Individual scale factor approach for the vibrational circular dichroism similarity-guided spectral and conformational analysis of perezone and dihydroperezone.

Evaluation of DFT calculated vibrational parameters for the IR and VCD spectra similarity of perezone (1) and dihydroperezone (2) was undertaken. Conformational sets were obtained using different search engines, and the parameters needed for spectra prediction were obtained using several combinations of commonly employed functionals and basis sets, and then weighted spectra were generated and compared with observed traces to provide infrared similarity (SIR ) and enantiomeric similarity index (ESI) values. These values evidenced a poor performance of the evaluated levels of theory that were overcome when using the individual scaling factors approach, providing 16% to 139% increases of the ESI values. The best performing level of theory was the B3LYP/DGDZVP2 with ESI values of 0.722 and 0.792 for 1 and 2. Moreover, a correlation analysis showed that the irregular DFT performance arises from rotational strength deviations, which suggests to discard conformational abundance accuracy as the main source of differences. Furthermore, a similarity guided conformational analysis showed that conformations with high ESI values prefer particular orientations of the CC bonds directly attached to the stereogenic carbon atom, with more distant dihedral angles having less influence. Additionally, folded and extended conformers appear to be equally capable to yield high individual ESI values, although abundances of folded conformers just account for 16% of the total population. Nevertheless, abundance optimization showed that a high ESI similarity value of 0.834, is possible when the population of these conformers is increased to 26%, suggesting that a larger abundance of these conformers might be present in solution.

Chiral NMR analysis reveals the environmental dependence of areolal scalemization in Piptothrix areolare.

The occurrence of racemic and enantiomerically enriched (scalemic) mixtures of secondary metabolites in their natural sources is a rare phenomenon. The unprecedent case of enantiomeric variations from levorotatory to dextrorotatory, and back to levorotatory, passing through an almost racemic mixture, was recently documented for areolal, the major epoxythymol of Piptothrix areolare. In an attempt to shed some light to understand the reasons for such an unusual behavior, herein, we evaluated this phenomenon by correlating the areolal enantiomeric purity with several environmental variables, including temperature, humidity, rain precipitation, wind speed, and radiation during over 1 year of the plant life cycle. The specific rotation and enantiomeric excess determined by 1 H-NMR-BINOL measurements provided the scalemic variations of areolal samples isolated from the roots collected from the same location along a 427-day period. The 1 H-NMR-BINOL methodology provided better sensitivity to enantiomeric variations than specific rotation measurements. Statistical data, including matrix correlation analysis, exploratory analysis by heatmap plotting, and the principal component analysis (PCA), suggested direct correlation of the scalemic variation with humidity, rain precipitation, and radiation variables with the best PCA explanation (78.4%) and noncritical or poor correlations in PCA explained in 60.2% and 48.4%, respectively. When variations in the optical activity parameter of any metabolite are observed, the search for scalemic mixtures along their host plant life cycle should be undertaken. Herein, this phenomenon could be associated with interactions with soil microorganisms and with evolutionary aspects of Piptothrix areolare which belongs to Asteraceae, one of the most successfully adaptable plant families.

Vibrational circular dichroism enantiomeric similarity index improvement for absolute configuration discrimination using individual infrared scaling factors.

The use of IR individual scaling factors (ISF) for the correction of DFT-calculated frequencies, and its effect on IR and VCD similarity functions, has been evaluated using (+)-(R)-3-methylcyclopentanone as a probe molecule. Contrary to using a single scaling factor to improve spectra matching, this approach sequentially searches for the optimal scaling factor for each calculated transition using a computational search algorithm to maximize the overlap of the calculated and observed IR spectra expressed as the IR similarity (SIR ) function. The obtained ISFs are then applied to the calculated frequencies, which are used to produce a scaled VCD spectrum for comparison with the observed trace, thereby yielding enantiomeric similarity index (ESI) values as a similarity measure. This procedure provides a significant improvement of the SIR and ESI values when compared with the use of a single scale factor, showing 15.1% and 34.1% in average increments, respectively, and values as high as 0.98 and 0.94, respectively. When a set of manually found ISFs is used, most differences in SIR and ESI performance disappear, and nearly perfect spectra matches are found throughout the levels of theory tested. This suggests that the observed differences in computed IR/VCD spectra with commonly used levels of theory are related to differences in frequency rather than to intensity accuracy. Finally, the use of ISFs is expected to enhance the ability to aide stereochemical assignments, particularly in cases where sufficiently accurate frequencies are difficult to obtain due to the system size or complexity.

