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Identification, library synthesis and anti-vibriosis activity of 4-chloro-2-benzylphenol from cultures of the marine bacterium Shewanella halifaxensis

Sarah L. Moore,a,b Lucile Berthomier,a,b Chriselle D. Braganza,a Joanna K. MacKichan,c Jason L. Ryan,d Gabriel Visnovsky,e and Robert A. Keyzers. a *

a Center for Biodiscovery, and School of Chemical and Physical Sciences, Victoria University of Wellington PO BOX 600, Kelburn, Wellington 6140, New Zealand.

b Contributed equally to this project.c Center for Biodiscovery, and School of Biological Sciences, Victoria University of Wellington PO BOX 600, Kelburn, Wellington 6140,

New Zealand.d Integrated Bioactive Technologies Group, Callaghan Innovation, Gracefield, PO Box 31310, Lower Hutt 5040, New Zealand.e Chemical and Process Engineering, University of Canterbury, Private bag 4800, Christchurch 8140, New Zealand.

SUPPLEMENTARY INFORMATION

General experimental procedures and characterization data 2

Figure S1: 1H NMR spectrum (600 MHz, CDCl3) of isolated 2-benzyl-4-chlorophenol (1). 4

Figure S2: 1H NMR spectrum (600 MHz, CDCl3) of synthetic 2-benzyl-4-chlorophenol (1). 5

Figure S3: 13C NMR spectrum (150 MHz, CD3OD) of synthetic 2-benzyl-4-chlorophenol (1). 5

Figure S4: 1H NMR spectrum (600 MHz, CD3OD) of synthetic 2-benzyl-4-chlorophenol (1). 6

Figure S5: 13C NMR spectrum (150 MHz, CD3OD) of synthetic 2-benzyl-4-chlorophenol (1). 6

Figure S6: 1H NMR spectrum (600 MHz, CDCl3) of synthetic 2,6-dibenzyl-4-chlorophenol (2). 7

Figure S7: 13C NMR spectrum (150 MHz, CDCl3) of synthetic 2,6-dibenzyl-4-chlorophenol (2). 7


Figure S9: 13C NMR spectrum (150 MHz, CDCl3) of synthetic 4-benzyl-2-chlorophenol (3). 8


Figure S11: 13C NMR spectrum (150 MHz, CDCl3) of synthetic 3,-4-benzyl-2-chlorophenol (4). 9






Figure S17: 13C NMR spectrum (150 MHz, CDCl3) of synthetic 2,4-benzyl-5-chlorophenol (7). 12

Figure S18: 1H NMR spectrum (600 MHz, CD3OD) of two separate cultures of Shewanella halifaxensis IRL548 showing the presence of 2-benzyl-4-chlorophenol (1). 13

General Experimental Procedures1H, 13C and 2D NMR spectra were acquired using a Varian 600 NMR DirectDrive spectrometer with spectra

referenced to residual solvent peaks (CDCl3: δC 77.0, δH 7.26; CD3OD: δC 49.0, δH 3.31). HRESIMS data were acquired using an Agilent 6530 Q-TOF system. HPLC purification was performed using an Agilent 1260 Infinity HPLC system connected to a QuicksplitTM flow splitter that directed 5% of flow to an Agilent 380-evaporative light scattering detector. Separation utilized a C-18 Phenomenex Prodigy column (4.6 x 250 mm, 5 μm particle size) using a 1 mL/min flow rate. All solvents used were HPLC grade purchased form Fisher Scientific. Bench top chromatography was performed using Supleco Diaion® HP20 polystyrene-divinylbenzene and Supelco Discovery®

DSC-DIOL functionalized silica 3-(2,3,-Dihydroxy-propoxy)-propyl-silica (Diol) resins. Fractions were monitored using TLC plates using a 4:1:2, butanol:acetic acid:water mix for the running solvent. TLC plates were visualized with 5% H2SO4 methanol solutions followed by 0.1% vanillin before charring. Chemicals for synthesis were purchased from Sigma Aldrich and were used without further purification.

Culture Conditions

S. halifaxensis IRL548 was grown using PYM medium, containing 10 g yeast extract, 2.5 g meat peptone, 5 g bactopeptone, and 15 g instant ocean® salt per liter of RO water. The production was conducted in 20L Sartorius DCU bioreactor, with 3 rushton impellors (0.3 di/dt) placed at 1 di intervals. A 100 mL culture was first grown in Erlenmeyer shake flask for 24 hours at 15 °C from a frozen stock of S. halifaxensis. This was then added to the 20 L and grown under aerobic conditons (0.5 vvm air, 100 rpm, 100 mbar backpressure) at 15 °C for 24 hours.

