Letter pubs.acs.org/OrgLett
Penibruguieramine A, a Novel Pyrrolizidine Alkaloid from the Endophytic Fungus Penicillium sp. GD6 Associated with Chinese Mangrove Bruguiera gymnorrhiza Zhen-Fang Zhou,† Tibor Kurtán,‡ Xiao-Hong Yang,§ Attila Mándi,‡ Mei-Yu Geng,† Bo-Ping Ye,*,§ Orazio Taglialatela-Scafati,*,∥ and Yue-Wei Guo*,† †
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, People’s Republic of China ‡ Department of Organic Chemistry, University of Debrecen, P.O. Box 20, 4010 Debrecen, Hungary § School of Life Science & Technology, China Pharmaceutical University, Nanjing 210009, Jiangsu, People’s Republic of China ∥ Dipartimento di Farmacia, Università di Napoli ‘Federico II’, Via D. Montesano, 49, 80131 Napoli, Italy S Supporting Information *
ABSTRACT: A novel pyrrolizidine alkaloid, penibruguieramine A (1), characterized by an unprecedented 1-alkenyl-2methyl-8-hydroxymethylpyrrolizidin-3-one skeleton, was isolated from the endophytic fungus Penicillium sp. GD6, associated with the Chinese mangrove Bruguiera gymnorrhiza. The absolute configuration of penibruguieramine A (1) was established by TDDFT ECD calculations of the vacuum and solution conformers, exploiting the transitions of the lactam chromophore. A plausible pathway for its biosynthesis has been proposed.
M
The whole culture of Penicillium sp., strain GD6 (30L) was extracted exhaustively with EtOAc at room temperature. Then, the extract was subjected to repeated chromatography over MCI gel, silica gel, and C-18 reversed-phase silica gel to yield penibruguieramine A (1) (Supporting Information). In addition, three known compounds, namely scalusamide A (2),8 meleagrin (3),9 and roquefortine F (4),10 were also isolated during the purification process of 1 (Figure 1).
arine microorganisms are now recognized as a source of structurally unique and biopharmacologically interesting natural products, and they have been receiving considerable attention.1 Among them, fungal strains from marine plants constitute a hotspot in the recent research on endophytic fungi; in particular, fungi from mangrove habitats have shown great potential in the production of biologically active secondary metabolites.2 The genus Penicillium is one of the largest and most intensively investigated mangrove endophytic fungal genera.3 A wide array of bioactive secondary metabolites from mangrove endophytic Penicillium species have been characterized, including meroterpenoid and diphenyl ether derivatives,4 cytotoxic polyphenols,5 azaphilones, and p-terphenyls.6 In the course of our continuing efforts aimed at finding new bioactive substances from marine microorganisms,7 a fungal strain Penicillium sp. GD6, recently isolated from the Chinese mangrove Bruguiera gymnorrhiza collected off the coasts of Zhanjiang, China, was selected for further investigation since its crude EtOAc extract showed potent antibacterial activity against Staphylococcus aureus with an MIC value of 6.4 μg/ mL. A chemical investigation of the whole culture of Penicillium sp. GD6 has resulted in the isolation of a novel pyrrolizidine alkaloid, named penibruguieramine A (1), characterized by an unprecedented 1-alkenyl-2-methyl-8-hydroxymethyl pyrrolizidin-3-one skeleton. Herein, we describe the isolation, structure elucidation, and postulated biogenetic origin of this novel compound. © 2014 American Chemical Society
Figure 1. Structures of compounds 1−4. Received: January 16, 2014 Published: February 17, 2014 1390
dx.doi.org/10.1021/ol5001523 | Org. Lett. 2014, 16, 1390−1393
Organic Letters
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Penibruguieramine A (1),11 was isolated as a colorless gum. Its molecular formula was deduced to be C16H27NO3 by HRESIMS 304.1883 [M + Na]+ (cald 304.1889), suggesting the presence of four degrees of unsaturation. The IR spectrum showed a broad absorption at 3425 cm−1, suggesting the presence of hydroxyl group(s), and an absorption at υmax 1680 cm−1, in agreement with the presence of a five-membered lactam carbonyl. The 13C NMR spectrum of 1 (Table 1),
Figure 2. Key HMBC and 1H−1H COSY correlations of penibruguieramine A (1).
