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Keywords:

  • absolute configuration;
  • vibrational circular dichroism;
  • density functional theory

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

The absolute configuration (AC) of the antiprotozoal lactone, Klaivanolide, 1, from Uvaria klaineana, has been determined using Vibrational Circular Dichroism (VCD) spectroscopy. The experimental VCD spectrum of the (+) enantiomer of 1 was measured. To analyze the AC of (+)-1, the conformationally-averaged VCD spectrum of 7-S-1 was calculated using density functional theory (DFT) and the GAUSSIAN 03 program. The B3PW91/TZ2P conformationally-averaged VCD spectrum of 7-S-1 proves that the AC of 1 is 7-S-(+). Chirality 21:E48–E53, 2009. © 2009 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Recently, the isolation and structural elucidation of an unusual 7-membered lactone (5-acetoxy-7-benzoyloxymethyl-7H-oxepin-2-one) 1, from the stems of Uvaria klaineana (Annonaceae), was reported.1 This natural substance, klaivanolide 1, showed potent in vitro antileishmanial activity against both sensitive and amphotericin B-resistant promastigote forms of Leishmania donovani, the parasite responsible for the visceral leishmaniasis, with IC50 (the concentration inhibiting parasite growth by 50% after a 3-day incubation period, compared to a non-treated culture) values of 1.75 and 3.12 μM, respectively. Klaivanolide 1 also showed promising in vitro trypanocidal activity against trypomastigote forms of Trypanosoma brucei brucei. As part of ongoing studies to discover new antiparasitic compounds2 klaivanolide 1 represents an interesting lead compound, which justifies further pharmacomodulation studies to optimize its antiparasitic activities. Full identification of klaivanolide 1 was thus required. In previous studies, its structure was unequivocally established from NMR spectra (see Table 1 of Ref.1), but not the absolute configuration (AC). We reasoned that it might be possible to determine its absolute configuration by comparison of its calculated and experimentally determined vibrational circular dichroism (VCD) spectra. The absolute configuration (AC) of a chiral molecule can be determined using VCD by comparison of its experimental VCD spectrum to the density functional theory (DFT)–calculated VCD spectra of the two enantiomers.3–10 In the case of a conformationally-flexible molecule, conformational analysis (CA) is required.

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Table 1. DFT relative energies, relative free energies and room-temperature populations of the conformations of 1
ConformerB3PW91/TZ2P
ΔEaΔGaP (%)b
  • a

    ΔE and ΔG in kcal/mole.

  • b

    Populations based on ΔG values, T = 298 K.

a0.000.0038.6
b−0.060.2724.5
c0.310.5515.3
d0.240.5814.5
e0.151.007.1

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

The IR and VCD spectra of a CDCl3 solution of (+)-1 were measured using Thermo Nicolet Nexus 670 and Bomem/BioTools ChiralIR fourier transform (FT) spectrometers, respectively. Harrick 597μ and 240μ cells with KBr windows were used. IR and VCD spectra were measured at resolutions of 1 and 4 cm−1, respectively. The baseline for the VCD spectrum of (+)-1 was the VCD spectrum of CDCl3. VCD scan times were 1 hour.

The DFT calculations of vibrational frequencies, dipole strengths and rotational strengths were done using the GAUSSIAN 03 program. The IR and VCD spectra were obtained thence using Lorentzian bandshapes (γ = 4.0 cm−1).

The (+)-klaivanolide sample was obtained as follows: air dried stems of Uvaria klaineana were extracted with MeOH at room temperature. After evaporation of the solvent under reduced pressure, the crude extract was dissolved in 80% aqueous MeOH and extracted with CH2Cl2. The CH2Cl2 extract was then purified by flash chromatography over silica gel with CH2Cl2-hexanes (70:30) as eluant to give the lead compound (+)-1. The purity of (+)-1 is >95% (as judged by 1H NMR); no HPLC analysis was performed.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

The IR and VCD spectra of (+)-1 were measured using a CDCl3 solution of concentration 0.03M in cells of pathlengths 597 μ and 240 μ. Since the racemate of 1 was not available, the baselines for the IR and VCD spectra were the spectra of CDCl3 in the 597 μ and 240 μ pathlength cells. The IR and VCD spectra in the frequency range 1900–800 cm−1 are shown in Figure 1.