Isolation

A 20 L liquid culture of Shewanella halifaxensis IRL 548 was centrifuged (5,777 x g, 20 min.) to give a cell mass fraction and a clarified supernatant. The supernatant was passed through a column of HP20 (100 mL) that had been prewashed with 300 mL portions of acetone, MeOH and H2O, and was then partitioned with 300 mL of 30% acetone(aq), 75% acetone(aq) and 100% acetone. The 75% acetone fraction (285 mg) was not completely soluble in MeOH and so was filtered through cotton wool in MeOH to give a yellow filtrate (94 mg) and a brown solid (140 mg). The filtrate was then chromatographed on a pre-washed and pre-equilibrated column of HP20ss (50 mL) eluted with 150 mL portions of 50%, 60%, 70%, 80%, 90% and 100% MeOH/H2O. The 50%, 60%, 90% and 100% elutions were collected as discrete bulk fractions while the 70% and 80% were collected across 45 test tubes. The 100% MeOH fraction (3.1 mg) was finally separated by C18 HPLC (1 mL/min isocratic 75% MeOH(aq), monitoring at 230 and 254 nm) to give 0.9 mg 2-benzyl-4-chlorophenol (1).

2-Benzyl-4-chlorophenol (1): white amorphous solid; HRESIMS: [M-H]- m/z obs: 217.0434; calcd for C13H11OCl 217.0426 (Δ +0.8 mmu); HRESIMS/MS: m/z obs: 181.0657, calcd for C10H10ClO: 181.0659 (Δ +0.2 mmu); m/z obs: 101.0391, calcd for: C8H5 101.0397 (Δ 0.6 mmu); m/z obs: 41.0038, calcd for C3H5: 41.0033 (Δ=0.5 mmu), m/z obs: 34.9690, calcd for Cl: 34.9694 (Δ=-0.4 mmu) ; 13C NMR (150 MHz, CD3OD): see table 1; 1H NMR (600 MHz, CD3OD): see table 1; 13C NMR (150 MHz, CDCl3): δC 152.5 (C-1), 138.9 (C-1”), 130.7 (C-3), 129.0 (C-2), 128.9 (C-3”), 128.8 (C-2”), 127.7 (C-6), 126.8 (C-4”), 125.8 (C-4), 117.1 (C-5), 36.4 (C-1’); 1H NMR (600 MHz, CDCl3): δH

7.31 (t, 7.6 Hz, 2H, H-3”) 7.24 (t, 7.3 Hz, 1H, H-4”), 7.22 (d, 7.3 Hz, 2H, H-2”), 7.08 (s, 1H, H-3), 7.07 (dd, 6.7, 2.4 Hz, 1H, H-5), 6.72 (d, 9.3 Hz, H-6), 4.75 (s, 1H, OH-1), 3.95 (s, 2H, H-1’).

Synthesis

In each case, o-, m- or p-chlorophenol (0.2 g, 1.56 mmol) was added to benzyl bromide (0.19 mL, 1.60 mmol, 1 equiv) under argon. ZnCl2(anhyd.) (0.045 g, 0.33 mmol, 0.2 equiv) was added and the reaction stirred at 60°C for 2 hours after which heating was increased to 90°C after which heating was halted and the reaction was allowed to cool to RT and the reaction was quenched with H2O (15 mL). The reaction mixture was extracted with ether (15 mL) that was then washed with 2 M HCl (15 mL) and H2O (15 mL). The ether layer was extracted with KOH(aq) (0.1 M, 3 x 15 mL), the alkaline layer was washed with ether (20 mL) and the aqueous phase acidified with HCl (2 M). Finally, the aqueous phase was extracted with ether (2 x 20 mL) that was dried with MgSO4 and purified in each case using silica gel to yield the relevant mono- or di-alkylated chlorophenol.

From reaction with para-chlorophenol, 2-benzyl-4-chlorophenol (1, 0.002 g, 0.6% yield) and 2,6-dibenzyl-4-chlorophenol (2, 0.006 g, 1.2% yield) were isolated.

2-Benzyl-4-chlorophenol (1): All spectroscopic data identical to natural product.

