Note pubs.acs.org/jnp
Absolute Configuration of Amphidinin A Takahiro Iwai, Takaaki Kubota, and Jun’ichi Kobayashi* Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan S Supporting Information *
ABSTRACT: The absolute configurations at six stereogenic centers in amphidinin A (1), a cytotoxic linear polyketide isolated from a symbiotic marine dinoflagellate, Amphidinium sp., were elucidated to be 2R, 4R, 6S, 9R 11R, and 12S by the combination of J-based configuration analysis, modified Mosher’s method, and density-functional theory calculations.
A
1,2-O-isopropylidene derivative (2) of 1. Treatment of 1 with 2,2-dimethoxypropane (DMP) and pyridinium p-toluenesulfonate (PPTS) afforded the desired 2 and the 2,4-Oisopropylidene derivative (3), which were separated by a SiO2 column (Scheme 1). The relative configuration for C-2 and C-4 of 1 was confirmed, as 13C chemical shifts for the two methyl carbons in acetonide 3 showed typical values (δC 19.9 and 30.5) for a syn-1,3-diol acetonide.11 The 1,2-O-isopropylidene derivative (2) of 1 was treated with (R)-(−)- and (S)-(+)-2-methoxy-2-trifluoromethyl-2phenylacetyl chloride (MTPACl) to get the (S)- and (R)MTPA esters (4a and 4b, respectively). Δδ values obtained from the 1H NMR data of 4a and 4b indicated that the absolute configuration at C-4 of 2 was R (Figure 2). Thus, the absolute configurations at C-2, C-4, and C-6 of 1 were assigned as R, R, and S, respectively. The absolute configurations at C-9, C-11, and C-12 of 1 were deduced by the verification of NOESY data on calculated stable conformers for each of two possible structures (1a and 1b) of 1 (Figure 3). The top 100 stable conformers for each of 1a and 1b, obtained by the conformational search carried out with Spartan 10 (MMFF force-field), were optimized by ab initio molecular orbital calculations at the HF/3-21G level. Furthermore, the stable conformers within 40 kcal/mol of the most stable conformer for 1a and 1b (32 and 30 conformers, respectively) were optimized by DFT calculations at the EDF2/ 6-31G* level. The stable conformers for each of 1a and 1b that account for >95% of the Boltzmann distribution (5 and 7 conformers, respectively) were selected for the following discussions. The tetrahydrofuran rings of the stable conformers adopted a C-11 endo envelope conformation with C-19 in an equatorial position. The middle part of the stable conformers for each of 1a and 1b were superimposable (Figure S20). The distances of all hydrogen atom pairs exhibiting NOEs in the most stable conformer for 1a were consistent (3.0 Å) for observing NOEs (Figure 4). These results suggested that the true structure of amphidinin A (1) was 1a. This prediction was supported by comparison of NMR chemical shifts of 1 with theoretical NMR chemical shifts for 1a and 1b. The 1H and 13C NMR chemical shifts of each stable conformer for 1a and 1b were computed at the same approximation level. The theoretical NMR chemical shifts for 1a and 1b were estimated from calculated chemical shifts for each of the stable conformers for 1a and 1b weighted to their Boltzmann distribution. Both 1H and 13C NMR chemical shifts of 1 showed better agreement with the theoretical NMR chemical shifts for 1a than those for 1b (Figure 5). This comparison was also judged by DP4 probabiliy.12,13 DP4 analysis identified 1a as the true structure of amphidinin A (1) from two possible structures (1a and 1b) with a probability of 100%. Therefore, the absolute configurations at the six asymmetric centers in 1 have been concluded to be 2R, 4R, 6S, 9R, 11R, and 12S, respectively.
