Penicimutamides A–C: rare carbamate-containing alkaloids from a mutant of the marine-derived Penicillium purpurogenum G59

Abstract

Three rare carbamate-containing alkaloids, penicimutamides A–C (1–3), were isolated from a fungal mutant from the diethyl sulfate (DES) mutagenesis of marine-derived Penicillium purpurogenum G59. Their structures, including their absolute configurations, were determined by spectroscopic methods, especially the X-ray crystallography and CD analyses. HPLC-UV and HPLC-MS analyses evidenced that 1–3 were only produced in the mutant strain via biosynthetic pathways that were silent in the parental strain and activated by DES mutagenesis.

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The activation of silent biosynthetic pathways for secondary fungal metabolites has been a promising route to discover new compounds. One-strain-many-compounds (OSMAC),¹ chemical epigenetics² and co-cultivation³ strategies for this purpose could activate silent pathways by variation of environmental factors for the growth of the producing strains. Ribosome engineering⁴ was able to activate silent bacterial pathways by selecting drug-resistant mutants with activated pathways that work under standard culture conditions.⁵ This strategy has recently come into use in fungi since we developed a new series of methods for fungi,^6a–c which subsequently resulted in the discovery of several new compounds.^6c–e During this study, we also developed a practical mutagenesis strategy using diethyl sulfate (DES) to activate silent fungal pathways.⁷ We previously reported that the DES mutagenesis of Penicillium purpurogenum G59 resulted in the production of a diverse range of secondary metabolites in mutants by activating pathways that were silent in the parent strain.^7a We also reported that a mutant AD-2-1, selected by treating G59 spores with 1% (v/v) DES in 50% (v/v) DMSO at 4 °C for 1 day, produced a series of novel lipopeptides in liquid culture via the activation of pathways that were silent in the G59 strain.^7d

Continuing our work in this area, we now report herein three new alkaloids, penicimutamides A–C (1–3 in Fig. 1), produced in solid culture by the mutant AD-2-1 by activating silent pathways in parent G59 strain. These alkaloids have a rare structural feature that has only ever been reported in one other compound to date.⁸

Fig. 1 Structures of penicimutamides A–C (1–3) and aspeverin (revised from 4a ⁸ to 4 in the present study).

Prenylated indole alkaloids (PIAs) are a broad class of secondary fungal metabolites with diverse structures.⁹ A subclass of the PIAs, including such as some of notoamides,¹⁰ brevianamides¹¹ and stephacidins¹² among others,^13–16 possess a bicyclo[2.2.2]diazaoctane ring system as their core structure. This subclass of PIAs has attracted much attention as targets for total¹⁷/biomimetic¹⁸ synthesis because of their fascinating structures.¹⁹ The biosynthesis of this subclass PIAs was also extensively studied for many years, with particular emphasis on the formation of their core bicyclo[2.2.2]diazaoctane ring system.^19–21 Despite considerable research in this area, only one carbamate-containing PIA, aspeverin, has been reported to date, which was isolated from an Aspergillus species.⁸ In this study, we have identified three new PIAs 1–3 with the same ring system as aspeverin. Notably, 1–3 were found to be biosynthetically related to some of the other PIAs listed above. We have also revised the absolute configuration of aspeverin (4a)⁸ to 4 (Fig. 1).

In this study, the mutant AD-2-1 and parental G59 strains were concurrently fermented under the same conditions at 28 °C for 50 days using rice as a solid-substrate fermentation medium to obtain methanol (MeOH) extracts of their cultures. The extract from the mutant inhibited K562 cells with an inhibition rate (IR%) of 62.5% at 100 μg mL⁻¹, whereas the parent extract exhibited no inhibition with an IR% of 6.1% at 100 μg mL⁻¹. The mutant MeOH extract contained many new metabolites in comparison with the parent extract, based on their HPLC-UV analysis. The separation of the mutant extract, which tracked new metabolites in the mutant extract, resulted in the isolation of compounds 1–3 along with various other new metabolites.