2,6-Dibenzyl-4-chlorophenol (2): white amorphous solid; HRESIMS: [M-H]- m/z obs: 307.0857, calcd for C20H17OCl: 307.8095 (Δ -3.8 mmu); 13C NMR (150 MHz, CDCl3): δC 150.9 (C-1), 138.9 (C-1”), 129.1 (C-2), 129.0 (C-3”), 128.9 (C-3), 128.8 (C-2”), 126.9 (C-4”), 125.4 (C-4), 36.6 (C-1’); 1H NMR (600 MHz, CDCl3): δH 7.31 (t, 7.5 Hz, 4H, H-3”), 7.24 (t, 7.4 Hz, 2H, H-4”), 7.20 (d, 7.6 Hz, 4H, H-2”), 7.00 (s, 2H, H-3), 4.60 (s, 1H, OH-1), 3.93 (s, 4H, H-1’).

From reaction with ortho-chlorophenol, 4-benzyl-2-chlorophenol (3, 0.009 g, 0.6% yield) and 3,4-dibenzyl-2-chlorophenol (4, 0.007 g, 0.4% yield) were isolated.

4-Benzyl-2-chlorophenol (3): white amorphous solid; HRESIMS: [M-H]- m/z obs: 217.0426, calcd for C13H11OCl 217.0426 (Δ = 0.0 mmu); 13C NMR (150 MHz, CDCl3): δC 149.8 (C-1), 140.8 (C-1”), 134.6 (C-4), 129.2 (C-3), 129.0 (C-5), 128.9 (C-2”), 128.1 (C-3”), 126.4 (C-4”), 116.3 (C-6), 40.9 (C-1’); 1H NMR (600 MHz, CDCl3): δH 7.30 (t, 7.7 Hz, 2H, H-3”), 7.22 (t, 7.3 Hz, 1 H, H-4”), 7.17 (d, 7.3 Hz, 2H, H-2”), 7.13 (d, 2.1 Hz, 1H, H-3), 7.00 (dd, 8.3, 2.0 Hz, 1H, H-5), 6.94 (d, 8.3 Hz, 1H, H-6), 5.41 (s, 1H, OH-1), 3.89 (s, 2H, H-1’).

3,4-Dibenzyl-2-chlorophenol (4): white amorphous solid; HRESIMS: [M-H]- m/z obs: 307.0891, calcd for C20H17OCl 307.0895 (Δ = -0.4 mmu); 13C NMR (150 MHz, CDCl3): δC 149.8 (C-1), 141.2 (C-1”), 139.2 (C-3), 138.6 (C-1””), 134.6 (C-4), 129.20 (C-2””), 129.1 (C-5), 128.9 (C-2”), 128.7 (C-3”), 126.4 (C-4”), 119.8 (C-2), 116.3 (C-6), 41.0 (C-1’), 40.5 (C-1’”); 1H NMR (600 MHz, CDCl3): δH 7.28 (t, 7.7 Hz, 2H, H-4”, H-4””), 7.19, (t, 8.2 Hz, 4H, H-3”, H-3””), 7.13 (s, 1H, OH-1), 7.11 (d, 8.1 Hz, 2H, H-2””), 7.07 (d, 7.6 Hz, 2H, H-2”), 6.94 (d, 8.3 Hz, 1H, H-6), 3.89 (s, 2H, H-1’).

From reaction with meta-chlorophenol, 2-benzyl-5-chlorophenol (5, 0.005 g, 0.5% yield), 4-benzyl-3-chlorophenol (6, 0.006 g, 0.6% yield) and 2,4-dibenzyl-5-chlorophenol (7, 0.005 g, 0.2% yield) were isolated.

2-Benzyl-5-chlorophenol (5): white amorphous solid, HRESIMS: [M-H]- m/z obs: 217.0394, calcd for C13H11OCl 217.0426 (Δ -3.2 mmu); 13C NMR (150 MHz, CDCl3): δC 154.5 (C-1), 139.3 (C-1”), 132.9 (C-5), 131.9 (C-3), 128.9 (C-3”), 129.7 (C-2”), 126.7 (C-4”), 125.8 (C-2), 121.2 (C-4), 116.3 (C-6), 36.1 (C-1’) ; 1H NMR (600 MHz, CDCl3): δH 7.30 (t, 7.7 Hz, 2H, H-3”),7.23 (t, 7.4 Hz, 1H, H-4”), &.20 (d, 7.6 Hz, 2H, H-2”), 7.03 (d, 7.0 Hz, 1H, H-3), 6.88 (dd, 8.1, 2.0 Hz, 1H, H-4), 6.81 (d, 2.1 Hz, 1H, H-6), 4.80 (s, 1H, OH-1), 3.95 (s, 2H, H-1’).

