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Abstract

Objective: The aim of this research was the isolation, structure elucidation and evaluation of the bioactivity of natural products produced by an Aureobasidium pullulans fungal endophyte of the medicinal plant Thuja occidentalis. Methods: The natural products were isolated from an extract of A. pullulans culture broth by reversed-phase chromatography and their structures were elucidated based on analysis of HRESIMS and 1D/2D NMR data. The absolute configurations of the compounds were assigned by comparison of their electronic circular dichroism (ECD) and optical rotatory dispersion (ORD) data with values obtained for Boltzmann-weighted conformer ensembles calculated by density functional theory. Results/Conclusion: Two new C₁₁-polyketides, aureobasidol A (1) and B (2), were isolated from the A. pullulans extract. Both polyketides showed selective but weak inhibitory activity against Mycobacterium tuberculosis in vitro.

Keywords

polyketide bioactivity endophyte antimycobacterial fungus medicinal plant

Introduction

The investigation of novel biological sources continues to be an important strategy in the discovery of new and biologically active chemical diversity.¹ Fungal endophytes represent a relatively new and largely unexplored source of biodiversity^2-4 that are currently providing a remarkable diversity of structurally unique bioactive natural products.^5-8 In our continuing search for biologically active natural products from endophytic fungi of Canadian medicinal plants,^9-12 we have investigated the EtOAc extract of an Aureobasidium pullulans endophyte (JAJ1-073) isolated from the needles of white cedar (Thuja occidentalis).

The JAJ1-073 extract selectively inhibited the growth of Mycobacterium tuberculosis in our bioassay screening and subsequent examination by NMR spectroscopy indicated that it was unique within our collection of fungal extracts and contained 1 as the major constituent. NMR-guided fractionation of the extract led to the isolation and characterization of two new C₁₁ fatty alcohols, (R,2E,5Z)-2-((E)-but-2-en-1-ylidene)-3-hydroxy-4-oxohept-5-enal (aureobasidol A; 1) and (R,2E,5E)-2-((E)-but-2-en-1-ylidene)-3-hydroxy-4-oxohept-5-enal (aureobasidol B; 2) that both exhibited weak selective activity against M. tuberculosis. Herein we report the isolation, characterization and the antimycobacterial activity of 1 and 2 (Figure 1).

Figure 1.

Aureobasidol A (1) and B (2) from the endophyte Aureobasidium pullulans (JAJ1-073) and the related polyketides mollipilin A (3) and B (4),¹³ R-avellaneol (5),¹⁴ and lachnellin A (6).¹⁵

Results and Discussion

Fungal isolate JAJ1-073 was obtained from needles of Thuja occidentalis collected in Saint John, NB, Canada through an indirect isolation protocol on malt extract agar. The isolate was identified as Aureobasidium pullulans by morphological examination of colonies on various solid media (supporting information, Figure S1) and ITS DNA sequencing. An extract of the isolate obtained from a 2-week liquid fermentation of JAJ1-073 in potato dextrose broth was subjected to reversed-phase flash chromatography and HPLC to afford 1 and 2. HRESIMS indicated that both compounds shared the molecular formula C₁₁H₁₄O₃ displaying sodium adduct ions at m/z 217.0834 for 1 and m/z 217.0835 for 2 (C₁₁H₁₄O₃Na⁺ requires m/z 217.0835), and NMR spectroscopy suggested that they were diastereomeric by virtue of differing configurations at one of the three carbon–carbon double bonds present in the structures (see Table 1).

Table 1.

¹H NMR (400 MHz) and ¹³C NMR (100 MHz) data for 1 and 2 in CDCl₃ (δ in ppm, J in Hz).