A new germacranolide from Ageratina vernalis.

The aerial parts of Ageratina vernalis provided the new germacranolide 1,10-epoxydeltoidin A (3), together with the known pentacyclic triterpenoid hopane-6α,22-diol (1), and the also known germacranolides deltoidin A (2) and 15-hydroxydeltoidin A (4). In addition, pTsOH catalyzed cyclization of 2 afforded the new guaianolide 5. The absolute configuration of 2, 4, and 5 was assigned by vibrational circular dichroism spectroscopy, while the complete 1H and 13C NMR data assignments of 2-5 followed from 1 D- and 2 D-NMR experiments.

Absolute configuration assignment of stigmasterol oxiranes.

Diastereoisomeric stigmasterol oxiranes 4, 5, 8, and 9 are known phytosterol oxidation products (POPs) that have been evaluated for their cytotoxicity, although the results are of limited significance since, in most cases, they were evaluated as mixtures. Consequently, to establish biological activity hierarchy of these oxides, it is critical to evaluate individual pure POPs. Therefore, we now describe the obtention of individual molecules and their absolute configuration (AC) determination. The two acetylated C-5-C-6 oxiranes 6 and 7; the two acetylated C-22-C-23 oxides 10 and 11, obtained by means of Δ5 double bond protection-deprotection; and the four C-5-C-6, C-22-C-23 diepoxystigmasteryl acetates 19-22 were now individually gained and their AC determined by vibrational circular dichroism. Vibrational modes associated with the C-5-C-6 and the C-22-C-23 bonds were identified in dioxiranes 19-22 and used to assign the AC of monoepoxides 6, 7, 10, and 11. The AC of biological active non-acetylated molecules follows immediately. Due to the scarce spectroscopic information available for these POPs, the 1 H and 13 C NMR chemical shifts of 3-22 were assigned using 1D- and 2D-NMR experiments.

Methodology for the Absolute Configuration Determination of Epoxythymols Using the Constituents of Piptothrix areolare.

Since epoxythymols occur in Nature either as scalemic mixtures or as pure enantiomers, the knowledge of their chiral composition and of the absolute configuration (AC) of the dominant enantiomer turns out to be mandatory. This task has already been faced using 1,1-bis-2-naphthol (BINOL), as a chiral solvating agent in accurate 1H NMR quantifications to determine the enantiomeric ratio, and vibrational circular dichroism (VCD) to evidence the AC of the dominant enantiomer. We now explore the use of electronic circular dichroism (ECD) to determine the AC of an epoxythymol for which time-expensive DFT calculations would be required unless the AC of a related molecule is already known, from either VCD studies or single-crystal X-ray diffraction analysis, since one could correlate the ECD Cotton effect with the AC because in ECD only chromophores and their neighborhoods are evidenced. This method is now applied by using the epoxythymols from Piptothrix areolare. Known areolal (1) and 10-cinnamoyloxy-8,9-epoxythymol isobutyrate (2) were isolated from the roots, while known 7-acetoxy-10-cinnamoyloxy-8,9-epoxythymol isobutyrate (3) and 10-cinnamoyloxy-7-hydroxy-8,9-epoxythymol isobutyrate (4), as well as the new enantiopure 7-acetoxy-10-cinnamoyloxy-6-hydroxy-8,9-epoxythymol isobutyrate (5) and 10-cinnamoyloxy-8,9-epoxy-6-hydroxy-7-northymol isobutyrate (6), were obtained from the extract of the flowers. Chemical correlation of epoxythymols 1 and 3 was achieved. Compounds 1-4 were obtained as scalemic mixtures, and 5 and 6 as the pure (8S) enantiomers. In addition, the new 10-cinnamoyloxy-7-oxo-8,9-dehydrothymol isobutyrate (7) was isolated from the roots. The structures of 5-7 followed from NMR and HRMS data, while enantiomeric compositions of 1-6 were determined by 1H NMR-BINOL measurements. The AC determination for 2-6 was done by ECD using a sample of 1 to reference the ECD Cotton effect. In turn, the AC of 1 was determined by VCD and extensive DFT calculations. The ECD-BINOL methodology turned out to be some 500 times more sensitive than that combining VCD and 1H NMR-BINOL.