4-Benzyl-3-chlorophenol (6): white amorphous solid; HRESIMS: [M-H]- m/z obs: 217.0426, calcd for C13H11OCl 217.0426 (Δ 0.0 mmu); 13C NMR (150 MHz, CDCl3): δC 154.7 (C-1), 140.1 (C-1”), 134.7 (C-3), 131.8 (C-5), 131.0 (C-4), 128.9 (C-2”), 128.6 (C-3”), 126.3 (C-4”), 116.6 (C-2), 114.3 (c-6), 38.5 (c-1’); 1H NMR (600 MHz, CDCl3): δH

7.28 (t, 7.5 Hz, 2H, H-3”), 7.20 (t, 7.3 Hz, 1H, H-4”), 7.17 (d, 7.8 Hz, 2H, H-2”), 7.00 (d, 8.4 Hz, 1H, H-5), 6.90 (d, 2.6 Hz, 1H, H-2), 6.67 (dd, 8.4, 2.6 Hz, 1H, H-6), 4.02 (s, 2H, H-1’).

2,4-Dibenzyl-5-chlorophenol (7): white amorphous solid; HRESIMS: [M-H]- m/z obs: 307.0891, calcd for C20H17OCl 307.0895 (Δ -0.4 mmu); 13C NMR (150 MHz, CDCl3): δC 152.9 (C-1), 140.2 (C-1””), 139.4 (C-1”), 133.3 (C-3), 132.6 (C-5), 131.0 (C-4), 128.9 (C-3”), 128.8 (C-4””), 128.6 (C-4””), 128.5 (C-2”), 126.7 (C-4”), 126.2 (C-1””), 126.0 (C-2), 116.9 (C-6), 38.5 (C-1’”), 36.2 (C-1’) ; 1H NMR (600 MHz, CDCl3): δH 7.28 (td, 7.3, 2.9 Hz, 4H, H-3”, H-3””), 7.21 (t, 7.5 Hz, 2H, H-4”, H-4””), 7.17 (m, 4H, H-2”, H-2””), 6.94 (s, 1H, H-3), 6.85 (s, 1H, H-6), 4.74 (s, 1H, OH-1), 4.01 (s, 2H, H-1’”), 3.91 (s, 2H, H-1’).

Bioassays

Bacterial strains used for antimicrobial testing were Escherichia coli (DH5; Life Technologies), Staphylococcus aureus (ATCC Strain 6538), and V. harveyi (IRL827, kindly provided by Jason Ryan at Callaghan Innovation, New Zealand). S. aureus was cultured in trypticase soy broth, while E. coli was cultured in lysogeny broth with 5g/L NaCl (LB–Lennox), both at 37C. V. harveyi was cultured in LB-Miller broth (10 g/L NaCl) at 30C. Overnight cultures of all the strains were grown with shaking (200 rpm). The optical density at 600 nm, O.D.600, was measured the next morning and a bacterial suspension of O.D.600 = 0.1 was made. Antimicrobial assays were carried out in 96-well plates. Briefly, 100 µg of each compound was dissolved in 1 ml of dimethyl sulfoxide (DMSO). In a sterile 96-well plate, 100 µl of the appropriate liquid medium was added to each column, apart from the last one, which was left empty. In one column of a 96-well plate, 50 µl of the liquid medium was added, as well as 50 l of DMSO containing the compound of interest; this column represented the highest concentration tested. A series of 1:1 dilutions were made in the remaining columns, resulting in 100 µl of medium in each column. The wells were then brought up to 200 µl by adding 100 µl of the bacterial suspension at O.D.600 = 0.1. A control column (unchallenged) was included for each plate, and the outside wells were excluded, to avoid any distortions due to drying at the edges. The O.D. 600

absorbance at T=0 was measured using a plate reader. The plates were covered and incubated overnight at the appropriate temperature, with shaking. After 20-22 hours, the final O.D.600 was measured. The relative growth for an individual well was calculated as a fraction of the average readings of the unchallenged wells. For each compound in the dilution series, the average relative growth of the replicate wells per plate were plotted over the respective concentration of that compound, then a dynamic curve was fitted to the data. The IC50 was calculated at half way between the top and bottom (y-axis) plateaus of the variable slope curve. Each compound was tested in triplicate, in at least two experiments.

