Collimonins A–D, Unstable Polyynes with Antifungal or Pigmentation

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Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX

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Collimonins A−D, Unstable Polyynes with Antifungal or Pigmentation Activities from the Fungus-Feeding Bacterium Collimonas fungivorans Ter331 Kenji Kai,*,† Mai Sogame,† Fumie Sakurai,‡ Norihiro Nasu,‡ and Makoto Fujita‡ †

Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan ‡ Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan S Supporting Information *

ABSTRACT: The isolation and structure elucidation of collimonins A−D (1−4) from the fungus-feeding bacterium Collimonas f ungivorans Ter331 are reported. Collimonins are new derivatives of polyoxygenated hexadecanoic acid, including an ene−triyne moiety. Their absolute configurations were fully determined by combining spectroscopic, chemical, and crystalline sponge methods. Collimonins showed antifungal or pigmentation activities against the fungus Aspergillus niger ATCC 9029.

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large number of natural polyynes, unique molecules with alternating triple and single carbon−carbon bonds, have been reported from plants, basidiomycetes, and insects.1 However, examples of polyynes of bacterial origin are very limited. The very few partially characterized bacterial polyynes, such as cepacins from the human pathogen Burkholderia cepacia, caryoynencin from the carnation pathogen Burkholderia caryophylli, and Sch 31828 from Microbispora sp., possess a conjugated triple bond started with a terminal alkyne (Figure 1A and Figure S1).2 Because of this structural feature, bacterial polyynes are very unstable and, thus, require caution when handled. In addition, these compounds possess one to several chiral centers, which are formed as a result of oxidation, although their absolute configurations are rarely determined. Recently, the biosynthetic machinery for bacterial polyynes was identified, and the wider distribution of such metabolites in bacterial clades was suggested.3 However, although three decades have passed since the identification of Sch 31828,2c novel bacterial polyynes have not been isolated and characterized. Hence, the stable isolation and structure determination of bacterial polyynes are challenging and important topics in chemistry. The genus Collimonas consists of mostly soil β-proteobacteria that are defined by their ability to grow at the expense of living fungal hyphae under nutrient-limited conditions and, thus, are known as “fungus-feeding bacteria”.4 So far, three species have been described, and all of them have a representative for which antifungal activity was demonstrated. The detailed analysis of the mutants of Collimonas f ungivorans Ter331 defective in antifungal activity suggested that a gene cluster named cluster K is involved in the production of © XXXX American Chemical Society

Figure 1. (A) Structures of collimonins A−D (1−4) and caryoynencin. (B) col and cay gene clusters from C. f ungivorans Ter331 (Cf) and B. caryophylli DSM50341 (Bc), respectively. Genes for transport and efflux proteins are not shown.

undescribed bacterial polyynes.5 These compounds may contribute to the unique interactions of C. f ungivorans Ter331 with soil fungi. Although the compounds were Received: April 25, 2018

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DOI: 10.1021/acs.orglett.8b01311 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters originally named collimomycins, the suffix “mycin” is typically limited for application to products derived from actinomycetes.6 Therefore, we renamed these compounds “collimonins”. The genes encoding the possible enzymes for collimonin biosynthesis within cluster K were named colABCDEFG (Figure 1B). In silico analysis suggested that the col gene cluster starts with a long chain fatty acyl-CoA ligase (colA) and possesses three desaturases (colB, colC, and colE), an acyl carrier protein (ACP, colD), and two hydrolases (colF and colG). This gene combination was also observed in the cay gene cluster of B. caryophylli, which is capable of producing caryoynencin (Figure 1B).3a Thus, the structures of collimonins were also expected to be polyyne fatty acids with a terminal alkyne and hydroxy group(s).3a,5 However, because of their high instability, collimonins have not been isolated, so their structures and biological activities remain elusive. Here, we isolated collimonins A−D (1−4) (Figure 1A) from the culture of C. f ungivorans Ter331, elucidated their structures and absolute configurations, and investigated their biological activity. First, we determined the HPLC peaks of antifungal polyynes in the culture extract of Ter331. The EtOAc extract of 1200 WYA plates in which Ter331 was grown for 5 days was separated on an ODS column by eluting with a mixture of H2O/MeOH. The fraction eluted with 60% MeOH in H2O showed antifungal activity against Aspergillus niger (Figure 2A)