Novel Sesquiterpenoid Skeletons by Wagner-Meerwein Rearrangements of Longipinane-9,13-diol-1-one Derivatives.

The partial or total hydrolysis of (3R,4S,5S,6S,9R,10R,11R)-9,13-diangeloyloxylongipinan-1-one (1), isolated from the roots of Stevia viscida, gave alcohols 2 or 3, respectively, which were subjected to molecular rearrangements with boron trifluoride etherate. Compound 2 afforded (3R,4R,5R,6S,9R,10S,11S)-11,13-oxyneomorelian-1-one (10) and (4S,5R,6S,8S,10R)-10,13-oxyneojiquilp-2-en-1-one (11), both possessing novel sesquiterpenoid skeletons. In turn, 3 provided (3R,4R,5S,6S,9R,11R)-13-hydroxymoreli-10(14)-en-1-one (7) and 10. Acetylation of 3 gave 4, thus allowing reduction of the C-1 carbonyl group to yield 5, which was rearranged to (1S,3R,4S,5S,6S,9R,10R,11R)-13-acetoxy-9,11-epoxyjiquilpane (6), while an attempt to mesylate 3 directly gave rearranged (3R,4R,5S,6S,9R,11R)-13-mesyloxymoreli-10(14)-en-1-one (8) through expulsion of the C-9 mesylate group by the antiperiplanar C-4-C-10 bond migration to C-4-C-9. In addition, treatment of 1 with boron trifluoride etherate generated (3R,4R,5S,6S,9R,11R)-13-angeloyloxymoreli-10(14)-en-1-one (9). The structures of 2-11 were elucidated by 1D and 2D NMR experiments and those of 2, 3, 8, 10, and 11 were confirmed by single-crystal X-ray diffraction analysis.

Absolute configuration by vibrational circular dichroism of anti-inflammatory macrolide briarane diterpenoids from the Gorgonian Briareum asbestinum.

The four new briarane diterpenoids 2-butyryloxybriarane B-3 (2), 9-acetylbriarenolide S (3), briarenolide W (4), and 12-isobriarenolide P (5), along with briarane B-3 (1) and the five known diterpenes 6-10 were isolated from the gorgonian coral Briareum asbestinum collected from the Mexican Caribbean Sea. The structures were elucidated by 1D and 2D NMR and MS measurements. Since the structure of briarane B-3 (1) was only suggested and published without any spectroscopic support, it was herein confirmed, and the supporting data are now provided. In addition, 1 provided the opportunity to explore the sensitivity of vibrational circular dichroism (VCD) to determine the configuration of a single stereogenic center in the presence of eight other stereogenic centers in a molecule possessing a highly flexible ten-member ring. A single-crystal X-ray diffraction study, in which the Flack and Hooft parameters of 1 were determined, further confirmed that briarane B-3 is (1S,2S,6S,7R,8R,9S,10S,11R,17R)-1. This paper reports for first time the use of VCD in briarane diterpenes and with the presence of chlorine atoms. Biological evaluation of seven isolated compounds evidenced a moderate anti-inflammatory activity for compounds 6 and 9 but it did not show any cytotoxic, antiviral, antibacterial, and topoisomerase inhibitory activity.

