were performed; the substrate and the microorganism control. The substrate control was carried out with culture medium and substrate under the same incubation conditions; on the other hand the microorganism control was done with microorganism and its culture medium without addition of substrate. Aliquots were withdrawn every 24 h during a period of 96 h. The samples were extracted with ethyl acetate and spotted on 0.25 mm thick silica gel GF254 plates (Whatman) and chromatographed with ethyl acetate:methanol (95:05). The TLC plates were visualized by fluorescence under 254 nm UV light and with iodine steam. The flasks were brought together to conduct the extraction, mycelium was filtrated and extracted with acetone. The filtrate was saturated with NaCl and filtered through CeliteÒ, proceeding by the extraction with ethyl acetate three times. The extracts were Intens. 8 x10 2.5

HO

475.27024 C27H39O7

OH HO

4"

Error = 240 ppb

3"

6"

5"

O

2"

OH

1

1"

O

3'

HO

4'

2'

2.0

2 1'

14

13 3

4

5

6

7

15

8

9 10

11

12

6' 5'

1.5

1.0

0.5

405.05859

419.07426 491.26523

455.05097 0.0

380

400

420

440

460

Figure 1. ESI() FT-ICR MS of 4-NRC b-glycoside.

480

500

m/z

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K. C. F. Araújo Cordeiro et al. / Tetrahedron Letters 54 (2013) 6656–6659

Table 1 H and 13C NMR spectral data for 4-Nerolidylcatechol and 4-Nerolidylcatechol-30 -b-O-glycoside

1

1

HO

HO

3'

4'

2'

2

14

13

15

4"

1'

3

4

5

6

7

8

9 10

11

HO

12

6"

OH HO 3" 5"

6'

O

5'

2"

OH

1

1"

4-Nerolidylcatechol (1)

O

HO

2'

3'

2 1'

14

13 3

4

5

6

15

7

9

8

10

11

12

6'

4'

5'

4-Nerolidylcatechol-3’-β-O-glycoside (2) d1H (multiplicity, J Hz) 5.01 (dd, 17.5, 1.4) 5.06 (dd, 10.8, 1.4) 5.97 (dd, 17.5, 10.8) — 1.75, 1.64 (m) 1.85, 1.77 (m) 5.10 (m) — 1.94 (t, 7.2) 2.04 (dt, 7.8, 7.2) 5.09 (m) — 1.67 (dq, 1.1, 0.4) 1.31 (s) 1.51 (d, 0.8) 1.59 (dq, 1.0, 0.4) — 6.84 (d, 2.3) — — 6.79 (d, 8.4) 6.74 (dd, 8.4, 2.3) — — — — — — — 5.64 (br s)

1a 1b 2 3 4 5 6 7 8 9 10 11 12 13 14 15 10 20 30 40 50 60 1’’ 2’’ 3’’ 4’’ 5’’ 6’’a 6’’b OH 1

H and

13

d13C 111.7 111.7 147.2 43.9 39.9 26.9 124.7 135.2 41.3 25.9 124.5 131.6 25.1 23.3 16.1 17.9 141.1 115.2 143.2 141.4 114.4 119.3 — — — — — — — —

d1H (multiplicity, J Hz) 5.00 (dd, 17.5, 1.4) 5.05 (dd, 10.8, 1.4) 6.02 (dd, 17.5, 10.8) — 1.75, 1.64 (m) 1.85, 1.77 (m) 5.09 (m) — 1.94 (t, 7.2) 2.04 (dt, 7.8, 7.2) 5.07 (m) — 1.65 (dq, 1.1, 0.4) 1.34 (s) 1.49 (d, 1.3) 1.59 (dq, 1.0, 0.4) — 7.17 (d, 2.3) — — 6.77 (d, 8.4) 6.88 (dd, 8.4, 2.3) 4.69 (d, 7.5) 3.50 (dd, 9.8, 7.5) 3.45 (m) 3.46 (m) 3.36 (ddd, 7.4, 5.1, 2.4) 3.74 (dd, 12.1, 2.4) 3.87 (dd, 12.1, 5.1) —

d13C 112.2 112.2 149.1 44.9 42.5 24.4 126.0 135.8 40.8 27.8 125.4 132.1 25.8 25.4 16.2 17.9 140.6 118.3 146.6 146.4 116.9 123.4 105.1 75.2 78.0 71.4 78.6 62.6 62.6 —

C NMR assignments are based on 1H and 1H–13C HMBC spectra at 500 MHz and 125 MHz. Measured in CDCl3 for 1 and CD3OD for 2.