Penicimutamide A (1), obtained from MeOH as colorless needles with mp 168–170 °C and [α]²⁰_D −35.4 (c 0.71, MeOH), was assigned the molecular formula C₂₁H₂₃N₃O₃ by HRESIMS (m/z 366.1814 [M + H]⁺, calcd for C₂₁H₂₄N₃O₃ 366.1818). The ¹³C NMR data of 1 (Table 1) were similar to those of aspeverin except for an additional amide carbonyl signal at δ_C 167.4 (C-11) in 1 instead of the signals attributed to the C-11 methine and cyano groups in aspeverin.⁸ The chemical shifts of some of carbons positioned in close proximity to C-11 in 1 also changed accordingly. These ¹³C NMR data indicated that both these two compounds had the same skeletal structure. Then, detailed consideration of the DEPT, ¹H–¹H COSY, HMQC and HMBC spectra of 1 in combination with its IR absorptions enabled us to deduce the planar structure of 1 based on the key NMR data shown in Fig. 2. The IR absorptions observed at 3257, 3128, 1717 and 1582 cm⁻¹ were attributed to the carbamate ring in 1 at C-12 and C-14,⁸ whereas the amide band-I absorption at 1623 cm⁻¹ was consistent with the presence of an amide group at C-11. The NOEs observed in the NOESY of 1 between H₃-21/H-4, H-4/H-6, H-4/Hβ-13 and Hα-13/H-16 allowed us to establish the relative stereochemistry of 1 (Fig. 2).

Table 1 600 MHz ¹H and 150 MHz ¹³C NMR data of 1–3 in CD₃OD

No.	1		2		3
	δH (J in Hz)	δC	δH (J in Hz)	δC	δH (J in Hz)	δC
2	—	187.4 s	—	188.5 s	—	188.5 s
3	—	40.6 s	—	40.2 s	—	40.5 s
4	2.04 dd (13.0, 1.8)	52.4 d	1.97–1.85 m	48.4 d	1.61 dd (12.6, 3.3)	54.16 d
5	Hα 1.73 td (13.0, 11.6)	25.7 t	Hα 1.52–1.40 m	28.9 t	Hα 1.51 td (12.6, 10.2)	28.6 t
	Hβ 2.23 ddd (13.0, 4.6, 1.8)		Hβ 1.97–1.85 m		Hβ 2.00–1.92 m
6	3.53 tt (11.6, 4.6)	61.6 d	2.80–2.73 m	55.2 d	2.00–1.92 m	65.4 d
7	Hα 1.65 qd (11.6, 7.8)	33.5 t	Hα 1.52–1.40 m	31.4 t	Hα 1.59–1.51 m	31.2 t
	Hβ 2.19–2.14 m		Hβ 1.97–1.85 m		Hβ 2.00–1.92 m
8	Hα 2.09–2.02 m	23.2 t	Hα 1.97–1.85 m	22.8 t	Hα 1.93–1.85 m	22.5 t
	Hβ 1.92–1.83 m		Hβ 1.79–1.72 m		Hβ 1.82–1.76 m
9	Hα 3.63 dt (12.2, 9.0)	46.6 t	Hα 3.06 td (8.4, 3.0)	49.3 t	Hα 3.06 td (9.0, 2.4)	54.19 t
	Hβ 3.39 ddd (12.2, 9.8, 1.9)		Hβ 2.87 q (8.4)		Hβ 2.17 q (9.0)
11	—	167.4 s	Hα 4.09 s	95.1 d	Hα 3.02 d (11.4)	62.9 t
					Hβ 2.21 d (11.4)
12	—	58.2 s	—	57.6 s	—	54.4 s
13	Hα 3.17 d (13.8)	36.9 t	Hα 2.57 d (13.8)	37.8 t	Hα 2.50 d (14.4)	38.8 t
	Hβ 1.87 d (13.8)		Hβ 1.88 d (13.8)		Hβ 1.76 d (14.4)
14	—	88.0 s	—	88.3 s	—	88.0 s
15	—	137.3 s	—	137.5 s	—	137.4 s
16	7.56 br d (7.8)	124.4 d	7.53 br s (7.8)	124.3 d	7.518 br d (7.8)	124.3 d
17	7.34 td (7.8, 1.2)	128.2 d	7.32 td (7.8, 1.2)	127.9 d	7.31 td (7.8, 1.2)	127.9 d
18	7.47 td (7.8, 1.2)	132.0 d	7.46 td (7.8, 1.2)	131.8 d	7.45 td (7.8, 1.2)	131.8 d
19	7.52 br d (7.8)	121.7 d	7.52 br d (7.8)	121.6 d	7.516 br d (7.8)	121.6 d
20	—	153.9 s	—	153.8 s	—	153.9 s
21	1.50 3H, s	27.7 q	1.43 3H, s	27.6 q	1.44 3H, s	27.6 q
22	1.28 3H, s	21.6 q	1.20 3H, s	22.6 q	1.22 3H, s	22.2 q
23	—	155.1 s	—	155.1 s	—	155.0 s

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Fig. 2 Structures and key NMR data for 1.

The crystal structure of 1 (Fig. 3), determined by X-ray crystallography using Cu Kα radiation [Flack parameter = 0.0 (3)], established its absolute configuration as 4R,6R,12S,14S. Further in the TDDFT electronic CD (ECD) calculations^22,23 for 1 and its enantiomer ent-1, the calculated ECD of 1 also matched the measured CD data (ESI, Fig. S1†).