Deuterium effects on the vibrational circular dichroism spectra of flavanone.

The minimum chemical modification that can be incorporated into an organic molecule is the replacement of a hydrogen atom for a deuterium atom. This change is not altering the pharmacological properties of a molecule, although it provides the possibility of making specific spectroscopic evaluations. Thus, in the present study, we explore how a stereogenic center is influenced by such an isotopic labeling. The studies were conducted on both enantiomers of flavanone (1 and 2) which is the parent molecule of a large group of pharmacologically active natural occurring secondary metabolites. Flavanone comprised 12 carbon atoms forming two benzene rings, a carbonyl group, an ethereal oxygen atom, a methylene group, and only one C-H stereogenic center, so it seems to be an ideal candidate for such studies. Density functional theory (DFT) calculations were used for the accurate prediction of vibrational circular dichroism (VCD) spectra of (R)-(3) and (S)-flavanone-2-d (4), of (R)-(5) and (S)-flavanone-3,3-d2 (6), and of (R)-(7) and (S)-flavanone-2',3',4',5',6'-d5 (8). To gain compounds that provide experimental VCD spectra for comparative purposes, the calculated spectra of both enantiomers of the corresponding flavanones, obtained after HPLC separation of the racemates by means of a chiral column, were contrasted, thereby revealing excellent agreements when using the CompareVOA software. In addition, the VCD spectra of both unlabeled enantiomeric flavanones (1 and 2) were also compared to the labeled molecules, revealing that the VCD spectra show significant variations induced by the deuterium incorporation.

Biological Properties and Absolute Configuration of Flavanones From Calceolaria thyrsiflora Graham.

Flavanones (-)-(2S)-5,4'-dihydroxy-7-methoxyflavanone (1) and (-)-(2S)-5,3',4'-trihydroxy-7-methoxyflavanone (2) were isolated from the extracts of Calceolaria thyrsiflora Graham, an endemic perennial small shrub growing in the central zone of Chile. The absolute configuration of these compounds was resolved by optical rotation experiments and in silico calculations. Three analogs (3, 4, and 5) were synthesized to do structure-activity relationships with the biological assays studied. Biological tests revealed that only flavanone 2 exhibited a moderate inhibitory activity against the methicillin-resistant strain S. aureus MRSA 97-77 (MIC value of 50 µg/ml). In addition, flavanone 2 showed a potent, selective, and competitive inhibition of 5-hLOX, which supports the traditional use of this plant as an anti-inflammatory in diseases of the respiratory tract. Also, 2 exhibited cytotoxic and selective effects against B16-F10 (8.07 ± 1.61 µM) but 4.6- and 17-fold lesser activity than etoposide and taxol.

Further galphimines from a new population of Galphimia glauca.

Both DNA barcoding and phylogenetic data of the studied botanical material suggested the existence a new population of Galphimia glauca. Their leaves afforded three new nor-3,4-seco-friedelanes named galphimines M-O, together with known galphimines D, E, G, and I. Galphimines M and N possess bicyclic orthoacetates which are the first examples of orthoesters found in the Malpighiaceae family, while galphimine O has a 27,20-δ-lactone moiety. The structures elucidation followed from spectroscopic means and the absolute configuration followed from single crystal X-ray diffraction analyses. Tests for antibacterial and antifungal activities of galphimines N and M showed no promising effects.

Configurational Variation of a Natural Compound within Its Source Species. The Unprecedented Case of Areolal in Piptothrix areolare.