Collimonin A (1) generated complicated ESI-MS data (Figure S3), so its molecular weight was not determined only from this data. The 1H NMR data (in DMSO-d6) accounted for 12 nonexchangeable protons, composed of one terminal alkyne proton, two (Z)-olefinic protons (3JH,H = 10.9 Hz), five oxymethine protons, and two methylene groups (Table S1). Analysis of 13C NMR and HMQC spectra accounted for 16 carbon signals and identified the presence of a carboxyl group, (Z)-olefinic methines, triyne, five oxymethines, and two methylenes. The characteristic UV spectrum suggested the presence of an ene−triyne moiety (Figure S2).5 Taking into consideration these spectroscopic data, we expected that an ion peak at m/z 331 would be the [M + HCOO]− ion of this molecule (Figure S3), so the molecular formula of 1 was deduced as C16H14O5 (10 degrees of unsaturation) by its HRESIMS data. The COSY and HMBC correlations constructed the planar structure of 1, which possessed ene− triyne, trans-epoxide, and γ-lactone moieties and, thus, satisfied the degrees of unsaturation (Figure 2C). Collimonin B (2) showed ion peaks at m/z 285 [M − H]− and m/z 331 [M + HCOO]− in the negative ESI-MS (Figure S3), and therefore, its molecular weight was identical to that of collimonin A (1). The UV spectrum of 2 was very similar to that of 1, suggesting the presence of an ene−triyne moiety also in 2 (Figure S2). The 1H NMR spectrum of 2 was similar to that of 1, except for the 3JH,H value of H-9/H-10 (16.0 Hz) (Table S2). Analysis of 2D NMR and HRESIMS data revealed that 2 is the 9E-isomer of 1 (Figure 2C). Collimonin C (3) gave an [M − H]− ion at m/z 273 in the negative ESI-MS (Figure S3). The 1H NMR spectrum of 3 indicated the presence of 15 nonexchangeable protons, composed of one terminal alkyne proton, two (E)-olefinic protons, two oxymethine protons, and five methylene protons (Table S3). Analysis of 13C NMR and HMQC spectra accounted for 16 carbon signals and identified the presence of a carboxyl group, (E)-olefinic methines, triyne, two oxymethines, and five methylenes. Taken together, the molecular formula of 3 was suggested to be C16H18O4 (eight degrees of unsaturation), which was confirmed by HRESIMS. The carboxyl group and ene−triyne moiety accounted for these degrees of unsaturation, so 3 was revealed to be a linear fatty acid. The COSY and HMBC correlations elucidated the planar structure of 3, which possessed vicinal diol and ene−triyne moieties (Figure 2C). The UV and ESI-MS data of collimonin D (4) were identical to those of 3 (Figures S2 and S3). The planar structure of 4 constructed by 1D and 2D NMR data (Table S4) was the same as that of 3 (Figure 2C). However, 4 showed a different HPLC retention time from 3 (Figure 2B), indicating that these compounds are a pair of C-6 or C-7 epimers. To elucidate the relative configurations of collimonins C (3) and D (4), we prepared the respective isomers of methyl 6,7dihydroxyhexadecanoate (5) from the collimonins by catalytic hydrogenation and subsequent methylation (Figure 3A) and compared their HPLC retention times with those of synthetic syn- and anti-5. The authentic standards (synthetic syn- and anti-5) were synthesized from decanal in accordance with Scheme S1. Compound 5 prepared from collimonins C (3) and D (4) showed identical retention times to synthetic anti- and syn-5, respectively (Figure S4). This revealed that the relative configurations of the 6,7-diol moiety in 3 and 4 are anti and syn, respectively.

Figure 2. (A) Antifungal activity of column fractions obtained from Ter331 culture extract. The 60% MeOH eluate showed antifungal activity against A. niger ATCC 9029. (B) HPLC analysis of the 60% MeOH eluate. Collimonins A−D (1−4) were detected as major peaks. (C) Key COSY and HMBC correlations of collimonins.

and gave four major HPLC peaks showing characteristic UV spectra for polyynes (Figure 2B and Figure S2). As previously reported, 5 these compounds were unstable and easily polymerized to insoluble matter. By avoiding drying and exposure to oxygen, higher than ambient temperature, and light, we could isolate those compounds by two-step HPLC separation. The HPLC eluates containing collimonins were evaporated without heating to remove only the solvent. Then DMSO-d6 (0.5 mL) was added to the aqueous concentrates prior to the evaporation of water, and the purified collimonins (in DMSO-d6) were subjected to the following spectroscopic analyses. B

DOI: 10.1021/acs.orglett.8b01311 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters

Figure 4. (A) Conversion of collimonin B (2) to 10 by CuAAC with benzyl azide. ΔδHRS values (ppm) of bis-MPA derivatives 11 and 12 used to assign the absolute configuration of C-5/C-8 in 10 are also shown. (B) Newman projections with 3JH,H and 2JC,H values used to suggest the relative configuration of C-7/C-8 in 10.

configuration.8 The 3JH,H of H-4/H-5 in 10 was 4.0 Hz, and thus, the relative configuration of C-4/C-5 in 10 was suggested to be syn. The 3JH,H of H-7/H-8 (6.3 Hz) was intermediate, thereby indicating that the configuration of C-7/C-8 is an interconverting conformation (Figure 4B). The large 2JC,H values of C-7/H-8 (−4.6 Hz) and C-8/H-7 (−4.0 Hz) measured by J-resolved HMBC suggested that the configuration of C-7/C-8 in 10 is syn, although several important long-range C−H coupling constants could not be determined. Taken together, the remaining stereogenic centers in 10 were expected to be 4S, 6S, and 7S. To unambiguously determine the absolute configuration of 10, the crystalline sponge (CS) method was applied to the structure analysis.9 Only 5 μg of 10 (in acetone) was treated with a single crystal of [(ZnCl2)3(tpt)2] complex [CS crystal; tpt = 2,4,6-tris(4-pyridyl)triazine],10 and the guest-absorbed CS crystal was subjected to a diffraction study. The molecular structure of 10, trapped in the pore of the CS host, was clearly observed (Figure 5). The planar structure agreed with that of

Figure 3. (A) Conversion of collimonins C (3) and D (4) to the corresponding compound 5. (B) Chiral LC/MS analysis of compound 5 prepared from collimonin C (3) and synthetic (+)/(−)-anti-5. (C) Chiral LC/MS analysis of compound 5 prepared from collimonin D (4) and synthetic (+)/(−)-syn-5. (D) ΔδHRS values (ppm) of the bisMPA derivatives of (−)-anti- and (+)-syn-5 used to assign their absolute configurations.

Enantiopure anti/syn-5 were prepared from the synthetic racemic mixtures by chiral LC reparation. We then compared the chiral LC retention times of 5 prepared from collimonins C (3) and D (4) with those of synthetic enantiopure 5. Compound 5 prepared from 3 showed a retention time identical to that of synthetic (−)-anti-5 (Figure 3B), whereas the retention time of 5 prepared from 4 matched that of synthetic (+)-syn-5 (Figure 3C). Subsequently, the absolute configurations of synthetic (−)-anti- and (+)-syn-5 were determined by NMR experiments on compounds 6−9 (Figure S5), which were synthesized by coupling with auxiliary reagents, (R)/(S)-α-methoxyphenylacetic acid (MPA). This method was reported by Riguera et al., and the absolute configuration of the vicinal diol moiety can be assigned by comparing the ΔδHRS (δHR−δHS) signs.7 The ΔδHRS signs of synthetic (−)-anti- and (+)-syn-5 were consistent with the cases of 6S,7R and 6R,7R, respectively (Figure 3D). Therefore, the absolute configurations of 3 and 4 were determined to be 6S,7R and 6R,7R, respectively. Collimonin B (2) was converted to cycloadduct 10 by copper(I)-catalyzed azide−alkyne cycloaddition (CuAAC) with benzyl azide (Figure 4A).3 The absolute configuration of the 5,8-diol moiety in 10 was also determined by Riguera’s method.7 Its bis(R)/bis(S)-MPA esters (11 and 12) were prepared, and their proton signals were assigned based on COSY and HMQC experiments (Figure 4A). For the ΔδHRS signs from H-5 to H-8, the absolute configuration of 10 was assigned as 5R,8R. The substructure δ-hydroxy γ-caprolactone was found in some natural products, and several of their absolute configurations were elucidated. In those studies, the 3 JH,H value of H-4/H-5 was 4.0−4.2 Hz for the synconfiguration, while it was 3.0−3.1 Hz for the anti-

Figure 5. X-ray crystal structures of cycloadducts 10 and 13 prepared from collimonins B (2) and A (1), respectively. The CS method was used to analyze these molecular structures. For crystallographic details, see the Supporting Information.

