Dihydroisatropolone C from Streptomyces and Its Implication in Tropolone-Ring Construction for Isatropolone Biosynthesis

Isatropolones/isarubrolones are actinomycete secondary metabolites featuring a tropolone-ring in their structures. From the isatropolone/isarubrolone producer Streptomyces sp. CPCC 204095, 7,12-dihydroisatropolone C (H2ITC) is discovered and identified as a mixture of two interchangeable diastereomers differing in the C-6 configuration. As a major metabolite in the mycelial growth period of Streptomyces sp. CPCC 204095, H2ITC can be oxidized spontaneously to isatropolone C (ITC), suggesting H2ITC is the physiological precursor of ITC. Characterization of H2ITC makes us propose dihydrotropolone-ring construction in the biosynthesis of isatropolones.


Introduction
Isatropolones/isarubrolones are a complex of actinomycete secondary metabolites featuring a tropolone-ring in their structures, with isarubrolones being the non-enzymatic conjugation products of isatropolones with amines or amino acids [1]. Rubrolones and rubterolones are also actinomycete secondary metabolites featuring a tropolone-ring in their structures [2][3][4]. These metabolites share very similar structures and biosynthetic pathways [1,5,6]. In particular, the tropolone-rings in these metabolites are all constructed by enzyme-catalyzed oxidative rearrangement of poly-β-ketoacyl intermediates from type-II polyketide synthase (PKS) pathways [5]. However, tropolone-ring construction in the biosynthesis of these secondary metabolites has not been chemically confirmed, possibly because intermediates appearing in the tropolone-ring construction process are rather unstable.
We are interested in novel secondary metabolites, together with their biosynthesis, from actinomycetes [7][8][9]. Previously, we identified an isatropolone/isarubrolone producer Streptomyces sp. CPCC 204095 and discovered autophagic activity for the isatropolones/isarubrolones characterized from the strain. In a recent study of isatropolones/ isarubrolones and their production by Streptomyces sp. CPCC 204095, we identified 7,12dihydroisatropolone C (H 2 ITC) as a novel component of isatropolones, and determined it as the precursor of isatropolone C (ITC). In particular, characterization of H 2 ITC led us to present a revised tropolone-ring construction process in isatropolone (and rubrolone) biosynthesis that had been proposed before [1,5]. Previously, we identified the isatropolone/isarubrolone producer Streptomyces sp. CPCC 204095, with isatropolone C (Figure 1a) as a major component [10]. In the exploration of more metabolites with a tropolone-ring from the producer, we observed one HPLC pair peaks with identical but novel UV-visible absorption from an acidified ethyl acetate (EtOAc-5% acetic acid) extract of fermentation culture of the producer (Figures S1, S4 and S5). In pair peaks with identical but novel UV-visible absorption from an acidified ethyl acetate (EtOAc-5% acetic acid) extract of fermentation culture of the producer (Figures S1, S4 and S5). In addition, the acidified EtOAc extract produced a lower isatropolone C (ITC) peak than the EtOAc extract that gave a dominant ITC peak ( Figure S2), suggesting a relationship between the pair peaks and the ITC peak. The pair peaks aroused our interest in their identities. Figure 1. The structure of 7,12-dihydroisatropolone C (H2ITC, 1ab) and its oxidation to isatropolone C (ITC). (a) 7,12-dihydroisatropolone C and isatropolone C. (b) Keto-enol tautomerization of 1a and 1b in H2ITC, and their spontaneous oxidation to isatropolone C. The HPLC trace showed the pair peaks of H2ITC (1ab) and a small isatropolone C peak.

