Chrysomycins, Anti-Tuberculosis C-Glycoside Polyketides from Streptomyces sp. MS751

A new dimeric C-glycoside polyketide chrysomycin F (1), along with four new monomeric compounds, chrysomycins G (2), H (3), I (4), J (5), as well as three known analogues, chrysomycins A (6), B (7), and C (8), were isolated and characterised from a strain of Streptomyces sp. obtained from a sediment sample collected from the South China Sea. Their structures were determined by detailed spectroscopic analysis. Chrysomycin F contains two diastereomers, whose structures were further elucidated by a biomimetic [2 + 2] photodimerisation of chrysomycin A. Chrysomycins B and C showed potent anti-tuberculosis activity against both wild-type Mycobacterium tuberculosis and a number of clinically isolated MDR M. tuberculosis strains.


Introduction
Tuberculosis (TB) has overtaken HIV to be ranked as the primary cause of mortality in infectious diseases globally.In 2022, the WHO reported 7.5 million TB cases, including an estimated 410,000 new cases of multidrug-resistant or rifampicin-resistant TB (MDR/RR-TB) [1], which emphasises the urgent need for novel drugs and therapies to control and ultimately clear this increasing public health threat, especially in developing countries.During the last forty years, only two new drugs, bedaquiline and delamanid, were approved for the treatment of MDR-TB and extensive drug-resistant TB (XDR-TB) in 2012 and 2014, respectively [2].However, M. tuberculosis has developed an acquired resistance to these drugs very quickly [3].
Natural sources exhibit a wide range of chemical diversity and serve as an exclusive reservoir for drug discovery, with approximately 50% of clinical drugs derived from natural products or their synthetic analogue [4].Microbial natural products have played an important role in delivering streptomycin, rifamycins, capreomycin, kanamycin, and dycloserine for the treatment of tuberculosis throughout human history.In our previous work, new antitubercular bioactive compounds, such as abyssomicins, brevianamides, lobophorins, and pluramycins, were characterised by our microbial natural library [5][6][7][8].More specifically, a crude extract from a Streptomyces strain MS751 (obtained from a sediment sample retrieved at a depth of −3000 m in the South China Sea) exhibited potent antitubercular activity by using Bacillus Calmette-Guérin (BCG Pasteur 1173P2, an attenuated Mycobacterium bovis strain, with GFP expression vector pUV3583c-GFP) as an indicator.

Identification, Fermentation of Strain MS751, and Purification of New Chrysomycins
The strain MS751 was identified as Streptomyces sp.By an analysis of the 16S rRNA gene sequence (Figure 1D, GenBank Accession Number: KY688100) and morphology (Figure 1A-C).A large-scale culture was performed with 30 × 1 L flasks, each charged with a 300 mL AM2 medium.After incubating for 10 d, the fermentation broth was combined and centrifuged (8000 rpm, 10 min) to yield the supernatant and mycelia.The supernatant was extracted with an equal volume of ethyl acetate (×3), and the mycelia was extracted with 1 L acetone (×3).All the organic extracts were dried in a vacuum and combined to yield the residue.The residue was subjected to reversed-phase C18 vacuum chromatography, followed by reversed-phase HPLC, to yield chrysomycins A-C and F-J (1-8) (Figure 2A).Chrysomycin F (1) was characterised as a new dimer of the chrysomycin class of type II polyketide by detailed spectroscopic analysis.Chrysomycins G-J (2-5) were identified as new monomeric chrysomycins, and 6-8 were identified as chrysomycins A-C, which were previously identified from Streptomyces sporoverrucosus [9].In fact, chrysomycins A and B were first isolated from Streptomyces A-419 in 1955 [10].
important role in delivering streptomycin, rifamycins, capreomycin, kanamycin, and dycloserine for the treatment of tuberculosis throughout human history.In our previous work, new antitubercular bioactive compounds, such as abyssomicins, brevianamides, lobophorins, and pluramycins, were characterised by our microbial natural library [5][6][7][8].More specifically, a crude extract from a Streptomyces strain MS751 (obtained from a sediment sample retrieved at a depth of −3000 m in the South China Sea) exhibited potent antitubercular activity by using Bacillus Calmette-Guérin (BCG Pasteur 1173P2, an attenuated Mycobacterium bovis strain, with GFP expression vector pUV3583c-GFP) as an indicator.

