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New #-Pyridones with Quorum Sensing Inhibitory Activity from DiversityEnhanced Extracts of a Marine Algae-Derived Streptomyces sp. Yuqi Du, Jian Sun, Qianhong Gong, Yi Wang, Peng Fu, and Weiming Zhu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05330 • Publication Date (Web): 05 Feb 2018 Downloaded from http://pubs.acs.org on February 8, 2018

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Journal of Agricultural and Food Chemistry

New α-Pyridones with Quorum Sensing Inhibitory Activity from Diversity-Enhanced Extracts of a Marine Algae-Derived Streptomyces sp. Yuqi Du,† Jian Sun,† Qianhong Gong,† Yi Wang,† Peng Fu†,* and Weiming Zhu†,‡,* †

Key Laboratory of Marine Drugs, Ministry of Education of China, School of Medicine and Pharmacy, Ocean

University of China, Qingdao 266003, China ‡

Laboratory for Marine Drugs and Bioproducts of Qingdao National Laboratory for Marine Science and

Technology; Qingdao 266003, China

*

To whom correspondence should be addressed. Tel: +86-532-82031268. Fax: +86-532-82031268. E-mail:

[email protected], [email protected]

1

ACS Paragon Plus Environment


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ABSTRACT: Four new α-pyrones (1−4) and eight known analogues (5−12) were identified from the secondary metabolites of Streptomyces sp. OUCMDZ-3436 derived from the marine green algae, Enteromorpha prolifera. Seven new α-pyridones (14−20) were constructed by diversity-oriented synthesis, which has been an effective approach to expanding the chemical space of natural-product-like compounds. Compounds 16, 17, 19 and 20 were found to have inhibitory effect on the gene expression controlled by quorum sensing in Pseudomonas aeruginosa QSIS-lasI.

KEYWORDS:

Pyridone,

QS-inhibition,

diversity-enhanced

extracts,

Streptomyces

sp.,

Enteromorpha prolifera

2


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■ INTRODUCTION A large number of new bioactive natural products have been isolated marine-derived microorganisms.1,2 Most of these microorganisms were isolated from segments, corals, and sponges.3,4 In recent years, the secondary metabolites of microbes living with marine algae have attracted attentions of researchers.5,6 Numerous biologically active compounds have been discovered from this special resource.7,8 Green tide is a kind of harmful algae which is common in the coastal countries of the world. Enteromorpha prolifera is one of the most common algae to cause green tide. The frequent outbreak of this algae in Qingdao provided an opportunity for us to research the secondary metabolites of microbes associated with it. Our previous studies revealed several bioactive natural products from E. prolifera associated strains, 9–11 such as wailupemycins. As part of our studies to find new special structures from microbes associated with Enteromorpha for further investigation on chemical diversity-enhanced synthesis, we identified four new α-pyrones, germicidin K−N (1−4), as well as the known germicidin A (5),12–16germicidin B (6),12,13 germicidin C (7),14,15 germicidin D (8),14 germicidin H (9),17 germicidin I (10),17 germicidin J (11),17 and isogermicidin A (12),15 from the metabolites of Streptomyces

sp. OUCMDZ-3436. Although we

did not find any activities of these α-pyrones in the antimicrobial and quorum sensing inhibitory bioassays, we found this skeleton could be easily transformed into pyridin-2(1H)-one, which have been verified to have a variety of biological activities.18–26 Thus, we utilized the approach of “diversity-enhanced extracts” 27,28 to obtain α-pyridones from the α-pyrones-containing fraction. The fraction in ammonia solution was refluxed and the crude products were purified to afford seven new α-pyridones (14−20). Compounds 16, 17, 19 and 20 could inhibit the gene expression controlled by quorum sensing (QS) in Pseudomonas aeruginosa QSIS-lasI biosensor at a dose of 6.35 µg per well. ■ MATERIALS AND METHODS 3



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General Experimental Procedures. NMR spectra were measured on a Varian System 500 spectrometer in the solution of DMSO-d6 (δH/C 2.50/39.52), CD3OD (δH/C 3.31/49.00) or CHCl3 (δH/C 7.26/77.16). HRESIMS data were collected on a LC mass spectrometer, Q-TOF ULTIMA GLOBAL GAA076. UV data were measured by a 2487 detector of Waters. ECD spectra were recorded by the JASCO J-815 spectrometer. IR spectra were obtained from the Nicolet NEXUS 470 spectrophotometer using KBr discs as blank. Optical rotations were measured by a JASCO P-1020 digital polarimeter, using a halogen lamp as light source (589 nm). An ODS column (YMC-pack ODS-A, 5 µm, 250 × 4.6 mm, 1.0 mL/min) and another column with some packing (YMC-pack ODS-A, 5 µm, 250 × 10 mm, 4.0 mL/min) were used for analytical HPLC and semi-preparative HPLC, respectively. Column chromatography (CC) were performed over silica gel (200−300 mesh, Marine Chemical Factory of Qingdao) and Sephadex LH-20 (Amersham Biosciences), respectively. TLC plates were pre-coated with silica gel GF254 (10−40 µm). Collection and Phylogenetic Analysis of Strain OUCMDZ-3436. The bacterial strain OUCMDZ-3436 was isolated from the green alga, Enteromorpha prolifera, which was collected from Zhanqiao Beach, Qingdao, Shandong Province, China, in August 2012. Suspension of the E. prolifera shatters was deposited on an ISP2 agar plate (10 g/L malt powder, 4 g/L glucose, 4 g/L yeast extract, 20 g/L agar, in sea water), nystatin (100 µg/mL) was used as a fungi inhibitor in the medium. Then, the culture was incubated at 28 °C for 8 d and bacterial colonies were selected and streaked to purity using the same agar media. 16S rRNA gene sequence helped to identify the genus of OUCMDZ-3436 (GenBank access no. MG437309). Cultivation and Extraction of OUCMDZ-3436. Bacterium OUCMDZ-3436 was cultured in three Erlenmeyer flasks (500 mL) each equipped with 150 mL of a seawater-based medium (10 g/L starch, 3 g/L beef extract, 20 g/L glucose, 10 g/L peptone, 10 g/L yeast extract, 0.5 g/L MgSO4, 2 4


