2-hex-Structure of Potential Dithiolopyrrolone Antibiotics Detected from the DART-ToF-MS Spectra of Saccharothrix algeriensis Extract

Dithiolopyrrolone antibiotics, produced by the Saharan mycelial bacterium Saccharothrix algeriensis , are known for their potent biological activities. Biochemical profiling of S. algeriensis culture extract was done by direct analysis in real time and time-of-flight mass spectrometry (DART-ToF-MS). No other study on dithiolopyrrolones by this technique has been published. Eleven dithiolopyrrolone derivatives: thiolutin, butyryl-pyrrothine/iso-butyryl-pyrrothine, senecioyl-pyrrothine/tigloyl-pyrroth-ine, valeryl-pyrrothine/iso-valeryl-pyrrothine, 2-methyl-3-pentenyl-pyrrothine/2-hexonyl-pyrrothine, iso-hexanoyl-pyrrothine and benzoyl-pyrrothine were characterised by their exact mass measurement and the corresponding molecular formula of each compound. The obtained results confirmed that DART-ToF-MS is an appropriate confirmatory technique for powerful and rapid screening, as well as characterisation of bacterial secondary metabolites.

Direct analysis in real time (DART) ion source is a recently developed method, which allows ionization of most organic molecules under atmospheric pressure. It has proven to be an efficient and reliable technique, allowing for screening complex mixtures such as biochemical samples without sample preparation or chromatographic separation. When combined with a high-resolution mass analyser, such as time-of-flight mass spectrometer, it produces a DART-ToF-MS system that is a powerful tool for characterising bioactive molecules. [28][29][30] DART-ToF-MS has rapidly emerged as a powerful technique for profiling the major constituents of various kinds of samples without need of prior separation or pretreatment. 31 Since this emerging technique was patented in the USA in 2005 32,33 , it has been successfully applied in various fields such as pharmaceuticals, health sciences, food products analysis, quality control, explosives, material analyses, phytochemicals, synthetic and organic compounds, forensic sciences, pesticides, and environmental studies. [34][35][36][37] The goal of this work was to develop a rapid and accurate method for characterisation of DTP antibiotics (all characterised by the possession of N 2 O 2 S 2 ) in S. algeriensis directly from its intact dichloromethane organic layer using DART ion source coupled to ToF-MS.

Producing actinobacterial strain
The actinobacterium Saccharothrix algeriensis NRRL B-24137 (= DSM 44581) was used throughout this study as reported by Bouras et al. 11 It was grown and maintained at 4 °C on slants of ISP2 solid medium containing (per litre of distilled water): 4 g dextrose (D-glucose), 4 g malt extract, 4 g yeast extract, and 18 g agar. The pH of the medium was adjusted to 7.0 with a 2 M NaOH solution prior to autoclaving at 121 °C for 20 min.

Culture medium
A basal semi-synthetic (BSS) medium was used for both preculture and production of antibiotics as reported by Bouras et al. 23 This medium consisted of (per litre of distilled water): 10 g dextrose, 2 g (NH 4 ) 2 SO 4 , 2 g NaCl, 0.5 g KH 2 PO 4 , 1 g K 2 HPO 4 , 0.2 g MgSO 4 • 7H 2 O, 5 g CaCO 3 , and 2 g yeast extract. The pH of the medium was adjusted to 7.0 using a 2 M NaOH solution before autoclaving. The dextrose was autoclaved separately to avoid the chemical reaction between nitrogen sources and reducing carbon sources that gives a brown colour (Millard reaction), and then added aseptically to the culture medium before inoculation.

Culture conditions
The DTP antibiotic production was investigated in the BSS medium. The preculture (250 ml Erlenmeyer flask containing 50 ml of the culture medium) was incubated for 48 h on a model G25 gyratory shaker (New Brunswick Scientific Co., New Jersey, USA) at 260 rpm and 30 °C. The preculture was then homogenised, and 5 ml was used to inoculate 100 ml of the same medium in 500-mm Erlenmeyer flask. The incubation temperature was kept at 30 °C throughout the 72 h fermentation period (in general, DTP antibiotic production reached a maximum at 72 h after inoculation).

