Sporulaminals A and B: a pair of unusual epimeric spiroaminal derivatives from a marine-derived fungus Paraconiothyrium sporulosum YK-03

Abstract

Sporulaminals A (1) and B (2), a pair of unusual epimeric spiroaminal derivatives, bearing a 6/4/5/5 tetracyclic ring system derived from bergamotane sesquiterpenoid, were isolated from a marine-derived fungus Paraconiothyrium sporulosum YK-03. Their structures including the absolute configurations were elucidated by extensive NMR experiments, crystal X-ray diffraction and computational electronic circular dichroism (ECD) method. Furthermore, the epimerization induced by pH, temperature and H₂O was revealed, together with the hypothetical biosynthetic pathway.

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Introduction

Paraconiothyrium is a new genus proposed by Verkley from the phylogenetic point of view in 2004.^1,2 In recent years, the species of the Paraconiothyrium genus have attracted much attention due to the discovery of structurally diverse and biologically active secondary metabolites, including a number of various terpenoids (brasilamides A–N,^3–5 hawaiinolides A–G,^6,7 epoxyphomalins A–E⁸ and isopimarane diterpene glycosides⁹) and polyketide (graminin B,¹⁰ mycosporulone,¹¹ paraconfuranones A–H,¹² ascotoxin¹³ and coniothyriol¹⁴).

In the course of our continuing study on marine-derived fungi for novel chemistry, Paraconiothyrium sporulosum YK-03 (Genbank Accession Number KC416199), which was also proposed by Verkley through removing some Coniothyrium species including C. sporulosum and C. fuckelii to the genus Paraconiothyrium,¹ was isolated from the sea mud collected from the intertidal zone of Bohai Bay in Liaoning Province of China. Previous study on this fungus is seldom, and only two polyketides^11,14 have been reported until now. Our intensive chemical investigation of this fungus led to the isolation of a pair of unusual epimeric spiroaminal derivatives, sporulaminals A (1) and B (2) (Fig. 1). Notably, sporulaminals A (1) and B (2) represent the first example of a new class of spiroaminal, bearing an unusual 6/4/5/5 tetracyclic ring system originated from bergamotane sesquiterpenoid. To the best of our knowledge, only one single and two pairs of known bergamotene lactones (massarinolin A,¹⁵ expansolides A, B^16,17 and decipienolides A, B¹⁸) possessed this kind of ring system have been previously reported. As a pair of epimers at ketal carbon C-4, compounds 1 and 2 appeared to be interconvertible. Herein, the isolation, the structure determination based on the analysis of extensive 1D/2D NMR, crystal X-ray diffraction and computational electronic circular dichroism (ECD) method, the epimerization induced by pH, temperature and H₂O, as well as the hypothetical biosynthetic pathway of sporulaminals A (1) and B (2) are reported.

Fig. 1 Structures of compounds 1 and 2.

Results and discussion

Sporulaminal A (1) was obtained as a white amorphous powder. Its molecular formula was determined as C₁₅H₂₁NO₂ (six degrees of unsaturation) on the basis of the HR ESI-TOF MS data at m/z 270.1468 [M + Na]⁺. Analysis of the ¹H, ¹³C NMR (Table 1) and HSQC data of 1 revealed the presences of one methyl, six methylenes (one oxygenated), three methines, two sp³ quaternary carbons (one ketal carbon), one 1,1-disubstituted olefin, and one carboxyl carbon, indicating a tetracyclic system to satisfy the requirement of unsaturation. The HMBC correlations (Fig. 2) from H-15a to C-10/C-11, from H-15b to C-9, from H-9 to C-10, from H-8a to C-7/C-9/C-12, and from H-12a to C-11 confirmed the presence of a methylene cyclohexane ring (C7–C12). Meanwhile, the HMBC correlations of H₂-5/H-7/H-8a/H-9/H₂-14 to C-6, H₂-14 to C-5/C-7/C-9, and H-9 to C-5 suggested the linkages of C-6 to C-5, C-7, C-9 and C-14 and the presence of a bridged cyclobutane ring (C6–C9) fused with the cyclohexane ring through C-6, C-7 and C-9. In addition, the correlations from H₃-13 to C-1, C-2 and C-3, from H-3b to C-2 and C-4, and from –NH– to C-2, C-3 and C-4, revealed the existence of a γ-lactam ring (C1–C2–C3–C4–NH–), which was further supported by the IR spectrum, absorptions for a secondary amide group (3418.8 and 1706.1 cm⁻¹). Ketal carbon C-4 was connected with C-5 and C-14 via an etherlinkage, and hence a tetrahydrofuran ring (C4–C5–C6–C14–O) was established on the basis of the key HMBC correlations from H₂-5 and H₂-14 to C-4. The deduction was further supported by the downfield shift at C-14 (δ_C 68.8) and the remaining degrees of unsaturation. Therefore, the gross structure of sporulaminal A (1) was established.