Identification, Fermentation of Strain MS751, and Purification of New Chrysomycins
The strain MS751 was identified as Streptomyces sp.By an analysis of the 16S rRNA gene sequence (Figure 1D, GenBank Accession Number: KY688100) and morphology (Figure 1A-C).A large-scale culture was performed with 30 × 1 L flasks, each charged with a 300 mL AM2 medium.After incubating for 10 d, the fermentation broth was combined and centrifuged (8000 rpm, 10 min) to yield the supernatant and mycelia.The supernatant was extracted with an equal volume of ethyl acetate (×3), and the mycelia was extracted with 1 L acetone (×3).All the organic extracts were dried in a vacuum and combined to yield the residue.The residue was subjected to reversed-phase C18 vacuum chromatography, followed by reversed-phase HPLC, to yield chrysomycins A-C and F-J (1-8) (Figure 2A).Chrysomycin F (1) was characterised as a new dimer of the chrysomycin class of type II polyketide by detailed spectroscopic analysis.Chrysomycins G-J (2-5) were identified as new monomeric chrysomycins, and 6-8 were identified as chrysomycins A-C, which were previously identified from Streptomyces sporoverrucosus [9].In fact, chrysomycins A and B were first isolated from Streptomyces A-419 in 1955 [10].S1a).Detailed analysis of the 1D and 2D NMR (DMSO-d 6 ) data for 1 (Table 1) confirmed a dimeric structure, and each of the dimeric elements incorporated a 1,2,3,4-tetrasubstituted benzene, a 1,2,3,5-tetrasubstituted benzene, an isolated aromatic proton, an isolated CH 2 CH moiety, two methoxy groups, and a dimethyl-substituted pyranose moiety (Figure 2A), which could be assembled to form a chrysomycin derivative.The side chain at C8 was the moiety of CH 2 CH, based on 2D HMBC NMR data (Figure S1b-f, Table 1).The HMBC correlations from H-18/18' to C-7/7', C-8/8' and C-9/9', from H 2 -19/19' to C-8/8', from H-18 to C-18', and from H-18' to C-18 confirmed the dimeric structure of 1 (Figure 2B).The ROESY signals from H-18/18' to H 2 -19/19' revealed the trans conformation for the four-membered ring moiety (Figure S1g,h).The H 2 -19/19' should show an identical signal in the 1 H-NMR spectrum, if it is a trans-1,3-substitued dimer.Interestingly, two different signals were observed (2.33 and 2.45 ppm), which indicated that the dimer is a trans-1,2-substitued metabolite.All of the above-mentioned spectrum data supported the assignment of the dimeric structure for 1, as shown in Figure 2A.However, two possible types of trans configuration (1a and 1b) remain to be further defined.conformation for the four-membered ring moiety (Figure S1g,h).The H2-19/19' should show an identical signal in the 1 H-NMR spectrum, if it is a trans-1,3-substitued dimer.Interestingly, two different signals were observed (2.33 and 2.45 ppm), which indicated that the dimer is a trans-1,2-substitued metabolite.All of the above-mentioned spectrum data supported the assignment of the dimeric structure for 1, as shown in Figure 2A.However, two possible types of trans configuration (1a and 1b) remain to be further defined.An HRESI(+)MS measurement of 2 detected a pseudo-molecular ion at m/z 513.1758 ([M+H] + ), which indicated a molecular formula of C 27 H 28 O 10 (Figure S2a) and implied 14 degrees of unsaturation, suggestive of an oxidised analogue of the monomer of chrysomycin B [9] (Figure S2b-f, Table 2).The 1D NMR data of the hydroxymethyl group [δ C /δ H 62.2/4.70 (d, J = 5.4 Hz)] and [δ H 5.54 (t, J = 5.4 Hz)], together with the characteristic HMBC correlations from H 2 -18 to C-7, C-8, and C-9, supported the hydroxyl group at C-18 (Figure 2B), which allowed the structure to be assigned as shown (Figure 2A).
An HRESI(+)MS measurement of 5 detected a pseudo-molecular ion at m/z 539.1913 ([M+H] + ), which indicated a molecular formula of C 29 H 30 O 10 (Figure S5a), requiring 15 degrees of unsaturation.A comparison of the NMR data for 5 with 2 (Figure S5b-f 2B), the assignment of structure to be shown was supported (Figure 2A).