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Figure 1. (a) IR spectrum of (+)-1, pathlength 597μ; (b) VCD spectrum of (+)-1, pathlength 597 μ, except over range 1248–1219 cm−1 where a 240μ pathlength is used. The gap at ∼900 cm−1 is due to strong absorption of the CDCl3 solvent.

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Since klaivanolide is conformationally flexible, CA is needed to analyze the IR and VCD spectra. CA is performed using SPARTAN 02 and the MMFF94 forcefield. Using a window of 20 kcal/mol, 33 MMFF94 conformers were found. Reoptimization using DFT at the B3LYP/6-31G* level led to 24 independent conformers. Calculations of harmonic vibrational frequencies for all 24 conformers confirmed their stability and gave their relative free energies. These in turn allowed the room-temperature equilibrium populations to be calculated. Only seven conformers have free energies <4.5 kcal/mol and populations >4%. Further optimization of these seven conformers, followed by vibrational frequency and free energy calculations, were then carried out using the B3PW91 functional, together with the TZ2P basis set. The energies, free-energies and room-temperature populations of the resulting five B3PW91/TZ2P-optimized conformations are listed in Table 1.

The structures of conformations a, b, c, d, and e at the B3PW91/TZ2P level are shown in Figure 2. Key dihedral angles of the B3PW91/TZ2P geometries of conformations a-e are given in Table 2. In each conformation, the 7-membered lactone ring has the same structure since the ring dihedral angles are all very similar. The acetate moiety C5O18C19O20C21 is expected to be planar with either cis or trans conformations of C5O18C19O20. In a previous study (see Fig. 1 of Ref.11), we have shown that the cis (C5O18C19O20 ≈ 0°) orientation is energetically more favorable than the trans (C5O18C19O20 ≈ 180°) orientation for the acetate group. Similarly, the benzoyloxy group is expected to prefer a cis (C8O9C10O17 ≈ 0°) arrangement rather than a trans (C8O9C10O17 ≈ 180°) orientation. The phenyl ring is essentially coplanar with its adjacent C[DOUBLE BOND]O group (O17C10C11C12 ≈ 0°). Thus, the main differences among the conformers results from rotation about the C7-C8, C8-O9 and C5-O18 bonds.

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Figure 2. B3PW91/TZ2P structures of conformations (ae) of 7-S-1. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

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Table 2. Dihedral anglesa of the conformations a–e of 7-S-1
Dihedralabcde
  • a

    Dihedral angles are in degrees.

O1C2C3C4−35.0−36.0−35.7−35.3−34.9
C2C3C4C511.811.512.112.011.9
C3C4C5C628.229.128.927.428.1
C4C5C6C70.50.2−0.51.20.2
C5C6C7O1−67.0−67.4−66.7−67.0−66.3
C6C7O1C278.878.679.177.777.8
C7O1C2C3−15.9−14.7−15.5−15.1−15.9
O1C7C8O966.863.0−179.1−67.6168.8
C6C7C8O9−171.1−175.3−57.456.5−69.1
C7C8O9C10176.784.8−84.6−174.7107.3
C8O9C10O17−1.8−0.51.22.0−3.0
C8O9C10C11178.5179.4−178.5−178.2177.0
O9C10C11C12175.1−176.9177.2−175.0−179.0
O17C10C11C12−4.73.0−2.54.81.0
C4C5O18C19−142.0−164.2−166.0−132.3−71.4
C6C5O18C1938.815.413.248.4111.3
C5O18C19O200.5−0.30.30.6−0.6
C5O18C19C21−179.7179.7−179.8−179.7179.4
C3C4C5O18−151.1−151.4−151.8−151.8−149.2
C7C6C5O18179.7−179.3−179.7−179.7177.3

The vibrational frequencies and dipole strengths androtational strengths of the conformations of 7-S-1 are calculated using DFT, with the functional B3PW91 and the basis set TZ2P; the results are listed in Table 3.

