2025 Volume 73 Issue 4 Pages 314-317
3-Acetyl-11-keto-β-boswellic acid is a pentacyclic triterpenoid. It is found in frankincense, which is the resin found in plants from the Boswellia genus. Single crystals of 3-acetyl-11-keto-β-boswellic acid methanol and acetonitrile solvates were obtained from the Boswellia serrata extract. X-Ray crystal structure analysis revealed the coexistence of a brominated derivative, 3-acetyl-11-keto-12-bromo-β-boswellic acid. Mass spectroscopy analysis confirmed the presence of the brominated derivative in the extract. These results provide a structural basis for insights into the chemical reactivity and possibly the biosynthesis of 3-acetyl-11-keto-β-boswellic acid and its related substances in B. serrata.
Natural products have numerous bioactive properties.1–3) 3-Acetyl-11-keto-β-boswellic acid4) (AKBA, C32H48O5, molecular weight 512.72, Fig. 1a) is a pentacyclic triterpenoid. It is found in frankincense, which is the resin of a plant in the Boswellia genus that has been used as an incense, as a raw material for perfumes, and in herbal and Chinese medicines. AKBA exhibits pharmaceutical properties, including anti-inflammatory,5) antitumor,6) and neuroprotective7) activities, and is expected to be applied for drug therapy and as a lead compound for therapeutic drug discovery. It is highly insoluble in water; therefore, improving its solubility is crucial for its application in pharmaceutical formulations.
(a) Chemical structure of AKBA and Br-AKBA. Molecular structures of (b) AKBA and Br-AKBA methanol solvate and (c) those of acetonitrile solvate. C, H, O, and N atoms are shown in gray, white, red, and pale violet, respectively. Displacement ellipsoids of non-hydrogen atoms are drawn at the 50% probability level. Hydrogen bonds are shown with blue dotted lines.
In this research, we discovered a brominated derivative of AKBA (3-acetyl-11-keto-12-bromo-β-boswellic acid; Br-AKBA) during the isolation and formulation design of AKBA from Boswellia serrata extract. We characterized Br-AKBA using single-crystal X-ray structure analysis and mass spectroscopy.
Single-crystal X-ray structure analysis revealed that the crystals of AKBA obtained from the methanol/acetone solution and the acetonitrile solution were the methanol solvate crystal and the acetonitrile solvate crystal, respectively. The methanol solvate crystal was isomorphous with that previously reported.4) Initially, the asymmetric unit of the methanol solvate crystal was thought to contain one AKBA and one methanol molecule, while that of the acetonitrile solvate crystal contained two AKBA and two acetonitrile molecules, one ordered and the other disordered (Figs. 1b and 1c). Further crystallographic refinement revealed an additional spherical electron density in the vicinity of the C12 atom of AKBA (Figs. 2a and 2b, Supplementary Fig. 1S). No atoms of AKBA could be attributed to the electron density. The height of the unattributed electron density was comparable to those of a carbon atom. This indicated that the atom was either carbon or that of an element with a higher atomic number than carbon. The distance between C12 and the unattributed electron density was approximately 1.8 Å, which was significantly longer than the typical carbon–carbon single bond distance, at approximately 1.5 Å. This indicated that the crystals were mixtures of AKBA and an AKBA derivative with an unknown atom covalently bound to C12 and that the atomic number of the unknown atom was higher than that of carbon. To investigate the characteristics of the unknown atom, the Bijvoet difference map was calculated.8) The Bijvoet difference map is a Fourier map with coefficients of [F(+) – F(–)] and phases of ϕ+ π/2, where [F(+) – F(–)] is a structure amplitude difference of Friedel pair reflections and ϕ is a phase calculated from the structure model without atoms with high imaginary parts of the anomalous scattering, Δf″. The Bijvoet difference map indicates the locations of the atoms with the high Δf″ as peaks.9) In this study, this map showed a distinct spherical peak at the same location as the unattributed electron density (Fig. 2c). The highest value in the peak was 174σ above average, where σ was the standard deviation of the map. This meant that the unknown atoms had a high Δf″ value at the X-ray wavelength of MoKα 0.71073 Å. The C12 atom was the carbon in the alkene. Together, these data suggested that the unknown atom might be a halogen. The presence of such a derivative was not reported in the previous X-ray structure analysis of AKBA methanol solvate.4) The previous analysis used the resin of B. carteri. Birdw. and B. bhau-dajiana Birdw. from a supplier different from that of our research and AKBA was purified from the resin by silica-gel chromatography and C18 reverse-phase chromatography. Br-AKBA was not detected in the previous analysis because the resin was originally devoid of Br-AKBA or because the Br-AKBA, if present, was removed during the chromatographic purification.