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g/L CaCO3, 0.5 g/L KH2PO4, pH 7.5−8.0, in seawater) and shaken for three days (28 ± 0.5°C, 180 rpm), which were used as the seed liquid. The seed liquid (3 mL) was then directly inoculated into an Erlenmeyer flask (500 mL) equipped with 150 mL of the same medium as above, cultivated at 28 ± 0.5 °C for 9 days with shaking at 180 rpm. By extracting the whole broth (100 L) with equal volumes of EtOAc repeatedly, 30 g crude extract was collected in total. Purification. The crude extract (30 g) was subjected to a silica gel VLC column and eluted gradiently with CH2Cl2–petroleum ether (50−100%) and then MeOH–CH2Cl2 (0−50%), and eight fractions (Fr.1−Fr.8) were obtained. Fr.4 (3 g) was subjected to Sephadex LH-20 eluting with MeOH to give six fractions (Fr.4.1−Fr.4.6). Fr.4.4 (105 mg) was further prepared by semi-preparative HPLC (40% MeOH–H2O, YMC-pack ODS-A column) to yield compounds 6 (4.0 mg, tR = 19.2 min), 7 (12.0 mg, tR = 19.6 min), 10 (9.0 mg, tR = 21.5 min), 5 (6.5 mg, tR = 29.3 min), 12 (3.0 mg, tR = 30.5 min) and 11 (2.0 mg, tR = 32.0 min). Fr.4.5 (72 mg) was further prepared by semi-preparative HPLC (40% MeOH–H2O, YMC-pack ODS-A column), and compounds 8 (7.0 mg, tR = 13.3 min) and 9 (4.5 mg, tR = 14.6 min) were obtained. Fraction 5 (4 g) was separated into six fractions (Fr.5.1−Fr.5.6) on Sephadex LH-20, eluting with MeOH. Fr.5.1 (207 mg) was then fractionated by VLC on silica gel (200−300 mesh). A step gradient of EtOAc–petroleum ether (20−100%) was used for eluting, and eight subfractions (Fr.5.1.1–Fr.5.1.8) were collected. Fr.5.1.7 (42 mg) was further subjected to semi-preparative HPLC (30% MeOH–H2O, YMC-pack ODS-A column), and compounds 2 (23.0 mg, tR = 14.3 min) and 3 (5.0 mg, tR = 15.3 min) were obtained. Fr.5.1.8 (21 mg) was subjected to the semi-preparative HPLC (30% MeOH–H2O, YMC-pack ODS-A column), and then compound 1 (8.0 mg, tR = 12.5 min) was obtained. Fr.5.2 (172 mg) was fractioned into Fr.5.2.1–Fr.5.2.4 by VLC on silica gel (200−300 mesh), eluting with a gradient mixture of EtOAc– petroleum ether (30−100%). Fr.5.2.3 (25 mg) was prepared by HPLC (30% MeOH–H2O, YMC-pack 5



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ODS-A column) to yield compound 4 (4.0 mg, tR = 25.7 min). Germicidin K (1): Pale-yellow powder; UV (MeOH) λmax (log ε) 285 (2.41), 207 (2.37) nm; IR (KBr) νmax 3279, 2973, 2924, 1676, 1588, 1416 cm−1; 1H and 13C NMR data, Table 1; HRESIMS m/z 199.0960 [M + H]+ (calcd for C10H15O4, 199.0965). Germicidin L (2): Colorless needles; m.p. 113−114 °C; [α]D25 +10.6 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 284 (2.29), 207 (2.27) nm; ECD (0.0038 M, MeOH) λmax (∆ε) 293 (+2.4) nm; IR (KBr) νmax 3181, 2976, 2928, 1680, 1591, 1414cm−1; 1H and

13

C NMR data, Table 1; HRESIMS m/z

199.0963 [M + H]+ (calcd for C10H15O4, 199.0965). Germicidin M (3): Colorless needles; m.p. 171−172 °C; [α]D25 +43.7 (c 0.1 MeOH); UV (MeOH) λmax (log ε) 284 (2.20), 207 (2.27) nm; ECD (0.0025 M, MeOH) λmax (∆ε) 290 (+11.8) nm; IR (KBr) νmax 3095, 2973, 2918, 1677, 1593, 1420cm−1; 1H and