Biomimetic [2 + 2] Photodimerisation of Chrysomycin A
Chrysomycin F (1) is the bis-chrysomycin derivative possessing a cyclobutane moiety formed from chrysomycin A. To further figure out whether the stereochemistry of C-18/18' on chrysomycin F was as indicated in 1a or 1b, chrysomycin A was subjected to various direct [2 + 2] photocycloaddition conditions (Table S1) [11][12][13][14][15].As shown in Figure 3, [2 + 2] photodimerisation of chrysomycin A could be realised under visible light at 75-80 • C with CH 2 Cl 2 as the solvent.After 60 h, chrysomycin F with the trans-1,2-substituted framework was obtained selectively in a 40% yield, and NMR spectra of the synthesised chrysomycin F fully matched with the ones for a natural isolate (Figure S6a,b, Table S2).At this point, two different trans-1,2-substitued isomers (1a and 1b) should be formed in the direct photodimerisation, since the C-8 vinyl group is remote from the chiral sugar moiety.After a careful screening of UPLC separation conditions, we found both natural and synthetic chrysomycin F contained two isomers, i.e., 1a:1b ratio 1:1 (Figure S7).Fortunately, these two isomers could be separated by preparative HPLC.This biomimetic [2 + 2] photodimerisation revealed that the cyclobutane ring of chrysomycin F could be directly generated from the C-8 vinyl group of chrysomycin A (Figure S8a,b, Table S3).
Chrysomycin F (1) is the bis-chrysomycin derivative possessing a cyclobutane moiety formed from chrysomycin A. To further figure out whether the stereochemistry of C-18/18' on chrysomycin F was as indicated in 1a or 1b, chrysomycin A was subjected to various direct [2 + 2] photocycloaddition conditions (Table S1) [11][12][13][14][15].As shown in Figure 3, [2 + 2] photodimerisation of chrysomycin A could be realised under visible light at 75-80 °C with CH2Cl2 as the solvent.After 60 h, chrysomycin F with the trans-1,2-substituted framework was obtained selectively in a 40% yield, and NMR spectra of the synthesised chrysomycin F fully matched with the ones for a natural isolate (Figure S6a,b, Table S2).At this point, two different trans-1,2-substitued isomers (1a and 1b) should be formed in the direct photodimerisation, since the C-8 vinyl group is remote from the chiral sugar moiety.After a careful screening of UPLC separation conditions, we found both natural and synthetic chrysomycin F contained two isomers, i.e., 1a:1b ratio 1:1 (Figure S7).Fortunately, these two isomers could be separated by preparative HPLC.This biomimetic [2 + 2] photodimerisation revealed that the cyclobutane ring of chrysomycin F could be directly generated from the C-8 vinyl group of chrysomycin A (Figure S8a,b, Table S3).

Anti-Tuberculosis Activity Evaluation
Chrysomycin A and its synthetic derivatives were reported to display significant antimicrobial activity against several M. tuberculosis indicators including BCG and M. tuberculosis H37Rv/Hr1/Hr2/Hr3/Hr4/Hr5 [16].Thus, we also evaluate the anti-TB activities of isolated natural chrysomycins.Chrysomycins B and C displayed reduced inhibition against several M. tuberculosis indicators compared to chrysomycin A, with MICs ranging from 1.56 to 6.25 µg/mL (Table 3).In addition, these two compounds also exhibited antibacterial activity against gram-positive bacteria Staphylococcus aureus (ATCC 6538), methicillin-resistant S. aureus (MRSA), and S. pneumoniae (ATCC 49619), with MICs ranging from 3.12 to25 µg/mL but no inhibition against C. albicans.

Anti-Tuberculosis Activity Evaluation
Chrysomycin A and its synthetic derivatives were reported to display significant antimicrobial activity against several M. tuberculosis indicators including BCG and M. tuberculosis H37Rv/Hr1/Hr2/Hr3/Hr4/Hr5 [16].Thus, we also evaluate the anti-TB activities of isolated natural chrysomycins.Chrysomycins B and C displayed reduced inhibition against several M. tuberculosis indicators compared to chrysomycin A, with MICs ranging from 1.56 to 6.25 µg/mL (Table 3).In addition, these two compounds also exhibited antibacterial activity against gram-positive bacteria Staphylococcus aureus (ATCC 6538), methicillin-resistant S. aureus (MRSA), and S. pneumoniae (ATCC 49619), with MICs ranging from 3.12 to25 µg/mL but no inhibition against C. albicans.