The exceptional case of a natural compound that shows drastic absolute configuration variations within the same species was examined. Sequential samples of areolal (1) isolated from Piptothrix areolare showed dextrorotatory (ee 32%), almost racemic (ee 4%), levorotatory (ee 82%), and again dextrorotatory (ee 10%) values. Enantiomeric compositions of this epoxythymol derivative were determined from individual plant specimens collected from the same geographical location over a 46-day period, which were processed using the same extraction and isolation methods. Detection of this unusual phenomenon was possible by analysis of NMR data recorded in the presence of BINOL as a chiral solvating agent. The absolute configuration of (-)-(8S)-areolal followed from vibrational circular dichroism data of an enantiomerically enriched sample, while single-crystal X-ray diffraction and supramolecular analyses revealed interactions that diminish the crystal entropy in rac-1. These results might be related with environmental factors and biochemical processes, suggesting the need of strict evaluations of enantiomeric composition of natural products that could be considered for human applications.

Novel Sesquiterpene Skeletons by Multiple Wagner-Meerwein Rearrangements of a Longipinane-1,9-diol Derivative.

The tricyclic sesquiterpene (1R,3R,4S,5S,7S,8S,9S,10R,11R)-7,8-diangeloyloxylongipinan-1,9-diol, or rasteviol (7), underwent multiple Wagner-Meerwein molecular rearrangements and several hydride shifts when treated with Et2O-BF3 to generate the six new compounds (1R,3R,4S,5R,7S,8S,9S,10R,11S)-7,8-diangeloyloxy-1,9-epoxyjiquilpane (8), (1R,3R,4S,5R,7R,8S,9S,11S)-8-angeloyloxy-1,7-epoxyzamor-10(14)-ene (11), (2S,3R,4R,5R,6R,7R,8S,9S,10S)-7,8-diangeloyloxy-6,9-epoxyjanitziane (14), (4R,5R,7S,8S,9S,10S,11S)-7,8-diangeloyloxy-9-hydroxyjiquilp-3(15)-ene (16), (2S,3S,5R,7S,8R,10S,11R)-7,8-diangeloyloxyiratzian-9-one (18), and (2S,3S,5R,10S,11R)-8-angeloyloxyiratzi-7-en-9-one (22), of which 8, 11, 14, and 18 possess new hydrocarbon skeletons. Their structures were determined by 1D and 2D NMR in combination with single-crystal X-ray diffraction analyses of derivatives 10, 15, 20, and 21, which allowed confirmation of their absolute configurations by means of the Flack and Hooft parameters. In addition, some reaction mechanism information was gained from deuterium labeling experiments.

Conformational, configurational, and supramolecular studies of podocephalol acetate from Lasianthaea aurea.

Although podocephalol (1) and its derived acetate 2 were found in Lasianthaea podocephala four decades ago, and 1 was later detected in the essential oils of several vegetal species, its absolute configuration (AC) and conformational preferences remained to be established. The structures of ar-himachalene 1, now isolated from Lasianthaea aurea, and its derived acetate 2, were herein confirmed by extensive 1D and 2D nuclear magnetic resonance (NMR) studies, while the conformational preferences of the cycloheptene was established by density functional theory (DFT) calculations, which in combination with vibrational circular dichroism measurements provided the (R) absolute configuration of the molecules. The structure and AC were further verified through the Flack and Hooft parameters calculations derived from single crystal X-ray diffraction data of 2. In addition, careful evaluation of the crystal data allowed observing supramolecular layers cell package, an uncommon property in natural terpenes that might have potential applications. A transmission electron microscopy analysis of crystal of 2 was also possible, providing its physical characteristics at the micrometric scale.

Absolute configuration of phomactatriene diterpenoids obtained by Wagner-Meerwein rearrangement of epimeric verticillols.