The cross-peaks of H-200 with C-100 and C-300 , H-300 with C-200 , H-400 with C-200 and C-300 , and H-600 with C-400 and C-500 were also observed. The correlation between anomeric hydrogen (H-100 ) and H-20 in the NOE spectrum reinforced the assignment of glycosylation at the C-30 position. In order to evaluate the antioxidant activity of 2 we performed the analysis using differential pulse voltammetry. Compound 2 (Fig. 2) also showed two anodic peaks, the first and second at peak potentials, Ep1a and Ep2a, respectively at 348 mV and 629 mV, and current spikes 17 and 13 lA. The negative shift of more than 200 mV shows that compound 2 results in greater reducing power. Though, the glycosylation process has occurred at 30 hydroxyl group, leading to the modification of the cathecol group to mono phenol pattern, the peak potential, 1a, at Epa <0.4 V is very low, thus having a great electron donor behavior and promising antioxidant activity. In conclusion, we show a promising application of filamentous fungi C. echinulata ATCC 9245, exemplified here by the glycosylation of 1. The differential pulse voltammetry results confirm that 2 maintains the antioxidant activity, which associated with their better solubility and stability can result in improved biological

1a 2a

500 μA

0.0

0.2

0.4

0.6

0.8

1.0

E / V vs (SCE) Figure 2. Differential pulse voltammetry analysis obtained in the solid state for 1 (——) and 2 (- - -) immobilized on a carbon paste electrode of 0.5 mm diameter in phosphate buffer, 0.1 M, pH 7.0. Pulse amplitude of 50 mV, scan rate of 10 mV/s.

activities than its precursor 1. These results were obtained in accordance with the principles of green chemistry, the experimental

K. C. F. Araújo Cordeiro et al. / Tetrahedron Letters 54 (2013) 6656–6659

conditions have little environmental impact, and the solvents are renewable and economical. References and notes 1. Barros, L. F.; Barros, P. S.; Röpke, C. D.; Silva, V. V.; Sawada, T. C. H.; Barros, S. M. B., ; Belfort, R., Jr. Dose-dependent in vitro inhibition of rabbit corneal matrix metalloproteinases by an extract of Pothomorphe umbellata after alkali injury Braz J. Med. Biol. Res. 2007, 40(8), 1129–1132. 2. Desmarchelier, C.; Barros, S.; Repetto, M.; Latorre, L. R.; Kato, M.; Coussio, J.; Ciccia, G. 4-Nerolidylcatechol from Pothomorphe spp. scavenges peroxyl radicals and inhibits Fe(II)-dependent DNA damage Planta Med. 1997, 63, 561–563. 3. Andrade-Neto, V. F.; Pohlit, A. M.; Pinto, A. C. S.; Silva, E. C. C.; Nogueira, K. L.; Melo, M. R. S.; Henrique, M. C.; Amorim, R. C. N.; Silva, L. F. R.; Costa, M. R. F., et al In vitro inhibition of Plasmodium falciparum by substances isolated from Amazonian antimalarial plants Mem. Inst. Oswaldo Cruz 2007, 102(3), 359–365. 4. Núñes, V.; Castro, V.; Murillo, R.; Ponce-Soto, L. A.; Merfort, I.; Lomonte, B. Inhibitory effects of Piper umbellatum and Piper peltatum extracts towards myotoxic phospholipases A 2 from Bothrops snake venoms: isolation of 4nerolidylcatechol as active principle Phytochemistry 2005, 66, 1017–1025. 5. Gustafson, K. R.; Cardellina, J. H.; Mcmahon, J. B.; Pannell, L. K.; Cragg, G. M.; Boyd, M. R. The peltatols, novel hiv-inhibitory catechol derivatives from Pothomorphe peltata J. Org. Chem. 1992, 6, 2809–2811. 6. Mukhopadhyay, B.; Kartha, K. P. R.; Russell, D. A.; Field, R. A. Streamlined synthesis of per-O-cetylated sugars, glycosyl iodides, or thioglycosides from unprotected reducing sugars J. Org. Chem. 2004, 69, 7758–7760. 7. Campo, V. L.; Carvalho, I. Síntese de glicoaminoácidos de interesse biológico Quim. Nova 2008, 31, 1027–1033. 8. Chen, G.; Yang, X.; Zhai, X.; Yang, M. Microbial transformation of 20(S)protopanaxatriol by Absidia corymbifera and their cytotoxic activities against two human prostate cancer cell lines Biotechnol. Lett. 2013, 35(1), 91–95. 9. Yang, X.; Hou, J.; Liu, D.; Deng, S.; Wang, Z. B.; Kuang, H. X.; Wang, C.; Yao, J. H.; Liu, K. X.; Ma, X. C. Biotransformation of isoimperatorin by Cunninghamella blakeslean AS 3.970 J. Mol. Catal. B-Enzym. 2013, 88, 1–6. 10. Sun, X.-H.; Man, F.; Pang, L.-Y.; Gao, G.-H.; Li, X.-Q.; Qi, X.-L.; Li, F.-M. Fungal biotransformation of mosapride by Cunninghamella elegans J. Mol. Catal. BEnzym. 2009, 59(1–3), 82–89. 11. Shibazaki, M.; Yamaguchi, H.; Sugawara, T.; Suzuki, K.; Yamamoto, T. Microbial glycosylation and acetylation of brefeldin A J. Biosci. Bioeng. 2003, 96(4), 344–348. 12. Rao, G. P.; Davis, P. J. Biotransformations of HP 749 (Besipirdine) Using Cunninghamella elegans Drug Metab. Dispos. 1997, 25(6), 709–715. 13. Zhang, D.; Yang, Y.; Leakey, J. E. A.; Cerniglia, C. E. Phase I and phase II enzymes produced by Cunninghamella elegans for the metabolism of xenobiotics FEMS Microbiol. Lett. 1996, 138, 221–226.