Structural Elucidation of 7,12-Dihydroisatropolone C (H2ITC)
The pair peaks exhibited the same molecular mass (m/z at 475 for [M+H] + ) and MS 2 fragmentation pattern in LC-MS analysis ( Figure S6), indicating two isomeric molecules in the pair peaks. When the pair peaks were separated by HPLC, each peak would change back instantly to the former pair peaks, and the latter peak was always higher than the former peak. Therefore, compounds in the pair peaks were purified as a whole (mixture of epimers) for structure elucidation ( Figure S3).
A procedure of silica gel column chromatography, ODS column chromatography and reverse phase HPLC was used to purify the compound (1ab) in the pair peaks. Specifically, elutes from column chromatography and HPLC were stored at low temperature (0 or −20 °C), as 1ab was able to change slowly to ITC at room temperature. The purified 1ab sample was kept at −20 °C, and its NMR assay was conducted at −4 °C.
Compound 1ab was obtained as light-yellow amorphous powder. HRESIMS established its molecular formula C24H26O10, two hydrogen atoms more than ITC ( Figure S7). Its NMR spectra indicated a major set of signals for 1ab, together with an expected minor set of signals for ITC ( Figure S8). Luckily, these minor signals did not cause much difficulty in recognizing the major signals. A close examination of major signals revealed that they were all paired ones, indicating that 1ab was a mixture of two diastereomers.
The 1 H and 13

Structural Elucidation of 7,12-Dihydroisatropolone C (H 2 ITC)
The pair peaks exhibited the same molecular mass (m/z at 475 for [M+H] + ) and MS 2 fragmentation pattern in LC-MS analysis ( Figure S6), indicating two isomeric molecules in the pair peaks. When the pair peaks were separated by HPLC, each peak would change back instantly to the former pair peaks, and the latter peak was always higher than the former peak. Therefore, compounds in the pair peaks were purified as a whole (mixture of epimers) for structure elucidation ( Figure S3).
A procedure of silica gel column chromatography, ODS column chromatography and reverse phase HPLC was used to purify the compound (1ab) in the pair peaks. Specifically, elutes from column chromatography and HPLC were stored at low temperature (0 or −20 • C), as 1ab was able to change slowly to ITC at room temperature. The purified 1ab sample was kept at −20 • C, and its NMR assay was conducted at −4 • C.
Compound 1ab was obtained as light-yellow amorphous powder. HRESIMS established its molecular formula C 24 H 26 O 10 , two hydrogen atoms more than ITC ( Figure S7). Its NMR spectra indicated a major set of signals for 1ab, together with an expected minor set of signals for ITC ( Figure S8). Luckily, these minor signals did not cause much difficulty in recognizing the major signals. A close examination of major signals revealed that they were all paired ones, indicating that 1ab was a mixture of two diastereomers.
Molecules 2022, 27, 2882 3 of 9 δH (7.12, s), δH (7.77, s)] in ITC. The structure of 1ab was established as 7,12-dihydrogen ated ITC (Figure 1a) based on detailed analysis of NMR spectra, especially the 1 H-1 H COSY correlations of H-7/H-12 and the HMBC correlations from H-7 to C-8 and C-9 (Fig  ure 2). Additionally, compared with ITC, the C-6, C-8, C-11 and C-9 resonances of 1ab were shielded by ΔδC −2.4, −1.6, −2.8 and −0.5 ppm, respectively, which further supported 1ab as 7,12-dihydroisatropolone C (H2ITC). The NMR data of H2ITC (1ab) were assigned as in Table 1 ( Figures S9-S21).    Keto-enol tautomerizations have been reported for rubrolones and rubterolones [5,6]. A keto-enol tautomerization at C-6 of H 2 ITC (1ab) may result in a mixture of two diastereomers (1a and 1b) differing in the chiral C-12 configuration (Figure 1b). The one with C-12 S configuration was designated as 1a, and the other one with C-12 R configuration as 1b. The ∆G calculated by Multiwfn at ωB97XD/TZVP level revealed only a small energy difference for 1a and 1b [11]. Population distribution was 66.29% for 1a and 33.71% for 1b when they reached equilibrium, approximately agreeing with peak area ratio of the pair peaks ( Figure 1b). Thus, 1a was assigned to the higher one, and 1b to the lower one, of the pair peaks.