Figure S1: 1H NMR spectrum (600 MHz, CDCl3) of isolated 2-benzyl-4-chlorophenol (1).

-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5f1 (ppm)

6.66.76.86.97.07.17.27.37.4f1 (ppm)

Figure S2: 1H NMR spectrum (600 MHz, CDCl3) of synthetic 2-benzyl-4-chlorophenol (1).

0102030405060708090100110120130140150160170180190200210f1 (ppm)

36.4

117.

112

5.8

126.

812

6.8

127.

712

7.7

128.

812

8.8

128.

912

8.9

129.

013

0.7

139.

1

152.

5

Figure S3: 13C NMR spectrum (150 MHz, CDCl3) of synthetic 2-benzyl-4-chlorophenol (1).

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5f1 (ppm)

6.76.86.97.07.17.27.3f1 (ppm)

Figure S4: 1H NMR spectrum (600 MHz, CD3OD) of synthetic 2-benzyl-4-chlorophenol (1).

0102030405060708090100110120130140150160170180190200210f1 (ppm)

36.3

9

40.4

0

117.

07

124.

8612

6.96

127.

8112

9.32

129.

9413

0.91

131.

30

141.

91

155.

13

Figure S5: 13C NMR spectrum (150 MHz, CD3OD) of synthetic 2-benzyl-4-chlorophenol (1).

0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5f1 (ppm)

7.007.057.107.157.207.257.307.357.40f1 (ppm)

Figure S6: 1H NMR spectrum (600 MHz, CDCl3) of synthetic 2,6-dibenzyl-4-chlorophenol (2).

0102030405060708090100110120130140150160170180190200210f1 (ppm)

36.6

118.

112

5.4

126.

512

6.9

128.

712

8.8

128.

812

8.9

129.

012

9.0

129.

012

9.1

129.

213

1.3

138.

9

150.

9

Figure S7: 13C NMR spectrum (150 MHz, CDCl3) of synthetic 2,6-dibenzyl-4-chlorophenol (2).

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5f1 (ppm)

6.97.07.17.27.37.4f1 (ppm)


0102030405060708090100110120130140150160170180190200210f1 (ppm)

40.9

116.

2

119.

812

6.4

128.

712

8.9

129.

012

9.2

134.

6

140.

8

149.

7


0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0f1 (ppm)

6.97.07.17.27.3f1 (ppm)


0102030405060708090100110120130140150160170180190200210f1 (ppm)

40.5

41.7

116.

2

119.

812

6.2

128.

612

9.0

129.

012

9.0

129.

212

9.2

134.

713

8.5

139.

214

1.2

149.

7

Figure S11: 13C NMR spectrum (150 MHz, CDCl3) of synthetic 3,-4-benzyl-2-chlorophenol (4).

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5f1 (ppm)

6.86.97.07.17.27.3f1 (ppm)


0102030405060708090100110120130140150160170180190200210f1 (ppm)

36.0

116.

212

1.2

125.

812

6.7

128.

712

8.9

131.

913

2.9

139.

3

154.

5


0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0f1 (ppm)

6.66.76.86.97.07.17.27.37.4f1 (ppm)


0102030405060708090100110120130140150160170180190200210f1 (ppm)

38.5

114.

311

6.6

126.

312

8.6

128.

913

1.0

131.

813

4.7

140.

1

154.

7


0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5f1 (ppm)

6.86.97.07.17.27.37.4f1 (ppm)


0102030405060708090100110120130140150160170180190200210f1 (ppm)

36.2

38.5

116.

912

6.0

126.

212

6.7

128.

512

8.6

128.

812

8.9

131.

013

2.6

133.

313

9.4

140.

2

152.

9

Figure S17: 13C NMR spectrum (150 MHz, CDCl3) of synthetic 2,4-benzyl-5-chlorophenol (7).

0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.5f1 (ppm)

6.76.86.97.07.17.27.37.4f1 (ppm)

Figure S18: 1H NMR spectrum (600 MHz, CD3OD) of original (top) and repeat (bottom) cultures of Shewanella halifaxensis IRL548 showing the presence of 2-benzyl-4-chlorophenol (1).

ars.els-cdn.com€¦ · web viewirl548 was grown using pym medium, containing 10 g yeast extract,...

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