13


199.0960 [M + H]+ (calcd for C10H15O4, 199.0965). Germicidin N (4): Colorless needles; m.p. 116−117 °C; [α]D25 +12.9 (c 0.1 MeOH); UV (MeOH) λmax (log ε) 285 (2.45), 207 (2.33) nm; ECD (0.0024 M, MeOH) λmax (∆ε) 297 (+4.5) nm; IR (KBr) νmax 3392, 2974, 2935, 1676, 1582, 1415 cm−1; 1H and

13


213.1118 [M + H]+ (calcd for C11H17O4, 213.1121). Methylation of 2. 2.0 mg Na2CO3 was added into a solution of 2 (3.0 mg) in DMF (anhydrous, 1 mL). 10 µL of CH3I was added into the solution after the above solution was stirred at –3 °C for 0.5 h. The reaction mixture was stirred continuously for 6 h at room temperature (rt). To quench the reaction, a saturated solution of NH4Cl (2.0 mL) was added. Then EtOAc (3 × 3.0 mL) was used to extract the reaction product. It was further purified by HPLC (40% MeOH–H2O, 0.5‰ trifluoroacetic acid, YMC-pack ODS-A column) to afford compound 13 (2.2 mg, tR = 11.2 min, 69% yield). 6


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4-O-Methylgermicidin L (13): pale-yellow powder; 1H NMR (500 MHz, CD3OD) δ 6.45 (s, 1H, H-5), 3.95 (dq, J = 6.4, 6.4 Hz, 1H, H-8); 3.95 (s, 3H, CH3O-4), 2.62 (dq, J = 6.4, 7.0 Hz, 1H, H-7), 1.86 (s, 3H, H-11), 1.28 (d, J = 7.0 Hz, 3H, H-10), 1.15 (d, J = 6.4 Hz, 3H, H-9); HRESIMS m/z 213.1124 [M + H]+ (calcd for C11H17O4, 213.1121). Preparation of the (S)- and (R)-MTPA Esters of 13 by Modified Mosher′s Method. To a solution of compound 13 (1.0 mg) in dry CH2Cl2 (500 µL) and Et3N (100 µL), (R)-MTPACl (10 µL) was added quickly. The mixture was stirred for 6 h at rt, and then added with 4 mL of H2O. The reaction product was extracted with CH2Cl2 three times (5 mL for each). Then, the organic layer was collected and subjected to semi-preparative HPLC (70% MeOH–H2O, YMC-pack ODS-A column) to afford the (S)-MTPA ester 13a (1.2 mg, tR 12.1 min). By the same method, the reaction of 13 (1.0 mg) with (S)-MTPACl (10 µL) afforded (R)-MTPA ester 13b (0.8 mg, tR 12.3 min). 4-O-Methylgermicidin L (S)-MTPA ester (13a): 1H NMR (500 MHz, CDCl3) δ 5.87 (s, 1H, H-5), 5.39 (dq, J = 6.0, 6.4 Hz, 1H, H-8); 3.72 (s, 3H, CH3O-4), 2.79 (dq, J = 6.0, 7.1 Hz, 1H, H-7), 1.89 (s, 3H, H-11), 1.37 (d, J = 6.4 Hz, 3H, H-9), 1.21 (d, J = 7.1 Hz, 3H, H-10); ESIMS m/z 428.9 [M + H]+. 4-O-Methylgermicidin L (R)-MTPA ester (13b): 1H NMR (500 MHz, CDCl3) δ 5.97 (s, 1H, H-5), 5.39 (dq, J = 6.0, 6.3 Hz, 1H, H-8); 3.73 (s, 3H, CH3O-4), 2.84 (dq, J = 6.0, 7.1 Hz, 1H, H-7), 1.92 (s, 3H, H-11), 1.34 (d, J = 6.3 Hz, 3H, H-9), 1.28 (d, J = 7.1 Hz, 3H, H-10); ESIMS m/z 428.9 [M + H]+. Preparation of α-Pyridones (14−20). A mixture of fraction 4 (300 mg) and 28% ammonium solution (10 mL) was stirred and refluxed at 100 °C for 40 h till the pyrones were consumed up. The reaction mixture was concentrated by the rotary evaporator to afford the crude products, Fr.4N. Fr.4N (282 mg) was subjected to a Sephadex LH-20 column eluted with MeOH, and six subfractions 7



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(Fr.4N1−Fr.4N6) were obtained. Fr.4N5 (38 mg) was then purified by semi-preparative HPLC (40% MeOH/H2O, YMC-pack ODS-A column) to yield compounds 18 (6.6 mg, tR = 10.1 min), 19 (3.4 mg, tR = 11.6 min) and 20 (2.8 mg, tR = 12.1 min). Fr.4N6 (46 mg) was prepared using semi-preparative HPLC (30% MeOH/H2O, YMC-pack ODS-A column) to yield compounds 14 (2.3 mg, tR = 8.0 min), 15 (8.1 mg, tR = 10.0 min), 16 (2.2 mg, tR = 11.1 min) and 17 (4.7 mg, tR = 16.5 min). 6-Ethyl-4-hydroxy-3-methylpyridin-2(1H)-one (14): Pale-yellow powder; UV (MeOH) λmax (log ε) 281 (2.23), 206 (2.34) nm; IR (KBr) νmax 3155, 2976, 2938, 1681, 1618, 1206, 1134 cm−1; 1H and 13C NMR data, Table 2; HRESIMS m/z 154.0863 [M + H]+ (calcd for C8H12NO2, 154.0863). 4-Hydroxy-6-isopropyl-3-methylpyridin-2(1H)-one (15): Pale-yellow powder; UV (MeOH) λmax (log ε) 280 (2.25), 206 (2.38) nm; IR (KBr) νmax 3108, 2967, 2932, 1641, 1571, 1430, 1127 cm−1; 1