Table 3. Calculated B3PW91/TZ2P frequencies, dipole strengths and rotational strengths for conformations a–e of 7-S-1a
Modeabcde
νDRνDRνDRνDRνDR
  • a

    Frequencies ν in cm−1; dipole strengths D in 10−40 esu2 cm2; rotational strengths R in 10−44 esu2 cm2.

881841452.282.91831432.853.41829442.342.81843453.3102.01828475.73.7
871792743.6185.31792870.847.717951080.4−22.01791808.0−309.217951037.2125.9
861782680.6−82.71777506.631.41782422.53.11783711.5275.11780499.539.0
851697170.7−29.81691149.2−32.11690125.5−24.01700154.3−33.41714135.6−42.6
84165669.21.31658109.0−17.31655114.9−12.8165538.6−1.6165618.10.3
83165542.4−1.2165547.1−2.5165542.92.6165554.75.8165544.92.9
82163612.0−1.0163613.80.9163512.4−0.8163611.60.8163613.31.7
8115284.2−0.115293.91.715283.4−0.115294.20.315293.50.2
80149215.4−2.3148646.86.7148656.58.3148922.84.5149447.36.5
79148753.03.5147027.60.0148438.6−28.7148755.4−5.0148744.24.4
78147128.60.0146544.32.3147126.91.3147127.8−1.5147124.8−2.5
77146439.31.3146553.921.2146541.41.1146539.12.4146543.54.5
761442116.3−75.91440136.4−92.01440134.6−85.61442107.0−57.51439132.2−96.5
7514245.933.014251.98.5141146.832.0141142.7−11.9141084.192.7
741394180.6−20.21394195.7−15.61394196.3−6.91394186.5−35.01396193.3−14.8
73136685.64.0136518.32.0138530.3−59.5138424.034.1139138.5−45.6
72136525.416.3136357.919.8136512.03.813658.2−1.5136611.9−1.6
7113560.8−11.613577.9−30.8134687.1−20.0135472.143.9134235.6−9.2
70134251.34.5134255.92.8134220.013.5134235.4−7.0133518.60.1
691308777.4421.7132049.836.1132283.089.2131846.646.51317114.321.9
68129352.190.013081007.5490.91300574.7380.112951131.5−379.01290443.4115.3
6712811346.6−370.81290179.9169.41285828.4213.01283544.759.412811338.2−94.5
66126942.2−45.21279899.0−534.21280527.9−444.81271501.166.3127577.7−138.3
6512201240.8−74.412291851.4−165.212301561.6−272.512171223.497.412351164.87.1
641203577.515.91208342.192.51209369.433.41199256.538.91211400.6−121.1
631197181.8−5.41198219.9−31.91198204.9−41.31197361.4−15.81198173.03.3
6211844.4−2.311841.7−0.211843.2−1.911842.7−2.311853.1−6.7
6111811071.4−82.11177409.017.91177407.4132.01172969.4246.51178580.7206.3
601157338.819.61145536.8−94.41150373.693.21151908.867.1115962.8−30.3
591134535.5−83.81131463.534.41136567.4−121.51148144.6−23.111391153.541.2
581110205.491.11107102.386.5110686.182.1110625.9−15.4110774.713.3
571103240.731.61101337.2−26.81099348.1−144.51093294.3−18.11099206.627.3
561070233.8−104.91068193.5−154.91061470.0−158.9106016.4−30.91064352.8−68.8
55106023.5−7.6105929.6−9.4106018.919.51056223.0−42.7106118.3−28.0
54105499.53.21053124.9−2.4105371.7−10.81050100.618.1105485.4−2.4
53103662.1−14.2102238.6−29.31026145.1−40.6102299.8−24.8102215.10.1
5210227.40.8102212.8−1.3102115.0−3.7102175.3−8.010180.10.0
51101815.41.6101821.8−2.510190.10.010170.10.0101896.2−40.2
5010170.5−0.1101839.4−2.2101818.65.110171.6−0.2101617.011.9
491015310.6−29.51015237.5−1.6101589.514.910051.2−1.1101589.8−17.2
4810051.00.910050.2−0.310060.20.21002178.4−5.710050.1−0.2
4798894.6−17.798897.0−8.297417.5−0.59648.4−2.298796.016.4
469634.4−0.59645.20.99644.0−2.295582.440.09646.30.0
45937220.8−60.0939204.4−75.093616.5−12.6925127.4−12.193026.8−8.6
44912128.4−17.491291.42.2915100.115.3918100.8−9.9920161.3−77.3
4388239.7−7.0873104.320.4879128.8−43.089083.6−28.588262.8−11.3
428671.1−1.98670.40.28670.42.486846.2−18.38670.3−0.5
4185487.226.3837195.423.1838182.1−0.586710.58.2835216.135.3
40837226.00.383340.9−46.483046.5−12.5836254.9−9.382831.633.8
3982411.31.282512.75.08254.7−24.582313.30.88238.4−2.4
3880711.9−17.680712.4−17.380832.428.68177.95.081043.2−7.2
3777831.3−31.376922.2−20.979513.1−14.679218.0−9.679930.215.9