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14. Smith, R. V.; Rosazza, J. P. Microbial Models of Mammalian Metabolism J. Pharm. Sci. 1975, 64(11), 1737–1759. 15. Zi, J.; Valiente, J.; Zeng, J.; Zhan, J. Metabolism of quercetin by Cunninghamella elegans ATCC 9245 J. Biosci. Bioeng. 2011, 112(4), 360–362. 16. Cerniglia, C. E.; Freeman, J. P.; Mitchum, R. K. Glucuronide and sulfate conjugation in the fungal metabolism of aromatic hydrocarbons Appl. Environ. Microb. 1982, 43(5), 1070–1075. 17. Lustosa, K. R. M. D.; Menegatti, R.; Braga, R. C.; Lião, L. M.; de Oliveira, V. Microbial b-glycosylation of entacapone by Cunninghamella echinulata ATCC 9245 J. Biosci. Bioeng. 2012, 113(5), 611–613. 18. P. umbellata roots were purchased from a herbarium supply company (Flora Medicinal, Vale do Ribeira, SP, Brazil). The certified roots (1 kg) were dried, milled, and exhaustively extracted by ultrasonication for 1 h with dichloromethane, yielding a crude extract (184.2 g) after solvent evaporation. The crude extract was initially chromatographed (TLC; SiO2; dichloromethane:acetone 99:1) in parallel with previous authentic P. umbellata extracts showing a characteristic composition profile. Next, a flash-filtering silica gel 60 chromatography (0.063–0.2 mm, Merck, USA) column was set (90  330 mm) for running an hexane:ethyl acetate gradient elution. The apolar fractions that were obtained were recombined and submitted to a flash chromatography silica gel (0.063–0.2 mm, Merck) column (90  330 mm) on a Buchi Sepacore prep-MPLC System (Büchi Labortechnik, Flawil, Switzerland). The sample constituents were eluted with dichloromethane:cyclohexane:methanol (5:2:1) on a Sephadex LH-20 (GE Healthcare Bio-Sciences, Tokyo, Japan) column (49  920 mm) at a flow rate of 40.0 mL/min and monitored at 220/280 nm (Büchi UV detector model C-660, Flawil, Switzerland). Fractions containing 4-NC were detected by thin layer chromatography (TLC) analysis and further purified on a Sephadex LH-20 GE column (49  920 mm; 220 nm) to yield a pale yellow oil (1.2 g) that was chemically identified as 4-NC by 1H and 13C NMR. Sample purity was assessed with a peak purity analysis using high-performance liquid chromatography with a photodiode array detector (HPLC-PDA). The chromatographic conditions were as follows: RP-C18 Phenomenex column (150  4.6 mm; 4 lm) using ACN:MeOH:H2O 54:20:26 as the mobile phase and a flow rate of 1.0 mL/min. Sample purity was assessed at three peak spectra (upslope, downslope, and apex) showing values greater than or equal to 0.99. 19. Vaz, B. G.; Abdelnur, P. V.; Rocha, W. F. C.; Gomes, A. O.; Pereira, R. C. L. Predictive Petroleomics: Measurement of the Total Acid Number by Electrospray Fourier Transform Mass Spectrometry and Chemometric Analysis Energy Fuels 2013, 27, 1873–1880. 20. Garrett, R.; Vaz, B. G.; Hovell, A. M. C.; Eberlin, M. N.; Rezende, C. M. Arabica and robusta coffees: identification of major polar compounds and quantification of blends by direct-infusion electrospray ionization ‘mass spectrometry’ J. Agric. Food Chem. 2012, 60, 4253–4258.