Table 1 ¹H NMR (600 MHz) and ¹³C NMR (150 MHz) data for sporulaminals A (1) and B (2) in DMSO-d₆ (δ in ppm)

No.	1		2
	δH	δC	δH	δC
NH-1	8.46 (s)		8.40 (s)
1		178.8		178.7
2	2.43 (m)	34.8	2.28 (m)	35.6
3a	2.26 (dd, 12.6, 9.6)	42.4	1.70 (m)	41.8
3b	1.73 (m)		2.33 (dd, 12.6, 3.6)
4		94.8		95.9
5a	2.25 (d, 13.2)	46.7	2.29 (d, 13.2)	47.5
5b	2.29 (d, 13.2)		2.25 (d, 13.2)
6		51.1		51.0
7	2.35 (m)	40.2	2.26 (m)	40.6
8a	2.19 (m)	27.6	2.18 (m)	27.6
8b	1.42 (d, 9.6)		1.42 (d, 9.6)
9	2.71 (t, 6)	51.9	2.84 (t, 6)	50.7
10		150.3		150.0
11a	2.14 (m)	23.1	2.16 (m)	23.0
11b	2.48 (m)		2.47 (m)
12a	1.73 (m)	23.4	1.82 (m)	23.4
12b	1.79 (m)		1.80 (m)
13	1.01 (d, 7.2)	15.5	1.09 (d, 7.2)	17.3
14a	3.49 (d, 9.6)	68.8	3.49 (d, 9.6)	68.9
14b	3.37 (d, 9.6)		3.35 (d, 9.6)
15a	4.69 (s)	107.8	4.69 (s)	107.7
15b	4.65 (s)		4.64 (s)

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Fig. 2 Key HMBC correlations of compounds 1 and 2.

The relative stereochemistry of compound 1 was deduced by the NOESY experiment (Fig. 3). The relative configurations of the cyclohexane ring and bridged cyclobutane ring were established by the NOESY correlations of H-8b/H-11b, H-9/H-15b, H-14b/H-15b, H-14b/H-9 and H-7/H-14a. Meanwhile, the NOESY correlations of H-9/H-5b, H-7/H-5a, H-14a/–NH-1, H-7/–NH-1, H-5b/H-3b and H-3b/H₃-13 indicated that H-7, H-5a, H-14a and –NH-1 oriented in the same direction, and placed H-3b and H₃-13 on the same face of γ-lactam ring. Accordingly, the relative configuration of sporulaminal A (1) was established.

Fig. 3 Key NOESY correlations of compounds 1 and 2.

