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Supplementary Material

Biotransformation of 4-sec-butylphenol by gram-positive bacteria of the genera Mycobacterium and Nocardia including modifications on the alkyl chain and the hydroxyl group

Veronika Hahn*I, Katharina Sünwoldt*, Annett Mikolasch*, and Frieder Schauer*

* Institute of Microbiology, Ernst-Moritz-Arndt-University of Greifswald, Friedrich-Ludwig-Jahn-Str. 15, 17487 Greifswald, Germany

I To whom correspondence should be addressed. e-mail: [email protected], tel.: +49 3834 864229, fax: +49 3834 864202

S1 List of contents

Additional Information to Experimental Section S3

Structural data of 4-sec-butylphenol S5

Structural data of P1 S6

Structural data of P2a-d S7

Structural data of P3 S9

S2 Additional Information to Experimental Section

General Methods. The product was characterized by mass spectrometry (MS) using electro spray ionization under atmospheric conditions (API-ES) (dry and nebulizer gas: nitrogen) on LC/MS (Agilent Series 1200 HPLC system with diode array detector and Agilent 6120 quadrupole mass spectrometer [Waldbronn, Germany]. For further analyses GC/MS (Agilent gas chromatograph [Waldbronn, Germany] equipped with a 30 m HP-5ms column [0.25 mm by 0.25 µm film] and a mass detector 5975C inert XL EI/CI MSD with quadrupole mass spectrometer), was used. The nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Avance 600 instrument (Rheinstetten, Germany) at

600 MHz. Solvent used was MeOH-d4. Chemical shifts are expressed in δ (ppm) calibrated on the resonances of the residual nondeuterated solvent. J values are given in Hz. For routine analysis, the biotransformation assays and the isolated product were analyzed using an HPLC system LC-10AT VP (Shimadzu, Germany) consisting of a FCV-10AL VP pump, SPD-M10A VP diode array detector, and a SCL-10A VP control unit controlled by Class-VP version 6.12 SP5. The separation of the substances was achieved on an endcapped, 5-µm, LiChroCART® 125-4 RP18 column (Merck, Darmstadt, Germany) at a flow rate of 1 mL/min. A solvent system consisting of methanol (eluent A) and 0.1% phosphoric acid (eluent B), starting from an initial ratio of 30% A and 70% B and reaching 100% methanol within 14 min, was used. Chemicals were purchased from commercial suppliers. All chemicals were used as received. Isolation steps were performed by solid-phase extraction with a RP18 silicagel column (60 mL, 10 g adsorbent material, phenomenex, Strata, Germany).

For the isolation of P1 and P2 a four liter biotransformation assay containing 0.025% 4-sec- butylphenol was shaken at 250 rpm and 30 °C for 96 h. Thereafter the material was centrifuged and filtered. The cell-free supernatant was used for solid phase extraction with a RP18 silica gel column (60 mL, 10 g adsorbent material, phenomenex, Strata, Germany). After charging the column with 40 mL cell-free supernatant, 60 mL of methanol/0.1% acetic acid (30:70 v/v) was used to remove impurities and hydrophilic products. Elution of the product fraction was performed with 60 mL of methanol/0.1% acetic acid (50:50 v/v).The separated products were concentrated by rotary evaporation to 1 ml and then dried to completion using a nitrogen stream. Separation of P1 and P2 was carried out by preparative HPLC on an Agilent Series 1260 Infinity HPLC system with diode array detector, two separate high- pressure pumps and a fraction collector (Waldbronn, Germany). The separation was performed on an Agilent Flow, Eclipse XDB-C18 Prep HT, 21.2 x 250 mm (7µm), (Agilent, Waldbronn, Germany) at a flow rate of 10 ml/min. A solvent system consisting of methanol (eluent A) and 0.1% aqueous acetic

S3 acid (eluent B), starting from an initial ratio of 30% A and 70% B and reaching 100% methanol within 14 min, was used. For HPLC, LC/MS and GC/MS analyses, the products were dissolved in methanol. For isolation of P3 one liter biotransformation assay with 0.025% 4-sec-butylphenol was incubated at 30 °C and 250 rpm. After 96 h the assay was centrifuged and filtered. The cell-free supernatant was used for solid phase extraction with a RP18 silica gel column (60 mL, 10 g adsorbent material, phenomenex, Strata, Germany). The separated product was concentrated by rotary evaporation to 1 ml and then evaporated to dryness under a nitrogen stream.

