The Isolation of a New S-Methyl Benzothioate Compound from a Marine-Derived Streptomyces sp.

The application of an HPLC bioactivity profiling/microtiter plate technique in conjunction with microprobe NMR instrumentation and access to the AntiMarin database has led to the isolation of a new 1. In this example, 1 was isolated from a cytotoxic fraction of an extract obtained from marine-derived Streptomyces sp. cultured on Starch Casein Agar (SCA) medium. The 1D and 2D 1H NMR and ESIMS data obtained from 20 μg of compound 1 fully defined the structure. The known 2 was also isolated and readily dereplicated using this approach.


Introduction
The HPLC bioactivity profiling/microtiter plate technique in conjunction with microprobe NMR instrumentation and access to the AntiMarin database [1] has been utilized by our group as a tool to enhance dereplication as well as to obtain a rapid NMR data acquisition for characterization of new metabolites by using less than 50 μg of purified material. The sensitivity of the technique to enhance structural elucidation by using only small amounts of a natural product has been described [2][3][4][5][6][7][8][9][10]. In our continuing efforts to rapidly characterize new bioactive metabolites, a marine-derived Streptomyces sp. was investigated for its bioactivity and chemical properties. Herein we report the structure of a new 1 by using 20 μg of material.
The first S-methyl benzothioate group of metabolites, 3, was produced in a broth culture of S. collinus [11,12]. S. collinus remained the only reported producer of this unusual structural type until a comparable structure, 4, was identified from a sclerotium-colonizing isolate of the fungus, Mortierella vinacea [13]. The production of 5 was also reported from a marine Streptomyces sp. [14] and, recently, from Phaeobacter gallaeciensis and Oceanibulbus indolifex [15]. To date, 3, 4, and 5 are the only secondary metabolites reported for the S-methyl benzothioate group of metabolites (see Scheme 1).

General Experimental
Procedures. NMR spectra were recorded on a Varian INOVA AS-500 spectrometer (500 and 125 MHz for 1 H and 13 C NMR, resp.), using the signals of the residual solvent protons and the solvent carbons as internal references (δ H 3.3 and δ C 49.3 ppm for CD 3 OD). A Protasis CapNMR microprobe was used for the microplate dereplication studies. HRESIMS were acquired using a Micromass LCT TOF mass spectrometer. MS/MS experiments were performed on a Bruker Daltonics Esquire 4000 system. Solvents used for extraction and isolation were distilled prior to use. Bioactivity assays were made using standard protocols [16,17]. The eluent from the DAD was split in a 1 : 10 ratio between the ELSD and the fraction collector configured to collect into a 96-well microtiter plate (15 s/well). A total of 88 wells were collected (2.5-24.5 min). A daughter plate was prepared by transferring an aliquot (5 μL) from each well of the master plate. After complete evaporation of the solvent, the wells in the daughter plate were analyzed for activity against P388 murine leukemia cells as described previously [16,17]. The assay established that cytotoxicity was correlated with the peak observed by HPLC/ELSD/UV. The well F10 of the dried master plate, containing the bioactive 1 was analyzed using capillary probe NMR spectroscopy. The content of well F10 was dissolved in CD 3 OD (7 μL) and transferred into the Protasis CapNMR microprobe. Calibrations have shown that this effectively transfers 6 μL of sample into the probe. Standard operating conditions were used to acquire 1D and 2D NMR spectra. The quantity of the compound was estimated according to the formula:

Isolation and
where MW is the actual molecular weight of the compound (ESMS), or an estimated value, #H is the number of protons included in the integration of the 1 H NMR spectrum, and CF is the calibration factor that had previously been determined from a standard solution containing quinine (30 μg in 6 μL) in the same CD 3 OD solvent.  [2]. An aliquot (750 μg) of the cytotoxic fraction was injected on to the HPLC, and the fractions were collected into a microtitre plate to yield 1 (4 μg; R t 15.5 min). In the second attempt, an aliquot (up to 1000 μg) of the cytotoxic fraction was injected on to the HPLC and the fractions were collected into a microtitre plate to yield reasonably pure 1 (20 μg; R t 15.5 min).