Sporulaminal B (2) obtained as a white amorphous powder was assigned the molecular formula C₁₅H₂₁NO₂ (six degrees of unsaturation) on the basis of the HR ESI-TOF MS data (m/z 270.1461 [M + Na]⁺), which was identical to that of 1. Analysis of the NMR data of 2 (Table 1; Fig. 2) led to the conclusion that 2 was an epimeric isomer of 1, and assigned the same gross structure as 1. The NOESY experiment (Fig. 3) resulted in the same relative stereochemistry for the cyclohexane ring and the cyclobutane ring of both compounds. The key NOESY correlations of H-7/H-14a, H-9/H-14b, H-14b/–NH-1, H-5b/–NH-1, H-9/–NH-1, H-14a/H-3a and H-3a/H₃-13 in 2 revealed that 2 is an epimer of 1 with a different chiral ketal carbon at C-4.

After many attempts, a co-crystal of compounds 1 and 2 suitable for single-crystal X-ray diffraction analysis (Fig. 4) was successfully obtained upon slow evaporation of the solvent mixture (methanol–water, 20 : 1) by keeping the sample at room temperature for nearly one month. Based on the relative stereochemistry of 1 and 2, the absolute configurations were unambiguously determined as 2S,4R,6S,7S,9S for sporulaminal A (1) and 2S,4S,6S,7S,9S for sporulaminal B (2) by using Cu Kα radiation. Furthermore, the assignment of absolute configuration was confirmed by ECD calculation (Fig. 5).

Fig. 4 Diamond plot of X-ray crystal structure for 1 and 2.

Fig. 5 Calculated and experimental ECD spectra of 1 and 2.

Interestingly, sporulaminals A (1) and B (2) were initially isolated as an inseparable solid mixture in a ratio of 5 : 2. Each of them obtained by column chromatography and HPLC employing a C₁₈ column could spontaneously gave rise to a mixture of both compounds, indicating that there is a transformation reaction between these two compounds. Moreover, similar epimerization phenomenon has also been observed in synthetic spiroaminal derivative, which was inevitably synthesized as an inseparable mixture.^19,20 In our study, two single epimers, sporulaminals A (1) and B (2), were successfully separated by HPLC employing a CHIRALPAK AD-H chiral column (250 × 4.6 mm, 5 μm) and using anhydrous ethanol as the mobile phase at a flow rate of 0.4 mL min⁻¹ (Fig. 6), and subsequently dried in vacuo at 25 °C.

Fig. 6 HPLC separation chromatogram of 1 and 2 on chiral AD-H column (250 × 4.6 mm, 5 μm).

The effect of pH, temperature- and H₂O-dependent epimerization between sporulaminals A (1) and B (2) were further studied. As shown in Fig. S3 (ESI†), the epimerization is quite sensitive to acid (pH = 6.0) and may be occurred during the fermentation, since that acid may be come into being along with the fermentation. The variable-temperature heating experiment (Fig. S4a and b†) revealed that the single compound 1 was stable in anhydrous ethanol solution below 30 °C. As temperature increased, the speed of epimerization accelerated. The constant-temperature heating experiment (Fig. S5†) suggested that H₂O could promote epimerization and the epimerization might be ongoing during the fermentation, because 28 °C was the exact temperature for the large scale fermentation of Paraconiothyrium sporulosum YK-03. As the amount of H₂O increased, the speed of epimerization also accelerated (Fig. S6†). Based on the above results, the epimerization of 1 and 2 can be induced by pH, temperature and H₂O. Meanwhile, the amount ratio of 1 and 2 was almost remained 2 : 1 constantly when they reached an equilibrium state under these three conditions (Fig. S3, S4a, b and S5†).

Sporulaminals A (1) and B (2) represent the first example of a new class of spiroaminal derivatives originated from bergamotane sesquiterpenoid. Accordingly, the plausible biosynthetic pathway to compounds 1 and 2 could be proposed as shown in Scheme 1. Firstly, a key biogenetic intermediate, β-tans-bergamotent, generated from farnesyl pyrophosphate (FPP) via nerolidyl diphosphate (NPP) followed by a bisabolyl cation and a bergamotane cation intermediately.^3,21 Oxidation of β-tans-bergamotent obtained intermediate A, subsequently underwent reduction and condensation with NH₃ to generate intermediate B. Then, two epimeric intermediates C1 and C2 were formed through an infer-molecule nucleophilic addition reaction of B, in which the carbonyl carbon cation group was attacked from the re- and si- face. Finally, compounds 1 and 2 could be yielded from C1 and C2 through the loss of H₂O, respectively.