10 resolved by NMR experiments. The relative configuration of 10 was revealed to be 4S*,5R*,6S*,7S*,8R*, although its absolute configuration could not be established. Together with the above Riguera’s analysis, we determined the absolute configuration of 10 to be 4S,5R,6S,7S,8R, and thus, the molecular structure of collimonin B (2) was revealed (Figure 1A). Collimonin A (1) was also converted into cycloadduct 13 (Figure S6) and analyzed by the CS method. The crystal structure clearly showed that the relative configuration of 13 is 4S*,5R*,6S*,7S*,8R* (Figure 5). Moreover, the sign of optical rotation of collimonin A (1) was consistent with that of C

DOI: 10.1021/acs.orglett.8b01311 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters collimonin B (2) (1: [α]D25 = −326, c 0.225, DMSO; 2: [α]D25 = −1614, c 0.054, DMSO). These results suggest that their absolute configurations from C-4 to C-8 are identical. To confirm this, (Z)-olefin in 1 was isomerized to (E)-olefin by using UV light (365 nm). The isomerized product showed an identical retention time with 2 (Figure S7A). Furthermore, this conversion was also confirmed by the 1H NMR spectrum of the reaction product (Figure S7B). These results showed that the absolute configuration of 1 from C-4 to C-8 is identical to that of 2 from C-4 to C-8 (Figure 1A). The biological activities of collimonins against A. niger were examined. In a noncontact confrontation assay, Ter331 caused growth inhibition, branching, and pigmentation of A. niger hyphae (Figure S8). Collimonins A (1), C (3), and D (4) showed antifungal and hyphal branching activities (Figures 6 and S9). Collimonin B (2) induced the pigmentation of A. niger hyphae, while not showing antifungal activity. The yellow pigment partially purified showed a λmax at 423 nm and an [M + H]+ ion at m/z 473 (Figure S10). These results suggested that the pigment may not be a derivative of naphthopyrones, pigments commonly found from Aspergillus sp.11 This is the first observation that a natural polyyne causes the pigmentation of fungal hyphae. The saturated analogue of 3, compound 14 (Figure S11), did not show antifungal activity, confirming the importance of ene-triyne moiety (Figures 6). Together, we concluded that collimonins are the causative agents of antifungal and pigment-inducing activities of Ter331.

pigment production in fungal hyphae. Therefore, this study uncovered chemical and biological features of these enigmatic metabolites. It is still, however, unclear how the bacterium organizes these compounds and other factors such as chitinase to feed/antagonize the surrounding fungi.13 We are now trying to answer this question by investigating the mode of action of collimonins.



ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.8b01311. Experimental details, additional figures and tables, and NMR spectra (PDF) Accession Codes

CCDC 1834384−1834385 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.



AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Kenji Kai: 0000-0002-4036-9959 Makoto Fujita: 0000-0001-6105-7340 Notes

Figure 6. Antifungal and pigmentation activities of collimonins A−D (1−4) and compound 14 against A. niger. The concentration of each compound was 60 μg/disk.

The authors declare no competing financial interest.



ACKNOWLEDGMENTS We thank Dr. Ritesh Dubey (The University of Tokyo) for critically reviewing the XRD data analysis. This work was supported by JSPS KAKENHI (Grant No. 18H04628) (K.K.) and the JST-ACCEL program (M.F.).

As mentioned above, the col gene cluster is composed of enzyme genes similar to those in the cay cluster. However, the structures of collimonins turned out to be different from that of caryoynencin, especially the degrees of desaturation and oxidation. Furthermore, the degree of oxidation within collimonins A−D (1−4) is also different. The structures of collimonins A (1) and B (2) are unique, and such polyoxygenated long-chain fatty acids are, to the best of our knowledge, the first examples as natural products. Therefore, we are interested in collimonin biosynthesis in Ter331. However, their biosynthetic route was difficult to be deduced only on the basis of their structures and possible biosynthetic genes. Nonenzymatic conversion(s) might be involved in collimonin biosynthesis, as observed in ralfuranone biosynthesis in Ralstonia solanacearum.12 To know them further, we need to elucidate the derivatives of collimonins produced by the deletion mutants of colF/colG hydrolase genes in a future study. In conclusion, we isolated collimonins from C. f ungivorans Ter331, elucidated their structures, including absolute configurations, and evaluated their biological activities. The present study is the first to reveal the absolute configurations of polyoxygenated bacterial polyynes. The isolation and analytical techniques used here will provide a workflow for the identification of polyoxygenated polyynes from other bacteria. Collimonins A (1), C (3), and D (4) are the major antifungal agents of Ter331, and collimonin B (2) uniquely induced



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DOI: 10.1021/acs.orglett.8b01311 Org. Lett. XXXX, XXX, XXX−XXX