Structural Data of 4-sec-butylphenol

S4 4-sec-Butylphenol mAU 1200 11.18 OH Spectrum at time 11.15 min.

1000 0 0 U 0 A 2 m

800 0 0 0 1

200 400 600 nm 400

0 2 4 6 8 10 12 14 16 18 minutes

Fig. S1 HPLC elution profile and UV-vis spectrum of 4-sec- butylphenol.

Rf (HPLC) 11.18 min, UV-vis (MeOH) λmax 222, 275 nm. MW 150.22 g/mol.

Structural Data of P1

P1: product resulted from biotransformation of 4-sec-butylphenol by Mycobacterium neoaurum and

Nocardia cyriacigeorgica

S5 P1 4-(2-Hydroxy-1-methylpropyl)-phenol mAU 4.85 200 4.71 min U 0 A 0 4 OH m 0

150 0 6 1 2 2

3 5 100 0 4 200 300 400 500 600 nm 7 OH 10 8 50

0 2 4 6 8 10 12 14 16 18 Zeit [min] minutes

Fig. S2 HPLC elution profile and UV-vis-spectrum of P1 formed during biotransformation of 4-sec-butylphenol by Mycobacterium neoaurum and Nocardia cyriacigeorgica 0 6 9 5 6 5 3 0 9 6 4 2 6 5 0 9 9 7 9 8 9 8 7 2 0 9 8 7 6 5 6 4 6 4 4 4 2 2 9 9 9 6 6 6 6 5 5 9 , , , , , , , , , , , , , , , ,

6 6 6 6 3 3 3 2 2 2 2 2 1 1 0 0 40000000

30000000

20000000

10000000

0 2 1 1 1 3 3 ...... 1 6 0 0 8 8 5 8 0 8 6 3

7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 ppm (t1)

Fig. S3 1H NMR spectrum of P1

Synthesis and isolation as described above. Yellow solid. Yield 0.14 % (1.4 mg). 1H NMR: δ 0.95 (d, J = 6.2 Hz, 3H, H-9), 1.26 (d, J = 7.0 Hz, 3H, H-10), 2.50 (m, J = 7.0 Hz, J = 7.1 Hz, 1H, H-7), 3.68 (m, J = 6.2 Hz, J = 7.1 Hz, 1H, H-8), 6.69 (d, J = 8.3 Hz, 2H, H-2/H-6), 6.98 (d, J = 8.3 Hz, 2H, H-3/H-5).

Rf (HPLC) 4.85 min, UV-vis (MeOH) λmax 222, 276 nm. GC/MS m/z 39 (5.6), 43 (5.8), 45 (8.0), 65 (7.6), 77 (24.8), 91 (19.6), 93 (6.8), 103 (15.2), 107 (43.6), 120 (6.8), 121 (100), 122 (44.7) 166 [M]+ (6.0).

Structural Data of P2a-d

P2a-d: products resulted from biotransformation of 4-sec-butylphenol by Mycobacterium neoaurum

and Nocardia cyriacigeorgica

P2a-d I 4-sec-Butylidenecyclohexa-2,5-dienone

S6 II 4-(1-Methylenepropyl)-phenol III 4-(1-Methylpropenyl)-phenol IV 4-(1-Methylallyl)-phenol ] 0 U 0 2 A 1 m

[ P2b 0 0 0 5 0

I 1 5 0 0 8 0 0 6

0 P2d 0 4

P2a P2c 9 4 . 8 0 4 0 2 8 2 . 9 5 . II OH 6 0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 [minutes] U U 0 A A 0 0 0 U U 0 5 m 0 m 0 A A 0 1 1 4 4 m m