H and 13C NMR data, Table 2; HRESIMS m/z 168.1021 [M + H]+ (calcd for C9H14NO2, 168.1019). 4-Hydroxy-3-methyl-6-propylpyridin-2(1H)-one (16): Pale-yellow powder; UV (MeOH) λmax

(log ε) 281 (2.18), 207 (2.30) nm; IR (KBr) νmax 3111, 2963, 2933, 1677, 1642, 1572, 1126 cm−1; 1H and 13C NMR data, Table 2; HRESIMS m/z 168.1021 [M + H]+ (calcd for C9H14NO2, 168.1019). 3-Ethyl-4-hydroxy-6-isopropylpyridin-2(1H)-one (17): Pale-yellow powder; UV (MeOH) λmax (log ε) 280 (2.35), 209 (2.36) nm; IR (KBr) νmax 3110, 2971, 2936, 1606, 1569, 1427, 1132 cm−1; 1H and 13C NMR data, Table 2; HRESIMS m/z 182.1175 [M + H]+ (calcd for C10H16NO2, 182.1176). (S)-6-(sec-Butyl)-3-ethyl-4-hydroxypyridin-2(1H)-one (18): Pale-yellow powder; [α]D25 +12.2 (c 0.1 MeOH); UV (MeOH) λmax (log ε) 282 (1.93), 205 (2.21) nm; IR (KBr) νmax 3423, 2968, 2933, 1682, 1610, 1430, 1131 cm−1; 1H and

13

C NMR data, Table 3; HRESIMS m/z 196.1336 [M + H]+

(calcd for C11H18NO2, 196.1332). 4-Hydroxy-6-isobutyl-3-methylpyridin-2(1H)-one (19): Pale-yellow powder; UV (MeOH) λmax (log ε) 284 (2.16), 206 (2.36) nm; IR (KBr) νmax 3106, 2960, 2924, 1607, 1571, 1427, 1127 cm−1; 1H 8


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and 13C NMR data, Table 3; HRESIMS m/z 182.1176 [M + H]+ (calcd for C10H16NO2, 182.1176). (S)-6-(sec-Butyl)-4-hydroxy-3-methylpyridin-2(1H)-one (20): Pale-yellow powder; [α]D25 +13.0 (c 0.4 MeOH); UV (MeOH) λmax (log ε) 281 (2.29), 206 (2.33) nm; IR (KBr) νmax 3422, 2968, 2929, 1607, 1431, 1125 cm−1; 1H and 13C NMR data, Table 3; HRESIMS m/z 182.1174 [M + H]+ (calcd for C10H16NO2, 182.1176). X-ray Crystal Data of Compounds 2−4. Colorless crystals of 2−4 were obtained in MeOH from the slow volatilization of the solvent. All of the crystal data were obtained on a Bruker Smart-1000 CCD area detector diffractometer with graphite monochromated Cu-Kα radiation (λ = 1.54178 Å). Structures were expanded using full-matrix least-squares difference Fourier techniques and solved by direct methods (SHELXS-97). The deposited numbers of compounds 2−4 in the Cambridge Crystallographic Data Centre are 1575276, 1579274 and 1579277, respectively. Crystal Data for Compound 2. C10H14O4, 293 (2) K, orthorhombic. Space group: P212121 with a = 15.4492 (5) Å, b = 6.8778 (3) Å, c = 10.0201 (4) Å, V = 1064.70 (7) Å3. Z = 4, Dcalcd = 1.237 mg/m3, µ = 0.798 mm−1, and F (000) = 424. Crystal size: 0.34 × 0.23 × 0.18 mm3. Reflections collected/unique: 7004/1863 [R(int) = 0.0268]; Final R indices [I > 2 sigma (I)]: R1 = 0.0347, wR2 = 0.0930. Absolute structure parameter: 0.0 (2). Crystal Data for Compound 3. C10H14O4, 293 (2) K, orthorhombic. Space group: P212121 with a = 15.2361 (8) Å, b = 6.9936 (4) Å, c = 9.8333 (5) Å, V = 1047.79 (10) Å3. Z = 4, Dcalcd = 1.257 mg/m3, µ = 0.811 mm−1, and F (000) = 424. Crystal size: 0.35 × 0.30 × 0.14 mm3. Reflections collected/unique: 3327/1756 [R(int) = 0.0150]; Final R indices [I > 2 sigma (I)]: R1 = 0.0359, wR2 = 0.0956. Absolute structure parameter: −0.1 (2). Crystal Data for Compound 4. C11H16O4, 293 (2) K, monoclinic. Space group: P212121 with a = 14.7249 (5) Å, b =10.7533 (6) Å, c = 15.1127 (5) Å, V = 2392.96 (18) Å3. Z = 8, Dcalcd = 1.178 9



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mg/m3, µ = 0.741 mm−1, and F (000) = 912. Crystal size: 0.35 × 0.27 × 0.18 mm3. Reflections collected/unique: 7627/7627 [R(int) = 0.0000]; Final R indices [I > 2 sigma (I)]: R1 = 0.0512, wR2 = 0.1198. Absolute structure parameter: 0.0 (2). ■ RESULTS AND DISCUSSION Germicidin K (1) was obtained as a pale-yellow powder. HRESIMS spectrum showed the molecular ion peak at m/z 199.0960 [M + H] +, indicating the molecular formula of C10H14O4. The 13