Scheme 1. Preparation of 1,2- and 2,4-O-Isopropylidene Derivatives (2 and 3, Respectively) from Amphidinin A (1)
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EXPERIMENTAL SECTION
General Experimental Procedures. The specific rotation was recorded on a JASCO P-1030 polarimeter. 1H and 13C NMR spectra were recorded on Bruker AMX-500 and Bruker AMX-600 spectrometers using 2.5 mm microcells (Shigemi Co., Ltd.) for CDCl3 and C6D6. The 7.26 and 7.20 ppm resonances of residual CHCl3 and C6D5H, respectively, were used as internal references for 1 H NMR spectra. The 77.0 and 128 ppm resonances of CDCl3 and C6D6, respectively, were used as internal references for 13C NMR spectra. MS spectra were recorded on Thermo Scientific Exactive and Thermo Scientific LTQ-OrbitrapXL spectrometers. Cultivation and Isolation. The dinoflagellate Amphidinium sp. (2012-7-4A) was separated from an Amphiscolops sp. flatworm, which was collected at Ishigaki Island, Okinawa, Japan. The voucher specimen was deposited at the Graduate School of Pharmaceutical Sciences, Hokkaido University. The dinoflagellates were cultured at 25 °C for 3 weeks under a 16 h light/8 h dark schedule in 500 mL of 4× Provasoli’s enriched seawater (PES) medium.14 The supernatant (25 L) was aspirated, passed through a filter paper, and subjected to a porous polymer gel column (Diaion HP-20, Mitsubishi Chemical Co., 8.5 × 35.0 cm). The column was washed with H2O (4 L), and the adsorbed materials were eluted with MeOH (4 L) and were concentrated in vacuo. The MeOH-eluted materials (2.3 g), obtained from 250 L of culture, were partitioned between n-hexane (500 mL × 3) and H2O (500 mL) to afford n-hexane-soluble materials (743.9 mg). The n-hexane-soluble materials were fractionated by a silica gel column (Wakosil C-300, Wako Pure Chemical Industries, Ltd., 2.5 × 30 cm; eluent, CHCl3/MeOH, 100:0 to 0:100), and a fraction (167.8
Figure 2. ΔδH values [ΔδH (in ppm) = δH of 4a − δH of 4b] obtained from the (S)- and (R)-MTPA esters (4a and 4b, respectively) of 1,2O-isopropylidene derivative (2) of amphidinin A (1).
Figure 3. Two possible structures (1a and 1b) for amphidinin A (1).
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Figure 4. Most stable conformers for each of two possible structures (1a and 1b) of amphidinin A (1). Selected NOESY correlations (indicated by blue dashed arrows) are shown with distances for hydrogen atom pairs. Hydrogen atoms of methyl groups are hidden.
Figure 5. Differences between NMR chemical shifts of amphidinin A (1) and theoretical NMR chemical shifts for 1a and 1b. (a) ΔδH (in ppm) = δH of 1 (600 MHz in CDCl3) − δH of 1a (blue bar) or 1b (red bar). (b) ΔδC (in ppm) = δC of 1 (150 MHz in CDCl3) − δC of 1a (blue bar) or 1b (red bar). The horizontal and vertical axes represent atom numbers of amphidinin A (1) and Δδ value [Δδ (in ppm) = δ of 1 − δ of 1a (blue bar) or 1b (red bar)], respectively. Protons with “R”, “S”, “E”, and “Z” are pro-R, pro-S, E, and Z protons, respectively. mg) was separated by a C18 column (Cosmosil 140 C18 OPN, 1.5 × 20 cm, Nakarai Tesque Inc.; eluent, MeOH/H2O, 60:40 to 100:0) and C18 HPLC (Mightysil RP-18 GP, Kanto Chemical Co., Inc., 20 × 250 mm; eluent, MeCN/H2O, 55:45; flow rate, 10.0 mL/min; UV detection at 210 nm) to afford amphidinin A (1, tR 32 min, 82.2 mg).5 The 13C-enriched amphidinin A (1, 6.4 mg) was obtained from 25 L of 4× PES medium, which was supplemented with sodium 13Cbicarbonate (0.5 mM) on the seventh day, by the same procedure as described above. Preparation of Acetonide Derivatives of 1. Amphidinin A (1, 1.0 mg) in acetone (100 μL) was treated with 2,2-dimethoxypropane (20 μL) and pyridinium p-toluenesulfonate (0.75 mg) at room temperature (rt). After 2 h, Et3N (0.75 μL) was added and the mixture was concentrated by N2 blowing. The residue was subjected to a silica gel column (hexane/EtOAc, 3:1) to afford the 1,2-O-isopropylidene derivative (2, 0.6 mg) of 1 and the 2,4-O-isopropylidene derivative (3, 0.4 mg) of 1.