Discussion
Chrysomycins belong to the benzol[d]naphthol [1,2b]pyran-6-one-C-glycoside antibiotics, which contain different side chains at the C-8 position and glycosides at the C-4 position.Fischer et al. reported the complete gene cluster for gilvocarcin V, of which there is a D-fucofuranose at the C-4 position [17].Shepherd et al. created more gilvocarcin analogues by engineering the glycosyltransferase of GilGT [18], and Pahari synthesised the defucogilvocarcin M by in vitro enzymatic methods [19].Previously, only chrysomycins A-C were reported with the confirmed structure, while chrysomycins D and E were only identified by LCMS/UV/DNP [9,10,20].
Chrysomycins exhibit diverse biological activities, including antibacterial [9], antitumor [21,22], anti-bacteriophage [10], and anti-neuroinflammatory activities [23].Notably, studies have shown that chrysomycin A and its natural congeners possess significant antibacterial properties, particularly against pathogenic bacteria such as M. tuberculosis and S. aureus [24,25].Among them, chrysomycin A demonstrated superior efficacy in treating methicillin-resistant S. aureus (MRSA) compared to vancomycin hydrochloride, with a MIC of 0.5 µg/mL versus 2.0 µg/mL for vancomycin hydrochloride [24].In our earlier research, chrysomycin A exhibits potent anti-TB activity, with a MIC of 0.4 µg/mL against MDR-TB strains [16].Structure-activity relationship studies of Chrysomycins have highlighted the crucial role of the sugar moiety in their anti-TB activity and drug resistance [16].In this study, we investigated the antimicrobial properties of isolated natural chrysomycins.Our findings that modifying the 8-vinyl group in chrysomycin A may lead to a reduction or complete loss of its antimicrobial efficacy.This observation is supported by the absence of inhibition against MTB and several other indicators when testing dimer chrysomycin F (1), as well as the 8-vinyl oxidised products chrysomycins H, I (3, 4).Chrysomycins B (7) and C ( 8) displayed reduced anti-TB activity compared to chrysomycin A, with MICs ranging from 1.56-6.25 µg/mL, due to the 8-vinyl group being reduced to ethyl or replaced with methyl [16].
Cyclobutane-containing natural products have been isolated from a wide range of species, including bacteria, fungi, plants, and marine invertebrates [26,27].Many of these natural products are thought to be generated by photochemical reactions [28].Recently, a cyclobutane-containing gilvocarcin-type aryl-C-glycoside dimer digilvocarcin A was discovered from the soil-derived Streptomyces sp.OUCMDZ-945.The structure closely resembles that of the chrysomycin F obtained in this study, with the exception of the varying C-glycosides [29].The unique 1,2-diaryl substituted cyclobutane core of chrysomycin F and digilvocarcin A rarely existed in known cyclobutane-containing natural products [26,27,30,31].Since chrysomycin F (1) displayed as a pair of trans-1,2-substitued isomers, and that it could be formed from chrysomycin A (6) through a light-induced [2 + 2] cycloaddition just like digilvocarcin A [29], it is more likely a non-enzymatic product generated during the isolation process.Investigations into the chrysomycin biosynthetic gene cluster of Streptomyces sp.MS-751 may gain further information about this question.

General Experimental Section
1 H NMR spectra were acquired using a Brucker 400 MHz, 600 MHz, or 700 MHz spectrometer (Billerica, MA, USA) with DMSO-d 6 as the solvent unless otherwise stated. 13C NMR spectra were acquired using a Brucker 176 MHz or 201 MHz spectrometer (with complete proton decoupling).Electrospray ionisation mass spectra (ESI-MS) were recorded on an Agilent 1100 Series LC-MS (Santa Clara, CA, USA), in both positive and negative ion modes, and UPLC-MS on a Waters (Milford, MA, USA) UPLC-MS system equipped with a Waters BEH C18 1.7 µm column (2.1 × 50 mm), eluted with HPLC-grade water (solvent A) and HPLC-grade CH 3 CN (solvent B) with a flow rate of 0.3 mL/min at room temperature.HPLC was performed using an Agilent 1200 Series (Santa Clara, CA, USA) separations module equipped with a DAD detector fraction collector, controlled using ChemStation.High-resolution mass spectra were obtained using an Agilent 1200 HPLC/6520QTOFMS high-resolution mass spectrometer (Santa Clara, CA, USA) or Bruker APEX Flash chromatography (Billerica, MA, USA).Optical rotations were recorded on an Insmark ® digital polarimeter (Shanghai, China) at 589 nm and are recorded as [α] 25 D .