C NMR spectrum showed ten signals of carbon including three methyl carbons, one sp3 methylene

carbon, one sp2 methine group, and five non-protonated carbons (Table 1). Five carbon signals at δC 169.1, 167.6, 161.8, 103.8, and 99.4 and the proton signal at δH 6.09 (Table 1), together with the correlative signals between H-5 (δH 6.09) and C-4 (δC 167.6), C-6 (δC 161.8) in the HMBC spectrum suggested the 4-hydroxy-2-pyrone skeleton (Figure 1). The remaining NMR signals and the HMBC correlative signals of H2-7 (δH 2.60) to C-8 (δC 71.0) and C-9 (δC 29.4) as well as H3-9 (δH 1.26) to C-10 (δC 29.4) constituted a methyl group and a 2-hydroxyisobutyl moiety. Basing on the HMBC correlative signals of H3-11 (δH 1.86) to C-2 (δC 169.1), C-3 (δC 99.4) and C-4 (Figure 1), the methyl group was connected to C-3 (δC 99.4). Basing on the HMBC correlative signals of H2-7 to C-5 (δC 103.8) (Figure 1), the 2-hydroxyisobutyl fragment should be connected to C-6 (δC 161.8). Therefore, 4-hydroxy-6-(2-hydroxy-2-methylpropyl)-3-methyl-2H-pyran-2-one was confirmed to be the structure of compound 1. Germicidin L (2) was obtained as colorless needles. HRESIMS spectrum showed the molecular ion peak at m/z 199.0960 [M + H] + (C10H14O4), indicating an isomer of 1. By comparing its 13C and 1

H NMR spectra (Table 1) with 1, it could be found that two methine signals at δC/H 69.9/3.92 and

46.8/2.52 in 2 replaced the corresponding non-protonated carbon signal at δC 71.0 and methylene signals at δC/H 48.0/2.60 in 1. These changes along with the COSY correlative signals of 10


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H3-10/H-7/H-8/H3-9 (Figure 1) supported that the 2-hydroxy-sec-butyl group in 2 replaced the 2-hydroxyisobutyl in 1. The X-ray crystal of 2 (Cu Kα, Flack parameter: 0.0 (2)) confirmed the structure and the 7S, 8R configuration (Figure 2). We also adapted the Mosher's modified method to confirm the absolute configuration of 2. In order to avoid the effect of 4-OH, the 4-O-methyl derivative (13) was prepared. Then the corresponding (R)- and (S)-MTPA esters (13b and 13a) of 13 were obtained by reacting with (S)- and (R)-MTPA chloride, respectively. The chemical shift differences of 13a and 13b (∆δS−R) in the 1H NMR spectra helped to define the 8R configuration (Figure

1).

Thus,

compound

2

was

clearly

identified

as

4-hydroxy-6-((2S,3R)-3-hydroxybutan-2-yl)-3-methyl-2H-pyran-2-one. Germicidin M (3) was obtained as colorless needles. The molecular ion peak at m/z 199.0960 [M + H] + (C10H14O4) in the HRESIMS spectrum indicated that it was an isomer of 1 and 2. The 1D NMR spectrum was similar to 2 except for some tiny differences in the hydroxy-sec-butyl group, indicating 3 as the epimer of 2. The data of 2D NMR (Figure 1) further confirmed the structure of 3. The X-ray diffraction of single crystal (Cu Kα, Flack parameter: −0.1 (2)) suggested the 7S, 8S configuration (Figure 2), that is 8-epimer of 2. Germicidin N (4) was confirmed to have the molecular formula C11H16O4 (m/z 213.1118 [M + H]+), with one CH2 more than compound 2. By comparing its 13C and 1H NMR spectra with those of 2, it could be found that the methyl group in 2 was replaced by an ethyl group in 4. This change could be confirmed by the COSY correlation of H2-11/H3-12 and the HMBC correlative signals of H2-11 to C-2/C-3 (Figure 1). The X-ray diffraction of single crystal (Cu Kα, Flack parameter: 0.0 (2)) suggested the 7S, 8R configuration (Figure 2). Thus, compound 4 was identified as the derivative of 2 with 2-ethyl replacement of 2-methyl. Compounds 1−12 showed no antibacterial activity against fifteen pathogenic organisms, 11



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Escherichia coli ATCC 11775, S. aureus subsp. aureus ATCC 43300, Bacillus subtilis CGMCC 1.3376, B. cereus ATCC 14579, Pseudomonas aeruginosa ATCC10145, Staphylococcus aureus ATCC 6538, Clostridium perfringens CGMCC 1.0876, V. vulnificus ATCC 27562, Candida albicans ATCC 10231, C. glabrate ATCC 2001, Edwardsiella tarda ATCC 15947, Vibrio parahaemolyticus ATCC 17802, V. alginolyticus, V. splendidus, and Pseudoalteromonas nigrifaciens. And no QS inhibitory activity for these compounds was detected. In order to explore the potential of these α-pyrones, we proposed to use a simple method to increase the chemical diversity, which could provide more natural-product-like compounds for further biological activity study. In view of the structural feature of pyrones, we decided to utilize the amination to transform α-pyrones into α-pyridones, an important class of active compounds. HPLC-UV revealed that the α-pyrone analogues were mainly concentrated in fraction 4. So, this fraction was chosen to react with ammonia solution under reflux. Then the products were purified and identified as α-pyridones 14−20 (Figure 3) from their NMR and HRESIMS data (Tables 2 and 3). By comparing the specific rotation values of 18 ([α]D25 +12.2) and 20 ([α]D25 +13.0) with those of known germicidin A (5) ([α]D25 +10.6)24 and germicidin C (7) ([α]D25 +17.5),24 the absolute configurations of 18 and 20 were both confirmed. Thus, the structures of 14−20 were identified as 6-ethyl-4-hydroxy-3-methylpyridin-2(1H)-one