Electron-density maps of the (a) methanol solvate crystal and (b) molecule A of the acetonitrile solvate crystal. (c) Bijvoet difference map of the methanol solvate crystal. These maps were countered at average + 1.2σ level, where σ is the standard deviation.
Mass spectroscopy of the B. serrata extract was performed to investigate the presence of AKBA derivatives. The base peak in the MS occurred at m/z 513 (Fig. 3). This peak corresponded to the protonated AKBA molecule [M + H]+, confirming that the major component in the extract was AKBA. Two small peaks with comparable heights were also found at m/z 591 and 593. This strongly suggested the presence of Br-AKBA in the extract, with the peaks corresponding to 79Br and 81Br, respectively. The peaks corresponding to AKBA derivatives with covalently bound fluorine, chlorine, or iodine atoms instead of a bromine atom were not observed in the MS spectrum.
M and M′ represent AKBA and 79Br-AKBA (nominal molecular mass of 511 and 591, respectively).
When a bromine atom was placed into the unattributed electron density, followed by further crystallographic refinements, the R-factors of the methanol solvate crystal and the acetonitrile crystal converged to low values of 0.0311 and 0.0340, respectively (Table 1). The Flack parameter10) can determine the absolute configuration of the structural model using the anomalous scattering effect. In this study, the values were almost zero, even when a bromine atom was introduced into the crystal structure model that had anomalously high scattering (Δf″ = 2.460, 0.002, and 0.006 electron at the wavelength of MoKα for bromine, carbon, and oxygen atoms, respectively). When the halogen atoms Cl, I, and F were located in the unattributed electron density near the C12 atom and crystallographic refinements were conducted, the R-factors were slightly worse than that of Br, the Flack parameters converged to abnormal negative values, and the halogen-C12 distances were significantly different from the typical distances (Supplementary Table S1). These results indicated that the presence of Br-AKBA mixed in the crystals was highly consistent with the observed X-ray diffraction data.
| AKBA | AKBA | |
|---|---|---|
| Methanol solvate | Acetonitrile solvate | |
| Chemical formula | C32H47.928O5Br0.072 · CH3OH | C32H47.844O5Br0.156 · C32H47.982O5Br0.018 · (CH3CN)1.654 |
| Temperature (°C) | −180 | −180 |
| Space group | P 212121 | P 21 |
| Cell parameter (Å) | ||
| a | 11.51237(8) | 13.37332(13) |
| b | 13.78006(9) | 15.97144(13) |
| c | 19.15109(13) | 15.32507(12) |
| α | 90 | 90 |
| β | 90 | 106.7346(8) |
| γ | 90 | 90 |
| Cell volume (Å3) | 3038.15(4) | 3134.67(5) |
| Z/Z′ | 4/1 | 4/2 |
| Data collection | ||
| Wavelength (Å) | 0.71073 | 0.71073 |
| (sinθ/λ) max (Å−1) | 0.692 | 0.697 |
| No. of reflections | ||
| Measured | 182832 | 156431 |
| Unique: all | 8127 | 16747 |
| F>σ(F) | 7787 | 16329 |
| Rint | 0.0489 | 0.0269 |
| Refinement | ||
| R (F) [F>4σ(F)] | 0.0311 | 0.0340 |
| wR (F2) (all) | 0.0836 | 0.0896 |
| Goodness-of-fit | 1.064 | 1.115 |
| Δρmax, Δρmin | 0.28, −0.18 | 0.30, −0.18 |
| Flack parameter | 0.010(27) | 0.022(15) |
| CCDC deposition number | 2484409 | 2404408 |
Standard errors of the last digits are shown in parentheses.
The C–Br bond distances in the refined crystal structures were 1.821–1.896 Å, which were close to the typical C-Br distances found in alkenes and significantly different from those of other halogens (Table 2). The occupancy of the bromine atom in the methanol solvate crystal was 7.2%. The bromine atom was located in the channel occupied by the methanol molecules and did not form an unfavorably close contact with other molecules in the methanol solvate crystal. Br-AKBA was incorporated into the crystal in proportion to its molar content during crystallization. In contrast, the occupancies of bromine atoms in the acetonitrile solvate crystal differed from the value estimated from the MS. The asymmetric unit of the acetonitrile solvate crystal contained two AKBA molecules, molecules A and B, and the occupancies of the bromine atoms were 15.6 and 1.8%, respectively. The bromine atom in molecule A protruded into the space where the disordered and partially occupied acetonitrile molecules were located, and no close unfavorable contact was formed. The bromine atom in molecule B formed close contacts that were smaller than the sum of the van der Waals radii with the methyl groups of the neighboring molecules in the crystal. Therefore, Br-AKBA may preferentially incorporate at the position of molecule A during crystallization of the acetonitrile solvate crystal because of the unfavorable contacts at the location of molecule B. Together, the crystallographic refinement statistics, interactions at the bromine atoms in the crystals, and mass spectroscopy results confirmed the presence of Br-AKBA in the crystals and in the B. serrata extract used in this study.
