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Isolation of a 2-oxetanone from The Fruits of Synsepalum Dulcificum

Ming-Jen Cheng , Wen-Li Lo , Lee-Yu Huang , Chyi-Jia Wang & Chung-Yi Chen


Synsepalum dulcificum Daniell (Sapotaceae) is an evergreen shrub, native to tropical West Africa, and its fruits, red berries, have the remarkable property of modifying sour taste into sweet taste. Interestingly, the active material of the berry is the protein miraculin, which has no taste in itself (Chen, Kuo, Yang, Z. Wang, & H. Wang, 2009a). The intensity of the sweetness induced by 0.02 mol citric acid after 0.4 mola purified miraculin solution was held in the mouth is equivalent to that of about 0.3 mol sucrose. This value is equal to the maximum sweetness induced by miraculin (Chen et al., 2009b). In the course of screening for chemically novel agents from plants (Chen, Y. Wang, & H. Wang, 2010a; Chen, Y. Wang, & H. Wang, 2010b; Wang et al., in press), S. dulcificum was chosen for further phytochemical investigation. In this article, we report the isolation and structural elucidation of this 2-oxetanone compound.

Results and discussion

Ficumone, obtained as a clear oil, was established to have the molecular formula C3H4O3 by HR-EIMS at m/z [M] þ 88.0161 (Calcd for C3H4O3, 88.0160). The appearance of an IR band at 1750 cm 1 and a signal appearing at ¼ 173.0 in the 13 C-NMR spectrum indicate the oxetanone skeleton of this compound (Sandrine, Isabelle, Karine, & Philippe, 1993). The IR spectrum revealed the presence of hydroxyl group absorption at ¼ 3300 cm 1 . The 1 H-NMR spectrum of 1 showed one methine proton at ¼ 4.51 (1H, dd, J ¼ 6.0, 4.4), and one methylene proton at ¼ 2.81 (1H, dd ¼ 16.8, 6.0)/2.88 (1H, dd ¼ 16.8, 4.4), indicating that 1 was probably a 4-hydroxy-2-oxetanone. The 13 C-NMR spectrum indicated that compound 1 had a total of three carbons, with the skeleton consisting of a 4-hydroxy-2-oxetanone. The carbons of the 4-hydroxy-2-oxetanone were assigned, from 13 C-NMR and DEPT experiments, as one methylene at ¼ 38.3 (C-3), one methine at ¼ 67.1 (C-4) and one quaternary carbon at ¼ 173.0 (C-2). The structure of 1 was also confirmed by 2- D NMR experiments. A COSY correlation was observed between the H-3 and H-4 (Figure 1). The HETCOR experiment showed that the carbon signals at ¼ 38.3 for C-3 and 67.1 for C-4 were correlated to the proton signals at ¼ 2.81/2.88 for H-3 and 4.51 for H-4, respectively. The configuration at C-4 in 1 was determined as 4R on the basis of the []D value [þ2.35 (c ¼ 0.55), CHCl3]. The relative configuration of 1 was determined through 2-D NOESY analysis. The observation of the NOESY correlation from H-4eq. to H-3 suggested that 4-OH was in the -configuration. Thus, the structure of 1 was determined to be (R * )-4-hydroxy-2- oxetanone, and has been named ficumone. Following the structural elucidation of (R*)-4-hydroxy-2-oxetanone (1) by spectral means, it was determined that this compound had been prepared synthetically by Hanessian and Girard. Comparison of the spectral data of natural (R*)-4-hydroxy-2-oxetanone (1) with that of the presented synthetic compound proved it to be identical.


3.1. General

Ultra violet (UV) spectra were obtained on a Jasco UV-240 spectrophotometer in CH3CN; infrared (IR) spectra were measured on a Hitachi 260-30 O O HO H. NOESY correlations of ficumone. O O HO HMBC COSY. COSY and HMBC correlations of ficumone. Natural Product Research 1851spectrophotometer. 1 H-NMR (400 MHz), heteronuclear correlation (HETCOR), heteronuclear multiple bond correlation (HMBC), correlation spectroscopy (COSY), nuclear overhauser enhancement spectroscopy (NOESY) and distortionless enhancement by polarisation transfer (DEPT) spectra were obtained on a Varian (Unity Plus) NMR spectrometer. Low-resolution electron ionisation mass spectros

copy (EIMS) was collected on a Jeol JMS-SX/SX 102A mass spectrometer or Quattro gas chromatography–mass spectrometry (GC–MS) device having a direct inlet system. The high-resolution EIMS (HR-EIMS) spectrum was measured on a Jeol JMS-HX 110 mass spectrometer. Silica gel 60 (Merck, 70–230 mesh and 230–400 mesh) was used for column chromatography. Precoated silica gel plates (Merck, Kieselgel 60 F-254), 0.20 and 0.50 mm, were used for analytical TLC and preparative

TLC, respectively, visualised with 50% H2SO4.

3.2. Plant material

The specimen of the fruits of S. dulcificum was collected from Kaohsiung County, Taiwan, in October 2007. A voucher specimen was identified by Dr Fu-Yuan Lu (Department of Forestry and Natural Resources College of Agriculture, National Chiayi University) and was deposited in the School of Medical and Health Science, Fooyin University, Kaohsiung County, Taiwan.

3.3. Extraction and isolation

The air-dried fruits of S. dulcificum (2.5 kg) were extracted with methanol (MeOH) (10 L 6) at room temperature, and a MeOH extract (28.2 g) was obtained upon concentration under reduced pressure. The MeOH extract (28.2 g) was chromato graphed over silica gel (700 g, 70–230 mesh), using CHCl3–MeOH mixtures as eluents to produce 13 fractions. A part of fraction 5 (1.32 g) was subjected to silica gel chromatography by eluting with CHCl3 : MeOH (110 : 1), enriched with MeOH, to furnish 10 further fractions (5-1–5-10). Fraction 5-2 (0.22 g) was then purified on a silica gel column using CHCl3–MeOH mixtures to obtain ficumone (12 mg).

3.3.1. Ficumone ((R*)-4-hydroxy-2-oxetanone)

Clear oil; ½ 25 D þ 2.35 (c 0.55, CHCl3); UV-vis (CH3CN): max (log "): 210 (3.11) nm; IR (neat) max: 3300 (br, OH), 1750 (C¼O) cm 1 ; MS (EI, 70 eV): m/z (%): 88 [M] þ (67), 72 (100); HRMS-EI: m/z [M] þ Calcd for C3H4O3: 88.0160; found: 88.0161; 1 H-NMR (400 MHz, CDCl3) 2.81 (1H, dd, J ¼ 16.8, 6.0 Hz, H-3a), 2.88 (1H, dd, J ¼ 16.8, 4.4 Hz, H-3b), 4.51 (1H, dd, J ¼ 6.0, 4.4 Hz, H-4); 13 C-NMR (100MHz, CDCl3): 38.3 (C-3, CH2), 67.1 (C-4, CH), 173.0 (C-2, C).

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