(14),

4-hydroxy-6-isopropyl-3-methylpyridin-2(1H)-one

(15),

4-hydroxy-3-methyl-6-propylpyridin-2(1H)-one

(16),

3-ethyl-4-hydroxy-6-isopropylpyridin-2(1H)-one

(17),

(S)-6-(sec-butyl)-3-ethyl-4-hydroxypyridin-2(1H)-one

(18),

4-hydroxy-6-isobutyl-3-methylpyridin-2(1H)-one

(19),

and

(S)-6-(sec-butyl)-4-hydroxy-3-methylpyridin-2(1H)-one (20), respectively. 12


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When tested by an agar diffusion method, compounds 16, 17, 19 and 20 showed inhibition of gene expression controlled by QS in Pseudomonas aeruginosa QSIS-lasI biosensors at a minimum dosage of 6.35 µg/well (Figure S2), although no antimicrobial activity for compounds 14−20 was observed. ■ ASSOCIATED CONTENT Supporting Information. 1H, 13C and 2D NMR spectra of compounds 1−4; 1H and spectra of compounds 14−20; 1H NMR spectrum of compound 13;

13

13

C NMR

C and 1H NMR data of

compounds 5−12; ECD curves of compounds 2−5 and 7; Bioassay protocols used in the experiment. The Supporting Information is available free of charge on the ACS Publications website at DOI: ■ AUTHOR INFORMATION Corresponding Author *

Tel:

+86-532-82031268.

Fax:

+86-532-82031268.

E-mail:

[email protected],

[email protected] Notes The authors declare no competing financial interest. ■ ACKNOWLEDGEMENT This work was financially supported by the grants from the NSFC (Nos. 41376148, 81561148012 & 81373298), from the NSFC-Guangdong Joint Fund for Key Projects (Nos. U1501221), from the NSFC-Shandong Joint Fund for Marine Science Research Centers (No. U1606403), and from the Special Fund for Marine Scientific Research in the Public Interest of China (No. 201405038). ■ REFERENCES (1) Lu, X.; Cao, X.; Liu, X.; Jiao, B. Marine microbes-derived anti-bacterial agents. Mini Rev. Med. 13



Page 14 of 21

Chem. 2010, 10, 1077−1090. (2) Corinaldesi, C.; Barone, G.; Marcellini, F.; Dell'Anno, A.; Danovaro, R. Marine microbial-derived molecules and their potential use in cosmeceutical and cosmetic products. Mar. Drugs 2017, 15, 118. (3) Blunt, J. W.; Copp, B. R.; Keyzers, R. A.; Munro, M. H. G.; Prinsep, M. R. Marine natural products. Nat. Prod. Rep. 2015, 32, 116−211. (4) Blunt, J. W.; Copp, B. R.; Keyzers, R. A.; Munro, M. H. G.; Prinsep, M. R. Marine natural products. Nat. Prod. Rep. 2016, 33, 382−431. (5) Krohn, K.; Dai, J.; Flörke, U.; Aust, H. J.; Dräger, S.; Schulz, B. Botryane Metabolites from the fungus Geniculosporium sp. isolated from the marine red alga Polysiphonia. J. Nat. Prod. 2005, 68, 400−405. (6) Nazir, M.; El Maddah, F.; Kehraus, S.; Egereva, E.; Piel, J.; Brachmann, A. O.; König, G. M. Phenalenones: insight into the biosynthesis of polyketides from the marine alga-derived fungus Coniothyrium cereale. Org. Biomol. Chem. 2015, 13, 8071−8079. (7) Wang, S.; Li, X. M.; Teuscher, F.; Li, D. L.; Diesel, A.; Ebel, R.; Proksch, P.; Wang, B. G. Chaetopyranin, a benzaldehyde derivative, and other related metabolites from Chaetomium globosum, an endophytic fungus derived from the marine red alga Polysiphonia urceolata. J. Nat. Prod. 2006, 69, 1622−1625. (8) Jo, M. J.; Bae, S. J.; Son, B. W.; Kim, C. Y.; Kim, G. D. 3,4-dihydroxyphenyl acetic acid and (+)-epoxydon isolated from marine algae-derived microorganisms induce down regulation of epidermal growth factor activated mitogenic signaling cascade in Hela cells. Cancer Cell Int. 2013, 13, 49. (9) Chen, Z.; Hao, J.; Wang, L.; Wang, Y.; Kong, F.; Zhu, W. New α-glucosidase inhibitors from 14


