STATAN A

Example 4

Structural elucidation of Statan A

Accurate mass analysis was done on a Agilent HP 1100 liguid chromatograph with a diode array detector (DAD) coupled to a LCT Micromass oaTOF instrument with Z-spray electro spray source (ESI) and a lockspray probe. 1 microL of sample was injected on an Agilent Hypersil BDS-C₁₈ 125x2 mm column with 3 micro m particles. A water-acetonitrile gradient, starting with 15% acetonitrile-water going to 100% acetonitrile in 40 min, maintaining 100% acetonitrile for 5 min, before returning to the start conditions in 8 min equilibrating for 5 min. TFA, 50 ppm was added to the water. 

The MS was operated in the positive ESI mode using leucineenkephalin as lockmass ([M+H]+ ion at 556.2771 Da/e). The molecular ion of protonated Statan A appeared at 301.1421 corresponding to the composition C₁₈H₂₀O₄ (Δ -6.3 ppm) corresponding to 9 DBE.

NMR spectra of statans were recorded in 5 mm tubes at 600.13 MHz for ¹H and at 150.92 MHz for ¹³C and at 300 K, using DMSO-cf₆, on a Bruker DRX 600 according to Larsen et al. ,2001, (J Agricult and Food Chem 49: 5081-5084). The ¹³C and the C.H-COSY spectra revealed the presence of one methyl, four methylene, and two methine groups, together with 5 aromatic protons, two hydroxy protons (9.95 and 5.20 ppm), 5 quartemary aromatic carbon atoms and finally a carboxy group at 170.2 indicating an ester or lactone. 

The ¹H and H,H- COSY spectra revealed the presence of one aliphatic (-CH₂-CHOH-CH₂-CH-CH₂-CH₂-) and two aromatic spin systems (AB and ABC) together with a single methyl group. Interpretation of the heteronuclear multiple bond coherence spectrum (HMBC) established the structure of Statan A.

 The chemical shift values of the carbons in the naphthalene part of the molecule were very similar to the values of the model compound 7,8-dimethyl-1-naphtol (Jung, K.-Y., Koreeda, M., 1989, J Organic Chem 54: 5667-5675) and similarly the chemical shift values of the aliphatic part of the molecule were very similar to those reported for solistatin (Sørensen et al., 1999, Phytochemistry 51 : 1027-1029) and other statins, altogether strongly supporting the proposed structure.

¹H NMR (DMSO-d6): δ 9.95 (1 H, bs, 8'-OH), 7.54 (1 H, d, J = 8.2 Hz, H-4'), 7.24 (1 H, d, J = 8.2 Hz, H-3'), 7.24 (1 H, d, J = 7.9 Hz, H-5'), 7.16 (1 H, dd, J = 7.9 and 7.5 Hz, H-6'), 6.83 (1 H, d, J = 7.5 Hz, H-7'), 5.20 (1 H, br s, 3-OH), 4.72 (1 H, m, 5-H), 4.16, br s, 3-H), 3.45 (1 H, br s, H-7a), 3.36 (1H, br s, H-7b), 2.68 (1 H, dd, J = 17.2 and 4.7 Hz, H-2a), 2.44 (3H, s, Me), 2.43 (1 H, d m, 17.2 Hz, H-2b), 1.92 (2H, m, H-6), 1.91 (1 H, m, H-4a), 1.79 (1 H, ddd, 14.3, 11.7 and 3.3 Hz, H-4b).

¹³C NMR (DMSO-d6): δ 170.2 (C-1), 154.8 (C-8"), 135.1 (C-1')_F 135.1 (C-4'a), 132.1 (C-2'), 129.0 (C-3'), 125.8 (C-4'), 124.7 (C-6'), 123.1 (C-8'a), 119.5 (C-5'), 109.9 (C-7'), 75.5 (C-5), 61.0 (C-3), 38.5 (C-2), 36.5 (C-6), 34.8 (C-4), 27.0 (C-7), 19.5 (Me).

http://www.google.com/patents/WO2003048148A2?cl=en