Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX
pubs.acs.org/OrgLett
Acaulins A and B, Trimeric Macrodiolides from Acaulium sp. H‑JQSF Ting Ting Wang,†,§ Ying Jie Wei,‡ Hui Ming Ge,† Rui Hua Jiao,† and Ren Xiang Tan*,†,‡ †
State Key Laboratory of Pharmaceutical Biotechnology, Institute of Functional Biomolecules, Nanjing University, Nanjing 210023, China ‡ State Key Laboratory Cultivation Base for TCM Quality and Efficacy, Nanjing University of Chinese Medicine, Nanjing 210023, China § State Key Laboratory of Elemento-organic Chemistry, Nankai University, Tianjin 300071, China S Supporting Information *
ABSTRACT: Acaulin A (1) and its macrolactone ring-opened congener acaulin B (2) were characterized from the culture of Acaulium sp. H-JQSF (an isopod-associated fungus) as architecturally undescribed trimeric macrodiolides, with the former being antiosteoporotic at 0.4 μM in the prednisoloneinduced osteoporotic zebrafish. Identification of acaudiolic acid (3) as the monomeric macrodiolide precursor facilitated the proposal of the acaulin biosynthetic pathway.
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acrodiolides are a class of structurally complex and biologically important microbe-derived natural products that trigger, or at least inspire, investigations relating to drug discovery,1 organic synthesis,2,3 and chemical ecology.4 According to the building block (un)identity, this family of natural products can be grouped into homodimers such as ficiolides from Pestalotiopsis f ici5 and heterodimers such as pamamycin from Streptomyces alboniger.6 Some unusual macrodiolide structures signify the occurrence of intriguing biosynthetic mechanisms1,7 and enzymes with promising substrate specificity and reactivity.8,9 Accordingly, the characterization of novel macrodiolides may spur or revitalize related endeavors. In continuation of our search for intriguingly structured bioactive molecules from fungi,10,11 Acaulium sp. H-JQSF, an isopod-associated fungus, was recently ascertained to be a producer of acaulide as a dimeric macrodiolide with lower abundance, but pronounced antiosteoporotic activity in the prednisolone-induced osteoporotic zebrafish.1 To have more acaulide material for validating its osteogenic action in a mammal (e.g., mouse) model, we were obligated to regrow the fungus in a modified medium in favor of the macrodiolide production. Instead of yielding an escalated abundance of acaulide, an architecturally undescribed trimeric macrodiolide named acaulin A (1) and its hydrated congener acaulin B (2) were obtained from the fungal culture effort. To our knowledge, 1 and 2 are the first examples of macrodiolide trimers, which are unique in the trimerization of the three 14membered heterodimeric macrodiolide units with two edited in, and one anchored on a cyclopentanone nucleus (Figure 1). Exploration for the biosynthetic precursor led to the identification of a new acyclic polyketide named acaudiolic © XXXX American Chemical Society
Figure 1. Structures of acaulins A (1) and B (2).
acid (3) (Scheme 1). In particular, acaulin A (1) was demonstrated to be osteogenic at 0.4 μM in the prednisolone-induced osteoporotic zebrafish. Acaulin A (1) was evidenced to have a molecular formula of C42H54O19 from the Na+-liganded molecular ion at m/z 885.3143 (calcd for C42H54O19Na, 885.3152) in its highresolution electrospray ionization mass spectrometry (HR-ESIMS). Inspection of the 1H and 13C NMR spectra of 1 indicated the presence of three olefins, three ketones, and six ester carbonyls. These substructures accounted collectively for 12 of 16 double bond equivalents, thus suggesting that 1 could be tetracyclic. Its 1H NMR spectrum displayed the characteristic signals arising from six methyls, six methylenes, nine oxyReceived: March 18, 2018
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DOI: 10.1021/acs.orglett.8b00883 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters Scheme 1. Plausible Biosynthetic Pathways toward Acaulins A (1) and B (2)
Figure 2. Key 1H−1H COSY, HMBC correlations of 1 and 2.