Spontaneous Oxidation of 7,12-Dihydroisatropolone C (H 2 ITC, 1ab) to Isatropolone C (ITC)
The mechanism for spontaneous oxidation of H 2 ITC to ITC was also proposed as in Figure 1b, in which keto-enol exchange occurred at both C-6 and C-8 to generate H 2 ITC double-enol form. Like phenols/hydroquinones [12], H 2 ITC double-enol form is sensitive to air (O 2 ) oxidation, becoming ITC upon abstraction of two hydrogen atoms. The oxidation process was sped up at high temperature and pH, or slowed down at low temperature and pH. A careful examination of the 1 H-NMR spectrum of H 2 ITC revealed two lowfield signals at δ H 11.5 and 11.4 for active hydrogen atoms that were further proved by deuterium exchange (Figure S22), thus confirming the existence of the H 2 ITC double-enol form in H 2 ITC.
Spontaneous oxidation of H 2 ITC to ITC was then compared at: (a) 20 • C plus pH7.0, (b) −20 • C plus pH7.0 and (c) 20 • C plus pH8.0. A proportion of ca. 23% H 2 ITC was oxidized to ITC in 60 h at 20 • C plus pH7.0, and all H 2 ITC was oxidized to ITC in 40 min at 20 • C plus pH8.0, while H 2 ITC remained nearly unchanged for 60 h at −20 • C plus pH7.0 ( Figures S25-S27). In addition, a time-course monitoring of Streptomyces sp. CPCC 204095 indicated a slightly higher titer of H 2 ITC than ITC in the mycelial growth period (26-32 h) of the strain, then a higher titer of ITC than H 2 ITC afterwards due to H 2 ITC oxidation to ITC and its accumulation (Figure 3; ITC titer declined after 45 h due to ITC conjugating amines and amino acids for isarubrolone production). These results indicate H 2 ITC is the physiological precursor of ITC.
difference for 1a and 1b [11]. Population distribution was 66.29% for 1a and 33.71% when they reached equilibrium, approximately agreeing with peak area ratio of peaks (Figure 1b). Thus, 1a was assigned to the higher one, and 1b to the lower on pair peaks.

Spontaneous Oxidation of 7,12-Dihydroisatropolone C (H2ITC, 1ab) to Isatropolone
The mechanism for spontaneous oxidation of H2ITC to ITC was also propos Figure 1b, in which keto-enol exchange occurred at both C-6 and C-8 to generate double-enol form. Like phenols/hydroquinones [12], H2ITC double-enol form is s to air (O2) oxidation, becoming ITC upon abstraction of two hydrogen atoms. The tion process was sped up at high temperature and pH, or slowed down at low te ture and pH. A careful examination of the 1 H-NMR spectrum of H2ITC revealed tw field signals at δH 11.5 and 11.4 for active hydrogen atoms that were further pro deuterium exchange ( Figure S22), thus confirming the existence of the H2ITC doub form in H2ITC.
Spontaneous oxidation of H2ITC to ITC was then compared at: (a) 20 °C plus (b) −20 °C plus pH7.0 and (c) 20 °C plus pH8.0. A proportion of ca. 23% H2ITC w dized to ITC in 60 h at 20 °C plus pH7.0, and all H2ITC was oxidized to ITC in 40 20 °C plus pH8.0, while H2ITC remained nearly unchanged for 60 h at −20 °C plu ( Figures S25-S27). In addition, a time-course monitoring of Streptomyces sp. CPCC indicated a slightly higher titer of H2ITC than ITC in the mycelial growth period (2 of the strain, then a higher titer of ITC than H2ITC afterwards due to H2ITC oxid ITC and its accumulation (Figure 3; ITC titer declined after 45 h due to ITC conj amines and amino acids for isarubrolone production). These results indicate H2IT physiological precursor of ITC.  Isatropolones are able to conjugate amines to form isarubrolones. H 2 ITC was explored for its conjugation with NH 3 for 7,12-dihydroisarubrolone C formation. However, 7,12dihydroisarubrolone C was not identified from H 2 ITC with NH 3 , while isarubrolone C was produced from the reaction. A possible reason for this may be that 7,12-dihydroisarubrolone C is much more sensitive to oxidation than H 2 ITC, so it is oxidized immediately to isarubrolone C after its formation ( Figure S28).
It is very interesting that H 2 ITC can be spontaneously oxidized to ITC. A similar case has been reported for kibdelone B, a heterocyclic polyketide from Kibdelosporangium. Keto-enol tautomerizations of kibdelone B lead to aromatization of its ring C. Then, the aromatized intermediate, as a hydroquinone, undergoes spontaneous oxidation to convert kibdelone B with a C-C single bond in ring C to kibdelone A with a corresponding C-C double bond [13].