No.	Aureobasidol A (1)		Aureobasidol B (2)
No.	δ_C, type	δ_H (J in Hz)	δ_C, type	δ_H (J in Hz)
1	198.1, C	9.43, s	197.0, C	9.44, s
2	134.9, C		134.9, C
3	71.5, CH	5.21, s	70.2, CH	5.34, s
4	193.3, C		193.2, C
5	122.8, CH	5.99, dq (11.4, 1.8)	125.9, CH	6.07, dq (15.6, 1.7)
6	147.7, CH	6.33, m	146.2, CH	7.06, dq (15.8, 7.0)
7	16.7, CH₃	2.18, dd (7.2, 1.8)	18.7, CH₃	1.86, dd (6.9, 1.7)
1′	153.7, CH	7.00, d (11.4)	153.9, CH	7.00, d (11.5)
2'	126.8, CH	6.60, ddq (14.7, 11.4, 1.6)	126.8, CH	6.59, ddd (14.0, 11.4, 1.6)
3'	145.7, CH	6.40, m	146.0, CH	6.39, dq (13.7, 6.8)
4'	19.5, CH₃	1.95, dd (6.9, 1.6)	19.5, CH₃	1.95, dd (6.9, 1.6)

The ¹H, ¹³C, and HSQC NMR data of compound 1 indicated the presence of two allylic methyl groups, [δ_H 2.18/δ_C 16.7 (CH₃-7), δ_H 1.95/δ_C 19.5 (CH₃-4′)], six vinylic carbons [δ_H 6.33/δ_C 147.7 (CH-6), δ_H 5.99/δ_C 122.8 (CH-5), δ_C 134.9 (C-2) δ_H 7.00/δ_C 153.7 (CH-1′), δ_H 6.60/δ_C 126.8 (C-2′), δ_H 6.40/δ_C 145.7 (CH-3′)], one oxymethine [δ_H 5.21/δ_C 71.5 (CH-3)], and two carbonyl carbons: an aldehyde [δ_H 9.43/δ_C 198.1 (CH-1)]; and a ketone [δ_C 193.3 (C-4)] (Table 1). ¹H–¹H COSY correlations revealed two distinct spin-coupled systems in 1: H₃-7/H-6/H-5 and H-1′/H-2′/H-3′/H₃-4′; and the remaining atom connectivities were readily elucidated based on the observed HMBC correlations from H-3 to C-1, C-2, C-4, and C-1′, H-1′ to C-1, C-2, and C-3, and H-1 to C-2 (Figure 2).

Figure 2.

Key COSY and HMBC correlations for 1 and 2.

The NMR data obtained for compound 2 was similar to that of 1 except for key differences at positions 5, 6, and 7, suggesting that 2 was diastereomeric with 1 at Δ⁵. Indeed, the configuration of Δ⁵ was found to be cis in 1 (J₅₋₆ 11.4 Hz; CH₃-7 δ_H 2.18/δ_C 16.7) and trans in 2 (J₅₋₆ 15.7 Hz; ; CH₃-7 δ_H 1.86/δ_C 18.7) with trans configurations being confirmed for Δ^2,1′ and Δ^2′ in 1 and 2 based on the presence of NOESY correlations between H-1/H-1′ and the H-2′/H-3′ coupling constants (J_2′−3′ 14.7 Hz in 1 and 14.0 Hz in 2) in both molecules.

Repeated attempts to prepare α-methoxy-α-trifluoromethylphenylacetoyl esters or a 2,4-dinitrophenylhydrazone derivative to assign the absolute stereochemistries of 1 and 2 by either Mosher's method or x-ray crystallography were unsuccessful. The absolute configuration at C-3 of 1 and 2 was therefore determined by comparison of the observed ECD and ORD data with values predicted for Boltzmann-weighted conformer ensembles of the diastereomers obtained by DFT calculations. Both compounds displayed a negative-positive-negative sequence of Cotton effects at approximately 220, 255 and 290 nm, respectively, in their ECD spectra (Figure 3) and large negative rotations at 436 to 633 nm by ORD (Table 2). Predicted ECD and ORD data were derived from weighted averages of the respective calculated properties (ECD data were calculated by TDDFT at the CAM-B3LYP-D3BJ/def2-TZVPD[CPCM]//B3LYP-D3BJ/def2-TZVPD[CPCM] level and ORD data were calculated by DFT at the CAM-B3LYP/def2-TZVPD[CPCM]//B3LYP-D3BJ/def2-TZVPD[CPCM] level) for conformers that comprised 99.9% of the Boltzmann distributions of 1 (18 conformers) and 2 (13 conformers) at the B3LYP-D3BJ/def2-TZVPD[CPCM] level after an exhaustive conformer search and geometry optimization protocol. While examination of the calculated and experimental ECD spectra strongly suggested an R configuration for both 1 and 2 (Figure 3), there remained some uncertainty in these assignments given the relatively low enantiomeric similarity indices between the experimental and calculated spectra (50% similarity to R and 18% similarity to S for 1; 55% similarity to R and 26% similarity to S for 2).