Fig. 3 The crystal structure of 1.

Penicimutamides B (2), a white crystalline powder with mp 164–166 °C and [α]²⁰_D +18.8 (c 0.6, MeOH) from MeOH, and C (3), a white wax with [α]²⁰_D −66.7 (c 1.73, MeOH) from MeOH, had the molecular formula C₂₁H₂₅N₃O₃ for 2 and C₂₁H₂₅N₃O₂ for 3 by HRESIMS. Both 2 and 3 had the same UV absorption pattern as 1, which indicated that the compounds 1–3 have the same chromophore. The IR spectra of 2 and 3 contained similar carbamate absorptions to those of 1, but the amide band-I absorption at 1623 cm⁻¹ in 1 was absent in 2 and 3. The IR spectra also indicated the presence of OH (3650–3150 cm⁻¹, br) in 2, but not in 3. The ¹H and ¹³C NMR data of 2 and 3 (Table 1) were similar to those of 1 except for the signals due to an O-bearing methine in 2 and a methylene in 3 instead of the C-11 amide carbonyl signal in 1. Detailed analysis of the DEPT, ¹H–¹H COSY, HMQC and HMBC spectra allowed us to deduce the planar structures of 2 and 3 based on the key NMR data shown in Fig. 4. The relative stereochemistry of 2 and 3 was determined to be as shown in Fig. 4 based on the key NOEs shown in the same figure.

Fig. 4 Structures and key NMR data for 2 and 3.

The absolute configuration of 2 and 3 was assigned based on their CD data. The CDs of 2 and 3 in MeOH showed Cotton effects [Δε (nm): −11.43 (223.5), +6.34 (256.5), −1.02 (278.5) and +1.56 (298) for 2; −8.49 (225), +6.96 (255.5), −1.39 (279.5) and +1.66 (298.5) for 3] that closely resembled the CD of 1 [Δε (nm) in MeOH: −6.06 (224.5), +5.20 (256), −1.16 (280) and +1.59 (298.5)], as shown in Fig. 5. These data indicated that 2/3 had the same absolute configuration 4R,6R,12S,14S as 1, and that the absolute configuration of C-11 in 2 would be R according to the relative configuration. We then performed TDDFT ECD calculations^22,23 on 2 (4R,6R,11R,12S,14S) and its enantiomer ent-2, and 3 (4R,6R,12S,14S) and its enantiomer ent-3. The results revealed that 2 and 3 produced ECDs that matched their measured CD data, whereas ent-2 and ent-3 gave opposite ECDs (Fig. S2 and S3 in the ESI†). The absolute configurations of compounds 2 and 3 were therefore assigned as 4R,6R,11R,12S,14S and 4R,6R,12S,14S, respectively.

Fig. 5 The CD spectra of 1–3 in MeOH.

The [α]²³_D +17.8 (c 0.18, MeOH) and the CD pattern [Δε (nm): −17.0 (221), +8.4 (256), −1.2 (278) and +2.5 (300)] of aspeverin in MeOH⁸ were almost identical to those of 2, indicating that these two compounds shared the same absolute configuration, as shown with 2 and 4 in Fig. 1. However, aspeverin was assigned as having the opposite configuration (4a, Fig. 1) in the literature.⁸ Though total synthesis of aspeverin has been accomplished,²⁴ the synthetic study did not give any additional evidence for the proposed absolute structure. We performed TDDFT ECD calculations^22,23 on 4a and 4. The results revealed that the calculated ECD of 4 matched the measured CD of aspeverin,⁸ whereas 4a gave the opposite ECD (Fig. S2 in the ESI†). We then calculated [α]_D for 4.²⁵ The sign of the calculated [α]_D +15.5 in MeOH for 4²⁵ consisted with the measured [α]²³_D +17.8 (c 0.18, MeOH) reported for aspeverin.⁸ These present results suggested that the absolute configuration of aspeverin reported as 4a in the literature⁸ had been assigned incorrectly. Based on these results, we revised the absolute configuration of aspeverin as 4R,6R,11S,12S,14S (4 in Fig. 1).

To determine whether 1–3 were also produced in the parent strain, the MeOH extracts from the mutant AD-2-1 and parent G59 strains were analyzed by the HPLC-photodiode array detector-UV scanning and HPLC-ESIMS using 1–3 as reference standards. The results revealed that 1–3 were only detected in the mutant extract and not the parent extract, as determined by the retention times and the UV and MS spectra (Fig. S5 and S6 in the ESI†). This result provided evidence that 1–3 were produced in mutant AD-2-1 by the activation of biosynthetic pathways that were originally silent in the parent strain and subsequently activated by the DES mutagenesis process in the mutant. Plausible biosynthetic pathways for the production of 1–3 that are likely activated in mutant AD-2-1 are proposed in Scheme 1.