Dihydrotropolone-Ring Construction in Isatropolone Biosynthesis Implicated by H 2 ITC
As the most interesting and intriguing part in the biosynthesis of some actinomycete secondary metabolites with a tropolone-ring, Yan et al. proposed a tropolone-ring construction for rubrolones based on feeding with [ 13 C]-acetate [5]. Specifically, a monocyclic/aromatic intermediate derived from a poly-β-ketoacyl chain underwent complex oxidative rearrangement to construct the tropolone-ring (route 1, Scheme S1), in which two oxidations at C-11 and C-12 occurred. A similar tropolone-ring construction using a bicyclic/aromatic intermediate was proposed for isatropolone biosynthesis by Cai et al. (route 2, Scheme S1) [1]. However, neither tropolone-ring constructions is chemically proved.
The discovery of H 2 ITC from Streptomyces sp. CPCC 204095 and its spontaneous oxidation to ITC made us propose a dihydrotropolone-ring construction in isatropolone biosynthesis (Scheme 1). The construction involved only one oxidation at C-11 in the oxidative rearrangement. The dihydrotropolone-ring in H 2 ITC could be converted to the tropolone-ring in ITC by spontaneous oxidation.

General Experimental Procedures
HPLC was conducted on an Agilent system with a 1260 Quat-Pump and DAD detector. For analytical HPLC, a reverse-phase C18 column (YMC-Pack ODS-A column: 250 mm × 4.6 mm, S-5 μm, 12 nm) was used with a gradient solvent system from 15% to 70% CH3CN-H2O (0.1% HAc, v/v), 1.0 mL/min. For semipreparative HPLC, a reverse-phase C18 column (YMC-Pack ODS-A column: 250 mm × 10 mm, S-5 μm, 12 nm) was used with an isocratic solvent system for 30% CH3CN-H2O (0.1% HAc, v/v), 1.5 mL/min. UV spectra Genetic studies have demonstrated that two oxygenase genes are essential for the oxidative rearrangement of tropolone-ring construction in rubrolone biosynthesis [14]. However, the biochemical roles of the two oxygenases are still not clear, as substrates (the mono-or bi-cyclic/aromatic intermediates with polyketoacyl chains in Scheme 1) for the two oxygenases are very unstable and difficult to prepare, which prevents in vitro characterization of the biochemical reactions they catalyze.
Cai et al. conducted a heterologous expression of istG-R from an isatropolone gene cluster for aglycone biosynthesis in Streptomyces lividans, which resulted in the identification of two aglycones (compound 10 and its reduced derivative compound 9) [1]. Recently, Yijun Yan et al. reported multifunctional and non-stereoselective oxidoreductase RubE7/IstO for oxidation and reduction of these aglycones [15]. Our discovery of H 2 ITC seems to suggest that the primary aglycone in isatropolone biosynthesis may be a dihydrogenated 10. Its glycosylation (and hydroxylation) in Streptomyces sp. CPCC 204095 leads to the production of H 2 ITC, whose spontaneous oxidation generates ITC (Scheme 1, Figure S29).