Scheme 1 Plausible biosynthetic pathways of compounds 1 and 2.

Conclusions

In summary, our current study describes the discovery of a pair of unusual epimeric spiroaminals, sporulaminals A (1) and B (2), from a marine-derived fungus Paraconiothyrium sporulosum YK-03. Compounds 1 and 2 represent the first examples of spiroaminal derivatives with 6/4/5/5 tetracyclic ring system derived from bergamotane sesquiterpenoid. The unambiguous determination of their absolute configurations will provide valuable data for future research on this type of spiroaminal with 6/4/5/5 tetracyclic ring system. Furthermore, the successful separation of compounds 1 and 2 and the epimerization effect triggered by pH, temperature and H₂O will be valuable for the study of this type of inseparable epimeric spiroaminal mixture.

Experimental

General experimental procedures

Optical rotations were obtained on a Perkin-Elmer Model 341 polarimeter. Melting points were obtained on a SGM X-4 apparatus (Shanghai Precision & Scientific Instrument Co., Ltd., P. R. China). Melting points were obtained on a SGM X-4 apparatus (Shanghai Precision & Scientific Instrument Co., Ltd., P. R. China). UV spectra were measured on a Shimadzu UV-1601. IR spectra were taken on a Bruker IFS-55 infrared spectrophotometer with KBr disks. Circular dichroroism were measured on Bio-logic MOS 450 spectropolarimeter. The NMR spectral data were recorded on Bruker ARX-300 and AV-600 with TMS as the internal standard. The HR ESI-TOF MS data were obtained on the Micross Mass Autospec-UltimaE TOF mass spectrophotometer. Column chromatography was performed with Silica gel (Qingdao Marine Chemical-Co., Ltd, China) and Sephadex LH-20 (Pharmacia, Piscataway, NJ, USA). HPLC (Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan) was performed using a Shimadzu LC-20AB HPLC pump equipped with an SPD-20A detector, employing a YMC-Pack ODS-A column (250 × 4.6 mm, 5 μm, YMC Co., Ltd. Japan) and a CHIRALPAK AD-H column (250 × 4.6 mm, 5 μm, Daicel Chiral Technologies Co., Ltd, China). Reversed-phase HPLC (Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan) was performed using a Shimadzu LC-8A HPLC pump equipped with SPD-10A detector, employing a YMC-Pack ODS-A column (250 × 10 mm, 5 μm, YMC Co., Ltd. Japan).

Strain and cultivation

Paraconiothyrium sporulosum YK-03 was isolated from the sea mud collected from the intertidal zone of Bohai River in Liaonign Province of China. It was identified based on the analysis of the ITS sequence and has been deposited in the School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University. The strain was cultured on PDA (potato 20%, glucose 2% and agar 2%) medium in Petri dishes at 28 °C for 3 days, and were inoculated in a 500 mL Erlenmeyer flask containing 150 mL of media (maltose 2%, monosodium glutamate 1%, glucose 1%, yeast cream 0.3%, corn steep liquor 0.1%, maltose 2%, KH₂PO₄ 0.05%, MgSO₄·7H₂O 0.03%). After incubation at 28 °C and 180 rpm for 4 days, a 5 mL culture liquid was transferred as a seed into each of 500 mL flask containing 150 mL liquid medium (maltose 2%, monosodium glutamate 1%, glucose 1%, yeast cream 0.5%, east cream 0.3%, corn steep liquor 0.1%, mannitol 2%, KH₂PO₄ 0.05%, MgSO₄·7H₂O 0.03%, CaCO₃ 2%, sea water element 3.3%, pH 6.5). The flasks were subsequently incubated at the same conditions for 8 days.

