Pericolactines A–C, a New Class of Diterpenoid Alkaloids with Unusual Tetracyclic Skeleton

Fusicoccane diterpenoids usually possess a fused 5-8-5 tricyclic ring system, which are biogenetically generated from geranylgeranyl diphosphate (GGDP). In our report, three novel diterpenoid alkaloids with fusicoccane skeleton, pericolactines A–C (1–3), were isolated from Periconia sp.. Their structures with absolute configurations were determined by spectroscopic analyses and quantum chemical ECD calculation. Pericolactines A–C (1–3) are a new class of diterpenoid alkaloids with an unusual fused 5-5-8-5 tetracyclic ring system, which derive from a geranylgeranyl diphosphate (GGDP) and serine conjugated biosynthesis. They belong to the atypical diterpenoid alkaloids.

Pericolactine B (2) was obtained as a white amorphous powder. It was assigned the molecular formula C 22 H 33 NO 4 (seven degrees of unsaturation) according to a quasi-molecular ion at m/z 376.2491 [M + H] + in its HRESIMS spectrum. The molecular weight of 2 was a 42 atomic mass unit (C 2 H 2 O) less than 1, which indicated that 2 may be a 19-deacetylated derivative of 1. The 1 H and 13 C NMR spectra of 2 showed resonances very similar to 1, except for the disappearance of one acetyl group. Further detailed NMR analyses involving 1 H-1 H COSY and HMBC spectra (see Supplementary Table S2 online) confirmed the above deduction and given the assignments of all proton and carbon resonances ( Table 1).
The fact that 2 is the deacetylated derivative of 1 was confirmed by acid hydrolysis. Pericolactine A (1) was treated with H 2 SO 4 in MeOH, and then the product prepared from 1 was compared with 2 using HPLC (see Supplementary Figure S1 online), which displayed that the retention times of the product prepared from 1 were identical to 2 isolated from fungal broth in three eluting systems. Based on the above mentioned fact, the relative configuration of 2 was assigned as 2S*, 3R*, 4R*, 5S*, 11R*, 15R*, which was the same as 1. The absolute configurations of C-2, C-3, C-4, C-5, C-11, and C-15 in 2 were determined by quantum chemical ECD calculation. The conformational analysis for a pair of enantiomers ((2S, 3R, 4R, 5S, 11R, 15R)-2 and (2R, 3S, 4S, 5R, 11S, 15S)-2) was carried out in CONFLEX version 7.0 with an energy window for acceptable conformers (0-3 kcal mol −1 ). The acceptable conformers were obtained, and continued to be optimized in Gaussian09. After that, five lowest energy conformers were found out. These lowest energy conformers (Fig. 4) were submitted to the ECD calculation at [B3P86/6-311+ + G (2d, p)] level, and the predicted ECD curve of (2S, 3R, 4R, 5S, 11R, 15R)-2 was similar to the experimental one ( Fig. 5 and see Supplementary information). Therefore, the absolute configuration of 2 was established as 2S, 3R, 4R, 5S, 11R, and 15R.
Since 1 and 2 possess the similar ECD curves (Fig. 5) and 1 and 2 coexist in the same strain, 1 and 2 possess the same absolute configurations. Thus, the absolute configuration of 1 was also assigned as 2S, 3R, 4R, 5S, 11R, and 15R.
Pericolactine C (3) was isolated as a white amorphous powder. Its molecular formula was established as C 23 H 35 NO 5 (seven degrees of unsaturation) by the quasi-molecular ion at m/z 428.2418 [M + Na] + in the HRESIMS. The 1 H and 13 C NMR spectra of 3 were very similar to 2, expect for the absence of C-5 methine and the appearance of an oxygenated sp 3 quaternary carbon (δ C 97.0) and a methoxy group (δ C 50.6/δ H 2.99). The key HMBC correlation from the additional methoxy group at δ H 2.99 to C-5 (δ C 97.0) indicated that H-5 in 2 was substituted by methoxy group in 3. On the basis of 2D NMR analysis (see Supplementary Table S3 online), the planar structure of 3 was established (Fig. 2), and the assignments of all proton and carbon resonances are shown in Table 1.

Materials and Methods
General experimental procedures. Optical rotations were measured on a JASCO P1020 digital polarimeter, and UV data were obtained with a JASCO V-550 UV/vis spectrometer. The CD spectra were recorded in MeOH using a JASCO J-810 spectrophotometer at room temperature. IR data were recorded using JASCO FT/IR-480 Plus spectrometer. HRESIMS spectra were obtained on Waters Synapt G2 TOF mass spectrometer. The NMR data were acquired with a Bruker AV 400 NMR spectrometer using solvent signals (CD 3 OD: δ H 3.30/δ C 49.0) as standards. Column chromatography (CC) was carried out on Sephadex LH-20 (Pharmacia, USA), and ODS (60-80 μ m, YMC). TLC was performed on precoated silica gel plate (SGF254, 0.2 mm, Yantai Chemical Industry Research Institute, China). Analytical HPLC was performed on a Dionex HPLC system equipped with an Ultimate 3000 pump, an Ultimate 3000 diode array detector, an Ultimate 3000 column compartment, an Ultimate 3000 autosampler (Dionex, USA), and an Alltech (Grace) 2000ES evaporative light scattering detector (Alltech USA) using a Phenomenex Gemini C18 column (4.6 × 250 mm, 5 μ m). Preparative HPLC was carried out on Shimadzu LC-6AD system equipped with UV detectors, using a Phenomenex Gemini C18 column (21.2 × 250 mm, 5 μ m). Semi-preparative HPLC was carried out on Shimadzu LC-6AD system equipped with UV detectors, using a YMC-Pack ODS-A column (10.0 × 250 mm, 5 μ m).  flasks (500 mL), each containing 70 g of rice. Distilled H 2 O (105 mL) was added to each flask, and the rice was soaked overnight before autoclaving at 120 °C for 30 min. After cooling to room temperature, each flask was inoculated with 5.0 mL of the spore inoculum and incubated at room temperature for 45 days.