Microbial Transformation of Dihydroxyphenylacetic Acid by the Marine-Derived Bacterium Stappia sp.

Experimental

General. UV/visible spectra were measured on a Hitachi U-2001 UV/Vis spectrometer. IR spectra were recorded on a Bruker FT-IR model IFS-88 spectrometer. 1H- (400 MHz) and 13C-NMR (100 MHz) spectra were obtained on a JEOL JNM-ECP 400 NMR spectrometer, using TMS or solvent peaks [DMSO-d6: 1H (δ 2.50) and 13C (δ 39.5)] as reference standard. LC-MS and MS spectra were obtained on API 2000 (Applied Bio System) and IT-TOF (Shimadzu, Japan) spectrometer, respectively. HPLC was performed on a Young Lin ACME HPLC system using a reversed-phase analytical column (Gemini C18, 4.6 × 250 mm, 5 μm) with UV detection. Incubations of microorganisms and microbial transformation were performed on an Incubator Shaker JS-FS-2500 (Johnsam Co., Inchon, Korea).

Isolation of the Marine-Derived Bacterium Stappia sp. The Stappia sp., isolated from the surface of the edible marine green alga Enteromorph compressa (Korean name: NapJag PaRae), had light gray, downy, and soft white vegetative mycelia, and was identified on the basis of morphology and 16S rRNA analysis (SolGent Co., Ltd., Daejeon, Korea), identity of 99%. The Stappia sp., designated as BAac008, is deposited at Pukyong National University, South Korea.

Microbial Transformation of 2-(3,4-Dihydroxyphenyl)-acetic Acid (1). A two-stage culture protocol9 was used to obtain metabolites 2 and 3 on a preparative scale.

The SS medium consisting of soytone (0.1%), soluble starch (1.0%), and seawater (100%) was autoclaved at 121℃ for 15 min after addition of NaBr and CaBr2 (each 50 mM).14,15 The preparative culture (stage 1) was incubated in 1 L sterile medium in a 3 L culture flask on a rotary shaker (130 rpm) at 29 ℃ for 1 week. A 10% inoculum derived from the 1-week-old preparative culture was used to initiate the stage II culture (1 L), which was incubated for a further 24 h under the same conditions before addition of 20.0 mg of compound 1 in 1.0 mL of N,N-dimethyl formamide (DMF)-MeOH (3:1 v/v). Incubation was continued for two weeks in the same manner as described above. The substrate control consisted of sterile medium and substrate incubated under the same conditions, but without microorganisms. The culture control consisted of the microorganism grown under the same condition, but without substrate. After three weeks, each control was harvested and analyzed by TLC. TLC analyses showed that the composition of the extract differed from those derived from two controls.

Extraction and Isolation. The mycelium and broth were separated by filtration through the cheesecloth, and the whole broth was extracted with EtOAc (1 liter) to afford broth extract (60 mg). A portion of this extract (55 mg) was subjected to Si gel flash chromatography. Elution was performed with n-hexane–EtOAc (stepwise, 0–100% EtOAc) to yield twenty collections (50 mL each). These collections were pooled on the basis of their TLC profiles to give five combined fractions. Fractions 2 and 4 were separated by medium pressure liquid chromatography (MPLC) (ODS) using a H2O–MeOH gradient elution to afford crude substrate (1) and two metabolites (2, 3), respectively. These were further purified by HPLC (Gemini C18, 4.6 × 250 mm, 1 mL/min) utilizing a 30 min gradient program of 50% to 100% MeOH in H2O to furnish compounds 1 (4.0 mg), 2 (6.5 mg), and 3 (5.5 mg), respectively.

Methyl 2-(5,6-dibromo-3,4-dihydroxyphenyl)acetate (2): A colorless solid; IR (KBr) νmax 3513, 3290, 1736, 1603, 1589, 1408, 1349, 1278, 1203, 1175, 1024, 983, 855, 736 cm−1; UV λmax (MeOH) (log ε) 294 (3.28) nm; 1H-NMR (d6- DMSO, 400 MHz) and 13C-NMR (d6-DMSO, 100 MHz): see Table 1 and Figures S2 and Figures S3 in the supplementary information; LR-EI-MS m/z: 342 [M (81Br2)]+ (8), 340 [M(79Br, 81Br)]+ (18), 338 [M(79Br2)]+ (9), 283 [342-CO2CH3]+ (44), 281 [340-CO2CH3]+ (90), 279 [338-CO2CH3]+ (44), 261 [342-81Br]+ (95), 259 [338-79Br]+ (100), 233(15), 231(17), 203 (6), 201 (13), 199 (7), 180 (14) (see Figure S1 in the supplementary information); HR-EI-MS m/z 341.8749 [M(81Br2)]+ (calcd for C9H8O4 81Br2, 341.8748) (+0.3 ppm), m/z 339.8772 [M(79Br, 81Br)]+ (calcd for C9H8O4 79Br81Br, 339.8769) (+0.9 ppm), m/z 337.8787 [M(79Br2)]+ (Calcd for C9H8O4 79Br2, 337.8789) (−0.7 ppm).

Methyl 2-(2,5-dibromo-3,4-dihydroxyphenyl)acetate (3): Spectroscopic data were virtually identical to those reported in the literature.8

Radical Scavenging Activity Against DPPH.16 Samples (compounds 1-3, and positive control, L-ascorbic acid) to be tested were dissolved in MeOH and the solution (160 mL) was dispensed into wells of a 96-well microtiter tray. 40 mL of the DPPH solution in MeOH (1.5 × 10−4 M) was added to each well. The mixture was shaken and left to stand for 30 min, and the absorbance of the resulting solution was measured at 520 nm with microplate reader (Packard Co., Spectra CountTM). The scavenging activity on DPPH radical was expressed as IC50, which is the concentration of the tested compound required to give a 50% decrease of the absorbance from that of the blank solution [consisting of MeOH (160 mL) and DPPH solution (40 mL)].