Figure 3.

Experimental and calculated ECD spectra of 1 and 2.

Table 2.

Experimental and calculated ORD values for R-1 and R-2.

Wavelength (nm)	Specific rotation [α]
Wavelength (nm)	Calcd R-1	Exp 1	Calcd R-2	Exp 2
633	−566.0	−134.6	−272.8	−40.8
589	−692.0	−160.8	−331.6	−41.6
546	−866.5	−195.1	−411.8	−58.0
436	−1932.8	−322.6	−872.2	−89.6

We therefore used ORD data to confirm the assignment of configurations (Table 2) and the calculated ORD data for the R-series enantiomers provided large, negative values at all four wavelengths that decrease in magnitude with increasing wavelength (Table 2). These data provide a good match with the trends seen in the experimental values of 1 and 2, particularly considering the challenges currently associated with obtaining quantitatively accurate optical rotations for ensembles of flexible molecules.¹⁶ Given that both computational approaches to assigning the configurations of 1 and 2 are in agreement, we are confident in assigning the absolute configuration and structure of aureobasidol A (1) as (R,2E,5Z)-2-((E)-but-2-en-1-ylidene)-3-hydroxy-4-oxohept-5-enal and aureobasidol B (2) as (R,2E,5E)-2-((E)-but-2-en-1-ylidene)-3-hydroxy-4-oxohept-5-enal.

Surprisingly few natural products that contain the highly functionalized structural motif present in the aureobasidols have been isolated, with all of the related polyketides being of fungal origin: mollipilin A (3; from Chaetomium mollipilium) contains the full carbinol interrupted conjugated enone/dienal sequence observed in 1 and 2,¹³ while conjugation of the ketone is absent in mollipilin B (4; from Chaetomium mollipilium),¹³ avellaneol (5; from Hypocrea avellanea),^14,15 and lachnellin A (6; from a Lachnellula sp. isolate).¹⁷ All four of these related polyketides have exhibited either weak cytotoxicity against cancer cell lines (3 and 4; IC₅₀ values from 2 to 4 µM),¹³ moderate to weak antimicrobial activity (5; MIC values from 100 to 200 µM),¹⁴ or both (6; IC₅₀ values from 1 to 10 µM and MIC values from 5 to 200 µM).¹⁷ Owing to these reports and the bioactivity observed for the JAJ1-073 extract, we assessed the antimycobacterial activity of 1 and 2 against Mycobacterium tuberculosis. Although the extract displayed moderate activity in our preliminary screening (55% inhibition at 100 µg mL⁻¹), both isolated natural products exhibited only weak M. tuberculosis inhibitory activity [1: IC₅₀ 37.9 (36.3-39.6) µM, MIC 128 µM and 2: IC₅₀ 75.3 (72.8-77.9) µM, MIC 257 µM] comparable to that reported for the related compounds 3-6. The significant activity exhibited for the Aureobasidium pullulans extract is therefore likely due to the large amounts of 1 and 2 present in extracts of A. pullulans isolate JAJ1-073 consistently ranging from 30% to 40% for 1 and 5% to 10% for 2 (estimated by ¹H NMR spectroscopy) over multiple fermentations in our hands. However, the possibility that the extract contains further bioactive constituents responsible augmenting the observed activity of the extract should not be discounted.

Conclusion

Two new highly functionalized C₁₁-polyketides, aureobasidol A (1) and B (2), were isolated from the A. pullulans extract. The aureobasidols represent the latest members of a limited group of small polyketides exclusively of fungal origin. Both 1 and 2 showed selective but weak inhibitory activity against Mycobacterium tuberculosis in vitro.