Extraction and isolation

Following incubation, the fermentation broth of Paraconiothyrium sporulosum YK-03 (70 L) was concentrated and extracted with ethyl acetate and n-butanol, successively. The ethyl acetate extract (20 g) was subjected to silica gel column, eluted with CHCl₃–CH₃OH (100 : 1–0 : 1), yielding 13 fractions (A–M). Fraction F (1.2 g) was further fractionated by silica gel column using a gradient of petroleum ether–acetone (100 : 1–100 : 10) to yield five fractions (F1–F5). F5 (600 mg) was separated by Sephadex LH-20 column chromatography (CH₃OH) and semi-preparative HPLC (CH₃OH–H₂O, 43/100, flow rate 4 mL min⁻¹) to afford an inseparable mixture of 1 (t_R 109.3 min) and 2 (t_R 91.2 min). Each of them, obtained as a pure compound spontaneously gave rise to a mixture of both compounds in various proportions when kept in solution.

Separation of 1 and 2

The purified single compounds 1 and 2 was achieved by chiral HPLC to yield 1 (8.0 mg) and 2 (3.8 mg).

Chiral chromatographic condition:

(1) Column: CHIRALPAK AD-H column (250 × 4.6 mm, 5 μm).

(2) Mobile phase: anhydrous ethanol.

(3) Wavelength: 210 nm.

(3) Flow rate: 0.4 mL min⁻¹.

(4) Retention time: compound 1 (17.69 min); compound 2 (14.72 min).

(5) Dried mobile phase: in vacuo at 25 °C.

Sporulaminal A (1). White powder; mp 164–166 °C; [α]²⁵_D −135.5 (c 0.380, anhydrous ethanol); UV (CH₃CN) λ_max 211.0 nm; IR (KBr) ν_max 3418.8, 2927.3, 2872.4, 1706.1, 1666.1, 1632.4, 1451.4, 1384.2, 1122.3 cm⁻¹; CD (CH₃CN) λ_max 204 (+61.6), 225 (−30.2) nm; ¹H NMR (600 MHz, DMSO-d₆) and ¹³C NMR (150 MHz, DMSO-d₆) spectroscopic data, see Table 1; HR ESI-TOF MS m/z 270.1466 [M + Na]⁺, (calcd for C₁₅H₂₁NO₂ 247.1465).

Sporulaminal B (2). White powder; mp 179–181 °C; [α]²⁵_D −80.0 (c 0.075, anhydrous ethanol); UV (CH₃CN) λ_max 211.4 nm; IR (KBr) ν_max 3418.4, 3086.7, 2931.2, 2872.4, 1694.1, 1641.6, 1452.5, 1384.0, 1115.0 cm⁻¹; CD (CH₃CN) λ_max 204 (+40.1), 225 (+22.2) nm; ¹H NMR (600 MHz, DMSO-d₆) and ¹³C NMR (150 MHz, DMSO-d₆) spectroscopic data, see Table 1; HR ESI-TOF MS m/z 270.1461 [M + Na]⁺, (calcd for C₁₅H₂₁NO₂ 247.1465).

X-ray crystallographic analysis for the mixture of 1 and 2

Co-crystal data of sporulaminal A (1) and sporulaminal B (2): C₁₅H₂₁NO₂, M = 247.33, monoclinic, a = 12.3508(3) Å, b = 12.2285(3) Å, c = 17.9527(3) Å, β = 94.4756(18)°, U = 2703.15(11) ų, T = 102.5, space group P2₁ (no. 4), Z = 8, μ(Cu Kα) = 0.634, 19 254 reflections measured, 10 188 unique (R_int = 0.0264) which were used in all calculations. The final wR(F₂) was 0.1153 (all data). The crystallographic data for the structure of the mixture have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication CCDC no. 1444235.†

Computational methods

Relative configurations of 1 and 2 were deduced from their NOESY data.