Scheme 1 Plausible biosynthetic pathways for 1–3.

A common precursor for the biosynthesis of 1–3 is proposed to be deoxybrevianamide E, a fungal metabolite derived from L-proline, L-tryptophan and isoprene.^20,21 This precursor would produce I-2 via the intramolecular Diels–Alder [4 + 2] cyclization of the intermediate I-1.^19–21 Then, epoxidation of I-2 followed by the ring-opening of the epoxide would afford I-4. After that, differing from the ring-contractive pinacol-type rearrangement of I-4, which gave rise to the formation of other spiro-oxindole PIAs possessing the bicycle[2.2.2]diazaoctane ring system,^19–21 further enzymatic chemical modifications of I-4 and following products, as shown in Scheme 1, would produce 1–3.

In our test for inhibitory effect on human cancer cell lines K562, HL-60, HeLa and BGC-823, 1–3 and 5-fluorouracil (5-FU) gave IR% values at 100 μg mL⁻¹, listed in Table 2, which was determined by the MTT method after treatment of the cells with samples for 24 h at 37 °C. Under these conditions, only 2 showed stronger effect than the positive control 5-FU [IC₅₀ > 100 μg mL⁻¹ (796.2 μM)]. The IC₅₀ values for 2 were determined as given in Table 3.

Table 2 IR% values for 1–3 and 5-FU at 100 μg mL⁻¹

Cell lines	K562	HL-60	HeLa	BGC-823
1	8.9	6.7	28.5	11.4
2	81.0	92.7	77.3	77.4
3	22.9	18.2	12.5	7.8
5-FU	45.0	42.5	48.7	38.1

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Table 3 IC₅₀ values for 2 [μg mL⁻¹ (μM)]

Cell lines	K562	HL-60	HeLa	BGC-823
IC50	26 (70.8)	20 (54.5)	48 (130.8)	52 (141.7)

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Conclusions

In a similar manner to the ribosome engineering strategy,^4–6 the DES mutagenesis strategy allowed for the facile selection of fungal mutants with activated biosynthetic pathways that worked under standard culture conditions.⁷ The discovery of 1–3 in the current study from the mutant AD-2-1 strain further demonstrates the effectiveness of our previously reported DES mutagenesis strategy⁷ for obtaining new bioactive compounds from silenced fungal pathways. The results of this study also suggest that several other promising secondary metabolites could be identified by the DES mutagenesis of fungal species to activate otherwise silent pathways.

Conflicts of interest

The authors declare no conflict interest.

Acknowledgements

This work was financially supported by the grants from the NSFC (30973631/81573300), NHTRDP (2013AA092901/2007A A09Z411), NSTMP (2009ZX09301-002/2012ZX09301-003) and AMMS (2008), China, and the NSFC-Shandong Joint Fund for Marine Science Research Centers (U1406402), China.

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23. All of the TDDFT ECD calculations for 1/ent-1, 2/ent-2, 3/ent-3 and 4a/4 were performed in the gas phase using the Gaussian 09 software package (Gaussian 09, Revision A.01, Gaussian: Wallingford, CT, USA, 2009). See details in the ESI.†.
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25. The specific optical rotation of 4 was calculated using the Gaussian 09 software package, using four different methods. The calculations at the B3LYP/6-311++G(2d,p) level using the B3LYP/6-311++G(2d,p) in gas phase and PCM(MeOH)/B3LYP/6-311++G(2d,p) in MeOH geometries afforded [α]_D values of +78.5 and +71.5, respectively. Further, the calculations at the IEFPCM(MeOH)/B3LYP/6-311++G(2d,p) level using the IEFPCM(MeOH)/B3LYP/6-311++G(2d,p) in MeOH geometry and at the SMD(MeOH)/B3LYP/6-311++G(2d,p) level using the SMD (MeOH)/B3LYP/6-311++G(2d,p) in MeOH geometry afforded [α]_D values of +32.0 and +15.5, respectively. The sign of all of these calculated [α]_D values were consistent with that of the measured data for aspeverin in the literature (ref. 8).

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Footnotes

† Electronic supplementary information (ESI) available: Experimental procedures, NMR data for 1–3, calculated ECD spectra and DFT-optimized structures of the low-energy conformers for 1–4, figures for the HPLC-UV and HPLC-MS analyses, various spectra for 1–3, and X-ray data of 1 (CIF file). CCDC 1480828. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c6ra14904a

‡ These authors contributed equally to this work.

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