The conformationally-averaged IR spectrum of 1, predicted by the vibrational frequencies and dipole strengths of the conformations, is compared to the experimental IR spectrum in Figure 3.

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Figure 3. Comparison of the conformationally-averaged B3PW91/TZ2P IR spectrum of 1 to the experimental IR spectrum of (+)-1. Room-temperature equilibrium populations were obtained from B3PW91/TZ2P relative free energies (Table 1). Numbers are attached to help assign some of the experimental bands.

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The predicted IR spectrum allows a partial assignment of the experimental IR spectrum of 1. In particular, it enables us to characterize the bands which give rise to measurable VCD features. The GaussView program12 was used to animate the vibrational modes of 1. Band 1, centered at 1786 cm−1, is due to the C[DOUBLE BOND]O stretch of the acetate moiety (C19O20). The C[DOUBLE BOND]O stretch of the carbonyl group attached to the lactone ring gives rise to band 2 at 1746 cm−1. The benzoyloxy C[DOUBLE BOND]O stretch (C10O17), band 3, occurs at 1724 cm−1. The pair of bands (4 and 5) at 1603 cm−1 and 1563 cm−1 are associated with the asymmetric and symmetric stretch combinations of the lactone ring C[DOUBLE BOND]C bonds (C3C4 and C5C6), respectively. Bands 6 and 7 at 1273 cm−1 and 1232 cm−1 are the symmetric and asymmetric stretch combinations of the benzoyloxy and lactone ring C[BOND]O bonds (C10O9 and C2O1). Finally, band 8 at ∼1200 cm−1 is primarily due to the C[BOND]O stretch of the acetate group (C19O18). It should be noted that bands 6–8 are not pure modes and they include other atomic displacements in addition to those mentioned earlier.

The conformationally-averaged VCD spectrum of 7-S-1, predicted by the vibrational frequencies and rotational strengths of the conformations, is compared to the experimental VCD spectrum of (+)-1 in Figure 4.

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Figure 4. Comparison of the conformationally-averaged B3PW91/TZ2P VCD spectrum of 7-S-1 to the experimental VCD spectrum of (+)-1. Room-temperature equilibrium populations were obtained from B3PW91/TZ2P relative free energies (Table 1). The peaks used to assign the AC are labeled 1–8.

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In the experimental VCD spectrum the bands associated with the C[DOUBLE BOND]O stretches are not resolved. A broad positive peak, centered ∼1745 cm−1, is observed for bands 1–3. No VCD could be detected from the lactone C[DOUBLE BOND]C bond stretches (bands 4 and 5). A positive/negative couplet was observed for the C[BOND]O bond stretches at 1275 cm−1 and 1232 cm−1 (bands 6 and 7). Finally, a smaller negative peak was observed at 1198 cm−1 due to the acetate C[BOND]O stretch (band 8). These experimental VCD features are in good agreement with the predicted VCD spectrum shown in Figure 4.