Table 1. 1H (500 MHz) and 13C NMR (125 MHz) Data for 1 in CDCl3 no. 1 2 3 5ax 5eq 6ax 6eq 7ax 7eq 8 9a 9b 10 1′a 1′b 2′a 2′b 3′a 3′b 4′ 5′ 6′ 7′
δH, mult. (J in Hz) 2.98, q (7.2) 2.94, 3.78, 1.86, 1.95, 2.28, 1.47,
m m ddd (12.2, 12.9, 6.3) m ddd (13.5, 11.0, 6.3) m
3.54, 3.71, 1.06, 1.61, 1.72, 1.54, 1.24, 1.38, 1.26, 2.00, 5.41, 5.41, 1.65,
d (11.9) d (11.9) d (7.2) dd (14.5, 4.5) m m m m m m m m d (4.9)
and of H3-10 with C-1 and the carbonyl C-3. These correlations allowed the location of the alkenyl chain at the oxygenated quaternary C-1 and linkage of this latter carbon to the methylene-bearing C-8, which, in turn, must be connected to the fragment c via C-7. The presence of an amide group and the still “loose end” of a quaternary carbon at δC 75.8 (C-8) were rationalized with the aid of the HMBC correlations of H25 with C-3 and C-8, indicating that C-5 and C-8 must be both attached to the amide nitrogen. The consequent formation of a pyrrolizidine ring system is in perfect agreement with the degrees of unsaturation. Thus, all the NMR data of 1 were unambiguously assigned in accordance with the planar structure of 1 as reported in Table 1. The relative configuration of stereogenic carbons around the pyrrolizidine core was established by detailed analysis of the ROESY spectrum of 1. As shown in Figure 3, the ROESY
δC 81.6, qC 49.1, CH 177.4, qC 42.8, CH2 26.5, CH2 27.2, CH2 75.8, qC 65.8, CH2 7.1, CH3 34.2, CH2 23.2, CH2 30.0, CH2 32.3, CH2 130.9, CH 125.2, CH 17.9, CH3
Figure 3. Key ROESY correlations for penibruguieramine A (1) (relative configuration shown).
analyzed by means of the 2D HSQC experiments, evidenced resonances of two methyls, eight sp3 methylenes (one oxygenated), two sp2 methines, one sp3 methine, two sp3 quaternary carbons, and one amide carbonyl. The presence of one disubstituted double bond was easily recognized by its 1H and 13C NMR resonances [δH 5.41, m, 2H, H-5′ and H-6′; δC 130.9 (C-5′) and 125.2 (C-6′)]. Resonances arising from two methyls, including one vinyl methyl (δ 1.65, d, J = 4.9 Hz) and one secondary methyl (δ 1.06, d, J = 7.2 Hz), were also observed in the 1H NMR spectrum of 1 (Table 1). Moreover, the appearance of a pair of AB-type proton signals at δ 3.71 (1H, d, J = 11.9 Hz) and 3.54 (1H, d, J = 11.9 Hz), coupled in the HSQC spectrum with the resonance at δ 65.8, clearly suggested the presence of an uncoupled oxymethylene group. Since the carbon−carbon double bond and the amide carbonyl accounted for only two of the required four sites of unsaturation, penibruguieramine A (1) must be bicyclic. Analysis of the 1H−1H COSY spectrum of 1, aided by HSQC experiment, revealed three proton connectivities shown bolded in Figure 2, and identified as a 2-hepten-7-yl moiety (a), −CHCH3 (b), and −CH2CH2CH2− (c) fragments. These subunits, the amide carbonyl, two quaternary carbons (δC 75.8; δC 81.6), and the oxygenated methylene (δC 65.8), were connected by careful interpretation of the well-resolved HMBC spectrum. Particularly diagnostic were the HMBC cross peaks of H-7 with C-8, C-1, and C-9, of H2-1′ with C-1, C-2, and C-8,
correlations of Ha-9/Hax-5, Heq-6/Hax-7, Hb-9/Hb-1′, and Ha1′/H-2 indicated that H2-9, H2-1′, H-2, Hax-5, Heq-6, and Hax-7 were all cofacial, arbitrarily assigned as α-oriented. The opposite (β) orientation of Hax-6 and Heq-5 was established by the NOE cross-peak of Hax-6/Heq-5. Thus, the relative configuration of 2 was determined as (1S*,2R*,8R*). The trans geometry of the Δ5′(6′) double bond was deduced by the 13C NMR chemical shift of C-7′, resonating at δC 17.9 (