Characterisation of Streptomyces sp. Strain MS751
Strain MS751 (Streptomyces sp.) was obtained from a sediment sample that was collected from the South China Sea and was characterised as a Streptomyces sp. using 16S rRNA gene sequence analysis (GenBank accession no.KY688100).The strain has been submitted to the China General Microbiological Culture Collection Centre (accession no.6299), which is a member of the World Data Centre for Microorganisms (WDCM 550).

Synthesis, Isolation, and Characterisation of trans-Dimers 1a and 1b
To a sealed tube were added chrysomycin A (4.5 mg, 0.0089 mmol) and 0.5 mL of CH 2 Cl 2 under argon.The solution obtained was agitated at a temperature range of 75-80 • C with the irradiation of an 11 W compact fluorescent lightbulb.After 60 h, the reaction was cooled and then concentrated.The residue was purified by silica gel column chromatography (CH 2 Cl 2 /MeOH = 50/1 to 10/1) to give chrysomycin F (1.8 mg, 40%, 1a:1b = 1:1) as a light-yellow powder.The two trans-dimers, 1a and 1b, were further separated by preparative HPLC (XBridge C18
The assay for anti-M.smegmatis was performed according to the Clinical and Laboratory Standards Institute, and some modifications were made.M. smegmatis mc2155 was precultured at 37 • C in Middlebrook 7H9 broth (Difco) for 24 h.Then, it was diluted with 7H9 broth to yield a broth of CFU of 10 6 .The compounds were prepared at 100× stocks in DMSO.Serial dilutions of the compounds were prepared in the same solvent and added to the wells in a 2 mL volume.Isoniazid (purchased from Amresco (Solon, OH, USA), ultrapure grade) was used as a positive control for this assay.The plates were placed in a 37 • C incubator for 16 h.Subsequently, 10 µL of AlamarBlue was added and the plates were further incubated for 2 h.The inhibitory effect was determined by measuring fluorescence using an envision 2103 multilabel reader (Perkin-Elmer Life Sciences, Shelton, CT, USA) with excitation at 530 nm and emission at 590 nm.MIC here is defined as the minimum concentration of compound that inhibits more than 90% of bacterial growth reflected by ODs.
Assays for anti-Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Staphylococcus pneumoniae ATCC 49619 screening assays were performed according to the CLSI Antimicrobial Susceptibility Testing Standards and our previous report [6].Antifungal bioassays were performed according to a modified protocol of the CLSI M-27A [33] methods using the fungus Candida albicans (SC 5314).

Conclusions
In conclusion, we have isolated and fully characterised a number of new C-glycoside polyketide chrysomycin natural products using the bioassay-guided separation from Streptomyces sp.MS751.Chrysomycin F (1) shows a unique dimeric structure containing a trans-1,2-disubstitued cyclobutane motif, which was further confirmed by extensive spectroscopic analysis, as well as a biomimetic transformation from the monomeric precursor through [2 + 2] photodimerisation.Chrysomycins B and C show potent anti-TB activities against a panel of clinically isolated MDR TB strains.Our efforts to develop novel anti-TB drug candidates sourced from chrysomycins are presently underway and will be disclosed in due course.

Figure 1 .
Figure 1.Characterisation of strain MS751.Colony front side (A), back side (B) of the strain MS751 plate, and (C) its scanning electron micrograph (Bar, 5 µm).(D) Neighbour-joining phylogenetic tree of strain MS751 based on 16S rRNA gene sequence generated by Mega4.0.Numbers at nodes indicate levels of bootstrap support (percent) based on a neighbour-joining analysis of 1000 resampled datasets; only values >50% are given.NCBI accession numbers are given in parentheses.Bar, 0.01 nucleotide substitutions per site.Also, Kutzneria albida was chosen as the outgroup.

Table 3 .
Antimicrobial activity of compounds 1