The epimeric diterpenes (+)-(1S,3E,7E,11S,12S)-verticilla-3,7-dien-12-ol (1), isolated from Bursera suntui, and (+)-(1S,3E,7E,11S,12R)-verticilla-3,7-dien-12-ol (2), isolated from Bursera kerberi, gave the same Wagner-Meerwein rearrangement product (-)-(1E,4Z,8Z,11S,12R)-phomacta-1,(15)4,8-triene (3). The Et2 O:BF3 -induced transformations evidence that verticillenes and phomactanes, both containing the bicyclo[9.3.1]pentadecane skeleton, are biogenetically related through the verticillen-12-yl cation (A+ ), which also is a key intermediate in the biosynthetic pathways to generate antitumor taxanes. Molecular modeling using the Monte Carlo protocol, followed by density functional theory (DFT) geometry optimization employing the hybrid functionals B3LYP and B3PW91, both with the DGDZVP basis set, secured the configuration of 3 as followed from the good agreement between the calculated and experimental vibrational circular dichroism spectra. Similar DFT calculations allowed determining the absolute configuration of (+)-(1R,4R,5R,8S,9S,11S,12R,15R)-1,15:4,5:8,9-triepoxyphomactane (9), which surprisingly derives from epoxidation of the second minimum energy conformer of 3.

In search of safe pain relief: The analgesic and anti-inflammatory activity of phytosteryl ibuprofenates.

ß-Sitosteryl (S)-ibuprofenate (2), stigmasteryl (S)-ibuprofenate (3), ergosteryl (S)-ibuprofenate (4), and cholesteryl (S)-ibuprofenate (5) were prepared in 70-75% yields by Steglich esterification and were characterized by 1D and 2D NMR, as well as by MS. The new esters were evaluated in in vivo pain models of antinociception and anti-inflammation using the writhing, formalin, and carrageenan tests, in mice and rats, and the results were compared with those of (S)-ibuprofen (1). Damage to the gastric mucosa of animals was also assessed. The results indicated that 2-5 have comparable or eventually better activity than 1 at the same mg/kg doses. Since the molecular weight ratio of esters 2-5 to ibuprofen is about 3-1, the amount of truly incorporated ibuprofen was roughly one third to achieve similar effects. This resulted in minimal gastrointestinal damage in the stomach of the animals, in contrast to the large gastric injury caused by (S)-ibuprofen.

Structure and Absolute Configuration of Abietane Diterpenoids from Salvia clinopodioides: Antioxidant, Antiprotozoal, and Antipropulsive Activities.

The aerial parts of Salvia clinopodioides afforded abietanes 1a, 2a, and 3 (clinopodiolides A-C), two of which possess an unusual lactol moiety at C-19-C-20, together with an icetexane named clinopodiolide D (4a). Their structures were established by spectroscopic means, mainly 1H and 13C NMR, including 1D and 2D homo- and heteronuclear experiments. The antioxidant, antiprotozoal, and antidiarrheal effects of the isolates were evaluated. Compounds 2a and 3 showed better effects than α-tocopherol in the inhibition of lipid peroxidation with IC50 (µM) = 5.9 ± 0.1 and 2.7 ± 0.2, respectively, and moderate activity in the DPPH assay. All tested compounds showed moderate antiamoebic and antigiardial activity, as well as a good antipropulsive effect.

Parvifoline Derivatives as Tubulin Polymerization Inhibitors.