| Crystal | C–Br bond distance (Å) |
|---|---|
| Methanol solvate crystal | 1.821(2) |
| Acetonitrile solvate crystal | |
| Molecule A | 1.896(2) |
| Molecule B | 1.82(1) |
| C–X typical bond distance* (Å) | |
| X = F | 1.343 ± 0.021 |
| X = Cl | 1.721 ± 0.024 |
| X = Br | 1.876 ± 0.022 |
| X = I | 2.079 ± 0.022 |
*Average and standard deviation values of C–X (X = halogen) bond distances calculated from the crystal structures deposited in CCDC using Mogul software.
It is unknown whether Br-AKBA was an unexpected artifact formed during the undisclosed process of B. serrata extract preparation, B. serrata was grown in a high bromine concentration environment, or Br-AKBA was biosynthesized by the plants similar to that of bromoterpenoids biosynthesized by red algae in the sea.11) The direct substitution reaction of a hydrogen atom with a bromine atom at the C12 atom seems to be difficult based on the chemical structure. Therefore, AKBA-related substances such as AKBA precursors and metabolites might have possibly undergone addition or substitution reactions of the bromine atom. The Br-AKBA could be detected by the mass spectroscopy and single crystal X-ray analysis in this study, and this does not exclude the possibility that other derivatives, including the halogenated derivatives at the C12 atom other than the brominated one, were also present in the B. serrata extract. Although the process by which Br-AKBA was produced remains to be determined, the study provides the structural basis for insights into the chemical reactivity and possibly the land plant biosynthesis of AKBA and its related substances, including precursors and metabolites.
A new brominated derivative of AKBA, Br-AKBA, was found in the B. serrata extract and characterized by single-crystal X-ray analysis and mass spectroscopy. The bromine atom was covalently bound to C12 of AKBA. This study would promote the further research to identify the cause and molecular mechanism of the formation of the Br-AKBA derivative.
B. serrata extract containing approximately 50% AKBA was supplied by BIO ACTIVES JAPAN (Tokyo, Japan). The preparation method of the extract is not disclosed by the supplier. All other reagents were of the highest commercial grade.
Preparing AKBA CrystalsFour grams of B. serrata extract was dissolved in 50 mL of ethyl acetate and the solution was filtered through filter paper. The filtrate was slowly evaporated at room temperature to obtain AKBA crystals. Single crystals of AKBA methanol solvate were recrystallized using the evaporation method from a mixture solution of methanol and acetone (1 : 1). Single crystals of AKBA acetonitrile solvate were recrystallized using the evaporation method from an acetonitrile solution.
Single-Crystal X-Ray Structure AnalysesSingle-crystal X-ray diffraction data were collected at –180°C using an XtaLAB P200 system diffractometer (Rigaku Co., Ltd., Tokyo, Japan) with MoKα X-rays. Structure determinations and crystallographic refinements were performed using SHELXT,12) Oscail,13) SHELXL,14) and shelXle.15) All hydrogen atoms were well defined in the difference Fourier maps, except for those of disordered acetonitrile molecules. Hydrogen atoms were refined and placed on their parent atoms using the riding model. The crystal structures and diffraction data were deposited in the Cambridge Crystallographic Data Centre (CCDC) database. The statistics of the X-ray crystallographic analyses and the CCDC numbers are summarized in Table 1.
Mass SpectroscopyFast atom bombardment MS of the B. serrata extract was obtained using a JEOL JMS700 MStation mass system. A 1 : 1 mixture of dithiothreitol and α-thioglycerol was used as the liquid matrix.
We thank BIO ACTIVES JAPAN for providing the B. serrata extract and Dr. Katsuyoshi Mitsunaga (Faculty of Pharmaceutical Sciences, Toho University) for assistance with mass spectrometry.
The authors declare no conflict of interest.
This article contains supplementary materials.