The best assignment of the experimental VCD spectrum of (+)-1, based on the DFT calculated VCD spectrum of 7-S-1, is shown in Figure 4. As a result, the AC of 1 is concluded to be 7-S-(+).

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

The authors thank Dr. B. Akendengue for her help in the isolation of klaivanolide. They also thank the USC High Performance Computing and Communication (HPCC) facility for computer time.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED
  • 1
    Akendengue B,Roblot F,Loiseau PM,Bories C,Ngou-Milama E,Laurens A,Hocquemiller R. Klaivanolide, an antiprotozoal lactone from Uvaria klaineana. Phytochemistry 2002; 59: 885888.
  • 2
    Campos Vieira N,Herrenknecht C,Vacus J,Fournet A,Bories C,Figadère B,Salmen Espindola L,Loiseau PM. Selection of the most promising 2-substituted quinoline as antileishmanial candidate for clinical trials. Biomedicine Pharmacotherapy 2008; 62: 684689.
  • 3
    Stephens PJ,Devlin FJ. Determination of the structure of chiral molecules using ab initio vibrational circular dichroism spectroscopy. Chirality 2000; 12: 172179.
  • 4
    Stephens PJ,McCann DM,Devlin FJ,Smith AB. Determination of the absolute configurations of natural products via density functional theory calculations of optical rotation, electronic circular dichroism and vibrational circular dichroism: the cytotoxic sesquiterpene natural products quadrone, suberosenone, suberosanone and suberosenol A acetate. J Nat Prod 2006; 69: 10551064.
  • 5
    Stephens PJ,Pan JJ,Devlin FJ,Urbanová M,Hájíček J. Determination of the absolute configurations of natural products via density functional theory calculations of vibrational circular dichroism, electronic circular dichroism and optical rotation: the schizozygane alkaloid schizozygine. J Org Chem 2007; 72: 25082524.
  • 6
    Stephens PJ,Pan JJ,Devlin FJ,Krohn K,Kurtán T. Determination of the absolute configurations of natural products via density functional theory calculations of vibrational circular dichroism, electronic circular dichroism and optical rotation: the iridoids plumericin and iso-plumericin. J Org Chem 2007; 72: 35213536.
  • 7
    Stephens PJ,Pan JJ,Krohn K. Determination of the absolute configurations of pharmacological natural products via density functional theory calculations of vibrational circular dichroism: the new cytotoxic iridoid prismatomerin. J Org Chem 2007; 72: 76417649.
  • 8
    Stephens PJ,Pan JJ,Devlin FJ,Urbanová M,Julinek O,Hájíček J. Determination of the absolute configurations of natural products via density functional theory calculations of vibrational circular dichroism, electronic circular dichroism and optical rotation: the isoschizozygane alkaloids isoschizogaline and isoschizogamine. Chirality 2008; 20: 454470.
  • 9
    Figadère B,Devlin FJ,Millar JG,Stephens PJ. Determination of the absolute configuration of the sex pheromone of the obscure mealybug by vibrational circular dichroism analysis. J Chem Soc Chem Comm 2008; 11061108.
  • 10
    Stephens PJ,Devlin FJ,Pan JJ. The determination of the absolute configurations of chiral molecules using vibrational circular dichroism (VCD) spectroscopy. Chirality 2008; 20: 643663.
  • 11
    Devlin FJ,Stephens PJ,Österle C,Wiberg KB,Cheeseman JR,Frisch MJ. Configurational and conformational analysis of chiral molecules using ir and vcd spectroscopies: spiropentylcarboxylic acid methyl ester and spiropentyl acetate. J Org Chem 2002; 67: 80908096.
  • 12
    GaussView, Gaussian Inc., Pittsburgh, PA.