1,2-O-Isopropylidene derivative (2) from 1: colorless oil; 1H NMR (600 MHz, C6D6) δ 5.63 (1H, dt, J = 15.2 and 6.9 Hz, H-14), 5.41 (1H, dd, J = 15.2 and 8.1 Hz, H-13), 4.98 (1H, s, H-21 a), 4.90 (1H, s, H-21b), 4.87 (1H, s, H-17a), 4.86 (1H, s, H-17b), 4.46 (1H, m, H-2), 4.28 (1H, m, H-12), 4.02−4.08 (2H, H-1a and H-4), 3.65 (1H, m, H1b), 2.89 (1H, m, H-6), 2.65 (2H, d, J = 6.8 Hz, H2-15), 2.29 (1H, d, J = 13.1 Hz, H-8a), 2.20 (1H, d, J = 13.1 Hz, H-8b), 2.14 (1H, m, H11), 2.10 (1H, m, H-3a), 1.70 (1H, m, H-3b), 1.70 (3H, s, H3-18), 1.56−1.67 (3H, m, H2-5 and H-10a), 1.49 (3H, s, acetonide-CH3), 1.38 (3H, s, acetonide-CH3), 1.28 (1H, dd, J = 12.3 and 9.6 Hz, H10b), 1.13 (3H, s, H3-20), 1.13 (3H, d, J = 6.7 Hz, H3-22), 0.79 (3H, d, J = 6.9 Hz, H3-19); 13C NMR (150 MHz, C6D6) δ 151.4 (C-7), 144.3 (C-16), 131.4 (C-14), 130.0 (C-13), 111.6 (C-21), 111.3 (C17), 108.5 (acetonide-C), 82.9 (C-9), 82.7 (C-12), 74.4 (C-2), 70.0 (C-1), 65.9 (C-4), 50.3 (C-8), 47.3 (C-10), 44.1 (C-5), 41.1 (C-3 and C-15), 36.3 (C-11), 35.5 (C-6), 27.3 (acetonide-CH3), 26.1 (acetonide-CH3), 23.7 (C-20 and C-22), 22.4 (C-18), 15.3 (C-19); 1543
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ESIMS m/z 429 [M + Na]+; HRESIMS m/z 429.29757 [M + Na]+ (calcd for C25H42O4Na, 429.29753). 2,4-O-Isopropylidene derivative (3) from 1: colorless oil; 1H NMR (600 MHz, C6D6) δ 5.71 (1H, dt, J = 15.3 and 6.7 Hz, H-14), 5.53 (1H, dd, J = 15.3 and 7.4 Hz, H-13), 5.06 (1H, s, H-21a), 5.01 (1H, s, H-21b), 4.87 (1H, s, H-17a), 4.86 (1H, s, H-17b), 4.42 (1H, m, H-12), 3.84 (1H, m, H-4), 3.69 (1H, m, H-2), 3.46 (1H, dd, J = 11.1 and 3.3 Hz, H-1a), 3.42 (1H, dd, J = 11.1 and 6.1 Hz, H-1b), 2.79 (1H, m, H6), 2.72 (2H, d, J = 7.0 Hz, H2-15), 2.54 (1H, d, J = 14.1 Hz, H-8a), 2.39 (1H, d, J = 14.1 Hz, H-8b), 2.30 (1H, m, H-11), 1.73 (1H, dd, J = 12.2 and 7.3 Hz, H-10a), 1.69 (3H, s, H3-18), 1.65 (1H, m, H-5a), 1.58 (1H, m, H-5b), 1.54 (3H, s, acetonide-CH3), 1.53 (1H, m, H10b), 1.38 (3H, s, acetonide-CH3), 1.25 (3H, s, H3-20), 1.25 (1H, m, H-3a), 1.14 (3H, d, J = 6.9 Hz, H3-22), 1.03 (1H, dt, J = 12.9 and 2.2, H-3b), 0.90 (3H, d, J = 6.9 Hz, H3-19); 13C NMR (150 MHz, C6D6) δ 152.3 (C-7), 144.5 (C-16), 131.4 (C-13), 129.6 (C-14), 111.5 (C-21), 111.2 (C-17), 98.6 (acetonide-C), 82.2 (C-9), 81.9 (C-12), 70.1 (C2), 67.1 (C-4), 66.3 (C-1), 48.8 (C-8), 46.2 (C-10), 43.6 (C-5), 41.2 (C-15), 36.9 (C-11), 35.8 (C-6), 33.2 (C-3), 30.5 (acetonide-CH3), 26.2 (C-20), 22.4 (C-18), 21.3 (C-22), 19.9 (acetonide-CH3), 15.8 (C-19); ESIMS m/z 429 [M + Na]+; HRESIMS m/z 429.29767 [M + Na]+ (calcd for C25H42O4Na, 429.29753). Preparation of MTPA Esters. DMAP (0.38 mg), Et3N (0.80 μL), and (R)-(−)-MTPACl (1.20 μL) were added to a solution of 2 (0.3 mg) in CH2Cl2 (50 μL). After stirring for 4 h at rt, N,N-dimethyl-1,3propanediamine (0.80 μL) was added, and the mixture was concentrated under a stream of N2. The residue was subjected to a silica gel column (hexane/EtOAc, 5:1) to afford the (S)-MTPA ester (4a, 0.3 mg). The (R)-MTPA ester (4b, 0.3 mg) was obtained from 2 (0.3 mg) by using (S)-(+)-MTPACl through the same procedure as described for the preparation of the (S)-MTPA ester (4a). (S)-MTPA ester (4a): colorless oil; 1H NMR (600 MHz, C6D6) δ 7.05−7.82 (5H), 5.64 (1H, m, H-14), 5.49 (1H, m, H-4), 5.46 (1H, dd, J = 15.0 and 7.1 Hz, H-13), 4.82−5.02 (4H, H2-17 