Sporothioethers: deactivated alkyl citrates from the fungus Hypomontagnella monticulosa

Submerged cultivation of Hypomontagnella monticulosa MUCL 54604 resulted in formation of a stereoisomeric mixture of new sulfur-containing sporothriolide derivatives named sporothioethers A and B. The presence of the 2-hydroxy-3-mercaptopropanoic acid moiety attenuates the antimicrobial activity in comparison to the precursor sporothriolide suggesting a detoxification mechanism. However, moderate effects on biofilms of Candida albicans and Staphylococcus aureus were observed for sporothriolide and sporothioethers A and B at concentrations below their MICs.

Alkyl citrates are a structurally broad class of natural products requiring an alkyl citrate synthase (ACS) for their biosynthesis. 18][9] The spo biosynthetic gene cluster (BGC) is responsible for production of 8 (Scheme 1B). 10,11Decanoyl-CoA, produced by a dedicated fatty acid synthase (FAS), is used by the ACS SpoE to form the alkyl citrate 4. Subsequent enzymatic dehydration to 5, decarboxylation to 6, hydroxylation to 7 and lactonization then forms the furofurandione sporothriolide 8 (Scheme 1B).Spontaneous Diels-Alder (DA) reaction with the polyene trienylfuranol A 9, then results in formation of the sporochartines 10-13 (Scheme 1B).Sporothriolide 8 itself possesses useful antifungal activity. 9or example it has been shown to protect pepper seedlings from the plant pathogen Botrytis cinerea. 12Sporochartines A-D 10-13, in turn, are potent cytotoxins active against human cancer cell lines. 11ere, we report the isolation, synthesis, structure elucidation, and biological testing of two new sulphur-containing sporothriolide derivatives, termed sporothioether A 14 and B 15 (Fig. 1) from H. monticulosa MUCL 54604. 10n previous work, sporothriolide 8 was isolated from extracts of H. monticulosa MUCL 54604 aer cultivation in PDB medium. 10However, when H. monticulosa was cultivated in DPY medium, sporothriolide 8 was not detected.DPY was therefore previously used as a non-producing condition during transcriptomic analysis of the spo BGC. 10 However, analysis of the transcriptomic data did not reveal signicant down-regulation of the spo genes in DPY media, and LCMS analysis of the culture extract revealed the presence of a new metabolite under these conditions.Purication of the new metabolite using preparative reversed-phase chromatography (ESI, Fig. S1 †) resulted in isolation of an inseparable 3 : 2 mixture of isomers  In the 1 H-NMR and HSQC spectra of the 14 and 15 mixture (Table S1  2D NMR spectra (ESI, Fig. S7-S10 †) showed the core structures of 14 and 15 to be the sporothriolide scaffold (Fig. 1A and  B).In addition to this core structure, both new compounds harbour a 2-hydroxy-3-mercaptopropanoic acid moiety attached to C-13 of the core via a thioether bond.This linkage was supported by the HMBC correlation between CH 2 -14 and C-13 (Fig. 1A and B).The presence of the thioether was additionally conrmed by the distinctive 1 H/ 13  ).However, the stereochemistry at position C-3 differs in sporothioether A 14 and B 15. ROESY correlations (Fig. 1, A, B) showed that H-3, H-2, H-5 and H-6 are all syn in 14, but in 15 H-3 is anti to H-2, H-5 and H-6, as no ROESY correlation is observed.An energy-minimised model structure of the anti diastereomer 15 suggested a dihedral angle of 105°for H-3/H-2 consistent with the observed 3 J 2,3 value of 1.5 Hz (Fig. 1C).In contrast, an energy-minimised model of diastereomer 14 has a dihedral angle of 42°consistent with the observed 3 J 2,3 value of 9 Hz (Fig. 1C).Assuming the same absolute stereochemistry at C-2, C-5 and C-6 as in the parent compound sporothriolide 8, we conclude that in syn diastereomer 14, C-3 is R-congured, and in anti diastereomer 15, C-3 is S-congured.
In order to determine the conguration of the stereocenter at C-15 we initially attempted to form Mosher's esters of the 15-OH. 13However, this approach led to inconclusive results due to the complexity of the spectra of the resulting mixed diastereomers.In an alternative strategy, 15S and 15R sporothioether derivates were chemically synthesized for comparison by NMR spectroscopy (Scheme 2A).
Commercially available oxirane carboxylate enantiomers 18 and 19 were subjected to regioselective ring opening over two steps by treatment with triphenylmethanethiol to give intermediates 20 and 21, followed by acidic deprotection to give 22 and 23.Sporothriolide 8 was puried from H. monticulosa and reacted under basic conditions with either 22 or 23.The resulting sporothioethers were puried by reversed-phase chromatography as inseparable mixtures of diastereomers in each case.Analysis of the 1 H NMR data (Scheme 2B) of the synthetic products revealed that both contain a mixture of sporothioether stereoisomers, as indicated by two sets of signals with an integral ratio of 3 : 2. The major compound in both mixtures is 3S and the minor component is 3R.This was indicated by the 3 J 2,3 value of 1.6 Hz for the 3S congured sporothioethers.For the 3R congured stereoisomers 3 J 2,3 values of ∼9 Hz were observed, the same as for the natural 3R sporothioether 14. 1 H NMR chemical shis of the synthetic (15S, 3RS)-sporothioether mixture matched the signals of the fungal sporothioether mixture 14 and 15, whereas the signals of the synthetic (15R, 3RS)-sporothioether mixture 24 and 25 differed (Scheme 2B).Therefore, sporothioether A 14, the major compound in the natural mixture, is identied as (3R, 15S) and the minor compound sporothioether B 15 is identied as (3S, 15S).The non-natural synthetic diastereomers were now named sporothioether C 24 (3R, 15R) and sporothioether D 25 (3S, 15R).