Results and Discussion
Streptomyces sp. was obtained from liquid portions of a New Zealand marine tunicate and grown on Starch Casein Agar medium (60 plates) for 30 days at 28 • C. Extraction with EtOAc yielded 45.7 mg of crude extract. This extract showed cytotoxic activity in a P388 assay (IC 50 383 μg/mL). Analysis by reverse-phase C 18 analytical HPLC revealed three main peaks, one major and two minor. The result from the HPLC MTT plate assay indicated that cytotoxicity was correlated with one of the minor peaks eluted over R t 15.0-18.5 min. In the first attempt, an aliquot of 750 μg of the cytotoxic fraction from the crude extract was chromatographed with collection of fractions into a microtitre plate. Well F10 of the microtitre plate, containing 4 μg of 1, was analysed using the CapNMR microprobe technique and ESIMS. The ESIMS  , an additional 20 μg of 1 from the cytotoxic fraction of the crude extract was obtained for further spectroscopic analysis. Although some minor impurities contributed to the 1 H NMR spectroscopic data, HSQC-DEPT, HMBC, and NOE spectroscopic data were sufficient to elucidate the structure of 1.
From the HSQC-DEPT spectrum, the chemical shifts of the protons at δ H 1.29, 2.07, 2.18, and 2.41 were correlated with the chemical shifts of their directly bonded carbons ( 1 J CH couplings). The HMBC spectrum clearly illustrated the presence of a hexasubstituted benzenoid system. The position of one aryl methyl group at δ H 2.07 was established by strong HMBC correlations with two oxygen-bearing carbons (C-2, δ C 149.2 and C-4, δ C 155.5) and with one higher field carbon (C-3, δ C 110.2), thus, placing this group between two oxygenated aromatic carbons. The position of the other aryl methyl group was further established by strong HMBC connections of the signal at δ H 2.18 with one oxygenbearing carbon (C-4, δ C 155.5) and two carbons (C5, δ C 115.5 and C-6, δ C 139.9). One of the two remaining aromatic carbons was substituted by an isopropyl group, proven by a long-range correlation of two methyl groups (δ H 1.29) with the carbon (C-6, δ C 139.9), leaving the C-1 position to be substituted by the carbonyl group (C-7, δ C 198.4), which had a long-range HMBC correlation to the methyl group (δ H 2.41). The long range couplings of this compound are shown schematically in Figure 1.
The positions of the isopropyl and the carbonyl group were further confirmed by an NOE experiment. When the methine proton (δ H 3.1, m) and methyl proton (δ H 2.18, s) signals were irradiated, the signal for CH 3   These data from the NOE experiment confirmed the relationship between the methine group (C-10, δ C 31.6) and the two methyls (C-11, δ C 20.7; C-12, δ C 20.7) and the attachment overall of the isopropyl group at C-6 (δ C 139.9). The 1 H and 13 C chemical shifts for the thiomethyl group of 1 were comparable to those reported for 3 [12] and 4 [13]. A complete list of the 1 H and 13 C chemical shifts for 1 is presented in Table 1. As there were no absolute matches for this structure found, the designated 1 was therefore considered a new structure and named S-methyl 2,4-dihydroxy-6-isopropyl-3,5-dimethylbenzothioate.
The major peak identified in the HPLC analysis of the crude extract was characterized as 2 as the 1 H NMR, UV, and MS data obtained matched those previously reported for bohemamine [19,20].

Conclusions
A new metabolite, S-methyl 2,4-dihydroxy-6-isopropyl-3,5dimethylbenzothioate, 1, and a known metabolite bohemamine, 2, were isolated and identified from Streptomyces sp. using the CapNMR technique. The structure of 1 was fully characterized by 1 H, HSQC, HMBC, and NOE NMR experiments. This new compound is only the fourth natural product reported to contain the S-methyl benzothioate group.