Compound 1. Stochastic conformational searches were firstly conducted under MMFF94 force field for 1a (2S,4R,6S,7S,9S), which gave two conformers. Their difference was that the orientation of methyl of the lactam ring was different. Conformers were optimized at the B3LYP/6-31G(d) basis set level in methanol and the frequencies were calculated by further time-dependent density functional theory (TDDFT) method, showing that these two conformers combined into one stable conformer, which was identical to the conformer of X-ray crystallographic analysis of compound 1. Using the conformers at the B3LYP/6-31G(d) basis set level in methanol, forty excitation states at the B3LYP/6-31G(d) basis set level were calculated, peak stretcher was 0.4 eV, and finally the calculation results were Boltzmann averaged to yield the depicted electronic circular dichroism (ECD) spectra of 1, which was identical to the calculation results at the cam-B3LYP/6-31G(d) basis set level. All calculations were performed by Gaussian 09 program package (Version C.01).

Compound 2. The Spartan 14.0 (Wavefunction Inc., Irvine, CA, USA) search using molecular mechanics MMFF was performed for 2a (2S,4S,6S,7S,9S), which gave 100 conformers. The low-energy conformers of 2a accounting for more than 5% Boltzmann distribution were further optimized successively in the gas phase by semi-empirical method in Gaussian 09 program package, which were reoptimized and analysed frequency, orderly, using the density functional theory (DFT) at the B3LYP/6-31G(d,p) level and the same way in the methanol, resulted in no imaginary frequencies. Solvent effects were taken into consideration by using the conductor polarizable continuum model (CPCM). The conformers of 2a were calculated electronic circular dichroism (ECD) by the time-dependent density functional theory (TD-DFT) method at the B3LYP/6-31++G(d,p) level with the CPCM model in methanol solution. The overall calculated ECD curves of 2a were generated by Boltzmann weighting of their selected low-energy conformers using SpecDis 1.51 with σ = 0.12 eV at −8 nm shift.

pH-, H₂O- and temperature-dependent epimerization effect

All of the following dynamic HPLC spectra were recorded by chiral HPLC. The chromatographic condition was in accordance with that of the separation of 1 and 2 by chiral HPLC.

pH-Dependent epimerization effect: sporulaminal A (1) was stored at pH 6 and pH 8 and analytical runs taken over time to clearly show the epimerization occurrence.

Temperature-dependent epimerization effect: sporulaminal A (1) was dissolved in anhydrous ethanol and heated for one hour at 25, 30, 35, 40, 50 °C, respectively. No obvious variation was observed under 30 °C. After heated for 24 hours at 30 °C, no transformation was detected for 1. Then compounds 1 and 2 dissolved in anhydrous ethanol were continuously heated at 40 °C, respectively, to reach an equilibrium state.

H₂O-dependent epimerization effect: sporulaminal A (1) dissolved in H₂O and continuously heated at 28 °C, which was the exact temperature for the large scale fermentation of Paraconiothyrium sporulosum YK-03. The influence of different ratios of H₂O to EtOH was subsequently performed.

Acknowledgements

The work was financially supported by the National Natural Science Foundation of China (Grant No. 81202425) and the project for Scientific Research Fund of Liaoning Provincial Education Department (No. L2011178). We gratefully acknowledge Mr Yi Sha and Mrs Wen Li, Department of Analytical Testing Center, Shenyang Pharmaceutical University, for measurements of the NMR data. We thank Mr Jian Hao, Department of Analytical Testing Center, Beijing University of Chemical Technology, for the test of the X-ray diffraction, and Dr Li Li, Institute of Materia Medica, Chinese Academy of Medical Science Peking Union Medical College for ECD calculation.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: 1D and 2D NMR, HR ESE-TOF MS, UV, IR, CD spectra, the co-crystal data (CIF) and calculated ECD of 1 and 2. CCDC 1444235. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c6ra01401a

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