General Experimental Procedures
HPLC was conducted on an Agilent system with a 1260 Quat-Pump and DAD detector. For analytical HPLC, a reverse-phase C18 column (YMC-Pack ODS-A column: 250 mm × 4.6 mm, S-5 µm, 12 nm) was used with a gradient solvent system from 15% to 70% CH 3 CN-H 2 O (0.1% HAc, v/v), 1.0 mL/min. For semipreparative HPLC, a reversephase C18 column (YMC-Pack ODS-A column: 250 mm × 10 mm, S-5 µm, 12 nm) was used with an isocratic solvent system for 30% CH 3 CN-H 2 O (0.1% HAc, v/v), 1.5 mL/min. UV spectra were acquired with a Thermo Scientific (New York, NY, USA) Evolution 201 UVvisible spectrophotometer. NMR data were collected using an ADVANCE HD 800 MHz and a Bruker Avance III HD 700 MHz spectrometer, where chemical shifts (δ) were reported in ppm and referenced to the acetone-d 6 solvent signal (δ H 2.05 and δ C 29.84, 206.33). ESIMS and MS 2 were conducted on a 1100-6410 Triple Quad from Agilent (Santa Clara, CA, USA). HRESIMS were conducted on a Thermo (New York, NY, USA) LTQ Orbitrap XL.

Extraction and Isolation of Compound 1ab
Fermentation culture (5 L) of Streptomyces sp. CPCC 204095 was extracted with an equal volume of EtOAc (5% HAc, v/v) two times. Specifically, each extraction took no longer than a few hours. The combined organic layer was vacuum dried below 30 • C, yielding a dark brown residue (4.85 g). The residue was loaded onto a preparative silica column for fractionation with CH 2 Cl 2 -MeOH (0% MeOH, 20 min; 1% MeOH, 20 min; 2% MeOH, 30 min; 3% MeOH, 60 min, v/v) at a constant flow rate of 35 mL/min, which yielded 14 fractions from F1-1 to F1-14. Each fraction was analyzed by TLC and HPLC. Fractions from F1-2 to F1-5 were found to contain compound 1ab.
Fractions from F1-2 to F1-5 were combined and concentrated under reduced pressure below 30 • C, yielding a dark brown residue.

Quantitative Assay of H 2 ITC (1ab) and Isatropolone C (ITC)
A freshly prepared 1ab solution was divided into two parts. One part was vacuumdried to obtain the quantity of 1ab in the solution. The other part was serially diluted for analytical HPLC, establishing a linear relationship of 1ab quantity with its pair peaks area. The linear relationship was used for HPLC assay of 1ab, or 1ab titer in Streptomyces sp. CPCC 204095. Quantitative assay of isatropolone C (ITC) was also conducted by HPLC in a way similar to H 2 ITC, according to Liu Xiaoyan et al. (Figures S23-S24) [16].

Spontaneous
Oxidation of H 2 ITC (1ab) to Isatropolone C (1) H 2 ITC (1ab) was dissolved in 3.0 mL 30% MeOH/H 2 O (pH7.0) at a concentration of 0.45 mg/mL. It was equally separated into two 2.0 mL glass vials. One vial was kept at 20 • C, and the other at −20 • C. The two vials were then analyzed by HPLC for 1ab and isatropolone C derived from spontaneous oxidation of 1ab in 20 and 60 h, respectively.
(2) H 2 ITC (1ab) was dissolved in 1.0 mL 30% MeOH/H 2 O plus 0.5 mL phosphate buffer (0.1 mol/L, pH8.0) at a concentration of 0.45 mg/mL within a 2.0 mL glass vial. It was kept at 20 • C, and then analyzed by HPLC for 1ab and isatropolone C derived from spontaneous oxidation of 1ab in 1.0 and 40.0 min, respectively.