and H2-21), 4.35 (1H, m, H-12), 3.96 (1H, m, H-2), 3.78 (1H, m, H-1a), 3.60 (3H, s), 3.27 (1H, m, H-1b), 2.75 (1H, m, H-6), 2.72 (2H, m, H2-15), 2.46 (1H, d, J = 14.5 Hz, H-8a), 2.32 (1H, d, J = 14.5 Hz, H-8b), 2.25 (1H, m, H-11), 1.96 (1H, m, H-5a), 1.95 (1H, m, H-3a), 1.77 (1H, m, H5b), 1.70 (3H, s, H3-18), 1.67 (1H, m, H-10a), 1.64 (1H, m, H-3b), 1.46 (1H, m, H-10b), 1.42 (3H, s, acetonide-CH3), 1.28 (3H, s, acetonide-CH3), 1.20 (3H, s, H3-20), 1.14 (3H, d, J = 6.8 Hz, H3-22), 0.86 (3H, d, J = 6.9 Hz, H3-19); ESIMS m/z 645 [M + Na]+; HRESIMS m/z 645.33777 [M + Na]+ (calcd for C35H49F3O6Na, 645.33734). (R)-MTPA ester (4b): colorless oil; 1H NMR (600 MHz, C6D6) δ 7.05−7.87 (5H), 5.64 (1H, m, H-14), 5.46 (1H, m, H-13), 5.46 (1H, m, H-4), 4.84−5.04 (4H, H2-17 and H2-21), 4.28 (1H, m, H-12), 4.15 (1H, m, H-2), 3.89 (1H, m, H-1a), 3.53 (3H, s), 3.41 (1H, m, H-1b), 2.72 (2H, m, H2-15), 2.59 (1H, m, H-6), 2.40 (1H, d, J = 14.4 Hz, H8a), 2.28 (1H, d, J = 14.4 Hz, H-8b), 2.25 (1H, m, H-11), 2.06 (1H, m, H-3a), 1.86 (1H, m, H-5a), 1.69 (3H, s, H3-18), 1.68 (1H, m, H-3b), 1.67 (1H, m, H-5b), 1.67 (1H, m, H-10a), 1.46 (1H, m, H-10b), 1.46 (3H, s, acetonide-CH3), 1.35 (3H, s, acetonide-CH3), 1.19 (3H, s, H320), 1.08 (3H, d, J = 6.9 Hz, H3-22), 0.86 (3H, d, J = 6.9 Hz, H3-19); ESIMS m/z 645 [M + Na]+; HRESIMS m/z 645.33780 [M + Na]+ (calcd for C35H49F3O6Na, 645.33734). Calculations. Conformational searches and chemical shift calculations were performed with Spartan 10 (Wavefunction, Inc.). Two possible structures (1a and 1b) of amphidinin A (1) were submitted to a conformational search, which was carried out with Spartan 10 (MMFF force-field). The top 100 stable conformers for each of 1a and 1b, selected from 10 000 conformers, were optimized by ab initio molecular orbital calculations at the HF/3-21G level. The stable conformers within 40 kcal/mol of the most stable conformer for 1a and 1b (32 and 30 conformers, respectively) were further optimized by DFT calculations at the EDF2/6-31G* level assuming solventless (vacuum) conditions. The stable conformers for each of 1a and 1b that account for >95% of the Boltzmann distribution (5 and 7 conformers, respectively) were subjected to chemical shift calculations. The 1H and 13C NMR chemical shifts of each stable conformer for 1a
and 1b were computed at the EDF2/6-31G* level. The theoretical NMR chemical shifts for 1a and 1b were estimated from calculated chemical shifts for each of the stable conformers for 1a and 1b with correction based on the Boltzmann distribution.
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ASSOCIATED CONTENT
S Supporting Information *
NMR data of 1, 2, 3, 4a, and 4b and superimposed views of each of the stable conformers for 1a and 1b. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +81 (11) 706-3922. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank Dr. E. Fukushi, Graduate School of Agriculture, Hokkaido University, for measurements of HETLOC and J-resolved HMBC-2 spectra, and Ms. S. Oka, Center for Instrumental Analysis, Hokkaido University, for measurements of ESIMS. This work was partly supported by The Naito Foundation and Grant-in-Aid for Sports, Science and Technology of Japan.
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REFERENCES
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