P2a 0 P2b P2c P2d 0 0 1 0 0 0 0 2 5 0 0 2 0 5 0 0 0 0 200 300 400 500 600 200 300 400 500 600 200 300 400 500 600 200 300 400 500 600 nm nm nm nm III OH Fig. S4 HPLC elution profile and UV-vis-spectra of P2a-d formed during biotransformation of 4-sec-butylphenol by Mycobacterium neoaurum and Nocardia cyriacigeorgica

Synthesis and isolation as described above. Yellow solid. Yield 0.18 % (1.8 mg). Rf (HPLC) P2a 5.24 min, UV-vis (MeOH) λmax 225, 276 nm; Rf (HPLC) P2b 5.50 min, UV-vis (MeOH) λmax 222, 275 nm; Rf

(HPLC) P2c 6.97 min, UV-vis (MeOH) λmax 222, 275 nm; Rf (HPLC) P2d 8.47 min, UV-vis (MeOH)

+ λmax 223, 275 nm. Rf (LC/MS) P2a 5.07 min m/z AP-ESI: pos. ion mode [M+H] 149.1 (100); Rf

+ (LC/MS) P2b 5.27 min m/z AP-ESI: pos. ion mode [M+H] 149.0 (100); Rf (LC/MS) P2c 6.75 min m/z

+ AP-ESI: pos. ion mode [M+H] 149.1 (100); Rf (LC/MS) P2d 8.35 min m/z AP-ESI: pos. ion mode

+ [M+H] 149.1 (100); Rf (GC/MS) 5.66 min, m/z 39 (14.6), 51 (13.3), 53 (8.5), 55 (10.5), 63 (10.0), 65

(13.6), 77 (25.0), 78 (6.0), 79 (11.5), 89 (7.3), 91 (26.6), 94 (7.6), 103 (14.3), 105 (42.7), 107 (14.4),

S7 115 (15.5), 119 (11.5), 121 (6.4), 131 (17.7), 132 (9.2), 133 (100), 134 (9.8), 147 (31.7), 148 [M] +

(90.9), 149 (9.5); Rf (GC/MS) 5.93 min, m/z 39 (15.9), 51 (13.5), 53 (8.2), 55 (7.7), 63 (13.0), 65 (25.0),

66 (6.7), 77 (20.4), 78 (5.6), 79 (8.2), 89 (11.0), 91 (48.0), 94 (16.7), 103 (9.8), 105 (23.2), 107 (11.0),

115 (9.9), 118 (6.6), 119 (94.9), 120 (11.3), 131 (12.2), 132 (5.3), 133 (82.3), 134 (8.6), 147 (26.5), 148

+ [M] (100), 149 (10.5); Rf (GC/MS) 6.26 min, m/z 39 (14.8), 51 (13.8), 53 (9.4), 55 (11.6), 63 (10.3), 65

(14.6), 66 (5.9), 77 (27.0), 78 (6.3), 79 (12.3), 91 (26.8), 94 (8.0), 103 (14.1), 105 (44.5), 107 (15.6),

115 (15.4), 119 (12.7), 121 (6.8), 131 (18.0), 132 (8.7), 133 (100), 134 (10.1), 147 (31.6), 148 [M] +

(90.5), 149 (9.6); Rf (GC/MS) 6.70 min, m/z 39 (9.1), 43 (81.1), 51 (5.3), 55 (5.0), 57 (11.6), 65 (13.5),

77 (14.3), 91 (11.8), 93 (6.3), 94 (5.4), 103 (5.7), 105 (5.3), 107 (5.9), 119 (8.0), 121 (33.0), 133 (11.6),

137 (100), 138 (8.5), 148 [M]+ (11.4).

Structural Data of P3

P3: product resulted from biotransformation of 4-sec-butylphenol by Mycobacterium neoaurum

P3 4-sec-Butylphenol-α -D-glucopyranoside

S8 OH mAU 6' 4' n 5' O i m 1000 HO 9.048 Spectrum at tim e 8.83 min. 1 HO . 2' 1' 9 3' 0 U OH 2 0 3 A O 3 800 m