A series of functionalized sesquiterpenoids derived from benzocyclooctene, including natural parvifoline (1), isoparvifoline (3), epoxyparvifoline (5), epoxyisoparvifoline (7), 8,12-oxyparfivoline (9), 8,14-oxyparvifoline (11), and the respective benzoyl derivatives 2, 4, 6, 8, 10, and 12, were prepared and tested for their inhibitory effect on the in vitro α,ß-tubulin polymerization process. The structural analysis and characterization of the new compounds 5-7 and 9-12 were achieved by 1D and 2D NMR spectroscopy, mass spectrometry, and X-ray diffraction analysis of 6, 7, and 9. Preparation of 9 and 12 involved molecular rearrangements of the epoxide group with transannular 1,5-hydride shifts. At 10 µM compounds 1, 5, and 8 inhibited the polymerization of the α,ß-tubulin heterodimer by 24%, 49%, and 90% as compared to colchicine. These compounds were subjected to docking analysis that supported their interactions in a colchicine binding site located in the α-tubulin subunit, in the pocket formed by Phe296, Pro298, Pro307, His309, Tyr312, Lys338, Thr340, Ile341, and Gln342. Competitive inhibition assays with colchicine were also performed for the three compounds, which supported their binding at the colchicine secondary site in α-tubulin. Also, evaluations of their cytotoxicity on MCF7 breast carcinoma, HeLa cervix carcinoma, and HCT 116 colon carcinoma cell lines were carried out and showed that 8 is active against the HeLa and HCT 116 cell lines with IC50 3.3 ± 0.2 and 5.0 ± 0.5 µM, respectively.

Biomimetic Transformation of p-Menthene Glucosides into p-Cymenes and Carvotanacetone.

A biomimetic transformation of p-menthene glucosides into aromatic monoterpenoids that alluded to mechanisms for essential oil metabolism, which lines up with the precepts of molecular economy, is described. Acid treatment of (-)-(3 S,4 S,6 R)-3,6-dihydroxy-1-menthene 3- O-ß-d-glucopyranoside (1) and (-)-(3 S,4 R,5 R,6 S)-3,5,6-trihydroxy-1-menthene 3- O-ß-d-glucopyranoside (2), from Ageratina glabrata, yielded p-cymene (7) and carvacrol (9). The stable oxidized intermediates (+)-(3 S,4 S,6 R)-3,6-dihydroxy-1-menthene (3), (+)-(1 S,4 S,6 R)-1,6-dihydroxy-2-menthene (4), (+)-(1 R,4 S,6 R)-1,6-dihydroxy-2-menthene (5), (+)-(4 S,6 R)-yabunikkeol (6), (+)-(4 S)-carvotanacetone (8), (+)-(1 S,4 S,5 R,6 R)-1,5,6-trihydroxy-2-menthene (15), (+)-(1 R,4 S,5 R,6 R)-1,5,6-trihydroxy-2-menthene (16), and the new (+)-(4 S,5 R,6 S)-1(7),2-menthadiene (17) permitted establishment of the reaction mechanisms. The reactivity of the hydroxy groups of 4 and 5, as well as those of 15 and 16, was compared by acetylation reactions and supported by DFT calculations, revealing diminished reactivity in 4 and 15 due to the cis configuration of their hydroxy groups at C-1 and C-6. In addition, p-cymene (7) was detected as one of the major constituents of the essential oil of A. glabrata, which matches well with the biomimetic study.

Complete 1H NMR assignment of cholesteryl benzoate.

The 750 MHz 1H NMR spectrum of cholesteryl benzoate (1b) could be assigned completely, which means all chemical shifts and all coupling constants, including some long-range values, were established. This task was possible by extracting many approximate coupling constant values in the overlapped spectrum region from an HSQC experiment, and using these values in the 1H iterative full spin analysis integrated in the PERCH NMR software. The task was facilitated using our published data for 3ß-acetoxypregna-5,16-dien-20-one (3), the assignment data of the sesquiterpene benzoquinone dihydroperezone (2), also performed in the present study, which contains the same carbon atoms chain than cholesterol (1a), and an HSQC study of (25R)-27-deuteriocholesterol (1c) we prepared some 40 years ago. The HSQC values of 1c in combination with the coupling constants of 1b also allowed to completely assigning the spectrum of 1c. The complete assignment of 1b and 1c further provided the opportunity to estimate the hydrogen shifts induced upon benzoylation of cholesterol. Comparison of the experimental vicinal coupling constants of 1b with the values calculated using the Altona software provides an excellent correlation. In addition, a single crystal X-ray diffraction study of 1b provided the molecular conformation in the solid state, which revealed the side chain adopts an extended conformation.