Experimental

General Experimental Procedures

Optical rotations were recorded in MeOH on a Bioevopeak POL-537 polarimeter at 633, 589, 546, and 436 nm. UV spectra were measured on a Shimadzu UV-1280 spectrophotometer. Electron circular dichroism was measured on a JASCO-810 spectropolarimeter at room temperature, using a 1-mm quartz cuvette. NMR spectra were recorded on an Agilent 400-MR DD2 instrument in deuterated solvents and were calibrated to residual protonated solvent resonances. HR-MS data were recorded on a Thermo LTQ Exactive instrument with an electrospray ionization (ESI) source. Solvents for extraction and isolation were purchased from Fisher Scientific (Ottawa, ON, Canada) and deuterated solvents for NMR spectroscopy were purchased from Sigma-Aldrich (Oakville, ON, Canada). Flash chromatography was performed using a Biotage Flash + chromatography system fitted with C₁₈ (reversed-phase) SiliaSep cartridges (40-63 µm, 60 Å, 25 g; SiliCycle, QC, Canada). Semi-preparative reversed-phase HPLC was performed on a Phenomenex Luna C₁₈ column (250 × 10 mm, 10 µm, 100 Å) using an Agilent 1100 HPLC system comprising a G1311A binary pump and a G1315C diode array detector.

Endophyte Isolation

JAJ1-073 was isolated from the needles of Thuja occidentalis collected from Millidgeville, Saint John, NB, Canada (45°18′22″ N, 66°5′4″ W) in Fall 2015 and a voucher specimen has been deposited at the New Brunswick Museum (voucher number: NBM VP 40880). Needle surfaces were sterilized by sequential immersion in 70% ethanol (EtOH) for 5 s and rinsed with sterile distilled water (H₂O) for 10 s and then immersed in 6% aqueous sodium hypochlorite (NaOCl) for 10 s and rinsed with sterile distilled H₂O and blotted dry on autoclaved paper towel. Sterile tissue was immediately aseptically cut into pieces (1 cm) that were placed onto 2.0% malt extract agar and incubated at room temperature under ambient light.

Endophyte Identification

Isolate JAJ1-073 was identified as Aureobasidium pullulans through the examination colony morphology when grown on cornmeal, Czapek-Dox, malt extract, and potato dextrose agar (Supplementary Figure S1). The taxonomic classification was confirmed by comparison of the internal transcribed spacer (ITS) and 5.8S rRNA gene deoxyribonucleic acid (DNA) regions with corresponding sequences available in the GenBank database (National Center for Biotechnology Information, US National Library of Medicine, Bethesda, MD, USA). The genomic DNA of JAJ1-073 was isolated using a DNEasy plant mini kit (Qiagen, Toronto, Ontario) as directed by the manufacturer; the ITS gene was amplified by polymerase chain reaction using the ITS1 and ITS4 universal fungal primers (Invitrogen, Burlington, Ontario) as previously described,¹⁸ and the amplified ITS DNA was sequenced by Genome Québec (Montreal, Québec). The JAJ1-073 DNA sequence was checked for ambiguity before being compared with existing GenBank sequence data using the Basic Local Alignment Search Tool. The ITS gene sequence of JAJ1-073 was found to have >99% homology with numerous conspecific A. pullulans isolates and has been deposited in GenBank (accession number: MT738220).

Fermentation and Isolation

JAJ1-073 was fermented in 1.2% potato dextrose broth at room temperature with shaking (150 rpm) for 2 weeks (5 L; 50 × 100 mL batches in 250 mL Erlenmeyer flasks stoppered with foam baffles). Fermentation cultures were sonicated for 30 s, the fungal material was removed by filtration, and the spent broth was extracted with ethyl acetate (EtOAc, 3 × 1.6 L). The organic fractions were combined and concentrated in vacuo (650 mg). The PDB extract was subjected to C₁₈ flash chromatography (stepwise gradient from 100% H₂O to 100% acetonitrile [CH₃CN] in 10% increments) to give 11 fractions. Fraction 3 (4:1 H₂O/CH₃CN; 146 mg) was subjected to further isocratic reversed-phase HPLC (7:3 H₂O/CH₃CN) to give 1 (56 mg) and 2 (49 mg).