1 0 0 2 6 7 4 8

600 1 10 0 200 400 600 400 nm

4 5 6 7 8 9 10 11 12 13 14 15 16 17 Zeit [min] minutes

Fig. S5 HPLC elution profile and UV-vis-spectra of P3formed during biotransformation of 4-sec-butylphenol by Mycobacterium neoaurum 9 5 9 4 1 6 6 9 5 9 4 3 7 9 9 0 0 0 9 1 0 0 8 6 4 2 1 7 5 4 2 0 9 2 1 9 7 4 1 7 3 7 8 4 3 8 8 6 5 4 4 3 3 2 1 2 1 0 9 7 7 6 5 4 2 1 9 8 6 5 3 0 9 0 9 8 0 4 9 9 9 9 9 7 7 6 6 5 5 5 5 5 5 2 7 4 9 9 9 9 9 7 7 6 6 5 5 5 5 5 5 1 8 7 ...... 7 5 5 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 2 2 2 2 2 2 1 1 1 1 1 1 1 1 0 0 0 500000000

400000000

300000000

200000000

100000000

0 3 0 3 2 1 2 2 3 ...... 5 0 9 1 8 8 2 1 7 7 3 8 0 5 1 4

7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 -1.0 ppm (t1)

Fig. S6 1H NMR spectrum of P3 4 3 2 5 0 3 9 2 5 1 8 7 2 6 2 0 6 0 2 5 1 0 3 4 7 2 4 0 9 8 8 8 0 8 3 1 ...... 0 4 9 1 5 3 3 6 6 5 7 2 8 8 0 ...... 2 5 1 3 1 0 0 2 2 7 4 0 2 2 2 1 1 1 1 1 1 7 7 7 7 6 4 3 2 1

5000000

150 100 50 0 ppm (t1)

Fig. S7 13C NMR spectrum of P3

ppm (t1)

7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 ppm (t2)

Fig. S8 HMBC spectrum of P3

150 ppm (t1)

7.0 6.0 5.0 4.0 3.0 2.0 1.0 ppm (t2)

Fig. S9 HSQC spectrum of P3

ppm (t1)

7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0 ppm (t2)

Fig. S10 HH Cosy spectrum of P3

Synthesis and isolation as described above. Yellow solid. Yield 7 % (14 mg). 1H NMR: δ 0.80 (t, J = 7.4 Hz, 3H, H-9), 1.20 (d, J = 6.9 Hz, 3H, H-10), 1.57 (m, J = 7.4 Hz, 2H, H-8), 2.54 (m, J = 6.9 Hz, 1H, H-7), 3.70 (m, 2H, H-6’), 3.94 (m, 4H, H-2’-5’), 5.44 (d, J = 3.3 Hz, 1H, H-1’), 7.09 (m, 4H, H- 2,3,5,6). 13C NMR: δ 12.6 (C-9), 22.6 (C-10), 32.4 (C-8), 42.4 (C-7), 62.5 (C-6’), 70.1, 71.5, 73.0, 70.9 (C-2’-5’), 100.1 (C-1’), 118.4 (C-2,6), 128.9 (C-3,5), 142.8 (C-4), 157.1 (C-1). HMBC correlations: H- 1’ (71.5, 73.0, C-1), H-2,3,5,6 (C-1’, C-2,6, C-3,5, C-4, C-7), H-2’-5’ (C-6’), H-6’ (70.9, 73.0), H-7 (C- 3/C-5, C-8, C-9, C-10), H-8 (C-4, C-7, C-9), H-9 (C-7, C-8), H-10 (C-4, C-7, C-8). 1H-1H-COSY correlations: H-1’ (H-2’-5’), H-2,3,5,6 (H-2,3,5,6), H-2’-5’ (H-1’, H-6’), H-6’ (H-2’-5’), H-7 (H-8, H-

10), H-8 (H-7, H-9), H-9 (H-8), H-10 (H-7). Rf (HPLC) 9.04 min, UV-vis (MeOH) λmax 221, 273 nm.

S10 + + LC/MS m/z AP-ESI: pos. ion mode [M+H2O] 330.1 (100), [M+H+H2O] 331.1 (17); GC/MS m/z 57 (5.2), 73 (6.5), 77 (6.0), 91 (9.7), 107 (5.9), 122 (8.5), 150 (31.2), 312 [M]+ (0.02).