lines (δC 212.1, 79.1, and 66.3) and a pair of downfield moved and mutually coupled methine doublets (J = 10.0 Hz) at δH 4.70 and 3.67, which showed HSQC correlations with the carbon signals at δC 46.8 (C-8) and 60.5 (C-9), respectively. Furthermore, the methine proton signal at δH 3.67 displayed HMBC correlation with the ketone carbon resonance at δC 212.1. These observations could only be explained by the presence of a 2,2,3,4,5,5-hexasubstituted cyclopentanone nucleus, in which the methine protons resonated downfield, owing to being geminal to the carbonyl groups (Figure 2A). Starting from the cyclopentanone moiety, the HMBC spectrum of 1 facilitated the conjunction between the two 14-membered macrodiolide substructures (ring A, C-1−C-14; and ring C, C-1″− C-14″) and the covalent C-8′/8″ bondage of ring C with ring B (C-1′−C-14′) (Figure 2A). To understand the relative stereochemistry, the NOESY spectrum of 1 was acquired. The NOESY correlations of H-4/H-4′/H-4″ with H6/H-6′/H-6″ indicated the same orientations of related signals in rings A−C (Figure 3). The H-9 showed NOESY correlations
methines, and three trans-disubstituted double bonds (J2,3 = 15.7 Hz, J2′,3′ = 15.8 Hz, J2″,3″ = 15.6 Hz). Further interpretation of the 1H NMR signals employing 1H−1H COSY and HMBC experiments pinpointed the coexistence of three 4,5-dioxygenated hex-2(E)-enoyl moieties (C-1/C-1′/C-1″ to C-6/C6′/C-6″) as well as 2-substituted, 2,3-disubstituted, and 2,3,4chimerized octanoyl (C-7/C-7′/C-7″ to C-14/C-14′/C-14″) substructures. Moreover, the HMBC correlations of C-1/1′/1″ with H-3/3′/3″ and H-13/13′/13″ and of C-7/7′/7″ with H5/5′/5″ and H-9/9′/9″ suggested that three 14-membered macrodiolide moieties are incorporated in the structure of 1 (Figure 2A).1 To address the incorporation manner of the substructures, the 1H and 13C NMR spectra of 1 were scrutinized, indicating that one (viz., ring B) of the macrodiolide moieties of 1 was identical to the 14-membered cycle of acaulide.1 However, indiscernible in the 1H and 13C NMR spectra of acaulide1 were the three quaternary carbon resonance
Figure 3. Selected NOESY correlations of 1.
with H-11a and H-11″a/H-11″b suggesting the β-orientation from the cyclopentanone ring (Figure 3). Thus, H-8 was αoriented since H-9 and H-8 located anti to allow a larger coupling constant (J8,9 = 10.0 Hz).12 However, the NOESY spectrum of 1 failed to signify the relative configuration of the other chiral carbons, owing to the conformational flexibility and the tandem connection of three or four quaternary carbons. Therefore, we tried, but failed unfortunately, to obtain the acaulin A single crystal suitable for X-ray diffraction analysis. The HR-ESI-MS spectrum of acaulin B (2) displayed the Na+-liganded molecular ion at m/z 903.3261, indicative of its molecular formula of C42H56O20 (calcd for C42H56O20Na, 903.3257), which possessed an extra “H2O” in comparison to that of 1. Regardless of the stereochemistry, this observation B
DOI: 10.1021/acs.orglett.8b00883 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters
through H-14 (Figure S5). In conjunction with the HMBC correlation of C-1 with H-3 and H-13, the planar structure of 3 was proposed to be identical to 7-hydrated derivative of 10ketoacaudiol.1 Since they are co-produced by the same fungal strain, 3 could share the (4R,5S,13S)-configuration with 10ketoacaudiol.1 This assumption gained support from the Keck macrolactonization14 of 3 into 10-ketoacaudiol (Figure S7) and close similarity in specific rotations [3, +44 (c = 0.05); 10ketoacaudiol, +48.6 (c = 0.05)]. Acaudiolic acid (3) and (10-keto)acaudiol1 are stereochemically identical and share the same skeleton with the fungal polyketide colletodiol.15,16 This suggested that they might be the polyketides constructed in the same bioassembly line. To substantiate their polyketide nature, the cultivation condition was reoptimized until one or two macrodiolides became dominant in the fungal culture. Under the condition in which acaudiol was richly produced, [1-13C]-acetate was supplemented in the fungal culture to harvest the isotopically labeled variant of acaudiol, whose 13C NMR spectrum underpinned the polyketide attribute (Table S3, Figure S8). The validation experiment encouraged our proposal for the fungal generation of 1 and 2. As illustrated in Scheme 1, the epoxidation of 10ketoacaudiol (the common precursor of acaulide and acaulones A and B1) would give intermediate I. An acid-catalyzed keto− enol tautomerization of I gave III via II. The methylene of III is co-activated by two adjacent carbonyls to be more susceptible for a Michael addition reaction with 10-ketoacaudiol to afford IV. Via a subsequent intramolecular aldol reaction, IV might form V, whose methine remains co-activated by the two carbonyls to be prone enough to undergo the second-round Michael addition reactions both with 10-ketoacaudiol to yield 1, and with 3 to form 2. It was noteworthy that the possible interconversion between 1 and 2 could not be ruled out. The structures of 1 and 2 are similar in part to that of acaulide, an antiosteoporotic dimeric macrodiolide found in the same fungal strain.1 This observation encouraged us to test these two polyketides for the osteogenic action in the same osteoporotic zebrafish model.1 To our anticipation, 1 reduced the prednisolone-induced skull bone loss of the osteoporotic zebrafish in a roughly dose-dependent manner at 0.4, 2.0, and 10.0 μM (Figures 5 and S9). However, 2 was found to be negligibly active in the model at 10.0 μM. The present reinvestigation of the secondary metabolites of the title fungus led to the characterization of acaudiolic acid (3), which failed to be isolated but was supposed to appear earlier.1 This finding pinpointed another option for the
suggested that 2 could be a hydrated congener of 1. This assumption was reinforced by the 1H and 13C NMR spectra of 2, which were well comparable to those of 1 (Table S1). However, in the case of 2, the most downfield-shifted carbon resonance at δC 176.8 (Table S1) signified the presence of an assumable α,β-saturated 7′-carboxylic group at C-8′ (Figure 2B). Moreover, the proton of the 7′-carboxylic acid might form intramolecular hydrogen bonds with 7-, 7″-, and 9″-carbonyl groups (Figure S1A). This explained why the 1H and 13C NMR spectral signals of 2 were broadened if acquired in acetone-d6 but sharpened upon subsequent addition of 0.2% NaOD in the NMR tube (Figure S2). The above assignment was confirmed by the 2D NMR spectra of 2, which allowed an exact assignment of all 1H and 13 C NMR signals (Table S1). In comparison with the spectra of 1, the downfield movement of C-7′ resonance line of 2 was accompanied by the upfield shifting of H-5′ signal (Table S1). As in the case of 1, the NOESY correlations of 2 could only address the relative configuration of some of the chiral carbons (Figure S1B). Fortunately, this frustration was overcome by the formation of single crystal of 2. Thus, the low-temperature, single-crystal X-ray crystallography of 2 (Cu Kα) pinpointed its (4R,5S,8S,9S,13S,4′R,5′S,8′R,13′S,4″R,5″S,8″S,10″S,13″S) configuration with a Flack parameter of 0.06(9) (Figure 4).13
Figure 4. X-ray structure of 2.