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marine algae-derived Streptomyces sp. OUCMDZ-3434. Sci. Rep. 2016, 6, 20004. (10) Liu, H.; Chen, Z.; Zhu, G.; Wang, L.; Du, Y.; Wang, Y.; Zhu, W. Phenolic polyketides from the marine alga-derived Streptomyces sp. OUCMDZ-3434. Tetrahedron 2017, 73, 5451−5455. (11) Sun, K.; Zhu, G.; Hao, J.; Wang, Y.; Zhu, W. Chemical-epigenetic method to enhance the chemodiversity of the marine algicolous fungus, Aspergillus terreus OUCMDZ-2739. Tetrahedron 2018, 74, 83−87. (12) Petersen, F.; Zahner, H. Germicidin, an autoregulative germination inhibitor of Streptomyces viridochromo genes NRRL B-1551. J. Antibiot. 1993, 46, 1126−1138. (13) Xu, Z.; Ding, L.; Hertweck, C. A branched extender unit shared between two orthogonal polyketide pathways in an Endophyte. Angew. Chem. Int. Ed. 2011, 50, 4667−4670. (14) Song, L.; Barona-Gomez, F.; Corre, C.; Xiang, L.; Udwary, D. W.; Austin, M. B.; Noel, J. P.; Moore, B. S.; Challis, G. L. Type III polyketide synthase β-ketoacyl-ACP starter unit and ethylmalonyl-CoA extender unit selectivity discovered by Streptomyces coelicolor genome mining. J. Am. Chem. Soc. 2006, 128, 14754−14755. (15) Aoki, Y.; Matsumoto, D.; Kawaide, H.; Natsume, M. Physiological role of germicidins in spore germination and hyphal elongation in Streptomyces coelicolor A3(2). J. Antibiot. 2011, 64, 607−611. (16) Zhang, H.; Saurav, K.; Yu, Z.; Mandi, A.; Kurtan, T.; Li, J.; Tian, X.; Zhang, Q.; Zhang, W.; Zhang, C. α Pyrones with diverse hydroxy substitutions from three marine-derived Nocardiopsis strains. J. Nat. Prod. 2016, 79, 1610−1618. (17) Ma, M.; Rateb, M. E.; Yang, D.; Rudolf, J. D.; Zhu, X.; Huang, Y.; Zhao, L.; Jiang, Y.; Duan, Y.; Shen, B. Germicidins H−J from Streptomyces sp. CB00361. J. Antibiot. 2017, 70, 200−203. (18)

Findlay,

J.

A.;

Tam,

W.

H.

J.;

Krepinsky,

J.

The

chemistry

of

some 15



Page 16 of 21

6-methyl-4-hydroxy-2-pyridones. Can. J. Chem. 1978, 56, 613−616. (19)

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Al-Deeb,

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Al-Afify,

A.;

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(aryl)-6-aryl-3-cyano-2(1H)-pyridinones and their 2-imino isosteres as nonsteroidal cardiotonic agents. Il Farmaco 1999, 54, 195−201. (20) Cocco, M.T.; Congiu, C.; Onnis, V. Synthesis and antitumour activity of 4-hydroxy-2-pyridone derivatives. Eur. J. Med. Chem. 2000, 35, 545−552. (21) Öztürk, G.; Erol, D. D.; Uzbay, T.; Aytemir, M. D. Synthesis of 4(1H)-pyridinone derivatives and investigation of analgesic and antiinflammatory activities. Il Farmaco 2001, 56, 251−256. (22) Cocco, M. T.; Congiu, C.; Onnis, V. New bis(pyridyl)methane derivatives from 4-hydroxy-2-pyridones: synthesis and antitumoral activity. Eur. J. Med. Chem. 2003, 38, 37−47. (23) Wakabayashi, K.; Böger, P. Phytotoxic sites of action for molecular design of modern herbicides (Part 2): Amino acid, lipid and cell wall biosynthesis, and other targets for future herbicides. Weed Biol. Manag. 2004, 4, 59−70. (24)

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4-hydroxy-3-nitro-2-pyridinones as an activation step in the construction of dihydropyrido[3,4-b] benzo[f][1,4]thiazepin-1-one based anti-HIV agents. Tetrahedron Lett. 2005, 46, 2919−2922. (25) Evidente, A.; Fiore, M.; Bruno, G.; Sparapano, L.; Motta, A. Chemical and biological characterisation of sapinopyridione, a phytotoxic 3,3,6-trisubstituted-2,4-pyridione produced by Sphaeropsis sapinea, a toxigenic pathogen of native and exotic conifers, and its derivatives. Phytochemistry 2006, 67, 1019−1028. (26) Macdonald, G. E.; Puri, A.; Shillinget, D. G. Interactive effect of photoperiod and fluridone on growth, reproduction, and biochemistry of dioecious hydrilla (Hydrilla Verticillata). Weed Sci. 2008, 56, 189−195. 16


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(27) Lopez, S. N.; Ramallo, I. A.; Sierra, M. G.; Zacchino, S. A.; Furlan, R. L. E. Chemically engineered extracts as an alternative source of bioactive natural product-like compounds. Proc. Natl. Acad. Sci. USA. 2007, 104, 441−444. (28) Ramallo, I. A.; Salazar, M. O.; Mendez, L.; Furlan, R. L. E. Chemically engineered extracts: source of bioactive compounds. Acc. Chem. Res. 2011, 44, 241−250.