It seems likely that the sporothioethers are biosynthesised by addition of 2S-2-hydroxy-3-mercaptopropanoic acid 22 to sporothriolide 8 itself in vivo.Formation of an epimeric mixture at C-3 may indicate that this is not an enzymatic process.To test this hypothesis sporothriolide 8 was incubated with 3-mercaptopropionic acid 26 under physiological conditions in phosphate buffer at pH 7.5.LCMS analysis revealed the formation of a new compound with an m/z of 343 [M − H] − , which corresponds to the mass of the expected product 30 and 31 (ESI, scheme S1 †).
The inseparable mixtures of 14 and 15, and 24 and 25, were assayed for biological activity (see ESI †).In cytotoxicity assays no activity was detected for the sporothioethers against KB-3-1 and L929 cell lines in the tested range (37-0.63mg mL −1 ).Similarly, in previous studies no cytotoxic activity was detected for sporothriolide 8 itself. 9Both, the sporothioether mixture 14 and 15 and sporothriolide 8, were tested in biolm inhibition and dispersion assays against Stapylococcus aureus and Candida albicans (Table 1).Sporothriolide 8 displayed signicant activity against the formation of S. aureus biolms at subtoxic concentrations (MIC (S. aureus) > 66.6 mg mL −1 ), meanwhile only weak inhibitory effects on preformed biolm of S. aureus were observed.
In addition, the sporothioether mixture 14 and 15 displayed moderate inhibitory activity against biolm formation of C. albicans (Table 1).In antimicrobial assays the mixtures of 14 and 15 and 24 and 25 were tested against selected bacteria and fungi in the same assays that were used previously to assess the bioactivity of sporothriolide 8. 9 The minimum inhibitory concentrations (MIC, Table 2) showed that sporothriolide 8 possesses moderate antimicrobial activity against the tested microorganisms as reported previously, 9 but sporothioethers 14 and 15 were inactive against most organisms and had signicantly attenuated effects vs. Mucor hiemalis and Schizosaccharomyces pombe.Sporothioethers 24 and 25 were inactive against all tested microorganisms.
The sporothioethers 14 and 15 appear to arise by spontaneous addition of 2S-2-hydroxy-3-mercaptopropionate to sporothriolide.This is supported by observation of facile addition of 3-mercaptopropionate to 8 in the absence of biological catalysts.This is also consistent with the S-conguration of the 2hydroxy-3-mercaptopropanoic acid moiety in other natural compounds, such as berkeleylactone A 28, 14 sumularin C 30, 15 and thiopleurotin 32 (Fig. 2). 16or the latter compound, cysteine was identied as the origin for the sidechain 22 and this is also likely to be the case for the sporothioethers. 16,17In our hands the 3-epimeric forms of the sporothioethers were inseparable.However, it is clear that material isolated from biological and synthetic sources contains differing proportions of the epimers (Scheme 2B).This may be explained by preferential extraction, or preferential degradation, of one epimer from the biological source.Some of the 2-hydroxy-3-mercaptopropanoic acid moietybearing natural compounds have potent bioactivities, whereas others are signicantly less toxic in comparison to their originating compounds.For example, sumalarin C 30 is a potent cytotoxin vs. tumor cell lines, comparable to its congener 10,11dehydrocurvularin 29 (Fig. 2). 15,18Berkeleylactone A 28 displays strong antibacterial effects against Gram-positive bacteria, even though it is denoted as pro-drug of antibiotic A26771B 27.Here it is hypothesised that the sulfur side chain disrupts the g-ketoa,b-unsaturated carboxyl, which is thought to be important for bioactivity of the macrolide antibiotic 27 (Fig. 2). 19,20n contrast, thiopleurotinic acid A 32, derived from the cytotoxic and antibacterial compound dihydropleurotinic acid 31 does not possess bioactivity (Fig. 2). 16Another case in which the addition of 2-hydroxy-3-mercaptopropanoic acid 22 is reported as a detoxication mechanism in fungi is antimicrobial a Microporenic acid A, 74 ± 12 (250 mg mL −1 ), 75 ± 6 (7.8 mg mL −1 ), 42 ± 7 (3.9 mg mL −1 ).b Farnesol: 75 ± 9 (250 mg mL −1 ), 51 ± 7 (31.3mg mL −1 ), 38 ± 10. c Microporenic acid A: 64 ± 7 (250 mg mL −1 ), 41 ± 15 (15.6 mg mL −1 ); SD: standard deviation; -no inhibition.
compound Sch-642305 33.It is converted into a sulfur derivative 34 (Fig. 2) harboring the 2-hydroxy-3-mercaptopropanoic acid moiety in Aspergillus niger, losing its bioactivity in the process. 21ur results are also consistent with the hypothesis that addition of 2-hydroxy-3-mercaptopropanoic acid may be a self-resistance mechanism, as sporothioethers A 14 and B 15 display signicantly reduced antifungal activity than the parent compound 8.
Submerged cultivation of Hypomontagnella monticulosa MUCL 54604 resulted in formation of a stereoisomeric mixture of new sulfur-containing sporothriolide derivatives named sporothioethers A and B. The presence of the 2-hydroxy-3-mercaptopropanoic acid moiety attenuates the antimicrobial activity in comparison to the precursor sporothriolide suggesting a detoxification mechanism.However, moderate effects on biofilms of Candida albicans and Staphylococcus aureus were observed for sporothriolide and sporothioethers A and B at concentrations below their MICs.