(R,2E,5Z)-2-((E)-But-2-en-1-ylidene)-3-hydroxy-4-oxohept-5-enal (1) $[α]_{D}^{25}$

-160.8 (c 4.00, MeOH); UV (MeOH) λ_max (log ε) 227 (2.84), 275 (2.68) nm; CD (c 3.4 mM, MeOH) λ_max (Δε) 200 (0.48), 217 (0), 234 (-0.46), 255 (0), 274 (0.17), 290 (0), 313 (-0.11), 353 (0) nm; ¹H and ¹³C NMR data in CDCl₃, see Table 1 and Supplementary Figures S2–S7; HR-ESI-MS m/z 217.0834 [M+Na]⁺ (calcd for C₁₁H₁₄O₃Na⁺, 217.0835).

(R,2E,5E)-2-((E)-But-2-en-1-ylidene)-3-hydroxy-4-oxohept-5-enal (2) $[α]_{D}^{25}$

-41.6 (c 1.00, MeOH); UV (MeOH) λ_max (log ε) 217 (2.87), 272 (2.64) nm; CD (c 20 mM, MeOH) λ_max (Δε) 207 (0.19), 227 (0), 239 (-0.03), 245 (0), 270 (0.09), 295 (0), 306 (-0.02), 341 (0), 362 (0.1) nm; ¹H and ¹³C NMR data in CDCl₃, see Table 1 and Supplementary Figures S9–S14; HR-ESI-MS m/z 217.0835 [M+Na]⁺ (calcd for C₁₁H₁₄O₃Na⁺, 217.0835).

ECD and ORD Computational Methods

An exhaustive molecular mechanics conformer search of 1 and 2 was performed through systematic (Spartan 20, Wavefunction Inc., Irvine, CA, USA), Monte Carlo (Spartan 20) and GMMX (PCModel 10, Serena Software, Bloomington, IN, USA) searches using the MMFF94 force field. These methods identified 125, 61, and 108 conformers, respectively, for 1 and 107, 101, and 73 conformers, respectively, for 2. The two series of conformers were consolidated into sets of unique conformers (124 for 1 and 101 for 2). The geometries of the unique conformers were optimized by calculation at the DFT level using the BP86/def2-SV(P) functional at ground state in polar solvent (Spartan 20) and refined by sequential DFT optimization and frequency calculation (ORCA 5.01, Max Planck Institute fuer Kohlenforschung, Mülheim an der Ruhr, Germany) at the HF-3c (124 and 101 conformers, respectively), BLYP-D3BJ/def2-SVPD (51 and 34 conformers, respectively), and B3LYP-D3BJ/def2-TZVPD (23 and 15 conformers, respectively) levels with duplicate conformations and high-energy conformers that were not represented in 99.9% of the Boltzmann distribution being removed at each step.^19,20 The conductor-like polarizable continuum (CPCM) implicit solvation model was used in all DFT calculations to account for the effect of solvation in methanol.

Electronic transition and rotational strength calculations were performed by TDDFT for 10 transitions/excited states at the CAM-B3LYP-D3BJ/def2-TZVPD[CPCM]//B3LYP-D3BJ/def2-TZVPD[CPCM] level (ORCA 5.01) and optical rotatory dispersion calculations were carried out by DFT for wavelengths unaffected by electronic absorptions²¹ (ie >420 nm) at the CAM-B3LYP/def2-TZVPD[CPCM]//B3LYP-D3BJ/def2-TZVPD[CPCM] (Gaussian16²²) on the conformers that made up 99.9% of the Boltzmann distribution at the B3LYP-D3BJ/def2-TZVPD level (18 for 1 and 13 for 2). None of the conformers in the DFT calculations displayed imaginary frequencies indicative of saddle-points demonstrating that they were all true minima of the potential energy surface. The calculated ECD spectra were constructed using the Gibbs free energy Bolzmann-weighted average of oscillator and rotational strength values obtained for each of the conformers’ excited states using SpecDis v1.71.²³ The bandwidths and UV shifts used to generate the calculated ECD spectra (0.29 eV and -4 nm for 1 and 0.29 eV and -13 nm for 2) were obtained by comparison of the experimental and calculated UV spectra using the SpecDis v1.71 cross-sectional algorithm.²³ Calculated ORD values are weighted averages of the specific rotations of conformers that comprised 99.9% of the Boltzmann distributions of 1 (18 conformers) and 2 (13 conformers) at the B3LYP-D3BJ/def2-TZVPD[CPCM] level.