Figure 1. Key correlations for the structural assignment of 1–4 and ∆δ (= δS−δR) values for (S)- and (R)-MTPA esters of 13.

17



Page 18 of 21

Figure 2. ORTEP drawing of 2–4. Pyrones-containing fraction from Streptomyces sp. OUCMDZ-3436 NH3 H 2O reflux

purification

9

O R1 HO 10

2

4

14 : R 1 = CH3, R 2 = NH 6

11

15: R 1 = CH3, R 2 =

R2

10

8

7

12

9 7

9 10

11

19: R 1 = CH 3, R2 =

9

18 : R 1 = CH 2CH 3, R2 =

8

7

16 : R 1 = CH3, R 2 =

11

17 : R 1 = CH 2CH 3, R2 =

8

7

10

7

11

7 9

20 : R 1 = CH3, R 2 =

9

7 9

10

10

Figure 3. Diversity-enhanced chemical transformation of the pyrones-containing fraction.

Table 1. 1H (500 MHz) and 13C (125 MHz) NMR Data for Compounds 1−4 in CD3OD 1

2

3

4

no.

δC

2

169.1, C

168.9, C

169.1, C

168.5, C

3

99.4, C

99.3, C

99.2, C

105.5, C

4

167.6, C

167.7, C

167.8, C

167.4, C

5

103.8, CH

6

161.8, C

δH, mult. (J in Hz)

6.09, s

δC

101.5, CH 166.4, C


6.03, s

δC


101.7, CH 166.3, C

6.03, s

δC

101.6, CH


6.04, s

166.6, C

18


Page 19 of 21

Journal of Agricultural and Food Chemistry 7

48.0, CH2

8

71.0, C

2.60, s

46.8, CH

2.52, dq (6.4, 7.0)

47.2, CH

2.55, dq (6.9, 7.3)

46.8, CH

2.51, dq (6.4, 7.0)

69.9, CH

3.92, dq (6.4, 6.3)

70.1, CH

3.93, dq (6.9, 6.2)

69.9, CH

3.92, dq (6.4, 6.4)

9

29.4, CH3

1.26, s

21.3, CH3

1.15, d (6.3)

20.7, CH3

1.20, d (6.2)

21.4, CH3

1.15, d (6.4)

10

29.4, CH3

1.26, s

13.7, CH3

1.25, d (7.0)

14.7, CH3

1.20, d (7.3)

13.7, CH3

1.24, d (7.0)

11

8.3, CH3

1.86, s

8.3, CH3

1.85, s

8.3, CH3

1.86, s

17.3, CH2

2.39, q (7.4)

12.8, CH3

1.03, t (7.4)

12

Table 2. 1H (500 MHz) and 13C (125 MHz) NMR Data for Compounds 14−17 in DMSO-d6 14

15

16

17

no.

δC

2

164.8, C

165.3, C

164.7, C

164.2, C

3

96.5, C

96.9, C

99.2, C

109.9, C

4

163.3, C

5

103.4, CH

6

147.4, C


δC


163.4, C 5.75, s

δC


163.7, C

103.8, CH

6.00, s

152.7, C

δC


163.5, C

103.7, CH

5.69, s

146.9, C

95.4, CH

5.69, s

152.0, C

7

25.3, CH2

2.36, q (7.3)

30.9, CH

2.74, qq (6.9, 6.9)

33.9, CH2

2.30, t (7.6)

30.9, CH

2.63, qq (6.9, 6.9)

8

12.9, CH3

1.09, t (7.3)

21.3, CH3

1.14, d (6.9)

21.6, CH2

1.52, tq (7.6, 7.4)

21.3, CH3

1.11, d (6.9)

9

8.3, CH3

1.75, s

21.3, CH3

1.14, d (6.9)

13.4, CH2

0.85, t (7.4)

21.3, CH3

1.11, d (6.9)

8.1, CH3

1.81, s

8.2, CH3

1.73, s

15.8, CH2

2.30, q (7.2)

13.0, CH3

0.92, t (7.2)

10 11

Table 3. 1H (500 MHz) and 13C (125 MHz) NMR Data for Compounds 18−20 in DMSO-d6 18

19

20

no.

δC

2

164.4, C

3

109.7, C

98.5, C

95.5, C

4

162.6, C

163.3, C

163.0, C

5

95.5, CH


δC


164.7, C

5.67, s

103.5, CH

δC


164.8, C

5.66, s

103.4, CH

145.1, C

5.70, s

6

150.4, C

7

38.1, CH

2.40, tq (6.9, 6.9)

41.1, CH2

2.19, d (7.2)

38.1, CH

150.2, C 2.41, tq (6.9, 6.9)

8

28, CH2

1.46, m; 1.54, m

27.8, CH

1.85, m

28.1, CH2

1.45, m; 1.53, m

9

11.6, CH3

0.78, t (7.3)

22.0, CH3

0.83, d (6.6)

11.6, CH3

0.77, t (7.3)

10

19.0, CH3

1.09, d (6.9)

22.0, CH3

0.83, d, (6.6)

19.0, CH3

1.10, d (6.9)

11

15.8, CH2

2.29, q (7.1)

8.3, CH3

1.73, s

8.3, CH3

1.74, s

19


Journal of Agricultural and Food Chemistry 12

13.0, CH3

Page 20 of 21

0.92, t (7.1)

20


Page 21 of 21


Table of Contents Graphic

21


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