Extraction of DTP antibiotics
The extraction of DTP antibiotics took place on the day of optimal production (after 3 days of fermentation). The culture broth was centrifuged for 20 min at 8000 ×g to remove the mycelium. The cell-free supernatant was extracted with an equal volume of dichloromethane. The organic layer was dehydrated with Na 2 SO 4 and evaporated to dryness by a rotary evaporator (Laborota 4000) under a vacuum at 40 °C. The resulting dry extracts were recuperated in 1 ml of methanol and subjected to analysis.

DART-ToF-MS conditions
The high-resolution mass spectra were recorded on an AccuTOF LC-plus JMS-T100 LP mass spectrometer from JEOL (Tokyo, Japan). This instrument consisted of a DART ion source from Ion Sense (Saugus, MA, USA) operated under atmospheric pressure, and a high-resolution time- tigloyl-pyrrothine (e) R: C 6 H 5 benzoyl-pyrrothine (f) R: (CH 2 ) 3 -CH 3 valeryl-pyrrothine (g) iso-hexonyl-pyrrothine (k) Fig. 1 -Structure of DTP antibiotics detected from the DART mass spectra of S. algeriensis extract of-flight mass analyser. All the obtained mass spectra were acquired using positive ionisation mode. The main parameters of DART ion source and ToF mass spectrometer were investigated and optimised in order to obtain the best mass resolution and signal intensity for the studied samples. The selected experimental conditions are given in detail further herein. The samples were vaporised and ionised using helium at a flow-rate of 4 l min −1 and heated at 250 °C. The discharge needle electrode of the DART ionisation source was set at a 3.0 kV potential, while the perforated and grid electrodes were set at 100 and 250 V voltages, respectively. For sample introduction using a glass rod, the gap distance separating the ionisation source outlet and the mass spectrometer inlet was 20 mm. For ion transfer from the DART source to the mass analyser, the potentials of orifice 1, orifice 2, and ring lens were set at 20, 5, and 13 V, respectively, and the potential of the radio-frequency guide was 500 V. The accurate mass spectra were acquired in the m/z range between 100 and 500 Da using a recording interval of 1 s. For mass drift compensation and accurate mass determination, a solution of polyethylene glycol (PEG 200) in methanol (200 µg ml −1 ) was used as calibration standard and injected before each sample. Data acquisition and processing were performed using the MassCenter (version 1.3) software from JEOL. In the investigated range, the mass spectra were obtained with a mass resolution between 3400 and 3900.
3 Results and discussion