Antimycobacterial Activity

Antimycobacterial activity was determined against Mycobacteria tuberculosis strain H37Ra (ATCC 25177) using a microplate resazurin assay as previously reported.²⁴

Supplemental Material

sj-docx-1-npx-10.1177_1934578X231210086 - Supplemental material for Aureobasidols A and B: New C₁₁-Polyketides From an Endophytic Aureobasidium pullulans Isolate

Supplemental material, sj-docx-1-npx-10.1177_1934578X231210086 for Aureobasidols A and B: New C₁₁-Polyketides From an Endophytic Aureobasidium pullulans Isolate by Nicholas J. Morehouse, John A. Johnson and Christopher A. Gray in Natural Product Communications

Footnotes

Acknowledgements

The authors are grateful to Hilary Jenkins (McMaster University), Larry Calhoun (UNB), and Josh Kelly (UPEI) for acquisition of 700 MHz NMR, 400 MHz NMR, and high resolution ESIMS data, respectively.

Authors’ Contributions

N.J.M. and J.A.J. isolated and identified JAJ1-073 and confirmed its taxonomy. N.J.M isolated and elucidated the structures of compounds 1 and 2. N.J.M. and C.A.G. performed the in silico calculations. N.J.M. evaluated the bioactivity of compound 1 and 2. The manuscript was written through contributions of all authors and they all approved the final version of the manuscript.

Declaration of Conflicting Interests

The authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Financial support for this research was provided by the Natural Sciences and Engineering Research Council of Canada (Discovery Grant 2019-04114 to CAG and CREATE Grant 510963 supporting the CREATE Training Program in BioActives) and the New Brunswick Innovation Foundation (Research Assistantship Initiative grants 2018-033 and 2019-024 to CAG).

Ethical Approval

Ethical Approval is not applicable for this article.

Statement of Human and Animal Rights

This article does not contain any studies with human or animal subjects.

Statement of Informed Consent

There are no human subjects in this article and informed consent is not applicable.

ORCID iD

Christopher A. Gray

Supplemental Material

Supplemental material for this article is available online.

Notes

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Supplementary Material

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Aureobasidols A and B: New C 11 -Polyketides From an Endophytic Aureobasidium pullulans Isolate

Abstract

Keywords

Introduction

Results and Discussion

Conclusion

Experimental

General Experimental Procedures

Endophyte Isolation

Endophyte Identification

Fermentation and Isolation

(R,2E,5Z)-2-((E)-But-2-en-1-ylidene)-3-hydroxy-4-oxohept-5-enal (1) [ α ] D 25

(R,2E,5E)-2-((E)-But-2-en-1-ylidene)-3-hydroxy-4-oxohept-5-enal (2) [ α ] D 25

ECD and ORD Computational Methods

Antimycobacterial Activity

Supplemental Material

sj-docx-1-npx-10.1177_1934578X231210086 - Supplemental material for Aureobasidols A and B: New C11-Polyketides From an Endophytic Aureobasidium pullulans Isolate

Footnotes

Acknowledgements

Authors’ Contributions

Declaration of Conflicting Interests

Funding

Ethical Approval

Statement of Human and Animal Rights

Statement of Informed Consent

ORCID iD

Supplemental Material

Notes

References

Supplementary Material

(R,2E,5Z)-2-((E)-But-2-en-1-ylidene)-3-hydroxy-4-oxohept-5-enal (1) $[α]_{D}^{25}$

(R,2E,5E)-2-((E)-But-2-en-1-ylidene)-3-hydroxy-4-oxohept-5-enal (2) $[α]_{D}^{25}$

sj-docx-1-npx-10.1177_1934578X231210086 - Supplemental material for Aureobasidols A and B: New C₁₁-Polyketides From an Endophytic Aureobasidium pullulans Isolate