The clarified absolute stereochemistry of 2 encouraged us to reconsider that of 1 (see above). Gratifyingly, both compounds resembled in CD curves (Figure S4) and optical rotations [1, + 60 (c = 0.05); 2, + 45 (c = 0.05)], suggesting their possession of the same absolute configurations. This assumption was reinforced by the Keck macrolactonization of 2 into 1 (Figure S3).14 Taken together, 1 shares with 2 the (4R,5S,8S,9S,13S,4′R,5′S,8′R,13′S,4″R,5″S,8″S,10″S,13″S)-configuration. The identity in the stereochemistry of 1 and 2 extended our curiosity about their biosynthetic relationship, which may have two options; namely, 2 could either be a hydrate of 1 or form via a shunt pathway in which an acyclic 14-carbon precursor might be involved. As anticipated, the LC−MS monitored fractionation of the combined mother liquor led to the characterization of the precursor-like acyclic molecule as an undescribed natural product we have named acaudiolic acid (3) (Scheme 1). The HR-ESI-MS spectrum of 3 exhibited an Na+liganded molecular ion at m/z 323.1092 corresponding to its molecular formula C 14 H 20 O 7 (calcd for C 14 H 20 O 7 Na, 323.1101). This molecular formula indicated that 3 is acyclic, in view of the α,β-unsaturated ester and acid motifs evidenced from its 1H and 13C NMR spectra (Table S2). The 1H−1H COSY spectrum of 3 disclosed the three coupling sequences from H-2 through H-6, from H-8 through H-9, and from H-11
Figure 5. Skull mineralization of zebrafishes exposed to prednisolone (PN) combined with 1 at 0.4, 2.0, and 10.0 μM, PN (model group), and DMSO (positive control). Columns: means of mineralized area (A) and integrated optical density (IOD, B) of three independent experiments. Bars: standard error of the mean (SEM) versus the model group; *p < 0.05, **p < 0.01. C
DOI: 10.1021/acs.orglett.8b00883 Org. Lett. XXXX, XXX, XXX−XXX
Organic Letters
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generation of (10-keto)acaudiol, the presumed precursor of acaulide and its shunt products acaulones A and B.1 The discovery of acaulins A (1) and B (2) suggested the oxidation of the α,β-unsaturated γ-ketonic macrolactone motif of 10ketoacaudiol. Such an oxidation played a key role in the assembly of 1 and 2, a pathway in parallel with that of acaulide, an antiosteoporotic dimeric macrodiolide.1 Furthermore, the striking difference between 1 and 2 in the osteogenic action highlighted that the ring B of the former is essential for the antiosteoporotic property. In conclusion, this work describes the characterization of acaulins A (1) and B (2) as skeletally undescribed trimeric fungal macrodiolides with the former exhibiting antiosteoporotic activity in a submicromolar range in the zebrafish model. They feature a unique natural product framework that forms from an unexpected conjunction of a cyclopentanone nucleus with two or three 14-membered macrolactone moieties. The difference in their biological activity underscores the osteogenic significance of the 14-membered macrolactone cycle (ring B of 1) anchored via a single bond on the cyclopentanone core. Furthermore, the osteogenic polyketide 1 can be prepared from 2 via the Keck macrolactonization approach.14 The capture of acaudiolic acid (3) as an early-stage acyclic monomeric precursor enables the postulation of the acaulin biosynthesis and the reconsideration of (10-keto)acaudiol generations.1 In aggregation, this investigation addresses the structure, bioactivity, and plausible biosynthetic pathway of two skeletally novel macrodiolide trimers from Acaulium sp. H-JQSF, an isopod-associated fungus.
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REFERENCES
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.8b00883. General methods, fractionation procedure, 1D and 2D NMR spectra (PDF) Accession Codes
CCDC 1565521 contains 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 data_
[email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Hui Ming Ge: 0000-0002-0468-808X Ren Xiang Tan: 0000-0001-6532-6261 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The work was cofinanced by the NSFC (Grant Nos. 81530089, 21672101, 81573833, and 21661140001). The fungus was identified by Prof. L. D. Guo at Institute of Microbiology, Chinese Academy of Sciences, Beijing, China. D
DOI: 10.1021/acs.orglett.8b00883 Org. Lett. XXXX, XXX, XXX−XXX