Characterisation of the compounds in the mass spectra
Mass spectrometry is one of the most powerful analytical methods available for determining the structure of mi-crobial (bacterial and fungal) secondary metabolites. This powerful technique could be very useful for investigation of bacterial secondary metabolites. DART-ToF-MS has been used for the rapid screening of various natural complex samples, but its use in the characterisation of DTP antibiotics has not been reported previously.
In this study, the high-resolution mass spectrum of the dichloromethane organic layer of S. algeriensis was recorded using DART-ToF-MS. The accurate molecular weight of the main constituents was determined and their formula deduced, allowing the characterisation of several DTPs. To confirm the results, the contribution of the minor isotopes of carbon and sulphur was also checked to establish the molecular formula of each DTP (all characterised by the possession of N 2 O 2 S 2 ). Fig. 2 shows the high-resolution mass spectrum of S. algeriensis dichloromethane organic layer in positive ionisation mode. The peaks corresponding to the main constituents of the investigated extract were observed in the mass range m/z 100 to 500 Da, and after calibration and processing of the experimental results, the accurate molecular weights were obtained with five decimals. The main results obtained from interpretation of this spectrum are reported in Table 1; they show the experimental (measured) mass, the calculated mass, the mass difference (in mmu or mDa), and the proposed molecular formula for each indexed peak. Also, the unsaturated degree of each compound helps to predict the chemical structure of the expected antibiotic. The peaks corresponding to DTP species in dichloromethane organic layer were assigned to: thiolutin (m/z 228), butyryl-pyrrothine/iso-butyryl-pyrrothine (m/z 256), senecioyl-pyrrothine/tigloyl-pyrrothine (m/z 268), valeryl-pyrrothine/iso-valeryl-pyrrothine (m/z 270), 2-hexonyl-pyrrothine/2-methyl-3-pentenyl-pyrrothine (m/z 282), iso-hexanoyl-pyrrothine (m/z 284), and benzoyl-pyrrothine (m/z 290). All the characterised eleven DTPs were already reported to be produced by S. algeriensis. 1,3,[24][25][26][27] It was established that the positive ionisation in DART ion source can occur through three possible mechanisms by interaction with the heated and excited metastable helium atoms, with no or little fragmentation. Generally, the main observed peak corresponds to a protonated adduct ion [M+H] + ; it results from protonation by interaction with atmospheric water molecules. A second possible mechanism can occur with highly unsaturated compounds by loss of an electron and formation of a radical molecular ion M + . The third ionisation process is less likely, it corresponds to a loss of hydride which leads to an [M-H] + ion. Indeed, the detected DTPs showed mainly the protonated molecular ion [M+H] + , beside the lower ion-radical M +. . Fig. 3 shows enlarged portions of the high-resolution mass spectrum of the dichloromethane extract of S. algeriensis. The most intense peak at m/z 229.01070 corresponds to the protonated molecular ion of thiolutin with the molecular formula C 8 H 9 N 2 O 2 S 2 and 5.5 as unsaturation degree. In the same peak cluster, the peak at m/z 228.00005 shows the same molecular formula as thiolutin C 8 H 8 N 2 O 2 S 2 ; it is due to its molecular ion-radical. Due to the high intensity of these two molecular peaks of thiolutin, the contribution of minor isotopes in carbon and sulphur can also be observed at m/z 230.01227 and 231.00995. They correspond to the protonated molecular ion of thiolutin [M+H] + including either a carbon 13 or a sulphur 34 isotope, respectively. Another intense peak at m/z 269.04328 corresponds to the protonated molecular ion of senecioyl-pyrrothine (tigloyl-pyrrothine). Such as for thiolutin, the ion-radical molecular species is also present at m/z 268.03510 with the formula C 11 H 12 N 2 O 2 S 2 . However, due to overlapping with other DTP clusters, the minor peaks could not be observed. Similarly, the other dithiolopyrrolones characterised in Fig. 3 showed the presence of one or two molecular peaks M + and [M+H] + corresponding to butyryl-pyrrothine, valeryl-pyrrothine, 2-hexonyl-pyrrothine and benzoyl-pyrrothine. When the ion intensity is low, the small peaks, due to the minor isotopes, are masked by the background signals.
On the other hand, it should be noted that some DTPs such as butyryl-pyrrothine/iso-butyryl-pyrrothine, and senecioyl-pyrrothine/tigloyl-pyrrothine, etc., have exactly the same molecular formula and exact mass m/z 256 and 268, respectively. Since DART ion source is a "soft" ionisation technique, which produces essentially the protonated molecular ion of each species with no or little fragmentation, it cannot differentiate between isomers that will correspond to the same peak in the spectrum. Therefore, they cannot be distinguished on the basis of high-resolution mass spectrometry such as DART-ToF-MS technique. However, all these isomers have been reported to be produced by S. algeriensis. 1,[24][25][26][27] As reported previously, some DTPs were detected by HPLC only after addition of some precursors to enhance their production. 1,[24][25][26][27] However, it is important to mention that, in the present work, all the detected DTPs were easily observed by DART-ToF-MS, without precursor feeding.

Conclusion
The dichloromethane organic layer of S. algeriensis was investigated by DART-ToF-MS in positive ionisation mode. The interpretation of the high-resolution mass spectrum allowed determination of the accurate molecular weight and the possible formula of its main organic constituents. Among them, eleven DTP derivatives were characterised in the S. algeriensis extract without separation or sample treatment (except extraction). To the best of our knowl-

DECLARATION OF INTEREST STATEMENT
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.